DNT细胞在肿瘤中的作用

近年来,通过细胞免疫治疗肿瘤的方法可以使肿瘤患者的愈后及生存质量得到很大提高和改善,细胞免疫治疗成为研究的热点.免疫细胞可以通过免疫调节或直接杀伤作用于肿瘤细胞.DNT细胞(CD4CD8 double-negative T cells)是新近发现的一类细胞表面既不表达CD4分子同时也不表达CD8分子的T细胞群.本文主要介绍DNT细胞的两个亚群中的双阴性调节T细胞(DN Tregs)在肿瘤中作用的进展情况.     

DNT细胞在免疫治疗中的研究进展

调节性T细胞在调节免疫反应中有着非常重要的作用,近来新发现一类特殊的调节性T细胞,它们的细胞表型为TCRαβ+ CD3+ CD4-CD8-NKl.1-,即DNT细胞.DNT细胞能够抑制CD4+、CD8+、B、NK和树突状细胞,从而在多种疾病免疫调节如自身免疫性疾病、感染、炎性疾病、移植免疫和肿瘤免疫等发挥着重要的作用,近年来DNT细胞已成为免疫治疗研究新的热点.本文主要就DNT细胞的可能来源及发挥调节作用的机制作进一步的阐述.     

乳腺癌患者B7-CD28共刺激通路的研究

目的探讨乳腺癌患者外周血中CD2 8表达阳性的T细胞数量和患者乳腺癌原代细胞表面B7 1(CD80 )、B7 2 (CD86 )的表达水平 ,以探讨乳腺癌患者共刺激通路是否异常。方法采用流式细胞技术检测乳腺癌患者外周血中CD2 8阳性T细胞与乳腺癌患者原代细胞B7分子的表达 ,并与乳腺良性疾病作比较。结果乳腺癌患者CD2 8阳性的T细胞数明显低于对照组 (t =2 879,P <0 0 5 ) ,特别是CD4/CD2 8双阳性T细胞数更低于对照组 (t=3 0 17,P <0 0 0 1)。乳腺癌患者原代细胞表面B7 1和B7 2表达水平都低于对照组。结论乳腺癌患者外周血中T细胞低表达CD2 8分子 ,乳腺癌细胞低表达B7分子 ,从而影响B7 CD2 8共刺激通路 ,使T细胞不能有效地清除肿瘤细胞。     

小鼠CD4T细胞转化的双阴性T细胞的效应分子研究

目的 研究小鼠CD4 T细胞转化的CD4-CD8-双阴性T细胞的功能特点,进一步明确双阴性T细胞的免疫抑制作用机制.方法 应用转录组测序和蛋白质质谱技术分析了小鼠转化的双阴性T细胞基因表达谱.应用流式细胞术等技术验证了细胞活化分子CD44、CD69和OX40的表达水平并应用CFSE染色法验证双阴性T细胞的杀伤功能.结果 小鼠转化的双阴性T细胞表达了与其他成熟CD4 T细胞不同的细胞表型.小鼠转化的双阴性T细胞明显高表达细胞表面活化分子CD44、CD69和OX40,以及颗粒酶、穿孔素等细胞杀伤相关基因.穿孔素基因敲除的双阴性T细胞抑制淋巴细胞增殖的能力明显降低.结论 小鼠CD4 T细胞转化的双阴性T细胞是与其他成熟CD4 T细胞不同的T细胞终末分化亚群.小鼠转化的双阴性T细胞高表达细胞活化分子和免疫杀伤类细胞因子,并主要通过穿孔素途径发挥免疫抑制功能.     

DN Treg细胞在免疫治疗中的研究进展

免疫系统是人体抵抗疾病的自身防卫系统.免疫治疗就是通过刺激人体自身免疫系统来抵抗各种疾病的治疗方法,它主要分为细胞免疫治疗及疫苗治疗两部分.近年来,细胞免疫治疗通过免疫调节或直接杀伤作用于肿瘤细胞,使肿瘤患者的预后及生存质量得到很大提高和改善,因此,细胞免疫治疗成为研究的热点.DN Treg(cD4cD8 double negative T cells)是新近发现的一类细胞,表面既不表达cD4分子同时也不表达cD8分子的调节性T细胞群.本文主要介绍DN Treg细胞在免疫治疗中的进展情况.     

RATG对CD4+细胞和CD8+细胞共刺激分子基因表达和细胞因子分泌的影响

目的 探讨兔抗人胸腺细胞多克隆抗体(RATG)在体外对CD4+细胞和CD8+细胞共刺激分子基因表达和细胞因子分泌的影响.方法 从正常成人外周血单个核细胞中分离和纯化CD4+细胞和CD8+细胞,加入RATG,37℃下培养.分别于24、48和72 h收集培养上清液和细胞,以不处理的细胞为正常对照,正常兔Ig处理的细胞作为阴性对照.采用实时聚合酶链反应技术检测培养细胞的细胞毒性T淋巴细胞相关抗原4(CTLA4)、CD154、Foxp3、OX40、γ干扰素(IFN-γ)、白细胞介素2(IL-2)、IL-10和IL-2受体(CD25)的基因表达水平,应用Multiplex检测技术测定培养上清液中IFIN-γ、IL-2、IL_4和IL-10的水平.结果 与正常CD4+细胞比较,加入RATG的CD4+细胞培养24 h,其CTLA-4、CD154、Foxp3、OX40、IFN-γ、IL-2、IL-10和CD25基因转录表达均上调,阴性对照CD4+细胞则无这些基因转录表达的上调.处理48 h后,CD4+细胞的CD154和IL-2的基因转录表达呈现下调现象,而CTLA4、Foxp3、0X40、IFN-γ、IL-10和CD25的基因转录水平均较24 h时明显降低.处理72 h后,CD4+细胞的CTLA4、Foxp3、OX40和CD25的基因转录表达再次呈现高水平,CD154和IFN-γ的基因转录表达上调表达不明显,而IL-2和IL-10的基因转录表达则呈现明显的下调.RATG处理的CD4+细胞培养上清液中的IFN-γ、IL-2、IL-4和IL-10浓度显著增加,以处理24 h的水平最高,而阴性对照者未测出.与正常CD8+细胞相比较,加入RATG处理的CD8+细胞培养24 h,其CTLA4、Foxp3、OX40、IFN-γ,、IL-2、IL-10和CD25的基因转录表达呈现明显上调,而CD154基因转录表达稍有下调.RATG处理48 h,CD8+细胞的CTLA4、Foxp3、OX40、IFN-γ和CD25基因转录表达仍维持在高水平,CD154基因转录表达仅仅呈现低水平的上调,IL-10基因转录表达水平显著下降,而IL-2的基因转录表达则明显下调.处理72 h,CD8+细胞的CTLA4、Foxp3、OX40、IFN-γ、IL-10和CD25的基因转录表达仍维持在高水平,CD154基因转录表达则呈现下调,而IL-2基因转录表达的下调更为显著.阴性对照CD8+细胞则未呈现这些基因转录的上调现象.RATG处理的CD8+细胞培养上清液中IFN-γ、IL-2和IL-10显著增多,以处理24 h的浓度最高,IL-4浓度升高的幅度较小,而正常CD8+细胞和阴性对照CD8+细胞几乎检测不到IFN-γ、IL-2、IL-4和IL-10.结论 在体外,RATG可以刺激CD4+细胞和CD8+细胞上调多种促进免疫抑制的共刺激分子基因表达,促进其分泌与免疫调节相关的IFN-γ、IL-2、IL-4和IL-10.     

Th1/Th2细胞因子与肝病关系的研究新进展

辅助T细胞(helper T cell,Th)是指能帮助B细胞分化成抗体产生细胞和放大细胞免疫应答的一个细胞群,它表达CD4而不表达CD8.Th活化后可释放细胞因子,调节T细胞、B细胞、单核细胞和其他免疫细胞活性,根据产生细胞因子的不同,Th可分为Th1、Th2及Th0三型,机体对细胞内与细胞外抗原的免疫应答主要受Th1和Th2调节.……     

CD4-CD8-双阴性T细胞在移植免疫耐受形成中的作用

体内移植物抗宿主反应和宿主抗移植物反应主要是由T淋巴细胞介导。在同种异体移植时,因移植物的组织相容性抗原与受者不符,而刺激受者免疫系统发生排斥反应。由于移植物的细胞构成不同,触发的免疫反应会有所差异。去除T细胞或抑制T细胞的功能均能使移植物的存活时间延长。最近研究发现一种T细胞亚群——αβTCR^＋CD3^＋CD4^- CD8^-双阴性T细胞（double-nega-tiveTcell,DNT）可通过抑制同系基因型CD4^＋ T细胞或CD8^＋ T细胞从而抑制移植物排斥反应的发生,因此研究DNT细胞在移植免疫耐受形成中的作用将有重要意义。     

DNT细胞对体内外胰腺癌生长的抑制作用

目的 探讨CD4 - CD8 - DNT细胞对体内、外胰腺癌生长的抑制作用.方法 采用四甲基偶氮唑盐法检测DNT细胞对人胰腺癌细胞株Panc-1生长的影响.将18只裸鼠随机分成3组,其中前两组皮下注射Panc-1细胞以建立荷瘤鼠人胰腺癌皮下移植瘤模型,第3组注射DNT与Panc-1细胞按数量5∶1共培养后的细胞混悬液.前两组待成瘤后,再随机分为对照组和治疗组.治疗组予塞来昔布4 mg/d口服,对照组每日给予等量蒸馏水口服.每2周测量裸鼠肿瘤体积一次,描绘肿瘤生长曲线.治疗结束后处死裸鼠,剥除瘤组织,测量肿瘤体积并计算抑瘤率.结果 (1)四甲基偶氮唑盐结果显示,DNT细胞能在体外抑制人胰腺癌细胞株Panc-1的生长,且DNT细胞对胰腺癌细胞的生长抑制呈剂量依赖性;(2) DNT细胞能够干预人胰腺癌裸鼠皮下移植瘤的生长.结论 DNT细胞能够抑制体内、外胰腺癌细胞的生长.     

人单核细胞共刺激分子在异种免疫反应中的表达及其作用机制

目的 揭示人单核细胞共刺激分子在异种免疫反应中的表达及其作用机制.方法 从猪的主动脉分离血管内皮细胞(PEC)并培养扩增;从人单个核细胞(PBMC)中纯化CD4+T淋巴细胞和单核细胞.建立PEC和人PBMC混合培养体系,培养后收集细胞,然后加入荧光标记的单克隆抗体,通过流式细胞术检测CD14+单核细胞表面共刺激分子表达情况.为了检测淋巴细胞增殖反应以及阻断共刺激分子对PEC免疫反应的作用,在PEC和人PBMC混合培养体系中分别加入抗CD154、CD80和CD86单克隆抗体.在培养的最后24 h加入同位素,于培养结束后收集细胞并经同位素计数仪进行检测.纯化的单核细胞经PEC刺激后与CD4+T淋巴细胞共培养来研究这些单核细胞诱导CD4+T淋巴细胞的增殖以及阻断共刺激分子的作用.结果 PEC和人PBMC混合培养后可检测到PBMC对异种PEC的高度免疫增殖反应;流式细胞术检测到PBMC中的CD14+单核细胞表面无CD40和CD80的表达,但表达CD86,经PEC刺激后,CD14+单核细胞膜表面显著上调CD40和CD80蛋白分子的表达,CD86表达上调.与未经刺激的单核细胞相比较,经PEC刺激后的单核细胞和CD4+T淋巴细胞共培养后可诱导CD4+T淋巴细胞明显增殖,抗人CD154、CD80、CD86单克隆抗体可以阻断CD4+T淋巴细胞对PEC的增殖反应.结论 人CD14+单核细胞在异种免疫反应过程的间接抗原提呈和共刺激信号传导中发挥重要作用,通过上调其共刺激分子的表达与CD4+T淋巴细胞共刺激分子CD154和CD28相互作用形成第二信号,并诱导CD4+T淋巴细胞对PEC的增殖反应;阻断共刺激分子可抑制异种细胞免疫反应.     

Adoptive transfer of DNT cells induces long-term cardiac allograft survival and augments recipient CD4(+)Foxp3(+) Treg cell accumulation

Regulatory T (Treg) cells play an important role in the regulation of immune responses but whether Treg will induce tolerance in transplant recipients in the clinic remains unknown. Our previous studies have shown that TCRαβ⁺CD3⁺CD4^?CD8^?NK1.1^? (double negative, DN) T cells suppress T cell responses and prolong allograft survival in a single locus MHC-mismatched mouse model. In this study, we investigated the role of DNT cells in a more robust, fully MHC-mismatched BALB/c to C57BL/6 transplantation model, which may be more clinically relevant. Adoptive transfer of DNT cells in combination with short-term rapamycin treatment (days 1–9) induced long-term heart allograft survival (101 ± 31 vs. 39 ± 13 days rapamycin alone, p < 0.01). Furthermore adoptive transfer DNT cells augmented CD4+Foxp3+ Treg cells accumulation in transplant recipients while depletion of CD4⁺ Treg cells by anti-CD25 inhibited the effect of DNT cells on long-term graft survival (48 ± 12 days vs. 101 ± 31 days, p < 0.001). In conclusion, DNT cells combined with short-term immunosuppression can prolong allograft survival, which may be through the accumulation of CD4⁺Foxp3⁺ Treg cells in the recipient. Our result suggests that allograft tolerance may require the co-existence of different type Treg cell phenotypes which are affected by current immunosuppression.     

Interaction Between Cell-Surface-Expressed 70-Kilodalton Heat Shock Protein-like Antigen and CD3 + 4-8- Killer T Cells

Abstract: The 70-kilodalton heat shock proteins may be expressed on the cell surface by an unknown mechanism and may interact with CD3 ⁺ 4^-8^- T cell receptor-αβ^- killer (DNT) cells. In this interaction, certain cellular nascent or mutant peptides may be important (the complexes of 70-kilodalton heat shock protein and cellular peptides directly interact with DNT cells). The results imply that the interaction between 70 kilodalton heat shock proteins and DNT cells may also work in graft rejection. By using antibodies that react with the cell surface-expressed 70-kilodalton heat shock proteins, one may overcome graft rejection. Key Words: Heat shock proteins–T cells–Cellular peptides–Molecular chaper-ones.     

Antigen‐dependent interactions between regulatory B cells and T cells at the T:B border inhibit subsequent T cell interactions with DCs

IL‐10+ regulatory B cells (Bregs) inhibit immune responses in various settings. While Bregs appear to inhibit inflammatory cytokine expression by CD4+ T cells and innate immune cells, their reported impact on CD8+ T cells is contradictory. Moreover, it remains unclear which effects of Bregs are direct versus indirect. Finally, the subanatomical localization of Breg suppressive function and the nature of their intercellular interactions remain unknown. Using novel tamoxifen‐inducible B cell–specific IL‐10 knockout mice, we found that Bregs inhibit CD8+ T cell proliferation and inhibit inflammatory cytokine expression by both CD4+ and CD8+ T cells. Sort‐purified Bregs from IL‐10‐reporter mice were adoptively transferred into wild‐type hosts and examined by live‐cell imaging. Bregs localized to the T:B border, specifically entered the T cell zone, and made more frequent and longer contacts with both CD4+ and CD8+ T cells than did non‐Bregs. These Breg:T cell interactions were antigen‐specific and reduced subsequent T:DC contacts. Thus, Bregs inhibit T cells through direct cognate interactions that subsequently reduce DC:T cell interactions.     

Anti-tumour activity of heat-shock protein 60-recognizing CD4+ T cells against syngeneic murine renal cell carcinoma

OBJECTIVES: To determine whether heat-shock protein (HSP) 60-recognizing CD4(+) T cells show antitumour activity against renal carcinoma (RENCA) cells, as HSP is highly expressed by tumour cells and induced in cells by various stresses, including transformation. MATERIALS AND METHODS: RENCA, a renal cortical adenocarcinoma cell line of BALB/c origin, was used. Expression of major histocompatibility complex (MHC) class I, class II and HSP-60 on RENCA tumour cells was analysed by flow cytometry. BASL1.1, an autoreactive T-helper type 1 type CD4(+) T cell clone established by us, and that recognises HSP-60, was also used for a tumour-neutralising assay. RESULTS: The RENCA cells were negative for MHC class II, but expressed intracellular HSP-60. In the tumour-neutralising assay, BASL1.1 cells significantly suppressed the in vivo growth of RENCA cells. Three of five mice rejected RENCA cells when co-inoculated with BASL1.1 cells. CONCLUSIONS: These results indicate that HSP-60-recognizing CD4(+) T cells have the potential to eliminate renal cell carcinoma in vivo and that the eliciting of an anti-self T cell response at the tumour site can lead to regression of renal cancer.     

DNT细胞分离鉴定及其在正常人外周血中含量的检测

目的 建立人DNT细胞的免疫磁性分离(megnetic activated cell sorting,MACS)方法,并检测其在正常人外周血中含量.方法 运用MACS法分离止常人外周血中DNT细胞,用台盼蓝染色流式细胞法检测选出细胞的活性和纯度,运用流式细胞法检测其在正常人外周血中含量.结果 MACS分选法分选出的DNT细胞活性＞97%,纯度为82.77%;DNT细胞占TCRαβ+T细胞的比例(6.25±2.61)%(n=45).结论 MACS法可简单迅速分选出高纯度DNT细胞,且不影响细胞活力.DNT细胞占TCRCD3+T细胞的比例为(6.25±2.61)%.     

CD4+ cells play a major role in xenogeneic human anti-pig cytotoxicity through the Fas/Fas ligand lytic pathway

BACKGROUND: In this study, the role of cell-mediated cytotoxicity by human leukocytes against pig endothelial cells was examined in vitro. The aim was to determine which cell subsets were responsible for this phenomenon and which pathways were involved in cell lysis. METHODS: Primed human peripheral blood mononuclear cells (PBMC) or purified CD4+ or CD8+ T cells were used in a cell-mediated cytotoxicity assay in which cytotoxicity of an SV40 transformed porcine endothelial cell (EC) line (SVAP) was determined by Annexin V binding. RESULTS: Human PBMC demonstrated specific lysis of porcine EC that was proportional to the effector: target ratio. CD4+ T cells accounted for >60% of this lysis, whereas CD8+ T cells accounted for <20%. CD4+ T cell-mediated lysis depended on direct recognition of porcine major histocompatibility complex class II molecules as inhibition of swine leukocyte antigen class II on porcine EC-inhibited CD4+ T cell cytotoxicity. This lysis was mediated through the Fas/FasL pathway as addition of anti-Fas and/or anti-FasL antibody profoundly inhibited antiporcine lysis. In addition, FasL gene expression was detected in primed PBMC and CD4+ T cells by RT-PCR, whereas granzyme B gene expression was not. Primed CD4+ T cells demonstrated high level FasL protein by Western blotting and two-color FACS analysis, whereas NK cells and CD8+ T cells did not. Finally, recombinant human FasL induced apoptosis in Fas expressing porcine EC cells, demonstrating that human FasL interacted with and activated Fas on porcine EC cells. CONCLUSIONS: In conclusion, human to pig cell-mediated cytotoxicity was mediated predominantly by CD4+ T cells through the Fas/FasL pathway of apoptosis. These results suggest that direct cytotoxicity by xenoreactive CD4+ T cells may be one of several effector mechanisms involved in cellular xenograft rejection.     

Interleukin-2 blocks the antitumour activity caused by depletion of CD25 cells in a murine renal adenocarcinoma model

OBJECTIVE: To test the effectiveness of antimouse CD25 monoclonal antibody (mAb) against murine renal adenocarcinoma (RENCA) cells, as immunoregulatory/suppressor cells are known to be involved in tumour development in vivo, but the functions of these cells are not yet clear, and eliminating naive CD25 (interleukin-2 receptor alpha)-positive T cells elicits potent immune responses to syngeneic tumours in vivo. MATERIALS AND METHODS: Aliquots of 1 x 10(4) or 1 x 10(5) RENCA cells were implanted into the subcapsule of the left kidney of syngeneic male Balb/c mice. Mice were injected with 125 micro g of antimouse CD25 mAb to deplete CD25(+) cells before RENCA implantation. Then 10(4) units of recombinant human interleukin-2 (rhIL-2) were subcutaneously injected twice daily for 7 days. Fourteen or 25 days later the tumour size was determined by laparotomy, and cells sorted using two-colour flow cytometry. RESULTS: Depletion of naive CD25(+) cells with anti-CD25 mAb and rhIL-2 administration effectively induced anti-RENCA tumour activity in Balb/c hosts. However, co-administration of anti-CD25 mAb and rhIL-2 abrogated this significant suppression of RENCA tumour growth. RENCA implantation reduced the proportion of CD4(+) cells among splenocytes, whereas anti-CD25 mAb treatment increased it. The proportion of CD25(+)CD8(+) cells among splenocytes and that of CD25(+) cells among CD8(+) cells were markedly reduced by co-administration of anti-CD25 mAb and rhIL-2 with RENCA implantation. Both CD4(+) and CD8(+) cells were stained around the remnant microscopic RENCA tumour after anti-CD25 mAb treatment. CONCLUSION: Either depletion of naive CD25(+) cells or rhIL-2 administration suppressed RENCA tumour growth in murine hosts. However, co-administration of anti-CD25 mAb and rhIL-2 abrogated this significant suppression of RENCA tumour growth.     

Differential modulation of CD4 and CD8 T-cell proliferation by induction of nitric oxide synthesis in antigen presenting cells

BACKGROUND: On antigenic stimulation, CD4 T cells generally proliferate more readily than CD8 T cells. The purpose of the present experiments was to determine whether nitric oxide (NO) might differentially modulate CD4 vs. CD8 T-cell proliferation. METHODS: Various concentrations of C57BL/6 iNOS +/+ and -/- bone marrow (BM)-derived antigen presenting cells (APC) (obtained by culture in granulocyte-macrophage colony-stimulating factor [GM-CSF] and interleukin [IL]-4) were cultured with purified BALB/c CD4 or CD8 T cells. RESULTS: Proliferation of CD4 T cells was similar in the presence of both NO synthase (iNOS) +/+ and -/- APC, whereas CD8 T cell proliferation was inhibited at the higher concentrations of iNOS +/+ dendritic cells (DC), coincident with increased levels of NO in the culture supernatant. Analysis of cytokine levels revealed that more interferon (IFN)-gamma, a potent inducer of NO synthesis in many cell types, was present in CD8 T cell than in CD4 T-cell-APC cultures. Addition of IFN-gamma to CD4 T-cell-APC cultures resulted in induction of NO synthesis and inhibition of proliferation at higher levels of NO than that required to inhibit CD8 T cell proliferation. However, CD4 T-cell proliferation was moderately inhibited in the presence of lipopolysaccharide (LPS)-stimulated CD11c DC, coincident with production of IFN-gamma and induction of NO synthesis. CONCLUSIONS: These findings indicate that CD8 T-cell proliferation can be inhibited by lesser amounts of APC-derived NO than is necessary to inhibit CD4 T cell proliferation. NO synthesis was not initiated in CD4 T cell-DC cultures unless costimulatory molecules were up-regulated and IFN-gamma was produced.     

The effect of burn injury on CD8+ and CD4+ T cells in an irradiation model of homeostatic proliferation

BACKGROUND: Homeostatic proliferation of T cells has recently been shown to be an important mechanism in the host response to infection. However, its role in the T cell response to burn injury is unknown. In this study, we examine the effect of burn injury on CD4+ and CD8+ T cell homeostatic proliferation after irradiation. METHODS: Wild-type C57BL/6 female mice were irradiated with six grays ionizing radiation and 48 hours later, syngeneic whole splenocytes or purified CD4+ or CD8+ T cells labeled with carboxy-fluorescein diacetate, succinimidyl ester were adoptively transferred. Two days later, mice underwent a 20% burn injury, followed by splenocyte harvest 3 and 10 days after injury. RESULTS: Burn mice demonstrate increased splenic cellularity and CD8+ T cell proliferation after adoptive transfer of either purified CD8+ cells or whole spleen populations compared with unburned (sham) mice. In contrast, CD4+ T cell proliferation after burn injury is unchanged after adoptive transfer of whole spleen cells and drastically decreased after adoptive transfer of a purified CD4+ population compared with sham mice. Ten days after burn injury CD8+ T cells continue to demonstrate greater proliferation than CD4+ T cells. CONCLUSIONS: CD8+ T cells are more robust than CD4+ T cells in their proliferative response after burn injury. In addition, CD8+ T cell proliferation appears less reliant on other immune cells than purified CD4+ T cell proliferation. These data reiterate the importance of CD8+ T cells in the initial immune response to burn injury.