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Maturation of antibody responses entails somatic hypermutation (SHM), class-switch DNA recombination (CSR), plasma cell differentiation, and generation of memory B cells, and it is thought to require T cell help. We showed that B cell Toll-like receptor 4 (TLR4)–B cell receptor (BCR) (receptor for antigen) coengagement by 4-hydroxy-3-nitrophenyl acetyl (NP)–lipopolysaccharide (LPS) ( Escherichia coli lipid A polysaccharide O-antigen) or TLR5-BCR coengagement by Salmonella flagellin induces mature antibody responses to NP and flagellin in Tcr β −/− Tcr δ −/− and NSG/B mice. TLR-BCR coengagement required linkage of TLR and BCR ligands, “linked coengagement.” This induced B cell CSR/SHM, germinal center–like differentiation, clonal expansion, intraconal diversification, plasma cell differentiation, and an anamnestic antibody response. In Tcr β −/− Tcr δ −/− mice, linked coengagement of TLR4-BCR by LPS or TLR5-BCR by flagellin induced protective antibodies against E. coli or Salmonella Typhimurium. Our findings unveiled a critical role of B cell TLRs in inducing neutralizing antibody responses, including those to microbial pathogens, without T cell help.

Supplementary Fig. S2 from Trabectedin (ET-743) promotes differentiation in myxoid liposarcoma tumors

Differentiation is a complex set of events that can be blocked by rearrangements of regulatory genes producing fusion proteins with altered properties. In the case of myxoid liposarcoma (MLS) tumors, the causative abnormality is a fusion between the CHOP transcription factor and the FUS or EWS genes. CHOP belongs to and is a negative regulator of the large CAAT/enhancer binding protein family whose α, β,and δ members are master genes of adipogenesis. Recent clinical data indicate a peculiar sensitivity of these tumors to the natural marine compound trabectedin. One hypothesis is that the activity of trabectedin is related to the inactivation of the FUS-CHOP oncogene. We find that trabectedin causes detachment of the FUS-CHOP chimera from targeted promoters. Reverse transcription-PCR and chromatin immunoprecipitation analysis in a MLS line and surgical specimens of MLS patients in vivo show activation of the CAAT/enhancer binding protein–mediated transcriptional program that leads to morphologic changes of terminal adipogenesis. The activity is observed in cells with type 1 but not type 8 fusions. Hence, the drug induces maturation of MLS lipoblasts in vivo by targeting the FUS-CHOP–mediated transcriptional block. These data provide a rationale for the specific activity of trabectedin and open the perspective of combinatorial treatments with drugs acting on lipogenic pathways. [Mol Cancer Ther 2009;8(2):449–57]

PDF file, 60KB, Progression-free (panel A) and overall (panel B) survival according to the mitosis count. HPF: high power field.

Supplementary Table S1 from Trabectedin (ET-743) promotes differentiation in myxoid liposarcoma tumors

Purpose: Platelet-derived growth factor receptor-alpha (PDGFRA) mutations are found in approximately 5% to 7% of advanced gastrointestinal stromal tumors (GIST). We sought to extensively assess the activity of imatinib in this subgroup.Experimental Design: We conducted an international survey among GIST referral centers to collect clinical data on patients with advanced PDGFRA-mutant GISTs treated with imatinib for advanced disease.Results: Fifty-eight patients were included, 34 were male (59%), and median age at treatment initiation was 61 (range, 19–83) years. The primary tumor was gastric in 40 cases (69%). Thirty-two patients (55%) had PDGFRA-D842V substitutions whereas 17 (29%) had mutations affecting other codons of exon 18, and nine patients (16%) had mutation in other exons. Fifty-seven patients were evaluable for response, two (4%) had a complete response, eight (14%) had a partial response, and 23 (40%) had stable disease. None of 31 evaluable patients with D842V substitution had a response, whereas 21 of 31 (68%) had progression as their best response. Median progression-free survival was 2.8 [95% confidence interval (CI), 2.6–3.2] months for patients with D842V substitution and 28.5 months (95% CI, 5.4–51.6) for patients with other PDGFRA mutations. With 46 months of follow-up, median overall survival was 14.7 months for patients with D842V substitutions and was not reached for patients with non-D842V mutations.Conclusions: This study is the largest reported to date on patients with advanced PDGFRA-mutant GISTs treated with imatinib. Our data confirm that imatinib has little efficacy in the subgroup of patients with D842V substitution in exon 18, whereas other mutations appear to be sensitive to imatinib. Clin Cancer Res; 18(16); 4458–64. ©2012 AACR.

The aim of the present study was to explore (1) informed consent (IC) representations, level of understanding, needs, and factors that influence the willingness of cancer patients to participate in randomized controlled trials (RCTs) (phase I) and (2) representations, experiences, and critical issues of physicians involved in the same process (phase II). Semi-structured interviews were conducted with 20 cancer patients who had been asked to enroll in a phase II/III RCT (phase I). Two focus groups were conducted with 13 physicians enrolled in the same process (phase II). The content produced was analyzed through a thematic analysis. The themes that emerged in the first phase I were grouped into six categories: IC representation, randomization, experimentation, meeting with the physician, factors that influence the willingness to participate, and trial participants’ needs. The themes emerged in the phase II were grouped into four: IC representation, critical issues of the IC, relationship, and recruitment of trial participants. Each theme is articulated into sub-themes and deeply discussed. This study highlights (1) the gap between what is ethically demanded in a RCT consultation and the reality of the situation and (2) the difference in perceptions between patients and physicians with reference to the meaning, objectives, and level of understanding of IC.

Short-chain fatty acids (SCFAs) butyrate and propionate are metabolites from dietary fiber's fermentation by gut microbiota that can affect differentiation or functions of T cells, macrophages and dendritic cells. We show here that at low doses these SCFAs directly impact B cell intrinsic functions to moderately enhance class-switch DNA recombination (CSR), while decreasing at higher doses over a broad physiological range, AID and Blimp1 expression, CSR, somatic hypermutation and plasma cell differentiation. In human and mouse B cells, butyrate and propionate decrease B cell Aicda and Prdm1 by upregulating select miRNAs that target Aicda and Prdm1 mRNA-3′UTRs through inhibition of histone deacetylation (HDAC) of those miRNA host genes. By acting as HDAC inhibitors, not as energy substrates or through GPR-engagement signaling in these B cell-intrinsic processes, these SCFAs impair intestinal and systemic T-dependent and T-independent antibody responses. Their epigenetic impact on B cells extends to inhibition of autoantibody production and autoimmunity in mouse lupus models., Dietary fiber-derived short-chain fatty acids (SCFA) act as histone deacetylase (HDAC) inhibitors on Tregs and innate immune cells, promoting immune tolerance by altering gene expression. Here the authors show that SCFA HDAC inhibitor activity impacts B cell differentiation, antibody responses and antibody-driven autoimmunity.

Artículo escrito por un elevado número de autores, solo se referencian el que aparece en primer lugar, el nombre del grupo de colaboración, si le hubiere, y los autores pertenecientes a la UAM, Pazopanib was assessed prospectively in the GEIS-32 phase II study (NCT02066285) on advanced solitary fibrous tumour (SFT), resulting in a longer progression-free survival (PFS) and overall survival (OS) compared with historical controls treated with chemotherapy. A retrospective analysis of peripheral inflammatory indexes in patients enrolled into GEIS-32 was performed to evaluate their prognostic and predictive value. Patients received pazopanib 800 mg/day as the first antiangiogenic line. The impacts of baseline neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), and red cell distribution width (RDW) on PFS, OS, and Choi response were evaluated by univariate and multivariate analysis. Metastasis-free interval (MFI), mitotic count, and ECOG were also included as potential prognostic factors. Sixty-seven SFT patients, enrolled in this study, showed a median age of 63 years and a female/male distribution of 57/43. The median follow-up from treatment initiation was 16.8 months. High baseline NLR, PLR, and standardised RDW were significantly associated with worse PFS and OS. NLR, RDW, MFI, and mitotic count were independent variables for PFS, while RDW and ECOG were independent for OS. Further, NLR and mitotic count were independent factors for Choi response. High baseline NLR and RDW values were independent prognostic biomarkers for worse outcome in advanced SFT patients treated with pazopanib

IgA is the predominant antibody isotype at intestinal mucosae, where it plays a critical role in homeostasis and provides a first line of immune protection. Dysregulation of IgA production, however, can contribute to immunopathology, particularly in kidneys in which IgA deposition can cause nephropathy. Class-switch DNA recombination (CSR) to IgA is directed by TGF-β signaling, which activates Smad2 and Smad3. Activated Smad2/Smad3 dimers are recruited together with Smad4 to theIgHα locusIαpromoter to activate germline Iα-Cα transcription, the first step in the unfolding of CSR to IgA. Epigenetic factors, such as non-coding RNAs, particularly microRNAs, have been shown to regulate T cells, dendritic cells and other immune elements, as well as modulate the antibody response, including CSR, in a B cell-intrinsic fashion. Here we showed that the most abundant miRNA in resting B cells, miR-146a targetsSmad2, Smad3andSmad4mRNA 3’UTRs and keeps CSR to IgA in check in resting B cells. Indeed, enforced miR-146a expression in B cells aborted induction of IgA CSR by decreasing Smad levels. By contrast, upon induction of CSR to IgA, as directed by TGF-β, B cells downregulated miR-146a, thereby reversing the silencing ofSmad2, Smad3andSmad4, which, once expressed, led to recruitment of Smad2, Smad3 and Smad4 to the Iα promoter for activation of germlineIα-Cαtranscription. Deletion of miR-146a inmiR-146a–/–mice significantly increased circulating levels of steady state total IgA, but not IgM, IgG or IgE, and heightened the specific IgA antibody response to OVA. InmiR-146a–/–mice, the elevated systemic IgA levels were associated with increased IgA+B cells in intestinal mucosae, increased amounts of fecal free and bacteria-bound IgA as well as kidney IgA deposition, a hallmark of IgA nephropathy. Increased germlineIα-Cαtranscription and CSR to IgA inmiR-146a–/–B cellsin vitroproved that miR-146a-induced Smad2, Smad3 and Smad4 repression is B cell intrinsic. The B cell-intrinsic role of miR-146a in the modulation of CSR to IgA was formally confirmedin vivoby construction and OVA immunization of mixed bone marrowμMT/miR-146a–/–chimeric mice. Thus, by inhibitingSmad2,Smad3andSmad4expression, miR-146a plays an important and B cell intrinsic role in modulation of CSR to IgA and the IgA antibody response.

While the biology of IgD begins to be better understood, the mechanism of expression of this phylogenetically old and highly conserved Ig remains unknown. In B cells, IgD is expressed together with IgM as transmembrane receptor for antigen through alternative splicing of long primaryVHDJH-Cμ-s-m-Cδ-s-mRNAs, which also underpin secreted (s)IgD. IgD is also expressed through class switch DNA recombination (CSR), as initiated by AID-mediated double-strand DNA breaks (DSBs) in Sμ and σδ, and resolution of such DSBs by a still unknown mechanism. This synapses Sμ with σδ region DSB resected ends leading to insertion of extensive S-S junction microhomologies, unlike Ku70/Ku86-dependent NHEJ which resolves DSB blunt ends in CSR to IgG, IgA and IgE with little or no microhomologies. Our previous demonstration of a novel role of Rad52 in a Ku70/Ku86-independent “short-range” microhomology-mediated synapsis of intra-Sμ region DSBs led us to hypothesize that this homologous recombination DNA annealing factor is also involved in short-range microhomology-mediated alternative endjoining (A-EJ) recombination of Sμ with σδ. We found that induction of IgD CSR by selected stimuli downregulated Zfp318 (the suppressor ofCμ-s-mtranscription termination), promoted Rad52 phosphorylation and Rad52 recruitment to Sμ and σδ, leading to Sμ-σδ recombination with extensive microhomologies,VHDJH-Cδstranscription and sustained IgD secretion. Rad52 ablation in mouseRad52−/−B cells aborted IgD CSRin vitroandin vivoand dampened the specific IgD antibody response to OVA. Further, Rad52 knockdown in human B cells virtually abrogated IgD CSR. Finally, Rad52 phosphorylation was associated with high levels of IgD CSR and anti-nuclear IgD autoantibodies in lupus-prone mice and lupus patients. Thus, Rad52 effects CSR to IgD through microhomology-mediated A-EJ and in concert with Zfp318 modulation. This is a previously unrecognized, critical and dedicated role of Rad52 in mammalian DNA repair that provides a mechanistic underpinning to CSR A-EJ.

Memory B cells (MBCs) are long-lived and produce high-affinity, generally, class-switched antibodies. Here, we use a multiparameter approach involving CD27 to segregate naïve B cells (NBC), IgD+ unswitched (unsw)MBCs and IgG+ or IgA+ class-switched (sw)MBCs from humans of different age, sex and race. Conserved antibody variable gene expression indicates that MBCs emerge through unbiased selection from NBCs. Integrative analyses of mRNAs, miRNAs, lncRNAs, chromatin accessibility and cis-regulatory elements uncover a core mRNA-ncRNA transcriptional signature shared by IgG+ and IgA+ swMBCs and distinct from NBCs, while unswMBCs display a transitional transcriptome. Some swMBC transcriptional signature loci are accessible but not expressed in NBCs. Profiling miRNAs reveals downregulated MIR181, and concomitantly upregulated MIR181 target genes such as RASSF6, TOX, TRERF1, TRPV3 and RORα, in swMBCs. Finally, lncRNAs differentially expressed in swMBCs cluster proximal to the IgH chain locus on chromosome 14. Our findings thus provide new insights into MBC transcriptional programs and epigenetic regulation, opening new investigative avenues on these critical cell elements in human health and disease., Human memory B cells differentiate from naïve B cells and can express different immunoglobulin (Ig) isotypes resulted from class-switch recombination. Here the authors describe, using transcriptional and epigenetic data from human memory B cells and integrated multi-omics analyses, the differentiation regulation and trajectory of IgG+, IgA+ and IgD+ memory B cells.

B cell differentiation driven by microbial antigens leads to production of anti-microbial antibodies, such as those neutralizing viruses, bacteria or bacterial toxin, that are class-switched (IgG and IgA) and somatically hypermutated (maturation of the antibody response) as well as secreted in large volume by plasma cells. Similar features characterize pathogenic antibodies to self-antigens in autoimmunity, reflecting the critical role of class switch DNA recombination (CSR), somatic hypermutation (SHM) and plasma cell differentiation in the generation of antibodies to not only foreign antigens but also self-antigens (autoantibodies). Central to CSR/SHM and plasma cell differentiation are AID, a potent DNA cytidine deaminase encoded by Aicda, and Blimp-1, a transcription factor encoded by Prdm1. B cell-intrinsic expression of Aicda and Prdm1 is regulated by epigenetic elements and processes, including DNA methylation, histone posttranslational modifications and non-coding RNAs, particularly miRNAs. Here, we will discuss: B cell-intrinsic epigenetic processes that regulate antibody and autoantibody responses; how epigenetic dysregulation alter CSR/SHM and plasma cell differentiation, thereby leading to autoantibody responses, as in systemic lupus; and, how these can be modulated by nutrients, metabolites and hormones through changes in B cell-intrinsic epigenetic mechanisms, which can provide to be therapeutic targets in autoimmunity.

B cells are exposed to innate and T cell stimuli during the antibody response, although whether and how they functionally integrate such signals are unclear. Here we have identified IL-27 as the cytokine specifically produced by murine B cells upon sequential stimulation by TLR ligands and then CD154 and IL-21, the hallmark factors of T follicular helper cells, and during the T-dependent antibody response to a conjugated hapten or virus infection. B-cell Il27p28 transcription is concomitant with increased locus accessibility and depends on newly induced BATF3 transcription factor. IL-27-producing B cells are inefficient in antibody secretion, but cooperate with IFNγ to promote proliferation, survival, class-switching and plasma cell differentiation of CD40-activated B cells, leading to optimal IgG2a and IgG1 responses. Overall, IL-27-producing B cells function as “helper” B cells that integrate the innate and adaptive stages of the antibody response.One-sentence summaryB cells integrate innate TLR and adaptive CD40 signals to induce BATF3 transcription factor for production of IL-27, which together with INFg optimizes antibody responses.

Estrogen contributes to females' strong antibody response to microbial vaccines and proneness to autoimmunity, particularly antibody-mediated systemic autoimmunity, in females. We have hypothesized that this is due to estrogen-mediated potentiation of class switch DNA recombination (CSR) and somatic hypermutation (SHM). As we have shown, estrogen boosts AID expression, which is critical for both CSR and SHM, through upregulation of HoxC4, which together with NF-κB critically mediates Aicda (AID gene) promoter activation. We contend here that additional regulation of Aicda expression by estrogen occurs through epigenetic mechanisms. As we have shown, histone deacetylase inhibitors (HDIs) short-chain fatty acid (SCFA) butyrate and propionate as well as the pharmacologic HDI valproic acid upregulate miRNAs that silence AID expression, thereby modulating specific antibody responses in C57BL/6 mice and autoantibody responses in lupus-prone MRL/Fas lpr/lpr mice. Here, using constitutive knockout Esr1 -/- mice and B cells as well as conditional knockout Aicda cre/cre Esr1 flox/flox mice and B cells, we showed that the HDI-mediated downregulation of Aicda expression as well as the maturation of antibody and autoantibody responses is reversed by estrogen and enhanced by deletion of ERα or E2 inhibition. Estrogen's reversion of HDI-mediated inhibition of Aicda and CSR in antibody and autoantibody responses occurred through downregulation of B cell miR-26a, which, as we showed, targets Aicda mRNA 3'UTR. miR-26a was significantly upregulated by HDIs. Accordingly, enforced expression of miR-26a reduced Aicda expression and CSR, while miR-26a-sponges (competitive inhibitors of miR-26a) increased Aicda expression and CSR. Thus, our findings show that estrogen reverses the HDI-mediated downregulation of AID expression and CSR through selective modulation of miR-26a. They also provide mechanistic insights into the immunomodulatory activity of this hormone and a proof-of-principle for using combined ER inhibitor-HDI as a potential therapeutic approach.

How host immune elements, particularly B cells, are hijacked to promote breast cancer (BCa) tumor progression and acquisition of resistance to current endocrine therapies remain largely unclear. Prompted by our recent findings that B cells are a major source of cytokine IL-27 (the heterodimer of IL-27p28 and EBI3), which regulates many immune functions as both a pro-inflammatory and anti-inflammatory cytokine, as well as the high expression of IL-27 receptor genes in the breast, elevated circulating IL-27 in BCa patients and the association of increased IL-27p28 expression with reduced survival in BCa patients overall and ER+ BCa patients after endocrine therapies, here we hypothesize that B cells promote BCa tumor progression and drug resistance and do so by producing IL-27. In C57 mice transplanted with ERa+ E0771 syngeneic medullary breast adenocarcinoma cells, deficiency in B cells in mMT mice resulted in virtually abolished tumor growth, resulting from significantly high tumor cell death. Remarkably, in residual tumors developed in mMT mice after transplantation with a higher load of E0771 cells, B cells, which were absent in the blood and lymph nodes, were accumulated within tumors – at an even higher frequency than that in wildtype C57 mice – and expressed IL-27. Further, like class-switching B cells that respond to IL-27 produced by neighboring B cells to express an IFNg-induced gene signature in the secondary lymphoid organ during the antibody response, a distinct population of tumor-infiltrating B cells expressed high levels of PD-L1, which could be induced by IL-27 in many immune cell types, including B cells, and several cancer cell types. Finally, IL-27 promoted the growth MCF-7 human BCa cells in the presence of tamoxifen in vitro, consistent with a role of B cell-produced IL-27 in the acquisition of drug resistance by BCa. These, together with the delayed tumor growth in mixed bone marrow chimeric mice with B cell-specific deficiency in IL-27 production, outline a potential role of B cell-produced IL-27 in BCa progression and drug resistance through both BCa cell-intrinsic mechanisms and induction of PD-L1+ B regulatory cells that participate in the immune checkpoint in BCa. Citation Format: Yan H, Viswanadhapalli S, Fernandez M, Rivera C, Sareddy G, Casali P, Vadlamudi R. Role of B lymphocytes and B cell-produced IL-27 in breast cancer progression and drug resistance [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-15.

We describe a novel B cell–associated cytokine, encoded by an uncharacterized gene (C17orf99; chromosome 17 open reading frame 99), that is expressed in bone marrow and fetal liver and whose expression is also induced in peripheral B cells upon activation. C17orf99 is only present in mammalian genomes, and it encodes a small (∼27-kDa) secreted protein unrelated to other cytokine families, suggesting a function in mammalian immune responses. Accordingly, C17orf99 expression is induced in the mammary gland upon the onset of lactation, and a C17orf99−/− mouse exhibits reduced levels of IgA in the serum, gut, feces, and lactating mammary gland. C17orf99−/− mice have smaller and fewer Peyer’s patches and lower numbers of IgA-secreting cells. The microbiome of C17orf99−/− mice exhibits altered composition, likely a consequence of the reduced levels of IgA in the gut. Although naive B cells can express C17orf99 upon activation, their production increases following culture with various cytokines, including IL-4 and TGF-β1, suggesting that differentiation can result in the expansion of C17orf99-producing B cells during some immune responses. Taken together, these observations indicate that C17orf99 encodes a novel B cell–associated cytokine, which we have called IL-40, that plays an important role in humoral immune responses and may also play a role in B cell development. Importantly, IL-40 is also expressed by human activated B cells and by several human B cell lymphomas. The latter observations suggest that it may play a role in the pathogenesis of certain human diseases.

IgA is the predominant antibody isotype at mucosal surfaces. It plays a critical role in mucosal homeostasis and provides a first line of defense in immune protection. Class switch DNA recombination (CSR) to IgA is initiated by TGF-β, which induces Smad2, Smad3, Smad4 and Runx3 binding to the Iα promoter. This promotes germline Iα-Cα transcription, thereby increasing accessibility to the Igα locus. Epigenetic modifications, such as microRNAs, have been shown to modulate the antibody response, although the role of microRNAs in B cell differentiaton processes, such as guiding CSR to specific Ig isotypes, remains unexplored. miR146a has been shown to regulate T cell, dendritic cell and macrophage functions in innate and adaptive immune responses. Here we show that miR146a, which targets Smad2, Smad3 and Smad4 3′UTRs and is the most abundant miRNA in resting B cells, is significantly downregulated in B cells undergoing CSR to IgA. Deletion of miR146a, as in miR146a−/− B cells, frees Smad2, Smad3 and Smad4 from transcriptional repression, thereby increasing recruitment of these transcription factors to the Iα promoter. This results in increased germline Iα-Cα transcription and CSR to IgA without alterations to Aicda and Prdm1 expression. Accordingly, miR146a−/− mice displayed an elevated gut, systemic IgA responses and kidney IgA deposition, a hallmark of hyper IgA nephropathy. This was recapitulated in μMT/miR146a−/− mixed bone marrow chimera mice, further demonstrating the B cell-intrinsic role of miR146a in the modulation of CSR to IgA. Thus, by silencing Smad2, Smad3 and Smad4 transcription, miR-146a plays an important B cell intrinsic role in the modulation of the IgA response.

Somatic hypermutation (SHM) and class-switch DNA recombination (CSR) together with plasma cell and memory B cell differentiation are central to the maturation of the antibody response. Prompted by our finding that CSR and plasma cell differentiation can be induced in vitro by a TLR ligand together with BCR crosslinking, we hypothesized that TLR and BCR coengagement can induce mature antibody responses in the absence of T cells. To test this hypothesis, we injected Tcrβ−/−Tcrδ−/− mice, which are devoid of T cells, with NP-LPS or Salmonella Typhimurium flagellin, which coengages B cell TLR4 or TLR5 with BCR. These mice generated NP or flagellin-specific IgG class-switched antibodies and plasma cells. The IgG response to NP evoked formation of germinal center microclusters, a high frequency of point-mutations in IgH V186.2 (region which encodes NP-binding BCRs), including the affinity-enhancing Trp33Leu mutation that underpins the production of high-affinity anti-NP antibodies, and generation of memory B cells. The T cell-independence of the class-switched antibody response was further emphasized by the anti-NP IgG response in immune-deficient NSG mice engrafted with purified mouse B cells. It was mimicked by the specific TLR5-dependent IgG response to S. Typhimurium flagellin in Tcrβ− /−Tcrδ−/− mice. The LPS-immunized and flagellin-immunized Tcrβ−/−Tcrδ−/− mice generated LPS-specific and flagellin-specific IgG antibodies that neutralized E. coli and S. Typhimurium in vitro and protected them from lethal infection in vivo. Thus, TLR-BCR coengagement can induce a class-switched, hypermutated and protective antibody response in the absence of T cells.

B cells frequently infiltrate breast cancer (BCa) tumors, but their roles are inadequately understood. As shown by our analyses of the TCGA datasets and human BCa arrays, tumoral expression of B cell hallmark factors (e.g., CD20 and PAX5) coincides with those of the two subunits of pleiotropic cytokine IL-27, EBI3 and IL-27p28, whose expression is associated with poor prognosis in patients. In syngeneic mouse models, B cells promote BCa progression and do so by producing IL-27, but not IL-35 (IL-12/EBI3 heterodimer). Upon treatment by recombinant IL-27 or co-culturing with IL-27-producing B cells, BCa cells as well as targeted B lymphocytesactivate STAT1/STAT3 and upregulate immune checkpoint PD-L1 and chemokine receptor CXCR3, blocking of which synergistically arrest BCa cell proliferation in vitro and tumor growth in vivo, concomitant with reduction in PD-L1+B cells. Finally, B cell-specific deficiency in IL-27 receptor or PD-L1 inhibits BCa tumorigenesis, showing the dual functions of tumor-infiltrating B cells, i.e., IL-27 production for paracrinic PD-L1 upregulation. Citation Format: Hui Yan, Suryavathi Viswanadhapalli, Dariela Perez, Daniel Chupp, Maria Fernandez, Jingwei Wang, Shuai Wu, Carlos E. Rivera, Justin B. Moroney, Julia Taylor, John Im, Yiliao Luo, Junhao Liu, Sareddy Gangadhara, Tyler Curiel, Paolo Casali, Ratna Vadlamudi, Zhenming Xu. B cells produce IL-27 in breast cancer to upregulate PD-L1 expression and promote tumor progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2654.

Upon activation by CD40 or TLR signaling, B lymphocytes activate NF-κB to induce activation-induced cytidine deaminase and, therefore, Ig class switch DNA recombination, as central to the maturation of the Ab and autoantibody responses. In this study, we show that NF-κB activation is boosted by colocalization of engaged immune receptors, such as CD40, with RAB7 small GTPase on mature endosomes, in addition to signals emanating from the receptors localized on the plasma membrane, in mouse B cells. In mature endosomes, RAB7 directly interacts with TRAF6 E3 ubiquitin ligase, which catalyzes K63 polyubiquitination for NF-κB activation. RAB7 overexpression in Cd19+/creRosa26fl-STOP-fl-Rab7 mouse B cells upregulates K63 polyubiquitination activity of TRAF6, enhances NF-κB activation and activation-induced cytidine deaminase induction, and boosts IgG Ab and autoantibody levels. This, together with the extensive intracellular localization of CD40 and the strong correlation of RAB7 expression with NF-κB activation in mouse lupus B cells, shows that RAB7 is an integral component of the B cell NF-κB activation machinery, likely through interaction with TRAF6 for the assembly of “intracellular membrane signalosomes.”

An effective response against a previously experienced pathogen is contingent upon the generation of memory B cells. These display intrinsic differences over naïve B cells, including extended lifespan and rapid response to antigen, with production of high-affinity class-switched antibodies. However, the transcriptional and regulatory programs that underpin the unique features of memory B cells remain unexplored. Here, we have developed an integrated analysis of differentially expressed (DE) mRNAs, miRNAs, lncRNAs and chromatin profiles to determine how gene expression intersects with epigenetic regulatory elements to shape the identity and functions of human memory B cells. DE analysis revealed an exclusive gene signature shared by CD27+IgG+ and CD27+IgA+ B cells that segregates these memory B cells from CD27−IgD+ naïve B cells – “unswitched” CD27+IgD+memory B cells displayed a transcriptional signature of transition between naïve and switched memory B cells. While changes in DNA accessibility typically predicted changes in mRNA expression, some genes of the “memory” signature deviated from this, suggesting a layer of post-transcriptional regulation mediated by ncRNAs. Indeed, probing the miRNAome revealed downregulation of miR-181 and concomitant upregulation of five target genes (RASSF6, TOX, TRERF1, TRPV3, RORA) in switched memory B cells. In these B cells, select DE lncRNAs clustered proximal to the IgH gene locus on chr 14, suggesting a role in their regulation of BCR expression. Thus, we have identified a set of genes characteristic of memory B cells and have provided evidence these genes are subject to integrated regulation by chromatin remodeling, cis-regulatory elements and distinct sets of miRNAs and lncRNAs.

How B cells integrate innate receptor and adaptive T cell signals in the class-switched antibody response needs be better understood. By using cytokine gene knockout and reporter mice, in vitro B cell cultures and single-cell analyses, we have identified a subset of B cells that produces IL-27 as signature cytokine upon priming by TLR ligands, which up-regulate CD40 expression, and exposure to Tfh cell stimuli CD154 and IL-21. B cell IL-27 induction depends on NF-kB activated by linear ubiquitin assembly complexes and up-regulated Baft3 transcription factor, which are recruited to the Il27p28 locus, as made accessible by CD154 and IL-21 stimulation. IL-27-producing B cells are bystander B cells that are inefficient in secreting antibodies, but express high levels of adhesion molecules which engage other activated B cells. Together with IFNg, they boost proliferation, survival and class-switching to IgG2a of B cells stimulated with CD154 and IL-21. Accordingly, mice with B cell-specific ablation of IL-27 and IFNg receptors display severe defects in germinal center B cell and plasma cell differentiation, leading to abrogation of IgG2a and markedly reduced IgG1 responses. Thus, IL-27-producing B cells function as “helper” cells to promote differentiation of antigen-specific B cells for optimal IgG responses. As such, they play a critical role in the production of effective anti-viral antibodies and, possibly, modulation of anti-tumoral immunity.

Somatic hypermutation (SHM) and class-switch DNA recombination (CSR) together with plasma cell and memory B cell differentiation are central to the maturation of the antibody response. Prompted by our finding that CSR and plasma cell differentiation can be induced in vitro by a TLR ligand together with BCR crosslinking, we hypothesized that TLR and BCR co-engagement can induce mature T-independent antibody responses in vivo. To test this hypothesis, we injected Tcrβ−/−Tcrδ−/− mice, which are devoid of T cells, with NP-LPS (co-engaging NP-specific BCR and B-cell TLR4) or Salmonella flagellin (co-engaging flagellin-specific BCR and B-cell TLR5). These mice readily generated class-switched B cells, plasma cells and NP- or flagellin-specific IgG antibodies. The response to NP-LPS, which was also elicited in NSG mice (as defective in most innate immune elements in addition to the absence of T and B cells) grafted with purified B cells, evoked frequent VH region mutations, including the Trp33Leu mutation that would underpin the production of high-affinity anti-NP antibodies even in the absence of sustained germinal center formation, as well as the generation of long-lived memory B cells. These, together with the strong in vivo bactericidal activity of Salmonella flagellin-specific IgG and IgA elicited in Tcrβ−/−Tcrδ−/− mice, demonstrated that TLR/BCR co-engagement can induce mature, anamnestic and protective antibody responses, thereby providing the basis for new TLR-based B cell-targeted vaccine strategies to overcome T cell deficiencies.

Mice are widely used models for research and drug discovery, yet provide limited predictive value due to their divergence from humans in immune elements and functions. This underscores the need for more faithful models of the in vivo human immune response. To address this shortcoming, we have generated a HuMice platform by grafting NSG KitW-41J mice with fresh human cord blood CD34+ stem cells via intracardiac injection. This yielded up to 98% human CD45+ cell reconstitution within all major lymphoid compartments, including GALT. Human leukocyte development and long-term persistence in CD34+ cell-engrafted HuMice revealed robust T and B cell maturation, leading to emergence of IgM, IgG, IgA and IgE forming cells. B cells from HuMice underwent in vitro CSR and plasma cell differentiation as efficiently as B cells isolated from healthy humans. Once conditioned with our immune potentiation treatment, HuMice supported T-dependent and T-independent antibody responses involving formation of germinal center-like structures, class-switching, somatic hypermutation, as well as plasma cell and memory B cell differentiation, this entailed further maturation of the B cell compartment resulting in high B2/B1 cell ratio. HuMice were amenable to hydrocarbon-induced autoimmunity leading to lupus-like disease and were susceptible to infection by and respond to human pathogens such as vaccinia virus and EBV. Thus, we have devised a novel in vivo HuMice platform, which allows for generation and maturation of human antibody responses to microbial agents and lends itself to in vivo modeling of autoimmunity, thereby offering opportunities for identification of therapeutic targets and vaccine development.

The host immune response can be evaded by cancer cells, e.g., through upregulation of the PD-L1/PD-1 immune checkpoint, and even hijacked to promote tumor progression. Here, we show that tumor-infiltrating B lymphocytes play an important role in PD-L1 expression using two different murine models of breast cancer (BCa). Mice deficient in B cells displayed virtually abrogatedtumor growth from transplanted syngeneic BCa cells and expressed less PD-L1 in residual tumors. Mechanistic studies showed that the impact of B cells was through their production of IL-27, a pleotropic cytokine composed of the IL-27p28/EBI3 heterodimer and possessing both pro- and anti-inflammatory functions. Accordingly, mice with B cell-specific deficiency in expressing IL-27p28/EBI3(but not Il12a, which can heterodimerize with EBI3 to form cytokine IL-35) were severely impaired in the BCa tumor growth and tumoral PD-L1 expression. IL-27-producing B cells were generated by priming of B cells with TLR ligands (that are abundant in the tumor microenvironment due to the release by apoptotic cells) followed by stimulation with CD154 and IL-21 (as likely expressed by activated tumor-infiltrating CD4+T cells). Single cell-RNA-sequencing analysis using Il27p28-Gfp reporter mice revealed that IL-27-producing B cells expressed a transcriptome that is distinct than other B cells and IL-27-producing non-B cells. B cell secreted IL-27, induced STAT1 and STAT3 activation, upregulated PD-L1 expression, and enhanced the growth of human and mouse BCa cells in vitro.It also activated STAT1/STAT3 and upregulated PD-L1 in B lymphocytes, which were the immune cell type with the highest PD-L1 expression in murine BCa tumors. In human primaryBCa tumors, stronger IL-27 expression was associatedwith more B cell infiltration and PD-L1 expression, and correlated with a poorer survival in BCa patients. Further, recombinant IL-27 supported human BCa tumor growth in mice with humanized immune system. Collectively, these results demonstrated that B cell production of IL-27 underpins how the host immune system is hijacked to promote BCa tumor progression. Supported by DOD/BCRP grant BC170448 (to R.V. and Z.X.), NIH/NIAID grant AI 135599 and UT Health Mays Cancer Center - Dew Foundation Pilot Grant (to Z.X.). Citation Format: Hui Yan, Suryavathi Viswanadhapalli, Daniel Chupp, Maria Fernandez, Shuai Wu, Jingwei Wang, Justin Moroney, Julia Taylor, John Im, Carlos Rivera, Yiliao Luo, Junhao Liu, Gangadhara Sareddy, Tyler Curiel, Paolo Casali, Ratna Vadlamudi, Zhenming Xu. Tumor-infiltrating B lymphocytes produce IL-27 to upregulate PD-L1 expression in the tumor microenvironment and promote breast cancer progression [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-05.

Ig class switch DNA recombination (CSR) in B cells is crucial to the maturation of antibody responses. It requires IgH germline IH-CH transcription and expression of AID, both of which are induced by engagement of CD40 or dual engagement of a Toll-like receptor (TLR) and B cell receptor (BCR). Here, we have addressed cross-regulation between two different TLRs or between a TLR and CD40 in CSR induction by using a B cell stimulation system involving lipopolysaccharides (LPS). LPS-mediated long-term primary class-switched antibody responses and memory-like antibody responses in vivo and induced generation of class-switched B cells and plasma cells in vitro. Consistent with the requirement for dual TLR and BCR engagement in CSR induction, LPS, which engages TLR4 through its lipid A moiety, triggered cytosolic Ca2+ flux in B cells through its BCR-engaging polysaccharidic moiety. In the presence of BCR crosslinking, LPS synergized with a TLR1/2 ligand (Pam3CSK4) in CSR induction, but much less efficiently with a TLR7 (R-848) or TLR9 (CpG) ligand. In the absence of BCR crosslinking, R-848 and CpG, which per se induced marginal CSR, virtually abrogated CSR to IgG1, IgG2a, IgG2b, IgG3 and/or IgA, as induced by LPS or CD154 (CD40 ligand) plus IL-4, IFN-γ or TGF-β, and reduced secretion of class-switched Igs, without affecting B cell proliferation or IgM expression. The CSR inhibition by TLR9 was associated with the reduction in AID expression and/or IgH germline IH-S-CH transcription, and required co-stimulation of B cells by CpG with LPS or CD154. Unexpectedly, B cells also failed to undergo CSR or plasma cell differentiation when co-stimulated by LPS and CD154. Overall, by addressing the interaction of TLR1/2, TLR4, TLR7 and TLR9 in the induction of CSR and modulation of TLR-dependent CSR by BCR and CD40, our study suggests the complexity of how different stimuli cross-regulate an important B cell differentiation process and an important role of TLRs in inducing effective T-independent antibody responses to microbial pathogens, allergens and vaccines.

Class-switch DNA recombination (CSR) and somatic hypermutation (SHM), which require activation-induced cytidine deaminase (AID), and plasma cell differentiation, which requires B lymphocyte–induced maturation protein-1 (Blimp-1), are critical for the generation of class-switched and hypermutated (mature) Ab and autoantibody responses. We show that histone deacetylase inhibitors valproic acid and butyrate dampened AICDA/Aicda (AID) and PRDM1/Prdm1 (Blimp-1) mRNAs by upregulating miR-155, miR-181b, and miR-361 to silence AICDA/Aicda, and miR-23b, miR-30a, and miR-125b to silence PRDM1/Prdm1, in human and mouse B cells. This led to downregulation of AID, Blimp-1, and X-box binding protein 1, thereby inhibiting CSR, SHM, and plasma cell differentiation without altering B cell viability or proliferation. The selectivity of histone deacetylase inhibitor–mediated silencing of AICDA/Aicda and PRDM1/Prdm1 was emphasized by unchanged expression of HoxC4 and Irf4 (important inducers/modulators of AICDA/Aicda), Rev1 and Ung (central elements for CSR/SHM), and Bcl6, Bach2, or Pax5 (repressors of PRDM1/Prdm1 expression), as well as unchanged expression of miR-19a/b, miR-20a, and miR-25, which are not known to regulate AICDA/Aicda or PRDM1/Prdm1. Through these B cell–intrinsic epigenetic mechanisms, valproic acid blunted class-switched and hypermutated T-dependent and T-independent Ab responses in C57BL/6 mice. In addition, it decreased class-switched and hypermutated autoantibodies, ameliorated disease, and extended survival in lupus MRL/Faslpr/lpr mice. Our findings outline epigenetic mechanisms that modulate expression of an enzyme (AID) and transcription factors (Blimp-1 and X-box binding protein 1) that are critical to the B cell differentiation processes that underpin Ab and autoantibody responses. They also provide therapeutic proof-of-principle in autoantibody-mediated autoimmunity.

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA recombination (CSR), and plasma cell differentiation. In recent years, epigenetic modifications or factors, such as histone deacetylation and microRNAs (miRNAs), have been shown to interact with B-cell genetic programs to shape antibody responses, while the dysfunction of epigenetic factors has been found to lead to autoantibody responses. Analyzing genome-wide miRNA and mRNA expression in B cells in response to epigenetic modulators is important for understanding the epigenetic regulation of B-cell function and antibody response. Here, we demonstrate a protocol for inducing B cells to undergo CSR and plasma cell differentiation, treating these B cells with histone deacetylase (HDAC) inhibitors (HDIs), and analyzing mRNA and microRNA expression. In this protocol, we directly analyze complementary DNA (cDNA) sequences using next-generation mRNA sequencing (mRNA-seq) and miRNA-seq technologies, mapping of the sequencing reads to the genome, and quantitative reverse transcription (qRT)-PCR. With these approaches, we have defined that, in B cells induced to undergo CSR and plasma cell differentiation, HDI, an epigenetic regulator, selectively modulates miRNA and mRNA expression and alters CSR and plasma cell differentiation.

Class switch DNA recombination (CSR) of the immunoglobulin heavy chain (IgH) locus crucially diversifies antibody biological effector functions. CSR involves the induction of activation-induced cytidine deaminase (AID) expression and AID targeting to switch (S) regions by 14-3-3 adaptors. 14-3-3 adaptors specifically bind to 5'-AGCT-3' repeats, which make up for the core of all IgH locus S regions. They selectively target the upstream and downstream S regions that are set to undergo S-S DNA recombination. We hypothesized that 14-3-3 adaptors function as scaffolds to stabilize CSR enzymatic elements on S regions. Here we demonstrate that all seven 14-3-3β, 14-3-3ε, 14-3-3γ, 14-3-3η, 14-3-3σ, 14-3-3τ and 14-3-3ζ adaptors directly interacted with AID, PKA-Cα (catalytic subunit) and PKA-RIα (regulatory inhibitory subunit) and uracil DNA glycosylase (Ung). 14-3-3 adaptors, however, did not interact with AID C-terminal truncation mutant AIDΔ(180-198) or AIDF193A and AIDL196A point-mutants (which have been shown not to bind to S region DNA and fail to mediate CSR). 14-3-3 adaptors colocalized with AID and replication protein A (RPA) in B cells undergoing CSR. 14-3-3 and AID binding to S region DNA was disrupted by viral protein R (Vpr), an accessory protein of human immunodeficiency virus type-1 (HIV-1), which inhibited CSR without altering AID expression or germline IH-CH transcription. Accordingly, we demonstrated that 14-3-3 directly interact with Vpr, which in turn, also interact with AID, PKA-Cα and Ung. Altogether, our findings suggest that 14-3-3 adaptors play important scaffold functions and nucleate the assembly of multiple CSR factors on S regions. They also show that such assembly can be disrupted by a viral protein, thereby allowing us to hypothesize that small molecule compounds that specifically block 14-3-3 interactions with AID, PKA and/or Ung can be used to inhibit unwanted CSR.

Toxoplasma gondii is an obligate intracellular parasite that can cause severe neurological disease in infected humans. CD40 is a receptor on macrophages that plays a critical role in controlling T. gondii infection. We examined the regulation of CD40 on the surface of T. gondii -infected bone marrow-derived macrophages (BMdMs). T. gondii induced CD40 expression both at the transcript level and on the cell surface, and interestingly, the effect was parasite strain specific: CD40 levels were dramatically increased in type II T. gondii -infected BMdMs compared to type I- or type III-infected cells. Type II induction of CD40 was specific to cells harboring intracellular parasites and detectable as early as 6 h postinfection (hpi) at the transcript level. CD40 protein expression peaked at 18 hpi. Using forward genetics with progeny from a type II × type III cross, we found that CD40 induction mapped to a region of chromosome X that included the gene encoding the dense granule protein 15 (GRA15). Using type I parasites stably expressing the type II allele of GRA15 (GRA15 II ), we found that type I GRA15 II parasites induced the expression of CD40 on infected cells in an NF-κB-dependent manner. In addition, stable expression of hemagglutinin-tagged GRA15 II in THP-1 cells resulted in CD40 upregulation in the absence of infection. Since CD40 signaling contributes to interleukin-12 (IL-12) production, we examined IL-12 from infected macrophages and found that CD40L engagement of CD40 amplified the IL-12 response in type II-infected cells. These data indicate that GRA15 II induction of CD40 promotes parasite immunity through the production of IL-12.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by the production of an array of pathogenic autoantibodies, including high-affinity anti-dsDNA IgG antibodies, which plays an important role in disease development and progression. Lupus preferentially affects women during their reproductive years. The pathogenesis of lupus is contributed by both genetic factors and epigenetic modifications that arise from exposure to the environment. Epigenetic marks, including DNA methylation, histone post-translational modifications and microRNAs (miRNAs), interact with genetic programs to regulate immune responses. Epigenetic modifications influence gene expression and modulate B cell functions, such as class switch DNA recombination (CSR), somatic hypermutation (SHM) and plasma cell differentiation, thereby informing the antibody response. Epigenetic dysregulation can result in aberrant antibody responses to exogenous antigens or self-antigens, such as chromatin, histones and dsDNA in lupus. miRNAs play key roles in the post-transcriptional regulation of most gene-regulatory pathways and regulate both the innate and the adaptive immune responses. In mice, dysregulation of miRNAs leads to aberrant immune responses and development of systemic autoimmunity. Altered miRNA expression has been reported in human autoimmune diseases, including lupus. The dysregulation of miRNAs in lupus could be the result of multiple environmental factors, such as sex hormones and viral or bacterial infection. Modulation of miRNA is a potential therapeutic strategy for lupus.

An effective response against previously experienced pathogens is contingent upon the unique functionality of memory B cells. These display intrinsic differences over naïve B cells, including longer lifespan and rapid response to antigen with production of high-affinity, class-switched antibodies. We have developed an integrated analysis of mRNA, miRNA and long non-coding RNA (lncRNA) profiles to determine how the mRNA transcriptome intersects with epigenetic regulatory non-coding RNAs to shape the functionality and phenotype of human memory B cells (CD27+IgG+ and CD27+IgA+) as compared to their naïve B cell counterparts (CD27−IgD+IgM+). Differential expression analysis revealed a novel gene signature shared by CD27+IgG+ and CD27+IgA+ cells, distinguishing memory from naïve B cells. Additionally, CD27+IgD+ B cells, which are not considered bona-fide memory, revealed a transcriptional identity straddling naïve and switched memory B cells. Signaling pathway analysis implicated the “memory” gene signature in the generation, homing and survival of class-switched memory B cells. Integrated analysis of mRNA and miRNA transcriptomes showed that in memory B cells the expression of select genes was dependent upon miRNA-mediated regulation. lncRNA analysis revealed that some of these RNAs clustered with IgH genes, thereby suggesting a role of such lncRNAs in the regulation of the IgH locus. Additional data suggested that select lncRNAs act as miRNA sponges. Thus, we have identified a novel set of genes that play a role in the generation and maintenance of memory B cells and have provided evidence that these genes are subjected to integrated regulation by distinct sets of miRNAs and lncRNAs.

Inbred mice, the most widely used surrogates for human biology, recapitulate many features of the human immune system and immune response. They, however, diverge from humans in important immune elements and functions, such as differential TLR expression, certain antibody and T cell responses, species-specific viral and/or bacterial infections and drug interactions, thereby underscoring the need for a human model that mimics the in vivo human immune response. Humanized mice have recently provided some data on human infections, autoimmunity and cancer, with limited results. We have undertaken a systematic approach to the generation of humanized mice by grafting NOD.Cg-KitW-41J Prkdcscid Il2rgtm1Wjl/WaskJ (NSGW41) mice within 48 hours of birth, devoid of prior irradiation, with human hematopoietic (CD34+) stem cells. Human umbilical cord hematopoietic stem cells were collected within 30 minutes post-partum and injected intracardially, consistently yielding up to 95% human cell peripheral reconstitution. Human leukocyte development, differentiation and long-term persistence in our CD34+ cell-grafted NSGW41 mice revealed B and T cell maturation as part of a full immune system development, which led to emergence of IgM, IgG, IgA and IgE antibody titers comparable to wild type mice. CD34+ cell-grafted NSGW41 mice supported specific T-dependent and T-independent antibody responses that include human B cell class switch DNA recombination, plasma cell and memory B cell differentiation. They also supported a hydrocarbon-induced autoantibody response leading to symptoms mimicking systemic lupus. Thus, we have devised a robust in vivo platform allowing for generation and maturation of human antibody and autoantibody responses.

Somatic hypermutation (SHM) and class-switch DNA recombination (CSR) together with plasma cell and memory B cell differentiation are central to the maturation of the antibody response. Prompted by our demonstrations that CSR and plasma cell differentiation can be induced in vitro by co-engagement of TLR4 and BCR, we tested the hypothesis that co-engagement of a TLR and BCR can induce mature T cell-independent antibody responses in vivo. To this end, we injected Tcrβ−/−Tcrδ−/− mice, which are devoid of all T cells, with TLR4-dependent NP-LPS or TLR5-dependent Salmonella flagellin. These mice readily generated NP-specific IgG antibodies and class-switched B cells and plasma cells. The high-affinity NP-LPS-induced IgG response was accompanied by a high-frequency of V186.2 region DNA mutations in class-switched B cells and NP-specific memory B cells. The TLR4-dependence and T cell-independence of this class-switched, anti-NP response was emphasized by immunedeficient NSG mice grafted with purified B cells, and mimicked in Tcrβ−/−Tcrδ−/− mice injected with TLR5-dependent flagellin. These mice generated flagellin-specific IgG and IgA, which displayed strong bactericidal activity and protected mice against Salmonella challenge. Thus, co-engagement of TLR4-BCR or TLR5-BCR, respectively, can effectively induce a mature and protective T cell-independent antibody response, thereby providing the basis for new TLR/BCR co-engagement-based B cell-targeted vaccine strategies.