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.     

Alterations in the signal-transducing molecules of T cells and nk cells in colorectal tumor-infiltrating,gut mucosal and peripheral lymphocytes: Correlation with the stage of the disease

T cells from mice bearing an experimental colon carcinoma, and from patients with colorectal and renal carcinomas, have atypical T-cell receptors (TCR). In the present study, further characterization of modulations in CD3- and CD 16-associated ζ chain in peripheral blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) from colorectal carcinomas was performed. Relative to PBL, the percentage of natural killer (NK) cells among fresh TIL was reduced, while a higher proportion of T cells expressing HLA-DR was found. As previously reported, we found significantly reduced levels of the CD3- and CD 16-associated ζ chain in TIL and, to a lesser extent, also in patients' PBL. Levels of ζ chain in T and NK cells from non-cancerous colorectal tissue from patients were lower than in PBL but higher than in TIL, with a direct relationship between levels of this signal-transducing molecule and the distance from the tumor. In addition, ζ levels correlated with the Dukes' stage of the disease, since PBL from patients with lymph-node involvement or distant organ metastases (Dukes' stages C and D) had significantly less CD3 ζ than patients with localized disease (stages A and B). Patients' T cells also had decreased levels of cell-surface and cytoplasmic CD3 ?. We also observed reduced levels of the TCR accessory molecules CD4 and CD8, mainly on TIL but to a lesser extent also on patients' PBL. Biochemical analysis of anti-CD3 ?-immunoprecipitated TCR complexes demonstrated that the CD3 complex was not associated with the ζ chain, either on TIL or on PBL or on lymphocytes from non-cancerous colon tissue, suggesting a defect in the assembly of the TCR complex. Following several days of in vitro culture with recombinant interleukin-2 and phytohemagglutinin, anti-CD3 or anti-CD2 monoclonal antibodies (MAbs), levels of CD3 ζ chain as well as of cell surface CD3 ? were normalized. Our findings suggest an abnormal expression as well as assembly of several different signal-transducing molecules of T cells and NK cells, which correlate with the stage of the disease in patients with colorectal carcinomas. © 1995 Wiley-Liss, Inc.     

Enhanced expression of IFN-gamma mRNA in CD4(+)or CD8(+)tumour-infiltrating lymphocytes compared to peripheral lymphocytes in patients with renal cell cancer

The mRNA expression of the cytokines IFN-gamma, IL-10 and TNF-alpha and the proapoptotic factor Fas ligand (FasL) was compared in freshly isolated CD4(+)and CD8(+)tumour-infiltrating lymphocytes (TIL) and simultaneously obtained autologous CD4(+)and CD8(+)peripheral blood lymphocytes (PBL) from 20 patients with renal cell carcinomas (RCC). TIL were isolated from mechanically disaggregated tumour material and PBL from peripheral blood by gradient centrifugation. The cells of the interphase were depleted from tumour cells with anti-human epithelial antigen magnetic beads and then positive selection was performed with anti-CD4 or anti-CD8 magnetic beads. In these pure lymphocyte preparations the constitutive expression of cytokine and FasL mRNAs was determined by using a PCR-assisted mRNA amplification assay. In the CD4(+)TIL from the 20 patients with RCC, levels of mRNAs encoding for IFN-gamma (P相似文献   

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.     

Correlation between CD4+CD25+Treg Cells and CCR4 in Nasopharyngeal Carcinoma

OBJECTIVE CD4+CD25+ T regulatory (Treg) cells are a population of T cells which suppress an overactive immune system. CCR4 is a chemokine receptor involved in the recruitment of lymphocytes. Nasopharyngeal carcinoma (NPC) is resistant to immunosurveillance, owing to the increased number of tumor-infiltrating Treg cells which are recruited to the tumor by CCR4.METHODS The percentage of CD4+CD25+Treg cells and CCR4+ cells in tissue or peripheral blood (PB) lymphocytes of patients with untreated NPC or normal subjects was analysed by flow cytometry. RESULTS In both tissue and PB lymphocytes, the percentage of CD4+CD25+ Treg cells and CCR4+ cells was significantly elevated in patients with NPC in comparison with that in the normal tissue of controls. Furthermore, in the patients with NPC, a higher percentage of CD4+CD25+ Treg cells was found in the tumor-infiltrating (T1) lymphocyte population than in the PB population. In the NPC patient group, a general trend towards an increased percentage of Tl Treg cells was found in the patients with advanced stage NPC. The number of CD4+CD25+ Treg cells was positively related to the number of CCR4+ cells in the tumor and in the PB of the patients with NPC, while the number of CD4+CD25+ Treg cells was negatively related to the number of CD4+CD25- T cells.CONCLUSION Immunosuppression was observed in NPC, especially at the tumor sites. CD4+CD25+ Treg cells may suppress CD4+CD25- T cells. CCR4 may have an important role in the recruitment of CD4+CD25+ Treg cells to tumor sites, thus causing resistance to immunosurveillance.     

Functional characterization of tumor-infiltrating lymphocytes, lymph-node lymphocytes and peripheral-blood lymphocytes from patients with breast cancer

Mononuclear cell infiltration is frequently seen within human solid tumors. Effector cells within the tumor site usually fail to exhibit cytotoxic or natural killer activity when freshly isolated; however, they develop potent and sometimes specific cytotoxicity after expansion in IL2. Thus, local tumor environment may influence lymphocyte function. In our study, we disaggregated human breast-cancer and lymph-node tissue to obtain lymphocyte-enriched cell fractions. Besides phenotypic analysis, functional characterization with regard to proliferation and cytokine production of tumor-infiltrating lymphocytes (TIL), peripheral-blood lymphocytes (PBL) and lymph-node lymphocytes (LNL) was the aim of our study. TIL showed an enrichment of CD8+ cells with a corresponding decrease in CD4+ cells in comparison with PBL and LNL. In response to PHA, TIL showed decreased 3H-thymidine uptake, but TIL were significantly stimulated by rhIL2. TIL produced low levels of IL2, TNF and IFN gamma upon mitogen/phorbol ester stimulation, while PBL produce high levels of TNF and IFN gamma but low levels of IL2. Under the same experimental conditions, LNL produce high levels of TNF and IL2 but low levels of IFN gamma. Mitogen-mediated TNF secretion was increased after addition of autologous tumor cells in TIL and LNL, whereas IFN gamma secretion tended to be suppressed. Our results indicate different patterns of activities of TIL, LNL and PBL from breast-cancer patients.     

Lymphokine-activated killer-cell function of lymphocytes from peripheral blood, regional lymph nodes and tumor tissues of patients with oral cancer

Lymphokine-activated killer (LAK) cells, generated from peripheral blood lymphocytes (PBL) from patients with oral cancer or oral leukoplakia and from healthy donors showed comparable lysis of 6 target tumor cell lines, including 3 derived from head and neck and oral cancers. The tumor burden of the host did not appear to influence the systemic LAK activity. LAK activity of lymphocytes infiltrating the tumor tissues (TIL) was also comparable to that of the PBL. Both TIL and PBL showed a parallel increase in proportion of HNK-I+ and CD-25+ cells upon activation with IL-2. The lymph-node lymphocytes (LNL) from metastatic (met) and non-metastatic (non-met) draining lymph nodes, however, showed reduced LAK activity and an increase in CD8+ cells, in addition to CD25+ and HNK-I+ cells, when cultured with IL-2. When IL-2-activated LNL were co-cultured with autologous PBL during IL-2 activation of the latter, a strong suppressive effect was exerted by LNL. In contrast, IL-2-activated PBL did not suppress autologous LAK generation in spite of an increase in CD8+ cells seen after activation with IL-2. Frequency distribution of LAK precursors was significantly lower in LNL than in PBL from oral cancer patients. LAK precursor frequency in TIL was comparable to that of PBL. The results show that, in oral cancer, regional lymph nodes may not have adequate IL-2-inducible cytotoxic potential, due to a reduced number of LAK progenitors and possible activation of suppressor cells. Alternatively, TIL can be a potential source for LAK cell function.     

Differential immune reactivity of tumour-intrinsic and peripheral-blood lymphocytes against autoplastic colorectal carcinoma cells.

Peripheral blood lymphocytes (PBL) were obtained from 13 patients and tumour-intrinsic lymphocytes (TIL) from 20 patients with colorectal cancer. The PBL were separated on a Ficoll-Isopaque gradient and the TIL by digestion of the tumour with collagenase-DNase. Both PBL and TIL were passed through nylon-wool columns and the eluted cells were co-cultured for 2 h with 51Cr-labelled tumour cells from the same patient. If patients in whom spontaneous 51Cr release from the tumour cells was greater than 33% were excluded, PBL showed cytotoxicity for the autoplastic tumour cells in 5/10 cases and TIL in 3/10 cases (NS). In 12 cases the cytotoxicity of the TIL was compared with that for TIL from the same tumour after the lymphocytes had been washed a further 6 times in Medium 199. Three effector: target (E/T) ratios, 5:1, 10:1 and 20:1, were used. The proportion of effector populations showing cytotoxicity was 2/12 for unwashed TIL and 9/12 for washed TIL (P less than 0.006). At the 5:1 E/T ratio the level of cytotoxicity was not significantly greater for washed TIL, but at the 10:1 ratio washed TIL showed significantly more cytotoxicity (P less than 0.025. At the 20:1 E/T ratio, a comparison was possible in 15 cases and the washed TIL again showed greater cytotoxicity (P less than 0.001).     

Clonal analysis of lymphocytes from tumor, peripheral blood, and nontumorous kidney in primary renal cell carcinoma

The immunological properties of lymphocytes from tumor, peripheral blood (PBL), and nontumorous kidney from 16 patients with renal cell carcinoma were characterized at the clonal level with respect to their clonogenic efficiency, phenotypic expression, and cytotoxicity against autologous and allogenic tumor cells. The objectives were to delineate: (a) the quantitative differences in the immunological properties of tumor-infiltrating lymphocytes (TIL) from patient to patient; and (b) the qualitative differences in immunological properties between TIL and lymphocytes from peripheral blood or nontumorous kidney from a single patient. A total of 926 clones were characterized for phenotype expression, and 465 clones were characterized for cytotoxicity. The clonogenic efficiency of TIL varied with individuals: high in one patient; relatively high to moderate in seven patients; low in seven patients, and extremely low in the remaining one patient. The levels of autologous tumor cell lysis by TIL clones also varied with individuals. More than one-third of the TIL clones established in 4 of 13 patients displayed significant (greater than or equal to 10%) lysis against autologous tumor cells, and in each of the four patients the average percentage of lysis in the total TIL clones was higher than 10%. In two patients, 5 of 26 or 3 of 13 TIL clones were cytotoxic, but averages of percentage of lysis in the total clones were less than 10%. One 1 or 2 TIL clones of 10-27 total clones were cytotoxic in each of 4 patients, while no cytotoxic TIL clones were found in the remaining 3 patients. Clonogenic efficiency did not correlate with the level of cytotoxicity, and TIL from no tumors displayed both high proliferation and high cytotoxicity at the clonal level. In a majority of patients (12 of 13), most cytotoxic TIL clones against autologous tumor cells also lysed allogenic tumor cells. In contrast, TIL clones lysed only autologous tumor cells in the remaining one patient (patient 2). The clonogenic efficiency of TIL was lower than that of PBL in 6 of 12 patients, while the opposite was true in the remaining 6 patients. The level of cytotoxicity in the PBL clones of these 12 patients primarily correlated with that of the TIL clones. With one exception (patient 2), most cytotoxic PBL clones against autologous tumor cells also lysed allogenic targets in a majority of patients. CD4+CD8-T-cell clones (70-85%) predominated in all patients regardless of the different lymphocyte sources.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lysis of fresh human tumor cells by autologous peripheral blood lymphocytes and tumor-infiltrating lymphocytes activated by PSK

The protein-bound polysaccharide PSK was tested for the ability to induce in vitro autologous tumor killing (ATK) activity in human cancer patients. Peripheral blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) demonstrated various levels of cytotoxicity against autologous, freshly isolated tumor cells. When PBL and TIL were cultured overnight with PSK, ATK activity was induced in previously non-reactive cases and augmented in previously reactive samples. The PSK effect was observed with PSK concentrations of 10-100 micrograms/ml that could be obtained in the blood of cancer patients who received standard oral administration of PSK. The manifestation of PSK-induced ATK required active cell metabolism and RNA and protein syntheses, but not DNA synthesis of lymphocytes. PSK-induced enhancement of ATK was not abrogated by monoclonal antibodies (mAb) directed against interferon (IFN) alpha or IFN gamma. In addition, mAb that neutralized interleukin-2 (IL-2) or mAb reactive with alpha-chain or beta-chain of IL-2 receptors (IL-2R) had no effect on PSK-induced ATK activity. Supernatants from PSK-stimulated lymphocyte cultures did not induce ATK. Cell fractionation experiments revealed that CD3-CD16+ large granular lymphocytes (LGL) and/or CD3+CD16- T lymphocytes were responsible for both spontaneous and PSK-induced ATK. PSK-activated LGL, but not T lymphocytes expressed lysis of fresh allogeneic tumor cells. These results indicate that PSK activates PBL and TIL to exhibit ATK independently of IL-2/IL-2R systems.     

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.     

Presence of in vivo-activated t-cells expressing hla-dr molecules and il-2 receptors in peripheral blood of patients with nasopharyngeal carcinoma

Epstein-Barr Virus (EBV) is associated with two malignant diseases, African Burkitt's Lymphoma (BL) and Undifferentiated Nasopharyngeal Carcinoma (UNPC). North Africa is a geographical area with a high incidence of NPC. Our purpose in this study was to explore cell-mediated immunity of peripheral blood lymphocytes (PBL) from patients with UNPC and DNPC. We found an elevated percentage of OKT8 cells and of large granular lymphocytes (LGL) (30 - 35% HNK-I-positive cells) compared to PBL from healthy matched individuals. PBL from NPC patients contained 35% HLA-DR- positive and 30% Interleukin-2 (IL-2) receptor-positive circulating lymphocytes. PBL from NPC patients exhibited a normal proliferative response to phytohemagglutinin (PHA) and Concanavalin A (Con A) and an increased response to pokeweed mitogen (PWM). Natural killer (NK) activity towards K562 cells was low in our patients who, in addition, exhibited no lytic activity against HLA-matched EBV-transformed B cells. This lack of cytotoxicity against an EBV-transformed B-cell line cannot be explained by an impairment of IL-2 secretion, and is probably a result of the presence of high numbers of OKT8 suppressor T cells.     

Lymphokine-activated killer activity induced by in vivo interleukin 2 therapy: predominant role for lymphocytes with increased expression of CD2 and leu19 antigens but negative expression of CD16 antigens

The phenotype and function of lymphocytes from cancer patients treated with repetitive weekly cycles of continuous i.v. infusions of recombinant interleukin 2 (IL-2) were examined. Peripheral blood lymphocytes (PBL) obtained after IL-2 therapy showed an increased percentage of cells bearing the CD16 and leu19 markers which are associated with natural killer cells. These PBL mediated significantly increased levels of IL-2-dependent lymphokine-activated killer (LAK) activity against the Daudi cell line. Depletion of CD16+ cells from PBL obtained after in vivo IL-2 caused only slight inhibition of their LAK activity or their proliferative response to IL-2 in vitro. This indicates that CD16+ cells are involved but play only a minor role in these responses. In contrast, depletion of leu19+ cells, from PBL activated in vivo with IL-2, virtually abrogated their LAK activity and their proliferative response to IL-2. Two-color flow cytometry studies showed that a leu19+/CD16- population was expanded by in vivo IL-2 therapy and was responsible for the majority of LAK activity by in vivo-activated PBL. Moreover, this CD16- population showed an increased density of leu19 and CD2 (E rosette receptor) antigens when compared to the resting PBL obtained prior to IL-2 treatment. These data show that the predominant population mediating in vitro LAK activity, induced by in vivo IL-2 therapy, consists of activated natural killer cells with a high density of leu19 and CD2 antigens but negative for the CD16 antigen.     

Ex vivo expansion of tumor-infiltrating lymphocytes from nasopharyngeal carcinoma patients for adoptive immunotherapy

Establishing Epstein-Barr virus (EBV)-specific cytolytic T lymphocytes (EBV-CTLs) from peripheral blood mononuclear cells (PBMCs) for adoptive immunotherapy has been reported in EBV-associated malignancies including Hodgkin's lymphoma and nasopharyngeal carcinoma (NPC). In the current study,we performed ex vivo expansion of tumor-infiltrating lymphocytes (TILs) obtained from NPC biopsy specimens with a rapid expansion protocol using anti-CD3 monoclonal antibody (OKT3), recombinant human interleukin (IL)-2, and irradiated PBMCs from healthy donors to initiate the growth of TILs. Young TIL cultures comprised of more than 90% of CD3+T cells, a variable percentage of CD3+CD8+and CD3+CD4+T cells, and less than 10% of CD3-CD16+natural killer cells, a similar phenotype of EBV-CTL cultures from PBMCs. Interestingly, TIL cultures secreted high levels of the Th1 cytokines, interferon gamma (IFNγ) and tumor necrosis factor-alpha (TNF-α), and low levels of the Th2 cytokines, IL-4 and IL-10. Moreover, young TILs could recognize autologous EBV-transformed B lymphoblast cell lines, but not autologous EBV-negative blast cells or allogeneic EBV-negative tumor cells. Taken together, these data suggest that ex vivo expansion of TILs from NPC biopsy tissue is an appealing alternative method to establish T cell-based immunotherapy for NPC.     

Restoration of Tumor-Specific HLA Class I Restricted Cytotoxicity in Tumor Infiltrating Lymphocytes of Advanced Breast Cancer Patients by in vitro Stimulation with Tumor Antigen-Pulsed Autologous Dendritic Cells

Breast tumor infiltrating lymphocytes (TIL) are enriched in tumor-specific cytotoxic T lymphocytes (CTL), and may represent a superior source of CTL compare to peripheral blood lymphocytes (PBL), for adoptive T cell immunotherapy of breast cancer. However, the immunocompetence of TIL and the possibility to consistently restore their tumor-specific lytic activity in vitro remains an open issue. In this study we evaluated the potential of tumor antigen-pulsed fully mature dendritic cell (DC) stimulation in restoring tumor-specific cytotoxicity in anergic TIL populations from advanced breast cancer patients. In addition we have compared tumor-specific T cell responses induced by tumor antigen-loaded DC stimulation of TIL to responses induced from PBL. Although TIL were consistently non-cytotoxic after isolation or culture in the presence of interleukin-2 (IL-2), in matched experiments from three consecutive patients, tumor-lysate-pulsed DC-stimulated CD8+ T cell derived from TIL were found to be significantly more cytotoxic than PBL (p < 0.05). In addition, cytotoxicity against autologous tumor cells was more significantly inhibited by an anti-HLA class I (W6/32) MAb in TIL compared to PBL (p < 0.05). CTL populations derived from TIL and PBL did not lyse autologous EBV-transformed lymphoblastoid cell lines, and showed negligible cytotoxicity against the NK-sensitive cell line K562. Furthermore, in both CD8+ T cell populations the majority of the tumor-specific CTL exhibited a Th1 cytokine bias (IFN-^high/IL-4^low). Taken together, these data show that tumor lysate-pulsed mature DC can consistently restore tumor-specific lytic activity in non-cytotoxic breast cancer TIL. These results may have important implications for the treatment of chemotherapy resistant breast cancer with active or adoptive immunotherapy.     

Immunosuppression in renal cancer: differential expression of signal transduction molecules in tumor-infiltrating, near-tumor tissue, and peripheral blood lymphocytes

Alterations in the expression of signal activation molecules, such as the T-cell receptor (TCR) ζ and ε chains and p56^lck tyrosine kinase, are described in tumor-infiltrating lymphocytes (TIL). The aim of this study was to ascertain if such molecules were present in near-tumor-tissue lmphocytes (NTTL) and peripheral blood lymphocytes (PBL), as well as TIL, of renal cell carcinoma patients, to verify whether this tumor induces immunosuppression only locally or affects distant lymphocytes as well. Tissue from the tumor and from healthy nearby sites, as well as blood samples, were obtained from 27 consecutive patients who had undergone radical nephrectomy for renal cell carcinoma. Phenotype analysis and immunohistochemical staining of the TCR ζ and ε chains and p56^lck were performed with standard techniques on TIL, NTTL, and PBL, and values were compared for each patient. Low expression of the TCR ζ chain and an almost complete absence of TCR ε chain and p56^lck expression was observed in TIL (median values: 10% for ζ chain and 0% for ε and p56^lck). In NTTL, these signal transduction molecules were expressed by a higher percentage of cells (60%, 50%, and 60%, respectively; p = 0.000 vs. TIL), whereas PBL showed an almost normal expression of ζ and ε chains (80% and 90%, respectively; p = 0.000 vs. TIL). Conversely, p56^lck was detected in a greater proportion of NTTL than PBL (50% vs. 10%; p = 0.001). The absence or the very low expression of signaling activation molecules in TIL compared with NTTL and PBL in renal cancer patients suggest that tumor-induced immunosuppression generally occurs or starts locally.     

The T-cell receptor V beta gene usage in tumor-infiltrating lymphocytes and blood of patients with hepatocellular carcinoma.

To determine a possibly restricted T-cell receptor (TCR) repertoire in tumor-infiltrating lymphocytes (TIL) in response to tumor-associated antigens in patients with hepatocellular carcinoma (HCC), freshly isolated TIL (n = 5) and peripheral blood lymphocytes (PBL; n = 6; 3 paired with TIL) were studied for expression of TCR variable (V) beta regions. RNA purified from TIL or PBL was reverse-transcribed into complementary DNA. This complementary DNA was amplified by quantitative polymerase chain reaction with 22 primers specific for 20 TCR V beta gene families and a 3' constant (C) beta primer. As a reference for later quantitation, a fragment of TCR C alpha was coamplified with each V beta region. Using 32P-labeled 3' primers, the percentage of total V beta expression was calculated by measuring the cpm of each of the amplified products. In contrast to PBL of 6 control, healthy individuals, whose range of expression of each TCR V beta gene varied from 0 to 13%, the expression of some V beta genes in HCC TIL was as high as 33%, indicating a restricted TCR V beta usage in HCC TIL. When polymerase chain reaction-amplified complementary DNAs of the V beta 1 or V beta 3 genes obtained from two TIL preparations were cloned and sequenced, the same rearrangements were found in the majority of DNA clones. The particular V beta genes that were over- or underrepresented in TIL varied among the patients. In 3 of 6 PBL and 3 of 5 TIL, the V beta 3 gene was expressed with a relatively high frequency. The V beta 4 gene expression was consistently low in patients' TIL or PBL. In 3 paired PBL and TIL, V beta expression was similar. In 5 of 6 cases, HCC PBL had different TCR V beta frequencies from those seen in normal PBL. This analysis of TCR V beta usage in freshly isolated TIL and in PBL indicated that T-lymphocytes in patients with HCC might have restricted immunological reactivity and that V beta 3-restricted TIL might represent antitumor effector cells.     

Control of apoptosis in Epstein Barr virus-positive nasopharyngeal carcinoma cells: opposite effects of CD95 and CD40 stimulation

The expression and function of CD95 and CD40 were investigated in malignant cells from EBV-positive undifferentiated nasopharyngeal carcinomas (NPCs). Large amounts of CD95 and CD40 expression were detected in 15 of 16 EBV-positive NPC specimens. In contrast, CD95 was not detected in two biopsies from patients with EBV-negative differentiated NPCs. We tested whether the CD95 apoptotic pathway was functional in NPC cells by treating two EBV-positive NPC tumor lines in vitro with a CD95 agonist. In both cases, NPC cells were extremely susceptible to CD95-mediated apoptosis, despite strong constitutive expression of Bcl-x. Combined CD40 and CD95 stimulation was used to investigate the possible anti-apoptotic activity mediated by CD40. The CD40 receptor was activated by incubating NPC cells with murine L cells producing CD154, the CD40 ligand. This treatment resulted in a strong inhibition of CD95-related cytotoxicity. Such an anti-apoptotic effect of CD40 is well known for B lymphocytes, but has not previously been reported for epithelial cells. These data suggest that NPC tumor-infiltrating lymphocytes, which often produce the CD40 ligand in situ, may increase the survival of malignant cells, thereby enhancing tumor growth in patients.     

Simultaneous generation of CD8+ and CD4+ melanoma-reactive T cells by retroviral-mediated transfer of a single T-cell receptor

Adoptive immunotherapy of cancer requires the generation of large numbers of tumor antigen-reactive T cells for transfer into cancer patients. Genes encoding tumor antigen-specific T-cell receptors can be introduced into primary human T cells by retroviral mediated gene transfer as a potential method of providing any patient with a source of autologous tumor-reactive T cells. A T-cell receptor-specific for a class I MHC (HLA-A2)-restricted epitope of the melanoma antigen tyrosinase was isolated from a CD4(+) tumor-infiltrating lymphocyte (TIL 1383I) and introduced into normal human peripheral blood lymphocytes by retroviral transduction. T-cell receptor-transduced T cells secreted various cytokines when cocultured with tyrosinase peptide-loaded antigen-presenting cells as well as melanoma cells in an HLA-A2-restricted manner, and could also lyse target cells. Furthermore, T-cell clones isolated from these cultures showed both CD8(+) and CD4(+) transduced T cells could recognize HLA-A2(+) melanoma cells, giving us the possibility of engineering class I MHC-restricted effector and T helper cells against melanoma. The ability to confer class I MHC-restricted tumor cell recognition to CD4(+) T cells makes the TIL 1383I TCR an attractive candidate for T-cell receptor gene transfer-based immunotherapy.     

Enhanced susceptibility of adriamycin-treated human renal cell carcinoma cells to lysis by peripheral blood lymphocytes and tumor infiltrating lymphocytes

Previous studies indicated that the anticancer agent adriamycin (ADR) could induce activation of cytotoxic T lymphocytes (CTL) and natural killer cells. In this study, we investigated the effect of ADR on the susceptibility of human renal cell carcinoma (RCC) cells to lysis by peripheral blood lymphocytes (PBL) and tumor infiltrating lymphocytes (TIL). Treatment of human RCC cell line ACHN and freshly derived RCC cells with ADR at 1 microg/ml or more for 3 h significantly enhanced their susceptibility to lysis by PBL (P<0.05). This ADR-induced enhancement of susceptibility of RCC cells to lysis by PBL was also observed when freshly derived TIL were used as effector cells (P<0.05). ADR up-regulated the expression of leukocyte function-associated antigen-3 (LFA-3) and intercellular adhesion molecule-1 (ICAM-1), which are critical in the binding and killing of CTL against cancer cells. Of the five fresh RCC cell cultures treated with ADR, LFA-3 was increased in all and ICAM-1 was increased in three of them, respectively (P<0.05). Up-regulation of LFA-3 and ICAM-1 was also observed in ACHN cells treated with two derivatives of ADR, epirubicin and pirarubicin. ADR further significantly increased the bindings of PBL to RCC cells (P<0.05). These findings suggest that treatment of RCC patients with low doses of ADR may sensitize the RCC cells to killing by PBL and TIL and may be a novel immunotherapeutic modality for the treatment of drug-resistant and/or immune-resistant RCC. The inducing of LFA-3 and ICAM-1 by ADR may be involved in the enhancement of susceptibility of PBL and TIL-mediated cytolysis in human RCC cells.