Temporal changes of suppressor T lymphocytes and cytotoxic T lymphocytes in syngeneic murine malignant gliomas

Summary The temporal activities of suppressor T lymphocytes (Ts) and cytotoxic T lymphocytes (CTL) were investigated in a syngeneic murine malignant glioma (a methylcholanthrene-induced ependymoblastoma of C57BL/6 mouse origin, 203-glioma).After the s.c. tumor inoculation, it was suggested that both Ts and CTL were generated with target specificity against 203-glioma cells, because neither Ts nor CTL activity were seen against syngeneic EL 4 (benzpyrene-induced thymoma), allogeneic P815 (methylcholanthrene-induced mastocytoma of DBA/2 mouse origin) or YAC-1 (Moloney leukemia-induced T -cell lymphoma of A/Sn mouse origin), but only against 203-glioma. It was found that the generation of Ts preceeded that of CTL and that the turnover was faster; furthermore, Ts were generated in the thymus and spleen, while CTL were distributed in regional lymph nodes and spleen. Surface marker analysis revealed that only Lyt-1^–.2.3⁺ T-cells participated in suppressor responses in contrast to both Lyt-1^–.2.3⁺ and Lyt-1⁺.2.3⁺ T-cells participating in cytotoxic responses.The effects of adult thymectomy (ATx) on the changes of the immunized Tcell subsets were also investigated. In mice thymectomized 3 weeks previously, the Ts activity was abrogated, whereas the CTL activity increased markedly and Lyt-1⁺ .2.3⁺ T-cells were not detected. The results suggest that CTL or their precursors bearing Lyt-1⁺ .2.3⁺ phenotype and Ts bearing Lyt-1^– .2.3⁺ phenotype are short-lived lymphocytes.Accordingly, it is suggested that in tumor-bearing mice short-lived Ts are generated earliest with target specificity and, due to the reciprocal relationships between Ts and CTL activities, may have a modulating influence on CTL; furthermore, AR may alter the patterns of generation of the precursor T-cells and Ts.     

Regulatory T cells control macrophage accumulation and activation in lymphoma

Strategies of manipulating immunosuppressive regulatory T cells (Treg) in cancer patients are currently evaluated in clinical trials. Treg suppress immune responses of tumor‐specific T cells; yet, relatively little is known about the impact of Treg on innate immune cells in tumor models in vivo. Many tumors lose expression of MHC class I. Therefore, our study aimed at defining strategies to strengthen immune responses against a high tumor burden of the MHC class I‐deficient mouse lymphoma RMA‐S. We demonstrate that Treg depletion in mice led to tumor rejection that was dependent on T cells, NK cells and IFN‐γ. In the absence of Treg elevated levels of IFN‐γ were produced by tumor‐infiltrating T cells and NK cells. Tumor rejection observed in the absence of Treg correlated with a substantial IFN‐γ‐dependent increase in the numbers of tumor‐infiltrating leukocytes. The most abundant cell population in the tumors was macrophages. Tumor‐infiltrating macrophages from Treg‐depleted mice expressed increased amounts of MHC class II, produced highly enhanced levels of pro‐inflammatory cytokines and inhibited tumor cell proliferation. It was reported that tumor‐infiltrating macrophages have multi‐faceted functions promoting or counteracting tumor growth. In our study, high numbers of macrophages infiltrating RMA‐S tumors in the absence of Treg correlated with tumor rejection suggesting that macrophages are additional targets for Treg‐mediated immune suppression in cancer.     

Trafficking of tumor peptide-specific cytotoxic T lymphocytes into the tumor microcirculation

The major histocompatibility complex class I-restricted CD8(+) cytotoxic T-lymphocyte (CTL) effector arm of the adaptive immune response can specifically recognize and destroy tumor cells expressing peptide antigens. Although adoptive T-cell therapy has been successfully used for the treatment of viral and malignant diseases, little is known of the trafficking and fate of adoptively transferred antigen-specific T cells. In the present study, splenocytes derived from mice that rejected their tumors (CT26 or CT26-clone 25 tumors) in response to direct intratumor injection of disabled infectious single-cycle herpes simplex virus (DISC-HSV) encoding murine GM-CSF were restimulated with peptide in vitro. CTLs specific for the AH-1 and beta-gal peptides expressed by CT26 and CT26-clone 25 tumor cells, respectively, were generated and used for adoptive cellular therapy and trafficking studies. Intravenous administration of AH-1-specific CTLs 3 days following i.v. injection of CT26 cells resulted in significant tumor growth inhibition, whereas administration of control CTLs generated against a bacterial beta-gal peptide did not inhibit the growth of tumors. Trafficking of AH-1-specific lymphocytes and their interaction with the CT26 tumor microcirculation was analyzed using real-time in vivo microscopy (IVM). AH-1-specific but not beta-gal-specific CTLs adhered and localized in the CT26 tumor microvasculature, but neither population adhered to the endothelium of the normal microcirculation. This study provides direct visual evidence suggesting that AH-1-specific CTLs that mediate a therapeutic response traffic to and localize within the tumor microenvironment.     

The detection of circulating human papillomavirus‐specific T cells is associated with improved survival of patients with deeply infiltrating tumors

A detailed analyses of HPV‐specific immunity was performed in a large group of patients with HPV‐induced cervical cancer (CxCa) in relation to HLA‐types and prognostic factors. Patients were HLA‐typed and HPV16/18‐specific T‐cell immunity was assessed by proliferation assay and cytometric bead array using freshly isolated PBMC and by phenotypic analysis of HPV‐specific T cells. The results were analyzed in relation to known disease‐related HLA‐types (DR7, DR13, DR15/DQ06), invasion‐depth and size of tumor, lymph node (LN) status and disease free survival. In total 119 HLA‐typed patients with CxCa were analyzed. Patients expressing the HLA‐DR13 haplotype were underrepresented as compared to the Dutch population (p = 0.014), whereas HLA‐DR7 was overrepresented in patients with HPV16+ CxCa (p = 0.006). In 29 of 94 patients (31%) from whom blood could be tested, a proliferative response to HPV16/18 was detected, which was associated with increased numbers of HPV‐specific CD4+CD25+ (activated) T cells (p = 0.03) and HPV‐specific CD4+CD25+FoxP3‐positive T cells (p = 0.04). The presence of both FoxP3‐positive and negative HPV‐specific CD4+CD25+ T cells was significantly correlated (p = 0.01). Interestingly, the detection of HPV‐specific proliferation was associated with invasion depth (p = 0.020) but not with HLA type, tumor size nor LN status. Moreover, the detection of HPV‐specific immunity was associated with an improved disease free survival (p = 0.04) in patients with deeply infiltrating tumors. In conclusion, HPV‐specific proliferative T‐cell response, comprising higher percentages of HPV‐specific CD25+ and CD25+FoxP3‐positive CD4+T cells, are more frequently detected in patients with deep infiltrating CxCa tumors and associated with an improved survival.     

Allorestricted T lymphocytes with a high avidity T‐cell receptor towards NY‐ESO‐1 have potent anti‐tumor activity

The cancer‐testis antigen NY‐ESO‐1 has been targeted as a tumor‐associated antigen by immunotherapeutical strategies, such as cancer vaccines. The prerequisite for a T‐cell‐based therapy is the induction of T cells capable of recognizing the NY‐ESO‐1‐expressing tumor cells. In this study, we generated human T lymphocytes directed against the immunodominant NY‐ESO‐1_157–165 epitope known to be naturally presented with HLA‐A*0201. We succeeded to isolate autorestricted and allorestricted T lymphocytes with low, intermediate or high avidity TCRs against the NY‐ESO‐1 peptide. The avidity of the established CTL populations correlated with their capacity of lysing HLA‐A2‐positive, NY‐ESO‐1‐expressing tumor cell lines derived from different origins, e.g. melanoma and myeloma. The allorestricted NY‐ESO‐1‐specific T lymphocytes displayed TCRs with the highest avidity and best anti‐tumor recognition activity. TCRs derived from allorestricted, NY‐ESO‐1‐specific T cells may be useful reagents for redirecting primary T cells by TCR gene transfer and, therefore, may facilitate the development of adoptive transfer regimens based on TCR‐transduced T cells for the treatment of NY‐ESO‐1‐expressing hematological malignancies and solid tumors. © 2009 UICC     

Administration of 6-gingerol greatly enhances the number of tumor-infiltrating lymphocytes in murine tumors

Tumor-infiltrating lymphocytes (TILs) play critical roles in host antitumor immune responses. It is known that cancer patients with tumor-reactive lymphocyte infiltration in their tumors have better prognoses, while patients with tumors infiltrated by immunosuppressive cells have worse prognoses. We found that administration of 6-gingerol, which is a component of ginger, inhibited tumor growth in several types of murine tumors, such as B16F1 melanomas, Renca renal cell carcinomas and CT26 colon carcinomas, which were established by inoculating tumor cells on the flanks of mice. However, administration of 6-gingerol did not lead to complete eradication of the tumors. 6-Gingerol treatment of tumor-bearing mice caused massive infiltration of CD4 and CD8 T-cells and B220(+) B-cells, but reduced the number of CD4(+) Foxp3(+) regulatory T-cells. The CD8 tumor-infiltrating T lymphocytes in 6-gingerol-treated mice strongly expressed IFN-γ, a marker of activation of cytotoxic T lymphocytes (CTL) CD107a and chemokine receptors that are expressed on T(H) 1 cells, such as CXCR3 and CCR5. To test whether 6-gingerol could promote infiltration of tumor antigen-specific CD8 T-cells into tumors, we adoptively transferred CFSE-labeled OT-1 CD8 T-cells into EG7 tumor-bearing mice. We found that CD8 T cells isolated from 6-gingerol pretreated OT-1 mice, but not from control OT-1 mice, massively infiltrated tumors and tumor draining lymph nodes and divided several times. Our results strongly suggest that 6-gingerol can be used in tumor immunotherapy to increase the number of TILs.     

NY‐ESO‐1 antigen‐reactive T cell receptors exhibit diverse therapeutic capability

The cancer‐testis antigen NY‐ESO‐1 has been used as a target for different immunotherapies like vaccinations and adoptive transfer of antigen‐specific cytotoxic T cells, as it is expressed in various tumor types and has limited expression in normal cells. The in vitro generation of T cells with defined antigen specificity by T cell receptor (TCR) gene transfer is an established method to create cells for immunotherapy. However, an extensive characterization of TCR which are candidates for treatment of patients is crucial for successful therapies. The TCR has to be efficiently expressed, their affinity to the desired antigen should be high enough to recognize low amounts of endogenously processed peptides on tumor cells, and the TCR should not be cross‐reactive to other antigens. We characterized three NY‐ESO‐1 antigen‐reactive cytotoxic T lymphocyte clones which were generated by different approaches of T cell priming (autologous, allogeneic), and transferred their TCR into donor T cells for more extensive evaluations. Although one TCR most efficiently bound MHC‐multimers loaded with NY‐ESO‐1 peptide, T cells expressing this transgenic TCR were not able to recognize endogenously processed antigen. A second TCR recognized HLA‐A2 independent of the bound peptide beside its much stronger recognition of NY‐ESO‐1 bound to HLA‐A2. A third TCR displayed an intermediate but peptide‐specific performance in all functional assays and, therefore, is the most promising candidate TCR for further clinical development. Our data indicate that multiple parameters of TCR gene‐modified T cells have to be evaluated to identify an optimal TCR candidate for adoptive therapy.     

Regulatory T cells in tumor immunity

Recent studies have revealed that Foxp3⁺CD25⁺CD4⁺ regulatory T cells (Tregs), which are physiologically engaged in the maintenance of immunological self‐tolerance, play critical roles for the control of antitumor immune responses. For example, a large number of Foxp3⁺Tregs infiltrate into tumors, and systemic removal of Foxp3⁺Tregs enhances natural as well as vaccine‐induced antitumor T‐cell responses. Tregs are recruited to tumor tissues via chemokines, such as CCL22 binding to CCR4 expressed by Tregs. They appear to expand and become activated in tumor tissues and in the draining lymph nodes by recognizing tumor‐associated antigens as well as normal self‐antigen expressed by tumor cells. These results indicate that cancer vaccines targeting tumor‐associated self‐antigens may potentially expand/activate Tregs and hamper effective antitumor immune responses, and that tumor immunity can therefore be enhanced by depleting Tregs, attenuating Treg suppressive function, or rendering effector T cells refractory to Treg‐mediated suppression. Recent attempts have indeed demonstrated that combinations of monoclonal antibodies capable of modulating Treg functions synergistically enhance antitumor activity and are more effective than a single monoclonal antibody therapy. Combination therapy targeting a variety of molecules expressed in antigen‐presenting cells, effector T cells and Tregs is envisaged to be a promising anticancer immunotherapy.     

T lymphocytes in hepatocellular carcinoma immune microenvironment: insights into human immunology and immunotherapy

Hepatocellular carcinoma (HCC) is characterized by poor outcome and shows limited drug-response in clinical trials. Tumor immune microenvironment (TIME) exerts a strong selection pressure on HCC, leading to HCC evolvement and recurrence after multiple therapies. T cell-mediated immunoreaction during cancer surveillance and clearance is central in cancer immunity. Heterogenous T cell subsets play multiple roles in HCC development and progression. The re-educated T cells in TIME usually lead to deteriorated T cell response and tumor progression. Investigation into immune system dysregulation during HCC development will shed light on how to turn immune suppressive state to immune activation and induce more efficient immune response. Emerging T cell-based treatment such as cancer vaccines, CAR-T cell therapy, adoptive cell therapy, and immune checkpoint inhibitors (ICIs), have been proved to cause tumor regression in some clinical and preclinical trials. In this review, we focused on recent studies that explored T cells involved in HCC and how they affect the course of disease. We also briefly outlined current T cell-based immunotherapies in HCC.     

不同细胞因子协同活化的T细胞抗肿瘤活性的研究

目的：探讨不同细胞因子协同活化的T细胞抗肿瘤的活性。方法：采用CD3AK法，CD3／CD28共刺激法，CIK法，并作细胞扩增能力检测。测定刺激细胞对肿瘤的杀伤活性，及用流式细胞仪检测共刺激后的T细胞表型变化。结果：抗CD3单克隆抗体可通过CD3成分启动T细胞，使淋巴细胞表型发生变化，联系细胞因子诱导的T淋巴细胞能有效提高T细胞对鼻咽癌细胞的杀伤活性，CIK方法诱导的T淋巴细胞在体外能保持良好的扩增活性。结论：不同细胞因子协同活化的T细胞抗肿瘤活性不同，CD28联合CD3诱导的T细胞抗肿瘤活性较高，CIK法效果最好。     

Regulatory T cells and the PD-L1/PD-1 pathway mediate immune suppression in malignant human brain tumors

The brain is a specialized immune site representing a unique tumor microenvironment. The availability of fresh brain tumor material for ex vivo analysis is often limited because large parts of many brain tumors are resected using ultrasonic aspiration. We analyzed ultrasonic tumor aspirates as a biosource to study immune suppressive mechanisms in 83 human brain tumors. Lymphocyte infiltrates in brain tumor tissues and ultrasonic aspirates were comparable with respect to lymphocyte content and viability. Applying ultrasonic aspirates, we detected massive infiltration of CD4⁺FoxP3⁺CD25^high CD127^low regulatory T cells (Tregs) in glioblastomas (n = 29) and metastatic brain tumors (n = 20). No Treg accumulation was observed in benign tumors such as meningiomas (n = 10) and pituitary adenomas (n = 5). A significant Treg increase in blood was seen only in patients with metastatic brain tumors. Tregs in high-grade tumors exhibited an activated phenotype as indicated by decreased proliferation and elevated CTLA-4 and FoxP3 expression relative to blood Tregs. Functional analysis showed that the tumor-derived Tregs efficiently suppressed cytokine secretion and proliferation of autologous intratumoral lymphocytes. Most tumor-infiltrating Tregs were localized in close proximity to effector T cells, as visualized by immunohistochemistry. Furthermore, 61% of the malignant brain tumors expressed programmed death ligand-1 (PD-L1), while the inhibitory PD-1 receptor was expressed on CD4⁺ effector cells present in 26% of tumors. In conclusion, using ultrasonic tumor aspirates as a biosource we identified Tregs and the PD-L1/PD-1 pathway as immune suppressive mechanisms in malignant but not benign human brain tumors.     

基于记忆性干性T细胞和中心记忆性T细胞的细胞免疫治疗在血液肿瘤治疗中的价值

近年来,以抗原嵌合受体T细胞(CAR-T)为代表的细胞免疫治疗在血液肿瘤治疗中取得重大突破.记忆性干性T细胞(Tb)和中心记忆性T细胞(TCM)是T细胞免疫治疗强有力的载体,如何在体内及体外调控该类细胞的分化、如何利用其构建强效的治疗系统而避免潜在的毒副作用受到研究者们的关注.文章主要总结了第58届美国血液学会(ASH)年会关于TSCM和TCM在血液肿瘤治疗中的价值和相关研究进展.     

Clinical significance of regulatory T cells and CD8+ effector populations in patients with human endometrial carcinoma

BACKGROUND:

A study was carried out to determine the functional attributes of CD4⁺ CD25⁺ regulatory T cells in cancer progression by suppressing antitumor immunity.

METHODS:

Triple‐color flow cytometry was used to study the phenotype expression of CD4⁺ CD25⁺ regulatory T cells and CD8⁺ T cells in the peripheral blood lymphocytes (PBLs) and tumor‐infiltrating lymphocytes (TILs) of 57 cases of stage I to IV endometrial carcinoma. The expression of T cell subsets was correlated with clinical prognostic parameters.

RESULTS:

The prevalence of CD4⁺ CD25⁺ T cells was significantly higher in the TILs than PBLs. The expression of CD4⁺ CD25⁺ regulatory T cells in cancer milieu correlated with the tumor grade, stage, and myometrium invasion. The expression of FOXP3 and GITR in CD4⁺ CD25⁺ regulatory T cells was lower in PBLs than TILs. Most tumor‐infiltrating CD8⁺ T cells were CD28^? CD45RA^? CD45RO⁺ CCR7^?, suggesting good terminal differentiation. Most of them had an activated role with CD69⁺ CD103⁺ CD152⁺. Functionally, both granzyme B and perforin were scarcely expressed in peripheral regulatory T cells but were highly expressed in peripheral regulatory T cells in the tumor microenvironment. In contrast, CD8⁺ cytotoxic T cells derived from PBLs expressed both granzyme B and perforin, and at significantly higher levels than in TILs. Further functional assays demonstrated that Th1 cytokines and cytotoxic molecules can be synchronously up‐regulated in CD8⁺ cytotoxic T cells.

CONCLUSIONS:

Regulatory T cells in the tumor microenvironment may abrogate CD8⁺ T cell cytotoxicity in a granzyme B‐ and perforin‐dependent conduit. Decreases in both Th1 cytokines and cytotoxic enzymes are relevant for regulatory T cell‐mediated restraint of tumor clearance in vivo. Of clinical significance, the expression of regulatory T cells in TILs may mediate T cell immune repression within cancer milieu and thus greatly correlate with cancer progression. Cancer 2010. © 2010 American Cancer Society.     

Identification of promiscuous HPV16‐derived T helper cell epitopes for therapeutic HPV vaccine design

Cervical carcinoma and several other human papillomavirus (HPV)‐induced malignancies are a global public health problem, thus novel treatment modalities are urgently needed. Immunotherapy is an attractive option for treatment of HPV infection and HPV‐mediated premalignant and malignant lesions. However, previous approaches—focusing on the induction of cytotoxic CD8+ T cells (CTLs)—have as yet not yielded clinical successes. Since CD4+ T cells have been shown to be crucial for the induction and maintenance of CTL responses, and more recently to be also important for direct anti‐tumor immunity, human leukocyte antigen (HLA) class II‐restricted epitopes are intensively investigated to improve the efficacy of peptide‐based HPV immunotherapy. We here present an approach to identify promiscuous HPV16‐derived CD4+ T helper epitopes, which are capable of inducing T cell immunity in a large proportion of the population. To this end, we combined HLA class II epitope prediction servers with in vitro immunological evaluation to identify HPV16 E2‐, E5‐, E6‐, and E7‐derived CD4+ T cell epitopes. Candidate selected HPV16‐derived epitopes were found to be restricted by up to nine HLA‐DR molecules. Furthermore, they were found to induce frequent and robust HPV16 peptide‐specific Th1 responses in healthy donors, as monitored by interferon (IFN)‐γ ELISPOT and cytokine secretion assays. Moreover, these selected peptides also induced specific IFN‐γ T cell responses in blood from HPV16+ CIN2/3 and cervical carcinoma patients. We thus conclude that the identified T helper epitopes are valuable candidates for the development of a comprehensive therapeutic HPV vaccine.     

记忆性干细胞样T细胞在肿瘤治疗应用中的研究进展

记忆性干细胞样T细胞(stem memory T cells,TSCM)是最近几年发现的一个细胞亚群,约占外周血中总的CD4+T细胞和CD8+T细胞的2％～4％,是记忆性T细胞的早期分化阶段,具有记忆性细胞和干细胞的特点,基因谱介于初始T细胞和中央记忆T细胞之间.记忆性干细胞样T细胞具有自我更新和长期存活能力,这些特点使它们成为肿瘤领域关注的热点.本文将简述记忆性干细胞样T细胞的一些特点,以及它们在肿瘤治疗中的应用.     

Induction of anti-leukemic cytotoxic T lymphocytes by fusion of patient-derived dendritic cells with autologous myeloblasts

Presentation of AML antigens by dendritic cells (DC) could potentially induce a T cell-mediated anti-leukemic immune response. In the present study, we generated DC from adherent (AD-DC) and non-adherent (NAD-DC) myeloblasts obtained from bone marrows of AML patients. Both cell populations displayed morphological, phenotypic and functional properties of DC. The functions of NAD-DC were compared to AD-DC that had been fused with autologous AML blasts (FC/AML). The FC/AML induced greater T cell proliferation and CTL activity against autologous AML blasts (9/10 cases) as compared to NAD-DC. FC/AML may thus represent a promising strategy for DC-based immunotherapy of patients with AML.     

幼稚和记忆T 细胞在NSCLC 放疗患者中的表达及其预后价值

  目的  探讨幼稚和记忆T细胞在非小细胞肺癌（non-small cell lung cancer,NSCLC）放疗患者外周血中的表达及其与预后的关系。  方法  采用流式细胞仪检测2014年9月至2016年5月解放军307医院40例接受放疗的NSCLC首诊患者,治疗前外周血中幼稚CD4+T细胞、记忆CD4+T细胞、幼稚CD8+T细胞、记忆CD8+T细胞,与14例健康人进行对比,并分析其与临床病理特征及预后的关系。  结果  与健康组相比,NSCLC患者幼稚CD4+T细胞下降（P=0.031）,记忆CD4+T细胞和记忆CD8+T细胞升高（P=0.014、0.005）。与不吸烟患者相比,吸烟患者幼稚CD4+T细胞较低（P=0.043）,记忆CD4+T细胞较高（P=0.024）。与ECOG评分1～2分者相比,0分者幼稚CD8+T细胞较低（P=0.017）,记忆CD8+T细胞较高（P=0.020）。单因素分析显示幼稚CD4+T细胞较高组放疗后中位无进展生存期（median progression free survival,mPFS）更长（17个月vs. 9个月,P=0.044）、记忆CD4+T细胞较高组放疗后mPFS可能更短（9个月 vs. 15个月,P=0.069）。Cox多因素分析显示幼稚CD4+T细胞与患者放疗后无进展生存期（progression free survival,PFS）独立相关（P=0.009）。  结论  NSCLC患者外周血中幼稚T细胞储备下降,记忆T细胞上升。幼稚CD4+T细胞较高可能预示放疗后PFS更长。      

Regulation of follicular T helper cell differentiation in antitumor immunity

Follicular T helper (Tfh) cells are a subset of CD4⁺ T cells that play an important role in the formation of germinal centers and the maturation and differentiation of affinity-matured B cells. Recent studies have demonstrated important functions of Tfh cells in tertiary lymphoid structures of tumors, revealing great potential of Tfh cells in tumor immunity. However, Tfh development is incompletely understood. The differentiation of Tfh cells is a complex, multistage process regulated at the DNA, RNA and protein levels. This review just summarizes current research on the molecular mechanisms of Tfh cell differentiation to better understand the role of Tfh cells in antitumor immunity.