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Summary 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. [ABSTRACT FROM AUTHOR]

Summary 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. [ABSTRACT FROM AUTHOR]

The developmental pathway of intestinal TCRαβ+CD4- CD8β- intraepithelial lymphocytes (unconventional iIELs) has remained elusive. Bendelac and colleagues demonstrate that expression of unconventional iIEL TCRs in thymocytes causes negative selection and that unconventional iIELs are derived from rare escapees of cortical negative selection. [ABSTRACT FROM AUTHOR]

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)