- Professor, Medicine
- Division Chief, Infectious Disease
- Professor, Immunobiology
- Professor, BIO5 Institute
- M.D. Medicine
- Harvard Medical School, Boston, Massachusetts, United States
- B.A. Anthropology (magna cum laude)
- Bryn Mawr College, Bryn Mawr, Pennsylvania, United States
- University of Arizona, Tucson, Arizona (2016 - Ongoing)
- University of Colorado Denver (2011 - 2016)
- University of Colorado Denver (2003 - 2011)
- University of Colorado Health Sciences Center (1996 - 2003)
- University of Colorado Health Sciences Center (1994 - 1996)
- University of Colorado Health Sciences Center (1991 - 1994)
- Columbia Presbyterian Hospital (1988 - 1991)
- The Constance B. Wofsy Women's Health Investigator Award
- ACTG - AIDS Clinical Trials Group, Fall 2020
- Castle Connolly's Exceptional Women in Medicine for 2019
- Castle Connolly, Fall 2019
- Chair of Session
- National Institues of Health/Bill and Melinda Gate Foundation Joint Workshop on "Finding and Characterizing HIV Reservoirs", Summer 2019
- National Institutes of Health, Fall 2018
- Castle Connolly Top Doctors Tucson
- Castle Connolly, Spring 2019
- Elected to Best Doctors in America
- Best Doctors, Spring 2018
- Top Doctors in Tucson, Infectious Disease 2018
- Castle Connolly Top Doctors, Spring 2018
- Chair, ACTG Women's Health Committee
- AIDS Clinical Trials Group, Winter 2017
- Chair, AIDS Immunopathogenesis Study Section
- NIH/NIAID, Summer 2017
- Outstanding Reviewer
- Journal of AIDS, Fall 2014
Licensure & Certification
- Physician, New York Medical Board (1990)
- Physician, Colorado Medical Board (1992)
- Diplomate, ABIM Infectious Disease (2014)
- Diplomate, National Board of Medical Examiners (1989)
- Diplomate, American Board of Internal Medicine (2004)
- Medical License, Arizona Medical Board (2016)
- Board Certified Infectious Disease, American Board of Internal Medicine (1994)
Clinical care of people with infectious diseases
HIV immunopathogenesis and cure; sex differences in HIV infection; mechanisms underlying accelerated heart disease in HIV infection;
Introduction to ResearchMCB 795A (Spring 2017)
- Baum, L. L., Mathieson, B. J., & Connick, E. (2016). Immunity to HIV. In Encyclopedia of Immunobiology(pp 342-354). Oxford: Academic Press.
- Ellis, K. E., Nawas, G. T., Chan, C., York, L., Fisher, J., Connick, E., & Zangeneh, T. T. (2020). Clinical Outcomes Following the Use of Archived Proviral HIV-1 DNA Genotype to Guide Antiretroviral Therapy Adjustment. Open forum infectious diseases, 7(1), ofz533.More infoEvidence regarding the safety of using proviral HIV-1 DNA genotype (DNA GT) to guide antiretroviral therapy (ART) is limited. We hypothesized that HIV RNA would not increase following ART adjustment guided by DNA GT in a university HIV clinic.
- Fantry, L. E., & Connick, E. (2020). The Internist's Role in Ending the HIV Epidemic in the United States. The American journal of medicine, 133(1), 12-13.
- Fichtenbaum, C. J., Ribaudo, H. J., Leon-Cruz, J., Overton, E. T., Zanni, M. V., Malvestutto, C. D., Aberg, J. A., Kileel, E. M., Fitch, K. V., Van Schalkwyk, M., Kumarasamy, N., Martinez, E., Santos, B. R., Joseph, Y., Lo, J., Siminski, S., Melbourne, K., Sponseller, C. A., Desvigne-Nickens, P., , Bloomfield, G. S., et al. (2020). Patterns of Antiretroviral Therapy Use and Immunologic Profiles at Enrollment in the REPRIEVE Trial. The Journal of infectious diseases, 222(Suppl 1), S8-S19.More infoPatterns of antiretroviral therapy (ART) use and immunologic correlates vary globally, and contemporary trends are not well described.
- Ollerton, M. T., Berger, E. A., Connick, E., & Burton, G. F. (2020). HIV-1-Specific Chimeric Antigen Receptor T Cells Fail To Recognize and Eliminate the Follicular Dendritic Cell HIV Reservoir. Journal of virology, 94(10).More infoThe major obstacle to a cure for HIV infection is the persistence of replication-competent viral reservoirs during antiretroviral therapy. HIV-specific chimeric antigen receptor (CAR) T cells have been developed to target latently infected CD4 T cells that express virus either spontaneously or after intentional latency reversal. Whether HIV-specific CAR-T cells can recognize and eliminate the follicular dendritic cell (FDC) reservoir of HIV-bound immune complexes (ICs) is unknown. We created HIV-specific CAR-T cells using human peripheral blood mononuclear cells (PBMCs) and a CAR construct that enables the expression of CD4 (domains 1 and 2) and the carbohydrate recognition domain of mannose binding lectin (MBL) to target native HIV Env (CD4-MBL CAR). We assessed CAR-T cell cytotoxicity using a carboxyfluorescein succinimidyl ester (CFSE) release assay and evaluated CAR-T cell activation through interferon gamma (IFN-γ) production and CD107a membrane accumulation by flow cytometry. CD4-MBL CAR-T cells displayed potent lytic and functional responses to Env-expressing cell lines and HIV-infected CD4 T cells but were ineffective at targeting FDC bearing HIV-ICs. CD4-MBL CAR-T cells were unresponsive to cell-free HIV or concentrated, immobilized HIV-ICs in cell-free experiments. Blocking intercellular adhesion molecule-1 (ICAM-1) inhibited the cytolytic response of CD4-MBL CAR-T cells to Env-expressing cell lines and HIV-infected CD4 T cells, suggesting that factors such as adhesion molecules are necessary for the stabilization of the CAR-Env interaction to elicit a cytotoxic response. Thus, CD4-MBL CAR-T cells are unable to eliminate the FDC-associated HIV reservoir, and alternative strategies to eradicate this reservoir must be sought. Efforts to cure HIV infection have focused primarily on the elimination of latently infected CD4 T cells. Few studies have addressed the unique reservoir of infectious HIV that exists on follicular dendritic cells (FDCs), persists during antiretroviral therapy, and likely contributes to viral rebound upon cessation of antiretroviral therapy. We assessed the efficacy of a novel HIV-specific chimeric antigen receptor (CAR) T cell to target both HIV-infected CD4 T cells and the FDC reservoir Although CAR-T cells eliminated CD4 T cells that express HIV, they did not respond to or eliminate FDC bound to HIV. These findings reveal a fundamental limitation to CAR-T cell therapy to eradicate HIV.
- Pampusch, M. S., Haran, K. P., Hart, G. T., Rakasz, E. G., Rendahl, A. K., Berger, E. A., Connick, E., & Skinner, P. J. (2020). Rapid Transduction and Expansion of Transduced T Cells with Maintenance of Central Memory Populations. Molecular therapy. Methods & clinical development, 16, 1-10.More infoChimeric antigen receptor (CAR)-T cells show great promise in treating cancers and viral infections. However, most protocols developed to expand T cells require relatively long periods of time in culture, potentially leading to progression toward populations of terminally differentiated effector memory cells. Here, we describe in detail a 9-day protocol for CAR gene transduction and expansion of primary rhesus macaque peripheral blood mononuclear cells (PBMCs). Cells produced and expanded with this method show high levels of viability, high levels of co-expression of two transduced genes, retention of the central memory phenotype, and sufficient quantity for immunotherapeutic infusion of 1-2 × 10 cells/kg in a 10 kg rhesus macaque. This 9-day protocol may be broadly used for CAR-T cell and other T cell immunotherapy approaches to decrease culture time and increase maintenance of central memory populations.
- Beatty, N. L., Hanzlik, T. P., & Connick, E. (2019). West African Man with a Cavitary Pneumonia and Cutaneous Nodule. The American journal of tropical medicine and hygiene, 100(1), 5-6.
- Flerin, N. C., Bardhi, A., Zheng, J. H., Korom, M., Folkvord, J., Kovacs, C., Benko, E., Truong, R., Mota, T., Connick, E., Jones, R. B., Lynch, R. M., & Goldstein, H. (2019). Establishment of A Novel Humanized Mouse Model To Investigate Activation and Depletion of Patient-Derived HIV Latent Reservoirs. Journal of virology.More infoCuring HIV infection has been thwarted by the persistent reservoir of latently-infected CD4 T cells, which reinitiate systemic infection after antiretroviral therapy (ART) interruption. To evaluate reservoir-depletion strategies, we developed a novel pre-clinical model consisting of immunodeficient mice intrasplenically injected with peripheral blood mononuclear cells (PBMC) from long-term ART-suppressed HIV-infected donors. In the absence of ART, these mice developed rebound viremia which, two weeks after PBMC injection, was 1,000-fold higher (mean=9,229,281 HIV copies/ml) in mice injected intrasplenically than in mice injected intraperitoneally (mean=6,838 HIV copies/ml) or intravenously (mean=591 HIV copies/ml). One week after intrasplenic PBMC injection, hybridization of the spleen demonstrated extensive disseminated HIV infection, likely initiated from reactivated primary latently infected cells. Time-to-viremia was delayed significantly by treatment with a broadly neutralizing antibody, 10-1074, as compared to treatment with 10-1074-FcR, suggesting that 10-1074 mobilized Fc-mediated effector mechanisms to deplete the replication-competent reservoir. This was supported by phylogenetic analysis of Env sequences from viral-outgrowth cultures and untreated, 10-1074-treated or 10-1074-FcR-treated mice. The predominant sequence cluster detected in viral-outgrowth cultures and untreated mouse plasma was significantly reduced in plasma of 10-1074-treated mice, while two new clusters emerged which were not detected in viral-outgrowth cultures or plasma from untreated mice. These new clusters lacked mutations associated with 10-1074 resistance. Taken together, these data indicated that 10-1074 treatment depletes the reservoir of latently infected cells harboring replication competent HIV. Furthermore, this mouse model represents a new approach for the preclinical evaluation of new HIV-cure strategies. Sustained remission of HIV infection is prevented by a persistent reservoir of latently infected cells capable of reinitiating systemic infection and viremia. To evaluate strategies to reactivate and deplete this reservoir, we developed and characterized a new humanized mouse model consisting of highly immunodeficient mice intrasplenically injected with peripheral blood mononuclear cells from long-term ART-suppressed HIV-infected donors. Reactivation and dissemination of HIV infection was visualized in the mouse spleens in parallel with the onset of viremia. The applicability of this model for evaluating reservoir depletion treatments was demonstrated by establishing, through delayed time-to-viremia and phylogenetic analysis of plasma virus, that treatment of these humanized mice with a broadly neutralizing antibody, 10-1074, depleted the patient-derived population of latently infected cells. This mouse model represents a new approach for the preclinical evaluation of new HIV-cure strategies.
- Gandhi, M., Smeaton, L. M., Vernon, C., Scully, E. P., Gianella, S., Poongulali, S., Sheth, A. N., Van Schalkwyk, M., Klingman, K. L., Short, W. R., Opollo, V. S., Cohn, S. E., Scarsi, K. K., Firnhaber, C., Bares, S., Swaminathan, S., Mngqibisa, R., Connick, E., & , W. H. (2019). Low Rate of Sex-specific Analyses in Presentations at the Conference on Retroviruses and Opportunistic Infections (CROI) Meeting, 2018: Room to Improve. Journal of acquired immune deficiency syndromes (1999), 81(5), e158-e160.
- Hijmans, J. G., Stockelman, K. A., Garcia, V., Levy, M. V., Brewster, L. M., Bammert, T. D., Greiner, J. J., Stauffer, B. L., Connick, E., & DeSouza, C. A. (2019). Circulating Microparticles Are Elevated in Treated HIV -1 Infection and Are Deleterious to Endothelial Cell Function. Journal of the American Heart Association, 8(4), e011134.More infoBackground Circulating microparticles have emerged as biomarkers and effectors of vascular disease. Elevated rates of cardiovascular disease are seen in HIV -1-seropositive individuals. The aims of this study were to determine: (1) if circulating microparticles are elevated in antiretroviral therapy-treated HIV -1-seropositive adults; and (2) the effects of microparticles isolated from antiretroviral therapy -treated HIV -1-seropositive adults on endothelial cell function, in vitro. Methods and Results Circulating levels of endothelial-, platelet-, monocyte-, and leukocyte-derived microparticles were determined by flow cytometry in plasma from 15 healthy and 15 antiretroviral therapy-treated, virologically suppressed HIV -1-seropositive men. Human umbilical vein endothelial cells were treated with microparticles from individual subjects for 24 hours; thereafter, endothelial cell inflammation, oxidative stress, senescence, and apoptosis were assessed. Circulating concentrations of endothelial-, platelet-, monocyte-, and leukocyte-derived microparticles were significantly higher (≈35%-225%) in the HIV -1-seropositive compared with healthy men. Microparticles from HIV -1-seropositive men induced significantly greater endothelial cell release of interleukin-6 and interleukin-8 (≈20% and ≈35%, respectively) and nuclear factor-κB expression while suppressing anti-inflammatory microRNAs (miR-146a and miR-181b). Intracellular reactive oxygen species production and expression of reactive oxygen species -related heat shock protein 70 were both higher in cells treated with microparticles from the HIV -1-seropositive men. In addition, the percentage of senescent cells was significantly higher and sirtuin 1 expression lower in cells treated with HIV -1-related microparticles. Finally, caspase-3 was significantly elevated by microparticles from HIV -1-seropositive men. Conclusions Circulating concentrations of endothelial-, platelet-, monocyte-, and leukocyte-derived microparticles were higher in antiretroviral therapy-treated HIV -1-seropositive men and adversely affect endothelial cells promoting cellular inflammation, oxidative stress, senescence, and apoptosis. Circulating microparticles may contribute to the vascular risk associated with HIV -1 infection.
- Hijmans, J. G., Stockelman, K., Levy, M., Brewster, L. M., Bammert, T. D., Greiner, J. J., Connick, E., & DeSouza, C. A. (2019). Effects of HIV-1 gp120 and TAT-derived microvesicles on endothelial cell function. Journal of applied physiology (Bethesda, Md. : 1985), 126(5), 1242-1249.More infoThe aims of this study were twofold. The first was to determine if human immunodeficiency virus (HIV)-1 glycoprotein (gp) 120 and transactivator of transcription (Tat) stimulate the release of endothelial microvesicles (EMVs). The second was to determine whether viral protein-induced EMVs are deleterious to endothelial cell function (inducing endothelial cell inflammation, oxidative stress, senescence and increasing apoptotic susceptibility). Human aortic endothelial cells (HAECs) were treated with recombinant HIV-1 proteins Bal gp120 (R5), Lav gp120 (X4), or Tat. EMVs released in response to each viral protein were isolated and quantified. Fresh HAECs were treated with EMVs generated under control conditions and from each of the viral protein conditions for 24 h. EMV release was higher ( < 0.05) in HAECs treated with R5 (141 ± 21 MV/µl) X4 (132 ± 20 MV/µl), and Tat (130 ± 20 MV/µl) compared with control (61 ± 13 MV/µl). Viral protein EMVs induced significantly higher endothelial cell release of proinflammatory cytokines and expression of cell adhesion molecules than control. Reactive oxygen species production was more pronounced ( < 0.05) in the R5-, X4- and Tat-EMV-treated cells. In addition, viral protein-stimulated EMVs significantly augmented endothelial cell senescence and apoptotic susceptibility. Concomitant with these functional changes, viral protein-stimulated EMVs disrupted cell expression of micro-RNAs 34a, 126, 146a, 181b, 221, and miR-Let-7a ( < 0.05). These results demonstrate that HIV-1 gp120 and Tat stimulate microvesicle release from endothelial cells, and these microvesicles confer pathological effects on endothelial cells by inducing inflammation, oxidative stress, and senescence as well as enhancing susceptibility to apoptosis. Viral protein-generated EMVs may contribute to the increased risk of vascular disease in patients with HIV-1. Human immunodeficiency virus (HIV)-1-related proteins glycoprotein (gp) 120 and transactivator of transcription (Tat)-mediated endothelial damage and dysfunction are poorly understood. Endothelial microvesicles (EMVs) serve as indicators and potent mediators of endothelial dysfunction. In the present study we determined if HIV-1 R5- and X4-tropic gp120 and Tat stimulate EMV release in vitro and if viral protein-induced EMVs are deleterious to endothelial cell function. gp120 and Tat induced a marked increase in EMV release. Viral protein-induced EMVs significantly increased endothelial cell inflammation, oxidative stress, senescence, and apoptotic susceptibility in vitro. gp120- and Tat-derived EMVs promote a proinflammatory, pro-oxidative, prosenescent, and proapoptotic endothelial phenotype and may contribute to the endothelial damage and dysfunction associated with gp120 and Tat.
- Li, S., Folkvord, J. M., Kovacs, K. J., Wagstaff, R. K., Mwakalundwa, G., Rendahl, A. K., Rakasz, E. G., Connick, E., & Skinner, P. J. (2019). Low levels of SIV-specific CD8+ T cells in germinal centers characterizes acute SIV infection. PLoS pathogens, 15(3), e1007311.More infoCD8+ T cells play an important role in controlling of HIV and SIV infections. However, these cells are largely excluded from B cell follicles where HIV and SIV producing cells concentrate during chronic infection. It is not known, however, if antigen-specific CD8+ T cells are excluded gradually as pathogenesis progresses from early to chronic phase, or this phenomenon occurs from the beginning infection. In this study we determined that SIV-specific CD8+ T cells were largely excluded from follicles during early infection, we also found that within follicles, they were entirely absent in 60% of the germinal centers (GCs) examined. Furthermore, levels of SIV-specific CD8+ T cells in follicular but not extrafollicular areas significantly correlated inversely with levels of viral RNA+ cells. In addition, subsets of follicular SIV-specific CD8+ T cells were activated and proliferating and expressed the cytolytic protein perforin. These studies suggest that a paucity of SIV-specific CD8+ T cells in follicles and complete absence within GCs during early infection may set the stage for the establishment of persistent chronic infection.
- Patterson, F., Connick, E., Brewer, B., & Grandner, M. A. (2019). HIV status and sleep disturbance in college students and relationship with smoking. Sleep health, 5(4), 395-400.More infoAdults with HIV have greater sleep difficulties and are more likely to smoke cigarettes. We tested whether current smoking plays a role in sleep difficulties experienced by young adults with HIV.
- Scarsi, K. K., Cramer, Y. S., Rosenkranz, S. L., Aweeka, F., Berzins, B., Coombs, R. W., Coughlin, K., Moran, L. E., Zorrilla, C. D., Akelo, V., Aziz, M., Friedman, R. K., Gingrich, D., Swaminathan, S., Godfrey, C., Cohn, S. E., & , A. C. (2019). Antiretroviral therapy and vaginally administered contraceptive hormones: a three-arm, pharmacokinetic study. The lancet. HIV, 6(9), e601-e612.More infoDrug-drug interactions between orally administered antiretroviral therapy (ART) and hormones released from an intravaginal ring are not known. We hypothesised that ART containing either efavirenz or ritonavir-boosted atazanavir would alter plasma concentrations of vaginally administered etonogestrel and ethinylestradiol but that ART concentrations would be unchanged during use of an intravaginal ring.
- Zangeneh, T. T., Connick, E., Ellis, K. E., Chan, C. B., & York, L. D. (2019). Clinical Outcomes Following the Use of Archived Proviral HIV-1 DNA Genotype to Guide Antiretroviral Therapy Adjustment. Open Forum Infectious Disease.
- Almodovar, S., Swanson, J., Giavedoni, L. D., Kanthaswamy, S., Long, C. S., Voelkel, N. F., Edwards, M. G., Folkvord, J. M., Connick, E., Westmoreland, S. V., Luciw, P. A., & Flores, S. C. (2018). Lung Vascular Remodeling, Cardiac Hypertrophy, and Inflammatory Cytokines in SHIVnef-Infected Macaques. Viral immunology, 31(3), 206-222.More infoFatal pulmonary arterial hypertension (PAH) affects HIV-infected individuals at significantly higher frequencies. We previously showed plexiform-like lesions characterized by recanalized lumenal obliteration, intimal disruption, medial hypertrophy, and thrombosis consistent with PAH in rhesus macaques infected with chimeric SHIVnef but not with the parental SIVmac239, suggesting that Nef is implicated in the pathophysiology of HIV-PAH. However, the current literature on non-human primates as animal models for SIV(HIV)-associated pulmonary disease reports the ultimate pathogenic pulmonary outcomes of the research efforts; however, the variability and features in the actual disease progression remain poorly described, particularly when using different viral sources for infection. We analyzed lung histopathology, performed immunophenotyping of cells in plexogenic lesions pathognomonic of PAH, and measured cardiac hypertrophy biomarkers and cytokine expression in plasma and lung of juvenile SHIVnef-infected macaques. Here, we report significant hematopathologies, changes in cardiac biomarkers consistent with ventricular hypertrophy, significantly increased levels of interleukin-12 and GM-CSF and significantly decreased sCD40 L, CCL-2, and CXCL-1 in plasma of the SHIVnef group. Pathway analysis of inflammatory gene expression predicted activation of NF-κB transcription factor RelB and inhibition of bone morphogenetic protein type-2 in the setting of SHIVnef infection. Our findings highlight the utility of SHIVnef-infected macaques as suitable models of HIV-associated pulmonary vascular remodeling as pathogenetic changes are concordant with features of idiopathic, familial, scleroderma, and HIV-PAH.
- Bronnimann, M. P., Skinner, P. J., & Connick, E. (2018). The B-Cell Follicle in HIV Infection: Barrier to a Cure. Frontiers in immunology, 9, 20.More infoThe majority of HIV replication occurs in secondary lymphoid organs (SLOs) such as the spleen, lymph nodes, and gut-associated lymphoid tissue. Within SLOs, HIV RNA cells are concentrated in the B-cell follicle during chronic untreated infection, and emerging data suggest that they are a major source of replication in treated disease as well. The concentration of HIV RNA cells in the B-cell follicle is mediated by several factors. Follicular CD4 T-cell subsets including T-follicular helper cells and T-follicular regulatory cells are significantly more permissive to HIV than extrafollicular subsets. The B cell follicle also contains a large reservoir of extracellular HIV virions, which accumulate on the surface of follicular dendritic cells (FDCs) in germinal centers. FDC-bound HIV virions remain infectious even in the presence of neutralizing antibodies and can persist for months or even years. Moreover, the B-cell follicle is semi-immune privileged from CTL control. Frequencies of HIV- and SIV-specific CTL are lower in B-cell follicles compared to extrafollicular regions as the majority of CTL do not express the follicular homing receptor CXCR5. Additionally, CTL in the B-cell follicle may be less functional than extrafollicular CTL as many exhibit the recently described CD8 T follicular regulatory phenotype. Other factors may also contribute to the follicular concentration of HIV RNA cells. Notably, the contribution of NK cells and γδ T cells to control and/or persistence of HIV RNA cells in secondary lymphoid tissue remains poorly characterized. As HIV research moves increasingly toward the development of cure strategies, a greater understanding of the barriers to control of HIV infection in B-cell follicles is critical. Although no strategy has as of yet proven to be effective, a range of novel therapies to address these barriers are currently being investigated including genetically engineered CTL or chimeric antigen receptor T cells that express the follicular homing molecule CXCR5, treatment with IL-15 or an IL-15 superagonist, use of bispecific antibodies to harness the killing power of the follicular CD8 T cell population, and disruption of the follicle through treatments such as rituximab.
- Haran, K. P., Hajduczki, A., Pampusch, M. S., Mwakalundwa, G., Vargas-Inchaustegui, D. A., Rakasz, E. G., Connick, E., Berger, E. A., & Skinner, P. J. (2018). Simian Immunodeficiency Virus (SIV)-Specific Chimeric Antigen Receptor-T Cells Engineered to Target B Cell Follicles and Suppress SIV Replication. Frontiers in immunology, 9, 492.More infoThere is a need to develop improved methods to treat and potentially cure HIV infection. During chronic HIV infection, replication is concentrated within T follicular helper cells (Tfh) located within B cell follicles, where low levels of virus-specific CTL permit ongoing viral replication. We previously showed that elevated levels of simian immunodeficiency virus (SIV)-specific CTL in B cell follicles are linked to both decreased levels of viral replication in follicles and decreased plasma viral loads. These findings provide the rationale to develop a strategy for targeting follicular viral-producing (Tfh) cells using antiviral chimeric antigen receptor (CAR) T cells co-expressing the follicular homing chemokine receptor CXCR5. We hypothesize that antiviral CAR/CXCR5-expressing T cells, when infused into an SIV-infected animal or an HIV-infected individual, will home to B cell follicles, suppress viral replication, and lead to long-term durable remission of SIV and HIV. To begin to test this hypothesis, we engineered gammaretroviral transduction vectors for co-expression of a bispecific anti-SIV CAR and rhesus macaque CXCR5. Viral suppression by CAR/CXCR5-transduced T cells was measured , and CXCR5-mediated migration was evaluated using both an transwell migration assay, as well as a novel tissue migration assay. The functionality of the CAR/CXCR5 T cells was demonstrated through their potent suppression of SIV and SIV replication in and migration to the ligand CXCL13 , and concentration in B cell follicles in tissues . These novel antiviral immunotherapy products have the potential to provide long-term durable remission (functional cure) of HIV and SIV infections.
- Hijmans, J. G., Stockleman, K., Reiakvam, W., Levy, M. V., Brewster, L. M., Bammert, T. D., Greiner, J. J., Connick, E., & DeSouza, C. A. (2018). Effects of HIV-1 gp120 and tat on endothelial cell sensescence and senescence-associated microRNAs. Physiological Reports, 6(6), e13647.More infoThe aim of this study was to determine, in vitro, the effects of X4 and R5 HIV-1 gp120 and Tat on: (1) endothelial cell senescence and (2) endothelial cell microRNA (miR) expression. Endothelial cells were treated with media without and with: R5 gp120 (100 ng/mL), X4 gp120 (100 ng/mL), or Tat (500 ng/mL) for 24 h and stained for senescence-associated β-galactosidase (SA-β-gal). Cell expression of miR-34a, miR-217, and miR-146a was determined by RT-PCR. X4 and R5 gp120 and Tat significantly increased (~100%) cellular senescence versus control. X4 gp120 significantly increased cell expression of miR-34a (1.60 ± 0.04 fold) and miR-217 (1.52 ± 0.18), but not miR-146a (1.25 ± 0.32). R5 gp120 significantly increased miR-34a (1.23 ± 0.07) and decreased miR-146a (0.56 ± 0.07). Tat significantly increased miR-34a (1.49 ± 0.16) and decreased miR-146a (0.55 ± 0.23). R5 and Tat had no effect on miR-217 (1.05 ± 0.13 and 1.06 ± 0.24; respectively). HIV-1 gp120 (X4 and R5) and Tat promote endothelial cell senescence and dysregulation of senescence-associated miRs.
- Melanson, E. L., Ritchie, H. K., Dear, T. B., Catenacci, V., Shea, K., Connick, E., Moehlman, T. M., Stothard, E. R., Higgins, J., McHill, A. W., & Wright, K. P. (2018). Daytime bright light exposure, metabolism, and individual differences in wake and sleep energy expenditure during circadian entrainment and misalignment. Neurobiology of sleep and circadian rhythms, 4, 49-56.More infoDaytime light exposure has been reported to impact or have no influence on energy metabolism in humans. Further, whether inter-individual differences in wake, sleep, 24 h energy expenditure, and RQ during circadian entrainment and circadian misalignment are stable across repeated 24 h assessments is largely unknown. We present data from two studies: Study 1 of 15 participants (7 females) exposed to three light exposure conditions: continuous typical room ~100 lx warm white light, continuous ~750 lx warm white light, and alternating hourly ~750 lx warm white and blue-enriched white light on three separate days in a randomized order; and Study 2 of 14 participants (8 females) during circadian misalignment induced by a simulated night shift protocol. Participants were healthy, free of medical disorders, medications, and illicit drugs. Participants maintained a consistent 8 h per night sleep schedule for one week as an outpatient prior to the study verified by wrist actigraphy, sleep diaries, and call-ins to a time stamped recorder. Participants consumed an outpatient energy balance research diet for three days prior to the study. The inpatient protocol for both studies consisted of an initial sleep disorder screening night. For study 1, this was followed by three standard days with 16 h scheduled wakefulness and 8 h scheduled nighttime sleep. For Study 2, it was followed by 16 h scheduled wake and 8 h scheduled sleep at habitual bedtime followed by three night shifts with 8 h scheduled daytime sleep. Energy expenditure was measured using whole-room indirect calorimetry. Constant posture bedrest conditions were maintained to control for energy expenditure associated with activity and the baseline energy balance diet was continued with the same exact meals across days to control for thermic effects of food. No significant impact of light exposure was observed on metabolic outcomes in response to daytime light exposure. Inter-individual variability in energy expenditure was systematic and ranged from substantial to almost perfect consistency during both nighttime sleep and circadian misalignment. Findings show robust and stable trait-like individual differences in whole body 24 h, waking, and sleep energy expenditure, 24 h respiratory quotient-an index of a fat and carbohydrate oxidation-during repeated assessments under entrained conditions, and also in 24 h and sleep energy expenditure during repeated days of circadian misalignment.
- Miles, B., & Connick, E. (2018). Control of the Germinal Center by Follicular Regulatory T Cells During Infection. Frontiers in immunology, 9, 2704.More infoFollicular regulatory T cells (Tfr) are a unique subset of CD4 T cells that control and impact adaptive immune responses in the lymphoid follicles and germinal centers (GC). Since their relatively recent discovery, several studies have revealed that Tfr interact with other cells within this niche and shape ensuing responses. Recent advances defining the functional and developmental characteristics of Tfr have revealed key characteristics of Tfr differentiation, GC recruitment and retention, and regulatory properties. Further, Tfr shape the GC response and balance tolerance through interactions with Tfh, by modifying Tfh number, diversity and function, as well as with B cells. Mechanisms by which Tfr regulate the GC include cell-to-cell interactions with Tfh and B cells, as well as altering their environment through cytokine production and sequestration. Tfr have been shown to have a diverse T cell receptor (TCR) repertoire and can be specific for immunizing agents, demonstrating a potential role in vaccine development. Due to these important characteristics and functions, Tfr play a major role in immune tolerance, response to infection, and vaccine efficacy.
- Namazi, G., Fajnzylber, J. M., Aga, E., Bosch, R. J., Acosta, E. P., Sharaf, R., Hartogensis, W., Jacobson, J. M., Connick, E., Volberding, P., Skiest, D., Margolis, D., Sneller, M. C., Little, S. J., Gianella, S., Smith, D. M., Kuritzkes, D. R., Gulick, R. M., Mellors, J. W., , Mehraj, V., et al. (2018). The Control of HIV After Antiretroviral Medication Pause (CHAMP) Study: Posttreatment Controllers Identified From 14 Clinical Studies. The Journal of infectious diseases, 218(12), 1954-1963.More infoHIV posttreatment controllers are rare individuals who start antiretroviral therapy (ART), but maintain HIV suppression after treatment interruption. The frequency of posttreatment control and posttreatment interruption viral dynamics have not been well characterized.
- Sharaf, R., Lee, G. Q., Sun, X., Etemad, B., Aboukhater, L. M., Hu, Z., Brumme, Z. L., Aga, E., Bosch, R. J., Wen, Y., Namazi, G., Gao, C., Acosta, E. P., Gandhi, R. T., Jacobson, J. M., Skiest, D., Margolis, D. M., Mitsuyasu, R., Volberding, P., , Connick, E., et al. (2018). HIV-1 proviral landscapes distinguish posttreatment controllers from noncontrollers. The Journal of clinical investigation, 128(9), 4074-4085.More infoHIV posttreatment controllers (PTCs) represent a natural model of sustained HIV remission, but they are rare and little is known about their viral reservoir. We obtained 1,450 proviral sequences after near-full-length amplification for 10 PTCs and 16 posttreatment noncontrollers (NCs). Before treatment interruption, the median intact and total reservoir size in PTCs was 7-fold lower than in NCs, but the proportion of intact, defective, and total clonally expanded proviral genomes was not significantly different between the 2 groups. Quantification of total but not intact proviral genome copies predicted sustained HIV remission as 81% of NCs, but none of the PTCs had a total proviral genome greater than 4 copies per million peripheral blood mononuclear cells (PBMCs). The results highlight the restricted intact and defective HIV reservoir in PTCs and suggest that total proviral genome burden could act as the first biomarker for identifying PTCs. Total and defective but not intact proviral copy numbers correlated with levels of cell-associated HIV RNA, activated NK cell percentages, and both HIV-specific CD4+ and CD8+ responses. These results support the concept that defective HIV genomes can lead to viral antigen production and interact with both the innate and adaptive immune systems.
- Strongin, Z., Sharaf, R., VanBelzen, D. J., Jacobson, J. M., Connick, E., Volberding, P., Skiest, D. J., Gandhi, R. T., Kuritzkes, D. R., O'Doherty, U., & Li, J. Z. (2018). Effect of Short-Term Antiretroviral Therapy Interruption on Levels of Integrated HIV DNA. Journal of virology, 92(12).More infoAnalytic treatment interruption (ATI) studies are required to evaluate strategies aimed at achieving ART-free HIV remission, but the impact of ATI on the viral reservoir remains unclear. We validated a DNA size selection-based assay for measuring levels of integrated HIV DNA and applied it to assess the effects of short-term ATI on the HIV reservoir. Samples from participants from four AIDS Clinical Trials Group ATI studies were assayed for integrated HIV DNA levels. Cryopreserved peripheral blood mononuclear cells (PBMCs) were obtained for 12 participants with available samples pre-ATI and approximately 6 months after ART resumption. Four participants also had samples available during the ATI. The median duration of ATI was 12 weeks. Validation of the HIV integrated DNA size-exclusion (HIDE) assay was performed using samples spiked with unintegrated HIV DNA, HIV-infected cell lines, and participant PBMCs. The HIDE assay eliminated 99% of unintegrated HIV DNA species and strongly correlated with the established Alu- assay. For the majority of individuals, integrated DNA levels increased during ATI and subsequently declined upon ART resumption. There was no significant difference in the levels of integrated HIV DNA between the pre- and post-ATI time points, with a median ratio of post- to pre-ATI HIV DNA levels of 0.95. Using a new integrated HIV DNA assay, we found minimal change in the levels of integrated HIV DNA in participants who underwent an ATI, followed by 6 months of ART. This suggests that short-term ATI can be conducted without a significant impact on the levels of integrated proviral DNA in the peripheral blood. Interventions aimed at achieving sustained antiretroviral therapy (ART)-free HIV remission require treatment interruption trials to assess their efficacy. However, these trials are accompanied by safety concerns related to the expansion of the viral reservoir. We validated an assay that uses an automated DNA size-selection platform for quantifying levels of integrated HIV DNA and is less sample- and labor-intensive than current assays. Using stored samples from AIDS Clinical Trials Group studies, we found that short-term ART discontinuation had minimal impact on integrated HIV DNA levels after ART resumption, providing reassurance about the reservoir effects of short-term treatment interruption trials.
- Webb, G. M., Li, S., Mwakalundwa, G., Folkvord, J. M., Greene, J. M., Reed, J. S., Stanton, J. J., Legasse, A. W., Hobbs, T., Martin, L. D., Park, B. S., Whitney, J. B., Jeng, E. K., Wong, H. C., Nixon, D. F., Jones, R. B., Connick, E., Skinner, P. J., & Sacha, J. B. (2018). The human IL-15 superagonist ALT-803 directs SIV-specific CD8 T cells into B-cell follicles. Blood advances, 2(2), 76-84.More infoSequestering of latent HIV in follicular helper T cells within B-cell follicles that largely exclude cytotoxic T cells is a major barrier to cellular immune-based approaches to eradicate HIV. Here, we show that the clinical-grade human interleukin-15 (IL-15) superagonist ALT-803 activates and redirects simian immunodeficiency virus (SIV)-specific CD8 T cells from the peripheral blood into B-cell follicles. In agreement with the increased trafficking of SIV-specific cytotoxic T cells to sites of cryptic viral replication, lymph nodes of elite controlling macaques contained fewer cells expressing SIV RNA or harboring SIV DNA post-ALT-803 treatment. These data establish ALT-803 as an immunotherapeutic for HIV and other chronic viral pathogens that evade host immunity by persisting in B-cell follicles.
- Wodarz, D., Skinner, P. J., Levy, D. N., & Connick, E. (2018). Virus and CTL dynamics in the extrafollicular and follicular tissue compartments in SIV-infected macaques. PLoS computational biology, 14(10), e1006461.More infoData from SIV-infected macaques indicate that virus-specific cytotoxic T lymphocytes (CTL) are mostly present in the extrafollicular (EF) compartment of the lymphoid tissue, with reduced homing to the follicular (F) site. This contributes to the majority of the virus being present in the follicle and represents a barrier to virus control. Using mathematical models, we investigate these dynamics. Two models are analyzed. The first assumes that CTL can only become stimulated and expand in the extrafollicular compartment, with migration accounting for the presence of CTL in the follicle. In the second model, follicular CTL can also undergo antigen-induced expansion. Consistent with experimental data, both models predict increased virus compartmentalization in the presence of stronger CTL responses and lower virus loads, and a more pronounced rise of extrafollicular compared to follicular virus during CD8 cell depletion experiments. The models, however, differ in other aspects. The follicular expansion model results in dynamics that promote the clearance of productive infection in the extrafollicular site, with any productively infected cells found being the result of immigration from the follicle. This is not observed in the model without follicular CTL expansion. The models further predict different consequences of introducing engineered, follicular-homing CTL, which has been proposed as a therapeutic means to improve virus control. Without follicular CTL expansion, this is predicted to result in a reduction of virus load in both compartments. The follicular CTL expansion model, however, makes the counter-intuitive prediction that addition of F-homing CTL not only results in a reduction of follicular virus load, but also in an increase in extrafollicular virus replication. These predictions remain to be experimentally tested, which will be relevant for distinguishing between models and for understanding how therapeutic introduction of F-homing CTL might impact the overall dynamics of the infection.
- Xu, Y., Ollerton, M. T., & Connick, E. (2018). Follicular T-cell subsets in HIV infection: recent advances in pathogenesis research. Current opinion in HIV and AIDS.More infoT cells within B-cell follicles of secondary lymphoid tissues play key roles in HIV immunopathogenesis. This review highlights recent findings and identifies gaps in current knowledge.
- Connick, E., Ritchie, H. K., Dear, T. B., Catenacci, V., Shea, K., Moehlman, T. M., Stothard, E. R., Higgins, J., McHill, A. W., & Wright Jr., K. P. (2017). Impact of Daytime Blue-Enriched Light Exposure on Cognition During the Workday. SLEEP.
- Hijmans, J., Levy, M., Greiner, J., Stauffer, B., Connick, E., & DeSouza, C. (2017). HIV-1 Infection and Inflammation-Related microRNAs. FASEB JOURNAL, 31.
- Miller, S. M., Miles, B., Guo, K., Folkvord, J., Meditz, A. L., McCarter, M. D., Levy, D. N., MaWhinney, S., Santiago, M. L., & Connick, E. (2017). Follicular Regulatory T Cells Are Highly Permissive to R5-Tropic HIV-1. Journal of virology, 91(17).More infoFollicular regulatory T (TFR) cells are a subset of CD4+ T cells in secondary lymphoid follicles. TFR cells were previously included in the follicular helper T (TFH) cell subset, which consists of cells that are highly permissive to HIV-1. The permissivity of TFR cells to HIV-1 is unknown. We find that TFR cells are more permissive than TFH cells to R5-tropic HIV-1 ex vivo TFR cells expressed more CCR5 and CD4 and supported higher frequencies of viral fusion. Differences in Ki67 expression correlated with HIV-1 replication. Inhibiting cellular proliferation reduced Ki67 expression and HIV-1 replication. Lymph node cells from untreated HIV-infected individuals revealed that TFR cells harbored the highest concentrations of HIV-1 RNA and highest levels of Ki67 expression. These data demonstrate that TFR cells are highly permissive to R5-tropic HIV-1 both ex vivo and in vivo This is likely related to elevated CCR5 levels combined with a heightened proliferative state and suggests that TFR cells contribute to persistent R5-tropic HIV-1 replication in vivoIMPORTANCE In chronic, untreated HIV-1 infection, viral replication is concentrated in secondary lymphoid follicles. Within secondary lymphoid follicles, follicular helper T (TFH) cells have previously been shown to be highly permissive to HIV-1. Recently, another subset of T cells in secondary lymphoid follicles was described, follicular regulatory T (TFR) cells. These cells share some phenotypic characteristics with TFH cells, and studies that showed that TFH cells are highly permissive to HIV-1 included TFR cells in their definition of TFH cells. The permissivity of TFR cells to HIV-1 has not previously been described. Here, we show that TFR cells are highly permissive to HIV-1 both ex vivo and in vivo The expression of Ki67, a marker of proliferative capacity, is predictive of expression of viral proteins, and downregulating Ki67 leads to concurrent decreases in expression of viral proteins. Our study provides new insight into HIV-1 replication and a potential new cell type to target for future treatment.
- Mohammad, H., Li, S., Sawahata, R., Mwakalundwa, G., Fraser, K., Mylvaganam, G., Martins, M., Masopust, D., Amara, R. R., Watkins, D., Connick, E., & Skinner, P. J. (2017). CTL-based vaccine-induced control is associated with induction of high follicular to extra-follicular ratios of virus-specific CDS plus T cells.. JOURNAL OF IMMUNOLOGY, 198(1).
- Moore, C. M., MaWhinney, S., Forster, J. E., Carlson, N. E., Allshouse, A., Wang, X., Routy, J. P., Conway, B., & Connick, E. (2017). Accounting for dropout reason in longitudinal studies with nonignorable dropout. Statistical methods in medical research, 26(4), 1854-1866.More infoDropout is a common problem in longitudinal cohort studies and clinical trials, often raising concerns of nonignorable dropout. Selection, frailty, and mixture models have been proposed to account for potentially nonignorable missingness by relating the longitudinal outcome to time of dropout. In addition, many longitudinal studies encounter multiple types of missing data or reasons for dropout, such as loss to follow-up, disease progression, treatment modifications and death. When clinically distinct dropout reasons are present, it may be preferable to control for both dropout reason and time to gain additional clinical insights. This may be especially interesting when the dropout reason and dropout times differ by the primary exposure variable. We extend a semi-parametric varying-coefficient method for nonignorable dropout to accommodate dropout reason. We apply our method to untreated HIV-infected subjects recruited to the Acute Infection and Early Disease Research Program HIV cohort and compare longitudinal CD4+ T cell count in injection drug users to nonusers with two dropout reasons: anti-retroviral treatment initiation and loss to follow-up.
- Kohler, S. L., Pham, M. N., Folkvord, J. M., Arends, T., Miller, S. M., Miles, B., Meditz, A. L., McCarter, M., Levy, D. N., & Connick, E. (2016). Germinal Center T Follicular Helper Cells Are Highly Permissive to HIV-1 and Alter Their Phenotype during Virus Replication. Journal of immunology (Baltimore, Md. : 1950), 196(6), 2711-22.More infoHIV-1 replication is concentrated within CD4(+) T cells in B cell follicles of secondary lymphoid tissues during asymptomatic disease. Limited data suggest that a subset of T follicular helper cells (TFH) within germinal centers (GC) is highly permissive to HIV-1. Whether GC TFH are the major HIV-1 virus-producing cells in vivo has not been established. In this study, we investigated TFH permissivity to HIV-1 ex vivo by spinoculating and culturing tonsil cells with HIV-1 GFP reporter viruses. Using flow cytometry, higher percentages of GC TFH (CXCR5(high)PD-1(high)) and CXCR5(+)programmed cell death-1 (PD-1)(low) cells were GFP(+) than non-GC TFH (CXCR5(+)PD-1(intermediate)) or extrafollicular (EF) (CXCR5(-)) cells. When sorted prior to spinoculation, however, GC TFH were substantially more permissive than CXCR5(+)PD-1(low) or EF cells, suggesting that many GC TFH transition to a CXCR5(+)PD-1(low) phenotype during productive infection. In situ hybridization on inguinal lymph node sections from untreated HIV-1-infected individuals without AIDS revealed higher frequencies of HIV-1 RNA(+) cells in GC than non-GC regions of follicle or EF regions. Superinfection of HIV-1-infected individuals' lymph node cells with GFP reporter virus confirmed the permissivity of follicular cells ex vivo. Lymph node immunostaining revealed 96% of CXCR5(+)CD4(+) cells were located in follicles. Within sorted lymph node cells from four HIV-infected individuals, CXCR5(+) subsets harbored 11-66-fold more HIV-1 RNA than CXCR5(-) subsets, as determined by RT PCR. Thus, GC TFH are highly permissive to HIV-1, but downregulate PD-1 and, to a lesser extent, CXCR5 during HIV-1 replication. These data further implicate GC TFH as the major HIV-1-producing cells in chronic asymptomatic HIV-1 infection.
- Li, S., Folkvord, J. M., Rakasz, E. G., Abdelaal, H. M., Wagstaff, R. K., Kovacs, K. J., Kim, H. O., Sawahata, R., MaWhinney, S., Masopust, D., Connick, E., & Skinner, P. J. (2016). Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8+ Cells In Vivo. Journal of virology, 90(24), 11168-11180.More infoHuman immunodeficiency virus (HIV)- and simian immunodeficiency virus (SIV)-specific CD8(+) T cells are typically largely excluded from lymphoid B cell follicles, where HIV- and SIV-producing cells are most highly concentrated, indicating that B cell follicles are somewhat of an immunoprivileged site. To gain insights into virus-specific follicular CD8(+) T cells, we determined the location and phenotype of follicular SIV-specific CD8(+) T cells in situ, the local relationship of these cells to Foxp3(+) cells, and the effects of CD8 depletion on levels of follicular SIV-producing cells in chronically SIV-infected rhesus macaques. We found that follicular SIV-specific CD8(+) T cells were able to migrate throughout follicular areas, including germinal centers. Many expressed PD-1, indicating that they may have been exhausted. A small subset was in direct contact with and likely inhibited by Foxp3(+) cells, and a few were themselves Foxp3(+) In addition, subsets of follicular SIV-specific CD8(+) T cells expressed low to medium levels of perforin, and subsets were activated and proliferating. Importantly, after CD8 depletion, the number of SIV-producing cells increased in B cell follicles and extrafollicular areas, suggesting that follicular and extrafollicular CD8(+) T cells have a suppressive effect on SIV replication. Taken together, these results suggest that during chronic SIV infection, despite high levels of exhaustion and likely inhibition by Foxp3(+) cells, a subset of follicular SIV-specific CD8(+) T cells are functional and suppress viral replication in vivo These findings support HIV cure strategies that augment functional follicular virus-specific CD8(+) T cells to enhance viral control.
- Miles, B., & Connick, E. (2016). TFH in HIV Latency and as Sources of Replication-Competent Virus. Trends in microbiology, 24(5), 338-44.More infoDuring untreated disease, HIV replication is concentrated within T follicular helper cells (TFH). Heightened permissiveness, the presence of highly infectious virions on follicular dendritic cells (FDCs), low frequencies of virus-specific cytotoxic T lymphocytes (CTLs) in B cell follicles, expansions in TFH, and TFH dysfunction, all likely promote replication in TFH. Limited data suggest that memory TFH play a role in the latent or subclinical reservoir of HIV during antiretroviral therapy (ART), potentially for many of the same reasons. A better understanding of the role of memory TFH and FDC-bound virions in promoting recrudescent viremia in the setting of ART cessation is essential. Studies that target follicular virus reservoirs are needed to determine their role in HIV latency and to suggest successful cure strategies.
- Miles, B., Miller, S. M., Folkvord, J. M., Levy, D. N., Rakasz, E. G., Skinner, P. J., & Connick, E. (2016). Follicular Regulatory CD8 T Cells Impair the Germinal Center Response in SIV and Ex Vivo HIV Infection. PLoS pathogens, 12(10), e1005924.More infoDuring chronic HIV infection, viral replication is concentrated in secondary lymphoid follicles. Cytotoxic CD8 T cells control HIV replication in extrafollicular regions, but not in the follicle. Here, we show CXCR5hiCD44hiCD8 T cells are a regulatory subset differing from conventional CD8 T cells, and constitute the majority of CD8 T cells in the follicle. This subset, CD8 follicular regulatory T cells (CD8 TFR), expand in chronic SIV infection, exhibit enhanced expression of Tim-3 and IL-10, and express less perforin compared to conventional CD8 T cells. CD8 TFR modestly limit HIV replication in follicular helper T cells (TFH), impair TFH IL-21 production via Tim-3, and inhibit IgG production by B cells during ex vivo HIV infection. CD8 TFR induce TFH apoptosis through HLA-E, but induce less apoptosis than conventional CD8 T cells. These data demonstrate that a unique regulatory CD8 population exists in follicles that impairs GC function in HIV infection.
- Miles, B., Miller, S., & Connick, E. (2016). CD4 T Follicular Helper and Regulatory Cell Dynamics and Function in HIV Infection. Frontiers in Immunology, https://doi.org/10.3389/fimmu.2016.00659. doi:https://doi.org/10.3389/fimmu.2016.00659
- Haas, M. K., Levy, D. N., Folkvord, J. M., & Connick, E. (2015). Distinct patterns of Bcl-2 expression occur in R5- and X4-tropic HIV-1-producing lymphoid tissue cells infected ex vivo. AIDS research and human retroviruses, 31(3), 298-304.More infoMost HIV-1 replication occurs in secondary lymphoid tissues in T cells within B cell follicles. Mechanisms underlying the accumulation of HIV-1-producing cells at these sites are not understood. Antiapoptotic proteins such as Bcl-2 could promote follicular CD4(+) T cell survival, contributing to sustained virus production. Tonsils obtained from subjects without known HIV infection were disaggregated and analyzed for Bcl-2 expression in follicular (CXCR5(+)) and extrafollicular (CXCR5(-)) CD3(+)CD4(+) cells by flow cytometry. Additional tonsil cells were cultured with phytohemagglutinin (PHA) and interleukin-2 (IL-2) for 2 days, infected with either CCR5(R5) or CXCR4-tropic (X4) GFP reporter viruses, and analyzed for Bcl-2 expression. In freshly disaggregated CD3(+)CD4(+) tonsil cells, mean florescence intensity (MFI) for Bcl-2 was higher in CXCR5(+) (median, 292) compared to CXCR5(-) cells (median, 194; p=0.001). Following in vitro stimulation with PHA and IL-2, Bcl-2 MFI was higher in both CXCR5(+) cells (median, 757; p=0.03) and CXCR5(-) cells (median, 884; p=0.002) in uninfected cultures compared to freshly isolated tonsil cells. Bcl-2 MFI was higher in GFP(+)CD3(+)CD8(-) R5-producing cells (median, 554) than in X4-producing cells (median, 393; p=0.02). Bcl-2 MFI was higher in R5-producing CXCR5(+) cells (median, 840) compared to all other subsets including R5-producing CXCR5(-) cells (median, 524; p=0.04), X4-producing CXCR5(+) cells (median, 401; p=0.02), and X4-producing CXCR5(-) cells (median, 332; p=0.008). Bcl-2 expression is elevated in R5 HIV-1-producing CXCR5(+) T cells in vitro, which may contribute to propagation of R5 virus in B cell follicles in vivo.
- Miles, B., Miller, S. M., Folkvord, J. M., Kimball, A., Chamanian, M., Meditz, A. L., Arends, T., McCarter, M. D., Levy, D. N., Rakasz, E. G., Skinner, P. J., & Connick, E. (2015). Follicular regulatory T cells impair follicular T helper cells in HIV and SIV infection. Nature communications, 6, 8608.More infoHuman and simian immunodeficiency viruses (HIV and SIV) exploit follicular lymphoid regions by establishing high levels of viral replication and dysregulating humoral immunity. Follicular regulatory T cells (TFR) are a recently characterized subset of lymphocytes that influence the germinal centre response through interactions with follicular helper T cells (TFH). Here, utilizing both human and rhesus macaque models, we show the impact of HIV and SIV infection on TFR number and function. We find that TFR proportionately and numerically expand during infection through mechanisms involving viral entry and replication, TGF-β signalling, low apoptosis rates and the presence of regulatory dendritic cells. Further, TFR exhibit elevated regulatory phenotypes and impair TFH functions during HIV infection. Thus, TFR contribute to inefficient germinal centre responses and inhibit HIV and SIV clearance.
- Connick, E., Folkvord, J. M., Lind, K. T., Rakasz, E. G., Miles, B., Wilson, N. A., Santiago, M. L., Schmitt, K., Stephens, E. B., Kim, H. O., Wagstaff, R., Li, S., Abdelaal, H. M., Kemp, N., Watkins, D. I., MaWhinney, S., & Skinner, P. J. (2014). Compartmentalization of simian immunodeficiency virus replication within secondary lymphoid tissues of rhesus macaques is linked to disease stage and inversely related to localization of virus-specific CTL. Journal of immunology (Baltimore, Md. : 1950), 193(11), 5613-25.More infoWe previously demonstrated that HIV replication is concentrated in lymph node B cell follicles during chronic infection and that HIV-specific CTL fail to accumulate in large numbers at those sites. It is unknown whether these observations can be generalized to other secondary lymphoid tissues or whether virus compartmentalization occurs in the absence of CTL. We evaluated these questions in SIVmac239-infected rhesus macaques by quantifying SIV RNA(+) cells and SIV-specific CTL in situ in spleen, lymph nodes, and intestinal tissues obtained at several stages of infection. During chronic asymptomatic infection prior to simian AIDS, SIV-producing cells were more concentrated in follicular (F) compared with extrafollicular (EF) regions of secondary lymphoid tissues. At day 14 of infection, when CTL have minimal impact on virus replication, there was no compartmentalization of SIV-producing cells. Virus compartmentalization was diminished in animals with simian AIDS, which often have low-frequency CTL responses. SIV-specific CTL were consistently more concentrated within EF regions of lymph node and spleen in chronically infected animals regardless of epitope specificity. Frequencies of SIV-specific CTL within F and EF compartments predicted SIV RNA(+) cells within these compartments in a mixed model. Few SIV-specific CTL expressed the F homing molecule CXCR5 in the absence of the EF retention molecule CCR7, possibly accounting for the paucity of F CTL. These findings bolster the hypothesis that B cell follicles are immune privileged sites and suggest that strategies to augment CTL in B cell follicles could lead to improved viral control and possibly a functional cure for HIV infection.
- McHill, A. W., Melanson, E. L., Higgins, J., Connick, E., Moehlman, T. M., Stothard, E. R., & Wright, K. P. (2014). Impact of circadian misalignment on energy metabolism during simulated nightshift work. Proceedings of the National Academy of Sciences of the United States of America, 111(48), 17302-7.More infoEating at a time when the internal circadian clock promotes sleep is a novel risk factor for weight gain and obesity, yet little is known about mechanisms by which circadian misalignment leads to metabolic dysregulation in humans. We studied 14 adults in a 6-d inpatient simulated shiftwork protocol and quantified changes in energy expenditure, macronutrient utilization, appetitive hormones, sleep, and circadian phase during day versus nightshift work. We found that total daily energy expenditure increased by ∼4% on the transition day to the first nightshift, which consisted of an afternoon nap and extended wakefulness, whereas total daily energy expenditure decreased by ∼3% on each of the second and third nightshift days, which consisted of daytime sleep followed by afternoon and nighttime wakefulness. Contrary to expectations, energy expenditure decreased by ∼12-16% during scheduled daytime sleep opportunities despite disturbed sleep. The thermic effect of feeding also decreased in response to a late dinner on the first nightshift. Total daily fat utilization increased on the first and second nightshift days, contrary to expectations, and carbohydrate and protein utilization were reduced on the second nightshift day. Ratings of hunger were decreased during nightshift days despite decreases in 24-h levels of the satiety hormones leptin and peptide-YY. Findings suggest that reduced total daily energy expenditure during nightshift schedules and reduced energy expenditure in response to dinner represent contributing mechanisms by which humans working and eating during the biological night, when the circadian clock is promoting sleep, may increase the risk of weight gain and obesity.
- Connick, E. (2019, January). The Role of B Cell Follicles in HIV Replication and Persistence. Second Annual ASU-UA Virology Symposium. Tucson, AZ: Arizona State University and University of Arizona.
- Connick, E. (2017, April). The Role of B Cell Follicles in HIV Replication and Persistence. DOM Research Seminar Series. College of Medicine, University of Arizona: Department of Medicine, University of Arizona.
- Connick, E. (2017, December). The Role of B Cell Follicles in HIV Replication and Persistence. HIV Persistence During Therapy. Reservoirs and Eradication Strategies Workshop, Miami, FL.
- Connick, E. (2017, May). Imaging B Cell Follicles to Investigate HIV/SIV Persistence. Harnessing Novel Imaging Approaches to Guide HIV Prevention and Cure Strategies - NIH Workshop. Rockville, MD: NIH/NIAID and Global Vaccines Enterprises.
- Connick, E. (2017, May). The Role of Follicular Regulatory T Cells in HIV and SIV Pathogenesis. Pharmaconeuroimmunology Seminar U MN. Minneapolis, MN: University of Minnesota.
- Connick, E., Miller, S., Miles, B., Folkvord, J., Meditz, A., McCarter, M., Santiago, M., Levy, D. N., & MaWhinney, S. (2017, February). High HIV Permissivity of T Follicular Regulatory Cells is Related to Ki67 Expression. 24th Conference on Retroviruses and Opportunistic Infections. Seattle, WA.
- Abdelaal, H. M., Li, S., Fraser, K. A., Mylvaganam, G. H., Martins, M. A., Folkvord, J., Masopust, D., Amara, R. R., Watkins, D. I., Connick, E., & Skinner, P. J. (2016, August). CTL-based vaccine-induced protection is associated with induction of high follicular to extra-follicular ratios of virus-specific CD8 T cells. HIV Vaccines. Olympic Valley, CA: Keystone Symposia.
- Connick, E. (2016, April). How HIV Evades the CTL Response: The Immune Privilege of B Cell Follicles. NIH/Division of AIDS. Washington, D.C.: NIH/NIAID.
- Connick, E. (2016, December). The Role of the B Cell Follicular Sanctuary in HIV Immunopathogenesis. World AIDS Day Symposium. Chapel Hill, NC: University of North Carolina.
- Connick, E. (2016, February). The Lymph Node, CTL and HIV/SIV Infection. 23rd Conference on Retroviruses and Opportunistic Infections. Boston.
- Connick, E. (2016, July). The Role of B Cell Follicles in HIV Replication and Persistence. 2016 Towards an HIV Cure Symposium.. Durban, South Africa: International AIDS Society.
- Connick, E. (2016, June). The Role of B Cell Follicles in HIV Infection. Department of Medicine Grand Rounds. Tucson, AZ: DOM.
- Connick, E. (2016, October). Why is the Immune System Unable to Suppress HIV Replication. Western Regions Ryan White Part C/D Recipient Meeting. Tucson, AZ: Western Regions Ryan White.
- Connick, E., Skinner, P. J., & Burton, G. (2016, November). Directing Immune Effectors to Viral Sanctuaries in Lymphoid Tissues. Strategies for an HIV Cure 2016. Washington, D.C.: NIH/NIAID.
- Folkvord, J. M., Thomas, M., Rakasz, E., Li, S., Skinner, P. J., & Connick, E. (2016, FEbruary). Lack of CTL Attenuates, but Does Not Ablate Compartmentalization of SIV Replication [oral abstract #23]. 23rd Conference on Retroviruses and Opportunistic Infections. Boston.
- Connick, E. (2019, March 2019). Circulating Microparticles are Elevated in Treated HIV-1 Infection and are Deleterious to Endotheilial Cell Function. CROI 2019 - Conference on Retroviruses and Opportunistic Infections. Seattle WA: CROI Foundation.
- Connick, E. (2019, March 2019). HIV-1 gp120 and Tat induced microparticles impair endothelial cell function. CROI 2019 - Conference on Retroviruses and Opportunistic Infections. Seattle WA: CROI Foundation.
- Connick, E., & DeSouza, C. A. (2019, January 2019). Exercise, Oxidative Stress and Fibrinolytic Function in HIV-1 Infected Adults. University of Arizona Department of Medicine PI Research Poster Night. University of Arizona Kiewit Auditorium: University of Arizona Department of Medicine.
- Connick, E., Gandhi, M., Smeaton, L. M., Vernon, C., Scully, E. P., Gianella, S., Poongulali, S., Sheth, A. N., Van Schalkwyk, M., Klingman, K. L., Short, W. R., Opollo, V. S., Cohn, S. E., Scarsi, K. K., & Mngqibisa, R. (2019, March). Low Rate of Sex-Specific Analyses in CROI Presentations in 2018: Room to Improve [abstract #916]. 26th Conference on Retroviruses and Opportunistic Infections. Seattle, WA: International Antiviral Society.
- Connick, E., Pampusch, M. S., Berrocal, J. A., Abdelaal, H. M., Davey, B. C., Hart, G. T., Rakasz, E. G., Berger, E. A., & Skinner, P. J. (2019, August). CAR/CXCR5 T cell immunotherapy shows promise in SIV infected ART suppressed rhesus macaques. 5th Conference on Cell and Gene Therapy for HIV Cure. Seattle, WA: Fred Hutchinson Cancer Research Center, The University of Washington Center for AIDS Research (CFAR), and the University of Washington Virology Division.
- Connick, E., Folkvord, J. M., Bronnimann, M., Skinner, P., & Rakasz, E. (2018, March 2018). Characterization of FDC-bound viral products during HIV/SIV infection. CROI - Conference on Retroviruses and Opportunistic Infections. Boston MA: CROI - Conference on Retroviruses and Opportunistic Infections.
- Connick, E., Pampusch, M. S., Haran, K. P., Rakasz, E. G., Hart, G., Berger, E. A., & Skinner, P. J. (2018, October). An HIV cure approach based on CAR optimized for potency, inescapability, and non-immunogenicity co-expressed with the follicular homing receptor CXCR6. 36th Annual Symposium on Nonhuman Primate Models for AIDS. Seattle, WA: National Institutes of Health and Washington National Primate Research Center.
- Connick, E., Shengbin, L., Folkvord, J. M., Kovacs, K. J., Wagstaff, R. K., Mwakalundwa, G., & Rakasz, E. G. (2018, May). SIV-specific CD8 T cells are largely excluded from B cell follicles during early SIV Infection. AAI - Immunology 2018. Austin, TX: American Association of Immunologists.
- Connick, E., Strongin, Z., Sharaf, R., VanBelzen, J., Jacobson, J., Volberding, P., Skiest, D., Gandhi, R., Kuritzkes, D., O'Doherty, U., & Li, J. Z. (2018, March 2018). Short-term ART interruption has little effect on levels of integrated proviral DNA. CROI - Conference on Retroviruses and Opportunistic Infections. Boston MA: CROI - Conference on Retroviruses and Opportunistic Infections.
- Connick, E., Abdelaal, H. M., Li, S., Sawahata, R., Fraser, K., Mylvaganam, G., Martins, M., Mwakalundwa, G., Masopust, D., Amara, R., Watkins, D., & Skinner, P. (2017, July). CTL-based vaccine-induced control is associated with induction of high follicular to extra-follicular ratios of virus-specific CD8 T cell.. 9th International AIDS Society Conference on HIV Science. Paris, FR: International AIDS Society.
- Connick, E., Greiner, J., Dow, C., Diehl, K., Stauffer, B., & DeSouza, C. (2017, February). Exercise, oxidative sterss, and fibrionolytic function in HIV-1 infected adults (abstract #616). 24th Conference on Retroviruses and Opportunistic Infections. Seattle, WA.
- Connick, E., Hijmans, J. G., Levy, M., Greiner, J. J., Stauffer, B. L., & DeSouza, C. A. (2017, April). HIV-1 infection and circulating inflammation-related microRNAs. Experimental Biology. Chicago, IL: FASEB.
- Connick, E., Hijmans, J. G., Stockelman, K. A., Reiakvam, W. R., Levy, M., Bruster, L. M., Goldthwaite, Z. A., Bammert, T. D., Greiner, J. J., & DeSouza, C. A. (2017, July). Effects of HIV-1 gp 120 viral proteins on endothelial senescence and microRNA expression. American Heart Association Basic Cardiovascular Science Meeting. Portland, OR: AHA.
- Connick, E., Mwakalundwa, G., Haran, P., Li, S., Pampusch, M., Abdelaal, R. E., & Skinner, P. (2017, July). CXCR5-transduced primary rhesus macaque CD8 T cells accumulate in B cell follicles in a novel ex vivo B cell follicle migration assay.. 9th International AIDS Society Conference on HIV Science. Paris, FR: International AIDS Society.
- Connick, E., Skinner, P. J., Hajduczki, A., Haran, P., Pampusch, M. S., Mwakalundwa, G., Bolivar-Wagers, S., Vargas-Inchaustegui, D. A., Rakasz, E. G., & Berger, E. A. (2017, August). Targeting CAR T cells to B cell follicles to cure HIV infection. Conference on Cell & Gene Therapy for HIV Cure. Seattle, WA.
- Miles, B., & Connick, E. (2016, February). Follicular Tregs Reduce HIV Replication and Reactivation From Latency Ex VIvo [abstract #378]. 23rd Conference on Retroviruses and Opportunistic Infections. Boston.
- Miles, B., Miller, S. M., & Connick, E. (2016, February). CD8 Follicular Tregs Impair TFH via HLA-E and Tim -3 Interactions in HIV INfection Ex Vivo.. T Follicular Helper Cells and Germinal Centers. Monterey, CA: Keystone Symposia.
- Miller, S. M., & Connick, E. (2016, February). T Follicular Regulatory Cells Are Highly Permissive to HIV ex vivo. T Follicular Helper Cells and Germinal Centers. Monterey, CA: Keystone Symposium.
- Miller, S., & Connick, E. (2016, February). Follicular Regulatory T Cells Are Highly Permissive to HIV-1 Ex Vivo. 23rd Conference on Retroviruses and Opportunistic Infections. Boston.
- Connick, E., Averitt, D., Conley, J., & Auerbach, J. (2018. The liberating message of 'Undetectable = Untransmittable.
- Miles, B., Miller, S. M., & Connick, E. (2016. CD4 T Follicular Helper and Regulatory Cell Dynamics and Function in HIV Infection(p. 659).More infoT follicular helper cells (TFH) are a specialized subset of CD4 T cells that reside in B cell follicles and promote B cell maturation into plasma cells and long-lived memory B cells. During chronic infection prior to the development of AIDS, HIV-1 (HIV) replication is largely concentrated in TFH. Paradoxically, TFH numbers are increased in early and midstages of disease, thereby promoting HIV replication and disease progression. Despite increased TFH numbers, numerous defects in humoral immunity are detected in HIV-infected individuals, including dysregulation of B cell maturation, impaired somatic hypermutation, and low quality of antibody production despite hypergammaglobulinemia. Clinically, these defects are manifested by increased vulnerability to bacterial infections and impaired vaccine responses, neither of which is fully reversed by antiretroviral therapy (ART). Deficits in TFH function, including reduced HIV-specific IL-21 production and low levels of co-stimulatory receptor expression, have been linked to these immune impairments. Impairments in TFH likely contribute as well to the ability of HIV to persist and evade humoral immunity, particularly the inability to develop broadly neutralizing antibodies. In addition to direct infection of TFH, other mechanisms that have been linked to TFH deficits in HIV infection include upregulation of PD-L1 on germinal center B cells and augmented follicular regulatory T cell responses. Challenges to development of strategies to enhance TFH function in HIV infection include lack of an established phenotype for memory TFH as well as limited understanding of the relationship between peripheral TFH and lymphoid tissue TFH. Interventions to augment TFH function in HIV-infected individuals could enhance immune reconstitution during ART and potentially augment cure strategies.