Anti-CD180 (RP105) activates B cells to rapidly produce polyclonal Ig via a T cell and MyD88-independent pathway

CD180 is homologous to TLR4 and regulates TLR4 signaling, yet its function is unclear. We report that injection of anti-CD180 mAb into mice induced rapid Ig production of all classes and subclasses, with the exception of IgA and IgG2b, with up to 50-fold increases in serum IgG1 and IgG3. IgG production after anti-CD180 injection was not due to reactivation of memory B cells and was retained in T cell-deficient (TCR knockout [KO]), CD40 KO, IL-4 KO, and MyD88 KO mice. Anti-CD180 rapidly increased both transitional and mature B cells, with especially robust increases in transitional B cell number, marginal zone B cell proliferation, and CD86, but not CD80, expression. In contrast, anti-CD40 induced primarily follicular B cell and myeloid expansion, with increases in expression of CD80 and CD95 but not CD86. The expansion of splenic B cells was due, in part, to proliferation and occurred in wild-type and TCR KO mice, whereas T cell expansion occurred in wild-type, but not in B cell-deficient, mice, indicating a direct role for B cells in CD180 stimulation in vivo. Combination of anti-CD180 with various MyD88-dependent TLR ligands biased B cell fate because coinjection diminished Ig production, but purified B cells exhibited synergistic proliferation. Anti-CD180 had no effect on cytokine production from B cells, but it increased IL-6, IL-10, and TNF-α production in combination with LPS or CpG. Thus, CD180 stimulation induces intrinsic B cell proliferation and differentiation, causing rapid increases in IgG, and integrates MyD88-dependent TLR signals to regulate proliferation, cytokine production, and differentiation.     

Superantigen-induced CD4 memory T cell anergy. I. Staphylococcal enterotoxin B induces Fyn-mediated negative signaling

Memory CD4 T cells must provide robust protection for an organism while still maintaining self-tolerance. Superantigens reveal a memory cell-specific regulatory pathway, by which signaling through the TCR can lead to clonal tolerance (anergy). Here we show that the src kinase Fyn is a critical regulator of anergy in murine memory CD4 T cells induced by the bacterial superantigen staphylococcal enterotoxin B (SEB). Exposure to SEB results in impaired TCR signaling due to failed CD3/ZAP-70 complex formation. Further, signal transduction through the TCR remains similarly blocked when anergic memory cells are subsequently exposed to agonist peptide antigen. Pharmacological inhibition or genetic elimination of Fyn kinase reverses memory cell anergy, resulting in SEB-induced cell proliferation. The mechanism underlying impaired TCR signaling and subsequent memory cell anergy must involve a Fyn signaling pathway given that the suppression of Fyn activity restores CD3/ZAP-70 complex formation and TCR proximal signaling.     

p59fyn (Fyn) promotes the survival of anergic CD4-CD8- alpha beta TCR+ cells but negatively regulates their proliferative response to antigen stimulation

T cell anergy is characterized by alterations in TCR signaling that may play a role in controlling the unresponsiveness of the anergic cell. We have addressed questions regarding the importance of the Src kinase p59(fyn) (Fyn) in this process by using Fyn null mice. We demonstrate that a mature population of CD4(-)CD8(-) alphabeta TCR(+) anergic T cells lacking Fyn have a substantial recovery of their proliferation defect in response to Ag stimulation. This recovery cannot be explained by ameliorated production of IL-2, and the improved proliferation correlates with an enhanced ability of the Fyn(-/-) anergic T cells to up-regulate the high affinity IL-2 receptor. We also observe that anergic CD4(-)CD8(-) alphabeta TCR(+) T cells have a heightened survival ability that is partially dependent on the elevated levels of Fyn and IL-2 receptor beta-chain expressed by these cells. The enhanced survival correlates with an increased capacity of the anergic cells to respond to IL-15. We conclude that Fyn plays an important role in aspects of T cell anergy pertaining to TCR signaling and to cell survival.     

Lck和Fyn对T细胞发育过程的影响

胸腺中T细胞的发育及次级淋巴组织中成熟T细胞的活化均需要细胞能够对环境信号分子做出适应性的反应。在共刺激分子及细胞因子受体介导的信号参与下通过TCR(T cell receptor)及其辅助受体CD4和CD8与MHC/抗原肽复合物相互作用,可以诱导TCR信号通路激活并最终导致T细胞免疫反应的发生。Src家族激酶Lck(Lymphocyte-specific protein tyrosine kinase)和Fyn(Proto-oncogene tyrosine-protein kinase)的激活是启动TCR信号通路的关键因素。在T细胞的发育、阳性选择、初始T细胞的外周存活及由淋巴细胞缺失诱导的细胞增殖中都起着关键性的作用。研究显示,虽然这两种信号分子紧密相关,但在某些条件下Lck发挥着比Fyn更重要的作用,并且Fyn仅可以补充Lck的部分功能。文章针对这两个激酶在T细胞发育的整个过程中的作用机制进行了论述。     

Lck和Fyn对T细胞发育过程的影响

胸腺中T细胞的发育及次级淋巴组织中成熟T细胞的活化均需要细胞能够对环境信号分子做出适应性的反应。在共刺激分子及细胞因子受体介导的信号参与下通过TCR(T cell receptor )及其辅助受体CD4和CD8与MHC/抗原肽复合物相互作用, 可以诱导TCR信号通路激活并最终导致T细胞免疫反应的发生。Src家族激酶Lck(Lymphocyte-specific protein tyrosine kinase)和Fyn (Proto-oncogene tyrosine-protein kinase)的激活是启动TCR信号通路的关键因素。在T细胞的发育、阳性选择、初始T细胞的外周存活及由淋巴细胞缺失诱导的细胞增殖中都起着关键性的作用。研究显示, 虽然这两种信号分子紧密相关, 但在某些条件下Lck发挥着比Fyn更重要的作用, 并且Fyn仅可以补充Lck的部分功能。文章针对这两个激酶在T细胞发育的整个过程中的作用机制进行了论述。     

Fyn regulates the duration of TCR engagement needed for commitment to effector function

In naive T cells, engagement of the TCR with agonist peptide:MHC molecules leads to phosphorylation of key intracellular signaling intermediates within seconds and this peaks within minutes. However, the cell does not commit to proliferation and IL-2 cytokine production unless receptor contact is sustained for several hours. The biochemical basis for this transition to full activation may underlie how T cells receive survival signals while maintaining tolerance, and is currently not well understood. We show here that for CD8 T cells commitment to proliferation and cytokine production requires sustained activation of the Src family kinase Lck and is opposed by the action of Fyn. Thus, in the absence of Fyn, commitment to activation occurs more rapidly, the cells produce more IL-2, and undergo more rounds of division. Our data demonstrate a role for Fyn in modulating the response to Ag in primary T cells.     

Central role for TCR/CD3 ligation in the differentiation of CD4+ T cells toward A Th1 or Th2 functional phenotype.

Activated CD4+ T cells can be classified into distinct subsets; the most divergent among them may be considered to be the IL-2 and IFN-gamma-producing Th1 clones and the IL-4 and IL-5-producing Th2 clones. Because Th1 and Th2 clones can usually be detected only after several months of culture, we used conditions that modulate the IL-2 and IL-4 production in short term culture. Here we show that freshly isolated and subsequently in vitro-activated CD4+ T cells that were cultured for 11 days with rIL-2 and restimulated showed a IFN-gamma+ IL-2+ IL-3+ IL-4- IL-5- pattern. Because these cells were not capable of providing B cell help for IgG1, IgG2a, or IgE in an APC- and TCR-dependent T-B cell assay, they expressed a phenotype typical for most Th1 clones. In contrast, activated T cells that were cultured for 11 days with IL-2 plus a mAb to CD3 and then restimulated produced a IFN-gamma- IL-2- IL-3+ IL-4+ IL-5+ pattern. These cells were capable of providing B cell help for IgG1, IgG2a, and IgE synthesis and thus presented a phenotype typical for Th2 clones. Similar results were observed when mitogenic mAb to Thy-1.2 or to framework determinants of the alpha beta TCR were used. The induction of Th1- and Th2-like cells did not depend on the relative expression of CD44 or CD45 by the T cells before activation in vitro. Because the incubation of activated T cells with anti-CD3/TCR mAb induced high unrestricted lymphokine production, the latter might be responsible for the Th2-like lymphokine pattern observed after restimulation. To address this point, TCR V beta 8+ and V beta 8- T cell blasts were co-cultured in the presence of mAb to V beta 8. After restimulation, V beta 8+ cells had a IL-4high IL-2low phenotype and V beta 8- cells had a IL-4low IL-2high phenotype. This demonstrates that TCR ligation but not lymphokines alone are capable of inducing Th2-like cells, and this points out a central role for the TCR in the generation of T cell subsets.     

Anergy in CD4 memory T lymphocytes. II. Abrogation of TCR-induced formation of membrane signaling complexes

Memory and naive CD4 T cells have unique regulatory pathways for self/non-self discrimination. A memory cell specific regulatory pathway was revealed using superantigens to trigger the TCR. Upon stimulation by bacterial superantigens, like staphylococcal enterotoxin B (SEB), TCR proximal signaling is impaired leading to clonal tolerance (anergy). In the present report, we show that memory cell anergy results from the sequestration of the protein tyrosine kinase ZAP-70 away from the TCR/CD3ζ chain. During SEB-induced signaling, ZAP-70 is excluded from both detergent-resistant membrane microdomains and the immunological synapse, thus blocking downstream signaling. We also show that the mechanism underlying memory cell anergy must involve Fyn kinase, given that the suppression of Fyn activity restores the movement of ZAP-70 to the immunological synapse, TCR proximal signaling, and cell proliferation. Thus, toleragens, including microbial toxins, may modulate memory responses by targeting the organizational structure of memory cell signaling complexes.     

Lyn but not Fyn kinase controls IgG-mediated systemic anaphylaxis

Anaphylaxis is a rapid, life-threatening hypersensitivity reaction. Until recently, it was mainly attributed to histamine released by mast cells activated by allergen crosslinking (XL) of FcεRI-bound allergen-specific IgE. However, recent reports established that anaphylaxis could also be triggered by basophil, macrophage, and neutrophil secretion of platelet-activating factor subsequent to FcγR stimulation by IgG/Ag complexes. We have investigated the contribution of Fyn and Lyn tyrosine kinases to FcγRIIb and FcγRIII signaling in the context of IgG-mediated passive systemic anaphylaxis (PSA). We found that mast cell IgG XL induced Fyn, Lyn, Akt, Erk, p38, and JNK phosphorylation. Additionally, IgG XL of mast cells, basophils, and macrophages resulted in Fyn- and Lyn-regulated mediator release in vitro. FcγR-mediated activation was enhanced in Lyn-deficient (knockout [KO]) cells, but decreased in Fyn KO cells, compared with wild-type cells. More importantly, Lyn KO mice displayed significantly exacerbated PSA features whereas no change was observed for Fyn KO mice, compared with wild-type littermates. Intriguingly, we establish that mast cells account for most serum histamine in IgG-induced PSA. Taken together, our findings establish pivotal roles for Fyn and Lyn in the regulation of PSA and highlight their unsuspected functions in IgG-mediated pathologies.     

IL-10-induced anergy in peripheral T cell and reactivation by microenvironmental cytokines: two key steps in specific immunotherapy.

Specific immunotherapy (SIT) is widely used for treatment of allergic diseases and could potentially be applied in other immunological disorders. Induction of specific unresponsiveness (anergy) in peripheral T cells and recovery by cytokines from the tissue microenvironment represent two key steps in SIT with whole allergen or antigenic T cell peptides (PIT). The anergy is directed against the T cell epitopes of the respective antigen and characterized by suppressed proliferative and cytokine responses. It is initiated by autocrine action of IL-10, which is increasingly produced by the antigen-specific T cells. Later in therapy, B cells and monocytes also produce IL-10. The anergic T cells can be reactivated by different cytokines. Whereas IL-15 and IL-2 generate Th1 cytokine profile and an IgG4 antibody response, IL-4 reactivates a Th2 cytokine pattern and IgE antibodies. Increased IL-10 suppresses IgE and enhances IgG4 synthesis, resulting in a decreased antigen-specific IgE:IgG4 ratio, as observed normally in patients after SIT or PIT. The same state of anergy against the major bee venom allergen, phospholipase A2, can be observed in subjects naturally anergized after multiple bee stings. Together, these data demonstrate the pivotal role of autocrine IL-10 in induction of specific T cell anergy and the important participation of the cytokine microenvironment in SIT. Furthermore, knowledge of the mechanisms explaining reasons for success or failure of SIT may enable possible predictive measures of the treatment.     

Impaired B cell development and function in the absence of IkappaBNS

IκBNS has been identified as a member of the IκB family of NF-κB inhibitors, which undergoes induction upon TCR signaling. Mice carrying a targeted gene disruption of IκBNS demonstrate dysregulation of cytokines in T cells, macrophages, and dendritic cells. IκBNS mediates both positive and negative gene regulation, depending on individual cell type and/or cytokine. In this study, we demonstrate an additional role for IκBNS in the B cell lineage. B cells from IκBNS knockout (KO) mice were impaired in proliferative responses to LPS and anti-CD40. IgM and IgG3 Igs were drastically reduced in the serum of IκBNS KO mice, although IκBNS KO B cells exhibited a higher level of surface IgM than that found in wild-type mice. Switching to IgG3 was significantly reduced in IκBNS KO B cells. The in vitro induction of plasma cell development demonstrated that progression to Ab-secreting cells was impaired in IκBNS KO B cells. In agreement with this finding, the number of Ab-secreting cells in the spleens of IκBNS KO mice was reduced and production of Ag-specific Igs was lower in IκBNS KO mice after influenza infection as compared with wild-type mice. Additionally, IκBNS KO mice lacked B1 B cells and exhibited a reduction in marginal zone B cells. Thus, IκBNS significantly impacts the development and functions of B cells and plasma cells.     

T cell substance P receptor governs antigen-elicited IFN-gamma production

Substance P (SP) enhances antigen-dependent T cell IFN-gamma production. It was determined if a T cell neurokinin-1 receptor (NK-1R) was critical for IFN-gamma regulation. T cells from schistosome-infected mice were mixed with splenocytes from uninfected NK-1R knockout (KO) animals. Thus only the schistosome egg antigen-specific T cells expressed NK-1R. The cells were cultured 18 h with or without SP. SP enhanced antigen-induced IFN-gamma production fourfold without affecting IL-4 or IL-5 secretion. NK-1R inhibitor blocked this stimulation. Neither purified T cells nor naive KO splenocytes cultured alone responded to antigen. To further define the importance of T cell NK-1R, we developed a T cell-selective NK-1R KO mouse by reconstituting T cell-deficient Rag mice with NK-1R KO T cells. These mice challanged with schistosomiasis developed abnormal liver granulomas. Granuloma size was smaller in T cell-selective NK-1R KO mice compared with granulomas in Rag reconstituted with normal T cells. Splenocytes and granuloma cells from NK-1R KO mice made less IFN-gamma. The mice also made less IgG2a. Thus T cell NK-1R is important for IFN-gamma regulation.     

Differential T-cell antigen receptor signaling mediated by the Src family kinases Lck and Fyn

Src family tyrosine kinases play a key role in T-cell antigen receptor (TCR) signaling. They are responsible for the initial tyrosine phosphorylation of the receptor, leading to the recruitment of the ZAP-70 tyrosine kinase, as well as the subsequent phosphorylation and activation of ZAP-70. Molecular and genetic evidence indicates that both the Fyn and Lck members of the Src family can participate in TCR signal transduction; however, it is unclear to what extent they utilize the same signal transduction pathways and activate the same downstream events. We have addressed this issue by examining the ability of Fyn to mediate TCR signal transduction in an Lck-deficient T-cell line (JCaM1). Fyn was able to induce tyrosine phosphorylation of the TCR and recruitment of the ZAP-70 kinase, but the pattern of TCR phosphorylation was altered and activation of ZAP-70 was defective. Despite this, the SLP-76 adapter protein was inducibly tyrosine phosphorylated, and both the Ras-mitogen-activated protein kinase and the phosphatidylinositol 4, 5-biphosphate signaling pathways were activated. TCR stimulation of JCaM1/Fyn cells induced the expression of the CD69 activation marker and inhibited cell growth, but NFAT activation and the production of interleukin-2 were markedly reduced. These results indicate that Fyn mediates an alternative form of TCR signaling which is independent of ZAP-70 activation and generates a distinct cellular phenotype. Furthermore, these findings imply that the outcome of TCR signal transduction may be determined by which Src family kinase is used to initiate signaling.     

SKAP55 recruits to lipid rafts and positively mediates the MAPK pathway upon T cell receptor activation

T cell receptor (TCR) engagement triggers a series of events including protein tyrosine kinase activation, tyrosine phosphorylation of adapter proteins, and multiple protein-protein interactions. We observed that adapter protein SKAP55, the Src kinase-associated phosphoprotein, formed homodimers through its SH3 domain and SK region. SKAP55 as a substrate interacted with Fyn kinase in vivo. In Jurkat cells, interaction between SKAP55 and Fyn kinase depended on TCR activation. Stable overexpression of SKAP55 in Jurkat cells caused mitogen-activated protein kinase activation following TCR engagement. Anti-CD3 stimulation also promoted the interaction of SKAP55 with Grb-2 in T cells. Mutational analysis revealed that tyrosine 271 in SKAP55 played a pivotal role for interaction with both Fyn kinase and adapter protein Grb-2, indicating that the Fyn-phosphorylated SKAP55 transiently associates with adapter Grb-2 to mediate mitogen-activated protein kinase activation. Intriguingly, T cell receptor engagement dramatically induced the translocation of endogenous SKAP55 to lipid rafts where SKAP55 was found to interact with Fyn kinase, suggesting that the positive function of SKAP55 via its association with Fyn and other signaling components may have been involved in raft-mediated T cell activation.     

Genetic evidence for Lyn as a negative regulator of IL-4 signaling.

IL-4 has multiple effects on B lymphocytes, many of which are concentration dependent. This is particularly so for Ig isotype switching, where different thresholds of IL-4 stimulation are needed to induce switching from IgM to either IgG1 or IgE. In this report we describe a critical role for the tyrosine kinase Lyn in setting IL-4 signaling thresholds in mouse B lymphocytes. Upon CD40 ligand stimulation of lyn-/- B cells, 10-fold less IL-4 was required to induce switching from IgM to IgG1 and IgE and an increased proportion of B cells isotype switched at each IL-4 concentration. These in vitro results correlate with the in vivo findings that in lyn-/- mice, IgG1 Ab-forming cells develop prematurely in ontogeny and that adult lyn-/- mice have an abnormally high proportion of IgG1-expressing B cells in their spleens. Adult lyn-/- mice also have significantly higher levels of IgE in their serum. These results identify Lyn as a molecule involved in modulating the IL-4 signal in B cells and provide insights into its regulation and how a B cell signaling imbalance may contribute to atopy.     

Normal development and activation but altered cytokine production of Fyn-deficient CD4+ T cells

The Src family kinase Fyn is expressed in T cells and has been shown to phosphorylate proteins involved in TCR signaling, cytoskeletal reorganization, and IL-4 production. Fyn-deficient mice have greatly decreased numbers of NKT cells and have thymocytes and T cells with compromised responses following Ab crosslinking of their TCRs. Herein we have addressed the role of Fyn in peptide/MHC class II-induced CD4(+) T cell responses. In Fyn-deficient mice, CD4(+) T cells expressing the DO11.10 TCR transgene developed normally, and the number and phenotype of naive and regulatory DO11.10(+)CD4(+) T cells in the periphery were comparable with their wild-type counterparts. Conjugation with chicken OVA peptide 323-339-loaded APCs, and the subsequent proliferation in vitro or in vivo of DO11.10(+) Fyn-deficient CD4(+) T cells, was virtually indistinguishable from the response of DO11.10(+) wild-type CD4(+) T cells. Proliferation of Fyn-deficient T cells was not more dependent on costimulation through CD28. Additionally, we have found that differentiation, in vitro or in vivo, of transgenic CD4(+) Fyn-deficient T cells into IL-4-secreting effector cells was unimpaired, and under certain conditions DO11.10(+) Fyn-deficient CD4(+) T cells were more potent cytokine-producing cells than DO11.10(+) wild-type CD4(+) T cells. These data demonstrate that ablation of Fyn expression does not alter most Ag-driven CD4(+) T cell responses, with the exception of cytokine production, which under some circumstances is enhanced in Fyn-deficient CD4(+) T cells.     

Defective T cell receptor-mediated signal transduction in memory CD4 T lymphocytes exposed to superantigen or anti-T cell receptor antibodies

Lymphocytes must promote protective immune responses while still maintaining self-tolerance. Stimulation through the T cell receptor (TCR) can lead to distinct responses in naive and memory CD4 T cells. Whereas peptide antigen stimulates both naive and memory T cells, soluble anti-CD3 antibodies and bacterial superantigens stimulate only naive T cells to proliferate and secrete cytokines. Further, superantigens, like staphylococcal enterotoxin B (SEB), cause memory T cells to become anergic while soluble anti-CD3 does not. In the present report, we show that signal transduction through the TCR is impaired in memory cells exposed to either anti-CD3 or SEB. A block in signaling leads to impaired activation of the kinase ZAP-70 so that downstream signals and cell proliferation do not occur. We further show that the signaling defect is unique to each agent. In anti-CD3-treated memory T cells, the src kinase Lck is only transiently activated and does not phosphorylate and activate ZAP-70. In SEB-treated memory T cells, ZAP-70 does not interact with the TCR/CD3 complex to become accessible to Lck. Finally, we provide evidence that alternative signaling pathways are initiated in SEB-treated memory cells. Altered signaling, indicated by an elevation in activity of the src kinase Fyn, may be responsible for memory cell anergy caused by SEB. Thus, differentiation of naive T cells into memory cells is accompanied by alterations in TCR-mediated signaling that can promote heightened recall immunity or specific tolerance.     

CD40 stimulation provides an IFN-gamma-independent and IL-4-dependent differentiation signal directly to human B cells for IgE production.

IgE induction from human cells has generally been considered to be T cell dependent and to require at least two signals: IL-4 stimulation and T cell/B cell interaction. In the present study we report a human system of T cell-independent IgE production from highly purified B cells. When human cells were co-stimulated with a mAb directed against CD40 (mAb G28-5), there was induction of IgE secretion from purified blood and tonsil B cells as well as unfractionated lymphocytes. Anti-CD40 alone failed to induce IgE from blood mononuclear cells or purified B cells. The effect of the combination of anti-CD40 and IL-4 on IgE production was very IgE isotype specific as IgG, IgM, and IgA were not increased. Furthermore, anti-CD40 with IL-5 or PWM did not co-stimulate IgG, IgM, or IgA and in fact strongly inhibited PWM-stimulated IgG, IgM and IgA production from blood or tonsil cells. IgE synthesis induced by anti-CD40 plus IL-4 was IFN-gamma independent as is the in vivo production of IgE in humans; the doses of IFN-gamma that profoundly suppressed IgG synthesis induced by IL-4, or IL-4 plus IL-6, had no inhibitory effect on anti-CD40-induced IgE production. Anti-CD23 and anti-IL-6 also could not block anti-CD40 plus IL-4-induced IgE production, but anti-IL-4 totally blocked their effect. IgE production via CD40 was not due to IL-5, IL-6 or nerve growth factor as none of these synergized with IL-4 to induce IgE synthesis by purified B cells. Finally, we observed that CD40 stimulation alone could enhance IgE production from in vivo-driven IgE-producing cells from patients with very high IgE levels; cells that did not increase IgE production in response to IL-4. Taken together, our data suggest that the signals delivered for IgE production by IL-4 and CD40 stimulation may mimic the pathway for IgE production seen in vivo in human allergic disease.