CD4~+CD25~+T细胞在CD8~+T细胞抗肿瘤免疫中的调节作用

实验旨在研究CD4+CD25+T细胞在CD8+T细胞抗肿瘤免疫中的调节作用。将小鼠脾脏中分离的单个核细胞分为两组,即去除CD4+CD25+T细胞组和未去除CD4+CD25+T细胞组,测定树突状细胞提呈的肿瘤抗原多肽刺激不同T细胞增殖活性、细胞因子IFN-γ分泌,以及多肽特异性CD8+T细胞对同源性胃癌细胞株MFC的杀伤活性。结果显示预先去除未致敏T细胞中的CD4+CD25+T细胞,所诱导的特异性CD8+CTL对肿瘤细胞免疫应答增强,表现为反应性T细胞对树突状细胞提呈的肿瘤抗原多肽增殖反应增强,IFN-γ分泌量提高及CD8+T细胞对MFC杀伤活性增强。这些结果表明,预先去除未致敏T细胞中的CD4+CD25+T细胞,肿瘤抗原多肽修饰的树突状细胞肿瘤疫苗效能可明显增加。CD4+CD25+T细胞在CD8+T细胞抗肿瘤免疫中起下调作用。     

R848协同CD3抗体诱导C57BL/6小鼠脾脏淋巴细胞功能改变

目的观察并初步分析1-[4-amino-2-(ethoxymethyl)imidazo[4,5-c]quinolin-1-yl]-2-methylpropan-2-ol(R848)协同CD3抗体诱导C57BL/6小鼠脾脏中淋巴细胞发生的功能改变。方法分离正常C57BL/6小鼠脾脏的淋巴细胞,分别在anti-CD3、R848及anti-CD3+R848的作用下培养72 h。通过MTT法观察淋巴细胞的增殖情况;采用ELISA的方法检测脾脏淋巴细胞的IFN-γ和IL-4的产生情况;使用细胞内细胞因子染色的方法检测CD4+T和CD8+T细胞的IFN-γ和IL-4的产生情况;再用细胞表面分子染色的方法检测R848诱导不同淋巴细胞群活化的情况。结果单独使用R848对淋巴细胞的增殖作用不明显,但anti-CD3+R848可促进淋巴细胞的增殖;R848能辅助anti-CD3明显诱导脾CD4+T和CD8+T细胞分泌IFN-γ和IL-4;R848能够使淋巴细胞活化,且以B细胞亚群为主。结论 R848能够协助CD3抗体促进脾脏淋巴细胞的增殖,分泌IFN-γ和IL-4,其机制与R848能明显促进B淋巴细胞活化有关。     

阻断CD40-CD40L共刺激途径对T细胞表型及细胞因子分泌的作用研究

目的探讨应用抗CD40L单克隆抗体阻断CD40-CD40L共刺激途径后对T细胞表型及其分泌的细胞因子的影响,为体外阻断该共刺激途径诱导T细胞对异体移植抗原的免疫耐受提供实验依据.方法供鼠(C57BL/6H-2b)脾T细胞作为反应细胞,受鼠(BALB/CH-2d)脾细胞作为刺激细胞,设单抗组(加抗CD40L单抗)和对照组(不加单抗),初次混合淋巴细胞培养(MLR)7天,在不同时间点采用3H-TdR掺入法检测细胞增殖率,以ELISA法测定培养上清液中IFN-γ、IL-2、IL-4、IL-10等的水平,第5天采用流式细胞仪检测CD4+T和CD8+T细胞上CD25、CD69、CD40L和CD45RA的表达.再次MLR 5天,第1、3、5天采用3H-TdR掺入法测定细胞的增殖情况和ELISA法测定培养上清液中的上述细胞因子的水平.结果初次和再次MLR结果均显示,单抗组细胞增殖反应率明显低于对照组.初次MLR单抗组中CD4+T和CD8+T细胞比例明显低于对照组(P＜0.05);单抗组中CD4+CD25+T、CD4+CD69+T、CD8+CD25+T、 CD4+CD40L+T和CD8+CD69+T细胞比例明显低于对照组(P＜0.05),而CD8+CD40L+T和CD4+CD45RA+T细胞的比例与对照组相比无明显差异(P＞0.05).初次MLR中单抗组和对照组培养上清中IL-4和IL-10几乎无法测出,而单抗组培养上清中IFN-γ和IL-2的水平均明显低于对照组(P＜0.01);再次MLR后培养上清中单抗组IFN-γ、IL-2和IL-4和IL-10的分泌水平明显低于对照组(P＜0.05),但处于低水平,仍明显低于对照组.结论在体外MLR体系中,应用抗CD40L单抗孵育供鼠脾T细胞,可同时作用于CD4+T和CD8+T细胞,使CD40L+,CD25+和CD69+表达下降,引起T细胞早期的活化和成熟障碍,T细胞增殖能力减低,抑制了Th1类细胞因子IFN-γ和IL-2及Th2类细胞因子IL-4和IL-10的分泌水平,可诱导供者T细胞免疫耐受.     

CpG-ODN联合抗CD40抗体活化小鼠调节性B细胞抑制CD4+T细胞增殖

目的分析CpG寡脱氧核苷酸(CpG-ODN)联合抗CD40抗体刺激后的调节性B细胞(Breg)对其免疫调节功能的影响。方法用流式细胞分选仪分选小鼠脾脏CD5+CD1 dhighBreg与CD5-CD1 dlowB细胞亚群,CpG-ODN联合抗CD40抗体激活24 h,再与纯化的CD4+T细胞共培养,流式细胞术检测活化后的Breg及对照CD5-CD1 dlowB细胞亚群白细胞介素10(IL-10)的表达,以及共培养抗CD3抗体联合抗CD28抗体激活的CD4+T细胞的增殖与γ干扰素(IFN-γ)的水平。结果 CpG-ODN联合抗CD40抗体激活的CD5+CD1 dhighBreg亚群与CD4+T细胞共培养时能明显抑制活化的CD4+T细胞的增殖,同时IFN-γ的水平降低;而CpG-ODN联合抗CD40抗体刺激后的对照B细胞亚群则不能抑制CD4+T细胞的增殖与IFN-γ分泌。CD5+CD1 dhigh Breg亚群在CpG-ODN联合抗CD40抗体刺激24 h,IL-10的表达水平明显高于CD5-CD1 dlowB细胞亚群。结论体外CpG-ODN联合抗CD40抗体刺激后的CD5+CD1 dhighBreg亚群通过分泌IL-10抑制CD4+T细胞的活化。     

B7H1-Ig诱导Tr1细胞向CD4~+CD25~+Foxp3~+Treg细胞转化的研究

为探讨Ⅰ型调节性T细胞(Tr1)与CD4+CD25+Foxp3+Treg之间的转化和相互关系,以预包被而固相化的B7H1-Ig融合蛋白加抗CD3单抗刺激初始CD4+CD62L+T细胞,分析细胞因子及Foxp3表达水平的变化,检测细胞功能;在B7H1-Ig开始刺激时或诱导细胞分化结束后加入重组人TGF-β,观察其对细胞分化的影响。结果显示,B7H1-Ig激活的CD4+T细胞产生高水平IL-10、IFN-γ和IL-5,极低水平的IL-2和IL-4,不表达Foxp3,通过分泌抑制性细胞因子IL-10发挥免疫抑制功能,证实B7H1-Ig可诱导Tr1细胞的产生。同时发现TGF-β不影响B7H1-Ig刺激的初始CD4+T的分化,却可促进B7H1-Ig诱导的已分化Tr1细胞向CD4+CD25+Foxp3+Treg转化,提示在特定条件下,Tr1细胞可转化的CD4+CD25+Foxp3+Treg。研究结果为将来临床应用CD4+Treg治疗免疫失调性疾病奠定了基础。     

成人麻疹细胞免疫与CD4+ CD25+调节性T细胞的关系

目的 了解CD4+CD25+调节性T细胞(Treg)与麻疹患者细胞免疫的关系.方法 采集34例成年麻疹患者和27例健康对照的外周血,采用流式细胞术进行CD4+CD25+细胞和FoxP3+细胞的检测.用免疫磁珠从外周血单个核细胞(PBMC)分选出CD4+CD25+T细胞和CD4+CD25-T细胞,分别用抗-CD3单克隆抗体、BCG和NV减毒株进行刺激培养,收集培养上清,用ELISA法检测培养上清中的IFN-γ和IL-10.结果 麻疹患者外周血白细胞、淋巴细胞及CD3+CC4·细胞比例明显降低,而CD4+CD25+细胞与FoxP3+细胞的比例则与对照无显著差别;CD4+CD25+细胞明显抑制抗CD3刺激下CD4+CD25-T细胞产生IFN-γ,且患者和对照没有差别,而IL-10的产生没有这种改变.结论 成人麻疹患者的的免疫抑制与Treg的抑制作用无关. 4+CD25+T细胞和CD4+CD25-T细胞,分别用抗-CD3单克隆抗体、BCG和NV减毒株进行刺激培养,收集培养上清,用ELISA法检测培养上清中的IFN-γ和IL-10.结果 麻疹患者外周血白细胞、淋巴细胞及CD3+CC4+细胞比例明显降低,而CD4+CD2 +细胞与FoxP3+细胞的比例则与对照无显著差别;CD4+CD25+细胞明显抑制抗CD3刺激下CD4+CD25-T细胞产生IFN-γ,且患者和对照没有差别,而IL-10的产生     

Th1、Th2样细胞因子对CD158+细胞比率的调节作用

目的:观察Th1、Th2样细胞因子对外周血CD158+细胞比率的调节作用,为寻找干细胞移植免疫耐受的方法提供论理依据。方法:将Th1样细胞因子(IL-2、IFN-γ)和Th2样细胞因子(IL-4、IL-6),单独或联合与人外周血单个核细胞(PBMC)培养72h,用流式细胞法分析总CD158a+、CD158b+细胞及CD3+、CD4+、CD8+和CD16+ CD56+细胞亚群中CD158a+、CD158b+细胞的比率。结果:①细胞因子对CD3+、CD4+、CD8+细胞及CD16+ CD56+细胞的影响:IL-2和IFN-γ均可增加上述细胞的百分率(P<0.05),但IL-2的作用大于IFN-γ(P<0.05)。IL-2+IFN-γ联合处理的效应高于IFN-γ单独处理(P<0.05)。IL-4+IL-6可降低上述细胞的百分率(P<0.05)。IL-2+IL-4对上述细胞百分率的影响,高于IL-4(P<0.05)但低于IL-2(P<0.05)。②细胞因子对CD158a+/b+细胞的影响:IL-2可增加总的CD158a+/b+细胞及CD3+、CD4+、CD8+和CD16+ CD56+细胞中的CD158a+/b+细胞的百分比率(P<0.05);IL-2+IFN-γ可增加CD158a+/b+细胞的百分率(P<0.05),但与IL-2单独处理无明显区别,IL-4+IL-6可降低CD158a+/b+细胞的百分率(P<0.05)。IL-2+IL-4可增加总的CD158a+/b+细胞及各亚群中CD158a+/b+细胞的百分率(P<0.05),但低于IL-2单独处理(P<0.05)。结论:IL-2有促进CD158基因表达或使CD158a+、CD158b+     

Ag85A DNA疫苗加强免疫显著提高卡介苗初免小鼠的抗结核T细胞免疫应答

目的 构建表达结核分枝杆菌(Mycobacterium tuberculosis,Mtb)免疫优势抗原Ag85A的DNA疫苗,分析其加强免疫后提高卡介苗(BCG)初免小鼠的抗结核T细胞免疫应答.方法 以Mtb毒株H37Rv基因组DNA为模板,PCR扩增Ag85A抗原编码的结构基因并克隆至真核表达载体pVAX1中构建其DNA疫苗;接着,将纯化后的该DNA疫苗加强免疫BCG初免小鼠2针,以BCG和DNA单独免疫小鼠为对照,免疫8周后无菌分离脾淋巴细胞,分别应用IFN-γ ELISPOT和多因子胞内流式细胞术(intracellular staining)分析免疫小鼠的Mtb抗原特异性效应细胞免疫水平与分泌IFN-γ/TNF-α/IL-2的多功能CD4+T细胞频率及其强度以及CD8+T细胞免疫应答.结果 与BCG免疫及DNA单独免疫组相比,Ag85A DNA加强免疫不仅能显著提高小鼠IFN-γ+TNF-α+IL-2+多功能T细胞,IFN-γ+IL-2+、IL-2+TNF-α+双功能T细胞与IL-2+单功能T细胞的频率以及IL-2的分泌能力,还能显著诱导小鼠产生更多分泌IFN-γ和IL-2的CD8+T细胞.结论 本研究成功构建了表达Mtb免疫优势抗原Ag85A的DNA疫苗并分析了其免疫原性,证实了BCG初免-DNA加强的免疫策略可同时显著增强实验小鼠的Mtb抗原特异性CD4+T和CD8+T细胞应答水平,有利于提高BCG的免疫原性,为增强BCG逐渐下降的抗结核保护效果提供新思路.     

天花粉蛋白合成肽通过诱导CD8抑制性T细胞治疗实验性自身反应性脑脊髓炎

为了探讨天花粉蛋白合成肽(M-Tk)治疗实验性自身反应性脑脊髓炎(EAE)的可能性及其作用机制,应用自身抗原MBP衍生肽MOG35-55免疫C57BL/6小鼠成功地诱发EAE后,以流式细胞术测定小鼠淋巴结细胞及脾细胞T亚群及细胞因子的表达,并通过临床评分观察M-Tk治疗EAE的有效性,包括以HE和LFB染色观察髓鞘病变。结果发现,M-Tk可抑制MOG35-55特异性T细胞的增殖反应,选择性诱导CD8+CD28-T调节细胞的扩增,明显提高IL-10的分泌和降低IFN-γ的产生,并有效改善EAE小鼠神经功能评分。采用M-Tk诱导的CD8 T细胞作体内输注可取得相似甚至更好的治疗效果(P>0.01)。提示M-Tk在体内诱导产生CD8+CD28-抑制性T细胞并上调IL-10分泌降低IFN-γ产生,是对EAE取得疗效的关键。     

B7-H1阻断对CD3AK细胞体外生物学活性的影响

目的: 探讨B7-H1阻断对CD3AK细胞增殖活化及其抗肿瘤免疫效应的影响.方法: 利用CD3单克隆抗体(mAb)刺激健康人外周血淋巴细胞诱导产生CD3AK细胞,然后利用B7-H1阻断型抗体阻断B7-H1通路,3H-TdR渗入法检测阻断后CD3AK细胞的增殖能力,ELISA法检测阻断后CD3AK细胞分泌IFN-γ、 TNF-α和IL-10的水平,同时将CD3AK细胞作用于膀胱肿瘤BIU-87细胞,MTT法检测阻断后CD3AK细胞的杀伤活性.结果: B7-H1阻断后,CD3AK细胞的增殖能力明显增强,体外存活时间明显延长;其分泌IFN-γ、 TNF-α的水平明显提高,而分泌IL-10的水平明显下降;同时CD3AK细胞对BIU-87细胞的杀伤活性亦明显升高.结论: 阻断B7-H1通路可以促进和维持CD3AK细胞的增殖和活化,并增强其抗肿瘤的免疫效应.阻断B7-H1通路将有望成为肿瘤免疫治疗的新策略.     

CD28 activation promotes Th2 subset differentiation by human CD4+ cells

Ligation of CD28 provides a costimulatory signal to T cells necessary for their activation resulting in increased interleukin (IL)-2 production in vitro, but its role in IL-4 and other cytokine production and functional differentiation of T helper (Th) cells remains uncertain. We studied the pattern of cytokine production by highly purified human adult and neonatal CD4⁺ T cells activated with anti-CD3, phorbol 12-myristate 13-acetate (PMA) and ionomycin, or phytohemagglutinin (PHA) in the presence or absence of anti-CD28 in repetitive stimulation-rest cycles. Initial stimulation of CD4⁺ cells with anti-CD3 (or the mitogens PHA or PMA+ionomycin) and anti-CD28 monoclonal antibodies induced IL-4, IL-5 and interferon-γ (IFN-γ) production and augmented IL-2 production (6- to 11-fold) compared to cells stimulated with anti-CD3 or mitogen alone. The anti-CD28-induced cytokine production corresponded with augmented IL-4 and IL-5 mRNA levels suggesting increased gene expression and/or mRNA stabilization. Most striking, however, was the progressively enhanced IL-4 and IL-5 production and diminished IL-2 and IFN-γ production with repetitive consecutive cycles of CD28 stimulation. The enhanced Th2-like response correlated with an increased frequency of IL-4-secreting cells; up to 70% of the cells produced IL-4 on the third round of stimulation compared to only 5% after the first stimulation as determined by ELISPOT. CD28 activation also promoted a Th2 response in naive neonatal CD4⁺ cells, indicating that Th cells are induced to express a Th2 response rather than preferential expansion of already established Th2-type cells. This CD28-mediated response was IL-4 independent, since enhanced IL-5 production with repetitive stimulation cycles was not affected in the presence of neutralizing anti-IL-4 antibodies. These results indicate that CD28 activation may play an important role in the differentiation of the Th2 subset in humans.     

Clonal analysis of CD4 mediated accessory function on the effector activity of human CD4+ T cell subsets

Background: It has been reported for the peripheral T cell repertoire that CD4 molecules may enhance adhesion between T cells and antigen presenting cells and, through their physical association with T cell antigen receptors, contribute to signal transduction. Objective: The aims of this study were to determine if the modulation of CD4 molecules had differential effects on T cell recognition, antigen induced cytokine (IL-4 and IFNγ), release and the induction of specific anergy for human TH-0, TH-1 and TH-2 cells. Methods: A panel of anti-CD4 antibodies was examined for its ability to modulate T cell proliferation, cytokine production and tolerance induction in house dust mite (TH-0 and TH-2) and influenza haemagglutinin (TH-1) specific human CD4+ T cell clones all restricted by DRB1^*1101 and isolated from dust mite allergic individuals. Results: We observed that anti-CD4 antibodies may inhibit or enhance antigen mediated T cell proliferation, which may reflect the differential requirements of T cells for selective functions of CD4. Furthermore, IFNγ and IL-4 production was differentially modulated depending on the specificity of the anti-CD4 antibody and the clone of T cells. However, pretreatment of T cells with anti-CD4 antibody alone neither induced nor enhanced the susceptibility of T cells to peptide mediated anergy. Conclusion: Antigen recognition by different subsets of human CD4+ T cells has differential requirements on CD4, whereas the induction of specific anergy appeared to be independent of the functions of CD4 molecules. Antigen induced IFNγ production was more susceptible than IL-4 to the inhibitory effects of anti-CD4 antibodies. Furthermore, it appeared that certain anti-CD4 antibodies can dissociate antigen induced IFNγ and IL-4 production, and may downregulate IFNγ synthesis without inhibiting antigen dependent proliferation.     

Production of IFN-γ by CD4(+) T cells in response to malaria antigens is IL-2 dependent

T-cell immune responses are critical for protection of the host and for disease pathogenesis during infection with Plasmodium species. We examined the regulation of CD4(+) T-cell cytokine responses during infection with Plasmodium berghei ANKA (PbA). CD4(+) T cells from PbA-infected mice produced IFN-γ, IL-4 and IL-10 in response to TCR stimulation at levels higher than those from uninfected mice. This altered cytokine response was dependent on parasitemia. To examine the specificity of the response, mice were adoptively transferred with CD4(+) T cells from OT-II TCR transgenic mice and were infected with PbA expressing OVA. Unexpectedly, CD4(+) T cells from the OT-II-transferred wild-type PbA-infected mice showed high levels of IFN-γ production after stimulation with OVA and the cells producing IFN-γ were not OT-II but were host CD4(+) T cells. Further investigation revealed that host CD4(+) T cells produced IFN-γ in response to IL-2 produced by activated OT-II cells. This IFN-γ response was completely inhibited by anti-CD25 mAbs, and this effect was not due to the block of the survival signals provided by IL-2. Furthermore, IFN-γ production by CD4(+) T cells in response to PbA antigens was dependent on IL-2. These findings suggest the importance of IL-2 levels during infection with malaria parasites and indicate that CD4(+) T cells can produce IFN-γ without TCR engagement via a bystander mechanism in response to IL-2 produced by other activated CD4(+) T cells.     

Downregulation of microRNA-24 and -181 parallels the upregulation of IFN-γ secreted by activated human CD4 lymphocytes

IFN-γ is a cytokine with important roles in the innate and adaptive immune responses. This cytokine is secreted by activated T cells, NK cells and macrophages. Studies on the regulation of human IFN-γ expression had been previously focused on the promoter region. Consequently, the role of microRNAs (miRs) in this regulation has not been investigated yet. As miR-24 and miR-181 were found to have potential target sites in IFN-γ mRNA 3′UTR, we assessed their impact on IFN-γ expression by co-stimulating PB CD4+ T cells with anti-CD3, anti-CD28, IL-12, and IL-18. This co-stimulation cocktail induced an abundant secretion of IFN-γ together with a down-regulation of miR-24, and miR-181. Existence of a link between these two phenomena was further substantiated by transfection and transduction assays that showed that these two miRs negatively regulate IFN-γ expression by directly binding to their target sites in the mRNA. Thus, identifying target sites for miR-24 and miR-181 in IFN-γ-3′UTR points to a novel regulatory mechanism of this crucial gene.     

Costimulation of CD3/TcR complex with either integrin or nonintegrin ligands protects CD4+ allergen-specific T-cell clones from programmed cell death

An optimal stimulation of CD4⁺ cells in an immune response requires not only signals transduced via the TcR/CD3 complex, but also costimulatory signals delivered as a consequence of interactions between T-cell surface-associated costimulatory receptors and their counterparts on antigen-presenting cells ‘APC). The intercellular adhesion molecule-1 ‘ICAM-1, CD54) efficiently costimulates proliferation of resting, but not antigen-specific, T cells. In contrast, CD28 and CD2 support interleukin ‘IL)-2 synthesis and proliferation of antigen-specific T cells more efficiently than those of resting T cells. The molecular basis for this differential costimulation of T cells is poorly understood. Cypress-specific T-cell clones ‘TCC) were generated from four allergic subjects during in vivo seasonal exposure to the allergen. Purified cypress extract was produced directly from fresh collected pollen and incubated with the patients' mononuclear cells. Repeated allergen stimulation was performed in T-cell cultures supplemented with purified extract and autologous APC. The limiting-dilution technique was then adopted to generate allergen-specific TCC, which were also characterized by their cytokine secretion pattern as ThO ‘IL-4 plus interferon-gamma) or Th2 ‘IL-4). Costimulation-induced proliferation or apoptosis was measured by propidium iodide cytofluorometric assay. By cross-linking cypress-specific CD4⁺ and CD8⁺ T-cell clones with either anti-CD3 or anti-CD2, anti-CD28, and anti-CD54 monoclonal antibodies, we demonstrated that CD4+ clones ‘with ThO- or Th2-type cytokine production pattern) undergo programmed cell death only after anti-CD3 stimulation, whereas costimulation with either anti-CD54 or anti-CD28 protects target cells from apoptosis. The costimulation-induced protection from apoptotic death was associated with a significant rise in IL-4 secretion in both Th0 and Th2-type clones. In contrast, cypress-specific Th0 CD8⁺ clones were more susceptible to stimulation-induced apoptosis via either anti-CD3 or anti-CD2, alone or in combination with anti-CD54 or anti-CD28, thus displaying only slight but nonsignificant modifications in the pattern of IL-4 secretion. The death-promoting costimulatory effects were not observed with highly purified normal resting CD4⁺ or CD8⁺ lymphocytes. Taken together, these results suggest that TcR engagement by an allergen in the context of functionally active APC induces activation-dependent cell death of some, perhaps less specific, cells, and this may be an important homeostatic mechanism through which functional expansion of allergen-specific T cells is regulated during an ongoing immune response.     

Effect of CD80 and CD86 blockade and anti-interleukin-12 treatment on mouse acute graftversus-host disease

We investigated the efficacy of a combination of anti-CD80 and CD86 (CD80 + 86) monoclonal antibodies (mAb), anti-interleukin (IL)-12 mAb, or both, for prophylaxis in a mouse acute graft-versus-host-disease (GVHD) model. The treatment with a combination of anti-CD80 + 86 mAb efficiently reduced the lethality of GVHD, whereas mAb against either CD80 or CD86 alone had an effect. A delay in lymphocyte reconstitution and GVHD-associated histological changes in organs was observed at 30 days post-bone marrow transplantation (BMT) even in the anti-CD80 + 86 mAb-treated mice, although these manifestations were resolved by 100 days. In vitro, host alloantigen-specific T cell proliferative responses and generation of CTL were significantly reduced by anti-CD80 + 86 treatment. Furthermore, anti-CD80 + 86 mAb preferentially inhibited the production of interferon (IFN)-γ, but not IL-4 and IL-10, when cultures were assayed at 21 days. Although the anti-IL-12 mAb treatment alone inhibited the generation of cytotoxic T lymphocytes and IFN-γ production in vitro, administration of anti-IL-12 mAb in vivo reversed the beneficial effects of anti-CD80 + 86 treatment on host survival post-BMT. The adverse effect of anti-IL-12 treatment seems to result from impairment of natural immunity and hematopoiesis, rather than as a consequence of an incomplete blockade of T helper (Th)1 responses. Our results suggest that the prevention of GVHD-induced death results from the efficient blockade of Th1 cell activation by the anti-CD80 + 86 treatment. However, further treatment is required for a complete prevention of GVHD, which seems to be partly mediated by Th2 cells.     

Human memory T cell differentiation into Th2-like effector cells is dependent on IL-4 and CD28 stimulation and inhibited by TCR ligation

Freshly isolated memory T cells primarily produced IL-2 and small amounts of IL-4 and IFN-γ after stimulation in vitro. Priming for 5 days in vitro with anti-CD28 monoclonal antibodies (mAb) alone markedly increased production of IL-4. In comparison to fresh cells, the increase in the amount of IL-4 secreted reflected a marked increase in the number of IL-4-producing cells. Stimulation with immobilized anti-CD3 mAb during priming limited subsequent IL-4 production. By contrast, IFN-γ production from in vitro primed memory T cells was directly correlated to the concentration of priming anti-CD3 mAb. IL-2 production by all restimulated cells was decreased. The differentiation of IL-4-producing cells could be blocked by antibody to IL-4 and enhanced by the addition of recombinant IL-4 as well as antibody to IFN-γ. Of note, the IL-4-producing effector cells induced from in vitro priming derived from the early CD27^pos memory T cell subset, whereas the small CD27^neg differentiated memory subset produced IL-4 without in vitro priming. The results indicate that memory T cells can be directed to differentiate into IL-4-producing effector cells by stimulation via CD28 and IL-4, whereas increasing engagement of the TCR limits Th2 memory cell differentiation.     

Interleukin-13 is produced by activated human CD45RA+ and CD45R0+ T cells: modulation by interleukin-4 and interleukin-12

Interleukin (IL)-13 is a cytokine originally identified as a product of activated T cells. Little is known, however, about IL-13 production by human T cells and its modulation by other cytokines. Here, we show that IL-13 is produced by activated human CD4⁺ and CD8⁺ CD45R0⁺ memory T cells and CD4⁺ and CD8⁺ CD45RA⁺ naive T cells. In contrast, IL-4, which shares many biological activities with IL-13, is only produced by CD45R0⁺ T cells following activation. Analysis of intracellular cytokine production by single CD45RA⁺ and CD45R0⁺ T cells indicated that IL-13 continued to be produced for more than 24 h after stimulation, whereas IL-4 could not be detected after 24 h. These data were confirmed by measurement of specific mRNA and suggest that IL-13, unlike IL-4, but like interferon-γ (IFN-γ), is a cytokine with long-lasting kinetics. The majority of human CD45R0⁺ T cells produced IL-4 and IL-13 simultaneously. In contrast, IFN-γ protein was generally not co-expressed with IL-4 or IL-13. IL-4 added to primary cultures of highly purified peripheral blood T cells activated by the combination of anti-CD3+anti-CD28 mAb enhanced IL-13 production by CD45RA⁺ and to a lesser extent by CD45R0⁺ T cells. Under these conditions, however, IL-12 inhibited IL-13 production by CD45RA⁺ T cells and to a lesser extent by CD45R0⁺ T cells in a dose-dependent fashion. These inhibiting effects were not related to enhanced IFN-γ production induced by IL-12, since IFN-γ by itself did not affect IL-13 production. Collectively, our data indicate that IL-13 is produced by peripheral blood T cells which also produce IL-4, but not IFN-γ, and by naive CD45RA⁺ T cells which, in contrast, fail to produce IL-4. These observations, together with the long-lasting production of IL-13, suggest that IL-13 may have IL-4-like functions in situations where T cell-derived IL-4 is still absent or where its production has already been down-regulated.     

Effect of CD80 and CD86 on T Cell Cytokine Production

Conjugation of the T cell receptor (TCR) with antigen/MHC proteins must be accompanied by conjugation of T cell counterreceptors (CD28 or CTLA-4) with costimulatory molecules CD80 or CD86 (B7-1 or B7-2) on antigen presenting cells (APC) to avert T cell anergy, and to provide essential signals for T cell activation and cytokine production. However, T cells and APC express changing patterns of counterreceptors and costimulatory molecules during the immune response. To determine the involvement of CD80 and CD86 in costimulation of T cell cytokine production, T cells were incubated with peritoneal exudate macrophages, which express CD80 and CD86, and stimulated in vitro for 48 or 72 hrs with anti-CD3 in the presence or absence of blocking antibody to CD80 or CD86. Alternatively, enriched anti-CD3 stimulated T cells were costimulated with antibody to CD28 and CTLA-4. Production of T cell IL-2, IL-4, and IL-5 was depressed in the presence of anti-CD86 but not anti-CD80. Production of IFN-γ was significantly blocked by either anti-CD80 and anti-CD86. Anti-CD28 was a potent costimulator of IFN-γ and IL-2 production, but a less potent costimulator of IL-4 and IL-5 production. The data suggest that T cell counterreceptors and APC costimulatory molecules act with varying efficacies at stimulating production of T cell cytokines.