Class-switched Antibody Responses Research Articles

Cholera toxin and O-specific polysaccharide immune responses after oral cholera vaccination with Dukoral in different age groups of Bangladeshi participants.

Vaccination is important to prevent cholera. There are limited data comparing anti-O-specific polysaccharide (OSP) and anti-cholera toxin-specific immune responses following oral whole-cell with cholera toxin B-subunit (WC-rBS) vaccine (Dukoral, Valneva) administration in different age groups. An understanding of the differences is relevant because young children are less well protected by oral cholera vaccines than older children and adults. We compared responses in 50 adults and 49 children (ages 2 to <18) who were administered two doses of WC-rBS at a standard 14-day interval. All age groups had significant IgA and IgG plasma-blast responses to the OSP and cholera toxin B-subunit (CtxB) antigens that peaked 7 days after vaccination. However, in adults and older children (ages 5 to <18), antibody responses directed at the OSP antigen were largely IgA and IgG, with a minimal IgM response, while younger children (ages 2 to <5) mounted significant increases in IgM with minimal increases in IgA and IgG antibody responses 30 days after vaccination. In adults, anti-OSP and CtxB memory B-cell responses were detected after completion of the vaccination series, while children only mounted CtxB-specific IgG memory B-cell responses and no OSP-memory B-cell responses. In summary, children and adults living in a cholera endemic area mounted different responses to the WC-rBS vaccine, which may be a result of more prior exposure to Vibrio cholerae in older participants. The absence of class-switched antibody responses and memory B-cell responses to OSP may explain why protection wanes more rapidly after vaccination in young children compared to older vaccinees.IMPORTANCEVaccination is an important strategy to prevent cholera. Though immune responses targeting the OSP of V. cholerae are believed to mediate protection against cholera, there are limited data on anti-OSP responses after vaccination in different age groups, which is important as young children are not well protected by current oral cholera vaccines. In this study, we found that adults mounted memory B-cell responses to OSP, which were not seen in children. Adults and older children mounted class-switched (IgG and IgA) serum antibody responses to OSP, which were not seen in young children who had only IgM responses to OSP. The lack of class-switched antibody responses and memory B-cell responses to OSP in younger participants may be due to lack of prior exposure to V. cholerae and could explain why protection wanes more rapidly after vaccination in young children.

Three doses of COVID-19 mRNA vaccine induce class-switched antibody responses in inflammatory arthritis patients on immunomodulatory therapies.

Patients with inflammatory arthritis (IA) are at increased risk of severe COVID-19 due to medication-induced immunosuppression that impairs host defenses. The aim of this study was to assess antibody and B cell responses to COVID-19 mRNA vaccination in IA patients receiving immunomodulatory therapies. Adults with IA were enrolled through the Johns Hopkins Arthritis Center and compared with healthy controls (HC). Paired plasma and peripheral blood mononuclear cell (PBMC) samples were collected prior to and 30 days or 6 months following the first two doses of mRNA vaccines (D2; HC=77 and IA=31 patients), or 30 days following a third dose of mRNA vaccines (D3; HC=11 and IA=96 patients). Neutralizing antibody titers, total binding antibody titers, and B cell responses to vaccine and Omicron variants were analyzed. Anti-Spike (S) IgG and S-specific B cells developed appropriately in most IA patients following D3, with reduced responses to Omicron variants, and negligible effects of medication type or drug withholding. Neutralizing antibody responses were lower compared to healthy controls after both D2 and D3, with a small number of individuals demonstrating persistently undetectable neutralizing antibody levels. Most IA patients respond as well to mRNA COVID-19 vaccines as immunocompetent individuals by the third dose, with no evidence of improved responses following medication withholding. These data suggest that IA-associated immune impairment may not hinder immunity to COVID-19 mRNA vaccines in most individuals.

Spontaneous class-switched antibody responses at endometrial cancer tumor bed drives superior patients’ outcome

Abstract The role of humoral responses in endometrial cancer remains insufficiently investigated. Using a cohort of 107 patients with different histological subtypes of endometrial carcinoma, we report that concomitant accumulation of T, B and plasma cells at tumor beds predicts better survival. However, only B cell markers predict survival specifically in high-grade endometrioid type and serous tumors. Accordingly, immune protection is associated with class-switched IgA and, to a lesser extent, IgG. Notably, expression of polymeric immunoglobulin receptor (pIgR) by tumor cells and its occupancy by IgA are superior predictors of outcome, and correlate with defects in methyl mismatch repair. Mechanistically, pIgR-dependent, antigen-independent IgA occupancy drives inflammatory pathways associated with IFN and TNF signaling in tumor cells, along with apoptotic and ER stress pathways, while thwarting DNA repair mechanisms. Therefore, coordinated humoral and cellular immune responses, characterized by IgA:pIgR interactions in tumor cells, determine the progression of human endometrial cancer, and therefore the potential for effective immunotherapies. Supported by grants from NIH (R01CA157664, R01CA124515, R01CA178687 and R01CA211913), and from Cancer Center Support Grant (CCSG) CA076292

CXCR5+CD8+ T Cells Shape Antibody Responses In Vivo Following Protein Immunisation and Peripheral Viral Infection.

Crosstalk between T and B cells is crucial for generating high-affinity, class-switched antibody responses. The roles of CD4+ T cells in this process have been well-characterised. In contrast, regulation of antibody responses by CD8+ T cells is significantly less defined. CD8+ T cells are principally recognised for eliciting cytotoxic responses in peripheral tissues and forming protective memory. However, recent findings have identified a novel population of effector CD8+ T cells that co-opt a differentiation program characteristic of CD4+ T follicular helper (Tfh) cells, upregulate the chemokine receptor CXCR5 and localise to B cell follicles. While it has been shown that CXCR5+CD8+ T cells mediate the removal of viral reservoirs in the context of follicular-trophic viral infections and maintain the response to chronic insults by virtue of progenitor/stem-like properties, it is not known if CXCR5+CD8+ T cells arise during acute peripheral challenges in the absence of follicular infection and whether they influence B cell responses in vivo in these settings. Using the ovalbumin-specific T cell receptor transgenic (OT-I) system in an adoptive transfer-immunisation/infection model, this study demonstrates that CXCR5+CD8+ T cells arise in response to protein immunisation and peripheral viral infection, displaying a follicular-homing phenotype, expression of cell surface molecules associated with Tfh cells and limited cytotoxic potential. Furthermore, studies assessing the B cell response in the presence of OT-I or Cxcr5-/- OT-I cells revealed that CXCR5+CD8+ T cells shape the antibody response to protein immunisation and peripheral viral infection, promoting class switching to IgG2c in responding B cells. Overall, the results highlight a novel contribution of CD8+ T cells to antibody responses, expanding the functionality of the adaptive immune system.

Intrinsic B cell TLR-BCR coengagement induces mature and protective antibody responses in the absence of T cells

Abstract 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-Cell Responses in the Lung during Acute Pneumocystis Infection

Abstract Pneumocystis jiroveci is an opportunistic fungal organism that causes Pneumocystis pneumonia (PCP) in humans with HIV-AIDS or other immunocompromised individuals. Pneumocystis can replicate in the alveoli where it disrupts gas exchange and promotes inflammation in the lung. Although Pneumocystis association is with T cell immunodeficiency, recent evidence shows that B cells play a key role as APCs in the lung to prime fungal T cell responses. We hypothesized that B cells are recruited and undergo tissue specific CSR during PCP infection and that these cells are critical to the priming of PCP specific T cells. To test this hypothesis we inoculated 4 to 6wk old male and female C57Bl/6 mice with Pneumocystis murina by oral pharyngeal aspiration. Flow cytometry analysis revealed that IgG+ and IgA+ B cells were at higher abundance in infected lung tissue at days 14 and 24. Single cell BCR-sequencing revealed an increase in class switched B cell clonotypes (both IgG and IgA) at day 14 compared to spleen. Our lab has previously shown that germ encoded IgM has specificity to PC antigens. To understand the role of germline encoded IgM and class-switched antibody responses, 4 to 6 wk old Aicda, Ighm −/− or C57Bl/6 mice were inoculated with P. murina for 14 days. We found at day 14 that fungal burden loads were slightly higher in the Aicda,Ighm −/− mice than C57Bl/6 mice, but H&amp;E staining showed that Aicda,Ighm −/− mice possessed high levels of immunopathology. Bulk RNA sequencing revealed a lack of Type II immune responses, which have been shown to control Pneumocystis lung burden. Our data suggests that both germline encoded IgM and class switched antibody responses are critical in controlling pneumocystis infection.

IL-27-producing B cells synergize with IFNγ to promote IgG2a and IgG1 antibody responses

Abstract 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.

Antibody Production in Murine Polymicrobial Sepsis-Kinetics and Key Players.

Although antigen-specific priming of antibody responses is impaired during sepsis, there is nevertheless a strong increase in IgM and IgG serum concentrations. Using colon ascendens stent peritonitis (CASP), a mouse model of polymicrobial abdominal sepsis, we observed substantial increases in IgM as well as IgG of all subclasses, starting at day 3 and peaking 2 weeks after sepsis induction. The dominant source of antibody-secreting cells was by far the spleen, with a minor contribution of the mesenteric lymph nodes. Remarkably, sepsis induction in splenectomized mice did not change the dynamics of the serum IgM/IgG reaction, indicating that the marginal zone B cells, which almost exclusively reside in the spleen, are dispensable in such a setting. Hence, in systemic bacterial infection, the function of the spleen as dominant niche of antibody-producing cells can be compensated by extra-splenic B cell populations as well as other lymphoid organs. Depletion of CD4+ T cells did not affect the IgM response, while it impaired IgG generation of all subclasses with the exception of IgG3. Taken together, our data demonstrate that the robust class-switched antibody response in sepsis encompasses both T cell-dependent and -independent components.

CD40 function in immune development and response: Cell type requirements and cytoplasmic domain function

Abstract CD40 is a TNFR family member and serves as a costimulatory molecule expressed on multiple antigen presenting cell (APC) lineages. The functional significance of CD40 expression is illustrated by human disease and mouse models of life-threatening immune deficiency. CD40-CD40L deficiency affects multiple aspects of immune development and response. However, cell type-specific requirements for CD40 and requirements for CD40 signaling domains have not been comprehensively analyzed. In this study, we use conditional CD40 knockout mice to study cell type requirements and generated CD40 intracellular domain mutants by CRISPR to determine domain-specific functions. CD40 expression on B cells but not DC is required for CD40 dependent superantigen-specific negative selection and for T-cell dependent class-switched antibody responses. TRAF2/3 binding domains of CD40 are critical for these CD40 functions, while deletion of putative TRAF6 binding domain had minimal effect. In contrast, in vivo priming of cytokine-producing T cells depends on CD40 expression on DC but not B cells and requires both TRAF2/3 and TRAF6 domains. CD40 expression on both B cells and DC is required for maximal autoimmune EAE disease induced by recombinant human MOG (rhMOG), and both TRAF2/3 and TRAF6 domains of CD40 are required for this autoimmune response. Initial results indicate that CD40 on thymic epithelial cells and DC contributes to negative selection of tissue restricted antigen-binding T cells. These results identify different CD40-dependent APC requirements and suggest distinct CD40 signaling pathways for specific immune responses. Analysis of the linkage between cell type-specific CD40 function and intracellular signaling is under study.

Murine models of Pneumocystis infection recapitulate human primary immune disorders.

Despite the discovery of key pattern recognition receptors and CD4+ T cell subsets in laboratory mice, there is ongoing discussion of the value of murine models to reflect human disease. Pneumocystis is an AIDS-defining illness, in which risk of infection is inversely correlated with peripheral CD4+ T cell counts. Due to medical advances in the control of HIV, the current epidemiology of Pneumocystis infection is predominantly due to primary human immunodeficiencies and immunosuppressive therapies. To this end, we found that every human genetic immunodeficiency associated with Pneumocystis infection that has been tested in mice recapitulated susceptibility. For example, humans with a loss-of-function IL21R mutation are severely immunocompromised. We found that IL-21R, in addition to CD4+ T cell intrinsic STAT3 signaling, were required for generating protective antifungal class-switched antibody responses, as well as effector T cell-mediated protection. Furthermore, CD4+ T cell intrinsic IL-21R/STAT3 signaling was required for CD4+ T cell effector responses, including IL-22 production. Recombinant IL-22 administration to Il21r-/- mice induced the expression of a fungicidal peptide, cathelicidin antimicrobial peptide, which showed in vitro fungicidal activity. In conclusion, SPF laboratory mice faithfully replicate many aspects of human primary immunodeficiency and provide useful tools to understand the generation and nature of effector CD4+ T cell immunity.

B lymphocytes are a major source of IL-27 that drives class-switched antibody responses and anti-viral immunity through paracrinic targeting of B cells and T follicular helper cells

Abstract In addition to generating antibodies against infections, B lymphocytes produce cytokines to regulate immune functions. Here, we have identified B cells as a major source of cytokine IL-27 (the heterodimer of IL-27p28 and EBI3) by using B cell-deficient mMT and JH mice, mice with B cell-specific deficiency in Ebi3 and NSG mice transplanted with highly purified B cells. IL-27 in turn targets B cells to undergo class-switching to IgG2a, as shown by decreased antigen-specific IgG2a in mice with B cell-specific deficiency IL-27 receptor signaling, leading to defective clearance of vaccinia virus infection. The paracrinic function of IL-27 is recapitulated in vitro by B cell production of IL-27 upon stimulation by a TLR ligand together with CD154 (CD40 ligand) and IL-21, two hallmark stimuli of TFH cells, and IL-27 induction of T-bet and a gene signature similar to that induced by IFNγ, IL-27 collaboration with IFNγ in inducing T-bet and IgG2a, and, finally, selective switching to IgG2a in primed B cells when mixed with IL-27-producing B cells, which display a metabolic program distinct from that in IL-27-responding B cells, i.e., heightened TCA cycle activities and enhanced lactate generation and glutaminolysis. B cell-produced IL-27 also promotes differentiation of TFH cells, as shown by reduced number of TFH cells and defective IL-21 expression in residual TFH cells in mice with B cell-specific deficiency in Ebi3 and, consequently, severe impairments in the germinal center development, class-switching to all Ig isotypes and protective antibody responses to vaccinia virus infection in these mice. Overall, B cell production of IL-27 is a previously unrecognized important regulatory mechanism in the antibody response and anti-viral immunity.

Human IgG repertoire of malaria antigen-immunized human immune system (HIS) mice

Humanized mouse models present an important tool for preclinical evaluation of new vaccines and therapeutics. Here we show the human variable repertoire of antibody sequences cloned from a previously described human immune system (HIS) mouse model that possesses functional human CD4+ T cells and B cells, namely HIS-CD4/B mice. We sequenced variable IgG genes from single memory B-cell and plasma-cell sorted from splenocytes or whole blood lymphocytes of HIS-CD4/B mice that were vaccinated with a human plasmodial antigen, a recombinant Plasmodium falciparum circumsporozoite protein (rPfCSP). We demonstrate that rPfCSP immunization triggers a diverse B-cell IgG repertoire composed of various human VH family genes and distinct V(D)J recombinations that constitute diverse CDR3 sequences similar to humans, although low hypermutated sequences were generated. These results demonstrate the substantial genetic diversity of responding human B cells of HIS-CD4/B mice and their capacity to mount human IgG class-switched antibody response upon vaccination.

IL2 production predicts Tfh differentiation

Abstract Naïve CD4 T cells differentiate into functionally distinct subsets in response to a set of extrinsic and intrinsic cues. Effector subsets, including Th1, Th2 and Th17 CD4s, are required for optimal cellular responses to invading pathogens, while class switched antibody responses depend on the differentiation of T follicular helper cells (Tfh). Development of both Tfh and non-Tfh occurs in parallel and involves two distinct but functionally related expression bifurcations. Upon formation of T cell receptor (TCR)/major histocompatibility (MHC) synapses with antigen-presenting cells (APC), a discrete subset of activated T cells secretes the cytokine IL2. Similarly, a portion of activated cells express the transcription factor Bcl6 and differentiate into Tfh cells, while the remainder express Blimp1 and develop into effector CD4s. However, the cellular, temporal and mechanistic relationships between these two bifurcations remain unclear. Here we show that the IL2 and Bcl6/Blimp1 expression bifurcations occur simultaneously, precede cell division, predict differentiation into Tfh and non-Tfh subsets, and are mechanistically related to antigen availability TCR density.

Murine models of Pneumocystis infection recapitulate human primary immune disorders

Abstract Despite the discovery of key pattern recognition receptors and CD4+ T-cell subsets in laboratory mice, there is ongoing discussion of the value of murine models to reflect human disease. We recognize a strong parallel between human and mouse CD4+ T-cell immunity using the hallmark example of CD4+ T-cell dependent immunity, Pneumocystis pneumonia. Pneumocystis jirovecii infection was the first AIDS-defining illness and most closely tied with peripheral CD4+ T-cell counts. Due to successful control of HIV, the epidemiology of Pneumocystis infection in children is now primarily due to primary human immunodeficiencies and immunosuppressive therapies. To this end, we found that every human genetic immunodeficiency associated with Pneumocystis infection, recapitulated susceptibility in Pneumocystis infection in mice bearing similar mutations. Similar to human IL-21R deficiency, we found that IL-21R and CD4+ T-cell STAT3 signaling were required for both protective anti-fungal class-switched antibody responses and CD4+ effector T-cell mediated protection. Furthermore, we discerned that CD4+ T-cell intrinsic IL-21R/STAT3 signaling was required for inducing a Pneumocystis-specific IL-22 response, and that IL-22 inhibited trophozoite binding to alveolar epithelial cells. Moreover, recombinant IL-22 administration to Il21r−/− mice induced the expression of a fungicidal peptide, cathelicidin anti-microbial peptide which showed in vitro fungicidal activity. In conclusion, SPF laboratory mice faithfully replicate many aspects of human primary immunodeficiency and provide useful tools to understand the generation and nature of effector CD4+ T-cell immunity.

Costimulatory pathways mediating T-dependent germinal center responses: distinct cellular requirements for CD40 and B7 costimulation

Abstract T cell-dependent (TD) germinal center (GC) and antibody responses require coordinated interactions of T cells with B cells and antigen-presenting dendritic cells (DCs). Although B7 and CD40 costimulatory pathways are critical for these responses, a comprehensive analysis of cell type-specific pathways has been hindered by the absence of models for conditional expression of B7 or CD40. Here we report generation of conditional knockout mice for both B7 and CD40, and the use of these lines together with conditional MHC class II (MHCII) knockouts and bone marrow chimeric strategies, to analyze pathways involved in TD GC and antibody responses. Our results indicate that MHCII-restricted recognition of both DCs and B cells is necessary for generation of antigen-specific TFH cells, GC B cells, and antibody response. Notably, the requirements for expression of B7 and CD40 were distinct, with B7 expression required on DCs but not on B cells, while CD40 was required on B cells but not DCs for generation of TFH cells, antigen-specific GC B cells, and antigen-specific class-switched antibody responses. These findings identify requirements for cell and costimulatory interactions in TD GC and antibody responses and support a unique model in which major costimulatory pathways function through interaction of T cells with distinct cell populations.

Regulation of the HSH2 adaptor protein turnover via an indirect NF-κB-dependent mechanism is involved in determining the nature of the class switched antibody response. (IRM10P.609)

Abstract The Hematopoietic SH2 containing adaptor protein (HSH2) is differentially expressed in mature, peripheral B cell subpopulations, exhibiting low level expression in germinal center (GC) B cells versus high-level expression in B1 and marginal zone B cells. Studies have demonstrated an inverse relationship between its expression and the production of class-switched antibodies due to effects on terminal differentiation. Therefore, delineation of molecular mechanism that controls its expression is critical for understanding the class-switched antibody response. Studies demonstrated that upregulation of HSH2 following TNF family receptor or TLR-mediated signals, as well as its maintenance, requires NF-κB activity. Using both pharmacological and genetic approaches, it was shown that the canonical pathway is critical for maintenance of HSH2 expression. Inhibition of NF-kB selectively results in the rapid loss of HSH2, whereas actinomycin D and cycloheximide treatment do not. Analysis of the canonical pathway revealed that maintenance of HSH2 requires translocation of NF-κB to the nucleus. Because HSH2 mRNA levels are not altered under any conditions, it is hypothesized that NF-κB indirectly regulates the function of a short-lived protease that regulates HSH2 expression at the protein. These results reveal a potentially novel mechanism, in which NF-κB regulates HSH2 expression via one or more proteases and may represent a novel paradigm for controlling GC B cell function.

Genetic control of isotype specific antibody responses to flagellin in mice (IRC4P.450)

Abstract Adjuvants can improve the efficacy of vaccines by inducing class-switched immune responses. Host factors controlling the magnitude of class-switched antibody responses are poorly understood. We have observed flagellin-immunized mouse strains differ in the magnitude of isotype specific antibody responses. Some strains are high responders (A/J, Swiss Webster) and other strains are low responders (C57BL6, Balb/c). All strains had comparable primary anti-flagellin IgM responses, suggesting common genetic variations influence isotype class switching. We hypothesized different genetic differences between A/J and C57BL/6 mice determine magnitude of responder phenotype. We bred C57BL/6 and A/J mice and tested F1 and F2 offspring for isotype specific antibody responses. Mice were immunized with a FliC protein mutated in the Naip5/6 recognition domain to overcome known differences in Naip5-dependent inflammasome activation between these mouse strains. We demonstrate anti-flagellin IgG1, IgG2a/c and IgA responses are inherited as a quantitative trait. Using consomic mice, we show the high responder phenotype is not controlled by the IgH locus on A/J chromosome 12. Using mutant flagellin proteins, we demonstrate the high responder phenotype is primarily controlled by the FliC D2/D3 domain. Understanding the host factors contributing to FliC D2/D3 induced antibody responses aid in the rationale design of vaccines, and help establish principles for future adjuvant design and development.

B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses (IRM10P.608)

Abstract CSR in B cells is crucial to the maturation of antibody responses. It is induced by engagement of CD40 or dual engagement of a TLR and 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 LPS. LPS mediated long-term primary class-switched antibody responses and induced generation of class-switched B cells and plasma cells. 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, virtually abrogated CSR, as induced by LPS or CD154, and reduced secretion of class-switched Igs, without affecting B cell proliferation or IgM expression. The CSR inhibition by TLR9 was associated with reduced 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 when co-stimulated by LPS and CD154. 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 T-independent antibody responses.

CTLA-4 controls follicular helper T-cell differentiation by regulating the strength of CD28 engagement

Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) is an essential regulator of T-cell responses, and its absence precipitates lethal T-cell hyperactivity. However, whether CTLA-4 acts simply to veto the activation of certain clones or plays a more nuanced role in shaping the quality of T-cell responses is not clear. Here we report that T cells in CTLA-4-deficient mice show spontaneous T-follicular helper (T(FH)) differentiation in vivo, and this is accompanied by the appearance of large germinal centers (GCs). Remarkably, short-term blockade with anti-CTLA-4 antibody in wild-type mice is sufficient to elicit T(FH) generation and GC development. The latter occurs in a CD28-dependent manner, consistent with the known role of CTLA-4 in regulating the CD28 pathway. CTLA-4 can act by down-regulating CD80 and CD86 on antigen presenting cells (APCs), thereby altering the level of CD28 engagement. To mimic reduced CD28 ligation, we used mice heterozygous for CD28, revealing that the magnitude of CD28 engagement is tightly linked to the propensity for T(FH) differentiation. In contrast, other parameters of T-cell activation, including CD62L down-regulation and Ki67 expression, were relatively insensitive to altered CD28 level. Altered T(FH) generation as a result of graded reduction in CD28 was associated with decreased numbers of GC B cells and a reduction in overall GC size. These data support a model in which CTLA-4 control of immunity goes beyond vetoing T-cell priming and encompasses the regulation of T(FH) differentiation by graded control of CD28 engagement.

B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: Modulation by BCR and CD40, and relevance to T-independent antibody responses

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.