T细胞过继免疫治疗技术的研究进展

随着科学技术的发展和治疗理念的转变,以免疫治疗为基础的肿瘤生物疗法作为现代肿瘤治疗的第四种模式,越来越受到重视,应用前景广阔。相比起传统的手术和放化疗,这种方法毒副作用小、安全稳定,适应性广泛,其原理是通过利用现代生物技术调     

调节性T细胞与恶性肿瘤的过继免疫治疗

过继免疫治疗利用自身免疫细胞进行体外扩增培养,然后回输到体内,它通过增强机体免疫力来对抗肿瘤,对肿瘤细胞具有靶向杀伤性作用,是最有可能彻底清除手术或放化疗后残存肿瘤细胞的治疗手段.但是,调节性T细胞(Regulatory T cells,Tregs)在多种恶性肿瘤患者外周血及微环境中普遍存在着比例上调的情况,这不仅会导致机体固有免疫的负性平衡,还会影响过继免疫治疗的疗效,使其不能发挥最大的抗肿瘤作用.通过对Tregs日渐深入的了解,人们已经研究出多种抑制及消除Tregs的治疗策略.     

CD4+CD25+CCR6+调节性T细胞在小鼠乳腺癌模型中对CD8+T细胞的抑制作用

目的：观察CD4+CD25+CCR6+调节性T细胞（简称CCR6+Tregs）体内对CD8+T细胞功能的抑制作用,并探讨其与肿瘤免疫逃逸的关系。方法：建立4T1乳腺癌细胞荷瘤裸鼠模型,FACS分选CCR6+Tregs,检测其Foxp3的表达;FACS分选4T1特异性CD8+T细胞,CFSE标记后分别与CCR6+Tregs或CCR6Tregs共同过继转输入4T1荷瘤裸鼠体内,观察荷瘤裸鼠肿瘤生长情况和小鼠存活时间;FACS检测肿瘤组织中CD8+T细胞的增殖、细胞因子IFNγ的产生和颗粒酶B的表达情况。结果：CCR6+Tregs和CCR6Tregs均高表达Foxp3;CCR6+Tregs和CD8+T细胞共转输组4T1荷瘤裸鼠肿瘤的生长明显快于CCR6Tregs共转输组和CD8+T细胞单转输组,同时该组荷瘤裸鼠生存时间也明显缩短（P<0.05）;CCR6+Tregs和CD8+T细胞共转输组CD8+T细胞的增殖、IFNγ的产生和颗粒酶B的表达均明显低于CCR6Tregs共转输组和CD8+T细胞单转输组（P<0.05）。结论：CCR6+Tregs在体内可以有效抑制CD8+T细胞的功能,其在肿瘤免疫逃逸和肿瘤发生、发展中发挥重要作用。     

结直肠癌患者外周血中CD4+CD45RA+ T和CD4+CD45RO+ T细胞的变化及其意义

目的 探讨外周血中CD4 CD45RA T细胞和CD4 CD45RO T细胞在结直肠癌中的变化及其临床意义。方法 采用流式细胞术检测60例结直肠癌患者手术前、术后1个月和3个月时,外周血的CD4 T细胞、CD4 CD45RA T细胞和CD4 CD45RO T细胞的比例。选取健康查体人群10例作为对照。结果 结直肠癌患者的CD4 T细胞与健康人群相比无差异。CD4 CD45RO T细胞比例明显增高,术后有显著下降,DukesA、B期患者尤其明显;而CD4 CD45RA T细胞比例正好相反。结论 CD4 CD45RA T细胞和CD4 CD45RO T细胞在肿瘤免疫中起重要作用,其表达同结直肠癌的分期和预后有密切关系。     

组织驻留CD8+T细胞在肿瘤中的研究进展

组织驻留记忆 T 细胞（ tissue-resident memory T cells,TRMs）作为一种特殊的记忆T细胞,在免疫应答中发挥着极其重要的作用。其特征是可表达归巢受体,从而具备驻留特性,因此能够驻留在外周组织器官中,当病原体侵袭时可以迅速反应。目前,TRMs（尤其是CD8+TRMs）与肿瘤的关系及其在抗肿瘤中的应用被愈加重视,一方面是CD8+TRMs可以通过分泌颗粒酶B、穿孔素和INF-γ等因子直接杀伤肿瘤细胞,另一方面一些抗肿瘤措施（如放化疗、免疫治疗等）可以使CD8+TRMs在肿瘤组织中富集,从而进一步提高治疗的效果。本文就CD8+TRMs的亚群分类、在肿瘤中如何调控形成以及其在肿瘤治疗中的作用等方面的研究进展进行综述。      

CD4+CD25+调节性T细胞与CD4+T、CD8+T细胞在结直肠癌组织中的分布

 目的 分析CD4+CD25+ FOXP3+调节性T细胞（Treg）与CD4+T、CD8+T在结直肠癌（colorectal carcinoma, CRC）组织中的分布及其与临床病理特征之间的关系。方法 收集42例CRC新鲜手术标本,应用冰冻切片、免疫组织化学SP法检测肿瘤组织和癌旁组织中FOXP3+、CD4+T和CD8+T阳性细胞数。结果 CRC患者肿瘤组织中FOXP3表达水平显著升高,与癌旁组织相比差异有统计学意义（P<0.01）;中低分化组Treg细胞数明显高于高分化组（P<0.01）;淋巴结转移组Treg细胞数明显高于无淋巴结转移组（P<0.05）;癌巢内CD4+、CD8+T细胞数及CD4+/CD8+值显著低于间质（P<0.01）;Ⅲ+Ⅳ期、淋巴结转移组癌巢内CD4+/CD8+比值显著低于Ⅰ+Ⅱ期及无淋巴结转移组（P<0.05）;CRC中Treg数量与癌巢内CD4+/CD8+比值显著负相关（r=-0.605, P<0.01）。结论 CRC的发生发展可能与其癌组织局部微环境中Treg数量变化相关,肿瘤局部Treg数量的增多与T淋巴细胞亚群比例失调可能成为肿瘤免疫逃逸的机制之一。     

CX3CR1+ CD8+ T细胞在肿瘤免疫治疗中作用的研究进展

趋化因子是免疫系统的重要组成部分,趋化因子CX3CL1因其独特的分子结构和双重的存在形式与其受体CX3CR1 共同影响免疫细胞的募集和归巢,参与多种肿瘤发生和肿瘤免疫过程。趋化因子受体CX3CR1响应CX3CL1的趋化作用。同 时,CX3CR1的表达指示CD8+ T细胞的效应分化状态,CX3CR1+ CD8+ T细胞具有细胞毒性和抗原特异性。在肿瘤微环境中,相比 CX3CR1- CD8+ T细胞,CX3CR1+ CD8+ T细胞表面的共抑制分子表达少、杀伤功能活跃。因此,CX3CR1对于CD8+ T细胞等淋巴 细胞的多重意义使其在肿瘤的细胞免疫治疗,如CAR-T细胞治疗中具有应用前景。在肾细胞癌、黑色素瘤和非小细胞肺癌的 PD-1靶向治疗中,外周血CX3CR1+ CD8+ T细胞的占比与疗效正相关,提示CX3CR1也是早期预测免疫治疗疗效的分子标志物。 尽管CX3CR1+ CD8+ T细胞功能活跃,但此群细胞处于终末分化状态,增殖能力和记忆效应差,不具有长久的保护作用。如何使 CAR-T 细胞在体内维持抗肿瘤作用一直是研究者积极探索的方向,也是将 CX3CR1 应用于 CAR-T 细胞免疫治疗需要克服的 难关。     

肿瘤过继免疫治疗临床级抗原特异性T细胞的研究进展

抗原特异性T细胞是肿瘤过继免疫治疗的核心,靶向性强、杀伤活性高、毒性作用小、疗效卓越的临床级细胞的制备是提高治疗效果的关键。近年来,全封闭自动化T细胞分离系统提高了安全性,新型人工抗原提呈方法和γc家族细胞因子增加了T细胞扩增效率,低分化表型T细胞的选择提高了有效性,而T细胞受体和嵌合抗原受体基因修饰赋予T细胞抗原特异性杀伤性能,病毒基因转导和理化基因转染T细胞方法的不断改进为基因修饰重定向T细胞的抗原特异性提供了保障。越来越多的临床试验展示了令人鼓舞的治疗效果,为肿瘤过继免疫治疗注入了新的生命力。     

干细胞样CD8+ T细胞：肿瘤免疫疗法的新生力量

CD8+ T 细胞是抗肿瘤免疫应答的主要执行者。通过重塑CD8+ T 细胞杀伤肿瘤细胞的能力,免疫疗法已在抗肿瘤领域取得重大突破,但临床获益仅局限于部分患者和癌症类型。如何克服CD8+ T细胞功能障碍是肿瘤免疫疗法亟待解决的关键问题。近年来,多项研究揭示了CD8+ T细胞的干性调控机制,发现了干细胞样CD8+ T细胞具有自我更新和增殖能力,阐明了该细胞亚群在维持持续性肿瘤免疫治疗应答中的重要性。本文论述了干细胞样CD8+ T 细胞的分子与功能特征、CD8+ T 细胞干性的细胞内外影响因素,归纳总结了目前靶向CD8+ T细胞的干性重编程策略,进一步展望了靶向CD8+ T细胞干性程序来提高肿瘤免疫疗法疗效的思路和方法。     

监测肾癌患者外周血CD4~+CD25~+调节性T细胞在术后免疫治疗中的作用

目的:探讨肾癌患者外周血CD4+CD25+T淋巴细胞的监测在术后辅助免疫治疗中的作用。方法:将24例肾癌患者随机分为两组:A组,根治性肾切除术后行IFN-α辅助免疫治疗;B组,单纯行根治性肾切除术;C组,10例健康对照。分别在术前、术后1周以及术后3个月免疫治疗结束时采集外周血样本,流式细胞仪检测CD4+、CD8+、CD4+CD25、CD4+CD25high T淋巴细胞亚群含量,并计算各部分比值。结果:A、B组术前CD4+CD25+、CD4+CD25high T淋巴细胞亚群总体上增加,并且其含量随肿瘤分期的进展而增加(P<0.05),但部分患者并不高于健康对照组。免疫治疗后,肾癌患者CD4+CD25+T淋巴细胞总体略有上升,但术前较高的患者中,约2/3下降。结论:利用流式细胞仪监测肾癌患者外周血CD4+CD25+T淋巴细胞亚群可反映机体抗肿瘤免疫状态,有助于预测免疫治疗的预后,为肾癌尤其是早期肾癌术后辅助免疫治疗的选择提供参考。     

CD4+ T cells support polyfunctionality of cytotoxic CD8+ T cells with memory potential in immunological control of tumor

Polyfunctionality/multifunctionality of effector T cells at the single cell level has been shown as an important parameter to predict the quality of T cell response and immunological control of infectious disease and malignancy. However, the fate of polyfunctional CD8⁺ CTLs and the factors that control the polyfunctionality of T cells remain largely unknown. Here we show that the acquisition of polyfunctionality on the initial stimulation is a sensitive immune correlate of CTL survival and memory formation. CD8⁺ T cells with high polyfunctionality, assessed with γ‐interferon and tumor necrosis factor‐α production and surface mobilization of the degranulation marker CD107a, showed enhanced Bcl‐2 expression, low apoptosis, and increased CD127^highKLRG1^low memory precursor phenotype. Consistent with these observations, CD8⁺ T cells were found to acquire high frequency of cells with polyfunctionality when stimulated in conditions known to enhance memory formation, such as the presence of CD4⁺ T cells, interleukin (IL)‐2, or IL‐21. Utilizing T‐cell receptor (TCR) transgenic mouse‐derived CD8⁺ T cells that express a TCR specific for a tumor‐derived neoantigen, we showed that polyfunctional tumor‐specific CTLs generated in the presence of CD4⁺ T cells showed long persistence in vivo and induced enhanced tumor regression when adoptively transferred into mice with progressing tumor. Acquisition of polyfunctionality thus impacts CTL survival and memory formation associated with immunological control of tumor.     

Generation and application of human induced‐stem cell memory T cells for adoptive immunotherapy

Adoptive T‐cell therapy is an effective strategy for cancer immunotherapy. However, infused T cells frequently become functionally exhausted, and consequently offer a poor prognosis after transplantation into patients. Adoptive transfer of tumor antigen‐specific stem cell memory T (T_SCM) cells is expected to overcome this shortcoming as T_SCM cells are close to naïve T cells, but are also highly proliferative, long‐lived, and produce a large number of effector T cells in response to antigen stimulation. We previously reported that activated effector T cells can be converted into T_SCM‐like cells (iT_SCM) by coculturing with OP9 cells expressing Notch ligand, Delta‐like 1 (OP9‐hDLL1). Here we show the methodological parameters of human CD8⁺ iT_SCM cell generation and their application to adoptive cancer immunotherapy. Regardless of the stimulation by anti‐CD3/CD28 antibodies or by antigen‐presenting cells, human iT_SCM cells were more efficiently induced from central memory type T cells than from effector memory T cells. During the induction phase by coculture with OP9‐hDLL1 cells, interleukin (IL)‐7 and IL‐15 (but not IL‐2 or IL‐21) could efficiently generate iT_SCM cells. Epstein–Barr virus‐specific iT_SCM cells showed much stronger antitumor potentials than conventionally activated T cells in humanized Epstein–Barr virus transformed‐tumor model mice. Thus, adoptive T‐cell therapy with iT_SCM offers a promising therapeutic strategy for cancer immunotherapy.     

IL‐27 improves adoptive CD8+ T cells’ antitumor activity via enhancing cell survival and memory T cell differentiation

IL‐27 is an anti‐inflammatory cytokine that triggers enhanced antitumor immunity, particularly cytotoxic T lymphocyte responses. In the present study, we sought to develop IL‐27 into a therapeutic adjutant for adoptive T cell therapy using our well‐established models. We have found that IL‐27 directly improved the survival status and cytotoxicity of adoptive OT‐1 CD8⁺ T cells in vitro and in vivo. Meanwhile, IL‐27 treatment programs memory T cell differentiation in CD8⁺ T cells, characterized by upregulation of genes associated with T cell memory differentiation (T‐bet, Eomes, Blimp1, and Ly6C). Additionally, we engineered the adoptive OT‐1 CD8⁺ T cells to deliver IL‐27. In mice, the established tumors treated with OT‐1 CD8⁺ T‐IL‐27 were completely rejected, which demonstrated that IL‐27 delivered via tumor antigen–specific T cells enhances adoptive T cells’ cancer immunity. To our knowledge, this is the first application of CD8⁺ T cells as a vehicle to deliver IL‐27 to treat tumors. Thus, this study demonstrates IL‐27 is a feasible approach for enhancing CD8⁺ T cells’ antitumor immunity and can be used as a therapeutic adjutant for T cell adoptive transfer to treat cancer.     

Frequency of CD45RO+ subset in CD4+CD25(high) regulatory T cells associated with progression of hepatocellular carcinoma

The purpose of this study was to assess the properties of CD4+CD25(high/low/negative) T cell subsets and analyze their relation with dendritic cells (DCs) in patients with hepatocellular carcinoma (HCC). In HCC patients, the prevalence of CD45RO+ cells in CD4+CD25(high) T cells was increased and associated with higher frequencies of plasmacytoid DCs. Larger proportions of this T cell subset were detected in the patients with larger tumor burdens. These results suggest that increased frequencies of the CD45RO+ subset in CD4+CD25(high) Tregs in HCC patients may establish the immunosuppressive environment cooperatively with tolerogenic plasmacytoid DCs to promote disease progression of liver cancer.     

Soluble γc receptor attenuates anti‐tumor responses of CD8+ T cells in T cell immunotherapy

Previous studies have shown that soluble common γ‐chain (sγc) modulates CD4⁺ T cell immunity with antagonistic functions in γc cytokine signaling. However, the role of sγc in functional properties of effector CD8⁺ T cells has not been fully defined. In this study, we report a new mechanism by which the anti‐tumor activity of mouse CD8⁺ T cells is suppressed in sγc of their own producing. While sγc significantly inhibits cytotoxicity of CD8⁺ T cells, blocking sγc production by genetic modification leads to potentiated effector function of CD8⁺ T cells, establishing persistent CD8⁺ T cells. This is due to the modulation of IL‐2 and IL‐15 signaling, which is required for expansion and survival of CD8⁺ T cells as well as for optimal cytotoxic activity. More efficient management of tumor growth was achieved by an adoptive transfer of sγc‐deficient CD8⁺ T cells than that of wild‐type or sγc‐overexpressing CD8⁺ T cells. Blocking of IL‐2 and IL‐15 signaling by sγc attenuates the capacity of CD8⁺ T cells to mount an optimal response to the tumor, with both quantitative and qualitative effects on antigen‐specific CD8⁺ T cells. These results could have a critical implication for the generation and survival of optimal effector T cells for adoptive immunotherapy of cancer.     

Human effector T cells derived from central memory cells rather than CD8T cells modified by tumor-specific TCR gene transfer possess superior traits for adoptive immunotherapy

Adoptive cell therapy provides an attractive treatment of cancer, and our expanding capacity to target tumor antigens is driven by genetically engineered human T lymphocytes that express genes encoding tumor-specific T cell receptors (TCRs). The intrinsic properties of cultured T cells used for therapy were reported to have tremendous influences on their persistence and antitumor efficacy in vivo. In this study, we isolated CD8⁺ central memory T cells from peripheral blood lymphocytes of healthy donors, and then transferred with the gene encoding TCR specific for tumor antigen using recombinant adenovirus vector Ad5F35-TRAV-TRBV. We found effector T cells derived from central memory T cells improved cell viability, maintained certain level of CD62L expression, and reacquired the CD62L⁺CD44^high phenotype of central memory T cells after effector T cells differentiation. We then compared the antitumor reactivity of central memory T cells and CD8⁺T cells after TCR gene transferred. The results indicated that tumor-specific TCR gene being transferred to central memory T cells effectively increased the specific killing of antigen positive tumor cells and the expression of cytolytic granule protein. Furthermore, TCR gene transferred central memory T cells were more effective than TCR gene transferred CD8⁺T cells in CTL activity and effector cytokine secretion. These results implicated that isolating central memory T cells rather than CD8⁺T cells for insertion of gene encoding tumor-specific TCR may provide a superior tumor-reactive T cell population for adoptive transfer.     

CD103+CD8+T细胞在结直肠癌组织中的浸润分布及其临床意义

[摘要] 目的:研究组织驻留CD8+T细胞（CD103+CD8+T细胞）在结直肠癌（colorectal cancer,CRC）组织中浸润程度及分布特征,分析其浸润程度与患者临床病理特征及预后的关系。方法:选用上海芯超生物科技有限公司的88 例结肠癌HColA180Su14和77 例直肠癌HRec-Ade180Sur-03 组织芯片,应用免疫荧光染色法分别检测CRC组织及相应癌旁组织中CD103+CD8+T细胞的浸润分布特征及程度,Wilcoxon 秩和检验比较CRC及癌旁组织中CD103+CD8+T细胞浸润程度,χ2检验分析CRC中CD103+CD8+T细胞浸润程度与患者临床病理特征的关系;Kaplan-Meier 生存分析CD103+CD8+T细胞浸润程度与患者预后的关系,拟合Cox 模型评价不同指标与患者预后的关系。结果: CRC组织中CD103+CD8+T 细胞浸润程度与癌旁组织比较差异无统计学意义（P>0.05）,有远处转移患者中CD103+CD8+T细胞高度浸润的比率显著低于无远处转移患者（P<0.01）,CD103+CD8+T细胞浸润程度与患者其他临床病理特征无明显相关（P>0.05）。Kaplan-Meier生存分析显示,CD103+CD8+T细胞高度浸润患者的OS较低度浸润患者显著延长（54.42% vs 25.00%,P<0.05）,多因素Cox 显示,病理分级（P<0.01）和CD103+CD8+T细胞高度浸润（P<0.05）均可作为CRC患者预后的独立影响因素。结论: CRC组织中CD103+CD8+T细胞浸润与预后相关,提示其在CRC发生发展过程中发挥重要作用。     

Impaired tumor rejection by memory CD8 T cells in mice with NKG2D dysfunction

Cytotoxic T cells are important effectors for robust antitumor immune responses. However, tumor-infiltrating CD8 T cells are often functionally impaired. Insufficient antitumor activity of CD8 T cells can be due to a lack of costimulatory signals. NKG2D is such a costimulatory receptor on CD8 T cells that facilitates immunorecognition of stressed and malignant cells, promotes tumor rejection by NK and CD8 T cells and contributes to immunosurveillance of spontaneous malignancies. Previous reports suggested an involvement of NKG2D in establishing CD8 T cell-mediated antitumor memory. However, the significance of NKG2D for the generation and effector phase of memory CD8 T cell responses is largely unknown. To address these issues, we made use of a transgenic mouse model (H2-K(b)-MICA mice) where the human NKG2D ligand MICA is ubiquitously and constitutively expressed resulting in a severe dysfunction of NKG2D. Both, ovalbumin (OVA)-specific (H2-K(b)/OVA(257-264)) memory CD8 T cells arisen from the endogenous T cell pool and adoptively transferred OVA-specific OT-I memory cells were unable to control growth of an OVA-expressing lymphoma in H2-K(b)-MICA mice. While expansion of memory T cells in these mice on antigen challenge was not different from controls, CD8 memory T cells of H2-K(b)-MICA mice did not effectively eliminate tumor cells in vivo. Altogether, our data suggest that NKG2D has no major role in the generation and expansion of memory CD8 T cells, but rather substantially enhances the cytolytic effector responses of reactivated memory T cells and thereby contributes to an efficacious tumor rejection.