CD1d-restricted help to B cells by human invariant natural killer T lymphocytes

Invariant natural killer T (NKT) cells are a highly conserved subset of T lymphocytes expressing a semi-invariant T cell receptor (TCR), which is restricted to CD1d and specific for the glycosphingolipid antigen alpha-galactosylceramide. Their ability to secrete a variety of cytokines, which in turn modulate the activation of cells of both innate and acquired immune responses, suggests that invariant NKT cells exert a regulatory role mainly via indirect mechanisms. A relevant question is whether invariant NKT cells can directly help B cells. We document here that human invariant NKT cells are as efficient as conventional CD4+ Th0 lymphocytes in promoting proliferation of autologous memory and naive B lymphocytes in vitro, and in inducing immunoglobulin production. Help to B cells by invariant NKT cells is CD1d-dependent and delivered also in the absence of alpha-galactosylceramide, suggesting that NKT cells recognize an endogenous ligand presented by CD1d on B cells. The two major subsets of invariant NKT cells, CD4+ and double negative (CD4-CD8-), express comparable levels of CD40 ligand and cytokines, but differ in helper functions. Indeed, both subsets induce similar levels of B cell proliferation, whereas CD4+ NKT cells induce higher levels of immunoglobulin production. These results suggest a direct role for invariant NKT cells in regulating B lymphocyte proliferation and effector functions.     

Functionally distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer staining

CD1d-restricted natural killer (NK)T cells are known to potently secrete T helper (Th)1 and Th2 cytokines and to mediate cytolysis, but it is unclear how these contrasting functional activities are regulated. Using lipid antigen-loaded CD1d tetramers, we have distinguished two subsets of CD1d-restricted T cells in fresh peripheral blood that differ in cytokine production and cytotoxic activation. One subset, which was CD4(-), selectively produced the Th1 cytokines interferon gamma and tumor necrosis factor alpha, and expressed NKG2d, a marker associated with cytolysis of microbially infected and neoplastic cells. This subset up-regulated perforin after exposure to interleukin (IL)-2 or IL-12. In contrast, CD4(+) CD1d-restricted NKT cells potently produced both Th1 and Th2 cytokines, up-regulated perforin in response to stimulation by phorbol myristate acetate and ionomycin but not IL-2 or IL-12, and could be induced to express CD95L. Further, for both CD1d-restricted NKT cell subsets, we found that antigenic stimulation induced cytokine production but not perforin expression, whereas exposure to inflammatory factors enhanced perforin expression but did not stimulate cytokine production. These results show that the various activities of CD1d-restricted T cells in tumor rejection, autoimmune disease, and microbial infections could result from activation of functionally distinct subsets, and that inflammatory and antigenic stimuli may influence different effector functions.     

CD1d-restricted human natural killer T cells are highly susceptible to human immunodeficiency virus 1 infection

Human natural killer (NK) T cells are unique T lymphocytes that express an invariant T cell receptor (TCR) Valpha24-Vbeta11 and have been implicated to play a role in various diseases. A subset of NKT cells express CD4 and hence are potential targets for human immunodeficiency virus (HIV)-1 infection. We demonstrate that both resting and activated human Valpha24(+) T cells express high levels of the HIV-1 coreceptors CCR5 and Bonzo (CXCR6), but low levels of CCR7, as compared with conventional T cells. Remarkably NKT cells activated with alpha-galactosylceramide (alpha-GalCer)-pulsed dendritic cells were profoundly more susceptible to infection with R5-tropic, but not X4-tropic, strains of HIV-1, compared with conventional CD4(+) T cells. Furthermore, resting CD4(+) NKT cells were also more susceptible to infection. After initial infection, HIV-1 rapidly replicated and depleted the CD4(+) subset of NKT cells. In addition, peripheral blood NKT cells were markedly and selectively depleted in HIV-1 infected individuals. Although the mechanisms of this decline are not clear, low numbers or absence of NKT cells may affect the course of HIV-1 infection. Taken together, our findings indicate that CD4(+) NKT cells are directly targeted by HIV-1 and may have a potential role during viral transmission and spread in vivo.     

CD4+CD25+ regulatory T cells inhibit natural killer cell functions in a transforming growth factor-beta-dependent manner

Tumor growth promotes the expansion of CD4+CD25+ regulatory T (T reg) cells that counteract T cell-mediated immune responses. An inverse correlation between natural killer (NK) cell activation and T reg cell expansion in tumor-bearing patients, shown here, prompted us to address the role of T reg cells in controlling innate antitumor immunity. Our experiments indicate that human T reg cells expressed membrane-bound transforming growth factor (TGF)-beta, which directly inhibited NK cell effector functions and down-regulated NKG2D receptors on the NK cell surface. Adoptive transfer of wild-type T reg cells but not TGF-beta-/- T reg cells into nude mice suppressed NK cell-mediated cytotoxicity, reduced NKG2D receptor expression, and accelerated the growth of tumors that are normally controlled by NK cells. Conversely, the depletion of mouse T reg cells exacerbated NK cell proliferation and cytotoxicity in vivo. Human NK cell-mediated tumor recognition could also be restored by depletion of T reg cells from tumor-infiltrating lymphocytes. These findings support a role for T reg cells in blunting the NK cell arm of the innate immune system.     

Tracking the response of natural killer T cells to a glycolipid antigen using CD1d tetramers

A major group of natural killer (NK) T cells express an invariant Valpha14(+) T cell receptor (TCR) specific for the lipoglycan alpha-galactosylceramide (alpha-GalCer), which is presented by CD1d. These cells may have an important immune regulatory function, but an understanding of their biology has been hampered by the lack of suitable reagents for tracking them in vivo. Here we show that tetramers of mouse CD1d loaded with alpha-GalCer are a sensitive and highly specific reagent for identifying Valpha14(+) NK T cells. Using these tetramers, we find that alpha-GalCer-specific T lymphocytes are more widely distributed than was previously appreciated, with populations of largely NK1.1(-) but tetramer-binding T cells present in the lymph nodes and the intestine. Injection of alpha-GalCer leads to the production of both interferon gamma and interleukin 4 by nearly all NK T cells in the liver and the majority of the spleen within 2 h. These cells mostly disappear by 5 h, and they do not reappear after 1 wk. Curiously, tetramer-positive thymocytes do not rapidly synthesize cytokines, nor do they undergo decreases in cell number after lipid antigen stimulation, although they express equivalent TCR levels. In summary, the data presented here demonstrate that alpha-GalCer-specific NK T cells undergo a unique and highly compartmentalized response to antigenic stimulation.     

T helper cell 1-type CD4+ T cells, but not B cells, mediate colitis in interleukin 10-deficient mice

Mice rendered deficient in the production of interleukin 10 (IL-10-/-) develop a chronic inflammatory bowel disease (IBD) that predominates in the colon and shares histopathological features with human IBD. Our aim was to identify which cell type(s) can mediate colitis in IL-10-/- mice. We detected an influx of immunoglobulin-positive cells into the colon and the presence of colon-reactive antibodies in the serum of IL- 10-/- mice. To assess a pathogenic role for B cells, we generated a B cell-deficient (B-/-) strain of IL-10-/- mice. B-/-IL-10-/- mice acquired a severe colitis analogous to that IL-10-/- mice, implying that B cells were not the primary mediator of IBD in this model. A series of cell transfer experiments was performed to assess a pathogenic role for T cells. When IL-10-/- T cell-enriched lamina propria lymphocytes (LPL) or intraepithelial lymphocytes (IEL) were transferred into immunodeficient recombinase-activating gene (RAG)-2-/- recipients, a mild to severe colitis developed, depending on the cell number transferred. Lymphocytes recovered from the colon of transplanted RAG-2-/- mice with colitis were predominantly alpha beta TCR+CD4+, including a large proportion of CD4+CD8 alpha + cells. These cells were also CD45RB-/low and CD44+, indicative of an activated/memory population. Individual populations of CD4+CD8 alpha-, CD4+CD8 alpha + and CD4-CD8 alpha + T cells were then isolated from the lamina propria compartment of IL-10-/- mice and transferred into RAG-2- /- recipients. Only IL-10-/- CD4-expressing LPL, including both the CD4+CD8 alpha- and CD4+CD8 alpha + populations, induced colitis in recipient mice. Interferon-gamma, but little to no IL-4, was produced by CD4+CD8 alpha- and CD4+CD8 alpha + LPL recovered from the inflamed colons of RAG-2-/- recipients implicating alpha T helper cell 1 (TH1)- mediated response. We thus conclude that colitis in IL-10-/- mice is predominantly mediated by TH1-type alpha beta TCR+ T cells expressing CD4 alone, or in combination with the CD8 alpha molecule.     

删除CD4^＋CD25^＋T细胞对OVA诱导的小鼠体液免疫应答的影响

目的观察删除CD4＋CD25＋T细胞对OVA免疫小鼠体液免疫应答的影响。方法小鼠腹腔注射抗CD25单克隆抗体,分别于3天、10天、27天采血流式检测外周血中CD4＋CD25＋T细胞的比例;注射抗CD25单克隆抗体3天后,OVA加铝佐剂免疫未删除和删除CD4＋CD25＋T细胞小鼠,7天后加强免疫一次,分别于初次免疫后7天,加强免疫后14天采血制备血清,ELISA法检测血清中OVA特异性IgE和IgG1的浓度。结果注射抗CD25单克隆抗体3天和10天,外周血中无CD4＋CD25＋T细胞;27天后CD4＋CD25＋T细胞的比例部分恢复。初次免疫7天后,删除CD4＋CD25＋T细胞小鼠总IgE、OVA特异性IgE和IgG1浓度较未删除组升高;加强免疫14天后,删除CD4＋CD25＋T细胞小鼠OVA特异性IgE较未删除组升高。结论删除CD4＋CD25＋T细胞能够影响小鼠OVA特异性体液免疫应答。     

CD4(+) Valpha14 natural killer T cells are essential for acceptance of rat islet xenografts in mice

Pancreatic islet transplantation represents a potential treatment for insulin-dependent diabetes mellitus. However, the precise cellular and molecular mechanisms of the immune reactions against allogeneic and xenogeneic transplanted islets remain unclear. Here, we demonstrate that CD4(+) Valpha14 natural killer T (NKT) cells, a recently identified lymphoid cell lineage, are required for the acceptance of intrahepatic rat islet xenografts. An anti-CD4 mAb, administrated after transplantation, allowed islet xenografts to be accepted by C57BL/6 mice, with no need for immunosuppressive drugs. The dose of anti-CD4 mAb was critical, and the beneficial effect appeared to be associated with the reappearance of CD4(+) NKT cells at around 14 days after transplantation. Interestingly, rat islet xenografts were rejected, despite the anti-CD4 mAb treatment, in Valpha14 NKT cell-deficient mice, which exhibit the normal complement of conventional lymphoid cells; adoptive transfer of Valpha14 NKT cells into Valpha14 NKT cell-deficient mice restored the acceptance of rat islet xenografts. In addition, rat islet xenografts were accepted by Valpha14 NKT mice having only Valpha14 NKT cells and no other lymphoid cells. These results indicate that Valpha14 NKT cells play a crucial role in the acceptance of rat islet xenografts in mice treated with anti-CD4 antibody, probably by serving as immunosuppressive regulatory cells.     

Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice

C57BL/6 mice genetically deficient in interleukin 15 (IL-15(-/-) mice) were generated by gene targeting. IL-15(-/-) mice displayed marked reductions in numbers of thymic and peripheral natural killer (NK) T cells, memory phenotype CD8(+) T cells, and distinct subpopulations of intestinal intraepithelial lymphocytes (IELs). The reduction but not absence of these populations in IL-15(-/-) mice likely reflects an important role for IL-15 for expansion and/or survival of these cells. IL-15(-/-) mice lacked NK cells, as assessed by both immunophenotyping and functional criteria, indicating an obligate role for IL-15 in the development and functional maturation of NK cells. Specific defects associated with IL-15 deficiency were reversed by in vivo administration of exogenous IL-15. Despite their immunological defects, IL-15(-/-) mice remained healthy when maintained under specific pathogen-free conditions. However, IL-15(-/-) mice are likely to have compromised host defense responses to various pathogens, as they were unable to mount a protective response to challenge with vaccinia virus. These data reveal critical roles for IL-15 in the development of specific lymphoid lineages. Moreover, the ability to rescue lymphoid defects in IL-15(-/-) mice by IL-15 administration represents a powerful means by which to further elucidate the biological roles of this cytokine.     

Impaired neutrophil response and CD4+ T helper cell 1 development in interleukin 6-deficient mice infected with Candida albicans

To define the role of interleukin (IL)6 in Candida albicans infection, IL-6 deficient mice were assessed for susceptibility to systemic or gastrointestinal infection, as well as for parameters of elicited T helper cell (Th) immunity. IL-6-deficient mice were more susceptible than wild-type mice to either type of infection caused by virulent C. albicans. In response to systemic challenge with a live vaccine strain of yeast, IL-6-deficient mice failed to mount Th1-associated protective immunity, but the resulting Th2-biased response could be redirected to the Th1 phenotype by IL-10 neutralization. Severe impairment of the macrophage and neutrophil response to infection was observed in IL-6- deficient mice, but administration of IL-6 would increase both neutrophil response and resistance to infection. IL-6 seems to oppose the Th2-promoting role of IL-10 in candidiasis, its early regulatory activity involving effects on neutrophil function.     

Inhibition of T helper cell type 2 cell differentiation and immunoglobulin E response by ligand-activated Valpha14 natural killer T cells.

Murine Valpha14 natural killer T (NKT) cells are thought to play a crucial role in various immune responses, including infectious, allergic, and autoimmune diseases. Because Valpha14 NKT cells produce large amounts of both interleukin (IL)-4 and interferon (IFN)-gamma upon in vivo stimulation with a specific ligand, alpha-galactosylceramide (alpha-GalCer), or after treatment with anti-CD3 antibody, a regulatory role on helper T (Th) cell differentiation has been proposed for these cells. However, the identity of the cytokine produced by Valpha14 NKT cells that play a dominant role on the Th cell differentiation still remains controversial. Here, we demonstrate by using Valpha14 NKT-deficient mice that Valpha14 NKT cells are dispensable for the induction of antigen-specific immunoglobulin (Ig)E responses induced by ovalbumin immunization or Nippostrongylus brasiliensis infection. However, upon in vivo activation with alpha-GalCer, Valpha14 NKT cells are found to suppress antigen-specific IgE production. The suppression appeared to be IgE specific, and was not detected in either Valpha14 NKT- or IFN-gamma-deficient mice. Consistent with these results, we also found that ligand-activated Valpha14 NKT cells inhibited Th2 cell differentiation in an in vitro induction culture system. Thus, it is likely that activated Valpha14 NKT cells exert a potent inhibitory effect on Th2 cell differentiation and subsequent IgE production by producing a large amount of IFN-gamma. In marked contrast, our studies have revealed that IL-4 produced by Valpha14 NKT cells has only a minor effect on Th2 cell differentiation.     

Expansion of natural killer cells but not T cells in human interleukin 2/interleukin 2 receptor (Tac) transgenic mice

Transgenic mice expressing both human IL-2 and the L chain of IL-2-R constitutively had an unusual expansion of Thy-1+/CD3-4-8- large granular lymphocytes, which bore the elevated NK activity. Unexpectedly, the transgenic mice had neither T cell expansion nor autoreactive antibodies. The increase in number and activity of NK cells seems to be responsible for both the severe interstitial pneumonia and lymphocyte depletion in the spleen that we found in these transgenic mice. In addition, we found the selective loss of Purkinje cells in the cerebellum of the mice, which gave rise to their disturbed gait. All the transgenic mice died by 4 wk of age.     

Allergen immunotherapy decreases interleukin 4 production in CD4+ T cells from allergic individuals

Allergen specific CD4+ T cell clones generated from allergic individuals have been shown to produce increased levels of the cytokine interleukin 4 (IL-4), compared to allergen specific clones generated from nonallergic individuals. This difference between CD4+ T cells from allergic and nonallergic individuals with regard to cytokine production in response to allergen is thought to be responsible for the development of allergic disease with increased IgE synthesis in atopic individuals. We examined the production of IL-4 in subjects with allergic rhinitis and in allergic individuals treated with allergen immunotherapy, a treatment which involves the subcutaneous administration of increasing doses of allergen and which is highly effective and beneficial for individuals with severe allergic rhinitis. We demonstrated that the quantity of IL-4 produced by allergen specific memory CD4+ T cells from allergic individuals could be considerably reduced by in vivo treatment with allergen (allergen immunotherapy). Immunotherapy reduced IL-4 production by allergen specific CD4+ T cells to levels observed with T cells from nonallergic subjects, or to levels induced with nonallergic antigens such as tetanus toxoid. In most cases the levels of IL-4 produced were inversely related to the length of time on immunotherapy. These observations indicate that immunotherapy accomplishes its clinical effects by reducing IL-4 synthesis in allergen specific CD4+ T cells. In addition, these observations indicate that the cytokine profiles of memory CD4+ T cells can indeed be altered by in vivo therapies. Thus, the cytokine profiles of memory CD4+ T cells are mutable, and are not fixed as had been suggested by studies of murine CD4+ memory T cells. Finally, treatment of allergic diseases with allergen immunotherapy may be a model for other diseases which may require therapies that alter inappropriate cytokine profiles of memory CD4+ T cells.     

CD3/Crry共刺激通路对小鼠CD4~+ T细胞的免疫调控

目的:探讨CD3/Crry共刺激通路对小鼠CD4+T细胞的免疫调控及其机制.方法:分离C57BL/6小鼠的脾脏CD4+T细胞,体外检测CD3、CD28、Crry、CD3/CD28、CD3/Crry、CD3/CD28/Crry共刺激对CD4+T细胞增殖的作用效果,并检测其细胞上清液中白细胞介素-2(IL-2)、γ-干扰素(IFN-γ)、白细胞介素-4(IL-4)和白细胞介素-10(IL-10)的水平.结果:与CD3组相比,CD3/CD28、CD3/Crry和CD3/CD28/Crry共刺激后.CIM+T细胞均出现显著增殖反应(P<0.05),而且CD3/CD28/Crry共刺激组的增殖活性较CD3/CD28或CD3/Crry共刺激组显著增高(P<0.05).CD3/CD28共刺激后,IL-2和IFN-γ水平较CD3、CD28、Crry组和CD3/Crry组显著升高(P<0.05),CD3/Crry共刺激后,IL-4水平较CD3、CD28、Crry组和CD3/CD28共刺激组显著升高(P<0.05),各组IL-10水平无显著差异(P>0.05).结论:CD3/Crry共刺激通路诱导CD4+T细胞产生分泌IL-4,抑制IL-2和IFN-γ的产生,有可能诱导同种移植免疫低反应性.     

Role of IFN-gamma in induction of Foxp3 and conversion of CD4+ CD25- T cells to CD4+ Tregs

IFN-gamma is an important Th1 proinflammatory cytokine and has a paradoxical effect on EAE in which disease susceptibility is unexpectedly heightened in IFN-gamma-deficient mice. In this study, we provide what we believe is new evidence indicating that IFN-gamma is critically required for the conversion of CD4+ CD25- T cells to CD4+ Tregs during EAE. In our study, the added severity of EAE in IFN-gamma knockout mice was directly associated with altered encephalitogenic T cell responses, which correlated with reduced frequency and function of CD4+ CD25+ Foxp3+ Tregs when compared with those of WT mice. It was demonstrated in both human and mouse systems that in vitro IFN-gamma treatment of CD4+ CD25- T cells led to conversion of CD4+ Tregs as characterized by increased expression of Foxp3 and enhanced regulatory function. Mouse CD4+ CD25- T cells, when treated in vitro with IFN-gamma, acquired marked regulatory properties as evidenced by suppression of EAE by adoptive transfer. These findings have important implications for the understanding of the complex role of IFN-gamma in both induction and self regulation of inflammatory processes.     

外周血自然杀伤T淋巴细胞和CD4＋自然杀伤T淋巴细胞对哮喘患儿发病的影响

目的：探讨外周血自然杀伤T淋巴细胞（NKT细胞）和CD4＋NKT细胞对哮喘患儿发病的影响。方法选取2008年1月至2012年12月住院治疗的哮喘病患儿150例（哮喘组）,另选160例体检健康的儿童作为健康对照组。收集研究对象外周静脉血标本,采用密度梯度离心法分离获得外周血单个核细胞,应用免疫荧光标记和流式细胞术检测哮喘组和健康对照组儿童外周血NKT细胞和CD4＋NKT细胞的比例,并观察两类细胞与过敏指标总免疫球蛋白E（IgE）、嗜酸性细胞阳离子蛋白（ECP）的相关性。酶联免疫吸附试验检测外周血细胞因子白细胞介素（IL）‐4、IL‐10、干扰素‐γ（IFN‐γ）水平。比较两组间的差异。结果哮喘组患儿的NKT细胞和CD4＋NKT细胞比例均较健康对照组明显下降（P＜0．05）。NKT细胞、CD4＋NKT细胞的比例与总IgE、ECP均无明显相关性（P＞0．05）。外周血IL‐4水平较健康对照组明显升高（P＜0．05）,IL‐10水平明显下降（P＜0．05）,而IFN‐γ水平与健康对照组比较差异无统计学意义（P＞0．05）。结论NKT细胞和CD4＋NKT细胞数量降低下及所分泌细胞因子功能变化是导致哮喘发病的重要原因。