Patterns of human tumor-infiltrating lymphocytes in 120 human cancers.

Tumor-infiltrating lymphocytes from 120 samples of human cancers, including melanoma, renal cell carcinoma, breast cancer, sarcoma, and colon cancer, were examined. The percentage of lymphocytes recovered from the cancer varied widely; that of renal cell carcinoma was higher than that of breast or colon cancer (65% vs 45%), which was higher than that of melanomas or sarcomas (30% to 35%). The types of lymphocytes before and after interleukin 2 activation showed specific patterns. CD4+ helper T cells predominated in all tumors except melanomas, which had more CD8+ cytotoxic T cells. CD16+ natural killer cells were recovered in renal cell carcinoma and sarcomas. Three different cytotoxic lymphocytes were identified among interleukin 2-activated tumor-infiltrating lymphocytes: (1) CD3+ CD16- cytotoxic T lymphocytes with cytotoxicity restricted to autologous tumor cells in melanomas, (2) CD3-CD16+ natural killer cells with vigorous major histocompatibility complex-nonrestricted cytotoxicity in renal cell carcinoma, and (3) CD3+ CD16- T cells with modest levels of major histocompatibility complex-nonstricted cytotoxicity in all cancers except melanomas. Thus, there was considerable diversity of tumor-infiltrating lymphocytes among these histologically distinct tumors with respect to magnitude of lymphocyte infiltration, phenotypic expression, and functional capacity.     

Immunohistochemical study of natural killer cells in tumor-infiltrating lymphocytes of primary intracranial germinomas.

A monoclonal antibody against the surface marker IOT-10 of natural killer (NK) cells was used to investigate the presence and distribution of these cells in a series of nine primary intracranial germinomas. In all of these tumors, IOT-10-positive NK cells were found in small numbers, mainly distributed among the tumor cells. The data obtained in the present study suggest that the presence of NK cells in primary intracranial germinomas can be influenced by factors other than the mere quantity of tumor-infiltrating lymphocytes.     

Improved in vivo efficacy of tumor-infiltrating lymphocytes after restimulation with irradiated tumor cells in vitro

Background: We investigated different culture conditions for tumor-infiltrating lymphocytes (TILs) with regard to proliferation, phenotypic changes, in vitro cytotoxicity, and in vivo therapeutic efficacy. Methods: After enzymatic digestion of the murine fibrosarcoma, MCA-105, TIL cultures were initiated as pure lymphocyte (groups 1 and 2) or mixed lymphocyte/tumor suspensions (groups 3 and 4). Group 1 TILs were grown in culture medium containing 100 IU/ml recombinant interleukin-2 (rIL-2). Group 2 TILs were stimulated with solid-phase anti-CD3 monoclonal antibody (mAb) for 48 h and cultured in rIL-2 (100 IU/ml)-containing medium. Group 3, which consisted initially of a surplus of tumor cells, received the same treatment as group 2. Group 4 was also activated with anti-CD3 mAb and rIL-2 but was additionally restimulated weekly with irradiated tumor cells (TILs to tumor, 20:1). Results: Groups 1 and 2 showed up to twofold higher increases in TIL numbers compared with groups 3 and 4 by the end of culture week 5. Although the original lymphocyte/tumor cell suspension consisted of 12.0 ± 3.8% CD4⁺ T cells and 5.3 ± 3.3% CD8⁺ T cells, all four TIL cultures showed 80% CD8⁺ TILs and no CD4⁺ TILs by the end of culture week 4. In vitro cytotoxicity did not correlate with in vivo efficacy of the examined TIL cultures. By using the MCA-105 pulmonary metastases model in C57BL/6 mice, only suboptimal doses of TILs (2 × 10⁶) from group 4, which had been restimulated weekly with irradiated tumor, showed significant tumor eradication compared with all other treatment groups (p<0.01). Conclusions: We conclude that in vitro tumor restimulation of TILs improves in vivo efficacy, most likely through the education of tumor-reactive T cells.Presented at the 48th Annual Meeting of The Society of Surgical Oncology, Boston, Massachusetts, March 23–26, 1995.     

人白细胞介素-15与18的协同刺激体外活化肿瘤浸润淋巴细胞的作用

目的 观察白细胞介素(IL)-15与IL-18协同刺激活化肿瘤浸润淋巴细胞(TIL)的作用.方法 用IL-15与IL-18共同刺激从结肠癌患者分离得到的肿瘤浸润淋巴细胞,通过测定其分泌转化生长因子(TGF)-β、IL-4、干扰素(IFN)-γ水平以及肿瘤浸润淋巴细胞的杀伤功能,观察IL-15与IL-18活化肿瘤浸润的淋巴细胞的功能.结果 IL-15与IL-18共同作用于肿瘤浸润淋巴细胞,能够抑制TGF-β、IL-4的分泌(P＜0.05),促进IFN-γ的分泌(P＜0.05),同时能提高肿瘤浸润淋巴细胞的杀伤活性.结论 IL-15与IL-18协同刺激能活化肿瘤浸润淋巴细胞.     

The effects of interleukin-6 on tumor-infiltrating lymphocytes derived from human renal cell cancer

Tumor-infiltrating lymphocytes (TIL) are a heterogeneous population of T cells with potent antitumor activity against a wide variety of tumors. TIL from renal cell cancer (RCC) typically exhibit diminished growth and antitumor activity after four weeks in vitro. We have therefore investigated effects of varying doses of interleukin-6 (IL-6) (0, 25, 100 units/ml.) on in vitro expansion, proliferation, cytotoxicity, and expression of cell surface phenotypes of long term renal TIL cultures from three RCC patients. Among the various conditions tested, three of three TIL cultures displayed a mild increase in cell expansion when grown in IL-2 with the addition of 100 units/ml. of IL-6. Two of three TIL cultures grown in IL-2 and 100 U/ml. of IL-6 demonstrated enhanced proliferation as determined by 3H-thymidine uptake. TIL could not be isolated or maintained in vitro when grown in the presence of IL-6 alone without IL-2. IL-6 was also found to enhance the long term non-specific cytotoxicity against an allogeneic nonrenal tumor target. No consistent effect on autologous tumor-specific cytotoxicity was demonstrated. We conclude that IL-6, when used in combination with IL-2, may modestly enhance the long-term growth of RCC-derived TIL.     

Clinical significance of tumor-infiltrating lymphocytes in neoplastic progression and lymph node metastasis of human breast cancer

To investigate the clinical significance of tumor-infiltrating lymphocytes (TILs) within the tumor milieu, we quantitatively measured and compared the subpopulations of TILs in 24 patients with stage I–III breast carcinoma. Peripheral blood mononuclear cells (PBMCs), normal breast parenchyma-infiltrating lymphocytes (NILs), and TILs were isolated from tissue specimens and quantified by flow cytometry. The results showed that increased proportion of CD8⁺ T cells, with decreased proportion of CD4⁺ T cells, was significant in gated CD3⁺ TILs as compared to autologous NILs or PBMCs (P < 0.001). The tumor-infiltrating CD8⁺ T cells significantly increased with stage progression, reflected in a more strongly decreased CD4/CD8 percentage (P = 0.003). The CD4/CD8 percentage of TILs was strongly correlated with lymphovascular permeation and subsequent lymph node metastasis (P < 0.001). Increased percentages of tumor-infiltrating CD8⁺ T cells with decreased CD4/CD8 percentages are of prognostic importance for cancer progression in human breast cancer.     

Immunohistochemical analysis of tumor-infiltrating lymphocytes and major histocompatibility antigens in human gliomas and metastatic brain tumors

A subpopulation of tumor-infiltrating lymphocytes (TILs) and the expression of major histocompatibility complex (MHC) antigens of the tumor cells were examined in 14 glioma and 13 metastatic brain tumor tissues. In both tumors, most of the TILs were T lymphocytes, and both phenotypes of the cytotoxic/suppressor and helper/inducer T lymphocyte were found. On examination of MHC antigens, β₂-microglobulin was shown intensely on tumor cells in all cases, and the monomorphic determinant of the human leukocyte antigen (HLA-DR) was shown in 10 glioma and in 5 metastatic cases. The correlation between the number of TILs and MHC antigen expression on tumor cells was equivocal as a whole in cases of both glioma and metastatic brain tumor.     

T-cell markers in tumor-infiltrating lymphocytes of head and neck cancer

Fresh suspensions of tumor-infiltrating lymphocytes (TIL) from 16 patients with squamous cell carcinoma of the head and neck (SCCHN) were examined for T-cell markers including CD4 (helper-inducer), CD8 (cytotoxic-suppressor), natural killer (NK) cell, and activation surface markers using monoclonal antibodies and two-color flow cytometry. Two of 8 (25%) patients with a CD4/CD8 ratio of less than 1 developed cervical lymph node metastases; none had extracapsular spread. Six of 8 (75%) patients with a CD4/CD8 ratio of greater than 1 developed cervical metastases; 5 of 6 (83%) exhibited extracapsular spread. An increased CD4/CD8 ratio was attributable to a decrease in CD8+ cells. A CD4/CD8 ratio of greater than 1 may be a useful prognostic indicator of the development of cervical metastases.     

人白细胞介素-15与18协同增强肿瘤浸润淋巴细胞体内对结肠癌细胞的杀伤功能

目的 观察白细胞介素(IL)-15与IL-18体内增强肿瘤浸润淋巴细胞(TIL)杀伤功能的效果.方法 用IL-15与IL-18共同刺激从结肠癌患者分离得到的TIL,通过测定其分泌转化生长因子(TGF)-β、IL-4、干扰素(IFN)-γ水平以及TIL的杀伤活性.用IL-15与IL-18共同刺激的TIL与结肠癌细胞SW480共孵育后,接种到SCID鼠皮下,测定肿瘤生长的大小、肝脏转移的肿瘤结节多少、血清中IL-4、IFN-γ水平以及腹腔巨噬细胞的吞噬能力.结果 IL-15与IL-18共同作用于TIL,能够抑制TGF-β、IL-4的分泌(P＜0.05),促进IFN-γ的分泌(P＜0.05).IL-15与IL-18共同作用于TIL后,能明显提高TIL对结肠癌细胞SW480的杀伤活性(P＜0.05).肝脏转移的肿瘤结节明显减少,血清中IFN-γ水平明显升高,腹腔巨噬细胞的吞噬能力增强.结论 IL-15与IL-18协同刺激体内能增强TIL对结肠癌细胞的杀伤功能.     

Regulatory effects of interleukin-4 on tumor-infiltrating lymphocytes derived from human renal cell carcinoma.

We have studied the effects of interleukin-4 (IL-4) on the expansion, proliferation, phenotype, and antitumor activity of tumor-infiltrating lymphocytes (TIL) derived from human renal cell carcinoma. Cultures were obtained from three primary renal tumors and one group of tumor-invaded, regional lymph nodes. IL-4 induced a significant increase in lymphocyte expansion and proliferation, but the response was dependent on the concurrent dose of IL-2 in culture. Increased growth activity was only observed in those cultures receiving low doses (20 U/ml) of IL-2 (average increase of fold expansion of 6.5, P < 0.01) with no changes in growth activity in the high dose (1000 U/ml) cultures. The combination of low dose IL-2 and IL-4 (200 U/ml) promoted lymphocyte growth significantly better than high dose IL-2 alone, the current standard growth regimen for in vitro expansion of TIL. TIL grown in the presence of IL-4 significantly reduced the level of non-specific, non-major histocompatibility complex-restricted antitumor activity (P < 0.01 for allogeneic renal, nonrenal, and NK-sensitive K562 cells), while exhibiting no effect on the level of autologous killing. This is in contrast to previous findings of significant enhancement of autologous antitumor activity using IL-4 on tumor-specific, melanoma-derived TIL. We also evaluated the effects of irradiated autologous tumor stimulation (TIL:tumor ratio, 10:1) on TIL cultures. Addition of autologous tumor cells increased expansion and proliferation of all cultures regardless of concurrent lymphokines present in the culture media (average increase fold expansion of 2.21, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of BCL-2 oncoprotein on tumor cells and tumor-infiltrating lymphocytes (TIL) in meningiomas

The Expression of the antiapoptotic oncoprotein BCL-2 and its correlation to tumor grade in 62 meningiomas (48 classic, 9 atypical, and 5 anaplastic) using single and double immunohistochemistry was investigated. BCL-2 expression was found in two different cell populations identified as lymphocytes (BCL-2+CD3+) and tumor cells (BCL+/CD3–). Tumor-infiltrating lymphocytes (TIL) (CD3+) were found within classic (9.5% of cells), atypical (2.4% of cells), and anaplastic (1.8% of cells) meningiomas. In classic meningiomas, 66.5% of TIL were BCL-2-positive, in atypical meningiomas 79.2%, and in anaplastic meningiomas 37.9%. In 33 (68.8%) of the classic meningiomas, medium to high counts of BCL-2+ tumor cells were detected. Atypical meningiomas showed nearly equal percentages of high (two patients), medium (five patients), and low (two patients) BCL-2+ tumor cell counts, whereas anaplastic meningiomas showed only medium (two patients) and low (three patients) BCL-2 tumor cell counts or were BCL-2-negative (one patient). In summary, a significant inverse correlation between the number of BCL-2-positive tumor cells and tumor grade in meningiomas was found. These findings support the hypothesis of cell survival prolongation by the antiapoptotic ability of BCL-2 proto- oncogenes and demonstrate the prognostic relevance of BCL-2 immunoreactivity in meningiomas. Received: 10 June 1998 / Accepted: 23 February 1999     

Characterization of tumor-infiltrating mononuclear cells in renal cell cancer: quantitative analysis by immunoperoxidase staining

In order to determine whether tumor-infiltrating mononuclear cells (TIM) of renal cell cancer (RCC) is suitable for adoptive immunotherapy, their number and characteristics were examined immunohistologically. TIM consisted of T cells and a smaller number of macrophages. Among T cells, CD8-positive cells were the dominant population which was reported to be more potent for the lysis of tumor cells. The number of T cells was variable: in 5 tumors no T cells were observed and in an other 5 the number of T cells were less than 2 x 10(6) cells/cm3. These results suggest that the TIM of RCC might be favorable for adoptive immunotherapy but TIM could not be available in some cases because of their small number.     

Characterization of effector cells mediating the augmentation of spontaneous cell-mediated cytotoxicity induced by therapeutic infarction of human renal carcinomas

Spontaneous cell-mediated cytotoxicity has been shown to be augmented after therapeutic infarction of human renal carcinomas. The effector cell responsible for the enhanced cytotoxicity was characterized by fractionation by adherence to nylon-wool and serum-coated plastic surfaces, density, and reactivity with certain monoclonal antibodies. The effector cell was nonadherent, sedimented in the lighter fractions of a 40-53% discontinuous Percoll gradient, and was not affected by preincubation with OKT 3 antibody selectively blocking cytotoxic T lymphocytes. About 70% of effector cells expressed the HNK-1 and 50% the OKM-1 antigenic markers. Cytotoxicity was high against the NK-sensitive target K-562 and low against the relatively NK-resistant target cell Daudi. These data define the effector cell mediating the increased cell-mediated cytotoxicity induced by infarction of renal carcinomas as a natural killer cell.     

Comparative analysis of tumor-infiltrating lymphocytes in a syngeneic mouse model of oral cancer

Objective To perform a comparative analysis of infiltrating immune cells in a newly developed C57BL/6 background syngeneic transplantable mouse oral cancer (MOC) model. Study Design/Setting Scientific study in an academic medical center. Methods Use of carcinogen-induced tumorigenesis, tissue culture, cell line transplantation, and flow cytometric analysis techniques. Results Previously, the authors established a series of cell line models that displayed dichotomous growth phenotypes when transplanted into immunocompetent mice. They now show that the indolent growth pattern of the MOC1-generated tumors is associated with increased baseline and inducible major histocompatibility complex class I expression and increased CD8(+) T-cell infiltration into the tumor microenvironment. Conversely, the aggressive and metastatic pattern of MOC2-generated tumors has decreased basal and inducible class I expression and is associated with FOXP3(+)CD4(+) regulatory T-cell infiltration. Delayed primary tumor growth after targeted monoclonal antibody therapy of these FOXP3(+) regulatory cells further suggests that these immune cells contribute to the aggressive phenotype of MOC2. Conclusion These data validate that key infiltrating immune cells identified here parallel findings in human head and neck cancer, making this newly developed syngeneic model a critical platform for the continued dissection of tumor-host interactions in head and neck cancer.     

CD8+肿瘤浸润淋巴细胞与乳腺癌预后的关系

目的：探讨CD8+肿瘤浸润淋巴细胞（TIL）与乳腺癌患者临床病理特征及生存期的关系。 方法：用免疫组化法检测112例乳腺癌肿瘤切片CD8+ TIL的表达;根据表达情况将患者分为CD8+ TIL阳性组与CD8+ TIL阴性组,分析CD8+ TIL与临床病理特征及生存期的关系。 结果：乳腺癌标本中CD8+ TIL阳性率为32.14%（36/112）。统计分析显示,CD8+ TIL的表达在肿瘤分化程度、TNM分期及组织学分类的组间差异有统计学意义(χ2=6.277,P=0.043;χ2=3.963,P=0.047;χ2=4.299,P=0.038）;CD8+ TIL阳性组5年生存率明显高于CD8+ TIL阴性组（88.89% vs. 71.05%;χ2=4.360,P=0.037）;Log-rank检验表明CD8+ TIL阳性组总生存率明显高于CD8+ TIL阴性组（P=0.043）。 结论：乳腺癌组织CD8+ TIL可以作为患者预后的指标,癌组织中CD8+ TIL低密度浸润提示预后不良。