BIOLOGY OF HUMAN TUMOR INFILTRATING LYMPHOCYTES

这项研究方案检验了这样一种假设，即识别 肿瘤浸润性原位激活的条件 淋巴细胞(TIL)将为人类免疫缺陷提供生物学基础 无体外输注的人实体瘤的免疫治疗 激活的淋巴细胞。具体目标如下：1. 黑色素瘤、肾癌和肉瘤TIL的特征。 至少有六种不同的TIL亚群如下：CD4-CD8+ T细胞与(I)自体肿瘤特异性或(Ii)MHC- 非限制性(LAK)细胞毒性；CD4+CD8-T细胞与(III) 自体肿瘤特异性活性或(Iv)LAK活性，(V) 具有LAK活性的CD3-CD16+NK细胞和(Vi)CD4-CD8-T细胞。 我们将(I)在前后提纯这些子集中的每一个 与rIL2孵育；(Ii)建立克隆细胞；(Iii) 调查其职能；(Iv)阐明以下要求 激活每个TIL亚群(IL-2、其他细胞因子、自体 肿瘤细胞或自体巨噬细胞)，以及(V)研究 参与TIL活化的分子(TCR、CD3、CD2、CD4、CD8、 TIL表面CD16和LFA抗原)。2.MHC-抗肿瘤的限制性研究 活动。我们将调查是否自体肿瘤特异性CTL或 黑色素瘤TIL的活性受MHC-Class I或Class的限制 通过测定肿瘤靶点上的II抗原表达 MHC-肿瘤的单倍型。3及4.规定的厘定 CD4、CD8-TIL产生IL-2及静息状态的分化 血淋巴细胞转化为TIL。巨噬细胞的作用。要求 检测内容包括：IL-L、IL-2、IL-2R诱导因子、抗CD_3单抗、 表达抗CD3单抗的杂交瘤、自体单核细胞、肿瘤 细胞，CD4+CD8-或CD4-CD8+克隆的TIL。5.抗肿瘤活性 黑色素瘤转移的LN淋巴细胞。LN淋巴细胞 亚群将在IL-2，抗CD3单抗， IL-L和/或自体单核细胞，然后检测其增殖能力。 发酵和抗肿瘤活性。6.识别及 白介素2受体诱导因子(IL2R-IF)的纯化文化 来自已建立的CD4+TIL系(TILH-11)和PBMC的上清液 本研究将使用PHA和PMA刺激。 对TIL免疫学特性的认识和对TIL的认识 TIL激活机制与宿主肿瘤生物学 人际关系将为发展 淋巴细胞原位激活及其治疗新策略 人类实体肿瘤的免疫治疗。

This research proposal tests the hypothesis that identification of the requirements for in situ activation of tumor-infiltrating lymphocytes (TIL) will provide the biological basis for immunotherapy of human solid tumors without infusion of ex vivo- activated lymphocytes. Specific aims are as follows: 1. Characterization of TIL from melanoma, renal carcinoma and sarcoma. There are at least six different TIL subsets as follows: CD4-CD8+ T cells with (i) autologous tumor-specific or (ii) MHC- nonrestricted (LAK) cytotoxicity; CD4+CD8- T cells with (iii) autologous tumor-specific The activity or (iv) LAK activity, (v) CD3-CD16+ NK cells with LAK activity and (vi) CD4-CD8- T cells. We will (i) purify each of these subsets before and after incubation with rIL2; (ii) establish cloned cells; (iii) investigate their functions; (iv) elucidate requirements for activation of each TIL subset (IL-2,other cytokines, autologous tumor cells or autologous macrophages), and (v) investigate molecules involved in the TIL activation (TCR, CD3, CD2, CD4, CD8, CD16 and LFA antigens on TIL). 2. MHC-restriction of antitumor activity. We will investigate if autologous tumor-specific CTL or The activity in melanoma TIL is restricted by MHC-Class I or Class II antigen expression on tumor targets respectively by determining MHC-haplotypes of tumors. 3 and 4. Determination of requirements for CD4 CD8- TIL to produce IL-2, and differentiation of resting blood lymphocytes into TIL. Role of macrophages. Requirements tested include: IL-l, IL-2, IL2R-inducing factor, anti-CD3 mAb., hybridomas expressing anti-CD3 mAb, autologous monocytes, tumor cells, CD4+CD8- or CD4-CD8+ cloned TIL. 5. Antitumor activity of lymphocytes from LN with melanoma metastasis. LN-lymphocyte subsets will be incubated in the presence of IL-2, anti-CD3 mAb, IL-l and/or autologous monocytes, followed by testing their proli- feration and antitumor activity. 6. Identification and purification of IL-2 receptor- inducing factor (IL2R-IF). Culture supernatants from the established CD4+ TIL line (TILh-Ll) and PBMC stimulated with PHA and PMA will be used for this study. Knowledge of immunological properties of TIL and understanding the mechanisms involved in TIL activation and the biology of host-tumor relationships will provide important findings for development of in situ lymphocyte activation and new strategies in the immunotherapy of human solid cancers.