Functional analysis of mononuclear cells infiltrating into tumors: lysis of autologous human tumor cells by cultured infiltrating lymphocytes

Lymphocytes separated from surgically resected tumor tissue, uninvolved lung tissue, and peripheral blood of lung cancer patients were investigated for cytotoxic potential and analyzed for their phenotypes at the time of surgery and after having been propagated for 4 to 5 wk in the presence of interleukin-2. Most of the tumor lymphocyte infiltrates examined were shown to have a shift in favor of T8 subsets from those found in peripheral blood. No natural killer activity and low cytotoxicity against the autologous tumor were found to characterize the tumor-derived lymphocyte population. Propagation of lymphocytes from the different tissues of the cancer patient in the presence of interleukin-2 preparation induced widespread lytic activity against K562 cells, autologous and allogeneic tumors, but not autologous normal lung or lymphoblasts. However, cytotoxic activity against autologous tumor cells exerted by cultured tumor-infiltrating lymphocytes was found to be significantly higher than the activity of cultured lymphocytes isolated from peripheral blood or uninvolved lung tissue of the same patient. The elevated lytic activity of cells derived from the tumor tissue indicates the accumulation at the tumor site of precursors of natural killer-like cells and specifically stimulated antitumor effectors. Our results suggest the coexistence of two types of anti-autotumor cytotoxic lymphocytes at the tumor site: natural killer-like and specific cytotoxic T-cells.     

Tumor infiltrating lymphocytes in ovarian cancer

The accumulation of tumor infiltrating lymphocytes (TILs) in ovarian cancer is prognostic for increased survival while increases in immunosuppressive regulatory T-cells (Tregs) are associated with poor outcomes. Approaches that bolster tumor-reactive TILs may limit tumor progression. However, identifying tumor-reactive TILs in ovarian cancer has been challenging, though adoptive TIL therapy in patients has been encouraging. Other forms of TIL immunomodulation remain under investigation including Treg depletion, antibody-based checkpoint modification, activation and amplification using dendritic cells, antigen presenting cells or IL-2 cytokine culture, adjuvant cytokine injections, and gene-engineered T-cells. Many approaches to TIL manipulation inhibit ovarian cancer progression in preclinical or clinical studies as monotherapy. Here, we review the impact of TILs in ovarian cancer and attempts to mobilize TILs to halt tumor progression. We conclude that effective TIL therapy for ovarian cancer is at the brink of translation and optimal TIL activity may require combined methodologies to deliver clinically-relevant treatment.     

Immunologic character of tumor infiltrating lymphocytes in ovarian carcinoma

Tumor-infiltratinglymphocytes(TIL)wasdirectlyisolatedfrompatient'stumortissues.ByrIL2activationandexpansioninvitro,TILwasagainimportedintothesamepatient'sbodytotreattumor,withapparenteffectsofanti-tumorandcomparativelessside-effect,withoutkillingothertumorcellsandnormalcells,allofwhichhavemadeitaeffectivewayoftreatingtumoratanadvancedstage.Inrecentyears,someresearchersfoundthattheeffectsofTIL'treatmentwerenotsofarapparent,withcomparativelyapparentdifference,whichmightbecloselyrelatedtothef…     

肿瘤浸润淋巴细胞的抗肿瘤免疫困境

体外肿瘤浸润淋巴细胞(TIL)具有一定的抗瘤活性,过继免疫治疗却未表现出理想效果,研究显示恶性肿瘤患者免疫系统处于系统性缺陷或耐受状态.在诱发阶段树突状细胞(DC)诱导TIL发生免疫偏倚,有的则因DC严重不足或状态不佳使TIL几无功能.肿瘤细胞的生理变化及肿瘤局部微环境也使TIL的杀伤活性受到严重抑制,甚至被反杀伤.体内TIL自身变化同样造成抗瘤免疫力低下.因此TIL的抗瘤免疫受到多方面困扰.     

肝癌肿瘤浸润淋巴细胞抗瘤活性及表型特征

目的 探讨肝癌肿瘤浸润淋巴细胞的抗瘤活性及表型特征。方法 13例肝癌患者(7例接受术前TACE，6例未接受)，MTT法检测TIL细胞抗肿瘤活性，APAAP法检测TIL细胞表型。结果 TACE组TIL细胞在第2周增殖达到高峰，至第3周平均扩增52倍，而未接受术前化疗组在第3周扩增达到高峰，平均扩增了12倍。术前介入化疗患者TIL细胞肿瘤杀伤活性增强，CD3^ 、CD4^ 、CD8^ 不同程度的增加。结论 术前介入化疗可缩短肝癌患者TIL细胞增殖时间，提高肿瘤杀伤活性。     

肿瘤浸润淋巴细胞的体外培养及临床应用研究

目的:研究肺癌患者胸腔积液中肿瘤浸润淋巴细胞(tumorinfiltratinglymphocytes,TIL)对瘤细胞的杀伤活性及其表型变化,并对其回输后毒副反应及患者免疫功能进行观察。方法:应用贴壁法分离恶性胸腔积液中TIL,并经rIL2诱导培养;用间接免疫荧光法测定CD3+、CD4+和CD8+比例;应用3HTdR释放法测定TIL对自体瘤细胞和801D细胞的杀伤作用;用活化的自体TIL注入患者胸腔内,观察胸腔积液变化。结果:第21天时CD3+和CD8+比例明显提高,而CD4+比例和CD4+/CD8+比值变化不大;对自体瘤细胞和801D细胞的杀伤力明显提高;40例自体回输治疗胸腔积液的患者,有效率为72.5%,且一般状况好,毒副反应小,辅助检查无异常改变,免疫功能增强。结论:肺癌TIL过继免疫治疗对恶性胸腔积液患者是一种安全、有效的免疫治疗方法。     

Tumor cytolysis by lymphocytes infiltrating ovarian malignant ascites

Tumor-associated lymphocytes (TAL) were isolated from the ascitic fluid of patients with adenocarcinoma of the ovary. These cells proliferated and expanded by 100-600-fold as either CD3+ CD4+ or CD3+ CD8+ cultures in the presence of moderate concentrations (50-200 cetus units/ml) of recombinant interleukin 2 and reached high numbers (5 x 10(8)-1 x 10(9)). After expansion of 16 TAL samples from 15 patients, 5 of the 7 isolated ovarian cytotoxic T-lymphocyte cell lines of T-cell receptor (TCR) (alpha beta)+ CD3+ CD8+ CD4- phenotype exhibited preferential cytolytic activity against autologous tumor targets and significantly lower cytolytic activity against allogeneic tumor targets and the natural killer-sensitive cell line K562. The cytolytic activity of the CD8+ TAL was inhibited by operationally anti-TCR (alpha beta) monoclonal antibody and monoclonal antibody specific for the CD3 differentiation antigen, indicating that the TCR and CD3 are involved in the cytolytic process. The other TAL cultures demonstrated similar cytolytic activity against both autologous and allogeneic tumors. The phenotype of these TAL was predominantly TCR (alpha beta)+ CD3+ CD4+ CD8-. Certain CD3+ CD8+ T-cell clones isolated from representative TAL exhibited preferential autologous tumor-specific cytotoxicity that may be major histocompatibility complex restricted. Other CD3+ CD8+ and CD3+ CD4+ clones exhibited nonmajor histocompatibility complex restricted cytotoxicity. These results demonstrate that CD3+ CD4+ and CD3+ CD8+ T-cells present in the ovarian malignant ascites can be propagated in large numbers and for long time intervals as T-cell lines in vitro. This finding may be significant for further investigation of ovarian tumor-specific cytotoxic T-lymphocytes and future adoptive specific immunotherapy studies.     

Flow cytometric analysis of tumour infiltrating lymphocytes in breast cancer

In 31 patients with carcinoma of the breast the phenotype and activation status of tumour infiltrating lymphocytes (TILs) was analysed by flow cytometry. The predominant cells, in all patients, were T lymphocytes and in the majority of cases CD8+ (cytotoxic/suppressor) T lymphocytes were present in greater numbers than CD4+ (helper) T lymphocytes. There was no relationship between the degree of lymphocytic infiltration and either tumour stage or grade but there appeared to be an inverse correlation with the levels of oestrogen receptor (ER) in the tumour (P less than 0.01). Both populations of T cells had significantly higher numbers of cells carrying HLA DR (class II major histocompatibility antigen) than the equivalent populations in peripheral blood from the same patient group (P less than 0.001). The transferrin receptor was found on similar numbers of CD8+ T cells in peripheral blood and among the tumour infiltrating lymphocytes while more of the CD4+ T cells infiltrating the tumour were found to carry this receptor (P = 0.034). The Tac (CD 25) antigen was also on similar numbers of CD8+ T cells from both peripheral blood and the tumour but was on fewer of the CD4+ T cells in the tumour with respect to peripheral blood (P = 0.029). In both TILs and blood lymphocytes, the Tac antigen was consistently present on greater numbers of CD4+ T lymphocytes than on the CD8+ T lymphocytes (P less than 0.001) and as this is a component of the interleukin 2 (IL-2) receptor this may be of relevance to the use of IL-2 in TIL cancer therapy.     

肝细胞性肝癌组织浸润淋巴细胞表型与分布研究

目的:肝细胞性肝癌组织浸润淋巴细胞与外周血T 细胞表型可能与肿瘤进展及预后相关,本研究检测肝癌患者组织及外周血T 细胞表型与分布,分析淋巴细胞表型变化与预后的关系。方法:分析2007年10月至12月中山医院147 例肝癌及癌旁组织浸润淋巴细胞表型（T 细胞或B 细胞表面标志物:CD3、CD8、CD4、CD20、CD19、Foxp 3）,表型与临床病理特征及预后的关系;检测26例肝癌外周血CD3、CD8、CD4 +T细胞数量并其比例变化。结果:癌巢内肿瘤浸润细胞明显少于癌周组织（P < 0.01）,癌周淋巴细胞主要分布于癌旁正常肝组织、汇管区,其与患者肝炎病史及肝硬化相关,表型以CD3 +T细胞为主,其中又以CD8 + 细胞毒性T 细胞为主;CD4 染色在多数病例为阴性,Foxp 3 仅在个别病例（15/ 109）呈阳性。肿瘤浸润淋巴细胞B 细胞标志CD20、CD19均为阴性。肿瘤组织内CD8 +T细胞浸润数量与预后正相关,而癌周浸润淋巴细胞数目与患者转移及复发无显著关系。结论:肝癌肿瘤浸润细胞在癌巢内明显少于癌周组织,肿瘤及癌周浸润细胞以CD8 + 细胞毒性T 细胞为主。肿瘤组织内CD8 +T细胞浸润数量与预后相关,而癌周浸润淋巴细胞数量与患者转移及复发无显著关系。      

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.     

鼻咽癌组织VEGF表达及淋巴细胞浸润对患者预后的影响

目的：研究鼻咽癌活检组织中血管内皮生长因子（VEGF）的表达及瘤巢中淋巴细胞的浸润程度对鼻咽癌患者远期预后的影响。方法：以免疫组化S-P法检测42例初诊无远处转移鼻咽癌患者活检组织中VEGF的表达情况,在HE切片上观察瘤巢中淋巴细胞的浸润程度,对其与患者临床病理特征及远期生存的关系进行分析。结果：VEGF低表达组与高表达组5年生存率分别为86．67％和37．04％（P=0．002）,瘤巢低淋巴细胞浸润组与中、高组5年生存率分别为23．5％和76．0％（P=0．0014）;VEGF低表达组与高表达组、瘤巢低淋巴细胞浸润组与中、高组的生存曲线分布差异有统计学意义。VEGF表达水平与N分期正相关,瘤巢淋巴细胞浸润程度与患者年龄呈负相关。结论：VEGF高表达、瘤巢低淋巴细胞浸润的鼻咽癌患者5年生存率降低,VEGF表达水平、瘤巢淋巴细胞浸润情况可以作为评价鼻咽癌患者预后的参考指标。     

Analysis of clonal immunoglobulin heavy chain rearrangements in ocular lymphoma

BACKGROUND: The morphologic diagnosis of primary and metastatic intraocular lymphoma (IOL) was made difficult by the paucicellular specimens with fragile populations of lymphocytes retrieved through pars plana vitrectomy (PPV). The analysis of immunoglobulin heavy chain (IgH) gene rearrangements (AIGHR) was used as an adjunct to cytopathology and flow cytometry in systemic lymphoma. In IOL, the sensitivity and specificity of AIGHR are unknown. METHODS: The authors reviewed the clinical records of patients who underwent PPV for suspicion of IOL at the Immunology and Uveitis Service of the Massachusetts Eye and Ear Infirmary (Boston, MA) between 2000 and 2002. AIGHR was performed as a routine diagnostic test on cell lysates isolated from < 0.5 mL of vitreous fluid. The authors used seminested polymerase chain reaction (PCR) with consensus primers for the VDJ-region of the IgH gene. PCR products were analyzed by polyacrylamide gel electrophoresis. RESULTS: Thirty patients (37 specimens) with chronic vitritis and 17 patients (23 specimens) with IOL were included. The specificity of vitreous fluid cytopathology, flow cytometry, and AIGHR was 1.0, and the sensitivity values were 0.24, 0.36, and 0.64, respectively. AIGHR was negative in two patients for whom cytopathology or flow cytometry revealed the diagnosis of lymphoma. Clonal IGHR was found in four specimens classified as negative for lymphoma based on cytopathology and flow cytometry. CONCLUSIONS: AIGHR supplemented cytopathology and flow cytometry to increase the diagnostic yield in IOL.     

大肠癌患者外周血与肿瘤组织中浸润淋巴细胞Fas及FasL表达的意义

目的 :探讨Fas/FasL在大肠癌患者外周血淋巴细胞及肿瘤浸润淋巴细胞的表达变化的意义。方法 :用流式细胞仪对 3 6例大肠癌患者外周血淋巴细胞与肿瘤浸润淋巴细胞(tumorinfiltratinglymphocyte ,TIL)的Fas及FasL进行定量分析。结果 :大肠癌患者外周血淋巴细胞上Fas/FasL表达明显高于正常对照 ,TIL上Fas/FasL表达高于外周血淋巴细胞上表达。结论 :Fas/FasL在大肠癌患者外周血淋巴细胞与TIL的不同分布说明 ,Fas/FasL不仅在肿瘤浸润区域而且在全身参与了免疫系统与肿瘤的相互斗争。     

Cytotoxic potential despite impaired activation pathways in T lymphocytes infiltrating nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is an epithelial tumor consistently associated with EBV. The histological picture is characterized by a strikingly abundant lymphocytic infiltrate. Furthermore, the epithelial tumor cells present several immunological characteristics which suggest an important role for tumor-infiltrating lymphocytes (TIL) in the biology of this tumor. The present study reports the phenotypic and functional characterization of TIL from NPC obtained after enzymatic digestion of 15 NPC biopsies. Flow cytometric analysis of TIL suspensions indicated that most TIL were mature CD3+ T lymphocytes (mean = 60%) with a variable CD4/CD8 ratio. Most TIL were TCR alpha/beta-positive (mean = 55%) and only a few TCR gamma-delta-positive cells could be identified. A small percentage (mean = 9%) displayed an activated phenotype (CD25+, HLA class II+). Using limiting dilution analysis, we found that the average frequency of proliferative T-lymphocyte precursors (PTL-P) is lower among TIL (1/40) than in autologous (1/7) or normal PBL (1/1.4). Moreover, sorting experiments have shown that this defect is significantly more pronounced in the CD8+ than in the CD4+ TIL subset. Accordingly, the TCR and the CD2-mediated antigen-independent pathways of activation were impaired. Different types of cytotoxic precursor could be detected. These included lectin-dependent cell cytotoxicity (LDCC) and NK-like or lymphokine-activated killer (LAK) activity. Interestingly, some TIL from NPC were able to lyse an NPC tumor (C15) maintained in nude mice. Thus, despite impaired activation pathways, the cytolytic potential of proliferating TIL in NPC is preserved.     

Tumor infiltrating lymphocytes in seminoma lesions comprise clonally expanded cytotoxic T cells

Seminoma lesions are characterized by a brisk inflammatory infiltrate containing both CD4 and CD8 T cells, which is of prognostic significance. However, whether seminoma cells express the HLA molecules required for classical T-cell recognition remains controversial. In the present study, we conducted a molecular, phenotypical and functional characterization of tumor infiltrating lymphocytes (TILs) from seminoma lesions. T-cell receptor clonotype mapping demonstrated the presence of clonally expanded T cells in the majority of the lesions. The cytotoxic capacity of TILs was indicated by expression of CD107a, which is a recently described surrogate marker for cytolytic activity. Indeed, the frequency of CD107a positive cells was substantially higher in TILs when compared to peripheral blood mononuclear cells. Moreover, fluorescence activated cell sorting of CD107a positive TILs allowed comparison of the clonotypic T-cell receptor fingerprint and demonstrated the ability of expanded clones to express this cytotoxic marker, suggesting cytotoxic activity at the tumor site. The cytotoxicity was confirmed by in situ granzyme B expression. Furthermore, by staining with multimeric HLA-peptide complexes, we could demonstrate the presence of Mage-3 specific T cells among TILs. In summary, specific and functional T-cell responses are operative in seminoma, indicating that the inflammatory infiltrate is indeed involved in the immunological control of the tumor.     

Human herpesvirus-6: tumorigenicity and tumor infiltrating lymphocytes.

We previously reported that human herpes-virus-6 (HHV-6) genome (strain GS) and a cloned subfragment (pZVH14) transfected NIH 3T3 cells, induced foci of transformation with a frequency significantly above the background level. The transformed cells produced tumors in nude mice and immunocompetent (Swiss) mice. In the current study, nude mice tumors were passed into Swiss mice and more aggressive tumors (G-2TS and 14-2TS derived from HHV-6 genome and pZVH14 DNA, respectively) were produced. 14-2TS tumors caused lung metastasis upon intravenous injection. In the case of subcutaneously growing aggressive tumors, tumor infiltrating lymphocytes (TIL) were isolated and characterized as T cells but lacked tumor specific killing as monitored by 51Cr-release assays. TIL lost activity at day 52 in a 4 h assay (but not in a 19 h assay) against the autologous tumor, but not a Maloney virus induced tumor (Yac-1). These studies indicate that an established nontumorigenic fibroblast cell line, when transfected with pZVH14 DNA of HHV-6, acquires both tumorigenic and metastatic potential and tumor bearing hosts mount immune response against such tumors.