恶性肿瘤胸腺水中TIL生物学活性研究

从卵巢癌腹水和肺癌胸水中分离出肿瘤浸润淋巴细胞，用ｒＩＬ－２诱导培养，观察其增殖活性。同时观察了ＰＪ－ＣＷ，ＰＯＧＰ，ＰＨＡ等对其杀伤活性的调节作用。结果表明，在培养５－１０天时ＴＩＬ增殖较快，其表面产生的ＣＤ３，ＣＤ４，ＣＤ８，Ｔａｃ等抗原，随培养时间的处长，表达量增加，尤以ＣＤ８增加较明显，ＣＤ４／ＣＤ８比值逐降。     

人体恶性实体瘤TIL增殖力、表型和杀伤力研究

在建立高活力肿瘤浸润淋巴细胞（ＴＩＬ）分离培养方法的基础上，对８３例恶性实体瘤ＴＩＬ的细胞增殖动力学、部份ＴＩＬ表型与杀伤力等作了较为系统的研究。结果提示，本实验室所建立分离培养的ＴＩＬ增殖能力较强且稳定，有６５％的增殖倍数大于１０００倍；３Ｈ－ＴｄＲ掺入高峰在培养的第４５～７５天；对其自身的原代肿瘤细胞杀伤力可以维持至５６天以上；对５６份经ｒＩＬ２诱导的ＴＩＬ（培养时间１７±５．２天）表型分析：ＣＤ３８０±２１％，ＣＤ４３７±２１％，ＣＤ８４４±１８％，ＨＬＡＤＲ６９±２４％，与ｒＩＬ２诱导前比较，ＣＤ３、ＣＤ４、ＣＤ８和ＨＬＡＤＲ均增多，尤以ＣＤ３与ＣＤ８表型增多明显。     

MHC-Ⅰ类分子在肿瘤浸润性淋巴细胞识别人黑色素瘤细胞中的作用

应用人白细胞抗原－Ａ２阳性（ＨＬＡ－Ａ＋２）黑色素瘤病人的瘤块中分离得到的肿瘤浸润性淋巴细胞（ＴＩＬ），进行ＨＬＡ－Ａ２限制性ＴＩＬ抗人黑色素瘤细胞作用的研究。在实验中发现，ＴＩＬ对自身和同种异体的ＨＬＡ－Ａ＋２黑色素瘤细胞具有杀伤作用，而对ＨＬＡ－Ａ－２黑色素瘤细胞及ＨＬＡ－Ａ＋２非黑色素瘤细胞无作用。选择重组白细胞介素４（ｒＩＬ－４）＋肿瘤坏死因子α（ＴＮＦα）能促进黑色素瘤细胞ＨＬＡ－Ａ２表达量，并增强ＴＩＬ对瘤细胞的杀伤活性。抗ＣＤ３、抗ＨＬＡ－ＡＢＣ和抗ＨＬＡ－Ａ２单抗具有明显抑制ＴＩＬ的抗瘤细胞活性。结果表明ＴＩＬ杀伤黑色素瘤细胞依赖于Ｔ细胞受体（ＴＣＲ）对瘤细胞共同抗原的识别，并有主要组织相容性复合体－Ⅰ类分子参与。     

肺鳞癌肿瘤浸润淋巴细胞的体外扩增,抗瘤活性及表型分析

对８例人原发性肺鳞癌的肿瘤浸润淋巴细胞（ＴＩＬ）进行分离培养并检测了ＴＩＬ的抗瘤活性及细胞表型，结果表明８例ＴＩＬ的平均扩增倍数为４３０；ＴＩＬ在１０～３０天之间显示出高水平的非特异性杀伤活性；１０天～时对Ａ１及Ｄａｕｄｉ细胞的杀伤百分率分别为７９．２％和４３．４％，２０天～时分别为５１．７％和２７．９％。非特异杀伤活性在３０天以后降至较低水平，所进行的几次实验表明肺鳞癌ＴＩＬ对自体癌细胞的杀伤活     

激素敏感型肾病综合征免疫遗传学研究

为了解激素敏感型肾病综合征是否有免疫遗传学背景，我们用病例，对照研究的方法研究了该病的家族遗传情况及ＨＬＡ抗原频率，患者组父母患病率（０．６２％）与对照组（０．１４％）比较差异无显著性。ＨＬＡ－ＤＲ７抗原频率患者组为４０．０％，对照组１１．２３偿（Ｐ＝２．５×１０＾－４）。患者组的ＨＬＡ－ＤＱ１抗原频率较对照组有显著性下降（Ｐ＝６．５×１０＾－３）。比较了８岁前与８岁后起病的患者的ＨＬＡ抗原频率，     

鉴别HCV C蛋白上HLA—A2限制的CTL识别表位的研究

本文先用计算机预测，后用＾５１Ｃｒ４ｈ释放分析证实的方法，对丙型肝炎病毒（ＨＣＶ）核心区蛋白（Ｃ蛋白）上，ＨＬＡ－Ａ２限制性ＣＴＬ识别表位进行了鉴别。结果发现，２名感染ＨＣＶ和ＨＬＡ－Ａ２阳性供血员的外周血单个核细胞（ＰＢＭＣ）对ＡＬＡＨＧＶＲＡＬ（ｃｏｒｅ１５０－１５８）肽段标记的自体靶细胞有溶解作用，杀伤率分别为３７．５％和１５．８％，用抗ＣＤ４ｍＡｂ阻断后杀伤率无明显变化，而用抗ＣＤ８ｍＡｂ     

肿瘤浸润淋巴细胞生物学性状初步研究

本研究发现，掺杂肿瘤细胞的早期ＴＩＬ培养上清液抑制自身淋巴结来源的淋巴细胞增殖作用；后期时，掺杂肿瘤细胞消失（即ＴＩＬ呈对数生长期），ＴＩＬ培养上清液却对自身淋巴结来源的淋巴细胞呈现生长刺激现象。首次发现消化酶对ＴＩＬ这些活力指标有影响。表型与淋巴细胞形态学对照研究时发现，初次分离的ＴＩＬ层细胞中，Ｇｉｅｍ－ｓａ染色的淋巴细胞比例为８３±６％；活细胞镜下计数淋巴细胞占７３±１２％；而ＦＡＣＳ分析ＣＤ３只有２３±８％，ＨＬＡＤＲ３１±２０％；是否属ＴＩＬ前体细胞，还须进一步研究。     

TIL识别的人黑色素瘤HLA-A2相关多肽的初步研究

从黑色素瘤病人的瘤块和淋巴结中制备黑色素瘤细胞和肿瘤浸润性淋巴细胞（ＴＩＬ）系,通过流式细胞仪对黑色素瘤细胞表型分析。经去垢剂处理、免疫沉淀和亲和层析法从ＨＬＡ Ａ２阳性患者黑色素瘤细胞中分离纯化ＨＬＡ Ａ２蛋白,其纯度通过４％～２０％梯度ＳＤＳ ＰＡＧＥ和Ｗｅｓｔｅｍｂｌｏｔ鉴定。１００℃沸水浴和弱酸处理ＨＬＡ Ａ２蛋白经反相 高压液相色谱层析（ＲＰ ＨＰＬＣ）得到不同组份多肽,将其与抗原加工缺陷的ＨＬＡ Ａ２阳性Ｔ２细胞反应,进行重建瘤细胞特异性表位测定。ＴＩＬ杀伤活性结果表明,来源于６２４ 黑色素瘤细胞的不同组份多肽中有三个活性峰值,而来源于Ｃｈａｐ 黑色素瘤细胞的不同组份多肽中仅一个与上述细胞组份位置相同的单一活性峰值。提示具有活性组份的多肽与ＨＬＡ Ａ２限制性ＴＩＬ识别的人黑色素瘤细胞抗原肽有关。     

反义寡聚硫代磷酸型、甲基磷酸型核苷酸体外抑制 HSV-Ⅰ增殖与抗原表达

以人单纯疱疹病毒（ＨＳＶ）基因组即刻早期ｍＲＮＡ４和５拼接区１０３４～１０５０密码子序列为靶位点，人工化学修饰、合成特异性８～１４ｍｅｒ反义寡聚硫代磷酸型、甲基磷酸型及未修饰型核苷酸（Ｓ－ＯＤＮ１４、Ｍ－ＯＤＮ８、Ｎ－ＯＤＮ８）体外抑制病毒活性。Ｓ－ＯＤＮ１４５～１０μｍｏｌ／Ｌ浓度即可抑制ＨＳＶ致ＣＰＥ和ＰＦＵ，Ｍ－ＯＤＮ８４０～６０μｍｏｌ／Ｌ表现明显抑制效应，ＥＬＩＳＡ检测Ｓ－ＯＤＮ１４１０μｍｏｌ／Ｌ接近５０％抑制病毒抗原表达。Ｍ－ＯＤＮ８的抑制病毒活性剂量呈现细胞毒性。     

滋养层细胞膜免疫特性研究

为探讨滋养层细胞膜的免疫调节作用及ＴＬＸ与ＣＤ４６的关系，我们用超声破碎、差速离心制备滋养层细胞膜（ＳｔＭＰＭ）。经酶学分析、电镜观察、ＭＨＣ抗原和β＿２微球蛋白测定及与标准抗ＴＬＸ抗体的反应，证实所分离的膜成份纯度、细胞特异性和抗原性。ＥＬＩＳＡ和ＦＡＣＳ竞争抑制试验表明兔抗ＭＣＰ对兔抗ＴＬＸ抗体的竞争抑制率为５２．３％；兔抗ＴＬＸ基本上能阻断ＴＲＡ－２－１０、ＣＢ－２４（均识别ＣＤ４６）与淋巴细胞的结合。但反之则不然。说明ＴＬＸ标准抗体能识别ＣＤ４６以外的抗原。向培养系统中加入ＳｔＭＰＭ．能特异地、剂量依赖性地抑制丝列原诱发的淋巴细胞增殖、ＩＬ－２与ｓＩＬ－２Ｒ分泌、ＩＬ－２Ｒ、ＨＬＡ－ＤＲ的表达；抑制混合淋巴细胞培养、Ｂ细胞ＩｇＧ的分泌、蜕膜ＮＫ和蜕膜、外周血ＬＡＫ的杀伤活性，但对外周血ＮＫ无抑制作用。核酸杂交显示对ＩＬ－２Ｒａ、ＴＣＲ－βｍＲＮＡ转录无显著影响，但能促进ＰＢＬ、ＪｕｒｋａｔＴＧＦ－βｍＲＮＡ的转录。从分子水平表明滋养层细胞膜具有广泛的免疫调节作用，为滋养层细胞在母胎免疫中的重要作用提供了新的实验依据。     

Selective expansion of a specific anti-tumor CD8+ cytotoxic T lymphocyte clone in the bulk culture of tumor-infiltrating lymphocytes from a melanoma patient: cytotoxic activity and T cell receptor gene rearrangements

In vitro culture of metastatic melanoma fragments with 150 units of recombinant interleukin 2 resulted in the successive expansion of CD4+ and then CD8+ tumor-infiltrating lymphocytes (TIL) throughout a 2-month period. TIL cultured for 43 days and consisting of 95% CD8+ and 10% CD4+ T lymphocytes were cloned by limiting dilution (LD). Thirteen CD8+ and thirty-one CD4+ clones were obtained, indicating that the frequency of clonogenic CD8+ proliferative T lymphocytes was much lower than that of their CD4+ homologues. When LD was performed in the presence of autologous melanoma cells the frequency of CD8+ clones was increased by factor 4. The DNA from TIL of day 43 bulk culture and of six CD8+ clones was hybridized with T cell receptor (TcR) beta and gamma probes. Identical configuration of the nonfunctional gamma and functional beta TcR genes was found in "bulk culture" and cloned TIL. The CD8+ clones therefore derived from a clonal population of CD8+ cells which had expanded in vitro before the LD. All the CD8+ clones tested were strongly cytotoxic for autologous melanoma cells but did not kill autologous fibroblasts or concanavalin A blasts, or any of the 10 allogeneic tumor targets tested, including 5 melanomas, 2 breast cell lines, 1 neuroblastoma, K-562 and the Epstein-Barr virus-transformed cell line used as a feeder. Furthermore, specific killing was inhibited by monoclonal antibodies against CD3, CD8, TcR alpha/beta and against class I major histocompatibility complex antigens indicating that these cytotoxic T lymphocyte clones recognized autologous tumor cells through the TcR, in an HLA class I-restricted manner. These data show that it is feasible to obtain tumor-specific cytotoxic T lymphocytes from melanoma TIL with a simple culture technique and that a single clone could be expanded to more than 10(10) cells which should allow testing of immunotherapeutic potential of these cells by adoptive transfer into melanoma patients.     

The emergence of non-cytolytic NK1.1+ T cells in the long-term culture of murine tumour-infiltrating lymphocytes: a possible role of transforming growth factor-beta.

The mechanism by which murine tumour-infiltrating lymphocytes (TIL) decreased their anti-tumour activity during an in vitro culture with interleukin-2 (IL-2) was investigated. A phenotype analysis revealed that the TIL cultured for 7 days (TIL-d7) were exclusively NKI.1- CD4- CD8+ CD3+ cells and that this population was replaced by natural killer (NK)1.1+ CD4- CD8 CD3+ cells by day 27 (TIL-d27) during the culture of TIL. The TIL-d7 cells showed a cytolytic activity against B16 melanoma, whereas the TIL-d27 cells had lost this activity, suggesting that the decrease in the anti tumour effect of TIL during the culture with IL-2 was due to their populational change. Analysis on the characteristics of the TIL-d27 cells revealed that they expressed skewed T-cell receptor (TCR) V beta 5 and increased mRNA expression of V alpha 14. In addition, they expressed transforming growth factor beta (TGF-beta) mRNA. Interestingly, TGF-beta augmented the proliferation of TIL-d27 cells under the presence of IL-2, but suppressed that of TIL-d7 cells. Moreover, the proliferation of TIL-d27 cells was suppressed by anti-TGF-beta monoclonal antibody. Collectively, these results suggest that, in contrast to its suppressive effect on anti-tumour effector T cells. TGF-beta could be an autocrine growth factor for NKL1.1+ T cells and thereby induce non-cytolytic NK1.1+ T cells in the long-term culture of TIL.     

Ultrastructure of IL2-stimulated tumor-infiltrating lymphocytes showing cytolytic activity against tumor cells.

Tumor-infiltrating lymphocytes (TIL) obtained from tumor tissue and pleural effusion of breast carcinoma were cultured with interleukin-2 (IL2) and thus activated. The ultrastructure of TIL stimulated by IL2 to kill various breast carcinoma cells was then investigated. Freshly isolated TIL cultured with autologous tumor cells for 48 h without IL2 were small, round and showed neither binding to nor killing of tumor cells. TIL stimulated to proliferate by IL2 became effector cells and showed cytotoxicity against tumor cells. Ultrastructurally, the effector TIL resembled large granular lymphocytes, and adhered to tumor cells through interdigitation or close apposition of the two plasma membranes accompanied by spot-like close membrane contacts. At the site of each spot-like contact, there was a 5-nm intercellular space. The morphology of the TIL processes did not differ from those of LAK and other CTL or NK cell processes during contact, invagination or the killing of target cells. The granules in TIL were considered to participate in the cytotoxic effect. Phenotypically heterogeneous TIL, CD8+/CD57- and CD8+/CD57+, adhered to autologous tumor cells and MCF7 (human breast carcinoma cell line). However, it was unclear which cell or cells acted as the effector for tumor-cell killing.     

Functional properties in Sézary cells with an unusual phenotype

The immunological and functional characteristics of Sézary cells with an unusual phenotype are reported. The clinical, histologic, and hematologic picture was typical for Sézary syndrome. Studies with monoclonal antibodies showed that 80% Sézary cells had an CD3+, CD4+, CD5+, CD7-, CD8-, Leu-7+, Leu-8-, Leu-11-, OKM1- phenotype. By two-color immunofluorescence assay 80% FACS-sorted Leu-7+ cells coexpressed CD4 antigen and did not express the myeloid antigen OKM1, CD8, and antigens characteristic of immature T cells. The cells had no NK activity but did display a high helper activity. Unseparated and FACS-sorted Leu-7+ and Leu-7- Sézary cells did not respond to mitogens but were able to grow in the presence of exogenous IL-2. FACS sorted Leu-7- cells, cultured for 7 days in the presence of 20% IL-2, acquired the receptors for Leu-7. IL-2 and IFN-gamma production was studied in unseparated Leu-7+ and Leu-7- FACS-sorted Sézary cells. IL-2 production was lower than in normal cells. The addition of PHA or PHA plus TPA led to an increase in IL-2 production. Also IFN-gamma production was marked lower than in normal controls but increased after 7-day culture in exogenous IL-2. In conclusion in this case the Sézary cells may represent a neoplastic expansion of the CD3+, CD4+, CD5+, Leu-7+, Leu-11- subpopulation which is equivalent to the 2-4% of the Leu-7+ population in normal lymphocytes.     

Sustained outgrowth of autotumor-reactive T lymphocytes from human ovarian carcinomas in the presence of tumor necrosis factor alpha and interleukin 2

Tumor-infiltrating lymphocytes (TIL) freshly isolated from human ovarian carcinomas were phenotyped (up to 75% CD3+ HLA-DR+ cells) and cultured in the presence of recombinant interleukin 2 and tumor necrosis factor alpha. In short-term cultures, an initial outgrowth of CD3+ CD8+ T lymphocytes resulted in the enrichment of autotumor cytotoxicity under these culture conditions. The early peak of autotumor cytotoxicity was accompanied by interleukin 1 beta and interferon gamma production. The RNA message for interferon gamma was detected by in situ hybridization in TIL induced with interleukin 2 and tumor necrosis factor alpha but not in freshly isolated TIL. A combination of interleukin 2 and tumor necrosis factor alpha was also advantageous for selective outgrowth of CD3+ CD8+ T lymphocytes with autotumor reactivity in long-term cultures of ovarian TIL. At days 30-40 of growth, total lytic units of autotumor cytotoxicity per culture increased from a mean of 59 to 2155, and the percentage of CD3+ CD8+ T lymphocytes rose to 98% in some of the cultures. Thus, the combination of interleukin 2 and tumor necrosis factor alpha provided conditions favorable for sustained growth of autotumor-reactive CD3+ CD8+ TIL in vitro.     

Mitogen-induced modulation of CD3, CD4, and CD8(1)

It is not clear whether CD3 contacts CD4 or CD8 directly, nor have the regulation and interregulation of expression of these three receptor molecules been determined. We explored these issues by first stimulating human peripheral blood lymphocytes in vitro with three well-characterized T-cell receptor-directed mitogens (phytohemagglutinin [PHA], concanavalin A [ConA], and anti-CD3 monoclonal antibody [alphaCD3]) and then using multiparameter flow cytometric techniques to investigate modulation of surface (sur) and cytoplasmic (c) CD3, CD4, and CD8. Cultures with alphaCD3 had a rapid, large, and persistent decline in surCD3; the cCD3 median fluorescent intensity (MFI) declined gradually, over the entire culture period. With alphaCD3, surCD4 MFI and cCD4 MFI declined by days 4 to 8 (31% of ex vivo value, p < 0.001 and 47%, p = 0.033), as did surCD8 MFI (58%, p = 0.010). PHA was associated with an increase in surCD8%, surCD8 MFI, and cCD8% at days 4 to 8 (178% of ex vivo, p = 0.003; 168%, p = 0.025; and 331%, p = 0.001). For PHA at days 4 to 8, cCD8 MFI was highly variable but always higher than in unstimulated cultures (5 of 5 experiments). With ConA, at 3 to 5 hours ex vivo, there was a decrease in surCD3 MFI relative to ex vivo (64%), surCD4% (83%), cCD4% (87%), surCD4 MFI (50%) and cCD4 MFI (48%), surCD8% (85%) and an increase in cCD8% (260%). As with PHA, at days 4 to 8, surCD8% was high relative to ex vivo (169%). Thus, we found that alphaCD3 had delayed effects on CD4 and CD8; PHA had delayed effects on CD8 only; and ConA had very rapid effects on CD3, CD4, and CD8, as well as a delayed effect on surface CD8. These effects involve both surface and cytoplasmic antigen expression and are more consistent with degradation or retention, rather than with shedding or increased production. They may reflect direct interactions between CD4 or CD8 and CD3 and/or interregulation of CD3 expression with expression of these coreceptor molecules.     

Enhancing of anti-viral activity against HIV-1 by stimulation of CD8+ T cells with thymic peptides.

HIV-1 can be neutralized by soluble factors produced and secreted by activated CD8+ T cells. Production of such anti-viral CD8 factors (including chemokines) can be induced with IL-2 or phytohaemagglutinin (PHA). In addition to PHA or IL-2, we have co-stimulated CD8+ T cells with PHA/IL-2 and a mixture of thymic peptides (TP) of molecular weights below 10 kD. For the activation, CD8+ T cells were purified from peripheral blood mononuclear cells of HIV-1- individuals and any resultant anti-viral activity was monitored using an HIV-1 neutralization assay. Using HIV-1 isolates highly resistant to chemokine inhibition we detected significantly higher levels of HIV-1 neutralizing activity in CD8+ T cell culture supernatants which had been co-activated with TP. When the TP-induced anti-viral activity was monitored, neutralization of both non-syncytia-inducing (NSI) and syncytia-inducing (SI) patient isolates was enhanced by 38% (NSI, PHA +/- TP), 66% (SI, PHA +/- TP), 28% (NSI, IL-2 +/- TP), and 57% (SI, IL-2 +/- TP) compared with the anti-viral activity present in supernatants from CD8+ T cell cultures stimulated only with PHA or IL-2. Peptide sequence analysis of purified TP showed that the TP mixture predominantly contains peptides with homology to human histone and collagen sequences. Our data demonstrate that CD8+ T cells are additionally activated by a mixture of TP. In this way, the production of HIV-1 neutralizing CD8 factors can be enhanced.     

Type 1 and type 2 tumor infiltrating effector cell subpopulations in progressive breast cancer

Effector T cells fall into two subpopulations based on cytokine-secretion. Type 1 cells secrete IFN-gamma, whereas type 2 cells secrete IL-4, IL-10, and GM-CSF. NKT cells represent a third subpopulation that secretes similar cytokines and have been associated with immunoregulation. Using the TS/A adenocarcinoma, we assessed the phenotype and kinetics of tumor-infiltrating lymphocytes (TIL) in mice challenged subcutaneously in the mammary region. Flow cytometric analysis shows that T cells do not infiltrate the primary tumor site until days 7-14 following tumor challenge. Both CD4 and CD8 TILs were predominantly CD44(High) and expressed CD25, CD69, and CD95 cell surface activation markers. Activated CD4/CD44(High) TIL numbers reached peak levels at day 21 that precipitously decreased by day 28 whereas corresponding CD8 cell numbers progressively increased, however, at lower levels and with later kinetics. Intracellular cytokine staining showed that greater numbers of IL-4-producing Th2 cells were elicited and with earlier kinetics than that of IFN-gamma-producing Th1 cells. T cells co-expressing DX5 (CD3(+)/DX5(+)) emerged (>21 days), suggesting a recruitment of NK-like T cells at later stages of tumor progression. Moreover, tumors selectively up-regulated TGF-beta, MIF, and IP-10 gene expression at times as early as day 4, with peak levels at day 7 in vivo. Such gene expression remained elevated and correlated with a continued progression in tumor growth suggesting that preferential effector cell recruitment and production of select factors during different stages of tumor maturation may aid in regulating effective endogenous antitumor responses in progressive breast cancer.     

PHA对人扁桃体T淋巴细胞亚群变化及活化抗原表达的效应

应用FITC和PE双色荧光试剂并经流式细胞计分析了人扁桃腺T淋巴细胞亚群组成,以及用PHA刺激后T淋巴细胞亚群的变化和活化抗原的表达情况。结果表明:1)人扁桃体T淋巴细胞以CD4~+细胞占优势,CD4~+与CD8~+细胞比值约为5.32±0.55;2),经PHA刺激培养72小时,CD4~+CD8~+细胞明显增加;3)经PHA刺激培养后,IL-2受体(CD25)和HLA-DR抗原表达增加。本实验结果为深入研究人扁桃体T淋巴细胞的表型变化提供了客观指标,对进一步探讨其在免疫系统中的作用具有一定的意义。     

Differential in situ expansion and gene expression of CD4+ and CD8+ tumor-infiltrating lymphocytes following adoptive immunotherapy in a murine tumor model system

In previous reports, we demonstrated that adoptively transferred T cells homed to the tumor site (among other sites) and that amplification of immune responses occurred in situ leading to the generation of cytotoxic CD8+ tumor-infiltrating lymphocytes (TIL) and macrophages. The present report extends these findings and shows that following adoptive immunotherapy (AIT) of mice bearing the immunogenic transplanted methylcholanthrene-induced rhabdomyosarcoma (MCA/76-9) there was a differential expansion of CD4+ and CD8+ TIL, the numbers peaking on days 6 and 8, respectively. At this time, CD8+ TIL accounted for the majority of Thy-1+ cells. Northern analyses of RNA extracted from positively selected (by panning) Thy-1+, CD8+ and CD4+ TIL isolated 8 days after AIT indicated the following: in five separate experiments, CD4+ cells expressed three- to sixfold more interleukin (IL)2 mRNA and six- to eightfold more IL6 mRNA than CD8+ cells, while CD8+ TIL expressed three- to sixfold more IL2 receptor (IL2R) mRNA and four- to sixfold more interferon-gamma mRNA than CD4+ cells. TIL cultured in 10% fetal bovine serum failed to release IL2 over a 24-h period, whereas both IL6 and interferon-gamma activities were demonstrable. The level of IL2R mRNA expression was reflected by a vigorous proliferative response of CD8+ TIL to exogenous recombinant IL2 and only a low response by CD4+ cells suggesting that most of the CD4+ TIL were in the resting stage. This was confirmed when it was shown that the incubation of panned CD4+ TIL with IL2 supplemented with irradiated spleen cells and "spent" 76-9 tumor culture supernatant (as a source of antigen) induced expansion of TIL resulting in a population consisting of greater than 90% CD4+ TIL. The overall data suggest that the relatively deactivated state of the CD4+ TIL at this particular time reflects the status of the rejection process in terms of the absence or low concentration of stimulating tumor-associated antigen.