Clonal analysis of tumor-infiltrating lymphocytes from human primary and metastatic liver tumors

Phenotypic and functional characteristics of tumor-infiltrating lymphocytes (TIL) obtained from human primary and metastatic liver tumors were studied. Lymphocytes isolated from 18 tumors and autologous (A) peripheral blood (6 cases) were phenotyped by 2-color flow cytometry and cloned in a limiting dilution system, which allows virtually all normal T lymphocytes to proliferate; 70-80% of fresh TIL were T cells (i.e., CD3+), and the ratio of CD4+/CD8+ cells was 1.2 in both primary and metastatic liver tumors. TIL contained significantly more CD56+ (NKHI+) cells, half of which were CD3+CD56+, CD3+CD25+ cells and CD3+HLA-DR+ cells, than A-PBL. The frequencies of proliferating T-cell precursors (PTL-p) and cytolytic T-lymphocyte precursors (CTL-p) reactive with K562, allogeneic tumor cells and autologous tumor cells, were determined. Mean PTL-p frequencies for TIL from hepatocellular carcinomas, cholangiocarcinomas and metastatic liver tumors were 0.52 (0.22-0.83), 0.10 (0.05-0.16) and 0.16 (0.01-0.30), respectively. The frequency of CTL-p with natural-killer-like activity was lower in TIL than in A-PBL. The frequency of CTL-p for autologous tumor cells in fresh TIL isolated from primary liver tumors was 0.02-0.13 and 12/81 clones were reactive against autologous tumor. In contrast, only 1/66 TIL clones obtained from colon carcinomas metastatic to liver showed autotumor reactivity. No clones reactive with autologous tumor were obtained from peripheral blood of patients with liver cancer. These data indicate that substantial differences in anti-tumor functions of TIL between primary and metastatic liver tumors exist, which can be detected at a clonal level.     

Gamma-delta T-cells in patients with squamous cell carcinoma of the head and neck

In our attempt to characterize a general immune-suppression found in patients with squamous cell carcinoma of the head and neck (SCCHN) we now focused on a subset of CD3 lymphocytes described as gamma/delta-T-cells, a cell type with potential relevance in non-MHC restricted anti-tumor immune responses. Peripheral blood of 33 SCCHN patients and 33 age-matched controls (CON) was evaluated for the frequency of gamma/delta-T-cells among CD3+ T-cells and their onset of apoptosis (Annexin V binding) by multicolor flow cytometry. Results were correlated with clinical parameters. Patients with SCCHN had a significantly higher proportion of gamma/delta-T-cells compared to healthy controls (4.4+/-0.4% for SCCHN vs. 3.0+/-0.3% for CON, p=0.01). However, this increase was not paralleled with a difference in the onset of apoptosis if compared to CON. There was also no correlation between the proportion of gamma/delta-T-cells and tumor stage. However, a significantly higher proportion of gamma/delta-T-cells was found in patients with recurrent or metachronous second primary SCCHN (6.0+/-1.0%) if compared to the other SCCHN (3.8+/-0.4%, p=0.02). In a follow up 3-6 months post-treatment patients showed a decrease of gamma/delta-T-cells among CD3+cells (2.7+/-0.4%, n=4) if they were operated only and an increase if primary radio-chemotherapy (6.7+/-1.7%, n=8) or a combination of operation plus radio-chemotherapy (6.8+/-2.3%, n=3) was applied. Furthermore, patients receiving palliative treatment including radio-chemotherapy had highest values of gamma/delta-T-cells (9.1+/-2.7%, n=4) overall implicating that the treatment modality significantly influences the proportion of gamma/delta-T-cells. Since patients with SCCHN, particularly those with recurrent or second primary disease after treatment, had a higher proportion of gamma/delta-T-cells without signs of a reduced onset of apoptosis this could be due to an increased de novo generation. The current study implies that increased frequencies of gamma/delta-T-cells in patients with SCCHN may not only be the result of tumor-host interactions but the consequence of applied treatment modalities.     

Tumor-reactive T-cells accumulate in lung cancer tissues but fail to respond due to tumor cell-derived factor.

The purpose of this study was to elucidate a possible immune response to tumor cells mediated by tumor-infiltrating lymphocytes (TIL) in lung cancer. In flow cytometry, the majority of T-cells of TIL were CD45RA-, CD45RO+, and CDw29high, and expressed HLA-DR. The expression of interleukin 2 receptor beta chain increased in both CD4+ and CD8+ TIL compared with both types of T-cells in peripheral blood. These results indicate that the major population of TIL is activated memory T-cells. The TIL preparation, which was usually contaminated with 5 to 10% tumor cells, did not exhibit any response in autologous mixed lymphocyte-tumor culture even in the presence of interleukin 2 (IL-2) in all five cases tested. Although purified T-cells from TIL showed the positive response in only 1 of 10 cases tested without addition of IL-2, it occurred in 7 of 10 cases in the addition of a low concentration of IL-2. The IL-2-dependent response to irradiated autologous tumor cells was suppressed when nonirradiated autologous tumor cells were added to the culture. Culture supernatants of four lung cancer cell lines and freshly prepared lung cancer cells obtained from 6 cases exhibited suppressive activity against anti-CD3 antibody-induced mitogenesis of peripheral blood mononuclear cells from healthy donors. We suggest that, taken together, (a) the major population of TIL in lung cancer are activated memory T-cells, and they include tumor-reactive ones, and that (b) the function of the TIL is impaired by unavailability of IL-2 and/or by suppression due to lung cancer cell-derived factor(s).     

Characterization of tumor infiltrating lymphocytes in paired primary and metastatic renal cell carcinoma specimens

Renal cell carcinoma (RCC) is one of the most chemo- and radio-resistant malignancies, with poor associated patient survival if the disease metastasizes. With recent advances in immunotherapy, particularly with PD-1/PD-L1 blockade, outcomes are improving, but a substantial subset of patients does not respond to the new agents. Identifying such patients and improving the therapeutic ratio has been a challenge, although much effort has been made to study PD-1/PD-L1 status in pre-treatment tumor. However, tumor infiltrating lymphocyte (TIL) content might also be predictive of response, and our goal was to characterize TIL content and PD-L1 expression in RCC tumors from various anatomic sites. Utilizing a quantitative immunofluorescence technique, TIL subsets were examined in matched primary and metastatic specimens. In metastatic specimens, we found an association between low CD8+ to Foxp3+ T-cell ratios and high levels of PD-L1. High PD-L1-expressing metastases were also found to be associated with tumors that were high in both CD4+ and Foxp3+ T-cell content. Taken together these results provide the basis for combining agents that target the PD-1/PD-L1 pathway with agonist of immune activation, particularly in treating RCC metastases with unfavorable tumor characteristics and microenvironment. In addition, CD8+ TIL density and CD8:Foxp3 T-cell ratio were higher in primary than metastatic specimens, supporting the need to assess distant sites for predictive biomarkers when treating disseminated disease.     

Tumor infiltrating lymphocytes and PD-L1 expression in brain metastases of small cell lung cancer (SCLC)

Brain metastases (BM) are frequent in small cell lung cancer (SCLC). Novel insights into their pathobiology are needed for development of better therapies. We investigated tumor-infiltrating lymphocyte (TIL) subsets (CD3+, CD8+, CD45RO+, FOXP3+ and PD-1+) and expression of PD-L1 in a series of 32 SCLC BM specimens and four matched primary tumor specimens using immunohistochemistry. 30/32 (93.8?%) BM specimens showed TIL infiltration. CD3+ TILs were observed in 30/32 (93.8?%) BM specimens, CD8+ TILs in 25/32 (78.1?%), CD45RO+ TILs in 15/32 (46.9?%), FOXP3+ TILs in 15/32 (46.9?%) and PD-1+ TILs in 1/32 (3.1?%) BM specimens. Patients with infiltration of CD45RO+ TILS had a significantly longer median survival time (11 months; 95?% CI 0.000–26.148) as compared to patients without the presence of CD45RO+ TILs (5 months; 95?% CI 0.966–9.034; p?=?0.007; log rank test). Membranous PD-L1 on tumor cells was observed in 24/32 (75.0?%) BM specimens, with 11/32 (34.4?%) cases showing PD-L1 expression in over 5?% of viable BM tumor cells. PD-L1 expression on TILs was seen in 8/32 (25.0?%) and on tumor infiltrating macrophages in 9/32 (28.1?%) cases. Patients with PD-L1 expression on TILs presented with improved survival prognosis (6 versus 29 months; p?=?0.002; log rank test). Among matched primary tumors, all (4/4; 100?%) specimens showed TIL infiltration, while PD-L1 expression found in only 1/4 (25.0?%) specimen. TIL infiltration and PD-L1 expression are commonly found in SCLC BM and presence of CD45RO+ memory T-cells and PD-L1+ TILs in SCLC BM seem to associate with favorable survival times. Our data suggest an active immune microenvironment in SCLC BM that may be targetable by immune-modulating drugs.     

Phenotypic analysis of tumor-infiltrating lymphocytes from human breast cancer.

Suspensions of fresh tumor-infiltrating lymphocytes (TIL) were prepared from 30 human breast ductal adenocarcinomas. To evaluate the phenotypic pattern of the isolated TIL, lymphocyte surface markers including CD19, CD3, CD4, CD8, CD16 and HLA-DR were examined by flow cytometry. Lymphocyte recovery ranged from 1.1% to 44%, independent of tumor size. TIL most often scored high for CD3+ with a varying number of CD4+ and CD8+ cells. Three samples out of 30 expressed up to 44% of CD19+ B cells, while CD3-CD16+ NK cells were rare. CD4 and CD8 expression was significantly different between the lymph node metastases group and the lymph node negative group (p < 0.01). 67% of the TIL with a CD4/CD8 ratio greater than 1 showed lymph node metastases. Furthermore, the CD4 expression of TIL and CD4/CD8 ratio correlated with tumor size (p < 0.01), but not with tumor differentiation and hormone receptor expression. Although there was considerable diversity of TIL among breast tumors, our data suggest that a high expression of CD4+ T cells may imply progression of the tumor, and an increased CD4/CD8 ratio of the TIL isolated from human breast adenocarcinoma may indicate development of metastases.     

Human melanoma-mediated inhibition of autologous CD4+ helper tumor-infiltrating lymphocyte growth in vitro.

Tumor-infiltrating lymphocytes (TIL) were isolated from a human melanoma metastatic to the abdomen. The TIL were 99% CD3+ and 99% CD4+ and CD8-. They were dependent on interleukin-2 (IL-2) for growth, as measured in a thymidine uptake assay, and were not cytotoxic to autologous or allogeneic melanoma or K562. When co-cultured with irradiated autologous tumor cells, or tumor cell supernatants, the TIL not only did not respond, but the IL-2-dependent growth was inhibited significantly. Inhibition occurred during the first 24 hours of co-culture and persisted as long as the tumor was present. After being washed free of inhibitory tumor cells, the TIL again were able to grow in the presence of IL-2, indicating that the inhibition was not caused by irreversible toxicity mediated by the tumor. Addition of excess IL-2 did not reverse the inhibitory effect, but addition of indomethacin, an inhibitor of cyclooxygenase and prostaglandin synthesis, partially blocked the inhibition. These data show melanoma-mediated inhibition of induction and expansion of human T-cells in vitro, which may reflect one of the mechanisms of inhibition of cellular responses in vivo. These results stress the need to examine the techniques for optimal in vitro expansion of tumor-specific TIL or cytotoxic T-cells for adoptive immunotherapy.     

Clinical results and characterization of tumor-infiltrating lymphocytes with or without recombinant interleukin 2 in human metastatic renal cell carcinoma

A Phase I trial of tumor-infiltrating lymphocytes (TIL) expanded in vitro and administered on Days 1 and 8, with or without continuous infusion recombinant interleukin 2 (rIL-2) in 25 patients with metastatic renal cell carcinoma, was conducted. Eighteen of the 25 eligible patients were treated with TIL and escalating doses of rIL-2 (0.0, 3.0, 4.5 x 10(6) units/m2) on Days 1 to 5 and 8 to 12. Dose-limiting toxicity was pulmonary, and the maximum tolerated dose of rIL-2 was 3.0 x 10(6) units/m2. No clinical responses were observed. Immunological monitoring of peripheral blood lymphocytes demonstrated significant increases in CD3+ and CD56+ cells, including the activated T-cell subsets. Phenotypic analysis of cultured TILs demonstrated significant heterogeneity and the presence of CD3+CD4+ and CD3+CD8+ T-cells, with CD3-CD56+ and CD3+CD56+ populations also present. The majority of cultured TILs expressed HLA-DR and CD45RO, with a variable number expressing CD25. The rIL-2-expanded TILs possessed cytotoxicity against allogeneic and autologous tumor, with cytolytic activity against only autologous tumor seen in one patient. Results demonstrate that in vitro expansion of TILs is possible, but further studies are needed to define the biology of TILs in renal cancer and to isolate and expand tumor-specific T-cells.     

Localization and density of immune cells in the invasive margin of human colorectal cancer liver metastases are prognostic for response to chemotherapy

Analysis of tumor-infiltrating lymphocytes (TIL) in primary human colorectal cancer (CRC) by in situ immunohistochemical staining supports the hypothesis that the adaptive immune response influences the course of human CRC. Specifically, high densities of TILs in the primary tumor are associated with good prognosis independent of other prognostic markers. However, the prognostic role of TILs in metastatic CRC lesions is unknown, as is their role in response or resistance to conventional chemotherapy. We analyzed the association of TIL densities at the invasive margin of CRC liver metastases with response to chemotherapy and progression-free survival in a set of 101 large section samples. High-resolution automated microscopy on complete tissue sections was used to objectively generate cell densities for CD3, CD8, granzyme B, or FOXP3 positive immune cells. A predictive scoring system using TIL densities was developed in a training set and tested successfully in an independent validation set. TIL densities at the invasive margin of liver metastases allowed the prediction of response to chemotherapy with a sensitivity of 79% and specificity of 100%. The association of high density values with longer progression-free survival under chemotherapy was statistically significant. Overall, these findings extend the impact of the local immune response on the clinical course from the primary tumor also to metastatic lesions. Because detailed quantification of TILs in metastatic lesions revealed a strong association with chemotherapy efficacy and prognosis, we suggest that the developed scoring system may be used as a predictive tool for response to chemotherapy in metastatic CRC.     

Immunization with haptenized, autologous tumor cells induces inflammation of human melanoma metastases

Twenty-four patients with metastatic melanoma were treated with a novel form of active immunotherapy, autologous tumor cell vaccine conjugated to the hapten, dinitrophenyl. This approach is based on the idea, well established in animal systems, that presentation of tumor antigens in the context of a strongly immunogenic hapten augments the development of immunity to those antigens. After being sensitized to dinitrophenyl, patients were given injections of dinitrophenyl-vaccine every 28 days following pretreatment with low dose cyclophosphamide. The vaccine induced a striking inflammatory response in superficial metastases in 14 of 24 patients, consisting of erythema, swelling, warmth, and tenderness over tumor masses. Immunohistochemistry and flow cytometric analysis of biopsy specimens showed marked infiltration with lymphocytes, the majority of which were CD8+, HLA-DR+ T-cells. These observations suggest that a T-cell-mediated immune response against melanoma-associated antigens was facilitated by the "helper" effect of the anti-hapten response.     

Phenotyping of human tumor-infiltrating lymphocytes before and after exposure to different in vitro stimulation conditions.

Tumor-infiltrating lymphocytes (TiL) and autologous peripheral blood lymphocytes (PBL), mainly from breast and kidney tumor patients were cultivated with high- and low-dose rIL-2 under addition of autologous tumor cells or nonmalignant epithelial cells. Tumor cells added to TIL-cultures induced an additional proliferative response. Before cultivation, CD8+ and CD25+ lymphocytes were more frequent in TIL when compared to PBL, while CD16+, CD19+ and CD56+ cells were rare. After culture, lymphocytes of both origins showed an increase of CD2+, CD25+, CD56+ and HLA-DR+ cells and a relative decrease of CD3+ cells. The CD4+/CD8+ ratios and a number of CD56+ cells were rIL-2 dose dependent.     

Prognostic value of the density of tumor-infiltrating lymphocytes in colorectal cancer liver metastases

Tumor-infiltrating lymphocytes (TILs) have been reported to reflect the anti-tumor immune status of patients and to be correlated with their prognosis and therapeutic outcomes. However, the characteristics of the local immune status in metastatic tumors is poorly understood, as primary tumors have been the focus in most previous studies. In addition, the local immune status may be influenced by preoperative chemotherapy. The present study aimed therefore to investigate the relationship between the degree of TIL infiltration and the prognosis in patients with curative resection of colorectal cancer liver metastases and to examine the effects of preoperative chemotherapy on the function of immune cells. A total of 108 patients who underwent curative resection of colorectal cancer liver metastases in our department between May 1996 and January 2017 were enrolled in the present study. Peripheral blood samples were obtained within two weeks before surgery. TIL infiltration was evaluated by immunohistochemical staining of surgically resected specimens of liver metastases using anti-CD8/CD3 antibodies. The mean number of TILs in five different fields was calculated, and patients were classified into a high-TIL group and a low-TIL group. Furthermore, patients were divided into three groups as follows: i) A group of patients who did not receive preoperative chemotherapy; ii) a group of patients who received short-term preoperative chemotherapy for <6 months; and iii) a group of patients who received long-term preoperative chemotherapy for ≥6 months. The results demonstrated that the density of TILs in colorectal liver metastases was not correlated with the absolute peripheral lymphocyte count in all patients. Furthermore, the degree of CD8⁺TIL infiltration in liver metastases was significantly lower in the recurrence group compared with the recurrence-free group following hepatectomy. In all patients with colorectal liver metastases, the degree of CD8⁺TIL infiltration was significantly associated with the relapse-free and overall survival. In patients without preoperative chemotherapy, the degree of CD8⁺TIL infiltration was significantly associated with the relapse-free survival, and a high CD8⁺TIL presence tended to have a better effect on the overall survival than a low CD8⁺TIL presence. In the short-term chemotherapy group, the degree of CD8⁺TIL infiltration was significantly associated with the relapse-free and overall survival. In the long-term chemotherapy group, there were no significant differences between the high- and low- CD8⁺TIL groups in the relapse-free and overall survival. In contrast to CD8⁺TILs, CD3⁺TILs showed a poor prognostic ability. In summary, the degree of CD8⁺TIL infiltration in colorectal cancer liver metastases may be correlated with patient prognosis. However, in patients who received long-term chemotherapy before surgery, the degree of TIL infiltration was not necessarily associated with prognosis as the anti-tumor effects of TILs may decrease. The degree of CD8⁺TIL infiltration may therefore be considered as a useful prognostic factor in patients with colorectal liver metastases, but the prognostic accuracy may decrease in patients who received long-term chemotherapy.     

Cytotoxic potential despite impaired activation pathways in T lymphocytes infiltrating nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is an epithelial tumor consistently associated with EBV. The histological picture is characterized by a strikingly abundant lymphocytic infiltrate. Furthermore, the epithelial tumor cells present several immunological characteristics which suggest an important role for tumor-infiltrating lymphocytes (TIL) in the biology of this tumor. The present study reports the phenotypic and functional characterization of TIL from NPC obtained after enzymatic digestion of 15 NPC biopsies. Flow cytometric analysis of TIL suspensions indicated that most TIL were mature CD3+ T lymphocytes (mean = 60%) with a variable CD4/CD8 ratio. Most TIL were TCR alpha/beta-positive (mean = 55%) and only a few TCR gamma-delta-positive cells could be identified. A small percentage (mean = 9%) displayed an activated phenotype (CD25+, HLA class II+). Using limiting dilution analysis, we found that the average frequency of proliferative T-lymphocyte precursors (PTL-P) is lower among TIL (1/40) than in autologous (1/7) or normal PBL (1/1.4). Moreover, sorting experiments have shown that this defect is significantly more pronounced in the CD8+ than in the CD4+ TIL subset. Accordingly, the TCR and the CD2-mediated antigen-independent pathways of activation were impaired. Different types of cytotoxic precursor could be detected. These included lectin-dependent cell cytotoxicity (LDCC) and NK-like or lymphokine-activated killer (LAK) activity. Interestingly, some TIL from NPC were able to lyse an NPC tumor (C15) maintained in nude mice. Thus, despite impaired activation pathways, the cytolytic potential of proliferating TIL in NPC is preserved.     

Effect of anti-CD3 antibody on the generation of interleukin-2-activated lymphocytes from tumor tissues of gastrointestinal cancer.

BACKGROUND. The efficiency of anti-CD3 antibody (OKT3) for adoptive immunotherapy using lymphokine-activated killer (LAK) cells generated from tumor-infiltrating lymphocytes (TIL), regional lymph node lymphocytes (RLNL), and peripheral blood lymphocytes (PBL) was investigated. METHODS. TIL, RLNL, and PBL derived from 39 patients with gastrointestinal cancers (16 gastric cancers, 17 colorectal cancers, and 6 esophageal cancers) were cultured for 4 weeks with 200 U/ml of recombinant interleukin-2. To one group, solid-phase 10 micrograms/ml OKT3 was added during the initial culture period (day 2 or 4). Cytotoxicity against K562 cells (NK-like activity) and Daudi cells (LAK activity) and the phenotypes of effector cells generated after culturing for 2-3 weeks were studied. RESULTS. Proliferative responses were significantly increased by OKT3 in each type of effector cell (P less than 0.01); in particular, TIL expanded more by OKT3 than PBL and RLNL (P less than 0.01). The population of CD8+ CD11b- cytotoxic T-cells in OKT3-stimulated groups was significantly larger than that in unstimulated groups (P less than 0.01), whereas no differences were observed with CD4+ cells (helper/inducer T-cells) and CD8+ CD11b+ cells (suppressor T-cells). OKT3 enhanced the NK-like activity of TIL and PBL but did not affect their LAK activity. OKT3 suppressed the NK and LAK activity of RLNL. CONCLUSIONS. OKT3 stimulation did not significantly enhance the LAK activity, but the authors propose that OKT3 could be an effective addition to adoptive immunotherapy using TIL due to an increased proliferation and generation of a large cytotoxic T-cell population.     

Effect of low dose cyclophosphamide on the immune system of cancer patients: depletion of CD4+, 2H4+ suppressor-inducer T-cells

We studied peripheral blood lymphocytes (PBL) from 42 patients with metastatic melanoma undergoing treatment with cyclophosphamide (CY) plus melanoma vaccine to determine whether CY immunopotentiation could be related to depletion of T-cells that function as inducers of suppression. Every 28 days, the patients were given CY, 300 mg/m2 i.v., followed 3 days later by the intradermal injection of autologous, irradiated melanoma cells mixed with Bacillus Calmette-Guérin. PBL were separated by density gradient centrifugation and cryopreserved until needed for testing. They were stained with monoclonal antibodies directly conjugated to fluorescein isothiocyanate or phycoerythrin and analyzed by two-color flow cytometry. At no time after the initiation of CY plus vaccine were there any significant changes in the percentages of helper-inducer T-cells (CD4+), suppressor-cytotoxic T-cells (CD8+), or the subpopulation of CD8+ cells expressing Leu 15, a marker for suppressor cells. Treatment of melanoma patients with CY plus vaccine resulted in a progressive fall in the proportion of CD4+ T-cells expressing the 2H4 (CD45) antigen, which identifies inducers of suppression. The reduction of CD4+, 2H4+ T-cells did not become apparent until day 28 after the first dose of CY and reached statistical significance only on days 49 (21 days after the second dose) and 105 (21 days after the fourth dose) (mean changes +/- SE: day 49, -5.4 +/- 1.4%, P less than 0.01; day 105, -9.1 +/- 2.2%, P less than 0.01; t test for nonindependent samples). In contrast, the proportion of CD4+ T-cells expressing the antigen 4B4 (CDw29), which are true helper cells, increased slightly, although not significantly, following the institution of CY plus vaccine (mean changes: day 49, +2.9 +/- 2.1%; day 105, +3.6 +/- 2.4%). Similar results were obtained when absolute numbers of circulating cells, rather than percentages, were analyzed. Thus the number of CD4+, 2H4+ T-cells fell from a mean of 395,000/ml on day 0 to 309,000/ml on day 49 (P less than 0.01) to 256,000/ml on day 105 (P less than 0.05). The absolute number of CD4+, 4B4+ cells remained unchanged at the same time points. These changes were not due to progression of metastatic disease, since a comparison of patients with progressive metastases with those who were rendered disease free by surgery showed no significant differences in the reduction of the percentage of CD4+, 2H4+ T-cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Depletion of T-cells with the CD4+CD45R+ phenotype in lymphocytes that infiltrate subcutaneous metastases of human melanoma

We studied the phenotypes of lymphocytes extracted from 22 specimens of human melanoma, 11 s.c. metastases and 11 lymph node metastases, by two-color flow cytometry. Lymphocytes extracted from s.c. metastases were characterized by a significantly reduced ratio of CD4+ to CD8+ T-cells, as compared with peripheral blood lymphocytes from the same patients. Ten of 11 tumor-infiltrating lymphocytes from s.c. metastases, but only 1 of 11 peripheral blood lymphocytes, had a CD4/CD8 ratio of less than 1.0. This alteration was not observed for lymphocytes obtained from nodal metastases. Furthermore, almost all of the CD4+ T-cells in s.c. metastases expressed the antigen CD29w and were negative for the complementary antigen CD45R. In contrast, the CD29w/CD45R ratio of tumor-infiltrating lymphocytes from lymph node metastases was similar to that of matched peripheral blood lymphocytes. Thus tumor-infiltrating lymphocytes from s.c. metastases have the phenotype associated with true helper or antigen-committed T-cells, which could reflect their sensitization to tumor antigens, while tumor-infiltrating lymphocytes from lymph node metastases may represent merely an expanded residua of lymph node lymphocytes. Since tumor-infiltrating lymphocytes expanded in vitro are being tested as therapy for patients with advanced cancer, these observations may have important therapeutic implications.     

MHC class I-restricted recognition of a melanoma antigen by a human CD4+ tumor infiltrating lymphocyte

It is generally considered that MHC class I-restricted antigens are recognized by CD8+ T cells, whereas MHC class II-restricted antigens are recognized by CD4+ T cells. In the present study, we report an MHC class I-restricted CD4+ T cell isolated from the tumor infiltrating lymphocytes (TILs) of a patient with metastatic melanoma. TIL 1383 I recognized HLA-A2+ melanoma cell lines but not autologous transformed B cells or fibroblasts. The antigen recognized by TIL 1383 I was tyrosinase, and the epitope was the 368-376 peptide. Antibody blocking assays confirmed that TIL 1383 I was MHC class I restricted, and the CD4 and CD8 coreceptors did not contribute significantly to antigen recognition. TIL 1383 I was weakly cytolytic and secreted cytokines in a pattern consistent with it being a Th1 cell. The avidity of TIL 1383 I for peptide pulsed targets is 10-100-fold lower than most melanoma-reactive CD8+ T cell clones. These CD4+ T cells may represent a relatively rare population of T cells that express a T-cell receptor capable of cross-reacting with an MHC class I/peptide complex with sufficient affinity to allow triggering in the absence of the CD4 coreceptor.     

Identification of DR9-restricted XAGE antigen on lung adenocarcinoma recognized by autologous CD4 T-cells

We previously demonstrated a dominant IgG response against XAGE-1b antigen in a lung cancer patient by serological analysis of antigens by recombinant expression cloning (SEREX) analysis using a cDNA library from the autologous OU-LU-6 tumor cell line. In this study, we investigated recognition of XAGE-1b on OU-LU-6 tumor by the patient CD4-expressing tumor-infiltrating lymphocytes (CD4 TIL). The response of CD4 TIL obtained from malignant pleural effusion was determined against autologous OU-LU-6 tumor cells and XAGE-1b mRNA-transfected PHA-stimulated CD4 T-cells (T-APC) from healthy individuals sharing HLA-DR with the patient by performing IFNgamma secretion and ELISPOT assays. The patient CD4 TIL recognized OU-LU-6 in an HLA-DR-restricted manner and XAGE-1b mRNA-transfected T-APC derived from DRB1 *0901-sharing healthy donor (HD)1 and HD2, but not DRB1 *1101-sharing HD3 or HD4. Epitope analysis using 17 18-mer peptides with 12 overlapping amino acids revealed that the CD4 TIL recognized XAGE-1b 33-49. The findings suggest that the patient CD4 T-cells recognized the XAGE-1b 33-49-related epitope on autologous OU-LU-6 tumor cells in a manner restricted by DR *0901.     

MHC-dependent cytolysis of autologous tumor cells by lymphocytes infiltrating urothelial carcinomas

Tumor-infiltrating lymphocytes (TIL) were grown from 23 urothelial carcinomas. Phenotyping analysis showed that the TIL cultures were mainly CD3⁺. Although CD4⁺ and CD8⁺ T-cell sub-sets were grown in culture, CD4⁺ T-cell sub-sets predominated over CD8⁺ T cells. Immunohistochemical studies performed on 5 tumor specimens confirmed this observation, and indicated that CD4⁺ T cells surrounded the tumor islets, whereas CD8⁺ T lymphocytes were localized among the tumor cells. Five short-term carcinoma cell lines established from these urothelial tumors were used as target cells in cytolysis assays in order to investigate the functional anti-tumor activity of autologous TIL. TIL from 4/5 tumors were lytic and 3 TIL lines displayed MHC-class-I-dependent cytotoxicity directed against autologous tumor cells. CD4⁺ T-cell-depletion experiments performed on TIL line 07 confirmed that CD8⁺ MHC-class-I-dependent CTL were the predominant effectors. Finally, experiments performed on 6 allogeneic urothelial-cancer cell lines matched for HLA-class-I molecules showed that TIL07 exhibited selective lytic activity toward tumor 07. These data indicate that CD8⁺ MHC-class-I-dependent CTL present in urothelial carcinomas are functional and may participate in the anti-tumor immune response. Int. J. Cancer 71:585-594, 1997. © 1997 Wiley-Liss, Inc.     

妇科肿瘤患者不同状态下的免疫功能评价

目的:研究妇科肿瘤患者的免疫功能变化情况,观察机体免疫功能与患者年龄、临床分期、治疗方式等因素的关系。方法:运用流式细胞术对151例患者外周血T淋巴细胞亚群及NK细胞进行检测。结果:与良性组比较,恶性肿瘤组CD3+下降 、CD4+下降极显著(P＜0.01), CD4+/CD8+下降显著（P＜0.05）,CD8+则略有升高;恶性肿瘤组CD4+、CD4+/CD8+表现出随临床分期提高逐渐降低的趋势,CD8+则反之。其中Ⅲ-Ⅳ期CD8+、CD4+/CD8+较I期存在极显著性差异（P＜0.01）;与未转移组比较,已转移组CD3+下降、CD4+下降显著(P＜0.05),CD4+/CD8+下降极显著（P＜0.01）,CD8+升高;术后组CD3+、CD4+、CD4+/CD8+较未治疗组升高,CD8+下降,并且CD4+/CD8+升高显著(P＜0.05);术后组CD4+、CD4+/CD8+较放化疗组升高、CD8+下降,差异均极显著（P＜0.01）;放化疗组CD4+、CD8+、CD4+/CD8+与未治疗组比较差异具有统计学意义(P＜0.05)。结论:妇科肿瘤患者的免疫功能受到不同程度的抑制,这种抑制与患者年龄、病情进展存在一定的相关性。在肿瘤治疗中解除瘤负荷和保护机体的免疫功能具有同等重要的意义。