The best-understood mechanisms for achieving antibody self/nonself discrimination discard self-reactive antibodies before they can be tested for binding microbial antigens, potentially creating holes in the repertoire. Here we provide evidence for a complementary mechanism: retaining autoantibodies in the repertoire displayed as low levels of IgM and high IgD on anergic B cells, masking a varying proportion of autoantibody-binding sites with carbohydrates, and removing their self-reactivity by somatic hypermutation and selection in germinal centers (GCs). Analysis of human antibody sequences by deep sequencing of isotype-switched memory B cells or in IgG antibodies elicited against allogeneic RhD+ erythrocytes, vaccinia virus, rotavirus, or tetanus toxoid provides evidence for reactivation of anergic IgM low IgD+ IGHV4-34+ B cells and removal of cold agglutinin self-reactivity by hypermutation, often accompanied by mutations that inactivated an N-linked glycosylation sequon in complementarity-determining region 2 (CDR2). In a Hy10 antibody transgenic model where anergic B cells respond to a biophysically defined lysozyme epitope displayed on both foreign and self-antigens, cell transfers revealed that anergic IgM low IgD+ B cells form twice as many GC progeny as naïve IgM hi IgD+ counterparts. Their GC progeny were rapidly selected for CDR2 mutations that blocked 72% of antigen-binding sites with N-linked glycan, decreased affinity 100-fold, and then cleared the binding sites of blocking glycan. These results provide evidence for a mechanism to acquire self/non-self discrimination by somatic mutation away from self-reactivity, and reveal how varying the efficiency of N-glycosylation provides a mechanism to modulate antibody avidity.self-tolerance | affinity maturation | clonal selection | autoimmunity

Kräutler et al. show that differentiation of antibody-producing plasma cells from germinal center (GC) B cell precursors is initiated by direct contact with high-affinity antigen within the GC but completed by separate signals delivered by collaborating, GC-resident T follicular helper cells.

Memory B cells (MBCs) and plasma cells (PCs) constitute the two cellular outputs of germinal center (GC) responses that together facilitate long-term humoral immunity. Although expression of the transcription factor BLIMP-1 identifies cells undergoing PC differentiation, no such marker exists for cells committed to the MBC lineage. Here, we report that the chemokine receptor CCR6 uniquely marks MBC precursors in both mouse and human GCs. CCR6 GC B cells were highly enriched within the GC light zone (LZ), were the most quiescent of all GC B cells, exhibited a cell-surface phenotype and gene expression signature indicative of an MBC transition, and possessed the augmented response characteristics of MBCs. MBC precursors within the GC LZ predominantly possessed a low affinity for antigen but also included cells from within the high-affinity pool. These data indicate a fundamental dichotomy between the processes that drive MBC and PC differentiation during GC responses.

B helper follicular T (Tfh) cells are critical for long-term humoral immunity. However, it remains unclear how these cells are recruited and contribute to secondary immune responses. Here we show that primary Tfh cells segregate into follicular mantle (FM) and germinal center (GC) subpopulations that display distinct gene expression signatures. Restriction of the primary Tfh cell subpopulation in the GC was mediated by downregulation of chemotactic receptor EBI2. Following collapse of the GC, memory T cells persisted in the outer follicle where they scanned CD169(+) subcapsular sinus macrophages. Reactivation and intrafollicular expansion of these follicular memory T cells in the subcapsular region was followed by their extrafollicular dissemination via the lymphatic flow. These data suggest that Tfh cells integrate their antigen-experience history to focus T cell help within the GC during primary responses but act rapidly to provide systemic T cell help after re-exposure to the antigen.

Antibodies have the specificity to differentiate foreign antigens that mimic self antigens, but it remains unclear how such specificity is acquired. In a mouse model, we generated B cells displaying an antibody that cross-reacts with two related protein antigens expressed on self versus foreign cells. B cell anergy was imposed by self antigen but reversed upon challenge with high-density foreign antigen, leading to germinal center recruitment and antibody gene hypermutation. Single-cell analysis detected rapid selection for mutations that decrease self affinity and slower selection for epistatic mutations that specifically increase foreign affinity. Crystal structures revealed that these mutations exploited subtle topological differences to achieve 5000-fold preferential binding to foreign over self epitopes. Resolution of antigenic mimicry drove the optimal affinity maturation trajectory, highlighting the value of retaining self-reactive clones as substrates for protective antibody responses.