Identification of target antigens in specific immunotherapy for renal cell carcinoma

PURPOSE: Effective immunotherapy against renal cell carcinoma has not yet been established despite recent advances in specific immunotherapy for various malignancies. A plausible reason is limited information about target antigens of renal cell carcinoma. We searched for useful cancer antigens applicable to immunotherapy for renal cell carcinoma by examining antigen expression in renal cell carcinoma cell lines and testing the ability to induce renal cell carcinoma reactive cytotoxic T lymphocytes. MATERIALS AND METHODS: mRNA expression of a panel of cancer associated antigens was examined using 5 renal cell carcinoma cell lines. Thereafter antigen derived peptides reported to induce cancer reactive cytotoxic T lymphocytes from human leukocyte antigen-A24+ patients with cancer were examined for their potential to induce cytotoxic T lymphocytes from peripheral blood mononuclear cells of human leukocyte antigen-A24+ patients with renal cell carcinoma. RESULTS: Three candidate antigens, including multidrug resistance-associated protein 3, polycomb group protein enhancer of zeste homologue 2 and Her2/neu, were expressed in all 5 renal cell carcinoma cell lines. Six peptides derived from these antigens, including multidrug resistance-associated protein 3(503-511), multidrug resistance-associated protein 3(1293-1302), polycomb group protein enhancer of zeste homologue 2(291-299), polycomb group protein enhancer of zeste homologue 2(735-743), Her2/neu342-350 and Her2/neu485-493, efficiently induced peptide specific and renal cell carcinoma reactive cytotoxic T lymphocytes from human leukocyte antigen-A24+ patients with renal cell carcinoma. Blocking and cold inhibition assays revealed that cytotoxicity against renal cell carcinoma depended on human leukocyte antigen class I restricted and peptide specific CD8+ T cells. CONCLUSIONS: This information could facilitate the development of effective immunotherapy against renal cell carcinoma.     

Tumor-infiltrating lymphocytes derived from human renal cell carcinoma: Clonal analysis of its characteristics

Aim:   To assess the characteristics of activated tumor-infiltrating lymphocytes (TIL), we report the isolation, growth response, and functional analysis of a CD4^- CD8⁺ TIL-clone derived from human renal cell carcinoma (RCC).
Methods:   Bulk TILs were expanded from a human RCC and the lymphocytes were separated into a CD8⁺ enriched population. Subsequently, using the limiting dilution technique, a TIL clone was established and its growth response, phenotype and cytotoxic activity were analyzed.
Results:   A clone, T16-13, by day 94 numbering 1 × 10⁷ cells, was harvested and characterized as a CD4^- CD8⁺ clone. On day 144, the cytotoxic activity of this clone against the autologous tumor was relatively high (2.3 ± 0.7 LU₃₀/10⁶ cells). Meanwhile, against allogeneic renal tumors, there was no cytotoxic activity (−0.1 LU₃₀/10⁶ cells).
Conclusions:   A TIL clone possessing modest autologous tumor-specific cytotoxicity can be isolated from human RCC. The characteristics analysis of various TIL clones may provide a better understanding of an RCC tumor microenvironment and may help to establish new modalities for the treatment of patients with metastatic kidney cancer.     

Adoptive transfer: The role of perforin in mouse cytotoxic T lymphocyte rejection of human tumor xenografts in vivo

ABSTRACT: The popliteal lymph node cells of immunocompetent mice generated a strong in vitro cytotoxic response to footpad injection of several human tumor cell lines and the resulting mouse effector cells predominantly used a perforin-mediated cytotoxic mechanism. A relatively minor FasL-dependent cytotoxic response to CEM-CCRF and Jurkat leukemias, but not colon carcinoma COLO 205 cells, was also detected in immunized perforin-deficient mice. In vitro depletion of CD3⁺ CD8⁺ T cells, but not CD4⁺ T or NK1.1⁺ cells, completely inhibited lysis of human tumor cells, suggesting that CD3⁺ CD8⁺ T cells were effectors of perforin-mediated xenospecific cytotoxicity. Xenospecific cytotoxic T cells from wild-type mice were extremely efficient at rejecting tumor when adoptively transferred into scid mice bearing established COLO 205, CEM-CCRF, or Jurkat tumor xenografts. By contrast, cytotoxic T lymphocytes of perforin-deficient mice had no effect on the growth of established tumor xenografts. These data indicate that perforin, and hence direct cytotoxicity, plays a key role in the ability of adoptively transferred CD8⁺ cytotoxic T lymphocytes to eradicate established xenografts.     

The immunophenotype and activation status of the lymphocytic infiltrate in human breast cancers, the role of the major histocompatibility complex in cell-mediated immune mechanisms, and their association with prognostic indicators

BACKGROUND: The aims of this study were to characterize, phenotypically, the immune infiltrate in human breast cancers, to assess the activation status of tumor-infiltrating lymphocytes (TIL), and to define the association of these findings with established prognostic indicators. METHODS: Immunohistochemistry was performed on frozen sections of 60 primary breast cancers by use of monoclonal antibodies to T lymphocytes (CD3), T-helper cells (CD4), cytotoxic T-cells (CD8), natural killer cells (CD56), interleukin-2 receptors (IL-2R), and major histocompatibility (MHC) class I antigen (HLA-ABC) and MHC class II antigen (HLA-DR). RESULTS: All tumors stained positive for CD3, CD4 and CD8, but with marked variation in the intensity of the infiltrate. In tumors with a moderate infiltrate of TIL, there was a trend toward a greater representation of T-helper cells. However, as the intensity of TIL increased, there was a decline in the proportion of T-helper cells and a concomitant rise in the relative proportion of cytotoxic T cells. There was a relative paucity of natural killer cells. A significant association was found between the intensity of TIL and the number of positive nodes (P=.02) and the intensity of the infiltrate of both T-helper cells and cytotoxic T cells with ER expression (P=.03 and.05, respectively). Most tumors stained positive for IL-2R. The expression of IL-2R was associated with the intensity of TIL (P<.0001), T-helper cells (P<.002), cytotoxic T cells (P=.01) and natural killer cells (P=0.04), and also with the degree of lymph node positivity (P=.02) and histologic tumor grade (P=.05). MHC class II expression was variable, and a large proportion of the tumors showed limited expression in individual cancer cells. There was an association between the expression of HLA-DR in tumor cells and the activation status of TIL (P=.03). CONCLUSION: An immune infiltrate is an invariable finding in breast cancers, and the intensity of the infiltrate is greater in node positive tumors. Additionally, TIL may well be activated, albeit partially, in most tumors, suggesting that cell-mediated immune mechanisms are functionally intact.     

A clinical study of immunological status in gastric cancer patients--with special reference to T-cell subsets in the lymphocytes of regional lymph nodes

Non-metastatic regional lymph node lymphocytes of 41 patients with gastric cancer were studied by using different monoclonal antibodies and flow cytometry. Used monoclonal antibodies were OKT3 (total T; CD3), OKT4 (helper/inducer T; CD4), OKT8 (suppressor/cytotoxic T; CD8) and Leu11 (NK/K cell; CD16). The results were as follows: 1. The percentage of CD3 cells and CD4 cells were about ten point fewer in lymph nodes than in peripheral blood. 2. CD8 cells were found to be one half or one third lesser in lymph nodes than in peripheral blood. 3. CD16 cells were found to be rare in lymph nodes. 4. The percentage of CD3, CD4 and CD8 cells were higher in distal lymph nodes than proximal ones. 5. The percentage of CD3, CD4 and CD8 cells were not different with progression of the cancer, whereas CD3 cells and CD8 cells were decreased in lymph nodes of stage IV. 6. The percentage of CD8 cells was higher in distal nodes of stage III. Regional lymph nodes are necessary to protect against cancer metastasis, and killer T cells and cytotoxic T cells were fewer in lymph nodes. These results suggested that killer activity and cytotoxicity of the lymph node lymphocytes are inactive and anergy.     

Lymphocyte subsets in renal allograft recipients with chronic hepatitis C virus infection.

BACKGROUND: The pathogenetic mechanisms of chronic hepatitis C virus (HCV) infection in renal allograft recipients are not well established. This study aimed to examine the relationship between altered immune status and HCV-related liver disease, by determining the changes in peripheral blood lymphocyte and natural killer (NK) cell subsets in these subjects. METHODS: Peripheral blood lymphocyte, NK cell and activation markers were detected by flow cytometry in renal allograft recipients with (TpC+) or without (TpC-) HCV infection, and compared with age- and sex-matched patients with post-transfusional chronic HCV infection (TfC+) and healthy controls. RESULTS: CD19+ cells were reduced in renal allograft recipients compared with controls. TpC+ subjects had increased CD3+CD8+ cells compared with controls, and increased CD3+DR+ cells but reduced CD4+ CD38+ and CD3-CD16/56+ cells compared with controls as well as TfC+ patients. TfC+ patients and controls had similar numbers and proportions for the lymphocyte subsets and NK cells. Chronic liver disease in HCV-infected renal allograft recipients was associated with increased CD3+CD16/56+ cells but reduced CD4+CD38+ cells. Reduction of CD3-CD16/56+ cells was noted in TpC+ subjects without liver disease. Yet among post-transfusional (TfC+) subjects this was associated with chronic hepatitis. CONCLUSIONS: Peripheral blood suppressor/cytotoxic T lymphocytes are increased, whereas activated helper/inducer T lymphocytes and NK cells are reduced, in renal allograft recipients with HCV infection. Increased non-MHC-restricted cytotoxic T cells and reduced NK cells are associated with the presence or absence of liver disease respectively. These data suggest that immune mechanisms are important in the pathogenesis of chronic hepatitis C after renal transplantation.     

Expression of the SART3 tumor rejection antigen in renal cell carcinoma

PURPOSE: We recently reported that SART3 tumor rejection antigen is recognized by HLA class I restricted cytotoxic T lymphocytes from patients with esophageal cancer. We now investigate the expression of SART3 antigen in renal cell carcinoma to identify an appropriate molecule that may be used in specific immunotherapy of renal cell carcinoma. MATERIALS AND METHODS: Renal cell carcinoma and nontumorous kidney tissues were obtained at surgery. A section of each sample was minced with scissors and stored at -80C until use. SART3 antigen expression was examined in uncultured renal cell carcinoma and nontumorous kidney tissues. We also evaluated the ability of derived peptides to include cytotoxic T lymphocytes in peripheral blood mononuclear cells from patients with renal cell carcinoma. RESULTS: The SART3 antigen was detected in all renal cell carcinoma cell lines, primary cultures of renal cell carcinoma and nontumorous kidney tissues, and in the cytosol of 57% and 15% of renal cell carcinoma and nontumorous kidney tissues, respectively. HLA-A2402 restricted and tumor specific cytotoxic T lymphocytes (KE4) used in cloning of the SART3 gene were significantly cytotoxic to cells from renal cell carcinoma cell lines and primary cultures of renal cell carcinoma tissue but they did not lyse normal cells, including those from primary cultures of nontumorous kidney tissue. The SART3 peptides derived from positions 109-118 and 315-323 induced HLA-A24 restricted cytotoxic T lymphocytes to renal cell carcinoma cells from peripheral blood mononuclear cells of patients with renal cell carcinoma. CONCLUSIONS: The SART3 antigen and derived peptides may be applied to the specific immunotherapy of HLA-A24+ renal cell carcinoma.     

Immuno-modulatory effects of relaxation training and guided imagery in women with locally advanced breast cancer undergoing multimodality therapy: A randomised controlled trial

Eighty women undergoing multimodality treatment for large (>4 cm) or locally advanced (T3, T4, Tx, N2), breast cancers participated in a randomised controlled trial (RCT) to evaluate the immuno-modulatory effects of relaxation training and guided imagery. Patients underwent chemotherapy followed by surgery, radiotherapy, and hormone therapy. Those in the intervention group were taught relaxation and guided imagery. Patients kept diaries of the frequency of relaxation practice and imagery vividness. On 10 occasions during the 37 weeks following the diagnosis, blood was taken for immunological assays CD phenotyping: T cell subsets (helper, cytotoxic), natural killer (NK) and lymphokine activated killer (LAK) cells, B lymphocytes and monocytes; cytotoxicity: NK and LAK cell activities; cytokines interleukin 1 beta (1β), 2, 4 and 6 and tumour necrosis factor alpha.Significant between-group differences were found in the number of CD25+ (activated T cells) and CD56+ (LAK cell) subsets. The number of CD3+ (mature) T cells was significantly higher following chemotherapy and radiotherapy, in patients randomised to relaxation and guided imagery. Using a median split, women who rated their imagery ratings highly had elevated levels of NK cell activity at the end of chemotherapy and at follow-up. Significant correlations were obtained between imagery ratings and baseline corrected values for NK and LAK cell activity, and IL1β. Relaxation frequency correlated with the number of CD4+ (T helper) cells, the CD4+:8+ (helper:cytotoxic) ratio, and IL1β levels.Relaxation training and guided imagery beneficially altered putative anti-cancer host defences during and after multimodality therapy. Such changes, to the best of our knowledge, have not been previously documented in a RCT.     

Effect of cardiopulmonary bypass on circulating lymphocyte function.

Extracorporeal cardiopulmonary bypass (CPB) has been associated with a wide variety of immunological derangements, including a transient postoperative impairment of lymphocyte function. We examined changes in phenotypic and nonspecific cytotoxicity of peripheral blood mononuclear cells after extracorporeal CPB. The peripheral blood samples obtained from 10 patients were subjected to natural killer and cytotoxic T lymphocyte activity assay before and at intervals after CPB. Phenotypic analysis of peripheral blood lymphocytes was performed in 5 patients before and immediately after CPB. We observed a significant increase in peripheral blood CD8+ cells (cytotoxic/suppressor T lymphocytes) (16.1% +/- 2.5% versus 22.5% +/- 2.1%; p less than .005) and a decrease in CD4+ cells (helper/inducer T lymphocytes) (46.1% +/- 3.5% versus 36.1% +/- 3.5%; p less than 0.02) immediately after extracorporeal circulation. The CD8/CD4 ratio in peripheral blood was significantly increased immediately after bypass (0.53 versus 0.80; p less than 0.001). No significant changes in percentages of other leukocyte subsets in peripheral blood were noted. The activity of cytotoxic T lymphocytes and natural killer cells in peripheral blood was impaired on postoperative days 1 and 3 but was restored to preoperative values by removal of mononuclear phagocytes from these cells. The decrease in natural killer cell and cytotoxic T lymphocyte activity in peripheral blood may signify a temporary impairment of the effector arm of the cell-mediated immunity in the post-operative period. The observed changes in peripheral blood phenotype and function may be involved in early organ injury and infectious complications after CPB.     

Lysis of autologous tumor cells by peripheral blood lymphocytes treated with interleukin 2 in patients with renal cell carcinoma

Peripheral blood lymphocytes derived from patients with renal cell carcinoma were stimulated with interleukin 2 and tested in a 4-hour 51chromium-release cytotoxicity assay against autologous cultured tumor and myeloid K562 cells. Of 23 patients autologous tumor lysis of more than 0 per cent was observed in 20 (range 5.3 to 82.8 per cent) and more than 10 per cent lysis was noted in 16. Since no significant correlation between the degree of cytotoxicity and the pathological findings of tumor stage, grade, cell type or lymphocyte infiltration in the tumor was found, it was impossible to predict from the pathological findings which tumor could be lysed by peripheral blood lymphocytes treated with interleukin 2. Comparison of natural killer cell activity against K562 of freshly prepared peripheral blood lymphocytes to that of interleukin 2-treated lymphocytes revealed significant augmentation from 23.6 +/- 9.7 to 65.2 +/- 29.1 per cent. From the kinetics study the lysis of autologous tumor cells was detectable on day 2 of interleukin 2 exposure and the peak activity was observed on day 5. A similar trend was noted in regard to K562 cell lysis. Measurements of peripheral blood lymphocyte subsets with monoclonal antibodies and argon ion laser flow cytometry resulted in a significant decrease of cells positive for OKT 4 (helper/inducer) and a significant increase of cells positive for OKT 8 (suppressor/cytotoxic) and Leu 7 (natural killer) by interleukin 2 treatment. A cold target cell inhibition test was performed in 2 patients with unlabeled autologous tumor and K562 cells as cold inhibitors. In 1 patient unlabeled K562 completely inhibited the tumor lysis but in 1 complete inhibition by autologous tumor and incomplete blockade by K562 were found. From this observation we concluded that peripheral blood lymphocytes treated with interleukin 2 lysed not only autologous tumor cells but also K562. Our results demonstrate that adoptive immunotherapy with peripheral blood lymphocytes activated by interleukin 2 in patients with renal cell carcinoma could be appropriate as a therapeutic procedure.     

Membrane antigens detected on human lung carcinoma cells by hybridoma monoclonal antibody

Hybridoma monoclonal antibodies were used to identify tumor cell membrane antigens on a new human lung carcinoma cell line. Hybridomas were constructed by fusing S194 mouse myeloma cells with lymphocytes from Balb/C mice immunized by the human carcinoma cell line UCLA-SO-P3. Of 768 hybridoma cultures tested 40 secreted antibody reacting with these P3 tumor cells by an indirect ¹²⁵I-protein A binding assay. Three produced antibody binding selectively to the P3 tumor cells, but not with a lymphoblastoid cell line from this same cancer patient. These 3 hybridomas were cloned by limiting dilution and antibody subclass was determined. The first monoclonal antibody reacted only with the P3 lung carcinoma and with one colon carcinoma line. The second cross-reacted with most human carcinomas tested including lung, colon, and breast carcinoma, but not with sarcomas, melanomas, embryonic cells, lymphoblastoid cells, or normal lymphocytes. The third reacted strongly with all the tumor cell lines tested except for sarcoma and with embryonic cells but not with lymphoblastoid cells or lymphocytes. These results show that monoclonal antibodies can be produced against a variety of different membrane antigens on this lung carcinoma cell line including several that may prove useful in diagnosis or treatment of human cancer.     

Differential sensitivity of head and neck cancers to non-major histocompatibility-restricted killer cell activity

Cell lines derived from squamous cell carcinoma of the upper aerodigestive tract (head and neck cancer) were phenotypically characterized with regard to differential sensitivity to nonmajor histocompatibility restricted (non-MHCr) killer cell activity. Requirements for detectable lysis of the cell lines in a standard chromium release assay included either isolation of fresh enriched Leu 19+ large granular lymphocytes (both Leu 19+CD3+ and Leu 19+CD3- populations) or interleukin-2 (IL-2) stimulation of peripheral blood lymphocytes (PBL). In neither circumstance could lytic activity be identified among Leu 19- populations. With PBL IL-2 stimulation significant differential sensitivity to lysis expressed by the head and neck cancer cell lines (P less than 0.001 by analysis of variance) was identified and maintained regardless of PBL source, i.e., PBL from healthy controls and three differing populations of head and neck cancer patients categorized by disease status and treatment. One factor associated with a cell line's increased sensitivity was degree of tumor differentiation, poorly differentiated tumors (as defined by intermediate filament cytochemical staining [decreased keratin and increased vimentin]) being more sensitive. Furthermore, as tumor cell lytic sensitivity increased, major histocompatibility complex (MHC)-class I antigen expression diminished concurrently. In 1 of 4 cell lines tested, however, pretreatment of tumor cells with interferon-gamma induced diminished lytic sensitivity independent of changes in MHC-class I expression, indicating factors not related to MHC-class I expression are likewise relevant. In previous studies we defined the in vivo prognostic significance of non-MHCr killer cell cytotoxicity activity against K562 targets, diminished activity being principally predictive of metastatic disease development in persons with poorly differentiated head and neck cancers. This report extends these observations by demonstrating in vitro that poorly differentiated head and neck cancer target cells are highly sensitive to changes in lytic function expressed by Leu 19+ non-MHCr effector cells.     

Fas ligand and TNF-related apoptosis-inducing ligand induction on infiltrating lymphocytes in bladder carcinoma by bacillus Calmette-Guérin treatment

AIM: To determine the molecular mechanisms underlying the efficacy of bacillus Calmette-Guérin (BCG) therapy against superficial carcinoma of the urinary bladder, we evaluated the expression of cytotoxic molecules on tumor-infiltrating lymphocytes before and after therapy. METHODS: Immunofluorescence staining allowed the specific detection of Fas, Fas ligand (FasL), and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) expression on tumor cells and the respective leukocyte populations in biopsy samples from 6 patients. RESULTS: Significant increases in the infiltration of FasL- and TRAIL-expressing CD4+ T cells and macrophages and FasL-expressing CD8+ T and NK cells were observed after BCG instillation in bladder carcinoma. Moreover, Fas expression was upregulated on tumor cells after BCG instillation. CONCLUSION: The data suggested that the enhanced infiltration of FasL- and/or TRAIL-expressing leukocytes (CD4+ T cells, CD8+ T cells, natural killer cells and macrophages) and the induction of Fas expression on tumor cells may play an important role in the therapeutic effect of BCG instillation.     

Depressed levels of granular lymphocytes with natural killer (NK) cell function in 247 cancer patients.

The HNK-1 (Leu-7) monoclonal antibody was used to enumerate and characterize the level of blood granular lymphocytes in 247 cancer patients. The results were compared to 146 control individuals. A fluorescence-activated cell sorter was used to purify blood HNK-1+ cells from cancer patients. The monoclonal antibody identified a homogeneous population of granular lymphocytes with greater than 95% purity. Conversely, virtually 100% of HNK-1- cells from cancer patients were agranular lymphocytes. These results were the same as previously observed in normal individuals, where the HNK-1+ cell fraction contained all the lymphocytes with spontaneous cytotoxicity in natural killer (NK) and killer (K) cell assays. The level of HNK-1+ cells in cancer patients correlated significantly with the patient's age and sex, with older individuals having higher levels and male patients containing a higher proportion than female patients. The levels in the cancer patients were significantly lower than normal controls (p = 0.04). When the results were subdivided by the histologic type of cancer, additional differences were noted. Compared to age and sex-matched controls, significantly depressed levels of HNK-1+ granular lymphocytes were observed in 49 patients with colon cancer (9.7% vs. 15.8%, p = 0.0001), 18 patients with lung carcinoma (11.7% vs. 27.0%, p = 0.0001), 24 patients with breast carcinoma (12.0% vs. 15.5%, p = 0.04) and 64 patients with head and neck carcinoma (15.9% vs. 19.1%, p = 0.05). However, there were no significant differences overall in the average HNK-1+ cell level of 66 patients with melanoma (13.0% vs. 13.5%, p = 0.75) and nine patients with sarcomas (15.8% vs. 14.3%, p = 0.71). Thus, this important subpopulation of granular lymphocytes with NK and K cell function was significantly depressed in most cancer patients. Accounting for the patient's age and sex and the histologic type of cancer was critical to interpreting the results.     

Cytotoxicity of lymphocytes sensitized by autologous tumor cells in vitro, against autologous lung cancer cell lines

In order to obtain killer lymphocytes that would have a strong cytotoxicity against autologous tumor cells, we conducted a cytotoxic assay of effector cells cultured from human peripheral blood lymphocytes (PBL) using 10 cultured lung cancer cell lines as target cells. We present this result. (1) Lymphocytes obtained by 17 days mixed lymphocyte tumor cell culture (MLTC: Fresh PBL from lung cancer patients was mixed with irradiated autologous tumor cells in a medium, and cultured for 3 days. Irradiated allogeneic lymphocytes were then added, and cultured for 14 days) were cytotoxic against 4 of the 10 autologous lung cancer cell lines. (2) Lymphocytes obtained by 14 days MLTC and 3 more days culture in a medium containing interleukin 2 were cytotoxic against all 10 of the autologous lung cancer cell lines. These lymphocytes proliferated to 4.13 times of the original cell number, and their surface marker was OKT3+. These killer lymphocytes are expected to be effective in adoptive immunotherapy as effector cells.     

人肝癌浸润淋巴细胞的分离培养及其CD8阳性亚群特性研究

目的从原发性肝癌（HCC）患者术后的肿瘤组织中提取、培养肿瘤浸润性淋巴细胞（TILs），进一步分选、扩增其CD8阳性亚群（CD8+TILs），并研究其特性。 方法标本取自38例肝癌根治术后的肿瘤组织，免疫组织化学染色观察癌巢组织和癌旁组织TILs的分布，通过机械剪碎、混合酶消化和不连续密度梯度离心法分离提取TIL前体细胞。采用重组人白介素2进行激活，抗CD3、抗CD28抗体进行共刺激扩增，用磁珠分选出CD8+TILs，CCK8法观察CD8+TILs对肝癌细胞Hep3B、HepG2生长的影响。 结果TILs富集于癌旁组织，12例成功提取TILs，6例培养并实现大量扩增，扩增17~50倍后细胞数为（1.2~2.5）×10⁸个，CD3+细胞比例为（77.53±16.37）%，CD3+CD4+比例为（27.08±21.56）%，CD3+CD8+比例（44.55±12.73）%，细胞活力为（90.5±3.0）%，CD8+TILs对肿瘤细胞系Hep3B、HepG2有较强的生长抑制作用。 结论本研究成功建立高效提取、培养CD8+TILs的方法，获得具有高抗肿瘤活性的CD8+TILs，为晚期肝癌等实体瘤的过继免疫治疗提供研究基础。     

Expansion of CD3+CD56+ lymphocytes correlates with induction of cytotoxicity by interleukin-2 gene transfer in human breast tumor cultures

Background: Mice immunized with murine mammary carcinoma cells genetically engineered to secrete interleukin-2 (IL-2) are rendered resistant to subsequent challenge with unmodified tumor cells, and in the case of mice bearing established tumors, the rate of development of pulmonary metastases is reduced. Despite these encouraging animal results, little is known about the induction of antitumor immunity by IL-2 gene transfer in human breast cancer. Methods: Adenovirally mediated IL-2 gene transfer was performed in 12 tumor fragment cultures established from seven primary breast cancers. Autologous tumor infiltrating lymphocytes (TILs) or peripheral blood mononuclear cells (PBMCs) were cocultured with transduced tumor fragments, and changes in phenotype and cytotoxicity were measured. Results: IL-2 was never detectable in the untransduced cultures, but it peaked at 5.0—1,324.8 ng/ml in the transduced cultures. Lymphocyte counts declined in all untransduced cultures, but they increased two- to sevenfold in four transduced cultures. CD4:CD8 ratios decreased from a mean of 2.11 at baseline to 1.27 after stimulation in coculture (p=0.03). Expansion of lymphocytes expressing the natural killer cell phenotype (CD3⁻CD56⁺) occurred in only one culture, but the CD3⁺CD56⁺ population increased in four of six cultures. Lymphocytes from four of 10 cocultures generated significant cytotoxicity against allogeneic breast cancer cells. Induction of cytotoxicity correlated with expansion of the CD3⁺CD56⁺ phenotype (R²=0.805, p=0.02). Conclusions: IL-2 gene expression by human breast cancer causes expansion of CD3⁺CD56⁺ cytotoxic lymphocytes. This phenotype is consistent with that of a non-major histocompatibility complex (MHC)-restricted cytokine induced killer cell population previously described. Opinions, interpretations, conclusions and recommendations are those of the author and are not necessarily endorsed by the U.S. Army. Presented at the 49th Annual Cancer Symposium of The Society of Surgical Oncology, Atlanta, Georgia, March 21–24, 1996.     

Human accessory cells activate fresh, normal, tumor-distant T lymphocytes but not tumor-infiltrating T lymphocytes to lyse autologous tumor cells in a primary cytotoxic T lymphocyte assay in renal cell carcinoma.

OBJECTIVES: Search for an ideal responder T-lymphocyte source for adoptive T-lymphocyte therapy in renal cell carcinoma (RCC). METHODS: Cytotoxic T-lymphocyte (CTL) activity of (a) normal, tumor-distant, renal T lymphocytes, (b) tumor-infiltrating T lymphocytes and (c) peripheral blood T lymphocytes against autologous tumor epithelial cells (EC) of 10 patients with organ-confined, primary RCC was analyzed in a primary CTL assay. Freshly enriched T lymphocytes were cultured with or without autologous, mitomycin-C-treated normal or tumor EC in the presence or absence of antigen-presenting cells (APC) for 7 days. RESULTS: Both tissue T-lymphocyte populations displayed a similar CD4:CD8 ratio (1:1). Elevated CD62L coexpression of CD4+ T lymphocytes in normal, tumor-distant, renal tissue resulted in a significantly higher transient T-cell activation than that seen in renal tumor tissue (46 vs. 27%; p = 0.002). All trials to induce significant lysis of autologous, renal tumor EC in tumor-infiltrating and peripheral blood T lymphocytes failed. Only when normal, tumor-distant, renal T lymphocytes were stimulated by autologous APC and tumor EC was significant autologous tumor EC lysis obtained (mean 14%; p<0.05). Costimulation by anti-CD3 (mean 21%; p<0.05) or interleukin-2 (mean 31%; p<0.05) further increased tumor EC lysis significantly. CONCLUSIONS: Increased turnover of T lymphocytes in normal, tumor-distant, renal tissue was associated with a higher yield of pre-CTL which can be transformed into a functionally active effector T-cell pool by stimulation via antigen plus APC. Thus, tumor-distant renal tissue has to be included in the tissue-sampling procedure for adoptive immunotherapy.     

T-cell responses during pig-to-primate xenotransplantation

Xenotransplantation using porcine organs may resolve a chronic shortage of donor organs for clinical transplantation if significant immunological barriers can be overcome. To determine the potential role of T lymphocytes in Xenograft (Xg) rejection, we transplanted transgenic hCD46 porcine hearts heterotopically into baboon recipients. METHODS: Recipients were treated to deplete anti-Gal antibody with a non-antigenic alpha-Gal polyethylene glycol polymer (TPC) (n = 2), TPC plus rituximab (anti-CD20) (n = 1) or were untreated (n = 1). None of the recipients received T-cell immunosuppression. RESULTS: All Xgs failed within 7 days and showed evidence of a mixed humoral and cellular rejection process. Cellular infiltration consisting primarily of CD4+ T cells and few CD8+ T cells. Proliferation and cytotoxicity assays showed sensitization of CD4+ and CD8+ T cells that reacted with porcine IFN-gamma (pIFN-gamma)-stimulated porcine aortic endothelial cells (PAEC). The CD4+ lymphocytes displayed greater cytotoxicity than CD8+ cells. An increased frequency of PAEC-specific interleukin (IL) 2 and IFN-gamma-secreting T cells was observed, suggesting a Th1 cytokine bias. An increase in the percentage of circulating CD4+CD28- cells was observed at the time of rejection and over 50% of the CD4+ cells recovered from residual pig tissue at necropsy lacked CD28 expression. CONCLUSIONS: These findings show that lymphocytes are efficiently stimulated by PAEC antigens and can mediate direct tissue destruction. These studies (1) provide an insight into the potential of cellular-mediated cardiac Xg rejection, (2) show for the first time the induction of cytotoxic pig-specific CD4+CD28- lymphocytes and (3) provide a rational basis for determining different modes of immunosuppression to treat Xg rejection.