Lymphoma Driver Mutations in the Pathogenic Evolution of an Iconic Human Autoantibody.

Pathogenic autoantibodies arise in many autoimmune diseases, but it is not understood how the cells making them evade immune checkpoints. Here, single-cell multi-omics analysis demonstrates a shared mechanism with lymphoid malignancy in the formation of public rheumatoid factor autoantibodies responsible for mixed cryoglobulinemic vasculitis. By combining single-cell DNA and RNA sequencing with serum antibody peptide sequencing and antibody synthesis, rare circulating B lymphocytes making pathogenic autoantibodies were found to comprise clonal trees accumulating mutations. Lymphoma driver mutations in genes regulating B cell proliferation and V(D)J mutation (CARD11, TNFAIP3, CCND3, ID3, BTG2, and KLHL6) were present in rogue B cells producing the pathogenic autoantibody. Antibody V(D)J mutations conferred pathogenicity by causing the antigen-bound autoantibodies to undergo phase transition to insoluble aggregates at lower temperatures. These results reveal a pre-neoplastic stage in human lymphomagenesis and a cascade of somatic mutations leading to an iconic pathogenic autoantibody.

Denisovan, modern human and mouse TNFAIP3 alleles tune A20 phosphorylation and immunity.

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.

Phosphorylation and linear ubiquitin direct A20 inhibition of inflammation.

Inactivation of the TNFAIP3 gene, encoding the A20 protein, is associated with critical inflammatory diseases including multiple sclerosis, rheumatoid arthritis and Crohn's disease. However, the role of A20 in attenuating inflammatory signalling is unclear owing to paradoxical in vitro and in vivo findings. Here we utilize genetically engineered mice bearing mutations in the A20 ovarian tumour (OTU)-type deubiquitinase domain or in the zinc finger-4 (ZnF4) ubiquitin-binding motif to investigate these discrepancies. We find that phosphorylation of A20 promotes cleavage of Lys63-linked polyubiquitin chains by the OTU domain and enhances ZnF4-mediated substrate ubiquitination. Additionally, levels of linear ubiquitination dictate whether A20-deficient cells die in response to tumour necrosis factor. Mechanistically, linear ubiquitin chains preserve the architecture of the TNFR1 signalling complex by blocking A20-mediated disassembly of Lys63-linked polyubiquitin scaffolds. Collectively, our studies reveal molecular mechanisms whereby A20 deubiquitinase activity and ubiquitin binding, linear ubiquitination, and cellular kinases cooperate to regulate inflammation and cell death.

Systems-guided forward genetic screen reveals a critical role of the replication stress response protein ETAA1 in T cell clonal expansion.

T-cell immunity requires extremely rapid clonal proliferation of rare, antigen-specific T lymphocytes to form effector cells. Here we identify a critical role for ETAA1 in this process by surveying random germ line mutations in mice using exome sequencing and bioinformatic annotation to prioritize mutations in genes of unknown function with potential effects on the immune system, followed by breeding to homozygosity and testing for immune system phenotypes. Effector CD8+ and CD4+ T-cell formation following immunization, lymphocytic choriomeningitis virus (LCMV) infection, or herpes simplex virus 1 (HSV1) infection was profoundly decreased despite normal immune cell development in adult mice homozygous for two different Etaa1 mutations: an exon 2 skipping allele that deletes Gly78-Leu119, and a Cys166Stop truncating allele that eliminates most of the 877-aa protein. ETAA1 deficiency decreased clonal expansion cell autonomously within the responding T cells, causing no decrease in their division rate but increasing TP53-induced mRNAs and phosphorylation of H2AX, a marker of DNA replication stress induced by the ATM and ATR kinases. Homozygous ETAA1-deficient adult mice were otherwise normal, healthy, and fertile, although slightly smaller, and homozygotes were born at lower frequency than expected, consistent with partial lethality after embryonic day 12. Taken together with recently reported evidence in human cancer cell lines that ETAA1 activates ATR kinase through an exon 2-encoded domain, these findings reveal a surprisingly specific requirement for this ATR activator in adult mice restricted to rapidly dividing effector T cells. This specific requirement may provide new ways to suppress pathological T-cell responses in transplantation or autoimmunity.

CARD11 is dispensable for homeostatic responses and suppressive activity of peripherally induced FOXP3+ regulatory T cells.

FOXP3+ regulatory T (Treg) cells are essential for immunological tolerance and immune homeostasis. Despite a great deal of interest in modulating their number and function for the treatment of autoimmune disease or cancer, the precise mechanisms that control the homeostasis of Treg cells remain unclear. We report a new ENU-induced mutant mouse, lack of costimulation (loco), with atopic dermatitis and Treg cell deficiency typical of Card11 loss-of-function mutants. Three distinct single nucleotide variants were found in the Card11 introns 2, 10 and 20 that cause the loss of CARD11 expression in these mutant mice. These mutations caused the loss of thymic-derived, Neuropilin-1+ (NRP1+ ) Treg cells in neonatal and adult loco mice; however, residual peripherally induced NRP1- Treg cells remained. These peripherally generated Treg cells could be expanded in vivo by the administration of IL-2:anti-IL-2 complexes, indicating that this key homeostatic signaling axis remained intact in CARD11-deficient Treg cells. Furthermore, these expanded Treg cells could mediate near-normal suppression of activated, conventional CD4+ T cells, suggesting that CARD11 is dispensable for Treg cell function. In addition to shedding light on the requirements for CARD11 in Treg cell homeostasis and function, these data reveal novel noncoding Card11 loss-of-function mutations that impair the expression of this critical immune-regulatory protein.

Inhibiting TLR9 and other UNC93B1-dependent TLRs paradoxically increases accumulation of MYD88L265P plasmablasts in vivo.

The MYD88(L265P) mutation is found in 2% to 10% of chronic lymphocytic leukemia, 29% of activated B-cell type diffuse large B-cell lymphoma and 90% of Waldenström macroglobulinemia, making it conceptually attractive to treat these malignancies with inhibitors of endosomal Toll-like receptors (TLR9, TLR7) that activate MYD88. Here we show that genetic inhibition of endosomal TLRs has the opposite effect on accumulation of MYD88(L265P) B cells in vitro and in vivo. Activated mature B cells from wild-type, Unc93b1(3d/3d)-mutant, or Tlr9-deficient mice were transduced with retrovirus encoding MYD88(L265P) and analyzed either in vitro or after transplantation into Rag1(-/-) recipient mice. Unc93b1(3d/3d) mutation, which blocks TLR9 and TLR7 signaling, or Tlr9 deficiency suppressed MYD88(L265P) B-cell growth in vitro but paradoxically increased in vivo accumulation of MYD88(L265P) B cells as CD19(low) plasmablasts by 10- to 100-fold. These results reveal an unexpected, powerful inhibitory effect of TLR9 on MYD88(L265P) B-cell proliferation and differentiation that appears independent of TLR7, and they provide a preclinical indicator for caution in clinical trials of TLR7/9 inhibitors for MYD88(L265P) B-cell malignancies.

Omenn syndrome associated with a functional reversion due to a somatic second-site mutation in CARD11 deficiency.

Omenn syndrome (OS) is a severe immunodeficiency associated with erythroderma, lymphoproliferation, elevated IgE, and hyperactive oligoclonal T cells. A restricted T-cell repertoire caused by defective thymic T-cell development and selection, lymphopenia with homeostatic proliferation, and lack of regulatory T cells are considered key factors in OS pathogenesis. We report 2 siblings presenting with cytomegalovirus (CMV) and Pneumocystis jirovecii infections and recurrent sepsis; one developed all clinical features of OS. Both carried homozygous germline mutations in CARD11 (p.Cys150*), impairing NF-κB signaling and IL-2 production. A somatic second-site mutation reverting the stop codon to a missense mutation (p.Cys150Leu) was detected in tissue-infiltrating T cells of the OS patient. Expression of p.Cys150Leu in CARD11-deficient T cells largely reconstituted NF-κB signaling. The reversion likely occurred in a prethymic T-cell precursor, leading to a chimeric T-cell repertoire. We speculate that in our patient the functional advantage of the revertant T cells in the context of persistent CMV infection, combined with lack of regulatory T cells, may have been sufficient to favor OS. This first observation of OS in a patient with a T-cell activation defect suggests that severely defective T-cell development or homeostatic proliferation in a lymphopenic environment are not required for this severe immunopathology.

Ndfip1 mediates peripheral tolerance to self and exogenous antigen by inducing cell cycle exit in responding CD4+ T cells.

The NDFIP1 (neural precursor cell expressed, developmentally down-regulated protein 4 family-interacting protein 1) adapter for the ubiquitin ligase ITCH is genetically linked to human allergic and autoimmune disease, but the cellular mechanism by which these proteins enable foreign and self-antigens to be tolerated is unresolved. Here, we use two unique mouse strains--an Ndfip1-YFP reporter and an Ndfip1-deficient strain--to show that Ndfip1 is progressively induced during T-cell differentiation and activation in vivo and that its deficiency causes a cell-autonomous, Forkhead box P3-independent failure of peripheral CD4(+) T-cell tolerance to self and exogenous antigen. In small cohorts of antigen-specific CD4(+) cells responding in vivo, Ndfip1 was necessary for tolerogen-reactive T cells to exit cell cycle after one to five divisions and to abort Th2 effector differentiation, defining a step in peripheral tolerance that provides insights into the phenomenon of T-cell anergy in vivo and is distinct from the better understood process of Bcl2-interacting mediator of cell death-mediated apoptosis. Ndfip1 deficiency precipitated autoimmune pancreatic destruction and diabetes; however, this depended on a further accumulation of nontolerant anti-self T cells from strong stimulation by exogenous tolerogen. These findings illuminate a peripheral tolerance checkpoint that aborts T-cell clonal expansion against allergens and autoantigens and demonstrate how hypersensitive responses to environmental antigens may trigger autoimmunity.

Redemption of autoantibodies on anergic B cells by variable-region glycosylation and mutation away from self-reactivity.

The best-understood mechanisms for achieving antibody self/non-self 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.

Enhancement and suppression of signaling by the conserved tail of IgG memory-type B cell antigen receptors.

Immunological memory is characterized by heightened immunoglobulin (Ig) G antibody production caused in part by enhanced plasma cell formation conferred by conserved transmembrane and cytoplasmic segments in isotype-switched IgG B cell receptors. We tested the hypothesis that the IgG tail enhances intracellular B cell antigen receptor (BCR) signaling responses to antigen by analyzing B cells from Ig transgenic mice with IgM receptors or chimeric IgMG receptors containing the IgG tail segment. The IgG tail segment enhanced intracellular calcium responses but not tyrosine or extracellular signal-related kinase (ERK) phosphorylation. Biochemical analysis and crosses to CD22-deficient mice established that IgG tail enhancement of calcium and antibody responses, as well as marginal zone B cell formation, was not due to diminished CD22 phosphorylation or inhibitory function. Microarray profiling showed no evidence for enhanced signaling by the IgG tail for calcium/calcineurin, ERK, or nuclear factor kappaB response genes and little evidence for any enhanced gene induction. Instead, almost half of the antigen-induced gene response in IgM B cells was diminished 50-90% by the IgG tail segment. These findings suggest a novel "less-is-more" hypothesis to explain how switching to IgG enhances B cell memory responses, whereby decreased BCR signaling to genes that oppose marginal zone and plasma cell differentiation enhances the formation of these key cell types.

Diffuse large B-cell lymphoma and red cell autoimmunity: clinical role and pathogenesis.

Diffuse large B-cell lymphoma (DLBCL) is the most common form of B-cell non-Hodgkin lymphoma (B-NHL) with significant morbidity and mortality despite advancements in treatment. Lymphoma and autoimmune disease both result from breakdowns in normal cell regulatory pathways, and epidemiological studies have confirmed both that B-NHL is more likely to develop in the setting of autoimmune diseases and vice versa. Red cell immunity, as evidenced by direct antiglobulin test (DAT) positivity, has been linked to DLBCL and more recently the pathogenic causes of this association have begun to be better understood using molecular techniques. This project aimed to explore the relationship between red cell autoimmunity and DLBCL. DAT positivity was more common in DLBCL as compared to healthy controls (20.4% vs 3.7%, p=0.0005). Univariate analysis found a non-significant trend towards poorer overall survival in the DAT positive (DAT+) compared to the DAT negative (DAT-) groups (p=0.087). High throughput sequencing was used to compare mutations in DLBCL from DAT+ and DAT- patients. The most frequently mutated genes in 15 patient samples were KMT2D (n=13), MYOM2 (n=9), EP300 (n=8), SPEN (n=7), and ADAMTSL3 (n=7), which were mutated in both DAT+ and DAT- groups. BIRC3 (n=3), FOXO1 (n=3) and CARD11 (n=2) were found to be mutated only in samples from the DAT+ group. These gene mutations may be involved in disease development and progression, and potentially represent targets for future therapy. The immunoglobulin genotype IGHV4-34 is seen more frequently in DLBCL clones than in normal B cells and has intrinsic autoreactivity to self-antigens on red cells, which is largely mediated by two motifs within the first framework region (FR1); Q6W7 and A24V25Y.26 These motifs form a hydrophobic patch which determines red cell antigen binding and are frequently mutated away from self-reactivity in normal B cells. If this does not occur this may provide constant B cell receptor signalling which encourages lymphoma development, a theory known as antigen driven lymphomagenesis. As with previous studies, IGHV4-34 was over-represented (15.6%) in our DLBCL cohort. Furthermore, of 6 IGHV4-34-expressing DLBCL samples five had unmutated hydrophobic patch mutations providing further evidence for antigen-driven lymphomagenesis. Mutation analysis of these five samples demonstrated high frequency of mutations in several genes, including CREBBP and NCOR2. Further research could explore if mutations in CREBBP and NCOR2 work in conjunction with the preserved QW and AVY motifs to promote lymphomagenesis in IGHV4-34-expressing B cells, and if so, could guide future targeted therapy.

CARD11 gain-of-function mutation drives cell-autonomous accumulation of PD-1+ ICOShigh activated T cells, T-follicular, T-regulatory and T-follicular regulatory cells.

Introduction: Germline CARD11 gain-of-function (GOF) mutations cause B cell Expansion with NF-κB and T cell Anergy (BENTA) disease, whilst somatic GOF CARD11 mutations recur in diffuse large B cell lymphoma (DLBCL) and in up to 30% of the peripheral T cell lymphomas (PTCL) adult T cell leukemia/lymphoma (ATL), cutaneous T cell lymphoma (CTCL) and Sezary Syndrome. Despite their frequent acquisition by PTCL, the T cell-intrinsic effects of CARD11 GOF mutations are poorly understood. Methods: Here, we studied B and T lymphocytes in mice with a germline Nethyl-N-nitrosourea (ENU)-induced Card11M365K mutation identical to a mutation identified in DLBCL and modifying a conserved region of the CARD11 coiled-coil domain recurrently mutated in DLBCL and PTCL. Results and discussion: Our results demonstrate that CARD11.M365K is a GOF protein that increases B and T lymphocyte activation and proliferation following antigen receptor stimulation. Germline Card11M365K mutation was insufficient alone to cause B or T-lymphoma, but increased accumulation of germinal center (GC) B cells in unimmunized and immunized mice. Card11M365K mutation caused cell-intrinsic over-accumulation of activated T cells, T regulatory (TREG), T follicular (TFH) and T follicular regulatory (TFR) cells expressing increased levels of ICOS, CTLA-4 and PD-1 checkpoint molecules. Our results reveal CARD11 as an important, cell-autonomous positive regulator of TFH, TREG and TFR cells. They highlight T cell-intrinsic effects of a GOF mutation in the CARD11 gene, which is recurrently mutated in T cell malignancies that are often aggressive and associated with variable clinical outcomes.

Synergistic cooperation and crosstalk between MYD88L265P and mutations that dysregulate CD79B and surface IgM.

CD79B and MYD88 mutations are frequently and simultaneously detected in B cell malignancies. It is not known if these mutations cooperate or how crosstalk occurs. Here we analyze the consequences of CD79B and MYD88L265P mutations individually and combined in normal activated mouse B lymphocytes. CD79B mutations alone increased surface IgM but did not enhance B cell survival, proliferation, or altered NF-κB responsive markers. Conversely, B cells expressing MYD88L265P decreased surface IgM coupled with accumulation of endoglycosidase H-sensitive IgM intracellularly, resembling the trafficking block in anergic B cells repeatedly stimulated by self-antigen. Mutation or overexpression of CD79B counteracted the effect of MYD88L265P In B cells chronically stimulated by self-antigen, CD79B and MYD88L265P mutations in combination, but not individually, blocked peripheral deletion and triggered differentiation into autoantibody secreting plasmablasts. These results reveal that CD79B and surface IgM constitute a rate-limiting checkpoint against B cell dysregulation by MYD88L265P and provide an explanation for the co-occurrence of MYD88 and CD79B mutations in lymphomas.

IgD attenuates the IgM-induced anergy response in transitional and mature B cells.

Self-tolerance by clonal anergy of B cells is marked by an increase in IgD and decrease in IgM antigen receptor surface expression, yet the function of IgD on anergic cells is obscure. Here we define the RNA landscape of the in vivo anergy response, comprising 220 induced sequences including a core set of 97. Failure to co-express IgD with IgM decreases overall expression of receptors for self-antigen, but paradoxically increases the core anergy response, exemplified by increased Sdc1 encoding the cell surface marker syndecan-1. IgD expressed on its own is nevertheless competent to induce calcium signalling and the core anergy mRNA response. Syndecan-1 induction correlates with reduction of surface IgM and is exaggerated without surface IgD in many transitional and mature B cells. These results show that IgD attenuates the response to self-antigen in anergic cells and promotes their accumulation. In this way, IgD minimizes tolerance-induced holes in the pre-immune antibody repertoire.

Toll-Like Receptors and Cancer: MYD88 Mutation and Inflammation.

Pattern recognition receptors (PRRs) expressed on immune cells are crucial for the early detection of invading pathogens, in initiating early innate immune response and in orchestrating the adaptive immune response. PRRs are activated by specific pathogen-associated molecular patterns that are present in pathogenic microbes or nucleic acids of viruses or bacteria. However, inappropriate activation of these PRRs, such as the Toll-like receptors (TLRs), due to genetic lesions or chronic inflammation has been demonstrated to be a major cause of many hematological malignancies. Gain-of-function mutations in the TLR adaptor protein MYD88 found in 39% of the activated B cell type of diffuse large B cell lymphomas and almost 100% of Waldenström's macroglobulinemia further highlight the involvement of TLRs in these malignancies. MYD88 mutations result in the chronic activation of TLR signaling pathways, thus the constitutive activation of the transcription factor NFκB to promote cell survival and proliferation. These recent insights into TLR pathway driven malignancies warrant the need for a better understanding of TLRs in cancers and the development of novel anti-cancer therapies targeting TLRs. This review focuses on TLR function and signaling in normal or inflammatory conditions, and how mutations can hijack the TLR signaling pathways to give rise to cancer. Finally, we discuss how potential therapeutic agents could be used to restore normal responses to TLRs and have long lasting anti-tumor effects.

Consequences of the recurrent MYD88(L265P) somatic mutation for B cell tolerance.

MYD88(L265P) has recently been discovered as an extraordinarily frequent somatic mutation in benign monoclonal IgM gammopathy, Waldenström's macroglobulinemia, and diffuse large B cell lymphoma. In this study, we analyze the consequences for antigen-activated primary B cells of acquiring MYD88(L265P). The mutation induced rapid B cell division in the absence of exogenous TLR ligands and was inhibited by Unc93b1(3d) mutation and chloroquine or TLR9 deficiency, indicating continued dependence on upstream TLR9 activation. Proliferation and NF-κB activation induced by MYD88(L265P) were nevertheless rapidly countered by the induction of TNFAIP3, an NF-κB inhibitor frequently inactivated in MYD88(L265P)-bearing lymphomas, and extinguished by Bim-dependent apoptosis. MYD88(L265P) caused self-reactive B cells to accumulate in vivo only when apoptosis was opposed by Bcl2 overexpression. These results reveal checkpoints that fortify TLR responses against aberrant B cell proliferation in response to ubiquitous TLR and BCR self-ligands and suggest that tolerance failure requires the accumulation of multiple somatic mutations.

B cell survival, surface BCR and BAFFR expression, CD74 metabolism, and CD8- dendritic cells require the intramembrane endopeptidase SPPL2A.

Druggable proteins required for B lymphocyte survival and immune responses are an emerging source of new treatments for autoimmunity and lymphoid malignancy. In this study, we show that mice with an inactivating mutation in the intramembrane protease signal peptide peptidase-like 2A (SPPL2A) unexpectedly exhibit profound humoral immunodeficiency and lack mature B cell subsets, mirroring deficiency of the cytokine B cell-activating factor (BAFF). Accumulation of Sppl2a-deficient B cells was rescued by overexpression of the BAFF-induced survival protein B cell lymphoma 2 (BCL2) but not BAFF and was distinguished by low surface BAFF receptor and IgM and IgD B cell receptors. CD8-negative dendritic cells were also greatly decreased. SPPL2A deficiency blocked the proteolytic processing of CD74 MHC II invariant chain in both cell types, causing dramatic build-up of the p8 product of Cathepsin S and interfering with earlier steps in CD74 endosomal retention and processing. The findings illuminate an important role for the final step in the CD74-MHC II pathway and a new target for protease inhibitor treatment of B cell diseases.

Human lymphoma mutations reveal CARD11 as the switch between self-antigen-induced B cell death or proliferation and autoantibody production.

Self-tolerance and immunity are actively acquired in parallel through a poorly understood ability of antigen receptors to switch between signaling death or proliferation of antigen-binding lymphocytes in different contexts. It is not known whether this tolerance-immunity switch requires global rewiring of the signaling apparatus or if it can arise from a single molecular change. By introducing individual CARD11 mutations found in human lymphomas into antigen-activated mature B lymphocytes in mice, we find here that lymphoma-derived CARD11 mutations switch the effect of self-antigen from inducing B cell death into T cell-independent proliferation, Blimp1-mediated plasmablast differentiation, and autoantibody secretion. Our findings demonstrate that regulation of CARD11 signaling is a critical switch governing the decision between death and proliferation in antigen-stimulated mature B cells and that mutations in this switch represent a powerful initiator for aberrant B cell responses in vivo.