Emmanuel Katsanis

Interests

Research

Tumor and Transplant Immunology, Hematopoietic Cell Transplantation

Courses

Scholarly Contributions

Chapters

• Ramos, K., & Katsanis, E. (2020). Recent advances in haploidentical hematopoietic cell transplantation for pediatric hematologic malignancies. In Cancer Immunology: Bench to Bedside Immunotherapy of Cancers(pp 157-168). Switzerland: Springer Nature. doi:doi: 10.1002/jha2.20

Journals/Publications

• Smith, K. A., Zúñiga, T. M., Baker, F. L., Batatinha, H., Pedlar, C. R., Burgess, S. C., Gustafson, M. P., Katsanis, E., & Simpson, R. J. (2024). COVID-19 vaccination produces exercise-responsive SARS-CoV-2 specific T-cells regardless of infection history. Journal of sport and health science, 13(1), 99-107.
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  The mobilization and redistribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specific T-cells and neutralizing antibodies (nAbs) during exercise is purported to increase immune surveillance and protect against severe coronavirus disease 2019 (COVID-19). We sought to determine if COVID-19 vaccination would elicit exercise-responsive SARS-CoV-2 T-cells and transiently alter nAb titers.
• Baker, F. L., Zuniga, T. M., Smith, K. A., Batatinha, H., Kulangara, T., Seckeler, M., Burgess, S. C., Katsanis, E., & Simpson, R. (2023). Exercise mobilizes diverse antigen specific T-cells and elevates neutralizing antibodies in humans with natural immunity to SARS CoV-2. Brain, Behavior, and Immunity – Health.
• Baker, F. L., Zúñiga, T. M., Smith, K. A., Batatinha, H., Kulangara, T. S., Seckeler, M. D., Burgess, S. C., Katsanis, E., & Simpson, R. J. (2023). Exercise mobilizes diverse antigen specific T-cells and elevates neutralizing antibodies in humans with natural immunity to SARS CoV-2. Brain, behavior, & immunity - health, 28, 100600.
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  Epidemiological data suggest that physical activity protects against severe COVID-19 and improves clinical outcomes, but how exercise augments the SARS-CoV-2 viral immune response has yet to be elucidated. Here we determine the antigen-specific CD4 and CD8 T-cell and humoral immunity to exercise in non-vaccinated individuals with natural immunity to SARS CoV-2, using whole-blood SARS-CoV-2 peptide stimulation assays, IFN-γ ELISPOT assays, 8-color flow cytometry, deep T-cell receptor (TCR) β sequencing, and anti-RBD-1 neutralizing antibody serology. We found that acute exercise reliably mobilized (∼2.5-fold increase) highly functional SARS-CoV-2-specific T-cells to the blood compartment in those with natural immunity to the virus. The mobilized cells reacted with spike protein (including alpha (α) and delta (δ)-variants), membrane, and nucleocapsid peptides in those previously infected but not in controls. Both groups reliably mobilized T-cells reacting with Epstein-Barr viral peptides. Exercise mobilized SARS-CoV-2 specific T-cells maintained broad TCR-β diversity with no impact on CDR3 length or V and J family gene usage. Exercise predominantly mobilized MHC I restricted (i.e. CD8) SARS-CoV-2 specific T-cells that recognized ORF1ab, surface, ORF7b, nucleocapsid, and membrane proteins. SARS-CoV-2 neutralizing antibodies were transiently elevated ∼1.5-fold during exercise after infection. In conclusion, we provide novel data on a potential mechanism by which exercise could increase SARS-CoV-2 immunosurveillance via the mobilization and redistribution of antigen-specific CD8 T-cells and neutralizing antibodies. Further research is needed to define the tissue specific disease protective effects of exercise as SARS-CoV-2 continues to evolve, as well as the impact of COVID-19 vaccination on this response.
• Barker, K., Koza, S., Katsanis, E., & Husnain, M. (2023). Hypophosphatemia and pre-infusion thrombocytopenia as biomarkers for CRS and ICANS after CAR T therapy. Bone marrow transplantation, 58(11), 1267-1269.
• Batatinha, H., Batatinha, H., Diak, D., Diak, D., Niemiro, G., Niemiro, G., Baker, F. L., Baker, F. L., Smith, K. A., Smith, K. A., Zuniga, T. M., Zuniga, T. M., Seckeler, M., Seckeler, M., Lau, B., Lau, B., LaVoy, E., LaVoy, E., Gustafson, M., , Gustafson, M., et al. (2023). Human lymphocytes mobilized with exercise have an anti-tumor transcriptomic profile and exert enhanced graft-versus-leukemia effects in xenogeneic mice. Frontiers in Immunology.
• Batatinha, H., Diak, D. M., Niemiro, G. M., Baker, F. L., Smith, K. A., Zúñiga, T. M., Mylabathula, P. L., Seckeler, M. D., Lau, B., LaVoy, E. C., Gustafson, M. P., Katsanis, E., & Simpson, R. J. (2023). Human lymphocytes mobilized with exercise have an anti-tumor transcriptomic profile and exert enhanced graft-versus-leukemia effects in xenogeneic mice. Frontiers in immunology, 14, 1067369.
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  Every bout of exercise mobilizes and redistributes large numbers of effector lymphocytes with a cytotoxic and tissue migration phenotype. The frequent redistribution of these cells is purported to increase immune surveillance and play a mechanistic role in reducing cancer risk and slowing tumor progression in physically active cancer survivors. Our aim was to provide the first detailed single cell transcriptomic analysis of exercise-mobilized lymphocytes and test their effectiveness as a donor lymphocyte infusion (DLI) in xenogeneic mice engrafted with human leukemia.
• Eylon, M., Prabhu, S., John, S., King, M. J., Bhatt, D., Curran, K. J., Erickson, C., Karras, N. A., Phillips, C. L., Satwani, P., Hermiston, M., Southworth, E., Baumeister, S. H., Talano, J. A., MacMillan, M. L., Rossoff, J., Bonifant, C. L., Myers, G. D., Rouce, R. H., , Toner, K., et al. (2023). Mediport use as an acceptable standard for CAR T cell infusion. Frontiers in immunology, 14, 1239132.
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  Mediport use as a clinical option for the administration of chimeric antigen receptor T cell (CAR T cell) therapy in patients with B-cell malignancies has yet to be standardized. Concern for mediport dislodgement, cell infiltration, and ineffective therapy delivery to systemic circulation has resulted in variable practice with intravenous administration of CAR T cell therapy. With CAR T cell commercialization, it is important to establish practice standards for CAR T cell delivery. We conducted a study to establish usage patterns of mediports in the clinical setting and provide a standard of care recommendation for mediport use as an acceptable form of access for CAR T cell infusions.
• Fox, R. S., Armstrong, G. E., Gaumond, J. S., Vigoureux, T. F., Miller, C. H., Sanford, S. D., Salsman, J. M., Katsanis, E., Badger, T. A., Reed, D. R., Gonzalez, B. D., Jim, H. S., Warner, E. L., Victorson, D. E., & Oswald, L. B. (2023). Social isolation and social connectedness among young adult cancer survivors: A systematic review. Cancer, 129(19), 2946-2965.
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  Social isolation and connectedness are social determinants of health that have demonstrated effects on cancer-related outcomes. These constructs have been systematically evaluated among pediatric and older adult cancer populations. In this review, the authors evaluated the prevalence, correlates, and psychosocial implications of social isolation and connectedness among young adult (YA) cancer survivors aged 18-39 years.
• Gilman, K. E., Matiatos, A. P., Cracchiolo, M. J., Moon, A. G., Davini, D. W., Simpson, R. J., & Katsanis, E. (2023). Multiagent Intratumoral Immunotherapy Can Be Effective in A20 Lymphoma Clearance and Generation of Systemic T Cell Immunity. Cancers, 15(7).
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  The use of immunotherapies has shown promise against selective human cancers. Identifying novel combinations of innate and adaptive immune cell-activating agents that can work synergistically to suppress tumor growth and provide additional protection against resistance or recurrence is critical. The A20 murine lymphoma model was used to evaluate the effect of various combination immunotherapies administered intratumorally. We show that single-modality treatment with Poly(I:C) or GM-CSF-secreting allogeneic cells only modestly controls tumor growth, whereas when given together there is an improved benefit, with 50% of animals clearing tumors and surviving long-term. Neither heat nor irradiation of GM-CSF-secreting cells enhanced the response over use of live cells. The use of a TIM-3 inhibitory antibody and an OX40 agonist in combination with Poly(I:C) allowed for improved tumor control, with 90% of animals clearing tumors with or without a combination of GM-CSF-secreting cells. Across all treatment groups, mice rejecting their primary A20 tumors were immune to subsequent challenge with A20, and this longstanding immunity was T-cell dependent. The results herein support the use of combinations of innate and adaptive immune activating agents for immunotherapy against lymphoma and should be investigated in other cancer types.
• Truscott, L., Pariury, H., Hanmod, S., Davini, M., de la Maza, M., Sapp, L. N., Staples, K., Proytcheva, M., & Katsanis, E. (2023). Busulfan, fludarabine, and melphalan are effective conditioning for pediatric and young adult patients with myeloid malignancies underdoing matched sibling or alternative donor transplantation. Pediatric blood & cancer, 70(2), e30102.
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  Allogeneic hematopoietic cell transplantation (allo-HCT) remains a curative option for patients with high-risk myeloid malignancies.
• Wu, C., Manchen, P., Edelman, A., Husnain, M., Katsanis, E., Fuchs, D., Stephens, L., & Khurana, S. (2023). Refractory Pure Red Blood Cell Aplasia Secondary to Major ABO-Incompatible Allogeneic Stem Cell Transplantation Successfully Treated With Daratumumab. Journal of hematology, 12(6), 277-282.
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  Pure red cell aplasia (PRCA) is a rare hematologic phenomenon that is usually associated with inherited genetic mutations such as in Diamond-Blackfan anemia. However, due to the emergence of allogenic stem cell transplantation in the treatment of various malignant and non-malignant disorders, the incidence of PRCA has increased. PRCA following hematopoietic stem cell transplant (HSCT) is more commonly seen in the setting of a major ABO-incompatible transplant. Treatment of allo-HSCT induced PRCA can be initially supportive as it takes time for the bone marrow to fully recover. However, prolonged and/or failure of the bone marrow to recover, significantly increases patient's risk of iron overload in the setting of frequent transfusions. Iron deposition can potentially lead to severe life-threatening multiorgan involvement which can be fatal. Therefore, earlier recognition and intervention with immunomodulators in patients who undergo frequent transfusions can be beneficial to mitigate this risk. Here, we present a case with severe transfusion-dependent PRCA following major ABO-incompatible allo-HSCT successfully treated with daratumumab.
• Zuniga, T. M., Baker, F. L., Lau, B., Gustafson, M. P., Katsanis, E., & Simpson, R. (2023). Clonal kinetics and single-cell transcriptional profiles of T-cells mobilized to blood by acute exercise. Medicine & Science in Sports & Exercise.
• Zúñiga, T. M., Baker, F. L., Smith, K. A., Batatinha, H., Lau, B., Burgess, S. C., Gustafson, M. P., Katsanis, E., & Simpson, R. J. (2023). Clonal Kinetics and Single-Cell Transcriptional Profiles of T Cells Mobilized to Blood by Acute Exercise. Medicine and science in sports and exercise, 55(6), 991-1002.
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  Acute exercise redistributes large numbers of memory T cells, which may contribute to enhanced immune surveillance in regular exercisers. It is not known, however, if acute exercise promotes a broad or oligoclonal T-cell receptor (TCR) repertoire or evokes transcriptomic changes in "exercise-responsive" T-cell clones.
• Gilman, K. E., Cracchiolo, M. J., Matiatos, A. P., Davini, D. W., Simpson, R. J., & Katsanis, E. (2022). Partially replacing cyclophosphamide with bendamustine in combination with cyclosporine A improves survival and reduces xenogeneic graft-versus-host-disease. Frontiers in immunology, 13, 1045710.
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  The use of allogeneic hematopoietic cell transplantation (allo-HCT) for treating hematological disorders is increasing, but the development of graft-versus-host disease (GvHD) remains a major cause of morbidity and mortality. The use of post-transplant cyclophosphamide (CY) has significantly improved outcomes following allo-HCT, but complications of viral reactivation due to delayed immune reconstitution or relapse remain. Other laboratories are evaluating the potential benefit of lowering the dose of CY given post-transplant, whereas our laboratory has been focusing on whether partially replacing CY with another DNA alkylating agent, bendamustine (BEN) may be advantageous in improving outcomes with allo-HCT.
• Katsanis, E., Hanley, P. J., & Simpson, R. J. (2022). Editorial: Advances in Pediatric Hematopoietic Cell Therapies and Transplantation. Frontiers in pediatrics, 10, 847288.
• Katsanis, E., Hennig, T., Robinson, J. E., Long, G., Chao, N., Horwitz, M., & Rizzieri, D. (2022). Revisiting a single day salvage conditioning following graft failure. Bone marrow transplantation, 57(12), 1845-1847.
• Katsanis, E., Stea, B., Kovacs, K., Truscott, L., Husnain, M., Khurana, S., Roe, D. J., & Simpson, R. J. (2022). Feasibility and Efficacy of Partially Replacing Post-Transplantation Cyclophosphamide with Bendamustine in Pediatric and Young Adult Patients Undergoing Haploidentical Bone Marrow Transplantation. Transplantation and cellular therapy, 28(7), 390.e1-390.e10.
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  Post-transplantation cyclophosphamide (PT-CY) is the most widely applied graft-versus-host disease (GVHD) prophylaxis regimen in T-cell replete haploidentical bone marrow transplantation (haplo-BMT). Although PT-CY has met with great success in the haplo-BMT arena by suppressing GVHD, patients without acute GVHD have high relapse rates. One strategy to reduce relapse rates being explored by others is a dosage reduction of PT-CY. We have taken a different approach in evaluating whether partially replacing PT-CY with post-transplantation bendamustine (PT-BEN) would be advantageous, an idea based on our preclinical research identifying several beneficial immunomodulatory properties of BEN. We therefore initiated and completed a Phase Ia trial to evaluate the progressive substitution of PT-CY with PT-BEN (ClinicalTrials.gov identifier NCT02996773). We compared outcomes between 13 patients with high-risk hematologic malignancies who received PT-CY/BEN and 31 contemporaneous haplo-BMT recipients treated with the same myeloablative conditioning regimens but receiving only PT-CY. We found that partial replacement of PT-CY with PT-BEN (PT-CY/BEN) on day +4 was well tolerated and associated with significantly earlier trilineage engraftment. We also report favorable trends toward significant improvements on univariate and multivariate analyses with PT-CY/BEN compared with PT-CY with respect to rates of chronic GVHD (hazard ratio [HR], .08; 95% confidence interval [CI], .005 to 1.11; P = .06), and GVHD-free relapse-free survival (GRFS) (HR, .22; 95% CI, .05 to .86; P = .039). Our human trial has now transitioned to Phase Ib, which will further evaluate the safety and potential benefits of PT-CY/BEN. Herein we also expand our pediatric, adolescent, and young adult experience to 31 patients, demonstrating overall survival, progression-free survival, and GRFS at 3 years of 85.6%, 76.1%, and 58.2%, respectively, in a largely racial/ethnic minority cohort. PT-CY/BEN appears to be a promising treatment option that requires further evaluation.
• Molina, M. S., Hoffman, E. A., Stokes, J., Kummet, N., Simpson, R. J., & Katsanis, E. (2022). Murine precursors to type 1 conventional dendritic cells induce tumor cytotoxicity and exhibit activated PD-1/PD-L1 pathway. PloS one, 17(8), e0273075.
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  The immediate precursor to murine type 1 conventional DCs (cDC1s) has recently been established and named "pre-cDC1s". Mature CD8α+ cDC1s are recognized for suppressing graft-versus-host disease (GvHD) while promoting graft-versus-leukemia (GvL), however pre-cDC1s have not previously been investigated in the context of alloreactivity or anti-tumor responses. Characterization of pre-cDC1s, compared to CD8α+ cDC1s, found that a lower percentage of pre-cDC1s express PD-L1, yet express greater PD-L1 by MFI and a greater percent PIR-B, a GvHD-suppressing molecule. Functional assays were performed ex vivo following in vivo depletion of CD8α+ DCs to examine whether pre-cDC1s play a redundant role in alloreactivity. Proliferation assays revealed less allogeneic T-cell proliferation in the absence of CD8α+ cDC1s, with slightly greater CD8+ T-cell proliferation. Further, in the absence of CD8α+ cDC1s, stimulated CD8+ T-cells exhibited significantly less PD-1 expression compared to CD4+ T-cells, and alloreactive T-cell death was significantly lower, driven by reduced CD4+ T-cell death. Tumor-killing assays revealed that T-cells primed with CD8α-depleted DCs ex vivo induce greater killing of A20 B-cell leukemia cells, particularly when antigen (Ag) is limited. Bulk RNA sequencing revealed distinct transcriptional programs of these DCs, with pre-cDC1s exhibiting activated PD-1/PD-L1 signaling compared to CD8α+ cDC1s. These results indicate distinct T-cell-priming capabilities of murine pre-cDC1s compared to CD8α+ cDC1s ex vivo, with potentially clinically relevant implications in suppressing GvHD while promoting GvL responses, highlighting the need for greater investigation of murine pre-cDC1s.
• Mylabathula, P. L., Diak, D. M., Baker, F. L., Niemiro, G. M., Markofski, M. M., Crucian, B. E., Katsanis, E., & Simpson, R. J. (2022). IL-2 and Zoledronic Acid Therapy Restores the Anti-Leukemic Activity of Human Lymphocytes Pre-Exposed to Simulated Microgravity. Frontiers in bioscience (Landmark edition), 27(7), 215.
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  We have previously shown that the anti-tumor activity of human lymphocytes is diminished after 12-hours pre-exposure to simulated microgravity (SMG). Here we used an immunocompromised mouse model to determine if this loss of function would extend , and to also test the efficacy of IL-2 and zoledronic acid (ZOL) therapy as a potential countermeasure against SMG-induced immune dysfunction. We adoptively transferred human lymphocytes that were exposed to either SMG or 1G-control into NSG-Tg (Hu-IL15) mice 1-week after they were injected with a luciferase-tagged human chronic myeloid leukemia (K562) cell line. Tumor growth was monitored 2x weekly with bioluminescence imaging (BLI) for up to 6-weeks.
• Niemiro, G. M., Coletta, A. M., Agha, N. H., Mylabathula, P. L., Baker, F. L., Brewster, A. M., Bevers, T. B., Fuentes-Mattei, E., Basen-Engquist, K., Katsanis, E., Gilchrist, S. C., & Simpson, R. J. (2022). Correction: Salutary effects of moderate but not high intensity aerobic exercise training on the frequency of peripheral T-cells associated with immunosenescence in older women at high risk of breast cancer: a randomized controlled trial. Immunity & ageing : I & A, 19(1), 30.
• Niemiro, G. M., Coletta, A. M., Agha, N. H., Mylabathula, P. L., Baker, F. L., Brewster, A. M., Bevers, T. B., Fuentes-Mattei, E., Basen-Engquist, K., Katsanis, E., Gilchrist, S. C., & Simpson, R. J. (2022). Salutary effects of moderate but not high intensity aerobic exercise training on the frequency of peripheral T-cells associated with immunosenescence in older women at high risk of breast cancer: a randomized controlled trial. Immunity & ageing : I & A, 19(1), 17.
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  Immunosenescence is described as age-associated changes within the immune system that are responsible for decreased immunity and increased cancer risk. Physically active individuals have fewer 'senescent' and more naïve T-cells compared to their sedentary counterparts, but it is not known if exercise training can rejuvenate 'older looking' T-cell profiles. We determined the effects of 12-weeks supervised exercise training on the frequency of T-cell subtypes in peripheral blood and their relationships with circulating levels of the muscle-derived cytokines (i.e. 'myokines') IL-6, IL-7, IL-15 and osteonectin in older women at high risk of breast cancer. The intervention involved 3 sessions/week of either high intensity interval exercise (HIIT) or moderate intensity continuous exercise (MICT) and were compared to an untrained control (UC) group.
• Oswald, L. B., Lyleroehr, M., Gudenkauf, L. M., Armstrong, G. E., Tometich, D. B., Sanford, S. D., Loecher, N., Geiss, C., Rodriguez, Y., Scheel, K. L., Nieves-Lopez, A., Jim, H. S., Gonzalez, B. D., Antoni, M. H., Penedo, F. J., Reed, D., Katsanis, E., Salsman, J. M., Victorson, D., & Fox, R. S. (2022). Development and initial testing of TOGETHER-YA: an eHealth-delivered and group-based psychosocial intervention for young adult cancer survivors. Supportive care in cancer : official journal of the Multinational Association of Supportive Care in Cancer, 30(12), 10067-10076.
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  This study aimed to (1) develop TOGETHER-YA, an e-Health-delivered and group-based health-related quality of life (HRQOL) intervention for young adult (YA) cancer survivors aged 18-39 (Part 1), and (2) determine its initial feasibility and acceptability in a single-arm pilot trial (Part 2).
• Rangarajan, H. G., Stanek, J. R., Abdel-Azim, H., Modi, A., Haight, A., McKinney, C. M., McKeone, D. J., Buchbinder, D. K., Katsanis, E., Abusin, G. A., Ahmed, I., Law, J., Silva, J. G., Mallhi, K. K., Burroughs, L. M., Shah, N., Shaw, P. J., Greiner, R., Shenoy, S., , Pulsipher, M. A., et al. (2022). Hematopoietic Cell Transplantation for Congenital Dyserythropoietic Anemia: A Report from the Pediatric Transplant and Cellular Therapy Consortium. Transplantation and cellular therapy, 28(6), 329.e1-329.e9.
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  Hematopoietic cell transplantation (HCT) is the sole curative option for congenital dyserythropoietic anemia (CDA), a rare type of hemolytic anemia characterized by anemia, ineffective erythropoiesis, and secondary hemochromatosis. In this retrospective multicenter study, we report the outcomes of children with CDA who underwent HCT at participating Pediatric Transplantation and Cellular Therapy Consortium centers. Clinical information on HCT and associated outcomes was collected retrospectively using a common questionnaire. Data were analyzed using descriptive statistics and appropriate analysis. Eighteen patients with CDA who underwent allogeneic HCT between 2002 and 2020 were identified. The majority of patients (n = 13) had CDA type II, and the remainder had either CDA type I (n = 2) or CDA of unknown type (n = 3). Mutations were identified in 7 patients (39%), including SEC23B in 5, GATA1 in 1, and abnormality of chromosome 20 in 1. Thirteen patients had evidence of iron overload pre-HCT and received chelation therapy for a median duration of 10 months (range, 2 months to 17 years) pre-HCT. The median age at the time of HCT was 5.5 years (range, 0.7 to 26 years). Donors were HLA-matched (sibling, 4; unrelated, 10) and mismatched (haploidentical, 1; unrelated, 3). Graft sources were bone marrow in 15 patients, umbilical cord blood in 2 patients, or both in 1 patient. Conditioning included busulfan-based myeloablative (67%), fludarabine-based reduced-intensity (27%), or nonmyeloablative (6%) regimens. Five patients developed veno-occlusive disease, and 4 had viral reactivation. The cumulative incidence of acute graft-versus-host disease (GVHD) was 33%, and that of chronic GVHD was 22%. Four patients (22%) experienced graft failure; all engrafted following either a second HCT (n = 2) or third HCT (n = 2) but sustained considerable morbidities (3 GVHD, 1 death, 2 viral reactivation). With a median follow-up of 3.2 years (range, 0.6 to 14 years)), the 2-year overall survival, event-free survival (EFS), and GVHD-free EFS were 88% (95% confidence interval [CI], 73% to 100%), 65% (95% CI, 45% to 92%), and 60% (95% CI, 40% to 88%), respectively. Univariate analysis did not identify any patient- or transplantation-related variables impacting outcomes. Our study indicates that HCT can be curative for patients with CDA. Strategies such as aggressive chelation, use of preconditioning therapy, and early HCT in the presence of a suitable donor before comorbidities occur are needed to improve engraftment without increasing the risk for toxicity and mortality.
• Staples, K., Sapp, L. N., Truscott, L., Pariury, H., Hanmod, S., Davini, M., de la Maza, M., Proytcheva, M., Katsanis, E., Staples, K., Sapp, L. N., Truscott, L., Pariury, H., Hanmod, S., Davini, M., de la Maza, M., Proytcheva, M., & Katsanis, E. (2022). Busulfan, fludarabine, and melphalan are effective conditioning for pediatric and young adult patients with myeloid malignancies underdoing matched sibling or alternative donor transplantation. Pediatric Blood & Cancer, 70(2). doi:10.1002/pbc.30102
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  Abstract: Background Allogeneic hematopoietic cell transplantation (allo-HCT) remains a curative option for patients with high-risk myeloid malignancies. Procedure We present our 10-year experience (October 2012 to October 2021) of consecutive allo-HCT in patients with myeloid malignancies treated on the pediatric HCT service and conditioned with myeloablative targeted dose—busulfan (BU), fludarabine (FLU), and melphalan (MEL). Twenty-three children, adolescents, and young adult patients (CAYA) (median age 15.4 years) with acute myeloid leukemia (AML, n = 17), myelodysplastic syndrome (MDS, n = 4), or chronic myeloid leukemia (CML, n = 2) underwent allo-HCT post-BU-FLU-MEL. Four patients had treatment-related AML/MDS. Donor/stem cell source was matched sibling donor (MSD) PBSC (n = 7), matched unrelated donor (MUD) PBSC (n = 2), umbilical cord blood (UCB) (n = 3), or haploidentical-BMT (n = 11). Risk stratification was low (n = 2), intermediate (n = 15), high (n = 3), and very high risk (n = 1). The two patients with CML had failed tyrosine kinase inhibitor therapies. Results With a median follow-up of 41.6 months, the relapse rate is only 4.5% with an overall survival (OS) 100%, progression-free survival (PFS) 95.5%, and graft-versus-host-free-relapse-free survival (GRFS) 67.8%. The donor source and the acute graft-versus-host disease (GvHD) prophylaxis regimen significantly impacted grade II–IV aGvHD 66.7% versus 19.2% (p = .039) and chronic graft-versus-host-disease (cGvHD) 66.7% versus 0% (p = .002) in the patients receiving MSD or MUD PBSC compared to haplo-BMT, respectively, resulting in improved GRFS in haplo-BMT, 83.3% compared to 40% matched donor peripheral blood stem cell transplant (PBSCT) (p = .025). Conclusions Our results demonstrate that BU-FLU-MEL is efficacious conditioning for disease control in young patients with myeloid malignancies undergoing MSD or alternative donor allo-HCT, but in the setting of PBSC grafts with cyclosporine A-methotrexate (CSA-MTX) GvHD prophylaxis, it results in an unacceptably high incidence of GvHD.
• Stokes, J., Simpson, R. J., & Katsanis, E. (2022). Commentary: Post-Transplantation Cyclophosphamide Uniquely Restrains Alloreactive CD4+ T-Cell Proliferation and Differentiation After Murine MHC-Haploidentical Hematopoietic Cell Transplantation. Frontiers in immunology, 13, 887648.
• Zúñiga, T. M., Baker, F. L., Smith, K. A., Batatinha, H., Lau, B., Gustafson, M. P., Katsanis, E., & Simpson, R. J. (2022). Acute exercise mobilizes NKT-like cells with a cytotoxic transcriptomic profile but does not augment the potency of cytokine-induced killer (CIK) cells. Frontiers in immunology, 13, 938106.
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  CD3/CD56 Natural killer (NK) cell-like T-cells (NKT-like cells) represent
• Baker, F. L., Smith, K. A., Zúñiga, T. M., Batatinha, H., Niemiro, G. M., Pedlar, C. R., Burgess, S. C., Katsanis, E., & Simpson, R. J. (2021). Acute exercise increases immune responses to SARS CoV-2 in a previously infected man. Brain, behavior, & immunity - health, 18, 100343.
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  Evidence is emerging that exercise and physical activity provides protection against severe COVID-19 disease in patients infected with SARS-CoV-2, but it is not known how exercise affects immune responses to the virus. A healthy man completed a graded cycling ergometer test prior to and after SARS-CoV-2 infection, then again after receiving an adenovirus vector-based COVID-19 vaccine. Using whole blood SARS-CoV-2 peptide stimulation assays, IFN-γ ELISPOT assays, flow cytometry, viral-specific T-cell expansion assays and deep T-cell receptor (TCR) β sequencing, we found that exercise robustly mobilized highly functional SARS-CoV-2 specific T-cells to the blood compartment that recognized spike protein, membrane protein, nucleocapsid antigen and the B.1.1.7 α-variant, and consisted mostly of CD3+/CD8+ T-cells and double-negative (CD4-/CD8-) CD3 T-cells. The magnitude of SARS-CoV-2 T-cell mobilization with exercise was intensity dependent and robust when compared to T-cells recognizing other viruses (e.g. CMV, EBV, influenza). Vaccination enhanced the number of exercise-mobilized SARS-CoV-2 T-cells recognizing spike protein and the α-variant only. Exercise-mobilized SARS-CoV-2 specific T-cells proliferated more vigorously to peptide stimulation and maintained broad TCR-β diversity against SARS-CoV-2 antigens both before and after expansion. Neutralizing antibodies to SARS-CoV-2 were transiently elevated during exercise after both infection and vaccination. Finally, infection was associated with an increased metabolic demand to defined exercise workloads, which was restored to pre-infection levels after vaccination. This case study provides impetus for larger studies to determine if these immune responses to exercise can facilitate viral clearance, ameliorate symptoms of long COVID syndrome, and/or restore functional exercise capacity following SARS-CoV-2 infection.
• Batatinha, H., Baker, F. L., Smith, K. A., Zúñiga, T. M., Pedlar, C. R., Burgess, S. C., Katsanis, E., & Simpson, R. J. (2021). Recent COVID-19 vaccination has minimal effects on the physiological responses to graded exercise in physically active healthy people. Journal of applied physiology (Bethesda, Md. : 1985).
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  Athletes are advised to receive the COVID-19 vaccination to protect them from SARS CoV-2 infection during major competitions. Despite this, many athletes are reluctant to get the COVID-19 vaccine due to concerns that symptoms of vaccinosis may impair athletic performance.
• Gustafson, M. P., Wheatley-Guy, C. M., Rosenthal, A. C., Gastineau, D. A., Katsanis, E., Johnson, B. D., & Simpson, R. J. (2021). Exercise and the immune system: taking steps to improve responses to cancer immunotherapy. Journal for immunotherapy of cancer, 9(7).
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  The remarkable success of cancer immunotherapies has provided new hope to cancer patients. Unfortunately, a significant proportion of patients remain unable to respond to immunotherapy or maintain durable clinical responses. The lack of objective responses likely results from profound immune dysfunction often observed in patients with cancer. There is substantial evidence that exercise and physical activity can reduce incidence and improve outcomes in cancer patients. As the immune system is highly responsive to exercise, one potential avenue to improve immune function is through exercise and physical activity. A single event of dynamic exercise results in the substantial mobilization of leukocytes with increased functional capacities into the circulation. Chronic, or long-term, exercise leads to higher physical fitness in terms of greater cardiorespiratory function and/or muscle strength and endurance. High aerobic capacity, as measured by maximal oxygen uptake, has been associated with the reduction of dysfunctional T cells and improvements in the abundance of some T cell populations. To be sure, however, the mechanisms of exercise-mediated immune changes are both extensive and diverse. Here, we examine the evidence and theorize how acute and chronic exercise could be used to improve responses to cancer immunotherapies including immune checkpoint inhibitors, dendritic cell vaccines, natural killer cell therapies, and adoptive T cell therapies such as chimeric antigen receptor (CAR) T cells. Although the parameters of optimal exercise to yield defined outcomes remain to be determined, the available current data provide a compelling justification for additional human studies and clinical trials investigating the adjuvant use of exercise in immuno-oncology.
• Molina, M. S., Hoffman, E. A., Stokes, J., Kummet, N., Smith, K. A., Baker, F., Zúñiga, T. M., Simpson, R. J., & Katsanis, E. (2021). Regulatory Dendritic Cells Induced by Bendamustine Are Associated With Enhanced Flt3 Expression and Alloreactive T-Cell Death. Frontiers in immunology, 12, 699128.
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  The growth factor Flt3 ligand (Flt3L) is central to dendritic cell (DC) homeostasis and development, controlling survival and expansion by binding to Flt3 receptor tyrosine kinase on the surface of DCs. In the context of hematopoietic cell transplantation, Flt3L has been found to suppress graft-versus-host disease (GvHD), specifically host DCs. We previously reported that the pre-transplant conditioning regimen consisting of bendamustine (BEN) and total body irradiation (TBI) results in significantly reduced GvHD compared to cyclophosphamide (CY)+TBI. Pre-transplant BEN+TBI conditioning was also associated with greater Flt3 expression among host DCs and an accumulation of pre-cDC1s. Here, we demonstrate that exposure to BEN increases Flt3 expression on both murine bone marrow-derived DCs (BMDCs) and human monocyte-derived DCs (moDCs). BEN favors development of murine plasmacytoid DCs, pre-cDC1s, and cDC2s. While humans do not have an identifiable equivalent to murine pre-cDC1s, exposure to BEN resulted in decreased plasmacytoid DCs and increased cDC2s. BEN exposure and heightened Flt3 signaling are associated with a distinct regulatory phenotype, with increased PD-L1 expression and decreased ICOS-L expression. BMDCs exposed to BEN exhibit diminished pro-inflammatory cytokine response to LPS and induce robust proliferation of alloreactive T-cells. These proliferative alloreactive T-cells expressed greater levels of PD-1 and underwent increased programmed cell death as the concentration of BEN exposure increased. Alloreactive CD4 T-cell death may be attributable to pre-cDC1s and provides a potential mechanism by which BEN+TBI conditioning limits GvHD and yields T-cells tolerant to host antigen.
• Pariury, H., Truscott, L., & Katsanis, E. (2021). Have CD19-directed immunotherapy and haploidentical hematopoietic cell transplantation transformed pediatric B-cell acute lymphoblastic leukemia into a chronic disease?. Oncoimmunology, 10(1), 1956125.
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  The treatment of pediatric B-cell acute lymphoblastic leukemia (B-ALL) has undergone several recent advancements, leading to an increased amount of treatment options for relapsed patients. The development of immunotherapies such as anti-CD19 chimeric antigen receptor(CAR) T cells and bispecific T-cell engagers has given clinicians therapeutic options with less expected toxicity when compared to standard re-induction chemotherapy. This is especially beneficial in patients with toxicities from their prior treatment. Along with this, the emergence of haploidentical hematopoietic cell transplantation (HCT) has increased opportunity for patients to receive HCT who may not have had an available matched donor. We present four patients who have received all of these therapies in different combinations to treat multiple relapses. Because of the success of achieving remission as well as decreasing toxicity, the patients are alive and well up to 15 y after the original B-ALL diagnosis, rendering this as a chronic disease for them.
• Sharma, A., Huang, S., Li, Y., Brooke, R. J., Ahmed, I., Allewelt, H. B., Amrolia, P., Bertaina, A., Bhatt, N. S., Bierings, M. B., Bies, J., Brisset, C., Brondon, J. E., Dahlberg, A., Dalle, J. H., Eissa, H., Fahd, M., Gassas, A., Gloude, N. J., , Goebel, W. S., et al. (2021). Outcomes of pediatric patients with therapy-related myeloid neoplasms. Bone marrow transplantation, 56(12), 2997-3007.
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  Long-term outcomes after allogeneic hematopoietic cell transplantation (HCT) for therapy-related myeloid neoplasms (tMNs) are dismal. There are few multicenter studies defining prognostic factors in pediatric patients with tMNs. We have accumulated the largest cohort of pediatric patients who have undergone HCT for a tMN to perform a multivariate analysis defining factors predictive of long-term survival. Sixty-eight percent of the 401 patients underwent HCT using a myeloablative conditioning (MAC) regimen, but there were no statistically significant differences in the overall survival (OS), event-free survival (EFS), or cumulative incidence of relapse and non-relapse mortality based on the conditioning intensity. Among the recipients of MAC regimens, 38.4% of deaths were from treatment-related causes, especially acute graft versus host disease (GVHD) and end-organ failure, as compared to only 20.9% of deaths in the reduced-intensity conditioning (RIC) cohort. Exposure to total body irradiation (TBI) during conditioning and experiencing grade III/IV acute GVHD was associated with worse OS. In addition, a diagnosis of therapy-related myelodysplastic syndrome and having a structurally complex karyotype at tMN diagnosis were associated with worse EFS. Reduced-toxicity (but not reduced-intensity) regimens might help to decrease relapse while limiting mortality associated with TBI-based HCT conditioning in pediatric patients with tMNs.
• Smith, J., Alfonso, J. H., Reddivalla, N., Angulo, P., & Katsanis, E. (2021). Case Report: Haploidentical Bone Marrow Transplantation in Two Brothers With Wiskott-Aldrich Syndrome Using Their Father as the Donor. Frontiers in pediatrics, 9, 647505.
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  Wiskott-Aldrich syndrome (WAS) is an X-linked genetic disorder with a variable phenotypic expression that includes thrombocytopenia, eczema, and immunodeficiency. Some patients may also exhibit autoimmune manifestations. Patients with WAS are at increased risk of developing malignancies such as lymphoma. Allogeneic hematopoietic cell transplantation remains the only curative treatment. Haploidentical bone marrow transplantation (haplo-BMT) with post-transplant cyclophosphamide (PT-CY) has more recently been applied in WAS. Here, we report two brothers who underwent successful T-cell replete haplo-BMT with PT-CY at ages 9 months and 4 years using their father as the donor. Our myeloablative regimen was well-tolerated with minimal organ toxicity and no acute or chronic graft vs. host disease (GvHD). Haplo-BMT may be considered as a safe and effective option for patients with WAS who do not have available human leukocyte antigen (HLA) matched donors.
• Smith, J., Kumar, A., Stanton, N. A., & Katsanis, E. (2021). Concurrent application of blinatumomab and haploidentical donor leukocyte infusions for refractory primary mediastinal large B-cell lymphoma. Therapeutic advances in hematology, 12, 2040620721994348.
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  Primary mediastinal large B-cell lymphoma (PMBCL) is a rare hematologic malignancy with distinct clinical and immunopathological features. We report a case of a young male with disease refractory to multiple lines of therapy, including chimeric antigen receptor-T cells, who achieved his first complete remission after haploidentical bone marrow transplantation (haplo-BMT), following donor leukocyte infusions (DLIs) given concurrently with blinatumomab. While DLI has been used after T-replete haplo-BMT with post-transplant cyclophosphamide, there are no reports on its use for PMBCL. Similarly, blinatumomab is active against B-cell lymphomas, but literature is lacking in patients with PMBCL. Our experience illustrates that blinatumomab can be used concurrently with DLI in a haploidentical setting to achieve disease response in PMBCL. Despite our encouraging experience with this case, we would not recommend this approach outside of a clinical trial as blinatumomab may exacerbate the graft host disease risks of DLI, especially in a haploidentical setting. Evaluating this treatment combination in high-risk patients in the setting of a clinical trial may be meaningful.
• Stanley, K., Hanmod, S., Simpson, R. J., & Katsanis, E. (2021). Haploidentical hematopoietic cell transplantation is even more advantageous during the COVID-19 pandemic. Pediatric transplantation, 25(3), e14004.
• Stokes, J., Molina, M. S., Hoffman, E. A., Simpson, R. J., & Katsanis, E. (2021). Immunomodulatory Effects of Bendamustine in Hematopoietic Cell Transplantation. Cancers, 13(7).
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  Bendamustine (BEN) is a unique alkylating agent with efficacy against a broad range of hematological malignancies, although investigations have only recently started to delve into its immunomodulatory effects. These immunomodulatory properties of BEN in the context of hematopoietic cell transplantation (HCT) are reviewed here. Pre- and post-transplant use of BEN in multiple murine models have consistently resulted in reduced GvHD and enhanced GvL, with significant changes to key immunological cell populations, including T-cells, myeloid derived suppressor cells (MDSCs), and dendritic cells (DCs). Further, in vitro studies find that BEN enhances the suppressive function of MDSCs, skews DCs toward cDC1s, enhances Flt3 expression on DCs, increases B-cell production of IL-10, inhibits STAT3 activation, and suppresses proliferation of T- and B-cells. Overall, BEN has a broad range of immunomodulatory effects that, as they are further elucidated, may be exploited to improve clinical outcomes. As such, clinical trials are currently underway investigating new potential applications of BEN in the setting of allogeneic HCT.
• Agha, N. H., Mehta, S. K., Rooney, B. V., Laughlin, M. S., Markofski, M. M., Pierson, D. L., Katsanis, E., Crucian, B. E., & Simpson, R. J. (2020). Exercise as a countermeasure for latent viral reactivation during long duration space flight. FASEB journal : official publication of the Federation of American Societies for Experimental Biology.
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  Latent viral reactivation is a commonly reported manifestation of immune system dysregulation during spaceflight. As physical fitness and exercise training have been shown to benefit multiple arms of the immune system, we hypothesized that higher levels of preflight physical fitness and/or maintaining fitness during a mission would protect astronauts from latent viral reactivation. Standardized tests of maximal strength, muscular endurance, flexibility, and cardiorespiratory fitness (CRF) were performed in 22 international space station (ISS) crewmembers before and after a ~6-month mission. Reactivation of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and varicella zoster virus (VZV) was determined in crewmembers and ground-based controls before, during, and after spaceflight. Crewmembers with higher CRF before spaceflight had a 29% reduced risk of latent viral reactivation compared to crew with lower CRF. Higher preflight upper body muscular endurance was associated with a 39% reduced risk of viral reactivation, a longer time to viral reactivation, and lower peak viral DNA concentrations, particularly for EBV and VZV. Latent viral reactivation rates were highest in crew with lower preflight CRF and higher levels of CRF deconditioning on return to Earth. We conclude that physical fitness may protect astronauts from latent viral reactivation during long duration spaceflight missions.
• Baker, F. L., Bigley, A. B., Agha, N. H., Pedlar, C. R., O'Connor, D. P., Bond, R. A., Bollard, C. M., Katsanis, E., & Simpson, R. J. (2020). Systemic β-Adrenergic Receptor Activation Augments the Expansion and Anti-Tumor Activity of Vγ9Vδ2 T-Cells. Frontiers in immunology, 10, 3082.
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  TCR-gamma delta (γδ) T-cells are considered important players in the graft-vs.-tumor effect following allogeneic hematopoietic cell transplantation (alloHCT) and have emerged as candidates for adoptive transfer immunotherapy in the treatment of both solid and hematological tumors. Systemic β-adrenergic receptor (β-AR) activation has been shown to mobilize TCR-γδ T-cells to the blood, potentially serving as an adjuvant for alloHCT and TCR-γδ T-cell therapy. We investigated if systemic β-AR activation, using acute dynamic exercise as an experimental model, can increase the mobilization, expansion, and anti-tumor activity of TCR-γδ T-cells isolated from the blood of healthy humans. We also sought to investigate the β-AR subtypes involved, by administering a preferential β-AR antagonist (bisoprolol) and a non-preferential β + β-AR antagonist (nadolol) prior to exercise as part of a randomized placebo controlled cross-over experiment. We found that exercise mobilized TCR-γδ cells to blood and augmented their expansion by ~182% compared to resting blood when stimulated with IL-2 and ZOL for 14-days. Exercise also increased the proportion of CD56+, NKG2D+/CD62L-, CD158a/b/e+ and NKG2A- cells among the expanded TCR-γδ cells, and increased their cytotoxic activity against several tumor target cells (K562, U266, 221.AEH) by 40-60%. Blocking NKG2D on TCR-γδ cells eliminated the augmented cytotoxic effects of exercise against U266 target cells. Furthermore, administering a β + β-AR (nadolol), but not a β-AR (bisoprolol) antagonist prior to exercise abrogated the exercise-induced enhancement in TCR-γδ T-cell mobilization and expansion. Furthermore, nadolol completely abrogated while bisoprolol partially inhibited the exercise-induced increase in the cytotoxic activity of the expanded TCR-γδ T-cells. We conclude that acute systemic β-AR activation in healthy donors markedly augments the mobilization, expansion, and anti-tumor activity of TCR-γδ T-cells and that some of these effects are due to β-AR signaling and phenotypic shifts that promote a dominant activating signal via NKG2D. These findings highlight β-ARs as potential targets to favorably alter the composition of allogeneic peripheral blood stem cell grafts and improve the potency of TCR-γδ T-cell immune cell therapeutics.
• Katsanis, E., Sapp, L. N., Reid, S. C., Reddivalla, N., & Stea, B. (2020). T-Cell Replete Myeloablative Haploidentical Bone Marrow Transplantation Is an Effective Option for Pediatric and Young Adult Patients With High-Risk Hematologic Malignancies. Frontiers in pediatrics, 8, 282.
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  Twenty-one pediatric and young adult patients (1.1-24.7 years) with hematologic malignancies underwent myeloablative T-cell replete haploidentical bone marrow transplant (haplo-BMT) between October 2015 to December 2019. Fifty-seven percent of the patients were ethnic or racial minorities. Thirteen patients had B-cell precursor acute lymphoblastic leukemia (B-ALL) with 10 receiving 1,200 cGy fractionated total body irradiation with fludarabine while the remaining 11 patients had targeted dose-busulfan, fludarabine, melphalan conditioning. Graft-vs.-host disease (GvHD) prophylaxis consisted of post-transplant cyclophosphamide (15 patients) or cyclophosphamide and bendamustine (six patients), with all patients receiving tacrolimus and mycophenolate mofetil. Twelve patients were in first or second remission at time of transplant with five in >2nd remission and four with measurable disease. Three patients had failed prior transplants and three CAR-T cell therapies. Only one patient developed primary graft failure but engrafted promptly after a second conditioned T-replete peripheral blood stem cell transplant from the same donor. An absolute neutrophil count of 0.5 × 10/L was achieved at a median time of 16 days post-BMT while platelet engraftment occurred at a median of 30 days. The cumulative incidence of grades III to IV acute GvHD and chronic GvHD was 15.2 and 18.1%, respectively. With a median follow-up of 25.1 months the relapse rate is 17.6% with an overall survival of 84.0% and a progression-free survival of 74.3%. The chronic graft-vs.-host-free relapse-free survival (CRFS) is 58.5% while acute and chronic graft-vs.-host-free relapse-free survival (GRFS) is 50.1%. Myeloablative conditioned T-replete haploidentical BMT is a viable alternative to matched unrelated transplantation for children and young adults with high-risk hematologic malignancies.
• Kunz, H. E., Agha, N. H., Hussain, M., LaVoy, E. C., Smith, K. A., Mylabathula, P., Diak, D., Baker, F. L., O'Connor, D. P., Bond, R. A., Katsanis, E., Bollard, C. M., & Simpson, R. J. (2020). The effects of β and β adrenergic receptor blockade on the exercise-induced mobilization and ex vivo expansion of virus-specific T cells: implications for cellular therapy and the anti-viral immune effects of exercise. Cell stress & chaperones, 25(6), 993-1012.
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  The adoptive transfer of donor-derived virus-specific T cells (VSTs) is an effective treatment for infections following allogeneic hematopoietic cell transplantation. Acute exercise mobilizes effector lymphocytes and VSTs to the circulation and augments the ex vivo manufacture of VSTs. This study determined if β adrenergic receptor (AR) signaling precipitated the VST response to acute exercise. Healthy participants (n = 12) completed 30 min of steady-state cycling exercise after ingesting a placebo, a β AR antagonist (nadolol) or a β AR antagonist (bisoprolol). Circulating VSTs to cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus (AdV) antigens were enumerated before and after exercise, and peripheral blood mononuclear cells were cultured with viral peptides for 8 days to expand multi-VSTs. Compared with placebo, nadolol blunted the exercise-induced mobilization of CMV-VSTs (Δ VSTs/100,000 CD3 T cells = 93 ± 104 vs. 22 ± 91 for placebo and nadolol, respectively; p = 0.036), while bisoprolol did not, despite both drugs evoking similar reductions in exercising heart rate and blood pressure. Circulating AdV and EBV VSTs (VSTs/mL blood) only increased after exercise with placebo. Although not significant, nadolol partially mitigated exercise-induced increases in multi-VST expansion, particularly in participants that demonstrated an exercise-induced increase in VST expansion. We conclude that exercise-induced enhancements in VST mobilization and expansion are at least partially β AR mediated, thus highlighting a role for the β AR in targeted therapy for the augmentation of VST immune cell therapeutics in the allogeneic adoptive transfer setting. Moreover, long-term regular exercise may provide additional viral protection in the host through frequent β AR-dependent mobilization and redistribution of VSTs cumulated with each bout of exercise.
• Molina, M. S., Stokes, J., Hoffman, E. A., Eremija, J., Zeng, Y., Simpson, R. J., & Katsanis, E. (2020). Bendamustine Conditioning Skews Murine Host DCs Toward Pre-cDC1s and Reduces GvHD Independently of Batf3. Frontiers in immunology, 11, 1410.
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  Graft-versus-host disease (GvHD) remains the second leading cause of death in allogeneic hematopoietic stem cell transplantation recipients, highlighting the need for improved preventative strategies. Our laboratory has previously demonstrated in an experimental bone marrow transplantation (BMT) model that bendamustine combined with total body irradiation (BEN+TBI) is a safer alternative to cyclophosphamide with TBI (CY+TBI). The biological mechanisms of action of BEN have not been fully elucidated and likely involve multiple cell populations. Host dendritic cells (DCs) can prime naïve donor T-cells immediately following transplantation, making host DCs critical for the initiation phase of GvHD. We hypothesized that BEN+TBI conditioning favorably alters host DC composition to reduce GvHD. We demonstrate that host DCs treated with BEN+TBI induce less allogeneic T-cell proliferation than those conditioned with CY+TBI. We further show that BEN+TBI conditioning results in greater total numbers of all host DC subsets but with a more favorable composition compared to CY+TBI with significantly larger proportions of type 1 conventional DCs (cDC1), a highly regulatory DC subset capable of suppressing GvHD. Our studies using recipient Batf3 KO mice indicate that CD8α+ cDC1s are largely dispensable for the reduced GvHD following BEN+TBI conditioning. We found a higher frequency of host pre-cDC1s with BEN+TBI conditioning in both wild-type (WT) and Batf3 KO mice, which was inversely associated with GvHD. Additionally, we observed that BEN treatment results in greater expression of Flt3 receptor (CD135) on host DCs compared to CY, potentially contributing to the skewing of host DCs toward cDC1s. Further, BEN+TBI conditioning results in host cDCs with greater expression of PIR-B, an inhibitory receptor capable of preventing lethal GvHD. We conclude that BEN+TBI is a safer alternative to CY+TBI, resulting in a greater frequency of host pre-cDC1s and limiting GvHD.
• Simpson, R. J., & Katsanis, E. (2020). Progressive substitution of post-transplant cyclophosphamide with bendamustine: A phase I study in haploidentical bone marrow transplantation. eJHaem, 1(1), 1-7. doi:doi: 10.1002/jha2.20
• Simpson, R. J., & Katsanis, E. (2020). The immunological case for staying active during the COVID-19 pandemic. Brain, behavior, and immunity, 87, 6-7.
• Stokes, J., Hoffman, E. A., Molina, M. S., Kummet, N., Simpson, R. J., Zeng, Y., & Katsanis, E. (2020). Bendamustine with total body irradiation conditioning yields tolerant T-cells while preserving T-cell-dependent graft-versus-leukemia. Oncoimmunology, 9(1), 1758011.
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  Graft-versus-host disease (GvHD) remains a significant impediment to allogeneic hematopoietic cell transplantation (HCT) success, necessitating studies focused on alleviating GvHD, while preserving the graft-versus-leukemia (GvL) effect. Based on our previous studies showing bendamustine with total body irradiation (BEN-TBI) conditioning reduces GvHD compared to the current clinical standard of care cyclophosphamide (CY)-TBI in a murine MHC-mismatched bone marrow transplantation (BMT) model, this study aimed to evaluate the role and fate of donor T-cells following BEN-TBI conditioning. We demonstrate that BEN-TBI reduces GvHD compared to CY-TBI independently of T regulatory cells (Tregs). BEN-TBI conditioned mice have a smaller proportion and less activated donor T-cells, with lower CD47 expression, early post-transplant, but no sustained phenotypic differences in T-cells. In BEN-TBI conditioned mice, donor T-cells gain tolerance specific to host MHC antigens. Though these T-cells are tolerant to host antigens, we demonstrate that BEN-TBI preserves a T-cell-dependent GvL effect. These findings indicate that BEN-TBI conditioning reduces GvHD without compromising GvL, warranting its further investigation as a potentially safer and more efficacious clinical alternative to CY-TBI.
• Macaraeg, M., Proytcheva, M., & Katsanis, E. (2019). Transfusion independence after repeated haploidentical hematopoietic cell transplants in a patient with congenital dyserythropoietic anemia type II and hemosiderosis. Pediatric transplantation, 23(8), e13587.
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  Matched related or unrelated donor allogeneic HCT has occasionally been applied in patients with severe CDA type II and proven to be curative. We report on the first patient with CDA to undergo haploidentical bone marrow transplantation with PT-CY. A 12-year-old boy with severe hemosiderosis, and a, consequently, disturbed BM microenvironment, developed recurrent graft failures and required salvage with two additional haploidentical HCTs. He achieved complete donor chimerism and transfusion independence after the third HCT. Our case underscores the risks associated with performing haploidentical HCT in older pediatric patients with CDA and severe chronic iron overload.
• Doane, C. J., Patil, K., Hoffman, E. A., Stokes, J., Katsanis, E., & Besselsen, D. G. (2018). Supernumerary Incisors in CB6F1 Mice Conditioned with Chemotherapy and Total Body Irradiation before Bone Marrow Transplantation. Comparative medicine, 68(5), 349-352.
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  Multiple adult female CB6F1 mice presented with supernumerary incisors after preconditioning with chemotherapy and total body irradiation for bone marrow transplantation (BMT). Mice received nonmyeloablative total body irradiation (3 Gy) and either cyclophosphamide or bendamustine, followed by BMT and posttransplantation cyclophosphamide or bendamustine. Here we describe the clinical presentation, μCT findings, and histopathologic evaluation of the affected mice. These analyses confirmed the gross diagnosis and revealed details of the abnormal tooth morphology. We surmise that the combination of total body irradiation and chemotherapy resulted in the abnormal formation of supernumerary incisors. Supernumerary teeth should be considered as a potential confounding factor in tracking weight loss after BMT. These conditions can be managed to allow animals to reach their intended scientific endpoint.
• Famoso, J. M., Grow, J. L., Laughlin, B., Katsanis, E., & Stea, B. (2018). The Impact of Low Dose Cranial Boost on the Long-Term Outcomes of Adult Patients with High-Risk Acute Lymphoblastic Leukemia Undergoing Total Body Irradiation and Allogeneic Hematopoietic Stem Cell Transplantation. Practical radiation oncology.
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  Total Body Irradiation (TBI) is an integral part of the conditioning regimen for patients with Acute Lymphoblastic Leukemia (ALL) undergoing allogeneic hematopoietic cell transplantation (allo-HCT). There are conflicting data in the literature regarding the utility of a cranial irradiation boost in high risk adult ALL without evidence of preexisting central nervous system involvement. This study investigates the post-transplant clinical outcomes of high-risk adult ALL patients undergoing TBI conditioning for allo-HCT with, or without, whole brain boost in those patients without overt CNS involvement at diagnosis.
• Graetz, R., Meyer, R., Shehab, K., & Katsanis, E. (2018). Successful resolution of hyperammonemia following hematopoietic cell transplantation with directed treatment of Ureaplasma parvum infection. Transplant infectious disease : an official journal of the Transplantation Society, 20(2), e12839.
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  Hyperammonemia following hematopoietic cell transplantation (HCT) has been characterized as idiopathic and is associated with a very high mortality. A causal relationship between Ureaplasma infection and hyperammonemia in immunocompromised lung transplant recipients has recently been described. We document the first case of hyperammonemia following HCT associated with Ureaplasma parvum. The initiation of appropriate antibiotics resulted in rapid resolution of hyperammonemic encephalopathy and eradication of the implicating organism.
• Graff, R. M., Kunz, H. E., Agha, N. H., Baker, F. L., Laughlin, M., Bigley, A. B., Markofski, M. M., LaVoy, E. C., Katsanis, E., Bond, R. A., Bollard, C. M., & Simpson, R. J. (2018). β-Adrenergic receptor signaling mediates the preferential mobilization of differentiated subsets of CD8+ T-cells, NK-cells and non-classical monocytes in response to acute exercise in humans. Brain, behavior, and immunity, 74, 143-153.
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  Acute exercise preferentially mobilizes cytotoxic T-cells, NK-cells and non-classical monocytes to the bloodstream under the influence of hemodynamic forces and/or β-adrenergic receptor (β-AR) signaling. However, the relative contribution of these mechanisms to the redeployment of the most exercise-responsive cell types is largely unknown. We determined the lymphocyte and monocyte subtypes mobilized to blood during exercise via β-AR signaling whilst controlling for β-AR mediated reductions in hemodynamic forces. In a randomized, double blind, complete cross-over design, 14 healthy cyclists exercised for 30-minutes at +10% of blood lactate threshold after ingesting: (1) a placebo, (2) a β-preferential antagonist (10 mg bisoprolol), or (2) a non-preferential β + β-antagonist (80 mg nadolol) across three trials separated by >7-days. Bisoprolol was administered to reduce hemodynamic forces (heart rate and blood pressure) during exercise to levels comparable with nadolol but without blocking β-ARs. The mobilization of total NK-cells, terminally differentiated (CD57+) NK-cells, central memory, effector memory and CD45RA+ effector memory CD8+ T-cells; non-classical monocytes; and γδ T-cells were significantly blunted or abrogated under nadolol compared to both bisoprolol and placebo, indicating that the exercise-induced mobilization of these cell types to the blood is largely influenced by β-AR signaling. Nadolol failed to inhibit the mobilization of classical monocytes, CD4+ T-cells (and their subsets) or naïve CD8+ T-cells, indicating that these cell types are mobilized with exercise independently of the β-AR. We conclude that the preferential mobilization of NK-cells, non-classical monocytes and differentiated subsets of CD8+ T-cells with exercise is largely dependent on catecholamine signaling through the β-AR. These findings provide mechanistic insights by which distinct lymphocyte and monocyte subtypes are preferentially mobilized to protect the host from anticipated injury or infection in response to an acute stress response.
• Kanate, A. S., DiGilio, A., Ahn, K. W., Al Malki, M., Jacobsen, E., Steinberg, A., Hamerschlak, N., Kharfan-Dabaja, M., Salit, R., Ball, E., Bashir, Q., Cashen, A., Couriel, D., Diez-Martin, J., Katsanis, E., Linhares, Y., Mori, S., Nash, R., Pawarode, A., , Perales, M. A., et al. (2018). Allogeneic haematopoietic cell transplantation for extranodal natural killer/T-cell lymphoma, nasal type: a CIBMTR analysis. British journal of haematology, 182(6), 916-920.
• Katsanis, E., Sapp, L. N., Varner, N., Koza, S., Stea, B., & Zeng, Y. (2018). Haploidentical Bone Marrow Transplantation with Post-Transplant Cyclophosphamide/Bendamustine in Pediatric and Young Adult Patients with Hematologic Malignancies. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation, 24(10), 2034-2039.
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  More than half of patients undergoing hematopoietic cell transplantation at our institution are ethnic or racial minorities, making the search for matched unrelated donors more challenging. Since the introduction of haploidentical bone marrow transplant (haplo-BMT) into our pediatric BMT program in 2015, 69.2% of recipients have been minorities. Herein, we describe our experience with the first 13 pediatric and young adult patients with hematologic malignancies who have undergone T cell-replete haplo-BMT after myeloablative conditioning (MAC) at our institution. We have previously documented that in experimental haplo-BMT, post-transplant bendamustine (PT-BEN) is at least as effective as post-transplant cyclophosphamide (PT-CY) against graft-versus-host disease (GVHD) and elicits superior graft-versus-leukemia (GVL) effects. We report on, for the first time in humans, 4 patients treated with PT-CY and PT-BEN after haplo-BMT as part of our ongoing institutional phase I/II study (NCT02996773). The remaining 9 patients reviewed in this report received PT-CY. Our findings indicate that MAC haplo-BMT is well tolerated by children and young adults with advanced hematologic malignancies with no observed nonrelapse mortality or grades III to IV GVHD. All patients who underwent haplo-BMT remain alive and disease-free with a median follow-up of 15.6 months (range, 1.5 to 31.2). Preliminary findings from our ongoing clinical trial demonstrate that partial substitution of PT-BEN for PT-CY is feasible and safe after haplo-BMT as an immune modulatory strategy to alleviate GVHD and potentially more effectively preserve GVL.
• Nicholls, L., Montez, J., Pelayo-Katsanis, L., & Katsanis, E. (2018). Esophageal Varices in Adolescent and Young Adult Males with Acute Lymphocytic Leukemia. Journal of adolescent and young adult oncology.
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  Hepatic late effects are not commonly reported in survivors of childhood leukemia. Four young male patients with acute lymphoblastic leukemia (ALL) were diagnosed with bleeding esophageal varices (EVs) during or shortly after completion of maintenance chemotherapy. EVs were identified from 0 to 60 months after completion of leukemia therapy. All four patients were men between 20 to 24 years old. Hematemesis was the most common presenting symptom. Associated features included splenomegaly, cytopenias, azole therapy, alcohol use, and hepatic iron overload. EVs may be an under-recognized complication of ALL therapy, with adolescent and young adult males at highest risk.
• Riley, G., Meyer, R., Shehab, K. W., & Katsanis, E. (2017). Successful resolution of hyperammonemia following hematopoietic cell transplantation with directed treatment of Ureaplasma parvum infection. Transplant Infectious Disease.
• Stokes, J., Hoffman, E. A., Molina, M. S., Eremija, J., Larmonier, N., Zeng, Y., & Katsanis, E. (2018). Bendamustine with Total Body Irradiation Limits Murine Graft-versus-Host Disease in Part Through Effects on Myeloid-Derived Suppressor Cells. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation.
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  Graft-versus-host disease (GVHD) remains a significant challenge in allogeneic hematopoietic cell transplantation (HCT). An underinvestigated strategy to reduce GVHD is the modification of the preparative conditioning regimen. In the present study, we aimed to evaluate GVHD associated with bendamustine (BEN) conditioning in conjunction with total body irradiation (TBI) as an alternative to the standard myeloablative regimen of cyclophosphamide (CY) and TBI. We demonstrate that BEN-TBI conditioning, although facilitating complete donor chimerism, results in significantly less GVHD compared with CY-TBI. In BEN-TBI-conditioned mice, suppressive CD11bGr-1 myeloid cells are increased in the blood, bone marrow, spleen, and intestines. When Gr-1 cells are depleted before transplantation, the beneficial effects of BEN-TBI are partially lost. Alternatively, administration of granulocyte colony-stimulating factor, which promotes CD11bGr-1 myeloid cell expansion, is associated with a trend toward increased survival in BEN-TBI-conditioned mice. These findings indicate a potential role of myeloid-derived suppressor cells in the mechanism by which BEN allows engraftment with reduced GVHD. BEN-TBI conditioning may present a safer alternative to CY-TBI conditioning for allogeneic HCT.
• Okolo, O. N., Katsanis, E., Yun, S., Reveles, C. Y., & Anwer, F. (2017). Allogeneic Transplant in ELANE and MEFV Mutation Positive Severe Cyclic Neutropenia: Review of Prognostic Factors for Secondary Severe Events. Case reports in hematology, 2017, 5375793.
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  Cyclic neutropenia (CyN) is a rare autosomal dominant inherited disorder due to the mutation ELANE primarily affecting bone marrow stem cells and is characterized by recurrent neutropenia every 2 to 4 weeks. Symptoms vary from benign to severe, including death. Postulations on the cause of wide spectrum in symptom presentation include the possibility of other genetic mutations, such as MEFV. Recommended treatment for CyN is G-CSF to keep ANC higher to minimize risk of infection. A 25-year-old male diagnosed with CyN, on G-CSF but worsening quality of life. Pretransplant investigations revealed ELANE mutation positive severe CyN along with familial Mediterranean fever (MEFV) mutation. Bone marrow transplantation as treatment for dual mutation (ELANE and MEFV mutation) positive severe CyN. BMT may be considered as an alternative treatment for severe CyN in patients who are refractory to G-CSF. It is postulated that in our patient the combined mutations (CyN and MEFV) may have contributed to the severity of this individual's symptoms. We suggest CyN patients who present with severe symptoms have evaluation with ELANE mutation testing, Periodic Fever Syndromes Panel, and routine marrow assessment with FISH, conventional cytogenetics, and morphological evaluation for MDS/AML.
• Zeng, Y., & Katsanis, E. (2017). Potential niche indications for blinatumomab as a bridge to hematopoietic cell transplantation. Bone marrow transplantation, 52(12), 1671-1673.
• Zeng, Y., Hahn, S., Stokes, J., Hoffman, E. A., Schmelz, M., Proytcheva, M., Chernoff, J., & Katsanis, E. (2017). Pak2 regulates myeloid-derived suppressor cell development in mice. Blood advances, 1(22), 1923-1933.
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  Myeloid-derived suppressor cells (MDSCs) are CD11bGr1 cells that induce T-cell hyporesponsiveness, thus impairing antitumor immunity. We have previously reported that disruption of Pak2, a member of the p21-activated kinases (Paks), in hematopoietic stem/progenitor cells (HSPCs) induces myeloid lineage skewing and expansion of CD11bGr1 cells in mice. In this study, we confirmed that CD11bGr1 cells suppressed T-cell proliferation, consistent with an MDSC phenotype. Loss of Pak2 function in HSPCs led to (1) increased hematopoietic progenitor cell sensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling, (2) increased MDSC proliferation, (3) decreased MDSC sensitivity to both intrinsic and Fas-Fas ligand-mediated apoptosis, and (4) promotion of MDSCs by Pak2-deficient CD4 T cells that produced more interferon γ, tumor necrosis factor α, and GM-CSF. Pak2 disruption activated STAT5 while downregulating the expression of , a well-described myeloid transcription factor. Together, our data reveal a previously unrecognized role of Pak2 in regulating MDSC development via both cell-intrinsic and extrinsic mechanisms. Our findings have potential translational implications, as the efficacy of targeting Paks in cancer therapeutics may be undermined by tumor escape from immune control and/or acceleration of tumorigenesis through MDSC expansion.
• Katsanis, E., Sapp, L. N., Pelayo-Katsanis, L., Whitney, K., Zeng, Y., & Kopp, L. M. (2016). Alternative Donor Hematopoietic Cell Transplantation Conditioned With Myeloablative Busulfan, Fludarabine, and Melphalan is Well Tolerated and Effective Against High-risk Myeloid Malignancies. Journal of pediatric hematology/oncology, 38(8), e315-e318.
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  Busulfan, fludarabine, and melphalan as hematopoietic cell transplant conditioning, was used in 6 patients aged 1 to 19 years with very high-risk myeloid malignancies. This dose regimen had an acceptable toxicity profile resulting in complete donor engraftment even following transplantation of small 2/6 antigen disparate umbilical cord blood grafts. It provided excellent disease control as all patients had high-risk features in terms of cytogenetics, therapy-related leukemia, and/or significant measurable disease before transplant. All patients remain in remission, without acute or chronic graft-versus-host disease with a median follow-up of 24 months. A larger study is indicated to confirm the efficacy and safety of this regimen.
• Menon, N. N., Jenkins, L. M., Cui, H., Jenkins, C., Anwer, F., Yeager, A. M., & Katsanis, E. (2016). Factors associated with improved outcomes after second allogeneic hematopoietic cell transplantation for relapsed pediatric leukemia. Annals of hematology, 95(4), 637-44.
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  A second allogeneic (allo) hematopoietic cell transplant (HCT) is an important therapeutic consideration for patients relapsing after their first. We conducted a retrospective review of 41 pediatric patients with leukemia that underwent a second allo-HCT at our institution. Overall, 53.7 and 43.9 % of patients were alive and disease-free at 1 and 5 years, respectively, after the second allo-HCT. The factors affecting outcome by both univariate and multivariate analysis were interval between transplants and the use of a myeloablative conditioning (MAC) regimen prior to second transplant. Outcomes were inferior in patients who received their second transplant
• Stokes, J., Hoffman, E. A., Zeng, Y., Larmonier, N., & Katsanis, E. (2016). Post-transplant bendamustine reduces GvHD while preserving GvL in experimental haploidentical bone marrow transplantation. British journal of haematology, 174(1), 102-16.
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  Advances in haploidentical bone marrow transplantation (h-BMT) have drastically broadened the treatment options for patients requiring BMT. The possibility of significantly reducing the complications resulting from graft-versus-host disease (GvHD) with the administration of post-transplant cyclophosphamide (PT-CY) has substantially improved the efficacy and applicability of T cell-replete h-BMT. However, higher frequency of disease recurrence remains a major challenge in h-BMT with PT-CY. There is a critical need to identify novel strategies to prevent GvHD while sparing the graft-versus-leukaemia (GvL) effect in h-BMT. To this end, we evaluated the impact of bendamustine (BEN), given post-transplant, on GvHD and GvL using clinically relevant murine h-BMT models. We provide results indicating that post-transplant bendamustine (PT-BEN) alleviates GvHD, significantly improving survival, while preserving engraftment and GvL effects. We further document that PT-BEN can mitigate GvHD even in the absence of Treg. Our results also indicate that PT-BEN is less myelosuppressive than PT-CY, significantly increasing the number and proportion of CD11b(+) Gr-1(hi) cells, while decreasing lymphoid cells. In vitro we observed that BEN enhances the suppressive function of myeloid-derived suppressor cells (MDSCs) while impairing the proliferation of T- and B-cells. These results advocate for the consideration of PT-BEN as a new therapeutic platform for clinical implementation in h-BMT.
• Houghtelin, A. B., Kopp, L. M., Pelayo-Katsanis, L., Kuo, P. H., Yeager, A. M., & Katsanis, E. (2015). Extramedullary Breast Relapse of Acute Lymphoblastic Leukemia Controlled with a Second Allogeneic/Autologous Hematopoietic Cell Transplant. Journal of adolescent and young adult oncology, 4(1), 50-3.
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  Relapse of acute lymphoblastic leukemia (ALL) in the breast is uncommon and often precedes systemic relapse, resulting in poor survival. We report the development of breast involvement of ALL in a 20-year-old woman 32 months after a related allogeneic peripheral blood hematopoietic cell transplantation (PBHCT) in first remission. This extramedullary relapse occurred in the continuous presence of complete donor chimerism. After systemic re-induction chemotherapy and a second PBHCT using donor cells that had been cryopreserved at first transplant, our patient has remained in second complete remission for more than 44 months.
• Menon, N. M., Katsanis, E., Khalpey, Z., & Whitlow, P. (2015). Pediatric secondary chronic myeloid leukemia following cardiac transplantation for anthracycline-induced cardiomyopathy. Pediatric blood & cancer, 62(1), 166-8.
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  Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder of the hematopoietic stem cell that is exceptionally rare in the first five years of life, particularly as a secondary malignancy. This report describes a case of secondary CML in a four-year-old female occurring after AML treatment. Interestingly, CML developed while on immunosuppression for a heart transplant due to anthracycline-induced cardiomyopathy.
• Trad, M., Gautheron, A., Fraszczak, J., Alizadeh, D., Larmonier, C., LaCasse, C. J., Centuori, S., Audia, S., Samson, M., Ciudad, M., Bonnefoy, F., Lemaire-Ewing, S., Katsanis, E., Perruche, S., Saas, P., & Bonnotte, B. (2015). T Lymphocyte Inhibition by Tumor-Infiltrating Dendritic Cells Involves Ectonucleotidase CD39 but Not Arginase-1. BioMed research international, 2015, 891236.
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  T lymphocytes activated by dendritic cells (DC) which present tumor antigens play a key role in the antitumor immune response. However, in patients suffering from active cancer, DC are not efficient at initiating and supporting immune responses as they participate to T lymphocyte inhibition. DC in the tumor environment are functionally defective and exhibit a characteristic of immature phenotype, different to that of DC present in nonpathological conditions. The mechanistic bases underlying DC dysfunction in cancer responsible for the modulation of T-cell responses and tumor immune escape are still being investigated. Using two different mouse tumor models, we showed that tumor-infiltrating DC (TIDC) are constitutively immunosuppressive, exhibit a semimature phenotype, and impair responder T lymphocyte proliferation and activation by a mechanism involving CD39 ectoenzyme.
• Zeng, Y., & Katsanis, E. (2015). The complex pathophysiology of acquired aplastic anaemia. Clinical and experimental immunology, 180(3), 361-70.
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  Immune-mediated destruction of haematopoietic stem/progenitor cells (HSPCs) plays a central role in the pathophysiology of acquired aplastic anaemia (aAA). Dysregulated CD8(+) cytotoxic T cells, CD4(+) T cells including T helper type 1 (Th1), Th2, regulatory T cells and Th17 cells, natural killer (NK) cells and NK T cells, along with the abnormal production of cytokines including interferon (IFN)-γ, tumour necrosis factor (TNF)-α and transforming growth factor (TGF)-β, induce apoptosis of HSPCs, constituting a consistent and defining feature of severe aAA. Alterations in the polymorphisms of TGF-β, IFN-γ and TNF-α genes, as well as certain human leucocyte antigen (HLA) alleles, may account for the propensity to immune-mediated killing of HSPCs and/or ineffective haematopoiesis. Although the inciting autoantigens remain elusive, autoantibodies are often detected in the serum. In addition, recent studies provide genetic and molecular evidence that intrinsic and/or secondary deficits in HSPCs and bone marrow mesenchymal stem cells may underlie the development of bone marrow failure.
• Alizadeh, D., Katsanis, E., & Larmonier, N. (2014). Chemotherapeutic targeting of myeloid-derived suppressor cells. Oncoimmunology, 3(1), e27359.
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  Myeloid-derived suppressor cells (MDSCs), which expand in cancer-bearing hosts, contribute to the escape of malignant cells from immune destruction and impair the efficacy of immunotherapeutic interventions. We have recently demonstrated that the conventional chemotherapeutic agent doxorubicin selectively eliminates MDSCs, hence promoting the activity of immune effector cells and improving the therapeutic profile of adoptively transferred helper T lymphocytes.
• Alizadeh, D., Trad, M., Hanke, N. T., Larmonier, C. B., Janikashvili, N., Bonnotte, B., Katsanis, E., & Larmonier, N. (2014). Doxorubicin eliminates myeloid-derived suppressor cells and enhances the efficacy of adoptive T-cell transfer in breast cancer. Cancer research, 74(1), 104-18.
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  Myeloid-derived suppressor cells (MDSC) expand in tumor-bearing hosts and play a central role in cancer immune evasion by inhibiting adaptive and innate immunity. They therefore represent a major obstacle for successful cancer immunotherapy. Different strategies have thus been explored to deplete and/or inactivate MDSC in vivo. Using a murine mammary cancer model, we demonstrated that doxorubicin selectively eliminates MDSC in the spleen, blood, and tumor beds. Furthermore, residual MDSC from doxorubicin-treated mice exhibited impaired suppressive function. Importantly, the frequency of CD4(+) and CD8(+) T lymphocytes and consequently the effector lymphocytes or natural killer (NK) to suppressive MDSC ratios were significantly increased following doxorubicin treatment of tumor-bearing mice. In addition, the proportion of NK and cytotoxic T cell (CTL) expressing perforin and granzyme B and of CTL producing IFN-γ was augmented by doxorubicin administration. Of therapeutic relevance, this drug efficiently combined with Th1 or Th17 lymphocytes to suppress tumor development and metastatic disease. MDSC isolated from patients with different types of cancer were also sensitive to doxorubicin-mediated cytotoxicity in vitro. These results thus indicate that doxorubicin may be used not only as a direct cytotoxic drug against tumor cells, but also as a potent immunomodulatory agent that selectively impairs MDSC-induced immunosuppression, thereby fostering the efficacy of T-cell-based immunotherapy.
• Graner, M. W., Lillehei, K. O., & Katsanis, E. (2014). Endoplasmic reticulum chaperones and their roles in the immunogenicity of cancer vaccines. Frontiers in oncology, 4, 379.
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  The endoplasmic reticulum (ER) is a major site of passage for proteins en route to other organelles, to the cell surface, and to the extracellular space. It is also the transport route for peptides generated in the cytosol by the proteasome into the ER for loading onto major histocompatibility complex class I (MHC I) molecules for eventual antigen presentation at the cell surface. Chaperones within the ER are critical for many of these processes; however, outside the ER certain of those chaperones may play important and direct roles in immune responses. In some cases, particular ER chaperones have been utilized as vaccines against tumors or infectious disease pathogens when purified from tumor tissue or recombinantly generated and loaded with antigen. In other cases, the cell surface location of ER chaperones has implications for immune responses as well as possible tumor resistance. We have produced heat-shock protein/chaperone protein-based cancer vaccines called "chaperone-rich cell lysate" (CRCL) that are conglomerates of chaperones enriched from solid tumors by an isoelectric focusing technique. These preparations have been effective against numerous murine tumors, as well as in a canine with an advanced lung carcinoma treated with autologous CRCL. We also published extensive proteomic analyses of CRCL prepared from human surgically resected tumor samples. Of note, these preparations contained at least 10 ER chaperones and a number of other residents, along with many other chaperones/heat-shock proteins. Gene ontology and network analyses utilizing these proteins essentially recapitulate the antigen presentation pathways and interconnections. In conjunction with our current knowledge of cell surface/extracellular ER chaperones, these data collectively suggest that a systems-level view may provide insight into the potent immune stimulatory activities of CRCL with an emphasis on the roles of ER components in those processes.
• Hanke, N. T., LaCasse, C. J., Larmonier, C. B., Alizadeh, D., Trad, M., Janikashvili, N., Bonnotte, B., Katsanis, E., & Larmonier, N. (2014). PIAS1 and STAT-3 impair the tumoricidal potential of IFN-γ-stimulated mouse dendritic cells generated with IL-15. European journal of immunology, 44(8), 2489-2499.
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  Primarily defined by their antigen-presenting property, dendritic cells (DCs) are being implemented as cancer vaccines in immunotherapeutic interventions. DCs can also function as direct tumor cell killers. How DC cytotoxic activity can be efficiently harnessed and the mechanisms controlling this nonconventional property are not fully understood. We report here that the tumoricidal potential of mouse DCs generated from myeloid precursors with GM-CSF and IL-15 (IL-15 DCs) can be triggered with the Toll-like receptor (TLR) 4 ligand lipopolysaccharide to a similar extent compared with that of their counterparts, conventionally generated with IL-4 (IL-4 DCs). The mechanism of tumor cell killing depends on the induction of iNOS expression by DCs. In contrast, interferon (IFN)-γ induces the cytotoxic activity of IL-4 but not IL-15 DCs. Although the IFN-γ-STAT-1 signaling pathway is overall functional in IL-15 DCs, IFN-γ fails to induce iNOS expression in these cells. iNOS expression is negatively controlled in IFN-γ-stimulated IL-15 DCs by the cooperation between the E3 SUMO ligase PIAS1 and STAT-3, and can be partially restored with PIAS1 siRNA and STAT-3 inhibitors.
• Karski, E. E., Dvorak, C. C., Leung, W., Miller, W., Shaw, P. J., Qayed, M., Katsanis, E., & Feusner, J. H. (2014). Treatment of hepatoblastoma with high-dose chemotherapy and stem cell rescue: the pediatric blood and marrow transplant consortium experience and review of the literature. Journal of pediatric hematology/oncology, 36(5), 362-8.
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  Children with high-risk or relapsed hepatoblastoma continue to represent treatment challenges. Multiple case reports have documented the use of high-dose chemotherapy with stem cell rescue (HDC) for this population; however, the efficacy and appropriate use of HDC remains unclear.
• Lund, T. C., Cathey, S. S., Miller, W. P., Eapen, M., Andreansky, M., Dvorak, C. C., Davis, J. H., Dalal, J. D., Devine, S. M., Eames, G. M., Ferguson, W. S., Giller, R. H., He, W., Kurtzberg, J., Krance, R., Katsanis, E., Lewis, V. A., Sahdev, I., & Orchard, P. J. (2014). Outcomes after hematopoietic stem cell transplantation for children with I-cell disease. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation, 20(11), 1847-51.
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  Mucolipidosis type II (MLII), or I-cell disease, is a rare but severe disorder affecting localization of enzymes to the lysosome, generally resulting in death before the 10th birthday. Although hematopoietic stem cell transplantation (HSCT) has been used to successfully treat some lysosomal storage diseases, only 2 cases have been reported on the use of HSCT to treat MLII. For the first time, we describe the combined international experience in the use of HSCT for MLII in 22 patients. Although 95% of the patients engrafted, overall survival was low, with only 6 patients (27%) alive at last follow-up. The most common cause of death post-transplant was cardiovascular complications, most likely due to disease progression. Survivors were globally delayed in development and often required complex medical support, such as gastrostomy tubes for nutrition and tracheostomy with mechanical ventilation. Although HSCT has demonstrated efficacy in treating some lysosomal storage disorders, the neurologic outcome and survival for patents with MLII were poor. Therefore, new medical and cellular therapies should be sought for these patients.
• Zeng, Y., Stokes, J., Hahn, S., Hoffman, E., & Katsanis, E. (2014). Activated MHC-mismatched T helper-1 lymphocyte infusion enhances GvL with limited GvHD. Bone marrow transplantation, 49(8), 1076-83.
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  DLI is traditionally used to provide graft-versus-leukemia (GvL) effects when given to patients relapsing post-hematopoietic cell transplantation (HCT). However, it is often associated with significant GvHD and has only modest efficacy against acute leukemias. Therefore, novel cellular therapies are needed to improve the outcome of high-risk or relapsed leukemia patients following HCT. Activated T helper-1 (aTh-1) lymphocytes are CD4(+)CD25(+)CD40L(+)CD62L(lo) effector memory cells that produce large amounts of IFN-γ and TNF-α. We demonstrate that post-transplant adoptive aTh-1 cell therapy enhances GvL with limited GvHD in an MHC-mismatched murine BMT model. aTh-1 infusions result in superior leukemia-free survival when compared with unstimulated splenocytes (SC), purified CD4(+) T-cells and T-cell-enriched SC. aTh-1 cells display cytotoxicity against A20 leukemia cells in vitro and persist in vivo for at least 2 months following adoptive transfer. Furthermore, in contrast to unstimulated SC, aTh-1 cell infusion is associated with only transient, mild suppression of donor-derived hematopoiesis. aTh-1 cell therapy is safe, effective and warrants further investigation as an alternative to DLI.
• Alizadeh, D., Katsanis, E., & Larmonier, N. (2013). The multifaceted role of Th17 lymphocytes and their associated cytokines in cancer. Clinical & developmental immunology, 2013, 957878.
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  While the role of T helper 17 lymphocytes (Th17) in the pathogenesis of autoimmune diseases and in infectious immunity has been relatively well defined, the impact of these cells and their associated cytokines on cancer development is still under debate. Although multiple reports have indicated that Th17 can promote anticancer immunity, others have argued that these cells may exhibit tumor-promoting properties. This dichotomy in the function of Th17 lymphocytes in cancer may be related to the versatile nature of these cells, being capable of differentiating into either proinflammatory Th1 or suppressive FoxP3-expressing Treg cells or hybrid T cell subsets depending on the underlying environmental conditions. In the current review, we examine the role of Th17 lymphocytes and Th17-associated cytokines in cancer and discuss how factors that control their final lineage commitment decision may influence the balance between their tumor-promoting versus tumor-suppressing properties.
• Epple, L. M., Bemis, L. T., Cavanaugh, R. P., Skope, A., Mayer-Sonnenfeld, T., Frank, C., Olver, C. S., Lencioni, A. M., Dusto, N. L., Tal, A., Har-Noy, M., Lillehei, K. O., Katsanis, E., & Graner, M. W. (2013). Prolonged remission of advanced bronchoalveolar adenocarcinoma in a dog treated with autologous, tumour-derived chaperone-rich cell lysate (CRCL) vaccine. International journal of hyperthermia : the official journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group, 29(5), 390-8.
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  This paper presents the treatment of a 12-year-old female spayed Great Dane who presented with vestibular signs (ataxia, nystagmus, hind end collapse). Thoracic radiographs revealed a discrete pulmonary nodule in the right cranial lung lobe. Ultrasound-guided fine needle aspirate detected primary bronchoalveolar adenocarcinoma, verified via computed tomography, with a second smaller nodule discovered in the right cranial lung lobe.
• Graner, M. W., Romanoski, A., & Katsanis, E. (2013). The 'peptidome' of tumour-derived chaperone-rich cell lysate anti-cancer vaccines reveals potential tumour antigens that stimulate tumour immunity. International journal of hyperthermia : the official journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group, 29(5), 380-9.
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  Tumour-derived chaperone-rich cell lysate (CRCL) when isolated from tumour tissue or when embedded with peptide antigens is a potent anti-cancer vaccine consisting of numerous chaperone/heat shock proteins, including the highly immunogenic Hsp70, Hsp90, glucose regulated protein 94, and calreticulin. We have previously documented that CRCL provides both a source of tumour antigens and danger signals triggering antigen presenting cell activation. In this report we describe the 'peptidome' of potential antigens extracted from CRCL prepared from a murine tumour. Using mass spectrometry techniques we identify almost 60 different proteins of origin for the CRCL peptides; we determine that the parental proteins come from essentially all parts of the cell, and are involved in a broad range of functions. Further in silico analysis demonstrates that the parental proteins are components of major signalling networks of vital importance for cancer cell survival, proliferation, and migration. In many instances the peptides identified possess amino acid sequences that would allow their putative binding and display by murine major histocompatibility complex class I and II molecules, and there are also predicted binding motifs for Hsp70-type chaperones. By mixing fractionated pools of peptides with antigen-free (normal liver) CRCL, we were able to reconstitute effective anti-tumour activity of the vaccine, showing that the peptides are indeed the major purveyors of CRCL vaccines' efficacy.
• Hanke, N., Alizadeh, D., Katsanis, E., & Larmonier, N. (2013). Dendritic cell tumor killing activity and its potential applications in cancer immunotherapy. Critical reviews in immunology, 33(1), 1-21.
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  Universally viewed as the sentinels and messengers of the immune system and traditionally referred to as professional antigen-presenting cells, dendritic cells (DCs) play a fundamental role in antitumor immunity. DCs are uniquely equipped with the ability to acquire, process, and present to T lymphocytes tumor-derived antigens. They can drive the differentiation of naive T cells into activated tumor-specific effector lymphocytes. DCs also dictate the type and regulate the strength and duration of T-cell responses. In addition, they contribute to natural killer and natural killer T-cell antitumoral function and to B-cell-mediated immunity. Besides this cardinal role as orchestrators of innate and adaptive immune responses, many studies have provided evidence that DCs can also function as direct cytotoxic effectors against tumors. This less conventional aspect of DC function has, however, raised controversy as it relates to the origin of these cells and the induction, regulation, and mechanisms underlying their tumoricidal activity. The possible impact of the cytotoxic function of DCs on their capability to present antigens also has been the focus of intensive research. This review examines these questions and discusses the biological significance of this nontraditional property and possible strategies to exploit the killing potential of DCs in cancer immunotherapy.
• Bravo, R., Pelayo-Katsanis, L. O., Shehab, Z. M., & Katsanis, E. (2012). Diagnostic and treatment challenges for the pediatric hematologist oncologist in endemic areas for coccidioidomycosis. Journal of pediatric hematology/oncology, 34(5), 389-94.
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  Coccidioidomycosis is a mycosis endemic to certain areas in the Southwest, mostly Arizona and California, Mexico, and parts of Central and South America. Disseminated coccidioidomycosis is much more common in immunocompromised hosts; therefore, it is frequently encountered by pediatric oncologists in endemic areas. Special attention is needed to diagnose, effectively treat the infection, and appropriately adjust chemotherapy treatment plans to minimize immunosuppression. We describe the presentation and course of 6 patients with coccidioidomycosis who were seen by the pediatric hematology-oncology service at the University of Arizona during the last 3 years. Coccidioidomycosis is a relatively common infection encountered by pediatric oncologists in the southwestern states and should be considered in the differential diagnosis of patients living or visiting these areas.
• Centuori, S. M., Trad, M., LaCasse, C. J., Alizadeh, D., Larmonier, C. B., Hanke, N. T., Kartchner, J., Janikashvili, N., Bonnotte, B., Larmonier, N., & Katsanis, E. (2012). Myeloid-derived suppressor cells from tumor-bearing mice impair TGF-β-induced differentiation of CD4+CD25+FoxP3+ Tregs from CD4+CD25-FoxP3- T cells. Journal of leukocyte biology, 92(5), 987-97.
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  MDSCs and Tregs play an essential role in the immunosuppressive networks that contribute to tumor-immune evasion. The mechanisms by which tumors promote the expansion and/or function of these suppressive cells and the cross-talk between MDSC and Treg remain incompletely defined. Previous reports have suggested that MDSC may contribute to Treg induction in cancer. Herein, we provide evidence that tumor-induced gr-MDSCs, endowed with the potential of suppressing conventional T Lc, surprisingly impair TGF-β1-mediated generation of CD4(+)CD25(+)FoxP3(+) iTregs. Furthermore, gr-MDSCs impede the proliferation of nTregs without, however, affecting FoxP3 expression. Suppression of iTreg differentiation from naïve CD4(+) cells by gr-MDSC occurs early in the polarization process, requires inhibition of early T cell activation, and depends on ROS and IDO but does not require arginase 1, iNOS, NO, cystine/cysteine depletion, PD-1 and PD-L1 signaling, or COX-2. These findings thus indicate that gr-MDSCs from TB hosts have the unanticipated ability to restrict immunosuppressive Tregs.
• Kopp, L. M., Gupta, P., Pelayo-Katsanis, L., Wittman, B., & Katsanis, E. (2012). Late effects in adult survivors of pediatric cancer: a guide for the primary care physician. The American journal of medicine, 125(7), 636-41.
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  Because of significant medical advances in the past 50 years, the number of adult survivors of childhood/adolescent cancer has increased dramatically. Unfortunately, more than 60% of these survivors will have at least 1 long-term side effect from treatment. This growing population requires dedicated care by their primary physicians because they have specific risk factors depending on their initial cancer diagnosis and the treatment modalities they received. Internists and family physicians play an integral role in providing appropriate screening, treatment, and counseling to prevent morbidity and mortality in these patients.
• Larmonier, N., Bonnotte, B., & Katsanis, E. (2012). Cytotoxic and antigen presenting functions of T helper-1-activated dendritic cells. Oncoimmunology, 1(4), 566-568.
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  Although primarily defined by their cardinal antigen-presenting function, dendritic cells (DCs) are also equipped with cytotoxic properties. We have recently reported that DCs activated by IFNγ-secreting Th-1 lymphocytes can kill cancer cells and subsequently present the acquired tumor-derived antigens to T lymphocytes both in vitro and in vivo.
• Audia, S., Samson, M., Guy, J., Janikashvili, N., Fraszczak, J., Trad, M., Ciudad, M., Leguy, V., Berthier, S., Petrella, T., Aho-Glélé, S., Martin, L., Maynadié, M., Lorcerie, B., Rat, P., Cheynel, N., Katsanis, E., Larmonier, N., & Bonnotte, B. (2011). Immunologic effects of rituximab on the human spleen in immune thrombocytopenia. Blood, 118(16), 4394-400.
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  Immune thrombocytopenia (ITP) is an autoimmune disease with a complex pathogenesis. As in many B cell-related autoimmune diseases, rituximab (RTX) has been shown to increase platelet counts in some ITP patients. From an immunologic standpoint, the mode of action of RTX and the reasons underlying its limited efficacy have yet to be elucidated. Because splenectomy is a cornerstone treatment of ITP, the immune effect of RTX on this major secondary lymphoid organ was investigated in 18 spleens removed from ITP patients who were treated or not with RTX. Spleens from ITP individuals had follicular hyperplasia consistent with secondary follicles. RTX therapy resulted in complete B-cell depletion in the blood and a significant reduction in splenic B cells, but these patients did not achieve remission. Moreover, whereas the percentage of circulating regulatory T cells (Tregs) was similar to that in controls, splenic Tregs were reduced in ITP patients. Interestingly, the ratio of proinflammatory Th1 cells to suppressive Tregs was increased in the spleens of patients who failed RTX therapy. These results indicate that although B cells are involved in ITP pathogenesis, RTX-induced total B-cell depletion is not correlated with its therapeutic effects, which suggests additional immune-mediated mechanisms of action of this drug.
• Janikashvili, N., Bonnotte, B., Katsanis, E., & Larmonier, N. (2011). The dendritic cell-regulatory T lymphocyte crosstalk contributes to tumor-induced tolerance. Clinical & developmental immunology, 2011, 430394.
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  Tumor cells commonly escape from elimination by innate and adaptive immune responses using multiple strategies among which is the active suppression of effector immune cells. Regulatory T lymphocytes (Treg) and tolerogenic dendritic cells play essential roles in the establishment and persistence of cancer-induced immunosuppression. Differentiating dendritic cells (DCs) exposed to tumor-derived factors may be arrested at an immature stage becoming inept at initiating immune responses and may induce effector T-cell anergy or deletion. These tolerogenic DCs, which accumulate in patients with different types of cancers, are also involved in the generation of Treg. In turn, Treg that expand during tumor progression contribute to the immune tolerance of cancer by impeding DCs' ability to orchestrate immune responses and by directly inhibiting antitumoral T lymphocytes. Herein we review these bidirectional communications between DCs and Treg as they relate to the promotion of cancer-induced tolerance.
• Janikashvili, N., LaCasse, C. J., Larmonier, C., Trad, M., Herrell, A., Bustamante, S., Bonnotte, B., Har-Noy, M., Larmonier, N., & Katsanis, E. (2011). Allogeneic effector/memory Th-1 cells impair FoxP3+ regulatory T lymphocytes and synergize with chaperone-rich cell lysate vaccine to treat leukemia. Blood, 117(5), 1555-64.
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  Therapeutic strategies combining the induction of effective antitumor immunity with the inhibition of the mechanisms of tumor-induced immunosuppression represent a key objective in cancer immunotherapy. Herein we demonstrate that effector/memory CD4(+) T helper-1 (Th-1) lymphocytes, in addition to polarizing type-1 antitumor immune responses, impair tumor-induced CD4(+)CD25(+)FoxP3(+) regulatory T lymphocyte (Treg) immunosuppressive function in vitro and in vivo. Th-1 cells also inhibit the generation of FoxP3(+) Tregs from naive CD4(+)CD25(-)FoxP3(-) T cells by an interferon-γ-dependent mechanism. In addition, in an aggressive mouse leukemia model (12B1), Th-1 lymphocytes act synergistically with a chaperone-rich cell lysate (CRCL) vaccine, leading to improved survival and long-lasting protection against leukemia. The combination of CRCL as a source of tumor-specific antigens and Th-1 lymphocytes as an adjuvant has the potential to stimulate efficient specific antitumor immunity while restraining Treg-induced suppression.
• LaCasse, C. J., Janikashvili, N., Larmonier, C. B., Alizadeh, D., Hanke, N., Kartchner, J., Situ, E., Centuori, S., Har-Noy, M., Bonnotte, B., Katsanis, E., & Larmonier, N. (2011). Th-1 lymphocytes induce dendritic cell tumor killing activity by an IFN-γ-dependent mechanism. Journal of immunology (Baltimore, Md. : 1950), 187(12), 6310-7.
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  Dendritic cells (DCs) encompass a heterogeneous population of cells capable of orchestrating innate and adaptive immune responses. The ability of DCs to act as professional APCs has been the foundation for the development and use of these cells as vaccines in cancer immunotherapy. DCs are also endowed with the nonconventional property of directly killing tumor cells. The current study investigates the regulation of murine DC cytotoxic function by T lymphocytes. We provide evidence that CD4(+) Th-1, but not Th-2, Th-17 cells, or regulatory T cells, are capable of inducing DC cytotoxic function. IFN-γ was identified as the major factor responsible for Th-1-induced DC tumoricidal activity. Tumor cell killing mediated by Th-1-activated killer DCs was dependent on inducible NO synthase expression and NO production. Importantly, Th-1-activated killer DCs were capable of presenting the acquired Ags from the killed tumor cells to T lymphocytes in vitro or in vivo. These observations offer new possibilities for the application of killer DCs in cancer immunotherapy.
• Lakomy, D., Janikashvili, N., Fraszczak, J., Trad, M., Audia, S., Samson, M., Ciudad, M., Vinit, J., Vergely, C., Caillot, D., Foucher, P., Lagrost, L., Chouaib, S., Katsanis, E., Larmonier, N., & Bonnotte, B. (2011). Cytotoxic dendritic cells generated from cancer patients. Journal of immunology (Baltimore, Md. : 1950), 187(5), 2775-82.
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  Known for years as professional APCs, dendritic cells (DCs) are also endowed with tumoricidal activity. This dual role of DC as killers and messengers may have important implications for tumor immunotherapy. However, the tumoricidal activity of DCs has mainly been investigated in animal models. Cancer cells inhibit antitumor immune responses using numerous mechanisms, including the induction of immunosuppressive/ tolerogenic DCs that have lost their ability to present Ags in an immunogenic manner. In this study, we evaluated the possibility of generating tumor killer DCs from patients with advanced-stage cancers. We demonstrate that human monocyte-derived DCs are endowed with significant cytotoxic activity against tumor cells following activation with LPS. The mechanism of DC-mediated tumor cell killing primarily involves peroxynitrites. This observed cytotoxic activity is restricted to immature DCs. Additionally, after killing, these cytotoxic DCs are able to activate tumor Ag-specific T cells. These observations may open important new perspectives for the use of autologous cytotoxic DCs in cancer immunotherapy strategies.
• Cantrell, J., Larmonier, C., Janikashvili, N., Bustamante, S., Fraszczak, J., Herrell, A., Lundeen, T., J LaCasse, C., Situ, E., Larmonier, N., & Katsanis, E. (2010). Signaling pathways induced by a tumor-derived vaccine in antigen presenting cells. Immunobiology, 215(7), 535-44.
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  We have previously reported on the anti-tumoral potential of a chaperone-rich cell lysate (CRCL) vaccine. Immunization with CRCL generated from tumors elicits specific T and NK cell-dependent immune responses leading to protective immunity in numerous mouse tumor models. CRCL provides both a source of tumor antigens and danger signals leading to dendritic cell activation. In humans, tumor-derived CRCL induces dendritic cell activation and CRCL-loaded dendritic cells promote the generation of cytotoxic T lymphocytes in vitro. The current study was designed to identify the signaling events and modifications triggered by CRCL in antigen presenting cells. Our results indicate that tumor-derived CRCL not only promotes the activation of dendritic cells, but also significantly fosters the function of macrophages that thus appear as major targets of this vaccine. Activation of both cell types is associated with the induction of the MAP kinase pathway, the phosphorylation of STAT1, STAT5 and AKT and with transcription factor NF-kappaB activation in vitro and in vivo. These results thus provide important insights into the mechanisms by which CRCL-based vaccines exert their adjuvant effects on antigen presenting cells.
• Fraszczak, J., Trad, M., Janikashvili, N., Cathelin, D., Lakomy, D., Granci, V., Morizot, A., Audia, S., Micheau, O., Lagrost, L., Katsanis, E., Solary, E., Larmonier, N., & Bonnotte, B. (2010). Peroxynitrite-dependent killing of cancer cells and presentation of released tumor antigens by activated dendritic cells. Journal of immunology (Baltimore, Md. : 1950), 184(4), 1876-84.
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  Dendritic cells (DCs), essential for the initiation and regulation of adaptive immune responses, have been used as anticancer vaccines. DCs may also directly trigger tumor cell death. In the current study, we have investigated the tumoricidal and immunostimulatory activities of mouse bone marrow-derived DCs. Our results indicate that these cells acquire killing capabilities toward tumor cells only when activated with LPS or Pam3Cys-SK4. Using different transgenic mouse models including inducible NO synthase or GP91 knockout mice, we have further established that LPS- or Pam3Cys-SK4-activated DC killing activity involves peroxynitrites. Importantly, after killing of cancer cells, DCs are capable of engulfing dead tumor cell fragments and of presenting tumor Ags to specific T lymphocytes. Thus, upon specific stimulation, mouse bone marrow-derived DCs can directly kill tumor cells through a novel peroxynitrite-dependent mechanism and participate at virtually all levels of antitumor immune responses, which reinforces their interest in immunotherapy.
• Janikashvili, N., Larmonier, N., & Katsanis, E. (2010). Personalized dendritic cell-based tumor immunotherapy. Immunotherapy, 2(1), 57-68.
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  Advances in the understanding of the immunoregulatory functions of dendritic cells (DCs) in animal models and humans have led to their exploitation as anticancer vaccines. Although DC-based immunotherapy has proven clinically safe and efficient to induce tumor-specific immune responses, only a limited number of objective clinical responses have been reported in cancer patients. These relatively disappointing results have prompted the evaluation of multiple approaches to improve the efficacy of DC vaccines. The topic of this review focuses on personalized DC-based anticancer vaccines, which in theory have the potential to present to the host immune system the entire repertoire of antigens harbored by autologous tumor cells. We also discuss the implementation of these vaccines in cancer therapeutic strategies, their limitations and the future challenges for effective immunotherapy against cancer.
• Larmonier, N., Fraszczak, J., Lakomy, D., Bonnotte, B., & Katsanis, E. (2010). Killer dendritic cells and their potential for cancer immunotherapy. Cancer immunology, immunotherapy : CII, 59(1), 1-11.
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  Known for years as the principal messengers of the immune system, dendritic cells (DC) represent a heterogeneous population of antigen presenting cells critically located at the nexus between innate and adaptive immunity. DC play a central role in the initiation of tumor-specific immune responses as they are endowed with the unique ability to take up, process and present tumor antigens to naïve CD4(+) or CD8(+) effector T lymphocytes. By virtue of the cytokines they produce, DC also regulate the type, strength and duration of T cell immune responses. In addition, they can participate in anti-tumoral NK and NKT cell activation and in the orchestration of humoral immunity. More recent studies have documented that besides their primary role in the induction and regulation of adaptive anti-tumoral immune responses, DC are also endowed with the capacity to directly kill cancer cells. This dual role of DC as killers and messengers may have important implications for tumor immunotherapy. First, the direct killing of malignant cells by DC may foster the release and thereby the immediate availability of specific tumor antigens for presentation to cytotoxic or helper T lymphocytes. Second, DC may participate in the effector phase of the immune response, potentially augmenting the diversity of the killing mechanisms leading to tumor elimination. This review focuses on this non-conventional cytotoxic function of DC as it relates to the promotion of cancer immunity and discusses the potential application of killer DC (KDC) in tumor immunotherapy.
• Bleifuss, E., Bendz, H., Sirch, B., Thompson, S., Brandl, A., Milani, V., Graner, M. W., Drexler, I., Kuppner, M., Katsanis, E., Noessner, E., & Issels, R. D. (2008). Differential capacity of chaperone-rich lysates in cross-presenting human endogenous and exogenous melanoma differentiation antigens. International journal of hyperthermia : the official journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group, 24(8), 623-37.
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  The goal of immune-based tumor therapies is the activation of immune cells reactive against a broad spectrum of tumor-expressed antigens. Vaccines based on chaperone proteins appear promising as these proteins naturally exist as complexes with various protein fragments including those derived from tumor-associated antigens. Multi-chaperone systems are expected to have highest polyvalency as different chaperones can carry distinct sets of antigenic fragments. A free-solution isoelectric focusing (FS-IEF) technique was established to generate chaperone-rich cell lysates (CRCL). Results from murine systems support the contention that CRCL induce superior anti-tumor responses than single chaperone vaccines. We established an in vitro model for human melanoma to evaluate the capacity of CRCL to transfer endogenously expressed tumor antigens to the cross-presentation pathway of dendritic cells (DC) for antigen-specific T cell stimulation. CRCL prepared from human melanoma lines contained the four major chaperone proteins Hsp/Hsc70, Hsp90, Grp94/gp96 and calreticulin. The chaperones within the melanoma cell-derived CRCL were functionally active in that they enhanced cross-presentation of exogenous peptides mixed into the CRCL preparation. Superior activity was observed for Hsp70-rich CRCL obtained from heat-stressed melanoma cells. Despite the presence of active chaperones, melanoma cell-derived CRCL failed to transfer endogenously expressed melanoma-associated antigens to DC for cross-presentation and cytotoxic T cell (CTL) recognition, even after increasing intracellular protein levels of tumor antigen or chaperones. These findings reveal limitations of the CRCL approach regarding cross-presentation of endogenously expressed melanoma-associated antigens. Yet, CRCL may be utilized as vehicles to enhance the delivery of exogenous antigens for DC-mediated cross-presentation and T cell stimulation.
• Larmonier, N., Cantrell, J., Lacasse, C., Li, G., Janikashvili, N., Situ, E., Sepassi, M., Andreansky, S., & Katsanis, E. (2008). Chaperone-rich tumor cell lysate-mediated activation of antigen-presenting cells resists regulatory T cell suppression. Journal of leukocyte biology, 83(4), 1049-59.
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  CD4(+)CD25(+) regulatory T lymphocytes (Tregs) critically contribute to the mechanisms of cancer-induced tolerance. These cells suppress anti-tumoral CD8(+) and CD4(+) T lymphocytes and can also restrain the function of APCs. We have previously documented the immunostimulatory effects of a chaperone-rich cell lysate (CRCL) anti-cancer vaccine. Tumor-derived CRCL induces tumor immunity in vivo, partly by promoting dendritic cell (DC) and macrophage activation. In the current study, we evaluated the effects of CD4(+)CD25(+)forkhead box P3(+) Tregs isolated from mice bearing 12B1 bcr-abl(+) leukemia on DC and macrophages that had been activated by 12B1-derived CRCL. CRCL-activated DC and macrophages resisted Treg suppression, as the production of proinflammatory cytokines, the activation of transcription factor NF-kappaB, and their immunostimulatory potential was unaffected by Tregs. Our results thus highlight CRCL as a powerful adjuvant endowed with the capacity to overcome tumor-induced Treg-inhibitory effects on APCs.
• Larmonier, N., Janikashvili, N., LaCasse, C. J., Larmonier, C. B., Cantrell, J., Situ, E., Lundeen, T., Bonnotte, B., & Katsanis, E. (2008). Imatinib mesylate inhibits CD4+ CD25+ regulatory T cell activity and enhances active immunotherapy against BCR-ABL- tumors. Journal of immunology (Baltimore, Md. : 1950), 181(10), 6955-63.
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  Imatinib mesylate (Gleevec, STI571), a selective inhibitor of a restricted number of tyrosine kinases, has been effectively used for the treatment of Philadelphia chromosome-positive leukemias and gastrointestinal stromal tumors. Imatinib may also directly influence immune cells. Suppressive as well as stimulating effects of this drug on CD4(+) and CD8(+) T lymphocytes or dendritic cells have been reported. In the current study, we have investigated the influence of imatinib mesylate on CD4(+)CD25(+)FoxP3(+) regulatory T cells (Treg), a critical population of lymphocytes that contributes to peripheral tolerance. Used at concentrations achieved clinically, imatinib impaired Treg immunosuppressive function and FoxP3 expression but not production of IL-10 and TGF-beta in vitro. Imatinib significantly reduced the activation of the transcription factors STAT3 and STAT5 in Treg. Analysis of Treg TCR-induced signaling cascade indicated that imatinib inhibited phosphorylation of ZAP70 and LAT. Substantiating these observations, imatinib treatment of mice decreased Treg frequency and impaired their immunosuppressive function in vivo. Furthermore, imatinib mesylate significantly enhanced antitumor immune responses to dendritic cell-based immunization against an imatinib-resistant BCR-ABL negative lymphoma. The clinical applications of imatinib mesylate might thus be expanded with its use as a potent immunomodulatory agent targeting Treg in cancer immunotherapy.
• Li, G., Andreansky, S., Helguera, G., Sepassi, M., Janikashvili, N., Cantrell, J., Lacasse, C. L., Larmonier, N., Penichet, M. L., & Katsanis, E. (2008). A chaperone protein-enriched tumor cell lysate vaccine generates protective humoral immunity in a mouse breast cancer model. Molecular cancer therapeutics, 7(3), 721-9.
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  We have documented previously that a multiple chaperone protein vaccine termed chaperone-rich cell lysate (CRCL) promotes tumor-specific T-cell responses leading to cancer regression in several mouse tumor models. We report here that CRCL vaccine generated from a mouse breast cancer (TUBO, HER2/neu positive) is also capable of eliciting humoral immunity. Administration of TUBO CRCL triggered anti-HER2/neu antibody production and delayed the progression of established tumors. This antitumor activity can be transferred through the serum isolated from TUBO CRCL-immunized animals and involved both B cells and CD4(+) T lymphocytes. Further evaluation of the mechanisms underlying TUBO CRCL-mediated humoral immunity highlighted the role of antibody-dependent cell-mediated cytotoxicity. These results suggest that tumor-derived CRCL vaccine has a wider applicability as a cancer vaccine because it can target both T-cell- and B-cell-specific responses and may represent a promising approach for the immunotherapy of cancer.
• Larmonier, N., Cathelin, D., Larmonier, C., Nicolas, A., Merino, D., Janikashvili, N., Audia, S., Bateman, A., Thompson, J., Kottke, T., Hartung, T., Katsanis, E., Vile, R., & Bonnotte, B. (2007). The inhibition of TNF-alpha anti-tumoral properties by blocking antibodies promotes tumor growth in a rat model. Experimental cell research, 313(11), 2345-55.
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  Tumor necrosis factor (TNF) antagonists represent a milestone in the therapy of autoimmune conditions. Anti-TNF antibodies have been approved for clinical use and during the last eight years thousands of patients have been treated. However, the long-term sequelae of anti-TNF agents in promoting carcinogenesis remain unclear. This study sought to define the role of intra-tumor TNF-alpha production on cancer cell progression and to determine whether TNF-alpha antibodies can suppress anti-tumoral immunity. Using an experimental animal tumor model we demonstrate that anti-TNF-alpha antibodies hinder anti-tumor immune responses and promote growth of immunogenic rat colon tumors (REG) that are always rejected by immunocompetent untreated rats. The major role of TNF-alpha in the anti-tumoral immune response was confirmed by transfecting progressive and tolerogenic rat colon tumor cells (PRO) with the TNF-alpha gene. PRO tumor cells secreting TNF-alpha induce tumor-infiltrating dendritic cell (DC) activation. This triggers a potent immune response leading to tumor rejection and long-lasting immunity. Therefore, the prominent role of TNF-alpha in anti-tumoral immune responses underscores the need for caution and close surveillance following the administration of TNF inhibitors.

Reviews

• Filioglou, D., Husnain, M., Khurana, S., Simpson, R. J., & Katsanis, E. (2023. Has the shortage of fludarabine altered the current paradigm of lymphodepletion in favor of bendamustine?(p. 1329850).
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  The most common lymphodepletion regimen used prior to infusion of chimeric antigen receptor-T cells (CAR-T) is cyclophosphamide (CY) in combination with fludarabine (Flu) (CY-FLU). While cyclophosphamide (CY) possesses lymphotoxic effects, it concurrently preserves regulatory T cell activity, potentially affecting the efficacy of CAR-T cells. Moreover, the use of fludarabine (FLU) has been linked to neurotoxicity, which could complicate the early detection of immune effector cell-associated neurotoxicity syndrome (ICANS) observed in CAR-T cell therapy. Given the ongoing shortage of FLU, alternative lymphodepleting agents have become necessary. To date, only a limited number of studies have directly compared different lymphodepleting regimens, and most of these comparisons have been retrospective in nature. Herein, we review the current literature on lymphodepletion preceding CAR-T cell therapies for lymphoid hematologic malignancies, with a specific focus on the use of bendamustine (BEN). Recent evidence suggests that administering BEN before CAR-T cell infusion yields comparable efficacy, possibly with a more favorable toxicity profile when compared to CY-FLU. This warrants further investigation through randomized prospective studies.
• Kopp, L. M., & Katsanis, E. (2016. Targeted immunotherapy for pediatric solid tumors(pp e1087637).
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  Metastatic and refractory pediatric solid tumor malignancies continue to have a poor outcome despite the > 80% cure rates appreciated in many pediatric cancers. Targeted immunotherapy is impacting treatment and survival in these aggressive tumors. We review current promising immunotherapeutic approaches in the pediatric oncology solid tumor setting.