IL-21-induced isotype switching to IgG and IgA by human naive B cells is differentially regulated by IL-4

Naive B cells can alter the effector function of their Ig molecule by isotype switching, thereby allowing them to secrete not only IgM, but also the switched isotypes IgG, IgA, and IgE. Different isotypes are elicited in response to specific pathogens. Similarly, dysregulated production of switched isotypes underlies the development of various diseases, such as autoimmunity and immunodeficiency. Thus, it is important to characterize mediators controlling isotype switching, as well as their contribution to the overall B cell response. Isotype switching in human naive B cells can be induced by CD40L together with IL-4, IL-10, IL-13, and/or TGF-beta. Recently, IL-21 was identified as a switch factor for IgG1 and IgG3. However, the effect of IL-21 on switching to IgA, as well as the interplay between IL-21 and other switch factors, remains unknown. We found that IL-4 and IL-21 individually induced CD40L-stimulated human naive B cells to undergo switching to IgG, with IL-4 predominantly inducing IgG1(+) cells and IL-21 inducing IgG3. Culture of naive B cells with CD40L and IL-21, but not IL-4, also yielded IgA(+) cells. Combining IL-4 and IL-21 had divergent effects on isotype switching. Specifically, while IL-4 and IL-21 synergistically increased the generation of IgG1(+) cells from CD40L-stimulated B cells, IL-4 concomitantly abolished IL-21-induced switching to IgA. Our findings demonstrate the dynamic interplay between IL-4 and IL-21 in regulating the production of IgG subclasses and IgA, and suggest temporal roles for these cytokines in humoral immune responses to specific pathogens.     

Signaling events involved in interleukin 27 (IL-27)-induced proliferation of human naive CD4+ T cells and B cells

IL-27 induces stronger proliferation of naive than memory human B cells and CD4(+) T cells. In B cells, this differential response is associated with similar levels of IL-27 receptor chains, IL-27Rα and gp130, in both subsets and stronger STAT1 and STAT3 activation by IL-27 in naive B cells. Here, we show that the stronger proliferative response of CD3-stimulated naive CD4(+) T cells to IL-27 is associated with lower levels of IL-27Rα but higher levels of gp130 compared with memory CD4(+) T cells. IL-27 signaling differs between naive and memory CD4(+) T cells, as shown by more sustained STAT1, -3, and -5 activation and weaker activation of SHP-2 in naive CD4(+) T cells. In the latter, IL-27 increases G0/G1 to S phase transition, cell division and, in some cases, cell survival. IL-27 proliferative effect on naive CD4(+) T cells is independent of MAPK, but is dependent on c-Myc and Pim-1 induction by IL-27 and is associated with induction of cyclin D2, cyclin D3, and CDK4 by IL-27 in a c-Myc and Pim-1-dependent manner. In BCR-stimulated naive B cells, IL-27 only increases entry in the S phase and induces the expression of Pim-1 and of cyclins A, D2, and D3. In these cells, inhibition of Pim-1 inhibits IL-27 effect on proliferation and cyclin induction. Altogether, these data indicate that IL-27 mediates proliferation of naive CD4(+) T cells and B cells through induction of both common and distinct sets of cell cycle regulators.     

IL-21 induces differentiation of human naive and memory B cells into antibody-secreting plasma cells

IL-21 is a type I cytokine that influences the function of T cells, NK cells, and B cells. In this study, we report that IL-21 plays a major role in stimulating the differentiation of human B cells. When human B cells were stimulated through the BCR, IL-21 induced minimal proliferation, IgD down-modulation, and small numbers of plasma cells. In contrast, after CD40 engagement, IL-21 induced extensive proliferation, class switch recombination (CSR), and plasma cell differentiation. Upon cross-linking both BCR and CD40, IL-21 induced the largest numbers of plasma cells. IL-21 drove both postswitch memory cells as well as poorly responsive naive cord blood B cells to differentiate into plasma cells. The effect of IL-21 was more potent than the combination of IL-2 and IL-10, especially when responsiveness of cord blood B cells was examined. IL-21 costimulation potently induced the expression of both B lymphocyte-induced maturation protein-1 (BLIMP-1) and activation-induced cytidine deaminase as well as the production of large amounts of IgG from B cells. Despite the induction of activation-induced cytidine deaminase and CSR, IL-21 did not induce somatic hypermutation. Finally, IL-2 enhanced the effects of IL-21, whereas IL-4 inhibited IL-21-induced plasma cell differentiation. Taken together, our data show that IL-21 plays a central role in CSR and plasma cell differentiation during T cell-dependent B cell responses.     

The tachykinins substance P and hemokinin-1 favor the generation of human memory Th17 cells by inducing IL-1β, IL-23, and TNF-like 1A expression by monocytes

The nervous system influences immune responses through the release of neural factors such as neuropeptides. Among them, the tachykinin substance P (SP) signals via the neurokinin 1 receptor (NK-1R), which is expressed by various immune cells. We thereby analyzed in this paper whether tachykinins may participate in human CD4(+) Th cell polarization. We report that SP and hemokinin-1 (HK-1) upregulate IL-17A and IFN-γ production by human memory CD4(+) T cells without affecting IL-4 and IL-10 production. SP and HK-1 switch non-Th17-committed CD4(+) memory T cells into bona fide Th17 cells and Th1/Th17 cells. In contrast, SP and HK-1 do not modulate the polarization of naive CD4(+) T cells. SP- and HK-1-induced Th17 cell generation is mediated through NK-1R and requires the presence of monocytes. SP and HK-1 trigger IL-1β, IL-6, and TNF-α production, upregulate IL-23 production, and enhance TNF-like 1A expression on monocyte surface. Neutralization experiments demonstrated that IL-1β, IL-23, and TNF-like 1A are involved in the SP- and HK-1-induced Th17 cell. The other members of the tachykinin family, neurokinins A and B, have no effect on the differentiation of naive and memory T cells. These results thereby show that SP and HK-1 are novel Th17 cell-inducing factors that may act locally on memory T cells to amplify inflammatory responses.     

Regulation of immune function by calorie restriction and cyclophosphamide treatment in lupus-prone NZB/NZW F1 mice

We compared the effects of calorie restriction (CR) and cyclophosphamide (CTX) on the progression of lupus nephritis and immunological changes in NZB/NZW F1 mice. Ad libitum (AL)/CTX and CR delayed onset of proteinuria and significantly decreased serum levels of anti-dsDNA, anti-histone, and circulating immune complex antibodies. CTX and CR prevented the increase in and activation of B cells, the decline in CD8(+) T cells, and maintained a higher proportion of na?ve CD4(+) and CD8(+) cells. MHC class I antigen and LFA-1 expression on CD8(+) T cells and MHC class II antigen on B cells were also decreased. AL/CTX and CR prevented the increase in production of IL-10 and up-regulated IL-2 production in T cells ex vivo. We concluded that both CR and CTX can delay the onset of autoimmune disease, in part by maintaining higher numbers of na?ve T cells and the immune responsiveness of T cells and decreasing the proportion of B cells.     

IgM and IgG but not cytokine secretion is restricted to the CD27+ B lymphocyte subset.

In a recent study we reported that CD27 is expressed on a subpopulation of human B lymphocytes and presented circumstantial phenotypic evidence that CD27 expression may be acquired late during B cell differentiation. Here we present functional data showing that, after in vitro stimulation, CD27+ but not CD27- B cells secrete large amounts of both IgM and IgG. Using double immunofluorescence staining of CD27 and IgD, three functionally different B cell subsets representing distinct stages of B cell differentiation can be isolated: 1) the CD27- IgD+ B cells, which do not secrete appreciable Ig; 2) the CD27+IgD+ B cells, which exclusively secrete IgM; and 3) the CD27+IgD- B cells, which comprise the IgG-producing cells. Furthermore, costimulation of CD27- B cells with low m.w. B cell growth factor, in the presence or in the absence of a CD40 mAb, does not induce these cells to become Ig-secreting cells. Although CD27- B cells hardly secrete Ig of any isotype in response to Staphylococcus aureus+IL-2, these cells proliferate vigorously and express the IL-2R alpha chain (CD25) under these stimulatory conditions. Furthermore, both CD27- and CD27+ B cells are capable of producing similar amounts of IL-6 and TNF-alpha. Taken together, these findings indicate that CD27 is a unique non-Ig surface marker discriminating naive from primed B lymphocytes. Furthermore, the capacity to proliferate and to secrete the B cell differentiation factors IL-6 and TNF-alpha already exists at an early B cell differentiation stage at which the cells lack CD27 expression and are not induced to produce Ig.     

1,25-dihydroxyvitamin D3 impairs NF-κB activation in human naïve B cells

1α,25-dihydroxyvitamin D(3) (calcitriol), the bioactive metabolite of vitamin D, modulates the activation and inhibits IgE production of anti-CD40 and IL-4 stimulated human peripheral B cells. Engagement of CD40 results in NF-κB p50 activation, which is essential for the class switch to IgE. Herein, we investigated by which mechanism calcitriol modulates NF-κB mediated activation of human na?ve B cells. Na?ve B cells were predominantly targeted by calcitriol in comparison with memory B cells as shown by pronounced induction of the VDR target gene cyp24a1. Vitamin D receptor activation resulted in a strongly reduced p105/p50 protein and mRNA expression in human na?ve B cells. This effect is mediated by impaired nuclear translocation of p65 and consequently reduced binding of p65 to its binding site in the p105 promoter. Our data indicate that the vitamin D receptor reduces NF-κB activation by interference with NF-κB p65 and p105. Thus, the vitamin D receptor inhibits costimulatory signal transduction in na?ve B cells, namely by reducing CD40 signaling.     

TSC1/2 signaling complex is essential for peripheral naïve CD8+ T cell survival and homeostasis in mice

The PI3K-Akt-mTOR pathway plays crucial roles in regulating both innate and adaptive immunity. However, the role of TSC1, a critical negative regulator of mTOR, in peripheral T cell homeostasis remains elusive. With T cell-specific Tsc1 conditional knockout (Tsc1 KO) mice, we found that peripheral na?ve CD8(+) T cells but not CD4(+) T cells were severely reduced. Tsc1 KO na?ve CD8(+) T cells showed profound survival defect in an adoptive transfer model and in culture with either stimulation of IL-7 or IL-15, despite comparable CD122 and CD127 expression between control and KO CD8(+) T cells. IL-7 stimulated phosphorylation of Akt(S473) was diminished in Tsc1 KO na?ve CD8(+)T cells due to hyperactive mTOR-mediated feedback suppression on PI3K-AKT signaling. Furthermore, impaired Foxo1/Foxo3a phosphorylation and increased pro-apoptotic Bim expression in Tsc1 KO na?ve CD8(+)T cells were observed upon stimulation of IL-7. Collectively, our study suggests that TSC1 plays an essential role in regulating peripheral na?ve CD8(+) T cell homeostasis, possible via an mTOR-Akt-FoxO-Bim signaling pathway.     

An indispensable role for STAT1 in IL-27-induced T-bet expression but not proliferation of naive CD4+ T cells

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation, induces proliferation of naive CD4+ T cells, and synergizes with IL-12 in IFN-gamma production. It has been recently reported that IL-27 induces T-bet and IL-12Rbeta2 expression through JAK1/STAT1 activation. In the present study, we further investigated the JAK/STAT signaling molecules activated by IL-27 and also the role of STAT1 in IL-27-mediated responses using STAT1-deficient mice. In addition to JAK1 and STAT1, IL-27-activated JAK2, tyrosine kinase-2, and STAT2, -3, and -5 in naive CD4+ T cells. The activation of STAT2 and STAT5, but not of STAT3, was greatly diminished in STAT1-deficient naive CD4+ T cells. Comparable proliferative response to IL-27 was observed between STAT1-deficient and wild-type naive CD4+ T cells. In contrast, IL-27 hardly induced T-bet and subsequent IL-12Rbeta2 expression, and synergistic IFN-gamma production by IL-27 and IL-12 was impaired in STAT1-deficient naive CD4+ T cells. Moreover, IL-27 augmented the expression of MHC class I on naive CD4+ T cells in a STAT1-dependent manner. These results suggest that IL-27 activates JAK1 and -2, tyrosine kinase-2, STAT1, -2, -3, and -5 in naive CD4+ T cells and that STAT1 plays an indispensable role in IL-27-induced T-bet and subsequent IL-12Rbeta2 expression and MHC class I expression as well but not proliferation, while STAT3 presumably plays an important role in IL-27-induced proliferation.     

Augmentation of effector CD8+ T cell generation with enhanced granzyme B expression by IL-27

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation. We have recently demonstrated that IL-27 has a potent antitumor activity, which is mainly mediated through CD8+ T cells, and also has an adjuvant activity to induce epitope-specific CTL in vivo. In this study, we further investigated the in vitro effect of IL-27 on CD8+ T cells of mouse spleen cells. In a manner similar to CD4+ T cells, IL-27 activated STAT1, -2, -3, -4, and -5, and augmented the expression of T-bet, IL-12Rbeta2, and granzyme B, and slightly that of perforin in naive CD8+ T cells stimulated with anti-CD3. IL-27 induced synergistic IFN-gamma production with IL-12 and proliferation of naive CD8+ T cells. Moreover, IL-27 enhanced proliferation of CD4+ T cell-depleted spleen cells stimulated by allogeneic spleen cells and augmented the generation of CTL. In STAT1-deficient naive CD8+ T cells, IL-27-induced proliferation was not reduced, but synergistic IFN-gamma production with IL-12 was diminished with decreased expression of T-bet, IL-12Rbeta2, granzyme B, and perforin. In T-bet-deficient naive CD8+ T cells, IL-27-induced proliferation was hardly reduced, but synergistic IFN-gamma production with IL-12 was diminished with decreased expression of IL-12Rbeta2, granzyme B, and perforin. However, IL-27 still augmented the generation of CTL from T-bet-deficient CD4+ T cell-depleted spleen cells stimulated by allogeneic spleen cells with increased granzyme B expression. These results suggest that IL-27 directly acts on naive CD8+ T cells in T-bet-dependent and -independent manners and augments generation of CTL with enhanced granzyme B expression.     

Homeostasis of naive and memory CD4+ T cells: IL-2 and IL-7 differentially regulate the balance between proliferation and Fas-mediated apoptosis

Cytokines play a crucial role in the maintenance of polyclonal naive and memory T cell populations. It has previously been shown that ex vivo, the IL-7 cytokine induces the proliferation of naive recent thymic emigrants (RTE) isolated from umbilical cord blood but not mature adult-derived naive and memory human CD4(+) T cells. We find that the combination of IL-2 and IL-7 strongly promotes the proliferation of RTE, whereas adult CD4(+) T cells remain relatively unresponsive. Immunological activity is controlled by a balance between proliferation and apoptotic cell death. However, the relative contributions of IL-2 and IL-7 in regulating these processes in the absence of MHC/peptide signals are not known. Following exposure to either IL-2 or IL-7 alone, RTE, as well as mature naive and memory CD4(+) T cells, are rendered only minimally sensitive to Fas-mediated cell death. However, in the presence of the two cytokines, Fas engagement results in a high level of caspase-dependent apoptosis in both RTE as well as naive adult CD4(+) T cells. In contrast, equivalently treated memory CD4(+) T cells are significantly less sensitive to Fas-induced cell death. The increased susceptibility of RTE and naive CD4(+) T cells to Fas-induced apoptosis correlates with a significantly higher IL-2/IL-7-induced Fas expression on these T cell subsets than on memory CD4(+) T cells. Thus, IL-2 and IL-7 regulate homeostasis by modulating the equilibrium between proliferation and apoptotic cell death in RTE and mature naive and memory T cell subsets.     

Tacrolimus potently inhibits human osteoclastogenesis induced by IL-17 from human monocytes alone and suppresses human Th17 differentiation

Tacrolimus (FK506, Prograf?) is an orally available, T cell specific and anti-inflammatory agent that has been proposed as a therapeutic drug in rheumatoid arthritis (RA) patients. It has been known that T cells have a critical role in the pathogenesis of RA. Recent studies suggest that Th17 cells, which mainly produce IL-17, are involved in many autoimmune inflammatory disease including RA. The present study was undertaken to assess the effect of tacrolimus on IL-17-induced human osteoclastogenesis and human Th17 differentiation. Human CD14(+) monocytes were cultured in the presence of macrophage-colony stimulating factor (M-CSF) and IL-17. From day 4, tacrolimus was added to these cultures. Osteoclasts were immunohistologically stained for vitronectin receptor 10days later. IL-17 production from activated T cells stimulated with IL-23 was measured by enzyme-linked immunosorbent assay (ELISA). Th17 differentiation from na?ve T cells was assayed by flow cytometry. Tacrolimus potently inhibited IL-17-induced osteoclastogenesis from human monocytes and osteoclast activation. Addition of tacrolimus also reduced production of IL-17 in human activated T cells stimulated with IL-23. Interestingly, the population of human IL-17(+)IFN-γ(-) CD4 T cells or IL-17(+)TNF-α(+) CD4 T cells were decreased by adding of tacrolimus. The present study demonstrates that the inhibitory effect of tacrolimus on IL-17-induced osteoclastogenesis from human monocytes. Tacrolimus also inhibited expression of IL-17 or TNF-α by reducing the proportion of Th17, suggesting that therapeutic effect on Th17-associated disease such as RA, inflammatory bowel disease, multiple sclerosis, psoriasis, or allograft rejection.     

CpG oligodeoxynucleotides discriminately enhance binding capacity of human naïve B cells to Hepatitis B virus epitopes

CpG oligodeoxynucleotides (CpG ODN) have the potential to enhance the antigen-presenting cells function of human na?ve B cells. In this study, we aim to define the effect of CpG ODNs on the binding capacity of human na?ve B cells for different Hepatitis B virus (HBV) epitopes. Three HLA-A2 restricted epitopes were selected to incubate with CpG ODN-primed human na?ve B cells. Binding capacity for each epitope and expression of CD80, CD86, class I major histocompatibility complex (MHC), and class II MHC of na?ve B cells was tested, respectively, by flow cytometry. CpG ODNs, especially ODN 2216, enhanced the binding capacity of human na?ve B cells for HBV epitopes (p < 0.01), and induced markedly higher expression of CD80, CD86, class I MHC, and class II MHC. The binding capacity of CpG-treated naive B cells for each epitope was significantly different. In all the 3 subjects, CpG ODN 2216-primed na?ve B cells showed the highest binding ability for Env172-180 compared with the other epitopes with a high expression of co-stimulatory and MHC molecules. CpG ODN showed the potential to selectively enhance the binding capacity of human na?ve B cells for HBV epitopes. These results suggest new strategies for development of vaccine design.