Adam S Buntzman

Interests

Research

Autoimmunity, Immunodeficiency, Cancer, Infectious Disease, Transplant Tolerance.

Courses

Scholarly Contributions

Journals/Publications

• Buntzman, A., Frelinger, J., Hackett, J., & Gibson, H. (2022). Using the Collaborative Cross and Diversity Outbred Mice in Immunology. Current Protocols, 2(9). doi:10.1002/cpz1.547
• Shubitz, L. F., Powell, D. A., Dial, S. M., Butkiewicz, C. D., Trinh, H. T., Hsu, A. P., Buntzman, A., Frelinger, J. A., & Galgiani, J. N. (2022). Reactivation of Coccidioidomycosis in a Mouse Model of Asymptomatic Controlled Disease. Journal of fungi (Basel, Switzerland), 8(10).
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  The majority of human coccidioidomycosis infections are asymptomatic or self-limited but may have sequestered spherules in highly structured granulomas. Under immunosuppression, reactivation of fungal growth can result in severe disease. B6D2F1 mice asymptomatically infected with strain 1038 were immunosuppressed with dexamethasone (DXM) in drinking water. Treated mice died 16-25 days later, while untreated mice survived ( < 0.001). Flow cytometry of lung granulomas on days 5, 10, 15, and 20 of DXM treatment showed immune cell populations decreased 0.5-1 log compared with untreated mice though neutrophils and CD19IgDIgM cells rebounded by day 20. Histopathology demonstrated loss of granuloma structure by day 5 and increasing spherules through day 20. On day 20, T-cells were nearly absent and disorganized pyogranulomatous lesions included sheets of plasma cells and innumerable spherules. Mice given DXM for 14 days then stopped (DXM stop) survived 6 weeks (9/10). Lung fungal burdens were significantly lower ( = 0.0447) than mice that continued treatment (DXM cont) but higher than untreated mice. Histopathologically, DXM stop mice did not redevelop controlled granulomas by sacrifice, though T-cells were densely scattered throughout the lesions. This demonstrates a mouse model suitable for further study to understand the immunologic components responsible for maintenance control of coccidioidomycosis.
• Buntzman, A. S. (2020). The ADC API: A Web API for the Programmatic Query of the AIRR Data Commons. Frontiers in Big Data, 3, 22. doi:10.3389/fdata.2020.00022
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  AIRR Community Publication
• Lees, W., Busse, C. E., Corcoran, M., Ohlin, M., Scheepers, C., Matsen, F. A., Yaari, G., Watson, C. T., , A. C., Collins, A., & Shepherd, A. J. (2020). OGRDB: a reference database of inferred immune receptor genes. Nucleic acids research, 48(D1), D964-D970.
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  High-throughput sequencing of the adaptive immune receptor repertoire (AIRR-seq) is providing unprecedented insights into the immune response to disease and into the development of immune disorders. The accurate interpretation of AIRR-seq data depends on the existence of comprehensive germline gene reference sets. Current sets are known to be incomplete and unrepresentative of the degree of polymorphism and diversity in human and animal populations. A key issue is the complexity of the genomic regions in which they lie, which, because of the presence of multiple repeats, insertions and deletions, have not proved tractable with short-read whole genome sequencing. Recently, tools and methods for inferring such gene sequences from AIRR-seq datasets have become available, and a community approach has been developed for the expert review and publication of such inferences. Here, we present OGRDB, the Open Germline Receptor Database (https://ogrdb.airr-community.org), a public resource for the submission, review and publication of previously unknown receptor germline sequences together with supporting evidence.
• Merritt, E. F., Johnson, H. J., Wong, Z. S., Buntzman, A. S., Conklin, A. C., Cabral, C. M., Romanoski, C. E., Boyle, J. P., & Koshy, A. A. (2020). Transcriptional Profiling Suggests T Cells Cluster around Neurons Injected with Toxoplasma gondii Proteins. mSphere, 5(5).
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  's tropism for and persistence in the central nervous system (CNS) underlies the symptomatic disease that causes in humans. Our recent work has shown that neurons are the primary CNS cell with which interacts and which it infects This predilection for neurons suggests that 's persistence in the CNS depends specifically upon parasite manipulation of the host neurons. Yet, most work on -host cell interactions has been done and in nonneuronal cells. We address this gap by utilizing our -Cre system that allows permanent marking and tracking of neurons injected with parasite effector proteins Using laser capture microdissection (LCM) and RNA sequencing using RNA-seq, we isolated and transcriptionally profiled -injected neurons (TINs), Bystander neurons (nearby non--injected neurons), and neurons from uninfected mice (controls). These profiles show that TIN transcriptomes significantly differ from the transcriptomes of Bystander and control neurons and that much of this difference is driven by increased levels of transcripts from immune cells, especially CD8 T cells and monocytes. These data suggest that when we used LCM to isolate neurons from infected mice, we also picked up fragments of CD8 T cells and monocytes clustering in extreme proximity around TINs and, to a lesser extent, Bystander neurons. In addition, we found that transcripts were primarily found in the TIN transcriptome, not in the Bystander transcriptome. Collectively, these data suggest that, contrary to common perception, neurons that directly interact with or harbor parasites can be recognized by CD8 T cells. Like other persistent intracellular pathogens, , a protozoan parasite, has evolved to evade the immune system and establish a chronic infection in specific cells and organs, including neurons in the CNS. Understanding 's persistence in neurons holds the potential to identify novel, curative drug targets. The work presented here offers new insights into the neuron- interaction By transcriptionally profiling neurons manipulated by , we unexpectedly revealed that immune cells, and specifically CD8 T cells, appear to cluster around these neurons, suggesting that CD8 T cells specifically recognize parasite-manipulated neurons. Such a possibility supports evidence from other labs that questions the long-standing dogma that neurons are often persistently infected because they are not directly recognized by immune cells such as CD8 T cells. Collectively, these data suggest we reconsider the broader role of neurons in the context of infection and neuroinflammation.
• Christley, S., Scarborough, W., Salinas, E., Rounds, W. H., Toby, I. T., Fonner, J. M., Levin, M. K., Kim, M., Mock, S. A., Jordan, C., Ostmeyer, J., Buntzman, A., Rubelt, F., Davila, M. L., Monson, N. L., Scheuermann, R. H., & Cowell, L. G. (2018). VDJServer: A Cloud-Based Analysis Portal and Data Commons for Immune Repertoire Sequences and Rearrangements. Frontiers in immunology, 9, 976.
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  Recent technological advances in immune repertoire sequencing have created tremendous potential for advancing our understanding of adaptive immune response dynamics in various states of health and disease. Immune repertoire sequencing produces large, highly complex data sets, however, which require specialized methods and software tools for their effective analysis and interpretation.
• Ross, P., Nemec, P. S., Kapatos, A., Miller, K. R., Holmes, J. C., Suter, S. E., Buntzman, A. S., Soderblom, E. J., Collins, E. J., & Hess, P. R. (2018). The canine MHC class Ia allele DLA-88*508:01 presents diverse self- and canine distemper virus-origin peptides of varying length that have a conserved binding motif. Veterinary immunology and immunopathology, 197, 76-86.
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  Ideally, CD8+ T-cell responses against virally infected or malignant cells are defined at the level of the specific peptide and restricting MHC class I element, a determination not yet made in the dog. To advance the discovery of canine CTL epitopes, we sought to determine whether a putative classical MHC class Ia gene, Dog Leukocyte Antigen (DLA)-88, presents peptides from a viral pathogen, canine distemper virus (CDV). To investigate this possibility, DLA-88*508:01, an allele prevalent in Golden Retrievers, was expressed as a FLAG-tagged construct in canine histiocytic cells to allow affinity purification of peptide-DLA-88 complexes and subsequent elution of bound peptides. Pattern analysis of self peptide sequences, which were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS), permitted binding preferences to be inferred. DLA-88*508:01 binds peptides that are 9-to-12 amino acids in length, with a modest preference for 9- and 11-mers. Hydrophobic residues are favored at positions 2 and 3, as are K, R or F residues at the C-terminus. Testing motif-matched and -unmatched synthetic peptides via peptide-MHC surface stabilization assay using a DLA-88*508:01-transfected, TAP-deficient RMA-S line supported these conclusions. With CDV infection, 22 viral peptides ranging from 9-to-12 residues in length were identified in DLA-88*508:01 eluates by LC-MS/MS. Combined motif analysis and surface stabilization assay data suggested that 11 of these 22 peptides, derived from CDV hemagglutinin, large polymerase, matrix, nucleocapsid, and V proteins, were processed and presented, and thus, potential targets of anti-viral CTL in DLA-88*508:01-bearing dogs. The presentation of diverse self and viral peptides indicates that DLA-88 is a classical MHC class Ia gene.
• Smithey, M. J., Venturi, V., Davenport, M. P., Buntzman, A. S., Vincent, B. G., Frelinger, J. A., & Nikolich-Žugich, J. (2018). Lifelong CMV infection improves immune defense in old mice by broadening the mobilized TCR repertoire against third-party infection. Proceedings of the National Academy of Sciences of the United States of America, 115(29), E6817-E6825.
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  Lifelong interactions between host and the ubiquitous and persistent cytomegalovirus (CMV) have been proposed to contribute to the age-related decline in immunity. Prior work from us and others found some support for that idea, yet evidence that this led to increased vulnerability to other infections was not obtained. Moreover, evidence has accumulated that CMV infection can be beneficial to immune defense in young/adult mice and humans, dominantly via enhanced innate immunity. Here, we describe an unexpected impact of murine CMV (MCMV) upon the T cell response of old mice to expressing the model antigen, OVA (Lm-OVA). Single-cell sequencing of the OVA-specific CD8 T cell receptor β (TCRβ) repertoire of old mice demonstrated that old MCMV-infected mice recruited many diverse clonotypes that afforded broad and often more efficient recognition of antigenic peptide variants. This stood in contrast to old control mice, which exhibited strong narrowing and homogenization of the elicited repertoire. High-throughput sequencing of the total naïve CD8 TCRβ repertoire showed that many of these diverse OVA-specific clonotypes were present in the naïve CD8 repertoire of mice in all groups (adult, old control, and old MCMV) yet were only recruited into the Lm-OVA response in MCMV old mice. These results have profound implications for our understanding of T cell immunity over a life span and suggest that our coevolution with CMV may include surprising, potentially positive impacts on adaptive heterologous immunity in late life.
• Vander Heiden, J. A., Marquez, S., Marthandan, N., Bukhari, S. A., Busse, C. E., Corrie, B., Hershberg, U., Kleinstein, S. H., Matsen Iv, F. A., Ralph, D. K., Rosenfeld, A. M., Schramm, C. A., , A. C., Christley, S., & Laserson, U. (2018). AIRR Community Standardized Representations for Annotated Immune Repertoires. Frontiers in immunology, 9, 2206.
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  Increased interest in the immune system's involvement in pathophysiological phenomena coupled with decreased DNA sequencing costs have led to an explosion of antibody and T cell receptor sequencing data collectively termed "adaptive immune receptor repertoire sequencing" (AIRR-seq or Rep-Seq). The AIRR Community has been actively working to standardize protocols, metadata, formats, APIs, and other guidelines to promote open and reproducible studies of the immune repertoire. In this paper, we describe the work of the AIRR Community's Data Representation Working Group to develop standardized data representations for storing and sharing annotated antibody and T cell receptor data. Our file format emphasizes ease-of-use, accessibility, scalability to large data sets, and a commitment to open and transparent science. It is composed of a tab-delimited format with a specific schema. Several popular repertoire analysis tools and data repositories already utilize this AIRR-seq data format. We hope that others will follow suit in the interest of promoting interoperable standards.
• Woodcock, M., Vincent, B. G., & Buntzman, A. (2018). Evidence for Role of Enzymatic Operations in Shared T-Cell Receptors. Blood, 132(Supplement 1), 3703-3703. doi:10.1182/blood-2018-99-118884
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  Abstract Background: There exist shared T cell receptors (TCR) found in the circulating repertoires of healthy individuals. The exact mechanism for why some sequences are more commonly shared is unclear, but it has been hypothesized that the number of recombination permutations of V, D and J segments that produce a particular shared sequence is predictive (i.e. the "convergent recombination" hypothesis). An alternative hypothesis is that sharing is driven by decreased biochemical energy required to generate the shared sequences (i.e. the "enzyme proxy" hypothesis). Methods: Circulating T cells were collected from 100 healthy donors and TCR repertoires were profiled. Using a novel algorithm, the number of recombination events that could produce each TCR was calculated ("path count"). Sequence features associated with enzymatic operations - such as V and J region chewback and non-templated nucleotide addition - were calculated as well ("enzymatic" parameters). Multivariable regression was used to identify TCR sequence features associated with TCR clonotype sharing between individuals. Results: Nearly 38 million unique TCR sequences from the donor cohort were profiled. Based on regression coefficient analysis of significance, enzymatic parameters were overall negatively associated with TCR sharing (betas [-1.24x10-2 - 3.88x10-3], average -5.67x10-3, maximum p
• Rubelt, F., Busse, C. E., Bukhari, S. A., Bürckert, J. P., Mariotti-Ferrandiz, E., Cowell, L. G., Watson, C. T., Marthandan, N., Faison, W. J., Hershberg, U., Laserson, U., Corrie, B. D., Davis, M. M., Peters, B., Lefranc, M. P., Scott, J. K., Breden, F., , A. C., Luning Prak, E. T., & Kleinstein, S. H. (2017). Adaptive Immune Receptor Repertoire Community recommendations for sharing immune-repertoire sequencing data. Nature immunology, 18(12), 1274-1278.
• Narra, H. P., Shubitz, L. F., Mandel, M. A., Trinh, H. T., Griffin, K., Buntzman, A. S., Frelinger, J. A., Galgiani, J. N., & Orbach, M. J. (2016). A Coccidioides posadasii CPS1 Deletion Mutant Is Avirulent and Protects Mice from Lethal Infection. Infection and immunity, 84(10), 3007-16.
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  The CPS1 gene was identified as a virulence factor in the maize pathogen Cochliobolus heterostrophus Hypothesizing that the homologous gene in Coccidioides posadasii could be important for virulence, we created a Δcps1 deletion mutant which was unable to cause disease in three strains of mice (C57BL/6, BALB/c, or the severely immunodeficient NOD-scid,γc(null) [NSG]). Only a single colony was recovered from 1 of 60 C57BL/6 mice following intranasal infections of up to 4,400 spores. Following administration of very high doses (10,000 to 2.5 × 10(7) spores) to NSG and BALB/c mice, spherules were observed in lung sections at time points from day 3 to day 10 postinfection, but nearly all appeared degraded with infrequent endosporulation. Although the role of CPS1 in virulence is not understood, phenotypic alterations and transcription differences of at least 33 genes in the Δcps1 strain versus C. posadasii is consistent with both metabolic and regulatory functions for the gene. The in vitro phenotype of the Δcps1 strain showed slower growth of mycelia with delayed and lower spore production than C. posadasii, and in vitro spherules were smaller. Vaccination of C57BL/6 or BALB/c mice with live Δcps1 spores either intranasally, intraperitoneally, or subcutaneously resulted in over 95% survival with mean residual lung fungal burdens of
• Scheuermann, R. H., Uduman, M., Toby, I. T., Scott, J. K., Scheuermann, R. H., Scarborough, W., Rubelt, F., Rounds, W., Rosenfeld, A. M., Rojas, M., Monson, N. L., Mock, S., Marthandan, N., Kleinstein, S. H., Kim, M. S., Jordan, C., Hershberg, U., Heiden, J. A., Gupta, N., , Fonner, J. M., et al. (2016). VDJML – tools for capturing the results of inferring immune receptor rearrangements. Journal of Immunology, 196(1_Supplement), 209.24-209.24. doi:10.4049/jimmunol.196.supp.209.24
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  Despite the widespread use of immune repertoire profiling, there is currently no standardized format for output files from VDJ analysis. Researchers utilize software such as IgBlast and IMGT/High V-Quest to perform VDJ analysis and infer germline rearrangements. Each of these software tools produces results in a different file format, and can identify the same result using different labels. These differences make it challenging for users to perform additional analysis using the output file from one software to the next. We have addressed this problem by developing a standardized file format for representing results. The purpose of VDJML is to provide a common standardized format for different VDJ analysis applications and to facilitate downstream processing of the results in an application-agnostic manner. The VDJML file format is accompanied by a suite of analysis tools, which are accessible via command line and written in C++ and python. The VDJML suite will allow users to streamline their VDJ analysis and facilitate the sharing of scientific knowledge within the community. The VDJML suite and documentation are available from https://www.vdjserver.org/software. We welcome participation from others in developing the file format standard, as well as code contributions.
• Toby, I. T., Levin, M. K., Salinas, E. A., Christley, S., Bhattacharya, S., Breden, F., Buntzman, A., Corrie, B., Fonner, J., Gupta, N. T., Hershberg, U., Marthandan, N., Rosenfeld, A., Rounds, W., Rubelt, F., Scarborough, W., Scott, J. K., Uduman, M., Vander Heiden, J. A., , Scheuermann, R. H., et al. (2016). VDJML: a file format with tools for capturing the results of inferring immune receptor rearrangements. BMC bioinformatics, 17(Suppl 13), 333.
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  The genes that produce antibodies and the immune receptors expressed on lymphocytes are not germline encoded; rather, they are somatically generated in each developing lymphocyte by a process called V(D)J recombination, which assembles specific, independent gene segments into mature composite genes. The full set of composite genes in an individual at a single point in time is referred to as the immune repertoire. V(D)J recombination is the distinguishing feature of adaptive immunity and enables effective immune responses against an essentially infinite array of antigens. Characterization of immune repertoires is critical in both basic research and clinical contexts. Recent technological advances in repertoire profiling via high-throughput sequencing have resulted in an explosion of research activity in the field. This has been accompanied by a proliferation of software tools for analysis of repertoire sequencing data. Despite the widespread use of immune repertoire profiling and analysis software, there is currently no standardized format for output files from V(D)J analysis. Researchers utilize software such as IgBLAST and IMGT/High V-QUEST to perform V(D)J analysis and infer the structure of germline rearrangements. However, each of these software tools produces results in a different file format, and can annotate the same result using different labels. These differences make it challenging for users to perform additional downstream analyses.
• Vincent, B., Buntzman, A., Hopson, B., McEwen, C., Cowell, L., Akoglu, A., Zhang, H., & Frelinger, J. (2016). iWAS - A novel approach to analyzing Next Generation Sequence data for immunology. Cellular immunology, 299, 6-13.
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  In this communication we describe a novel way to use Next Generation Sequence from the receptors expressed on T and B cells. This informatics methodology is named iWAS, for immunonome Wide Association Study, where we use the immune receptor sequences derived from T and B cells and the features of those receptors (sequences themselves, V/J gene usage, length and character each of the CDR3 sub-regions) to define biomarkers of health and disease, as well as responses to therapies. Unlike GWAS, which do not provide immediate access to mechanism, the associations with immune receptors immediately suggest possible and plausible entrée's into disease pathogenesis and treatment.
• Hunsucker, S. A., McGary, C. S., Vincent, B. G., Enyenihi, A. A., Waugh, J. P., McKinnon, K. P., Bixby, L. M., Ropp, P. A., Coghill, J. M., Wood, W. A., Gabriel, D. A., Sarantopoulos, S., Shea, T. C., Serody, J. S., Alatrash, G., Rodriguez-Cruz, T., Lizée, G., Buntzman, A. S., Frelinger, J. A., , Glish, G. L., et al. (2015). Peptide/MHC tetramer-based sorting of CD8⁺ T cells to a leukemia antigen yields clonotypes drawn nonspecifically from an underlying restricted repertoire. Cancer immunology research, 3(3), 228-35.
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  Testing of T cell-based cancer therapeutics often involves measuring cancer antigen-specific T-cell populations with the assumption that they arise from in vivo clonal expansion. This analysis, using peptide/MHC tetramers, is often ambiguous. From a leukemia cell line, we identified a CDK4-derived peptide epitope, UNC-CDK4-1 (ALTPVVVTL), that bound HLA-A*02:01 with high affinity and could induce CD8⁺ T-cell responses in vitro. We identified UNC-CDK4-1/HLA-A*02:01 tetramer⁺ populations in 3 of 6 patients with acute myeloid leukemia who had undergone allogeneic stem cell transplantation. Using tetramer-based, single-cell sorting and T-cell receptor β (TCRβ) sequencing, we identified recurrent UNC-CDK4-1 tetramer-associated TCRβ clonotypes in a patient with a UNC-CDK4-1 tetramer⁺ population, suggesting in vivo T-cell expansion to UNC-CDK4-1. In parallel, we measured the patient's TCRβ repertoire and found it to be highly restricted/oligoclonal. The UNC-CDK4-1 tetramer-associated TCRβ clonotypes represented >17% of the entire TCRβ repertoire-far in excess of the UNC-CDK4-1 tetramer⁺ frequency-indicating that the recurrent TCRβ clonotypes identified from UNC-CDK-4-1 tetramer⁺ cells were likely a consequence of the extremely constrained T-cell repertoire in the patient and not in vivo UNC-CDK4-1-driven clonal T-cell expansion. Mapping recurrent TCRβ clonotype sequences onto TCRβ repertoires can help confirm or refute antigen-specific T-cell expansion in vivo.
• Kidd, J. A., Ross, P., Buntzman, A. S., & Hess, P. R. (2015). Development of an ELISA to detect circulating anti-asparaginase antibodies in dogs with lymphoid neoplasia treated with Escherichia coli l-asparaginase. Veterinary and comparative oncology, 13(2), 77-88.
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  Resistance to Escherichia coli l-asparaginase in canine lymphoma occurs frequently with repeated administration, a phenomenon often attributed, without substantiation, to the induction of neutralizing antibodies. To test the hypothesis that treated dogs develop antibodies against the drug, we created an enzyme-linked immunosorbent assay (ELISA) to measure plasma anti-asparaginase immunoglobulin G responses. Using samples from dogs that had received multiple doses, specific reactivity against l-asparaginase was demonstrated, while naïve patients' samples were negative. The optimized ELISA appeared sensitive, with endpoint titers >1 600 000 in positive control dogs. Intra- and inter-assay coefficients of variation were 3.6 and 14.5%. The assay was supported by the observation that ELISA-positive plasma could immunoprecipitate asparaginase activity. When clinical patients were evaluated, 3/10 dogs developed titers after a single injection; with repeated administration, 4/7 dogs were positive. l-asparaginase antibodies showed reduced binding to the PEGylated drug formulation. The ELISA should prove useful in investigating the potential correlation of antibody responses with resistance.
• Buntzman, A. S. (2014). Meeting report: advancing practical applications of biodiversity ontologies. Standards in Genomic Sciences, 9, 17.
• Buntzman, A. S. (2014). Overcoming the Limitations Posed by TCRbeta Repertoire Modeling Through a GPU-Based In-Silico DNA Recombination Algorithm. IEEE IPDPS, 231-240.
• Towne, J. W., Wagner, A. M., Griffin, K. J., Buntzman, A. S., Frelinger, J. A., & Besselsen, D. G. (2014). Elimination of Pasteurella pneumotropica from a mouse barrier facility by using a modified enrofloxacin treatment regimen. Journal of the American Association for Laboratory Animal Science : JAALAS, 53(5), 517-22.
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  Multiple NOD. Cg-Prkdc(scid)Il2rg(tm1Wjl)Tg(HLA-A2.1)Enge/Sz (NSG/A2) transgenic mice maintained in a mouse barrier facility were submitted for necropsy to determine the cause of facial alopecia, tachypnea, dyspnea, and sudden death. Pneumonia and soft-tissue abscesses were observed, and Pasteurella pneumotropica biotype Jawetz was consistently isolated from the upper respiratory tract, lung, and abscesses. Epidemiologic investigation within the facility revealed presence of this pathogen in mice generated or rederived by the intramural Genetically Engineered Mouse Model (GEMM) Core but not in mice procured from several approved commercial vendors. Epidemiologic data suggested the infection originated from female or vasectomized male ND4 mice obtained from a commercial vendor and then comingled by the GEMM Core to induce pseudopregnancy in female mice for embryo implantation. Enrofloxacin delivered in drinking water (85 mg/kg body weight daily) for 14 d was sufficient to clear bacterial infection in normal, breeding, and immune-deficient mice without the need to change the antibiotic water source. This modified treatment regimen was administered to 2400 cages of mice to eradicate Pasteurella pneumotropica from the facility. Follow-up PCR testing for P. pneumotropica biotype Jawetz remained uniformly negative at 2, 6, 12, and 52 wk after treatment in multiple strains of mice that were originally infected. Together, these data indicate that enrofloxacin can eradicate P. pneumotropica from infected mice in a less labor-intensive approach that does not require breeding cessation and that is easily adaptable to the standard biweekly cage change schedule for individually ventilated cages.
• Hess, S. M., Young, E. F., Miller, K. R., Vincent, B. G., Buntzman, A. S., Collins, E. J., Frelinger, J. A., & Hess, P. R. (2013). Deletion of naïve T cells recognizing the minor histocompatibility antigen HY with toxin-coupled peptide-MHC class I tetramers inhibits cognate CTL responses and alters immunodominance. Transplant immunology, 29(1-4), 138-45.
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  Alloreactive T-cell responses directed against minor histocompatibility (H) antigens, which arise from diverse genetic disparities between donor and recipient outside the MHC, are an important cause of rejection of MHC-matched grafts. Because clinically significant responses appear to be directed at only a few antigens, the selective deletion of naïve T cells recognizing donor-specific, immunodominant minor H antigens in recipients before transplantation may be a useful tolerogenic strategy. We have previously demonstrated that peptide-MHC class I tetramers coupled to a toxin can efficiently eliminate specific TCR-transgenic T cells in vivo. Here, using the minor histocompatibility antigen HY as a model, we investigated whether toxic tetramers could inhibit the subsequent priming of the two H2-D(b)-restricted, immunodominant T-cell responses by deleting precursor CTL. Immunization of female mice with male bone marrow elicited robust CTL activity against the Uty and Smcy epitopes, with Uty constituting the major response. As hypothesized, toxic tetramer administration prior to immunization increased survival of cognate peptide-pulsed cells in an in vivo CTL assay, and reduced the frequency of corresponding T cells. However, tetramer-mediated decreases in either T-cell population magnified CTL responses against the non-targeted epitope, suggesting that D(b)-Uty(+) and D(b)-Smcy(+) T cells compete for a limited common resource during priming. Toxic tetramers conceivably could be used in combination to dissect manipulate CD8(+) T-cell immunodominance hierarchies, and to prevent the induction of donor-specific, minor H antigen CTL responses in allotransplantation.
• Holmes, J. C., Holmer, S. G., Ross, P., Buntzman, A. S., Frelinger, J. A., & Hess, P. R. (2013). Polymorphisms and tissue expression of the feline leukocyte antigen class I loci FLAI-E, FLAI-H, and FLAI-K. Immunogenetics, 65(9), 675-89.
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  Cytotoxic CD8+ T-cell immunosurveillance for intracellular pathogens, such as viruses, is controlled by classical major histocompatibility complex (MHC) class Ia molecules, and ideally, these antiviral T-cell populations are defined by the specific peptide and restricting MHC allele. Surprisingly, despite the utility of the cat in modeling human viral immunity, little is known about the feline leukocyte antigen class I complex (FLAI). Only a few coding sequences with uncertain locus origin and expression patterns have been reported. Of 19 class I genes, three loci--FLAI-E, FLAI-H, and FLAI-K--are predicted to encode classical molecules, and our objective was to evaluate their status by analyzing polymorphisms and tissue expression. Using locus-specific, PCR-based genotyping, we amplified 33 FLAI-E, FLAI-H, and FLAI-K alleles from 12 cats of various breeds, identifying, for the first time, alleles across three distinct loci in a feline species. Alleles shared the expected polymorphic and invariant sites in the α1/α2 domains, and full-length cDNA clones possessed all characteristic class Ia exons. Alleles could be assigned to a specific locus with reasonable confidence, although there was evidence of potentially confounding interlocus recombination between FLAI-E and FLAI-K. Only FLAI-E, FLAI-H, and FLAI-K origin alleles were amplified from cDNAs of multiple tissue types. We also defined hypervariable regions across these genes, which permitted the assignment of names to both novel and established alleles. As predicted, FLAI-E, FLAI-H, and FLAI-K fulfill the major criteria of class Ia genes. These data represent a necessary prerequisite for studying epitope-specific antiviral CD8+ T-cell responses in cats.
• Woolard, M. D., Barrigan, L. M., Fuller, J. R., Buntzman, A. S., Bryan, J., Manoil, C., Kawula, T. H., & Frelinger, J. A. (2013). Identification of Francisella novicida mutants that fail to induce prostaglandin E(2) synthesis by infected macrophages. Frontiers in microbiology, 4, 16.
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  Francisella tularensis is the causative agent of tularemia. We have previously shown that infection with F. tularensis Live Vaccine Strain (LVS) induces macrophages to synthesize prostaglandin E(2) (PGE(2)). Synthesis of PGE(2) by F. tularensis infected macrophages results in decreased T cell proliferation in vitro and increased bacterial survival in vivo. Although we understand some of the biological consequences of F. tularensis induced PGE(2) synthesis by macrophages, we do not understand the cellular pathways (neither host nor bacterial) that result in up-regulation of the PGE(2) biosynthetic pathway in F. tularensis infected macrophages. We took a genetic approach to begin to understand the molecular mechanisms of bacterial induction of PGE(2) synthesis from infected macrophages. To identify F. tularensis genes necessary for the induction of PGE(2) in primary macrophages, we infected cells with individual mutants from the closely related strain F. tularensis subspecies novicida U112 (U112) two allele mutant library. Twenty genes were identified that when disrupted resulted in U112 mutant strains unable to induce the synthesis of PGE(2) by infected macrophages. Fourteen of the genes identified are located within the Francisella pathogenicity island (FPI). Genes in the FPI are required for F. tularensis to escape from the phagosome and replicate in the cytosol, which might account for the failure of U112 with transposon insertions within the FPI to induce PGE(2). This implies that U112 mutant strains that do not grow intracellularly would also not induce PGE(2). We found that U112 clpB::Tn grows within macrophages yet fails to induce PGE(2), while U112 pdpA::Tn does not grow yet does induce PGE(2). We also found that U112 iglC::Tn neither grows nor induces PGE(2). These findings indicate that there is dissociation between intracellular growth and the ability of F. tularensis to induce PGE(2) synthesis. These mutants provide a critical entrée into the pathways used in the host for PGE(2) induction.
• Bunztman, A., Vincent, B. G., Krovi, H., Steele, S., & Frelinger, J. A. (2012). The LCMV gp33-specific memory T cell repertoire narrows with age. Immunity & ageing : I & A, 9(1), 17.
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  The memory response to LCMV in mice persists for months to years with only a small decrease in the number of epitope specific CD8 T cells. This long persistence is associated with resistance to lethal LCMV disease. In contrast to studies focused on the number and surface phenotype of the memory cells, relatively little attention has been paid to the diversity of TCR usage in these cells. CD8+ T cell responses with only a few clones of identical specificity are believed to be relatively ineffective, presumably due to the relative ease of virus escape. Thus, a broad polyclonal response is associated with an effective anti-viral CD8+ T cell response.
• Frelinger, J. A., Buntzman, A., Biffa, D., & Barrigan, L. M. (2012). Intradermal route of infection with Francisella tularensis subspp novicida elicits differential induction of a wide array of cytokines and chemokines. Journal of Immunology, 188(1_Supplement), 117.7-117.7. doi:10.4049/jimmunol.188.supp.117.7
• Gojanovich, G. S., Murray, S. L., Buntzman, A. S., Young, E. F., Vincent, B. G., & Hess, P. R. (2012). The use of peptide-major-histocompatibility-complex multimers in type 1 diabetes mellitus. Journal of diabetes science and technology, 6(3), 515-24.
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  Major histocompatibility complex (MHC) class I and MHC class II molecules present short peptides that are derived from endogenous and exogenous proteins, respectively, to cognate T-cell receptors (TCRs) on the surface of T cells. The exquisite specificity with which T cells recognize particular peptide-major-histocompatibility-complex (pMHC) combinations has permitted development of soluble pMHC multimers that bind exclusively to selected T-cell populations. Because the pathogenesis of type 1 diabetes mellitus (T1DM) is driven largely by islet-reactive T-cell activity that causes β-cell death, these reagents are useful tools for studying and, potentially, for treating this disease. When coupled to fluorophores or paramagnetic nanoparticles, pMHC multimers have been used to visualize the expansion and islet invasion of T-cell effectors during diabetogenesis. Administration of pMHC multimers to mice has been shown to modulate T-cell responses by signaling through the TCR or by delivering a toxic moiety that deletes the targeted T cell. In the nonobese diabetic mouse model of T1DM, a pMHC-I tetramer coupled to a potent ribosome-inactivating toxin caused long-term elimination of a specific diabetogenic cluster of differentiation 8+ T-cell population from the pancreatic islets and delayed the onset of diabetes. This review will provide an overview of the development and use of pMHC multimers, particularly in T1DM, and describe the therapeutic promise these reagents have as an antigen-specific means of ameliorating deleterious T-cell responses in this autoimmune disease.
• Holl, E. K., Roney, K. E., Allen, I. C., Steinbach, E., Arthur, J. C., Buntzman, A., Plevy, S., Frelinger, J., & Ting, J. P. (2012). Plexin-B2 and Plexin-D1 in dendritic cells: expression and IL-12/IL-23p40 production. PloS one, 7(8), e43333.
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  Plexins are a family of genes (A,B,C, and D) that are expressed in many organ systems. Plexins expressed in the immune system have been implicated in cell movement and cell-cell interaction during the course of an immune response. In this study, the expression pattern of Plexin-B2 and Plexin-D1 in dendritic cells (DCs), which are central in immune activation, was investigated. Plexin-B2 and Plexin-D1 are reciprocally expressed in myeloid and plasmacytoid DC populations. Plasmacytoid DCs have high Plexin-B2 but low Plexin-D1, while the opposite is true of myeloid DCs. Expression of Plexin-B2 and Plexin-D1 is modulated upon activation of DCs by TLR ligands, TNFα, and anti-CD40, again in a reciprocal fashion. Semaphorin3E, a ligand for Plexin-D1 and Plexin-B2, is expressed by T cells, and interestingly, is dramatically higher on Th2 cells and on DCs. The expression of Plexins and their ligands on DCs and T cells suggest functional relevance. To explore this, we utilized chimeric mice lacking Plxnb2 or Plxnd1. Absence of Plexin-B2 and Plexin-D1 on DCs did not affect the ability of these cells to upregulate costimulatory molecules or the ability of these cells to activate antigen specific T cells. Additionally, Plexin-B2 and Plexin-D1 were dispensable for chemokine-directed in-vitro migration of DCs towards key DC chemokines, CXCL12 and CCL19. However, the absence of either Plexin-B2 or Plexin-D1 on DCs leads to constitutive expression of IL-12/IL-23p40. This is the first report to show an association between Plexin-B2 and Plexin-D1 with the negative regulation of IL-12/IL-23p40 in DCs. This work also shows the presence of Plexin-B2 and Plexin-D1 on mouse DC subpopulations, and indicates that these two proteins play a role in IL-12/IL-23p40 production that is likely to impact the immune response.
• Ross, P. S., Miller, K. R., Holmes, J. C., Hess, P. R., Gojanovich, G. S., Frelinger, J. A., Collins, E. J., & Buntzman, A. (2012). The canine MHC class I allele, DLA-88*50801, presents peptides of varying lengths with three key anchor residues. Journal of Immunology, 188(1_Supplement), 160.14-160.14. doi:10.4049/jimmunol.188.supp.160.14
• Ross, P., Buntzman, A. S., Vincent, B. G., Grover, E. N., Gojanovich, G. S., Collins, E. J., Frelinger, J. A., & Hess, P. R. (2012). Allelic diversity at the DLA-88 locus in Golden Retriever and Boxer breeds is limited. Tissue antigens, 80(2), 175-83.
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  In the dog, previous analyses of major histocompatibility complex class I genes suggest a single polymorphic locus, dog leukocyte antigen (DLA)-88. While 51 alleles have been reported, estimates of prevalence have not been made. We hypothesized that, within a breed, DLA-88 diversity would be restricted, and one or more dominant alleles could be identified. Accordingly, we determined allele usage in 47 Golden Retrievers and 39 Boxers. In each population, 10 alleles were found; 4 were shared. Seven novel alleles were identified. DLA-88*05101 and *50801 predominated in Golden Retrievers, while most Boxers carried *03401. In these breeds, DLA-88 polymorphisms are limited and largely non-overlapping. The finding of highly prevalent alleles fulfills an important prerequisite for studying canine CD8+ T-cell responses.
• Walsh, J., Vincent, B. G., Steele, S., Krovi, S. H., Kepler, T. B., Frelinger, J. A., & Buntzman, A. (2011). Degeneracy analysis of T cell receptors in mice.. Journal of Immunology, 186(1_Supplement), 160.17-160.17. doi:10.4049/jimmunol.186.supp.160.17
• Washburn, M. L., Bility, M. T., Zhang, L., Kovalev, G. I., Buntzman, A., Frelinger, J. A., Barry, W., Ploss, A., Rice, C. M., & Su, L. (2011). A humanized mouse model to study hepatitis C virus infection, immune response, and liver disease. Gastroenterology, 140(4), 1334-44.
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  Studies of hepatitis C virus (HCV) infection, immunopathogenesis, and resulting liver diseases have been hampered by the lack of a small animal model. We developed humanized mice with human immune system and liver tissues to improve the studies of hepatitis C virus pathogenesis and treatment.
• Buntzman, A., Steele, S., Kawula, T. H., Hensley, L. L., Frelinger, J. A., Buntzman, A., & Bixby, L. M. (2010). Shaping the immune response to Francisella tularensis by the first infected cell. Journal of Immunology, 184.
• Buntzman, A., Young, E., Tisch, R., Talmage, G., Larry, F., Hess, P. R., Frelinger, J. A., & Buntzman, A. (2010). Naive T cells found in the islets of NOD mice do not cause Type I diabetes. Journal of Immunology, 184.
• Vincent, B. G., Young, E. F., Buntzman, A. S., Stevens, R., Kepler, T. B., Tisch, R. M., Frelinger, J. A., & Hess, P. R. (2010). Toxin-coupled MHC class I tetramers can specifically ablate autoreactive CD8+ T cells and delay diabetes in nonobese diabetic mice. Journal of immunology (Baltimore, Md. : 1950), 184(8), 4196-204.
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  There is compelling evidence that self-reactive CD8(+) T cells are a major factor in development and progression of type 1 diabetes in animals and humans. Hence, great effort has been expended to define the specificity of autoimmune CD8(+) T cells and to alter their responses. Much work has focused on tolerization of T cells using proteins or peptides. A weakness in this approach is that residual autoreactive T cells may be activated and exacerbate disease. In this report, we use a novel approach, toxin-coupled MHC class I tetramers. Used for some time to identify Ag-specific cells, in this study, we use that same property to delete the Ag-specific cells. We show that saporin-coupled tetramers can delete islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-reactive T cells in vitro and in vivo. Sequence analysis of TCRbeta-chains of IGRP(+) cells reveals the repertoire complexity in the islets is markedly decreased as NOD mice age and significantly altered in toxic tetramer-treated NOD mice. Further tetramer(+) T cells in the islets are almost completely deleted, and, surprisingly, loss of tetramer(+) T cells in the islets is long lasting. Finally, we show deletion at 8 wk of age of IGRP(+) CD8(+) T cells, but not dystophia myotonica kinase- or insulin B-reactive cells, significantly delays diabetes in NOD mice.
• Walsh, J., Vincent, B. G., Steele, S., Kepler, T. B., Frelinger, J. A., & Buntzman, A. (2010). Large Scale T cell receptor repertoire sequencing of murine CD8+ T cells.. Journal of Immunology, 184.
• Buntzman, A., Young, E. F., Steele, S., Murray, S. L., Hess, P. R., Frelinger, J. A., Collins, E. J., & Buntzman, A. (2009). Selective deletion of HY-reactive CD8+ T cells by saporin-coupled MHC class I tetramers. Journal of Immunology, 182.
• Buntzman, A., Young, E. F., Vincent, B., Tisch, R., Hess, P. R., Frelinger, J. A., & Buntzman, A. (2009). Long term elimination of epitope-specific diabetogenic CD8+ T cells using toxin coupled tetramers in NOD mice. Journal of Immunology, 182.
• Young, E. F., Vincent, B. G., Stevens, R., Kepler, T. B., Frelinger, J. A., & Buntzman, A. S. (2008). Diversity of a diabetegenic T cell population decreases with age in pre-diabetic NOD mice. The FASEB Journal, 22(S2), 462-462. doi:10.1096/fasebj.22.2_supplement.462

Proceedings Publications

• Buntzman, A. S. (2019, December). Bit-Wise and Multi-GPU Implementations of the DNA Recombination Algorithm. In 2019 IEEE 26th International Conference on High Performance Computing, Data, and Analytics (HiPC), 131-140.

Presentations

• Buntzman, A. S. (2023, November). Profiling the Coccidioides granulomatous response in a murine model of fungal recrudescence. Seminar during Valley 20th Annual Fever Awareness Week and Dr Thomas Monath’s Farness Lectureship. University of Arizona.
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  During the 20th Annual Fever Awareness Week, one lecture was given by Dr Thomas Monath in Phoenix and one lecture was given by myself at the University of Arizona to highlight the work done by the Valley Fever Center for Excellence.
• Buntzman, A. S. (2023, September). Immunosuppression induced Coccidioides Granuloma Reactivation Reveals Gene expression profile of Recrudescence. Annual International Autoimmune Conference. Banff, Canada: Praespero Autoimmunity Fund.
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  The lecture discussed our research analyzing the expression profiles of chronic Coccidioides induced granulomas and how those granulomas then respond to autoimmune therapeutics that lead to transient  pharmacological immunosuppression leading to Coccidioides reactivation.
• Buntzman, A. S., & VFCE members, V. m. (2022, April).

  “Valley Fever Vaccine Workshop” 

  “REACTIVATION OF COCCIDIOIDAL DISEASE PROGRESSION IN ASYMPTOMATIC, STABLY INFECTED MICE” 

  ”IMMUNOGENETICS ASSOCIATED WITH SEVERE COCCIDIOIDOMYCOSIS”

  “CONTRIBUTION OF BIOLOGIC RESPONSE MODIFIERS TO THE RISK OF COCCIDIOIDOMYCOSIS SEVERITY”. Cocci Study Group Annual Meeting. Bakersfield, CA: CSG.
• Buntzman, A. S., & VFCE members, V. m. (2022, November). Impact and Control of Valley Fever. Workshop National Academies of Sciences, Engineering, and Medicine. Irvine, CA: National Academies of Sciences.
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  The National Academies of Sciences, Engineering, and Medicine public workshop reviewed the epidemiology of coccidioidomycosis (Valley Fever), its impact on populations, and currently available diagnostic and treatment options. The workshop will explore prospects for control of the disease through vaccination. Specifically, the workshop featured presentations and discussions on the following topics:-Overview of valley fever in human and animal populations to include current surveillance programs and diagnostic and treatment options;-Epidemiology of valley fever in the United States and throughout the Americas;-Impact of climate change on persistence and spread of Coccidioides; -Risk of disease by occupation, ethnicity, and immunogenetic susceptibility and impact on endemic communities;-Economic impact of valley fever;-Immunology of valley fever and potential prevention through vaccination;-Potential vaccine platforms and current animal models;-Strengthening research and research partnerships on overall fungal diseases for therapeutics or vaccine development;-Regulatory pathways for a fungal disease vaccine;-Vaccine acceptance and communication.
• Buntzman, A. S. (2022, September). RA Therapy-Associated Mycosis Reveals Novel Genetic Signature. Praespero Autoimmunity Summit Annual Conference. Vancouver, BC, CAN: Praespero Autoimmunity FUnd.

Poster Presentations

• Mandel, M. A., Shubitz, L., Lewis, L., Trinh, H., Buntzman, A. S., Frelinger, J. A., Galgiani, J. N., & Orbach, M. J. (2017, March, 2017). Construction and Efficacy of delta-cps1, a live attenuated vaccine for coccidioidomycosis. 29th Fungal Genetics Conference. Asilomar Conference Center, Pacific Grove, CA: Genetics Society of America.
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  A poster was presented, and was selected for a poster talk in the Concurrent Session "Human Pathogenic Fungi", which was presented by Dr. Mandel.The abstracts are published as a supplement of Fungal Genetics Reports.