Jeffrey Bunker

All Publications

• B cell superantigens in the human intestinal microbiota SCIENCE TRANSLATIONAL MEDICINE Bunker, J. J., Drees, C., Watson, A. R., Plunkett, C. H., Nagler, C. R., Schneewind, O., Eren, A., Bendelac, A. 2019; 11 (507)

  Abstract

  IgA is prominently secreted at mucosal surfaces and coats a fraction of the commensal microbiota, a process that is critical for intestinal homeostasis. However, the mechanisms of IgA induction and the molecular targets of these antibodies remain poorly understood, particularly in humans. Here, we demonstrate that microbiota from a subset of human individuals encode two protein "superantigens" expressed on the surface of commensal bacteria of the family Lachnospiraceae such as Ruminococcus gnavus that bind IgA variable regions and stimulate potent IgA responses in mice. These superantigens stimulate B cells expressing human VH3 or murine VH5/6/7 variable regions and subsequently bind their antibodies, allowing these microbial organisms to become highly coated with IgA in vivo. These findings demonstrate a previously unappreciated role for commensal superantigens in host-microbiota interactions. Furthermore, as superantigen-expressing strains show an uneven distribution across human populations, they should be systematically considered in studies evaluating human B cell responses and microbiota during homeostasis and disease.

  View details for DOI 10.1126/scitranslmed.aau9356

  View details for Web of Science ID 000483483300001

  View details for PubMedID 31462512

  View details for PubMedCentralID PMC6758550

• IgA Responses to Microbiota IMMUNITY Bunker, J. J., Bendelac, A. 2018; 49 (2): 211-224

  Abstract

  Various immune mechanisms are deployed in the mucosa to confront the immense diversity of resident bacteria. A substantial fraction of the commensal microbiota is coated with immunoglobulin A (IgA) antibodies, and recent findings have established the identities of these bacteria under homeostatic and disease conditions. Here we review the current understanding of IgA biology, and present a framework wherein two distinct types of humoral immunity coexist in the gastrointestinal mucosa. Homeostatic IgA responses employ a polyreactive repertoire to bind a broad but taxonomically distinct subset of microbiota. In contrast, mucosal pathogens and vaccines elicit high-affinity, T cell-dependent antibody responses. This model raises fundamental questions including how polyreactive IgA specificities are generated, how these antibodies exert effector functions, and how they exist together with other immune responses during homeostasis and disease.

  View details for DOI 10.1016/j.immuni.2018.08.011

  View details for Web of Science ID 000442354500008

  View details for PubMedID 30134201

  View details for PubMedCentralID PMC6107312

• Natural polyreactive IgA antibodies coat the intestinal microbiota SCIENCE Bunker, J. J., Erickson, S. A., Flynn, T. M., Henry, C., Koval, J. C., Meisel, M., Jabri, B., Antonopoulos, D. A., Wilson, P. C., Bendelac, A. 2017; 358 (6361)

  Abstract

  Large quantities of immunoglobulin A (IgA) are constitutively secreted by intestinal plasma cells to coat and contain the commensal microbiota, yet the specificity of these antibodies remains elusive. Here we profiled the reactivities of single murine IgA plasma cells by cloning and characterizing large numbers of monoclonal antibodies. IgAs were not specific to individual bacterial taxa but rather polyreactive, with broad reactivity to a diverse, but defined, subset of microbiota. These antibodies arose at low frequencies among naïve B cells and were selected into the IgA repertoire upon recirculation in Peyer's patches. This selection process occurred independent of microbiota or dietary antigens. Furthermore, although some IgAs acquired somatic mutations, these did not substantially influence their reactivity. These findings reveal an endogenous mechanism driving homeostatic production of polyreactive IgAs with innate specificity to microbiota.

  View details for DOI 10.1126/science.aan6619

  View details for Web of Science ID 000413251000031

  View details for PubMedID 28971969

  View details for PubMedCentralID PMC5790183

• Crossreactive alpha beta T Cell Receptors Are the Predominant Targets of Thymocyte Negative Selection IMMUNITY McDonald, B. D., Bunker, J. J., Erickson, S. A., Oh-Hora, M., Bendelac, A. 2015; 43 (5): 859-869

  Abstract

  The precise impact of thymic positive and negative selection on the T cell receptor (TCR) repertoire remains controversial. Here, we used unbiased, high-throughput cloning and retroviral expression of individual pre-selection TCRs to provide a direct assessment of these processes at the clonal level in vivo. We found that 15% of random TCRs induced signaling and directed positive (7.5%) or negative (7.5%) selection, depending on strength of signal, whereas the remaining 85% failed to induce signaling or selection. Most negatively selected TCRs exhibited promiscuous crossreactivity toward multiple other major histocompatibility complex (MHC) haplotypes. In contrast, TCRs that were positively selected or non-selected were minimally crossreactive. Negative selection of crossreactive TCRs led to clonal deletion but also recycling into intestinal CD4(-)CD8β(-) intraepithelial lymphocytes (iIELs). Thus, broadly crossreactive TCRs arise at low frequency in the pre-selection repertoire but constitute the primary drivers of thymic negative selection and iIEL lineage differentiation.

  View details for DOI 10.1016/j.immuni.2015.09.009

  View details for Web of Science ID 000366846000008

  View details for PubMedID 26522985

  View details for PubMedCentralID PMC4654978

• Innate and Adaptive Humoral Responses Coat Distinct Commensal Bacteria with Immunoglobulin A IMMUNITY Bunker, J. J., Flynn, T. M., Koval, J. C., Shaw, D. G., Meisel, M., McDonald, B. D., Ishizuka, I. E., Dent, A. L., Wilson, P. C., Jabri, B., Antonopoulos, D. A., Bendelac, A. 2015; 43 (3): 541-553

  Abstract

  Immunoglobulin A (IgA) is prominently secreted at mucosal surfaces and coats a fraction of the intestinal microbiota. However, the commensal bacteria bound by IgA are poorly characterized and the type of humoral immunity they elicit remains elusive. We used bacterial flow cytometry coupled with 16S rRNA gene sequencing (IgA-Seq) in murine models of immunodeficiency to identify IgA-bound bacteria and elucidate mechanisms of commensal IgA targeting. We found that residence in the small intestine, rather than bacterial identity, dictated induction of specific IgA. Most commensals elicited strong T-independent (TI) responses that originated from the orphan B1b lineage and from B2 cells, but excluded natural antibacterial B1a specificities. Atypical commensals including segmented filamentous bacteria and Mucispirillum evaded TI responses but elicited T-dependent IgA. These data demonstrate exquisite targeting of distinct commensal bacteria by multiple layers of humoral immunity and reveal a specialized function of the B1b lineage in TI mucosal IgA responses.

  View details for DOI 10.1016/j.immuni.2015.08.007

  View details for Web of Science ID 000370965900006

  View details for PubMedID 26320660

  View details for PubMedCentralID PMC4575282

• Biochemical and biophysical characterization of natural polyreactivity in antibodies. Cell reports Borowska, M. T., Boughter, C. T., Bunker, J. J., Guthmiller, J. J., Wilson, P. C., Roux, B., Bendelac, A., Adams, E. J. 2023; 42 (10): 113190

  Abstract

  To become specialized binders, antibodies undergo a process called affinity maturation to maximize their binding affinity. Despite this process, some antibodies retain low-affinity binding to diverse epitopes in a phenomenon called polyreactivity. Here we seek to understand the molecular basis of this polyreactivity in antibodies. Our results highlight that polyreactive antigen-binding fragments (Fabs) bind their targets with low affinities, comparable to T cell receptor recognition of autologous classical major histocompatibility complex. Extensive mutagenic studies find no singular amino acid residue or biochemical property responsible for polyreactive interaction, suggesting that polyreactive antibodies use multiple strategies for engagement. Finally, our crystal structures and all-atom molecular dynamics simulations of polyreactive Fabs show increased rigidity compared to their monoreactive relatives, forming a neutral and accessible platform for diverse antigens to bind. Together, these data support a cooperative strategy of rigid neutrality in establishing the polyreactive status of an antibody molecule.

  View details for DOI 10.1016/j.celrep.2023.113190

  View details for PubMedID 37804505

• The molecular characterization of antibody binding to a superantigen-like protein from a commensal microbe PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Borowska, M. T., Drees, C., Yarawsky, A. E., Viswanathan, M., Ryan, S. M., Bunker, J. J., Herr, A. B., Bendelac, A., Adams, E. J. 2021; 118 (39)

  Abstract

  Microorganisms have coevolved diverse mechanisms to impair host defenses. A major one, superantigens, can result in devastating effects on the immune system. While all known superantigens induce vast immune cell proliferation and come from opportunistic pathogens, recently, proteins with similar broad specificity to antibody variable (V) domain families were identified in a commensal microbiota. These proteins, identified in the human commensal Ruminococcus gnavus, are called immunoglobulin-binding protein (Ibp) A and B and have been shown to activate B cells in vitro expressing either human VH3 or murine VH5/6/7. Here, we provide molecular and functional studies revealing the basis of this Ibp/immunoglobulin (Ig) interaction. The crystal structure and biochemical assays of a truncated IbpA construct in complex with mouse VH5 antigen-binding fragment (Fab) shows a binding of Ig heavy chain framework residues to the Ibp Domain D and the C-terminal heavy chain binding domain (HCBD). We used targeted mutagenesis of contact residues and affinity measurements and performed studies of the Fab-IbpA complex to determine the stoichiometry between Ibp and VH domains, suggesting Ibp may serve to cluster full-length IgA antibodies in vivo. Furthermore, in vitro stimulation experiments indicate that binding of the Ibp HCBD alone is sufficient to activate responsive murine B cell receptors. The presence of these proteins in a commensal microbe suggest that binding a broad repertoire of immunoglobulins, particularly in the gut/microbiome environment, may provide an important function in the maintenance of host/microbiome homeostasis contrasting with the pathogenic role of structurally homologous superantigens expressed by pathogens.

  View details for DOI 10.1073/pnas.2023898118

  View details for Web of Science ID 000704004200022

  View details for PubMedID 34548394

  View details for PubMedCentralID PMC8488583

• Refined protocol for generating monoclonal antibodies from single human and murine B cells JOURNAL OF IMMUNOLOGICAL METHODS Ho, I. Y., Bunker, J. J., Erickson, S. A., Neu, K. E., Huang, M., Cortese, M., Pulendran, B., Wilson, P. C. 2016; 438: 67-70

  Abstract

  Generating monoclonal antibodies from single B cells is a valuable tool for characterizing the specificity and functional properties of humoral responses. We and others developed protocols that have facilitated major advances in our understanding of B cell development, tolerance, and effector responses to HIV and influenza. Here, we demonstrate various refinements and dramatically reduce the time required to produce recombinant antibodies. Further, we present new methods for cloning and isolating antibodies from cells with lower immunoglobulin mRNA levels that may be resistant to traditional techniques. Together, these refinements significantly increase single-cell antibody expression efficiency and are easily integrated into established and novel pipelines.

  View details for DOI 10.1016/j.jim.2016.09.001

  View details for Web of Science ID 000386738700009

  View details for PubMedID 27600311

  View details for PubMedCentralID PMC5322767

• Histone reader BRWD1 targets and restricts recombination to the Igk locus NATURE IMMUNOLOGY Mandal, M., Hamel, K. M., Maienschein-Cline, M., Tanaka, A., Teng, G., Tuteja, J. H., Bunker, J. J., Bahroos, N., Eppig, J. J., Schatz, D. G., Clark, M. R. 2015; 16 (10): 1094-+

  Abstract

  B lymphopoiesis requires that immunoglobulin genes be accessible to RAG1-RAG2 recombinase. However, the RAG proteins bind widely to open chromatin, which suggests that additional mechanisms must restrict RAG-mediated DNA cleavage. Here we show that developmental downregulation of interleukin 7 (IL-7)-receptor signaling in small pre-B cells induced expression of the bromodomain-family member BRWD1, which was recruited to a specific epigenetic landscape at Igk dictated by pre-B cell receptor (pre-BCR)-dependent Erk activation. BRWD1 enhanced RAG recruitment, increased gene accessibility and positioned nucleosomes 5' to each Jκ recombination signal sequence. BRWD1 thus targets recombination to Igk and places recombination within the context of signaling cascades that control B cell development. Our findings represent a paradigm in which, at any particular antigen-receptor locus, specialized mechanisms enforce lineage- and stage-specific recombination.

  View details for DOI 10.1038/ni.3249

  View details for Web of Science ID 000361686500015

  View details for PubMedID 26301565

  View details for PubMedCentralID PMC4575638

• Elevated T Cell Receptor Signaling Identifies a Thymic Precursor to the TCR alpha beta(+)CD4(-)CD8 beta(-) Intraepithelial Lymphocyte Lineage IMMUNITY McDonald, B. D., Bunker, J. J., Ishizuka, I. E., Jabri, B., Bendelac, A. 2014; 41 (2): 219-229

  Abstract

  The origin and developmental pathway of intestinal T cell receptor αβ(+) CD4(-)CD8β(-) intraepithelial lymphocytes (unconventional iIELs), a major population of innate-like resident cytolytic T cells, have remained elusive. By cloning and expressing several TCRs isolated from unconventional iIELs, we identified immature CD4(lo)CD8(lo)(DP(lo))CD69(hi)PD-1(hi) thymocytes as the earliest postsignaling precursors for these cells. Although these precursors displayed multiple signs of elevated TCR signaling, a sizeable fraction of them escaped deletion to selectively engage in unconventional iIEL differentiation. Conversely, TCRs cloned from DP(lo)CD69(hi)PD-1(hi) thymocytes, a population enriched in autoreactive thymocytes, selectively gave rise to unconventional iIELs upon transgenic expression. Thus, the unconventional iIEL precursor overlaps with the DP(lo) population undergoing negative selection, indicating that, concomitant with the downregulation of both CD4 and CD8 coreceptors, a balance between apoptosis and survival signals results in outcomes as divergent as clonal deletion and differentiation to the unconventional iIEL lineage.

  View details for DOI 10.1016/j.immuni.2014.07.008

  View details for Web of Science ID 000341455300010

  View details for PubMedID 25131532

  View details for PubMedCentralID PMC4188477

• A negative feedback loop mediated by the Bcl6-cullin 3 complex limits Tfh cell differentiation JOURNAL OF EXPERIMENTAL MEDICINE Mathew, R., Mao, A., Chiang, A. H., Bertozzi-Villa, C., Bunker, J. J., Scanlon, S. T., McDonald, B. D., Constantinides, M. G., Hollister, K., Singer, J. D., Dent, A. L., Dinner, A. R., Bendelac, A. 2014; 211 (6): 1137-1151

  Abstract

  Induction of Bcl6 (B cell lymphoma 6) is essential for T follicular helper (Tfh) cell differentiation of antigen-stimulated CD4(+) T cells. Intriguingly, we found that Bcl6 was also highly and transiently expressed during the CD4(+)CD8(+) (double positive [DP]) stage of T cell development, in association with the E3 ligase cullin 3 (Cul3), a novel binding partner of Bcl6 which ubiquitinates histone proteins. DP stage-specific deletion of the E3 ligase Cul3, or of Bcl6, induced the derepression of the Bcl6 target genes Batf (basic leucine zipper transcription factor, ATF-like) and Bcl6, in part through epigenetic modifications of CD4(+) single-positive thymocytes. Although they maintained an apparently normal phenotype after emigration, they expressed increased amounts of Batf and Bcl6 at basal state and produced explosive and prolonged Tfh responses upon subsequent antigen encounter. Ablation of Cul3 in mature CD4(+) splenocytes also resulted in dramatically exaggerated Tfh responses. Thus, although previous studies have emphasized the essential role of Bcl6 in inducing Tfh responses, our findings reveal that Bcl6-Cul3 complexes also provide essential negative feedback regulation during both thymocyte development and T cell activation to restrain excessive Tfh responses.

  View details for DOI 10.1084/jem.20132267

  View details for Web of Science ID 000337364500011

  View details for PubMedID 24863065

  View details for PubMedCentralID PMC4042651

• N-5-Phosphonoacetyl-L-ornithine (PALO): A convenient synthesis and investigation of influence on regulation of amino acid biosynthetic genes in Saccharomyces cerevisiae BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Johnson, B., Steadman, R., Patefield, K. D., Bunker, J. J., Atkin, A. L., Dussault, P. 2011; 21 (8): 2351-2353

  Abstract

  A scalable four-step synthesis of the ornithine transcarbamylase inhibitor N(5)-phosphonoacetyl-l-ornithine (PALO) is achieved through boroxazolidinone protection of ornithine. Investigations in the model organism Saccharomyces cerevisiae found that, in contrast to a previous report, PALO did not influence growth rate or expression of genes involved in arginine metabolism.

  View details for DOI 10.1016/j.bmcl.2011.02.081

  View details for Web of Science ID 000289074700037

  View details for PubMedID 21421312