PD‐1+ TIM‐3+ T cells in malignant ascites predict prognosis of gastrointestinal cancer

The liquid biopsy of ascites fluid could be an excellent source of tumor and microenvironment for the study of prognostic biomarkers because of its accessibility. Tumor‐infiltrating lymphocytes (TILs) can predict prognosis in multiple malignancies, including the response to immune checkpoint inhibitors, a breakthrough cancer therapy. However, TILs’ profiles from malignant ascites have not been extensively studied. Using flow cytometric analysis, we quantified the proportion of exhausted T cells and memory/naive/effector T‐cell subsets, among the CD4+ and CD8+ T‐cell populations of paired TILs and peripheral blood T cell samples (n = 22). The correlation between CD4+ and CD8+ subset profiles suggested that the combined analysis of CD4+ and CD8+ cells in malignant ascites was clinically significant. We found that cells positive for the exhaustion markers programmed cell death‐1 (PD‐1), and T‐cell immunoglobulin and mucin domain 3 (TIM‐3), and cells coexpressing PD‐1 and TIM‐3 abundantly exist among malignant ascites TILs. Furthermore, patients with high frequency of PD‐1+ TIM‐3+ cells among the CD4+ and CD8+ T‐cell population showed worse clinical outcome in multivariate analysis (n = 27). We propose that exhausted ascites TILs represent a clinically significant prognostic biomarker in advanced gastrointestinal cancer and represent an important target for immune checkpoint inhibitors.     

Peripheral blood and tumor infiltrating lymphocytes in non-small cell lung cancer: analysis at the population and clonal level

Tumor infiltrating (TIL) and peripheral blood lymphocytes (PBL) were isolated from 18 patients with non-small cell lung cancer undergoing radical surgery. Surface marker analysis revealed that TILs and PBLs mainly consisted of CD3+ T cells and that TILs generally displayed a lower CD4/CD8 ratio. Differences were found in the expression of CD25 (IL-2 receptor) and DR (MHC class II) antigens, which were increased in TILs, and in the percentage of CD16+ natural killer (NK) cells, which was reduced in TILs as compared to PBLs. Accordingly, the NK activity of TILs was lower than that of PBLs, whereas neither TILs nor PBLs expressed spontaneous cytolytic activity against fresh autologous tumor cells, melanoma cells and the "NK-resistant" A549 lung carcinoma cell line. After 4 days of culture in medium with recombinant-interleukin-2 (rIL-2), TILs and PBLs acquired cytolytic activity against all cell targets, but TILs expressed higher levels of cytotoxicity than autologous PBLs only in 3 patients out of 16 tested. More importantly, both TILs and PBLs displayed similar levels of cytotoxic activity against autologous tumor cells. TILs and PBLs from 8 patients were also analyzed by a limiting dilution microculture system. Cloning efficiency was remarkably lower in TILs, and surface marker analysis of T cell clones confirmed that an accumulation of CD8+ lymphocytes, which displayed cytolytic activity in a lectin-dependent assay, occurred at the tumor site. The non-MHC-restricted cytolytic activity of TIL- and PBL-derived T cell clones against K562, A549, and allogeneic melanoma cells and the cytolytic activity against autologous tumor cells showed no significant differences. Only 53% of TIL clones released IL-2 in response to PHA + TPA stimulation, whereas 68% of PBL-derived clones were IL-2 producers. Moreover, most PBL- and TIL-derived clones released tumor necrosis factor alpha in response to mitogen stimulation.     

Properties of recombinant interleukin 2-cultured tumor-infiltrating lymphocytes in human lung cancer

It is thought that TIL can be activated in vitro by rIL-2 and acquire specific anti-tumor activity. In this study, we investigated this possibility, using lymphocytes isolated from primary lung cancer tissues. In a first series of experiments, TILs and autologous PBLs from 16 patients were cultured in rIL-2 from 7 to 14 days under identical conditions, and were compared for proliferation (16 cases), cytolytic activity (11 cases), gamma interferon (IFN-gamma) production (8 cases), and phenotypes (10 cases). TILs grew in response to rIL-2 as well as PBLs. However, the induced cytolytic activity of TIL was significantly lower than that of PBL against autologous tumor cells and 2 human tumor cell lines. IL-2-mediated IFN-gamma production by TILs was also significantly lower than that of PBLs. TILs were phenotypically characterized by their high CD4/CD8 ratio and lack of Leu11-positive cells. Further investigations with 7 other cases showed that exogenous addition of IFN-gamma to rIL-2 cultures of TILs enhanced cytolytic activity in 4 cases. Our results indicate that IL-2 alone is sufficient for TILs to proliferate but not to acquire new functions (cytotoxicity and production of IFN-gamma).     

Flow cytometric measurement of intracellular cytokines detects immune responses in MUC1 immunotherapy.

The detection of tumor-specific T cells in immunized cancer patients usually relies on lengthy and difficult CTL assays; we now report on flow cytometry to detect the intracellular cytokines interleukin 2 (IL-2), IL-4, IFN-gamma, and tumor necrosis factor alpha (TNF-alpha) produced by CD4+CD69+ and CD8+CD69+ activated T cells after MUC1 antigen stimulation. Peripheral blood mononuclear cells were obtained from 12 patients with adenocarcinoma injected with mannan-MUC1; cells were exposed in vitro for 18 h to MUCI peptide in the presence of CD28 monoclonal antibody and Brefeldin; permeabilized cells were used for the expression of cytokines. After stimulation in vitro with MUC1-variable number of tandem repeats peptides, CD8+CD69+ T cells from all immunized patients generated 3-9 times higher levels of TNF-alpha(P < 0.038) and IFN-gamma (P <0.010) than did cells from 12 normal subjects; minor increases in IL-4 occurred. By contrast, CD4+CD69+ cells showed no overall alteration in TNF-alpha and IFN-gamma cytokine production, although in some patients, their measurement was informative; the measurement of IL-2 was not useful in either CD4+CD69+ or CD8+CD69+ cells. We conclude that in MUC1-immunized patients, the measurement of TNF-alpha and IFN-gamma in activated CD69+CD8+ T cells may be indicative of their immune status.     

非小细胞肺癌原发灶PET/CT SUVmax 与免疫细胞亚群浸润的相关性及其临床意义

  目的  通过分析18F-脱氧葡萄糖（fluorodeoxyglucose,FDG）在非小细胞肺癌（non-small cell lung cancer,NSCLC）原发灶最大标准摄取值（maximum standardized uptake value,SUVmax）与免疫细胞亚群浸润的相关性,初步探讨其在肺癌治疗及判断预后中的指导价值。  方法  回顾性分析2008年4月至2014年8月在天津医科大学肿瘤医院收治且接受正电子发射断层扫描/计算机体层摄影（positron emission tomography/computed tomography,PET/CT）检查及肺癌根治术的NSCLC患者119例,其中鳞癌32例、腺癌87例,应用免疫组织化学方法检测肿瘤原发灶CD3+肿瘤浸润淋巴细胞（tumor infiltrating lymphocytes,TILs）、CD8+TILs、CD68+肿瘤相关树巨噬细胞（tumor-associated macrophages,TAMs）、CD163+TAMs及CD11c+树突状细胞（dendritic cells,DCs）的浸润水平,采用Pearson法分析各免疫细胞亚群与SUVmax的相关性,同时采用Kaplan-Meier法及多因素Cox回归模型分析患者的生存状况。  结果  肿瘤原发灶SUVmax与CD8+TILs（r=0.332,P＜0.001）和CD163+TAMs（r=0.223,P=0.015）的浸润水平呈正相关,而与CD3+TILs、CD68+TAMs和CD11c+DCs的浸润水平无明显相关性。单因素分析提示TNM分期、SUVmax、肿瘤大小及免疫浸润细胞CD163+TAMs和CD11c+DCs与患者的预后有关,多因素分析仅TNM分期和肿瘤大小为患者的独立预后因素。  结论  肿瘤原发灶PET/CT SUVmax与部分免疫细胞亚群浸润水平显著相关,提示18F-FDG PET/CT可间接反应肿瘤微环境的免疫状态,这对NSCLC患者预后判断以及采取及时有效的干预治疗措施具有重要的指导价值。      

乳腺癌微环境中T细胞与临床病理因素的相关性

目的:探讨乳腺癌中间质肿瘤浸润淋巴细胞（TILs）包括CD8+TILs、CD4+TILs、三维淋巴结构（TLS）与临床病理因素和分子亚型的相关性。方法:收集我院2015年至2017年乳腺癌标本200例,经常规石蜡包埋制片,判读HE染色切片间质TLS个数,采用免疫组织化学SP法检测200例病例中CD8+TILs和CD4+TILs浸润密度。结果:乳腺癌间质CD8+TILs、CD4+TILs、TLS与SBR分级、病理TNM分期、Ki67、p53呈正相关;CD8+TILs和CD4+TILs与淋巴结转移呈正相关;与患者年龄呈负相关（均P＜0.05）。CD8+TILs和CD4+TILs呈正相关（P＜0.05）。TLS以CD4+TILs为主,分别与CD8+TILs和CD4+TILs呈正相关（均P＜0.05）。在乳腺癌不同分子亚型中CD8+TILs、CD4+TILs和TLS存在显著差异。结论:乳腺癌中浸润的CD8+TILs、CD4+TILs、TLS反映局部肿瘤微环境的免疫状态,三者密切相关。乳腺癌组织中增多的CD4+TILs诱导CD8+TILs产生CD8+Treg细胞参与乳腺癌的免疫抑制,导致乳腺癌局部免疫功能下降,促进乳腺癌进展及转移。结合乳腺癌分子亚型和局部免疫状态的分析将更有利于预测乳腺癌进展的生物学潜能和对分子靶向性疗效和预后的评估。     

Presentation of renal tumor antigens by human dendritic cells activates tumor-infiltrating lymphocytes against autologous tumor: implications for live kidney cancer vaccines.

The clinical impact of dendritic cells (DCs) in the treatment of human cancer depends on their unique role as the most potent antigen-presenting cells that are capable of priming an antitumor T-cell response. Here, we demonstrate that functional DCs can be generated from peripheral blood of patients with metastatic renal cell carcinoma (RCC) by culture of monocytes/macrophages (CD14+) in autologous serum containing medium (RPMI) in the presence of granulocyte macrophage colony-stimulating factor and interleukin (IL) 4. For testing the capability of RCC-antigen uptake and processing, we loaded these DCs with autologous tumor lysate (TuLy) using liposomes, after which cytometric analysis of the DCs revealed a markedly increased expression of HLA class I antigen and a persistent high expression of class II. The immunogenicity of DC-TuLy was further tested in cultures of renal tumor infiltrating lymphocytes (TILs) cultured in low-dose IL-2 (20 Biologic Response Modifier Program units/ml). A synergistic effect of DC-TuLy and IL-2 in stimulating a T cell-dependent immune response was demonstrated by: (a) the increase of growth expansion of TILs (9.4-14.3-fold; day 21); (b) the up-regulation of the CD3+ CD56- TcR+ (both CD4+ and CD8+) cell population; (c) the augmentation of T cell-restricted autologous tumor lysis; and (d) the enhancement of IFN-gamma, tumor necrosis factor-alpha, granulocyte macrophage colony-stimulating factor, and IL-6 mRNA expression by TILs. Taken together, these data implicate that DC-TuLy can activate immunosuppressed TIL via an induction of enhanced antitumor CTL responses associated with production of Thl cells. This indicates a potential role of DC-TuLy vaccines for induction of active immunity in patients with advanced RCC.     

CD8+ lymphocyte infiltration and apoptosis in hepatocellular carcinoma.

AIMS: Tumour-infiltrating lymphocytes (TILs) play a role in local anti-tumour immunity. Tumour cells may escape from immune surveillance by expressing RCAS1, a receptor-binding cancer antigen expressed on SiSo cells, which inhibits T cell growth. In this study, the correlation between the density of CD8+ TILs, tumour cell apoptosis, and tumour RCAS1 expression was investigated in hepatocellular carcinoma (HCC). METHODS: We obtained tissues from 60 patients with surgically resected HCCs. CD8+ TILS, apoptotic cancer cells, and RCAS1 expressing cancer cells were identified by immunohistochemistry. RESULTS: The density of CD8+ T cells in tumours (mean: 9.5/HPF, HPF: high power field) was significantly less than in non-cancerous hepatic lobules (17.8/HPF, p<0.001) and in relation to the progression of tumour stage. The density of CD8+ T cells in tumours positively correlated with the occurrence of tumour cell apoptosis, but did not correlate with RCAS1 protein expression. CONCLUSIONS: CD8+ TILs may play a role in the occurrence of tumour cell apoptosis in HCC, but CD8+ TILs may not be controlled by RCAS1 in HCC.     

Oligoclonal T lymphocytes infiltrating human lung cancer tissues.

To clarify the nature of tumor-infiltrating lymphocytes (TILs), we investigated the possible clonality of the T cells in TILs freshly isolated from human primary lung cancer tissues by assessing the rearrangement pattern of the T-cell receptor (TCR) gene beta locus using Southern blotting. First, in phenotypic analysis, TILs represented different populations among corresponding peripheral blood lymphocytes (PBLs) with an increased proportion of CD20+ (B) cells as well as a decreased proportion of CD16+ (natural killer) cells, and a variable CD4/CD8 ratio. Considering the central role of T cells in immune responses, we analyzed TCR beta gene rearrangement patterns in TILs and corresponding PBLs from 12 patients. In 10 of the 12 cases, TILs showed one or more TCR gene rearrangement bands with a predominance of the C beta 2 gene, in which 2 types of common rearranged band were observed among the cases with different clinical profiles in terms of histological types and disease stage, with bands at about 9.5 kb in 7 and at 11.5 kb in 8 patients. On the other hand, predominant rearranged bands were hardly detected in corresponding PBLs except in 2 cases. From these results, we conclude that TILs in lung cancer tissues frequently contain oligoclonal T-cell populations, which were probably sensitized by relatively common antigens at the tumor sites.     

CD8+ TILS Expression in Invasive Breast Carcinoma (No Special Type)

Invasive breast carcinoma (IBC) is the most cancer in women (24%) and the cause of cancer death in women worldwide. Based on data from globocan 2018 shows the incidence of breast cancer is around 2.08 million (11.6%) which is the second rank of all cancers after lung cancer with a mortality rate of 626.6 thousand (6.6%) which is also the most common cause of death in women. The basic role of the immune system in maintaining homeostasis by immunosurveillance and initiation of the inflammatory reactions that include coordination of innate and adaptive immune cells activation against tumor cells is one of the most important in the mechanism of tumor cell elimination. Prognosis of IBC were influenced by several factors, including tumor histology grade and Tumor Infiltrating Lymphocytes (TILs). Infiltration of lymphocytes in the tumor microenvironment/TILs through CD8+ T lymphocytes is known to be an important component of adaptive immunity that eliminates tumor cells. CD8+ T cells present tumor antigens on the surface of the major histocompatibility complex class I (MHC class I). Based on its importance in clinical application and it’s role in biological concepts, this study was conducted to determine the expression of CD8+ in Invasive Breast Carcinoma of No Special Type (IBC-NST) grades 1,2 and 3. Analytical study with a cross-sectional design on IBC-NST samples from 2017 until 2020 at the Laboratory of Anatomic Pathology, Faculty of Medicine, Hasanuddin University Makassar from May to August 2021. Immunoexpression data were analyzed to determine its relationship with grade. Eighty samples met the inclusion and exclusion criteria, there were 17 samples (21.3%) with grade 1, 32 samples (40%) with grade 2, and 31 samples (38.8%) with grade 3. In the high CD8+ TILs group, from a total of 27 samples, 10 samples with grade 1, 6 samples with grade 2, and as many as 11 samples with grade 3. In the low CD8+ TILs group, from a total of 53 samples, there were 7 samples with grade 1, 26 samples with grade 2, and 20 samples with grade 3. Based on the Chi-square test, p value = 0.017 (p <0.05). There is a significant difference in CD8+ TILS in Invasive Breast Carcinoma of No Special Type grade 1, grade 2 and grade 3.     

Regulatory CD4(+)CD25(+) T cells in tumors from patients with early-stage non-small cell lung cancer and late-stage ovarian cancer.

Immunosuppression may contribute to the progression of cancer. In this study we assessed the structural and functional status of T cells from tumor specimens obtained from patients with early stage non-small cell lung cancer and late-stage ovarian cancer. Although some groups have described structural alterations in the TCR in patients with other malignancies, we did not observe decreased expression of the CD3zeta subunit in the tumor-associated T cells. However, increased percentages of CD4(+)CD25(+) T cells were observed in the non-small cell lung cancer tumor-infiltrating lymphocytes and ovarian cancer tumor-associated lymphocytes. Furthermore, these CD4(+)CD25(+) T cells were found to secrete transforming growth factor-beta, consistent with the phenotype of regulatory T cells. Despite a generalized expression of lymphocyte activation markers in the tumor-associated T-cell populations, the CD8(+) T cells expressed low levels of CD25. To determine whether expression of CD25 could be restored on the CD8 cells, tumor-associated T cells were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies. After stimulation, nearly all of the CD8 T cells expressed CD25. Furthermore, despite the low levels of interleukin 2, IFN-gamma, and tumor necrosis factor-alpha secretion by the tumor-associated and peripheral blood T cells at baseline, stimulation with anti-CD3 and anti-CD28 monoclonal antibodies significantly increased the fraction of cells producing these cytokines. Thus, tumor-associated T cells from patients with early and late-stage epithelial tumors contain increased proportions of CD4(+)CD25(+) T cells that secrete the immunosuppressive cytokine transforming growth factor-beta. Furthermore, our results are consistent with previous reports showing impaired expression of CD25 on CD8(+) T cells in cancer patients. Finally, increased lymphocyte costimulation provided by triggering the CD28 receptor is able to increase CD25 expression and cytokine secretion in tumor-associated T cells. These observations provide evidence for the contribution of regulatory T cells to immune dysfunction in cancer patients.     

Differing phenotypes between intraepithelial and stromal lymphocytes in early-stage tongue cancer

The significance of tumor-infiltrating lymphocytes (TIL) has attracted much attention in relation to the prognosis of patients. We herein examined the activation status of the TILs in relation to the tumor microenvironment. By using frozen sections of human early-stage tongue cancers (n = 22), the TILs in the cancer nests and those in the cancer stroma were compared for the expression of PD-1, NKG2A, NKG2D, CD69, and Ki-67. The lymphocytes in oral lichen planus, an active immune response-mediated mucosal disease, were also analyzed for comparison purposes. All of the cancer specimens were abundantly infiltrated by CD8(+) T cells and CD56(+) natural killer (NK) cells in the stroma, as well as in the tumor nest. The tumor nest-infiltrating (intraepithelial) CD8(+) T cells frequently expressed PD-1, an inhibitory receptor, in sharp contrast to those in the stroma or in the lichen planus. Conversely, the intraepithelial CD8(+) T cells only infrequently expressed NKG2D, an activating receptor, in contrast to those in the stroma or in the lichen planus. No intraepithelial CD8(+) T cells expressed Ki-67, a proliferation-associated marker, whereas those in the stroma frequently expressed it. Furthermore, the intraepithelial NK cells expressed NKG2A, an inhibitory receptor, more frequently than those in the stroma or the lichen planus. Collectively, the intraepithelial CD8(+) T cells and NK cells are phenotypically inactivated, whereas stromal counterparts are phenotypically just as active as those in the lichen planus. These results suggest the first-step occurrence of an immune evasion mechanism in the tumor nest of oral squamous cell carcinoma.     

Associations Between Infiltrating Lymphocyte Subsets and Hepatocellular Carcinoma

Aims: We aimed to analyze the phenotype of tumor-infiltrating lymphocytes (TILs) and non-tumor infiltrating lymphocytes (NILs) in HCC and non-tumor tissues, and evaluate relationships between changes in these cells and the prognosis of HCC. Methods: Lymphocytes were isolated from HCC and corresponding non-tumor tissues and tested by flow cytometry. For comparison, clinical parameters were analyzed. Results: Compared with the non-tumor tissue, tumor tissue had a lower intensity of NK, NKT andCD8+T cell infiltration. TILs had higher intensity of CD4+CD25+Foxp3+regulatory T cell (Treg cells) infiltration compared with that in NILs. The prevalence of Treg cells was associated with fewer CD8 + T lymphocytes in the HCC immune microenvironment. The frequencies of NK cells and CD8+T cells in TILs of HCC patients with metastasis less than 12 months were lower than those without metastasis. However, the frequency of Treg cells was higher than those without metastasis. Conclusion: These results suggest that the frequencies of CD8+T, NK and NKT cells as well as Treg cells in the tumor tissue of HCC are significantly associated with patient survival, and could be applied as predictive indicators for HCC prognosis.     

Increased populations of regulatory T cells in peripheral blood and tumor-infiltrating lymphocytes in patients with gastric and esophageal cancers.

PURPOSE: It is well known that tumor-infiltrating lymphocytes (TILs) and, to a lesser extent, peripheral blood lymphocytes from patients with advanced-stage cancer have a poor immune response. Regulatory T cells (T-regs), characterized by coexpression of CD4 and CD25 markers, can inhibit the immune response mediated by CD4+/CD25- and CD8+ T cells. In the present study, we evaluated the prevalence of T-regs in peripheral blood and TILs in patients with gastric and esophageal cancers. EXPERIMENTAL DESIGN: The population of CD4+/CD25+ cells as a percentage of total CD3+ cells was evaluated by flow cytometric analysis with triple-color staining. To assess the functional activity of CD4+/CD25+ cells, CD4+/CD25+ or CD4+/CD25- cells were purified from peripheral blood mononuclear cells with magnetic beads. The cytokine production [interleukin (IL)-10 and IFN-gamma] from the CD4+/CD25+ cells in response to anti-CD3 stimulation was evaluated. Also, the antiproliferative function of CD4+/CD25+ cells was measured by evaluating the proliferative activity of CD4+/CD25- cells in response to anti-CD3 plus anti-CD28 in the presence of autologous CD4+/CD25+ cells. RESULTS: The prevalence of peripheral blood CD4+/CD25+ cells in both gastric (n = 20; 14.2 +/- 4.9%) and esophageal cancer patients (n = 10; 19.8 +/- 6.9%) was significantly higher than that in healthy donors (n = 16; 7.2 +/- 2.1%). The population of CD4+/CD25+ cells in the TILs of gastric cancer patients with advanced disease (19.8 +/- 4.5%) was significantly higher than that in TILs of patients with early-stage disease (4.8 +/- 2.1%) or that in intraepithelial lymphocytes of normal gastric mucosa (4.0 +/- 1.2%). As a functional consequence, CD4+/CD25+ cells did not produce IFN-gamma, whereas CD4+/CD25- cells secreted IFN-gamma. Moreover, CD4+/CD25+ cells produced large amounts of IL-10, whereas CD4+/CD25- cells secreted little IL-10. The proliferation of CD4+/CD25- cells was inhibited in the presence of CD4+/CD25+ cells in a dose-dependent manner, confirming that CD4+/CD25+ has an inhibitory activity corresponding to T-regs. CONCLUSIONS: The populations of CD4+/CD25+ T-regs in peripheral blood and TILs in patients with gastric and esophageal cancers were significantly higher in comparison with those in healthy donors or normal mucosa.     

Expanded tumor-reactive CD4+ T-cell responses to human cancers induced by secondary anti-CD3/anti-CD28 activation.

Generation of tumor-reactive T cells in large numbers ex vivo is a requisite step in the adoptive immunotherapy of patients. We examined the immune responses of T cells derived from tumor vaccine-primed lymph nodes activated with anti-CD3 alone and with an anti-CD3/anti-CD28 combination. Nylon wool-purified CD3+ cells were isolated from vaccine-primed lymph nodes obtained from melanoma, renal cell, and head and neck cancer patients. In the absence of antigen-presenting cells, activation with anti-CD3/anti-CD28 greatly enhanced subsequent T-cell expansion in interleukin 2 (>100-fold), compared to anti-CD3 alone. CD4+ T cells were preferentially stimulated. In four of eight patients, we found evidence of CD4+ cellular responses to autologous tumors by cytokine release assays. Positively selected CD4+ cells activated with anti-CD3/anti-CD28 released greater amounts of cytokine (IFN-gamma and granulocyte macrophage colony-stimulating factor) in response to autologous tumors compared to cells activated by anti-CD3 alone. The CD4+ reactivity was MHC class II restricted and appeared to be associated with the expression of class II molecules on the vaccinating tumor cells. The CD4+ T-cell responses to class II-restricted tumor-associated antigens in patients with renal cell cancers represent unique findings.     

Flow cytometric analysis of tumour infiltrating lymphocyte activation and tumour cell MHC class I and II expression in breast cancer patients.

The primary tumour cells and tumour infiltrating lymphocytes (TILs) of 31 breast cancer patients have been analysed by dual colour flow cytometry to determine whether the phenotype and/or activation status of the TILs bears any relationship to the expression of MHC antigens on the tumour cells. The phenotype and activation status of 5000 TILs were studied using Mabs to CD4, CD8, HLA DR, CD25 (the low affinity inducible IL-2 receptor) and the transferrin receptor and related to Class I and Class II MHC expression on 5000 primary tumour cells. On the tumour cells, Class I MHC expression ranged from 1-74%, averaging 12.9%. HLA DR expression ranged from 1-69% averaging 14.3%. When the phenotypic proportions of the lymphocytic infiltrate were analysed there was found to be a correlation between tumour expression of Class I MHC and the proportion of both CD4+ (P less than 0.05) and CD8+ (P less than 0.02) T cells within the tumour. No such relationship was found with the MHC Class II antigen. When TIL activation markers were analysed, the percentage of CD8+ TILs positive for HLA DR expression correlated strongly with the expression of Class I (P less than 0.001) and Class II (P less than 0.001) antigens on the tumour cells. The percentage of CD4+ TILs positive for HLA DR expression also correlated significantly, but less strongly with the expression of Class I (P less than 0.01) and Class II (P less than 0.02) antigen expression on the tumour cells. The percentage of CD4+ TILs positive for CD25 expression correlated with both Class I (P less than 0.05) and Class II (P less than 0.03) expression on the tumour cells while the percentage of CD8+ TILs positive for CD25 did not. The percentage of TILs bearing the transferrin receptor showed no measurable correlation with the expression of either class of MHC antigen on the tumour. The data suggest that MHC expression on the tumour cells has a selective effect on the response capacity of different parts of the immune system.     

肺癌患者免疫功能变化测定及其临床意义

目的 探讨肺癌患者机体免疫功能变化及临床意义.方法 采用流式细胞术测定68例肺癌患者治疗前及20例健康志愿者外周静脉血中CD+3T、CD+4T、CD+8T、NK细胞以及Th1类细胞因子干扰素(INF)-γ、白细胞介素(IL)-2、肿瘤坏死因子(TNF)-α;Th2类细胞因子IL-4、IL-6、IL-10的水平.结果 肺癌患者外周血中CD+3T、CD+4T细胞、CD4/CD8值下降;CD+8T、NK细胞升高;TNF-α、IL-4、IL-6、IL-10水平均升高,Th1/Th2值降低,与对照组比较差异均有统计学意义,(P分别＜0.05～0.001).肺癌患者NK与CD3、CD4、CD8均呈负相关;CD4与Th2类细胞因子呈负相关.化疗前肺癌患者外周血淋巴细胞,Th1、Th2类细胞因子水平与病理类型、淋巴结转移、远处转移无关均(P＞0.05).结论 肺癌患者细胞免疫功能普遍低下,其中CD+4T细胞的数量和功能降低比较显著,其功能的降低可能与Th1/Th2因子失衡有关.     

胃癌患者调节性T细胞胞内外细胞因子的检测及其意义

背景与目的:目前认为CD4 CD25 调节性T细胞与胃癌患者的免疫功能抑制密切相关,但CD4 CD25 调节性T细胞发挥免疫抑制功能的作用机制并不十分清楚.本研究通过检测胃癌患者CD4 CD25 调节性T细胞产生具有不同生物活性的细胞因子干扰素-γ(interferon-γ,IFN-γ)、白介素4(interleukin-4,IL-4)、IL-10及肿瘤生长因子-β(tumor growth factor-β,TGF-β)的分泌情况,进一步探讨这些细胞因子在胃癌患者CD4 CD25 调节性T细胞发挥免疫抑制功能的作用.方法:按常规方法制备患者外周血单个核淋巴细胞,采用免疫磁珠分选方法分离CD4 CD25 T细胞及CD4 CD25-T细胞后,用细胞内细胞因子染色法及ELISA法分别研究CD4 CD25 T细胞在胞内及胞外产生具有不同生物活性的细胞因子IFN-γ、IL-4、IL-1O及TGF-β的水平.结果:(1)与健康对照组比较,胃癌患者分泌内细胞因子IFN-γ、IL-4及IL-10的CD4 CD25 T细胞占CD4 细胞的百分比均显著增高(P＜0.05).(2)培养96 h后,上清液的各种细胞因子水平,无论是胃癌患者还是健康对照组,CD4 CD25 T细胞分泌的IL-10及TGF-β均显著高于CD4 CD25-T细胞(P＜0.05).CD4 CD25 T细胞分泌的IFN-γ显著低于CD4 CD25-T细胞(P＜0.05).结论:CD4 CD25 调节性T细胞体外免疫抑制作用的发挥可能与一些抑制性细胞因子有关,特别是细胞因子TGF-β在胃癌CD4 CD25 调节性T细胞的免疫抑制功能中起着相当重要的作用.     

The increase of CD57+ T cells in the peripheral blood and their impaired immune functions in patients with advanced gastric cancer

In addition to natural killer (NK) cells, T cells expressing natural killer cell markers, CD56 or CD57 (NK type T cells), have been considered to play an important role in antitumor immunity. We examined the proportion of NK cell and NK type T cell subsets in the peripheral blood from patients with gastric cancer. The IFN-gamma production capacity and population of cytoplasmic perforin positive cells in peripheral blood mononuclear cells (PBMC) were evaluated. Peripheral blood samples were obtained from 56 patients with gastric cancer and 21 healthy volunteers. The proportion of CD56- CD57+ T cells (CD57+ T cells) was significantly higher in advanced gastric cancer patients than those in healthy volunteers and patients with early stage gastric cancer, whereas no correlation was observed between the proportion of CD56+ T cells or NK cells and tumor progression. Furthermore, a significant decrease of CD8+ CD57+ T cells was found in patients with advanced gastric cancer. The proportion of CD57+ T cells did not correlate with interferon-gamma (IFN-gamma) production from PBMC in gastric cancer patients, although a significant correlation was found between them in healthy volunteers. The proportion of perforin positive CD57+ T cells, especially CD8+ CD57+ T cells, in patients with gastric cancer was markedly lower than that in healthy volunteers. Collectively, although the proportion of CD57+ T cells in PBMC was found to increase with tumor progression, their function in antitumor immunity is impaired in patients with gastric cancer.