Miescher S, Whiteside T L, Moretta L, von Fliedner V
J Immunol. 1987 Jun 1;138(11):4004-11.
A limiting dilution analysis (LDA) was used to assess the functional profiles of tumor-infiltrating lymphocytes (TIL) recovered from 15 human solid tumors. The microculture system applied in this study has been shown to allow virtually all normal peripheral blood T lymphocytes (PBL-T) to undergo clonal proliferation and was applied to obtain estimates of the frequency of both proliferating and cytolytic cells among the TIL population. A total of 624 microcultures proliferating in the presence of irradiated allogeneic spleen cells and interleukin 2 (IL 2) were expanded for clonal analysis. These TIL microcultures were assessed for surface antigen phenotype, IL 2 production (helper function) and for their cytolytic capabilities against the human erythroleukemic line K562 (natural killer (NK)-like activity) and P815, a mouse mastocytoma line, in the presence of phytohemagglutinin (PHA), i.e., lectin-dependent cell cytotoxicity (LDCC) which allows the detection of cytolytic activity irrespective of the antigenic specificity of the effector cells. Whenever feasible, cytolytic activity against autologous and allogeneic tumor cells was tested. LDA first demonstrated that the proliferative potential was decreased in T lymphocytes infiltrating human solid tumors (approximately 1 in 50 to 1 in 2 proliferating T lymphocyte precursors (PTL-P) in this series) as compared to normal PBL-T (1 in 2 to 1 in 1 PTL-P). The growth pattern in the titration cultures showed a remarkable agreement with the single-hit Poisson model implying that third party cells are unlikely to be involved in the reduced proliferative potential. Quantitative estimates of functional precursors showed that, in spite of reduced proliferative potential, cytolytic T lymphocyte precursors (CTL-P) against unknown antigens (LDCC-reactive) accounted for a considerable part of the microcultures in many cases. The precursor frequency of T lymphocytes with NK-like activity was usually low in situ (with the exception of glioma), whereas it was in the normal range in the patient's autologous PBL-T. In four evaluable cases, quantitative assessment showed that 1 in 200 to 1 in 1000 T lymphocytes from TIL was cytolytic against allogeneic tumor cells, which is in the range of alloreactive cytolytic T lymphocytes (CTL) generated in the mixed lymphocyte culture from normal PBL. Cytolytic activity against autologous target cells could not be quantitatively estimated but out of 88 clones from 4 patients, 3 clones originating from 2 glioma patients showed high lytic values against autologous tumor.(ABSTRACT TRUNCATED AT 400 WORDS)
采用极限稀释分析（LDA）评估从15例人类实体瘤中回收的肿瘤浸润淋巴细胞（TIL）的功能谱。本研究中应用的微量培养系统已被证明几乎能使所有正常外周血T淋巴细胞（PBL-T）进行克隆增殖，并用于估计TIL群体中增殖细胞和细胞溶解细胞的频率。在照射的同种异体脾细胞和白细胞介素2（IL-2）存在下增殖的624个微量培养物被扩增用于克隆分析。对这些TIL微量培养物进行表面抗原表型、IL-2产生（辅助功能）评估，以及在植物血凝素（PHA）存在下针对人红白血病细胞系K562（自然杀伤（NK）样活性）和小鼠肥大细胞瘤细胞系P815的细胞溶解能力评估，即凝集素依赖性细胞毒性（LDCC），这可检测效应细胞的细胞溶解活性，而不论其抗原特异性如何。只要可行，就检测针对自体和同种异体肿瘤细胞的细胞溶解活性。LDA首先证明，与正常PBL-T（每2至1个增殖性T淋巴细胞前体（PTL-P）中有1个）相比，浸润人类实体瘤的T淋巴细胞的增殖潜力降低（本系列中每50至2个增殖性T淋巴细胞前体（PTL-P）中有1个）。滴定培养中的生长模式与单打击泊松模型显著一致，这意味着第三方细胞不太可能参与增殖潜力的降低。功能前体的定量估计表明，尽管增殖潜力降低，但针对未知抗原（LDCC反应性）的细胞溶解T淋巴细胞前体（CTL-P）在许多情况下占微量培养物的相当一部分。具有NK样活性的T淋巴细胞的前体频率在原位通常较低（胶质瘤除外），而在患者的自体PBL-T中处于正常范围。在4例可评估的病例中，定量评估显示，TIL中每200至1000个T淋巴细胞中有1个对同种异体肿瘤细胞具有细胞溶解作用，这在正常PBL混合淋巴细胞培养中产生的同种异体反应性细胞溶解T淋巴细胞（CTL）范围内。针对自体靶细胞的细胞溶解活性无法进行定量估计，但在来自4例患者的88个克隆中，来自2例胶质瘤患者的3个克隆对自体肿瘤显示出高溶解值。（摘要截短于400字）
