Ligation of TLR7 on CD19+CD1dhi B cells suppresses allergic lung inflammation via regulatory T cells

B cells have been described as having the capacity to regulate cellular immune responses and suppress inflammatory processes. One such regulatory B‐cell population is defined as IL‐10‐producing CD19⁺CD1d^hi cells. Previous work has identified an expansion of these cells in mice infected with the helminth, Schistosoma mansoni. Here, microarray analysis of CD19⁺CD1d^hi B cells from mice infected with S. mansoni demonstrated significantly increased Tlr7 expression, while CD19⁺CD1d^hi B cells from uninfected mice also demonstrated elevated Tlr7 expression. Using IL‐10 reporter, Il10^?/? and Tlr7^?/‐ mice, we formally demonstrate that TLR7 ligation of CD19⁺CD1d^hi B cells increases their capacity to produce IL‐10. In a mouse model of allergic lung inflammation, the adoptive transfer of TLR7‐elicited CD19⁺CD1d^hi B cells reduced airway inflammation and associated airway hyperresponsiveness. Using DEREG mice to deplete FoxP3⁺ T regulatory cells in allergen‐sensitized mice, we show that that TLR7‐elicited CD19⁺CD1d^hi B cells suppress airway hyperresponsiveness via a T regulatory cell dependent mechanism. These studies identify that TLR7 stimulation leads to the expansion of IL‐10‐producing CD19⁺CD1d^hi B cells, which can suppress allergic lung inflammation via T regulatory cells.     

CD21−/low B cells associate with joint damage in rheumatoid arthritis patients

Depletion of B cells is beneficial in rheumatoid arthritis (RA) patients with autoantibodies to citrullinated proteins (ACPA) and/or the Fc portion of immunoglobulins (rheumatoid factor [RF]), suggesting a role for B cells in disease pathogenesis. To date, however, the identity of specifically pathogenic B cell subsets has not been discovered. One candidate population is identified by the low expression or absence of complement receptor 2 (CD21^?/low B cells). In this study, we sought to determine whether there was any correlation between CD21^?/low B cells and clinical outcome in patients with established RA, either ACPA⁺/RF⁺ (n = 27) or ACPA^?/RF^? (n = 10). Healthy donors (n = 17) were included as controls. The proportion of the CD21^?/low CD27^?IgD^? memory B cell subset in peripheral blood (PB) was significantly increased in ACPA⁺/RF⁺ RA patients compared with healthy donors, and the frequency of this subset correlated with joint destruction (r = 0.57, P < 0.04). The levels of the chemokines CXCL‐9 and CXCL‐10 were higher in synovial fluid than in plasma, and PB CD21^?/low cells expressed the receptor, CXCR3. In synovial fluid, most of the B cells were CD21^?/low, approximately 40% of that population was CD27^?IgD^?, and a third of those expressed the pro‐osteoclastogenic factor receptor activator of the nuclear factor κB ligand (RANKL). This subset also secreted RANKL, in addition to other factors such as IL‐6, even in the absence of stimulation. We interpret these data as reason to propose the hypothesis that the CD27^?IgD^? subset of CD21^?/low B cells may mediate joint destruction in patients with ACPA⁺/RF⁺ RA.     

CXCR7 influences the migration of B cells during maturation

The atypical chemokine receptor CXCR7 binds the chemokines CXCL12 and CXCL11. The receptor is widely expressed and was shown to tune CXCR12‐induced responses of CXCR4. Here, the function of CXCR7 was examined at late stages of human B‐cell maturation, when B cells differentiate into Ab‐secreting plasmablasts. We identified two populations of CXCR7⁺ cells in tonsillar lymphocytes, one being presumably memory B cells or early plasmablasts (FSC^lowCD19⁺CD38^mid) and the other being plasmablasts or early plasma cells (FSC^highCD19⁺CD38⁺). CXCR7 is expressed on CD19⁺CD27⁺ memory B cells, on CD19⁺CD38⁺CD138^? and intracellular immunoglobulin high plasmablasts, but not on CD19⁺CD138⁺icIg^high plasma cells. The differential expression pattern suggests a potential contribution of the scavenger receptor in final B‐cell maturation. On in vitro differentiating B cells, we found a marked inverse correlation between CXCR7 and CXCR5 cell surface levels, whereas expression of CXCR4 remained almost constant. Migration assays performed with tonsillar mononuclear cells or in vitro differentiated cells revealed that inhibition of CXCR7 markedly increases chemotaxis toward CXCL12, especially at late stages of B‐cell maturation. Chemotaxis was attenuated in the presence of CXCR4 antagonists, confirming that migration is CXCR4 mediated. Our findings unequivocally demonstrate a novel role for CXCR7 in regulating the migration of plasmablasts during B‐cell maturation.     

Renal‐infiltrating CD11c+ cells are pathogenic in murine lupus nephritis through promoting CD4+ T cell responses

Lupus nephritis (LN) is a major manifestation of systemic lupus erythematosus (SLE), causing morbidity and mortality in 40–60% of SLE patients. The pathogenic mechanisms of LN are not completely understood. Recent studies have demonstrated the presence of various immune cell populations in lupus nephritic kidneys of both SLE patients and lupus‐prone mice. These cells may play important pathogenic or regulatory roles in situ to promote or sustain LN. Here, using lupus‐prone mouse models, we showed the pathogenic role of a kidney‐infiltrating CD11c⁺ myeloid cell population in LN. These CD11c⁺ cells accumulated in the kidneys of lupus‐prone mice as LN progressed. Surface markers of this population suggest their dendritic cell identity and differentiation from lymphocyte antigen 6 complex (Ly6C)^low mature monocytes. The cytokine/chemokine profile of these renal‐infiltrating CD11c⁺ cells suggests their roles in promoting LN, which was confirmed further in a loss‐of‐function in‐vivo study by using an antibody‐drug conjugate (ADC) strategy targeting CX₃CR1, a chemokine receptor expressed highly on these CD11c⁺ cells. However, CX₃CR1 was dispensable for the homing of CD11c⁺ cells into lupus nephritic kidneys. Finally, we found that these CD11c⁺ cells co‐localized with infiltrating T cells in the kidney. Using an ex‐ vivo co‐culture system, we showed that renal‐infiltrating CD11c⁺ cells promoted the survival, proliferation and interferon‐γ production of renal‐infiltrating CD4⁺ T cells, suggesting a T cell‐dependent mechanism by which these CD11c⁺ cells promote LN. Together, our results identify a pathogenic kidney‐infiltrating CD11c⁺ cell population promoting LN progression, which could be a new therapeutic target for the treatment of LN.     

Follicular regulatory T cells repress cytokine production by follicular helper T cells and optimize IgG responses in mice

Follicular helper T (Tfh) cells provide crucial help to germinal center B (GCB) cells for proper antibody production, and a specialized subset of regulatory T cells, follicular regulatory T (Tfr) cells, modulate this process. However, Tfr‐cell function in the GC is not well understood. Here, we define Tfr cells as a CD4⁺ Foxp3⁺ CXCR5^hi PD‐1^hi CD25^low TIGIT^high T‐cell population. Furthermore, we have used a novel mouse model (“Bcl6FC”) to delete the Bcl6 gene in Foxp3⁺ T cells and thus specifically deplete Tfr cells. Following immunization, Bcl6FC mice develop normal Tfh‐ and GCB‐cell populations. However, Bcl6FC mice produce altered antigen‐specific antibody responses, with reduced titers of IgG and significantly increased IgA. Bcl6FC mice also developed IgG antibodies with significantly decreased avidity to antigen in an HIV‐1 gp120 “prime‐boost” vaccine model. In an autoimmune lupus model, we observed strongly elevated anti‐DNA IgA titers in Bcl6FC mice. Additionally, Tfh cells from Bcl6FC mice consistently produce higher levels of Interferon‐γ, IL‐10 and IL‐21. Loss of Tfr cells therefore leads to highly abnormal Tfh‐cell and GCB‐cell responses. Overall, our study has uncovered unique regulatory roles for Tfr cells in the GC response.     

Enhancing immunity prevents virus‐induced T‐cell‐mediated immunopathology in B cell‐deficient mice

Hyper‐activated or deviated immune responses can result in immunopathological diseases. Paradoxically, immunodeficiency represents a frequent cause of such immune‐mediated pathologies. Immunopathological manifestations are commonly treated by immunosuppression, but in situations in which immunodeficiency is the basis of disease development, enhancing immunity may represent an alternative treatment option. Here, we tested this counterintuitive concept in a preclinical model using infection of mice with lymphocytic choriomeningitis virus (LCMV). Firstly, we demonstrate that infection of B‐cell‐deficient (B^?/?) but not of wild‐type (WT) mice with the LCMV strain Docile induced a rapid and fatal CD8⁺ T‐cell‐mediated immunopathological disease. Similar to WT mice, LCMV‐infected B^?/? mice generated a potent, functional LCMV‐specific CD8⁺ T‐cell response but exhibited prolonged viral antigen presentation and increased vascular leakage in liver and lungs. Secondly, we were able to prevent this virus‐induced immunopathology in B^?/? mice by active or passive T‐cell immunizations or by treatment with LCMV‐specific virus neutralizing or non‐neutralizing monoclonal antibodies (mAb). Thus, boosting antiviral immunity did not aggravate immunopathology in this model, but prevented it by decreasing the formation of target structures for damage‐causing CD8⁺ T cells.     

NR4A1‐dependent Ly6Clow monocytes contribute to reducing joint inflammation in arthritic mice through Treg cells

Monocytes are central to the physiopathology of arthritis, but their roles in progression and resolution of the disease remain to be clarified. Using NR4A1^?/? mice, which lack patrolling lymphocyte antigen 6C (Ly6C^low) monocytes, we found that inflammatory Ly6C^high monocytes contribute to rapid development of arthritis in a serum transfer‐induced arthritis (STIA) model. Our experiments suggest that patrolling monocytes do not promote the initiation and progression of arthritis in mice, as severity of symptoms was amplified in NR4A1^?/? mice. Moreover, we show that treatment of arthritic wild type (WT) mice with cytosporone B (Csn‐B), a NR4A1‐specific agonist, significantly reduces severity of disease. Effects of Csn‐B were absent in monocyte‐depleted mice treated with clodronate until Ly6C^low monocytes were restored. Adoptive transfer of Ly6C^low monocytes in arthritic NR4A1^?/? mice treated with Csn‐B reduces joint inflammation, supporting the regulatory role of Ly6C^low subset on disease development. Our results also reveal that administration of Csn‐B to arthritic mice enhances levels of circulating CD4⁺CD25⁺FoxP3⁺ Treg cells, a process requiring the presence of Ly6C^low monocytes. Together, these data indicate that Ly6C^high monocytes are involved in the initiation and progression of arthritis and Ly6C^low monocytes contribute to reduce joint inflammation through the mobilization of Treg cells.     

CD21–/low B cells in human blood are memory cells

The complement receptor 2 (CR2, CD21) is part of a complex (CD21/CD19/CD81) acting as a co‐receptor to the B cell receptor (BCR). Simultaneous triggering of the BCR and CD21 lowers the threshold for B cell activation. Although CD21 is important, B cells that express low amounts or lack surface CD21 (CD21^–/low) are increased in conditions with chronic inflammation, e.g. autoimmune diseases. However, little is known about the CD21^–/low B cell subset in peripheral blood from healthy donors. Here, we show that CD21^–/low cells represent approximately 5% of B cells in peripheral blood from adults but are barely detectable in cord blood, after excluding transitional B cells. The CD21^–/low subset can be divided into CD38^–24⁺ and CD38^–24^low cells, where most of the CD38^–24⁺ are CD27⁺immunoglobulin (Ig)M⁺IgD⁺ and the CD38^–24^low are switched CD27^–. Expression levels of additional markers, e.g. CD95 and CD62L, are similar to those on classical memory B cells. In contrast to naive cells, the majority of CD21^–/low cells lack expression of the ABCB1 transporter. Stimulation with a combination of BCR, Toll‐like receptor (TLR)?7/8 and interleukin (IL)?2 induces proliferation and differentiation of the CD21^–/low B cells comparable to CD21⁺CD27⁺ memory B cells. The response excluding BCR agonist is not on par with that of classical memory B cells, although clearly above that of naive B cells. This is ascribed to a weaker response by the CD38^–24^low subset, implying that some memory B cells require not only TLR but also BCR triggering. We conclude that the CD21^–/low cells in healthy donors are memory B cells.     

IL‐7 is essential for lymphopenia‐driven turnover of colitogenic CD4+ memory T cells in chronic colitis

We previously demonstrated that IL‐7 is essential for the persistence of T‐cell‐mediated colitis, by showing that adoptive transfer of CD4⁺CD45RB^high T cells into IL‐7^?/?×RAG‐1^?/? mice did not induce colitis; and that intestinal IL‐7 is not essential for this colitis model, by showing that IL‐7^?/?×RAG‐1^?/? mice parabiosed with colitic CD4⁺CD45RB^high T‐cell‐transferred RAG‐1^?/? mice developed colitis. Here, we investigated the role of IL‐7 in the maintenance of colitogenic CD4⁺ T cells by surgically separating these parabionts. Surprisingly, the separated IL‐7^?/?×RAG‐1^?/? mice were consistently diseased after separation, although no IL‐7 mRNA was detected in the tissues of separated IL‐7^?/?×RAG‐1^?/? partners. CD4⁺ T cells isolated from the separated RAG‐1^?/? or IL‐7^?/?×RAG‐1^?/? mice were then transferred into new RAG‐1^?/? or IL‐7^?/?×RAG‐1^?/? mice. Regardless of the source of donor cells, RAG‐1^?/? recipients developed colitis, whereas IL‐7^?/?×RAG‐1^?/? recipients did not. Collectively, these results demonstrate that IL‐7 is essential for lymphopenia‐driven turnover of colitogenic CD4⁺ T cells rather than the maintenance of those cells in established colitic mice. They also provide a basis for the timing of IL‐7/IL‐7R blockade for the treatment of inflammatory bowel diseases.     

Residual LCMV antigen in transiently CD4+ T cell‐depleted mice induces high levels of virus‐specific antibodies but only limited B‐cell memory

Infection of C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV) strain Armstrong (Arm) induces an acute infection with rapid virus clearance by CD8⁺ T cells independently of CD4⁺ T cell help. Residual viral antigen may, however, persist for a prolonged time. Here, we demonstrate that mice that had been transiently depleted of CD4⁺ T cells during acute LCMV Arm infection generated high levels of virus‐specific IgG antibodies (Ab) after viral clearance. Robust induction of LCMV‐specific IgG after transient CD4⁺ T cell depletion was dependent on Fcγ receptors but not on the complement receptors CD21/CD35. In contrast to the potent production of LCMV‐specific IgG, the generation of LCMV‐specific isotype‐switched memory B cells after transient CD4⁺ T cell depletion was considerably reduced. Moreover, mice depleted of CD4⁺ T cells during acute infection were strongly impaired in generating a secondary LCMV‐specific B cell response upon LCMV rechallenge. In conclusion, our data indicate that LCMV antigen depots after viral clearance were capable of inducing high levels of virus‐specific IgG. They failed, however, to induce robust virus‐specific B cell memory revealing a previously unappreciated dichotomy of specific Ab production and memory cell formation after priming with residual antigen.     

TACI‐dependent APRIL signaling maintains autoreactive B cells in a mouse model of systemic lupus erythematosus

Autoantibodies contribute to the development of systemic lupus erythematosus (SLE). APRIL (a proliferation‐inducing ligand), a member of the TNF superfamily, regulates plasma‐cell survival and binds to TACI (transmembrane activator CAML interactor) and BCMA (B‐cell maturation antigen). We previously showed that APRIL blockade delayed disease onset in lupus‐prone mice. In order to evaluate the role of APRIL receptors in the development of SLE, APRIL, TACI, BCMA , or double TACI.BCMA null mutations were introduced into the Nba2.Yaa (Y‐linked autoimmune acceleration) spontaneous lupus mouse model. Mortality as a consequence of glomerulonephritis (GN) was reduced in Nba2.APRIL^?/?.Yaa , Nba2.TACI^?/?.Yaa and double‐KO mice compared with Nba2.Yaa mice and correlated with lower levels of circulating antibodies, while splenic populations remained unchanged. In contrast, the appearance of symptoms was accelerated in BCMA‐deficient mice, in which TACI signaling was increased. Finally, lupus‐prone mice deficient for the APRIL‐TACI axis produced less pathogenic antibodies and developed less GN. Disease reduction was attributed to impaired T‐independent type 2 responses when the APRIL‐TACI signaling axis was disrupted. Collectively, our results have identified and confirmed APRIL as a new target involved in B‐cell activation, in the maintenance of plasma cell survival and subsequent increased autoantibody production that sustains lupus development in mice.     

Differential requirements for IRF4 in the clonal expansion and homeostatic proliferation of naive and memory murine CD8+ T cells

Interferon regulatory factor 4 (IRF4) has critical roles in immune cell differentiation and function and is indispensable for clonal expansion and effector function in T cells. Here, we demonstrate that the AKT pathway is impaired in murine CD8⁺ T cells lacking IRF4. The expression of phosphatase and tensin homolog (PTEN), a negative regulator of the AKT pathway, was elevated in Irf4^?/? CD8⁺ T cells. Inhibition of PTEN partially rescued downstream events, suggesting that PTEN constitutes a checkpoint in the IRF4‐mediated regulation of cell signaling. Despite the clonal expansion defect, in the absence of IRF4, memory‐like CD8⁺ T cells could be generated and maintained, although unable to expand in recall responses. The homeostatic proliferation of naïve Irf4^?/? CD8⁺ T cells was impaired, whereas their number eventually reached a level similar to that of wild‐type CD8⁺ T cells. Conversely, memory‐like Irf4^?/? CD8⁺ T cells underwent homeostatic proliferation in a manner similar to that of wild‐type memory CD8⁺ T cells. These results suggest that IRF4 regulates the clonal expansion of CD8⁺ T cells at least in part via the AKT signaling pathway. Moreover, IRF4 regulates the homeostatic proliferation of naïve CD8⁺ T cells, whereas the maintenance of memory CD8⁺ T cells is IRF4‐independent.     

Differential requirement for CARMA1 in agonist‐selected T‐cell development

Caspase recruitment domain‐containing membrane‐associated guanylate kinase protein‐1 (CARMA1) is a critical component of the NF‐κB signaling cascade mediated by TCR engagement. In addition to activation of naïve T cells, TCR signaling is important for the development of agonist‐selected T‐cell subsets such as Treg, NKT cells, and CD8‐αα T cells. However, little is known about the role of CARMA1 in the development of these lineages. Here we show that CARMA1‐deficient mice (CARMA1^?/?) have altered populations of specific subsets of agonist‐selected T cells. Specifically, CARMA1^?/? mice have impaired natural and adaptive Treg development, whereas NKT cell numbers are normal compared with wild‐type mice. Interestingly, CD8‐αα T cells, which may also be able to develop through an extrathymic selection pathway, are enriched in the gut of CARMA1^?/? mice, whereas memory‐phenotype CD4⁺ T cells (CD62L^low/CD44^high) are present at reduced numbers in the periphery. These results indicate that CARMA1 is essential for Treg development, but is not necessary for the development of other agonist‐selected T‐cell subsets. Overall, these data reveal an important but differential role for CARMA1‐mediated TCR signaling in T‐cell development.     

C5a receptor‐deficient dendritic cells promote induction of Treg and Th17 cells

C5a is a proinflammatory mediator that has recently been shown to regulate adaptive immune responses. Here we demonstrate that C5a receptor (C5aR) signaling in DC affects the development of Treg and Th17 cells. Genetic ablation or pharmacological targeting of the C5aR in spleen‐derived DC results in increased production of TGF‐β leading to de novo differentiation of Foxp3⁺ Treg within 12 h after co‐incubation with CD4⁺ T cells from DO11.10/RAG2^?/? mice. Stimulation of C5aR^?/? DC with OVA and TLR2 ligand Pam₃CSK₄ increased TGF‐β production and induced high levels of IL‐6 and IL‐23 but only minor amounts of IL‐12 leading to differentiation of Th cells producing IL‐17A and IL‐21. Th17 differentiation was also found in vivo after adoptive transfer of CD4⁺ Th cell into C5aR^?/? mice immunized with OVA and Pam₃CSK₄. The altered cytokine production of C5aR^?/? DC was associated with low steady state MHC class II expression and an impaired ability to upregulate CD86 and CD40 in response to TLR2. Our data suggest critical roles for C5aR in Treg and Th17‐cell differentiation through regulation of DC function.     

Swiprosin‐1/EFhd2 limits germinal center responses and humoral type 2 immunity

Activated B cells are selected for in germinal centers by regulation of their apoptosis. The Ca²⁺‐binding cytoskeletal adaptor protein Swiprosin‐1/EFhd2 (EFhd2) can promote apoptosis in activated B cells. We therefore hypothesized that EFhd2 might limit humoral immunity by repressing both the germinal center reaction and the expected enhancement of immune responses in the absence of EFhd2. Here, we established EFhd2^?/? mice on a C57BL/6 background, which revealed normal B‐ and T‐cell development, basal Ab levels, and T‐cell independent type 1, and T‐cell independent type 2 responses. However, T cell‐dependent immunization with sheep red blood cells and infection with the helminth Nippostrongylus brasiliensis (N.b) increased production of antibodies of multiple isotypes, as well as germinal center formation in EFhd2^?/? mice. In addition, serum IgE levels and numbers of IgE⁺ plasma cells were strongly increased in EFhd2^?/? mice, both after primary as well as after secondary N.b infection. Finally, mixed bone marrow chimeras unraveled an EFhd2‐dependent B cell‐intrinsic contribution to increased IgE plasma cell numbers in N.b‐infected mice. Hence, we established a role for EFhd2 as a negative regulator of germinal center‐dependent humoral type 2 immunity, with implications for the generation of IgE.