灭活HIV-1颗粒对人CD4+T细胞活化和全血Th1/Th2细胞因子分泌的影响

目的：体外研究AT-2灭活的HIV-1颗粒对人CD4+T细胞活化和全血(whole blood，WB) Th1/Th2细胞因子分泌的影响。方法:AT-2灭活HIV-1ⅢB型病毒颗粒，运用ELISA法测定所制备的灭活病毒中p24抗原的含量，按照1/500、1/50和1/5 (V/V)的浓度加入到WB中，以植物血凝素(phytohemagglutinin，PHA)组为阳性对照；24 h后，收集WB培养上清，运用流式微球分析法（cytometric bead array，CBA）检测WB分泌Th1 (IL-2、IFN-γ和TNF-α)和Th2 (IL-4、IL-6和IL-10)细胞因子水平；同时运用免疫荧光抗体染色技术结合流式细胞术检测WB中CD4+T细胞早期活化标记分子CD69的表达百分率。结果:我们所制备的灭活病毒中p24抗原的含量为85.5 μg/L；24 h后，空白对照组中，CD4+T细胞CD69的表达百分率为(1.62±0.63)%，PHA组为(38.82±6.00)%，HIV-1(1/500)组为(3.83±1.07)%，HIV-1(1/50)组为(5.94±0.85)%，HIV-1(1/5)组为(9.30±1.22)%；空白对照组WB培养上清中细胞因子主要为IL-6和TNF-α，PHA组中Th1和Th2细胞因子全部升高，3个浓度的HIV-1组中Th1和Th2细胞因子也全部升高。结论:AT-2灭活的HIV-1ⅢB颗粒能够明显引起WB中CD4＋T细胞活化，并上调WB培养上清中Th1和Th2细胞因子的水平，其机制可能是除了HIV-1病毒蛋白的作用外，HIV-1出胞时，许多宿主细胞来源的免疫分子整合到病毒颗粒包膜中，而模拟抗原提呈细胞，从而产生免疫调节作用。     

Induction and function of virus-specific CD4+ T cell responses

CD4+ T cells - often referred to as T-helper cells - play a central role in immune defense and pathogenesis. Virus infections and vaccines stimulate and expand populations of antigen-specific CD4+ T cells in mice and in man. These virus-specific CD4+ T cells are extremely important in antiviral protection: deficiencies in CD4+ T cells are associated with virus reactivation, generalized susceptibility to opportunistic infections, and poor vaccine efficacy. As described below, CD4+ T cells influence effector and memory CD8+ T cell responses, humoral immunity, and the antimicrobial activity of macrophages and are involved in recruiting cells to sites of infection. This review summarizes a few key points about the dynamics of the CD4+ T cell response to virus infection, the positive role of pro-inflammatory cytokines in the differentiation of virus-specific CD4+ T cells, and new areas of investigation to improve vaccines against virus infection.     

Induction of CD4 T cell proliferation and in vitro Th1-like cytokine responses to measles virus

Mechanisms that lead to induction of life-long immunity to measles virus (MV) are poorly understood. In the present study, we have assessed the activation, proliferation and cytokine secreting function of peripheral blood T cells from MV immune individuals. Expression of cell blastogenesis markers, such as increased forward light scatter and CD38 expression, peaked 5-7 days after infection of peripheral blood mononuclear cells (PBMC) with the live attenuated Edmonston strain of MV. Subset analysis revealed that both CD3- and CD3+ cells expressed activation markers but that the CD3+ T cells predominated late in the culture period corresponding to maximal proliferation and cell recovery. The majority of CD3+ T cells consisted of CD4+CD8- cells. IFN-gamma and IL-4 production similarly showed optimal production late in culture. Depletion of CD4 cells prior to culture and MV stimulation completely abrogated both IFN-gamma and IL-4 production, whereas depletion of CD8 cells did not diminish production, suggesting that CD4+CD8- T cells were principally involved in production of these cytokines. Finally, optimal IFN-gamma production was elicited at high MV doses and IL-4 at much lower doses. These results suggest that among MV immune individuals, in vitro responses to measles are dominated by CD4+ T cells that, depending on antigen dose, primarily produce a Th1-like and, to a lesser extent, a Th1/Th2-mixed pattern of cytokine release.     

CD8+ suppressor-mediated regulation of human CD4+ T cell responses to glutamic acid decarboxylase 65

Although potentially autoreactive T cells are present even in healthy subjects, most individuals do not develop autoimmune disease. It has been well demonstrated that CD4+ CD25+ regulatory T cells play a significant role in controlling the expansion of autoreactive T cells in the periphery. However, some healthy individuals exhibit measurable responses to self peptide even in the presence of CD4+ CD25+ regulatory cells. This article describes the regulation of human CD4+ T cell responses to glutamic acid decarboxylase 65 (GAD65), an autoantigen implicated in type-1 diabetes, by autologous CD8+ suppressor T cells. In cells cultured from healthy individuals, the inclusion of autologous CD8+ T cells at physiological levels resulted in a dramatic decrease in the magnitude of in vitro CD4+ T cell responses to GAD65 peptide. Based on transwell experiments, the observed suppression was cell contact-dependent. However, antibody blocking studies indicated that suppression was mediated by IL-10. Cell fractionation studies suggested that CD8+ suppressor T cells originate from the CD45RA+ CD27- population. The suppression of CD4+ T cell responses to GAD65 in healthy individuals raises the possibility that CD8+ suppressor T cells play an important role in controlling potentially autoreactive T cells in the general population.     

IL-15 exacerbates collagen-induced arthritis with an enhanced CD4+ T cell response to produce IL-17

IL-15 is thought to be involved in the pathogenesis of rheumatoid arthritis (RA). We found that IL-15 plays an important role in the development of murine collagen-induced arthritis (CIA). The incidence and severity of CIA were slightly decreased in IL-15 KO mice but were increased in IL-15 Tg mice compared with wild-type (WT) mice. The levels of type II collagen (CII)-specific IL-17 production were significantly increased in IL-15 Tg mice compared with WT mice with CIA. Expression of IL-23R was up-regulated in CD4(+) T cells in IL-15 Tg mice but down-regulated in IL-15 KO mice compared with WT mice. In correlation with the expression levels of IL-23R, IL-17 production by CD4(+) T cells in response to exogenous IL-23 was increased in IL-15 Tg mice compared with WT mice. Furthermore, exogenous IL-15 synergized with IL-23 to induce CII-specific IL-17 production by CD4(+) T cells in vitro. Taken together, these results indicate that IL-15 plays an important role in the progression of CIA through increasing antigen-specific IL-17 production by CD4(+) T cells.     

Mucosal immunization of CD4+ T cell-deficient mice with an inactivated virus induces IgG and IgA responses in serum and mucosal secretions

Immunoglobulin (Ig) class switching can occur in the absence of alphabeta+ or gammadelta+ T cells when mice are infected with certain live viruses, although CD4 T helper cells are believed to be essential for induction of a high-affinity antibody response and for efficient isotype switching from IgM to IgG and IgA production. However, little information is available about the immune responses after mucosal immunization of CD4+ T cell-deficient mice with inactivated virus. In this study, we show that intranasal immunization with formalin-inactivated influenza A/PR8/34 virus induces IgG and IgA responses in serum and IgA responses in mucosal secretions in CD4+ T cell-deficient mice. All four subclasses of IgG were produced. IgG1/IgG2a ratios were found to be from 1 to 1.75, indicating that both Th1 and Th2 immune responses are induced by the inactivated influenza virus. The sera and mucosal secretions were found to have neutralizing activity against influenza virus in vitro. In addition, the mucosally immunized CD4+ T cell-deficient mice were protected completely from challenge with a lethal dose of live, pathogenic influenza virus. To our knowledge, this is the first demonstration that mucosal immunization with an inactivated virus induces immune responses in serum and mucosal secretions in CD4+ T cell-deficient mice.     

Fine-tuning CD4+ central memory T cell heterogeneity by strength of stimulation

The memory T cell pool serves as a relatively long-lived heterogeneous repository of antigen-experienced T cells that "remember" previous encounters with antigen. While heterogeneity in the memory T cell pool is now well established, signals regulating the generation of this memory T cell heterogeneity are not fully understood. Two articles in this issue of the European Journal of Immunology highlight the importance of the strength of antigenic stimulation in regulating the generation of phenotypically and functionally distinct CD4(+) T cell memory subsets. New insights are also provided into key molecular players that likely mediate differences in homeostatic and secondary expansion between the memory subsets.     

Controlling mature CD4+ T cell responses

Immune responses are by necessity highly regulated to achieve the appropriate balance of aggression and restraint. A mong the many factors involved in maintaining this balance are the interactions between accessory molecule receptorse xpressed on T cells and their ligands on antigen-presenting cells. Our studies during the past several years have focused on defining how particular accessory molecule interactions influence the activation of naive CD4⁺ T cells and the subsequent development of effector function. In this article, we discuss our findings on the effects of distinct accessory molecules with particular attention to the unique roles of LFA-1 and CD28 during different phases of the native CD4⁺ cell response.     

T cell receptor specificities of Toxoplasma gondii-reactive mouse CD4+ T lymphocytes and Th1 clones

The Toxoplasma gondii-directed CD4⁺ T cell response in chronically infected mice was studied with respect to both T cell receptor diversity and antigen specificities. T cell receptor chains Vβ4, 6, 8, 10, and 14 were predominantly found on toxoplasma-reactive CD4⁺ splenocytes. This repertoire was also detected among T. gondii-specific CD4⁺ T cell clones. Analysis of clonotypic cytokine profiles revealed typical Th1 clones secreting interleukin-2, interferon-γ and tumour necrosis factor activity and Th2 clones producing interleukin-4 and interleukin-10. Five distinct toxoplasma antigens (p26, p40, p55, p58 and p60) were detected in electrophoretically separated toxoplasma lysate by five individual Th1 clones. Parallel testing of CD4⁺ T lymphocytes from infected mice confirmed that these specificities constitute the peak immunogenic fractions of toxoplasma lysate. The expression patterns of two clonotypic, T cell-stimulatory parasite antigens were studied in detail. While p55 was expressed by mouse-virulent and avirulent T. gondii isolates and in both the tachyzoite and bradyzoite stages, p58 was detected only in virulent strains from intraspecies subgroup I. Thus, we describe the heterogeneity of toxoplasmic immunodominant T cell antigens including a 58-kDa group I-restricted molecule which may provide a marker for virulent isolates. Received: 3 February 1997     

Differences in maintenance of CD8+ and CD4+ bacteria-specific effector-memory T cell populations

Our knowledge about the kinetics and dynamics of complex pathogen-specific CD8(+) T cell responses and the in vivo development of CD8(+) memory T cells has increased substantially over the past years; in comparison, relatively little is known about the CD4(+) T cell compartment. We monitored and directly compared the phenotypical changes of pathogen (Listeria monocytogenes)-specific CD8(+) and CD4(+) T cell responses under conditions leading to effective and long-lasting protective immunity. We found that the general kinetics of bacteria-specific CD8(+) and CD4(+) T cells during the effector and post-effector phases are synchronized. However, later during the memory phase, CD8(+) and CD4(+) T cell populations differ substantially. Whereas CD8(+) memory T cell populations with immediate effector function are readily detectable in lymphoid and non-lymphoid tissues and remain remarkably stable in size, antigen-specific CD4(+) effector-memory T cells decline continuously in frequency over time. These findings have important implications for the better understanding of the in vivo development of protective immunity towards intracellular pathogens.     

Enhanced murine CD4+ T cell responses induced by the CD2 ligand CD48

The physiological functions of murine CD2 and its ligand CD48 are uncertain. We have examined the role of the CD2-CD48 interaction in murine T cell activation using a series of Chinese hamster ovary (CHO) cell transfectants. CHO cells expressing I-A^d together with CD48 induced more potent activation of OVA-specific, I-A^d -restricted DO11.10-transgenic T cells than CHO cells expressing I-A^d alone. CD48 augmented proliferation and IL-2 production in response to antigen. The enhancing effect of CD48 was of the same magnitude as that seen for CD80 (B7-1). Conjugate assays revealed the ability of CD48 to increase adhesion between T cells and CHO transfectants. The enhancing effects of CD48 on T cell-antigen-presenting cell adhesion and T cell activation were inhibited by anti-CD2 monoclonal antibody. This report provides the first evidence that the CD2 ligand CD48 contributes to the interactions of murine CD4⁺ T cells with antigen-presenting cells.     

CD4+ T cell help improves CD8+ T cell memory by retained CD27 expression

CD4+ T cell help during the priming of CD8+ T lymphocytes imprints the capacity for optimal secondary expansion upon re-encounter with antigen. Helped memory CD8+ T cells rapidly expand in response to a secondary antigen exposure, even in the absence of T cell help and, are most efficient in protection against a re-infection. In contrast, helpless memory CTL can mediate effector function, but secondary expansion is reduced. How CD4+ T cells instruct CD8+ memory T cells during priming to undergo efficient secondary expansion has not been resolved in detail. Here, we show that memory CTL after infection with lymphocytic choriomeningitis virus are CD27(high) whereas memory CTL primed in the absence of CD4+ T cell have a reduced expression of CD27. Helpless memory CTL produced low amounts of IL-2 and did not efficiently expand after restimulation with peptide in vitro. Blocking experiments with monoclonal antibodies and the use of CD27(-/-) memory CTL revealed that CD27 ligation during restimulation increased autocrine IL-2 production and secondary expansion. Therefore, regulating CD27 expression on memory CTL is a novel mechanism how CD4+ T cells control CTL memory.     

苯扎贝特对原发性胆汁性肝硬化患者外周血CD4＋T细胞的抑制作用研究

目的：观察苯扎贝特（Bezafibrate,BF）对原发性胆汁性肝硬化（Primary biliary cirrhosis,PBC）患者外周血CD4＋T细胞活化增殖和分化的影响,探讨其免疫抑制作用机理,为BF治疗PBC的作用靶点提供实验依据。方法：分离PBC患者的PBMCs,磁珠分选CD4＋T细胞,加入anti-CD3、anti-CD28及不同浓度的BF,以流式细胞方法和ELISA分析BF对CD4＋T细胞活化、增殖及Th1/Th17细胞因子产生的影响。结果：（1）BF能抑制PBC患者外周血CD4＋T细胞活化;（2）BF呈浓度依赖性抑制PBC外周血CD4＋T细胞增殖（P＜0.05）;（3）BF也能够抑制PBC患者外周血CD4＋T细胞产生IFN-γ和IL-17（P＜0.05）。结论：BF可通过抑制CD4＋T细胞活化增殖及细胞因子分泌,发挥免疫抑制作用。     

Multifunctional CD4+ T cells correlate with active Mycobacterium tuberculosis infection

Th1 CD4⁺ T cells and their derived cytokines are crucial for protection against Mycobacterium tuberculosis. Using multiparametric flow cytometry, we have evaluated the distribution of seven distinct functional states (IFN‐γ/IL‐2/TNF‐α triple expressors, IFN‐γ/IL‐2, IFN‐γ/TNF‐α or TNF‐α/IL‐2 double expressors or IFN‐γ, IL‐2 or TNF‐α single expressors) of CD4⁺ T cells in individuals with latent M. tuberculosis infection (LTBI) and active tuberculosis (TB). We found that triple expressors, while detectable in 85–90%TB patients, were only present in 10–15% of LTBI subjects. On the contrary, LTBI subjects had significantly higher (12‐ to 15‐fold) proportions of IL‐2/IFN‐γ double and IFN‐γ single expressors as compared with the other CD4⁺ T‐cell subsets. Proportions of the other double or single CD4⁺ T‐cell expressors did not differ between TB and LTBI subjects. These distinct IFN‐γ, IL‐2 and TNF‐α profiles of M. tuberculosis‐specific CD4⁺ T cells seem to be associated with live bacterial loads, as indicated by the decrease in frequency of multifunctional T cells in TB‐infected patients after completion of anti‐mycobacterial therapy. Our results suggest that phenotypic and functional signatures of CD4⁺ T cells may serve as immunological correlates of protection and curative host responses, and be a useful tool to monitor the efficacy of anti‐mycobacterial therapy.     

A correlation between function and selected measures of T cell avidity in influenza virus-specific CD8+ T cell responses

Activation of mature CD8+ T cells requires recognition, via the T cell receptor (TCR), of peptide + MHC (pMHC) complexes with an avidity that exceeds a designated threshold. Multiple indicators of T cell avidity have been described that provide unique information on the characteristics of T cell interactions. However, these indicators are routinely used in isolation, and, consequently, little is known about correlations between these measures or which measure, if any, correlates with the quality of the T cell response. Following influenza virus infection of C57BL/6J mice, we analyzed the relative avidities of five epitope-specific CD8+ T cell populations using five different measures. We demonstrated that the quality of CD8+ T cell responses, in terms of cytokine profiles, correlates with TCR dissociation rate and CD8 dependence, but not with the sensitivity to tetramer binding or peptide stimulation. Thus, we propose that, despite significant differences in TCR dissociation rate, the stimulation threshold of influenza-specific CD8+ T cell populations may be equivalent due to compensatory mechanisms largely provided by the CD8 coreceptor. Furthermore, this study shows that different indicators of avidity do not necessarily provide similar information and should be used in combination to obtain an overall picture of the characteristics of TCR binding.     

卵巢癌细胞培养上清诱导CD4+CD25-T细胞分化为CD4+CD25+调节性T细胞

目的研究卵巢癌细胞培养上清液是否能诱导外周血CD4^＋CD25^- T细胞转变为CD4^＋CD25^＋调节性T细胞。方法将外周血CD4^＋CD25^- T细胞分离后，对照组用CD3和CD28单抗活化，实验组在对照基础上加用卵巢癌细胞株SKOV3培养上清，72h后分离各组的CD25^＋和CD25^-T细胞，溴化脱氧尿嘧啶掺入标记法测定增殖能力及对静息的自体同源CD4^＋CD25^- T细胞的增殖抑制能力，流式细胞仪测定细胞糖皮质激素诱发型TNF受体（glucocorticoid-induced TNFR,GITR）与CTLA-4分子的表达，RT-PCR检测细胞卿mRNA的表达。结果与对照组相反，实验组的CD4^＋CD25^＋T细胞具有免疫抑制功能，自身增殖能力下降，GITR和CTLA-4分子的表达和CD4^＋CD25^＋调节性T细胞相似，并被诱导表达转录因子Foxp3 mRNA。结论卵巢癌细胞分泌的可溶性物质能诱导外周血CD4^＋CD25^-T细胞转化为CD4^＋CD25^＋调节性T细胞。     

CD4+CD25+调节性T细胞的发育与胸腺CD4-CD25+细胞关联性的探讨

目的:探讨CD4 CD25 调节性T细胞(CD4 CD25 regulatoryTcells,CD4 CD25 Tr)的发育及其与胸腺CD4-CD25 细胞的关系。方法:以流式细胞术检测小鼠从出生至发育成熟过程中,胸腺、脾脏、淋巴结和外周血中CD4 CD25 Tr比例变化,以及胸腺CD4-CD25 细胞比例变化;通过磁激活细胞分选(MACS)从小鼠淋巴结纯化CD4 CD25 T和CD4 CD25-T细胞,经CFDA-SE标记,以多种刺激形式诱导增殖。结果:小鼠出生1d到10周的发育过程中,胸腺CD4 CD25 Tr比例一直比较恒定,但在脾脏、淋巴结和外周血,随鼠龄增加而不断升高,从1d龄到1周时升高最迅速,其后的升高速度逐渐减慢,10周龄时达平台期。胸腺中CD4-CD25 细胞在出生1d的小鼠比例非常高,1d龄到1周龄期间迅速下降,10周龄时达平台期。ConA不能诱导CD4 CD25 Tr和CD4 CD25-T细胞增殖,但CD4 CD25 Tr出现一过性细胞增大;佛波醇酯加离子霉素能诱导CD4 CD25 Tr和CD4 CD25-T细胞增殖;包被的抗CD3抗体加可溶性抗CD28抗体能刺激CD4 CD25-T细胞增殖,但CD4 CD25 Tr不增殖,加入高浓度IL-2,CD4 CD25-T细胞增殖更强,CD4 CD25 Tr出现增殖。结论:胸腺CD4-CD25 细胞很有可能是CD4 CD25 Tr的前体。     

Co-expression of CD8α in CD4+ T cell receptor αβ+ T cells migrating into the murine small intestine epithelial layer

We investigated the surface phenotype of CD3⁺CD4⁺ T cell receptor (TCR) αβ⁺ T cells repopulating the intestinal lymphoid tissues of C.B-17 scidlscid (severe-combined immunodeficient; scid) (H-2^d, L^d+) mice. CD4⁺ CD8^? T cells were cell sorter-purified from various secondary and tertiary lymphoid organs of congenic C.B-17 +/+ (H-2^d, L^d+) or semi-syngeneic dm2 (H-2^d, L^d?) immunocompetent donor mice. After transfer of 10⁵ cells into young scid mice, a mucosa-homing, memory CD44^hi CD45RB^lo CD4⁺ T cell population was selectively engrafted. Large numbers of single-positive (SP) CD3⁺ CD2⁺ CD28⁺ CD4⁺ CD^8? T cells that expressed the α₄ integrin chain CD49d were found in the spleen, the mesenteric lymph nodes, the peritoneal cavity and the gut lamina propria of transplanted scid mice. Unexpectedly, large populations of donor-type doublepositive (DP) CD4⁺ CD8α⁺ CD8β^? T cells with high expression of the CD3/TCR complex appeared in the epithelial layer of the small intestine of transplanted scid mice. In contrast to SP CD4⁺ T cells, the intraepithelial DP T cells showed low expression of the CD2 and the CD28 co-stimulator molecules, and of the α₄ integrin chain CD49d, but expressed high levels of the α_IEL integrin chain CD103. The TCR-Vβ repertoire of DP but not SP intraepithelial CD4⁺ T cells was biased towards usage of the Vβ6 and Vβ8 viable domains. Highly purified populations of SP and DP CD4⁺ T cell populations from the small intestine epithelial layer of transplanted scid mice had different abilities to repopulate secondary scid recipient mice: SP CD4⁺ T cells repopulated various lymphoid tissues of the immunodeficient host, while intraepithelial DP CD4⁺ T cells did not. Hence, a subset of CD3⁺ CD4⁺ TCRαβ⁺ T cells apparently undergoes striking phenotypic changes when it enters the microenvironment of the small intestine epithelial layer.