Salmonella Infection Drives Promiscuous B Cell Activation Followed by Extrafollicular Affinity Maturation

SummaryThe B cell response to Salmonella typhimurium (STm) occurs massively at extrafollicular sites, without notable germinal centers (GCs). Little is known in terms of its specificity. To expand the knowledge of antigen targets, we screened plasmablast (PB)-derived monoclonal antibodies (mAbs) for Salmonella specificity, using ELISA, flow cytometry, and antigen microarray. Only a small fraction (0.5%–2%) of the response appeared to be Salmonella-specific. Yet, infection of mice with limited B cell receptor (BCR) repertoires impaired the response, suggesting that BCR specificity was important. We showed, using laser microdissection, that somatic hypermutation (SHM) occurred efficiently at extrafollicular sites leading to affinity maturation that in turn led to detectable STm Ag-binding. These results suggest a revised vision of how clonal selection and affinity maturation operate in response to Salmonella. Clonal selection initially is promiscuous, activating cells with virtually undetectable affinity, yet SHM and selection occur during the extrafollicular response yielding higher affinity, detectable antibodies

Compromised fidelity of B-cell tolerance checkpoints in AChR and MuSK myasthenia gravis

Myasthenia gravis () is an autoimmune condition in which neurotransmission is impaired by binding of autoantibodies to acetylcholine receptors (hR) or, in a minority of patients, to muscle specific kinase (Mu). There are differences in the dominant IgG subclass, pathogenic mechanisms, and treatment responses between the two subtypes (hR or Mu). The antibodies are thought to be T-cell dependent, but the mechanisms underlying their production are not well understood. One aspect not previously described is whether defects in central and peripheral tolerance checkpoints, which allow autoreactive B cells to accumulate in the naive repertoire, are found in both or either form of . An established set of assays that measure the frequency of both polyreactive and autoreactive B cell receptors () in naive populations was applied to specimens collected from patients with either hR or Mu and healthy controls. Radioimmuno- and cell-based assays were used to measure binding to hR and Mu. The frequency of polyreactive and autoreactive s (n = 262) was higher in both hR and Mu patients than in healthy controls. None of the -derived s bound hR or Mu. The results indicate that both these subtypes harbor defects in central and peripheral B cell tolerance checkpoints. Defective B cell tolerance may represent a fundamental contributor to autoimmunity in and is of particular importance when considering the durability of myasthenia gravis treatment strategies, particularly biologics that eliminate B cells

Salmonella Infection Drives Promiscuous B Cell Activation Followed by Extrafollicular Affinity Maturation.

The B cell response to Salmonella typhimurium (STm) occurs massively at extrafollicular sites, without notable germinal centers (GCs). Little is known in terms of its specificity. To expand the knowledge of antigen targets, we screened plasmablast (PB)-derived monoclonal antibodies (mAbs) for Salmonella specificity, using ELISA, flow cytometry, and antigen microarray. Only a small fraction (0.5%-2%) of the response appeared to be Salmonella-specific. Yet, infection of mice with limited B cell receptor (BCR) repertoires impaired the response, suggesting that BCR specificity was important. We showed, using laser microdissection, that somatic hypermutation (SHM) occurred efficiently at extrafollicular sites leading to affinity maturation that in turn led to detectable STm Ag-binding. These results suggest a revised vision of how clonal selection and affinity maturation operate in response to Salmonella. Clonal selection initially is promiscuous, activating cells with virtually undetectable affinity, yet SHM and selection occur during the extrafollicular response yielding higher affinity, detectable antibodies

Maturational characteristics of HIV-specific antibodies in viremic individuals

Despite the rare appearance of potent HIV-neutralizing mAbs in infected individuals requiring prolonged affinity maturation, little is known regarding this process in the majority of viremic individuals. HIV-infected individuals with chronic HIV viremia have elevated numbers of nonconventional tissue-like memory (TLM) B cells that predominate in blood over conventional resting memory (RM) B cells. Accordingly, we investigated affinity maturation in these 2 memory B cell populations. Analysis of IgG-expressing TLM B cells revealed a higher number of cell divisions compared with RM B cells; however, TLM B cells paradoxically displayed significantly lower frequencies of somatic hypermutation (SHM). To assess Ab reactivity in TLM and RM B cells, single-cell cloning was performed on HIV envelope CD4–binding site–sorted (CD4bs-sorted) B cells from 3 individuals with chronic HIV viremia. Several clonal families were present among the 127 cloned recombinant mAbs, with evidence of crosstalk between TLM and RM B cell populations that was largely restricted to non-VH4 families. Despite evidence of common origins, SHM frequencies were significantly decreased in TLM-derived mAbs compared with SHM frequencies in RM-derived mAbs. However, both cell populations had lower frequencies of SHMs than did broadly neutralizing CD4bs–specific mAbs. There was a significant correlation between SHM frequencies and the HIV-neutralizing capacities of the mAbs. Furthermore, HIV neutralization was significantly higher in the RM-derived mAbs compared with that seen in the TLM-derived mAbs, and both SHM frequencies and neutralizing capacity were lowest in TLM-derived mAbs with high polyreactivity. Thus, deficiencies in memory B cells that arise during chronic HIV viremia provide insight into the inadequacy of the Ab response in viremic individuals

PTPN22 inhibition resets defective human central B cell tolerance

The 1858T protein tyrosine phosphatase nonreceptor type 22 (PTPN22 T) allele is one of the main risk factors associated with many autoimmune diseases and correlates with a defective removal of developing autoreactive B cells in humans. To determine whether inhibiting PTPN22 favors the elimination of autoreactive B cells, we first demonstrated that the PTPN22 T allele interfered with the establishment of central B cell tolerance using NOD-scid-common γ chain knockout (NSG) mice engrafted with human hematopoietic stem cells expressing this allele. In contrast, the inhibition of either PTPN22 enzymatic activity or its expression by RNA interference restored defective central B cell tolerance in this model. Thus, PTPN22 blockade may represent a therapeutic strategy for the prevention or treatment of autoimmunity

Compromised fidelity of B-cell tolerance checkpoints in AChR and MuSK myasthenia gravis

Myasthenia gravis () is an autoimmune condition in which neurotransmission is impaired by binding of autoantibodies to acetylcholine receptors (hR) or, in a minority of patients, to muscle specific kinase (Mu). There are differences in the dominant IgG subclass, pathogenic mechanisms, and treatment responses between the two subtypes (hR or Mu). The antibodies are thought to be T-cell dependent, but the mechanisms underlying their production are not well understood. One aspect not previously described is whether defects in central and peripheral tolerance checkpoints, which allow autoreactive B cells to accumulate in the naive repertoire, are found in both or either form of . An established set of assays that measure the frequency of both polyreactive and autoreactive B cell receptors () in naive populations was applied to specimens collected from patients with either hR or Mu and healthy controls. Radioimmuno- and cell-based assays were used to measure binding to hR and Mu. The frequency of polyreactive and autoreactive s (n = 262) was higher in both hR and Mu patients than in healthy controls. None of the -derived s bound hR or Mu. The results indicate that both these subtypes harbor defects in central and peripheral B cell tolerance checkpoints. Defective B cell tolerance may represent a fundamental contributor to autoimmunity in and is of particular importance when considering the durability of myasthenia gravis treatment strategies, particularly biologics that eliminate B cells

Activation-Induced Cytidine Deaminase Expression in Human B Cell Precursors Is Essential for Central B Cell Tolerance

SummaryActivation-induced cytidine deaminase (AID), the enzyme-mediating class-switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes, is essential for the removal of developing autoreactive B cells. How AID mediates central B cell tolerance remains unknown. We report that AID enzymes were produced in a discrete population of immature B cells that expressed recombination-activating gene 2 (RAG2), suggesting that they undergo secondary recombination to edit autoreactive antibodies. However, most AID+ immature B cells lacked anti-apoptotic MCL-1 and were deleted by apoptosis. AID inhibition using lentiviral-encoded short hairpin (sh)RNA in B cells developing in humanized mice resulted in a failure to remove autoreactive clones. Hence, B cell intrinsic AID expression mediates central B cell tolerance potentially through its RAG-coupled genotoxic activity in self-reactive immature B cells