Renal-cell carcinoma-specific lysis by cytotoxic T-lymphocyte clones isolated from peripheral blood lymphocytes and tumor-infiltrating lymphocytes

Melanoma and renal-cell carcinoma (RCC) are generally considered to be relatively immunogenic tumor types in humans. In the case of melanoma, many major histocompatibility complex (MHC) class I-restricted tumor-specific cytotoxic T lymphocytes (CTL) have been isolated from either tumor-infiltrating lymphocytes (TIL) or autologous peripheral blood lymphocytes (PBL). In contrast, such CTL have only incidentally been described in the case of RCC. It has often been reported that TIL lines isolated from RCC display non-MHC-restricted and non-specific activity. Here, we report the isolation and characterization of tumor-specific CTL from PBL of one RCC patient and from TIL of another RCC patient. CTL clones 263/17 and 263/45, isolated from the PBL of patient LE-9211, were restricted by HLA-B7. CTL clone 5E, isolated from the TIL of patient LE-8915, was restricted by HLA-B37. The autologous RCC cell lines were efficiently lysed by the CTL clones, whereas normal epithelial cells of the proximal tubuli matched for the restriction element and K562 were not. From a panel of allogeneic RCC cell lines, CTL 5E recognized MZ-1940-RCC. Reactivity to allogeneic RCC sharing HLA-B7 was also observed with CTL 263/17 and 263/45, both of which could lyse the HLA-B7-positive cell line MZ-1851-RCC. Our data provide evidence that common tumor antigens are recognized by CTL on RCC. © 1996 Wiley-Liss, Inc.     

Characterization of T-cell memory phenotype after in vitro expansion of tumor-infiltrating lymphocytes from melanoma patients

Memory T-cell populations in human antitumor tumor-infiltrating lymphocytes (TILs) for adoptive cell transfer have not been fully characterized. Our studies demonstrated that CD62L, CD27 and CD28 positive effector memory T-cells were present in the TIL samples from the tumor tissues of melanoma patients and T-cell expansion led to the significant loss of memory T-cells. CD27- and CD28-positive T-cells had high levels of CD44 expression. T-Cell expansion resulted in significant down-regulation of CD44 expression. Interleukin-2 (IL-2) and anti-CD3 antibody stimulation may be responsible for CD44 down-regulation on CD8(+) T-cells during expansion. Furthermore, CD44 down-regulation using small interfering RNA (siRNA) on TILs dramatically reduced interferon-gamma and IL-2 release upon tumor stimulation. These results suggest that the regulation of CD44 expression in TILs may play an important role in memory T-cell maintenance and antitumor immune response.     

In vitro antitumor activity of tumor-infiltrating lymphocytes from human gastric carcinoma

Tumor-infiltrating lymphocytes (TIL) isolated from 11 gastric carcinoma were studied. TIL could grow for a long-term in medium containing recombinant interleukin-2(rIL-2). The mean expansion fold achieved in 6 long-term cultures of 11 specimens was 15.1 RIL-2 expanded gastric TIL exhibited significant cytotoxicity against K562, BGC823, MCF-7 and more effective antitumor cytotoxicity against fresh autologous tumor targets and human gastric cancer cell line. Peak cytotoxicity was shown in the third or fourth week after cultures. Cryopreservation of gastric TIL didn’t influence their expansion capacity and antitumor activity. Phenotypic analysis was demonstrated in this study. The results of present study indicate that TIL from human gastric carcinoma could be expanded and reach high levels of antitumor effector function in long-term cultures with rIL-2. Their function may be of clinical importance.     

Characterization of human autotumor-reactive T-cell clones obtained from tumor-infiltrating lymphocytes in liver metastasis of gastric carcinoma.

Three autotumor-reactive T-cell clones have been established from tumor-infiltrating lymphocytes isolated from a metastatic lesion of human gastric carcinoma in the liver. The clones all were shown to be CD3+, CD8+, CD4-, CD16-, T-cell receptor alpha/beta +, and T-cell receptor gamma/delta-, and they have retained both their autotumor reactivity and the same phenotype for over a year in culture. Each clone had a different rearrangement of the T-cell receptor gamma chain genes as indicated by Southern blot analysis. Tested against a panel of 18 tumor cell targets, the clones preferentially lysed autologous tumor (AuTu) cells, but each clone also showed weak cytotoxicity against one allogeneic cholangiocarcinoma cell line. At the same time, each clone showed appreciable cytotoxicity against K562 targets. In blocking experiments, anti-CD3, anti-WT31, anti-CD8, or anti-HLA class I monoclonal antibodies blocked AuTu cytotoxicity but not cytotoxicity against K562. In contrast, allocytotoxicity against the cholangiocarcinoma was blocked only by anti-CD3 monoclonal antibody. All 10 subclones of one T-cell clone had high levels of AuTu cytotoxicity but variable levels of anti-K562 cytotoxic activity. Proliferation of the T-cell clones was significantly stimulated by the addition of irradiated autologous but not allogeneic tumor cells. Preincubation of cultured AuTu cells with tumor necrosis factor alpha or gamma-interferon (IFN-gamma), but not with IFN-alpha, increased their susceptibility to lysis by the T-cell clones; however, it increased resistance of AuTu to lysis by interleukin 2-activated natural killer cells. The expression of an adhesion molecule, intercellular adhesion molecule 1, on the surface of AuTu was also up-regulated by tumor necrosis factor alpha or IFN-gamma, but not by IFN-alpha. All three cytokines up-regulated HLA-class-I antigens on AuTu. Pretreatment of K562 targets or allogeneic cholangiocarcinoma cells with the same cytokines increased their resistance to lysis by the T-cell clones. Overall, the results indicate that these T-cell clones show specificity for AuTu but also independently recognize a limited number of allogeneic tumor targets and lyse K562 targets. The mechanisms involved in the recognition by the T-cell clones of autologous, allogeneic, and K562 tumor targets appeared to be distinct.     

In vivo antitumor activity of tumor-infiltrating lymphocytes expanded in recombinant interleukin-2

A method was described for the generation of cells from tumor-bearing mice; these cells were capable of exhibiting significant antitumor reactivity when adoptively transferred into tumor-bearing hosts. Tumor cell suspensions from a variety of tumors were able to be separated using enzymatic techniques and they were cultured in medium containing recombinant interleukin-2. Activated infiltrating lymphocytes within these tumors expanded; and, by 6-8 days after initiation of culture, lymphocytes predominated and were able to grow to large numbers. The adoptive transfer of these tumor-infiltrating lymphocytes (TILs) made possible mediation of the reduction of established 3-day pulmonary micrometastases from 5 of 7 tumors tested, including two 3-methylcholanthrene (CAS: 56-49-5)-induced sarcomas, one 1,2-dimethylhydrazine (CAS: 540-73-8)-induced colon carcinoma, and the B16 melanoma, all in C57BL/6 mice, as well as the 1660 bladder carcinoma in BALB/c mice. Approximately 2-4 X 10(6) transferred cells were capable of totally eliminating 3-day established metastases. These cells were thus 50 to 100 times more effective than lymphokine-activated killer cells in reducing established metastases; however, they could not be generated from all tumors. The concomitant administration of recombinant interleukin-2 enhanced, by approximately fivefold, the in vivo activity of these cells. The specificity of action of TILs in vivo was different from that determined by classic amputation rechallenge experiments. The tumor-infiltrating lymphocytes that developed this antitumor reactivity appeared to be Thy-1+ and did not bear the asialo GM1 antigen. The potent antitumor effect of these TILs, when transferred in vivo to tumor-bearing hosts, raises the possibility of utilizing similar approaches for the isolation and therapeutic use of lymphocytes with antitumor reactivity from human tumors.     

TNFalpha stimulates NKG2D-mediated lytic activity of acute myeloid leukemic cells.

The mechanism by which leukemic cells interfere with normal hematopoiesis remains unclear. We show here that, whereas the leukemic KG1a cells are naturally devoid from cellular cytotoxicity, once activated by TNFalpha, they display cytolytic activity toward various cellular targets including CFU-GM. This mechanism is dependent on stimulation of the granzyme B/perforin system. In addition, KG1a cells expressed the NKG2D receptor and its signal-transducing adaptator DAP 10, which were functional as confirmed by redirected lysis experiments. Interestingly, flow cytometry analysis of 20 samples of patients with acute myeloid leukemia (AML) (FAB M0-M5) revealed the expression of NKG2D (40%) and other natural cytotoxicity receptors (40% for NKp30, 74% for NKp44, 39% for NKp46) by a pool >15% of leukemic cells. Furthermore, CD34+ hematopoietic progenitors undergoing granulomonocytic differentiation expressed NKG2D ligands. Altogether, we propose a model in which, upon stimulation by TNFalpha, leukemic cells may exert cytotoxicity against myeloid progenitors. This finding may have important clinical implications in the context of diseases characterized by TNFalpha accumulation, such as AML or myelodisplasic syndromes.     

T-cell activation marker expression on tumor-infiltrating lymphocytes as prognostic factor in cutaneous malignant melanoma.

The central role of T cells in antitumor immunity is well established. However, tumor progression, often seen in the presence of substantial lymphocytic infiltration, suggests that these T cells are not capable of mounting an effective immune response to control tumor growth. Evidence has accumulated that T lymphocytes infiltrating human neoplasms are functionally defective, incompletely activated, or anergic. Therefore, when characterizing the immune competent cells within lymphoid infiltrates of tumors, it is important to assess their activation state. We investigated the expression of two T-cell activation markers, interleukin 2 receptor alpha (CD25) and OX40 (CD134), by immunohistochemistry in primary cutaneous melanoma samples of 76 patients and analyzed it in relation to tumor stage and tumor progression (>5 years follow-up), as well as to patients' survival. We found that the degree of infiltration by CD25(+) and intratumoral OX40(+) lymphocytes showed a tendency to decrease in thicker melanomas. The frequency of samples with high numbers of peritumoral CD25(+) and OX40(+) cells was significantly lower (P = 0.0009 and P = 0.0087, respectively) in melanomas developing distant visceral metastases, compared with nonmetastatic or lymph node metastatic tumors. For both activation markers studied, high peritumoral densities were associated with longer survival by univariate analysis (P = 0.0028 and P = 0.0255 for CD25 and OX40, respectively), whereas peritumoral OX40(+) lymphocyte infiltration had an impact on survival also in multivariate analysis (P = 0.035). The results suggest that the presence of lymphocytes expressing the T-cell activation markers CD25 or OX40 shows correlation with tumor progression as well as with patients' survival in cutaneous malignant melanoma.     

Usage of T-cell receptor Vβ chain genes in fresh and cultured tumor-infiltrating lymphocytes from human melanoma

Tumor-infiltrating lymphocytes (TIL) freshly obtained from human malignant melanomas as well as the same TIL grown in the presence of interleukin 2 (IL2) were studied for gene expression of the T-cell receptor (TCR) variable β regions (Vβ). To perform the TCR-Vβ analysis, total RNA was isolated from TIL and reverse-transcribed into cDNA, which was then amplified by PCR using 22 different 5′ primers specifically recognizing the sequences of 20 Vβ gene families and a 3′ primer annealing to the constant region of the β chain. The TCR-α constant region (Cα) gene was co-amplified as a standard for the calculation of the percentage of each TCR-Vβ gene expressed. The frequency of individual Vβ regions expressed on TIL was computed from the ratio of cpm Vβ to cpm Ca for each Vβ region in relation to the total of all 22 ratios. With fresh TIL obtained from 8 different melanomas, oligoclonal distribution of Vβ genes expressed on TIL was observed, in comparison with a broader and unrestricted distribution seen with peripheral-blood T cells of 8 normal individuals. The oligoclonal patterns of Vβ-gene expression in fresh melanoma TIL were distinct in every tumor. Several of the Vβ-genes usually expressed in normal PBL were not expressed in fresh TIL. In melanoma TIL cultured in the presence of IL2 and IL4 and in the absence of autologous tumor (AuTu) or antigen-presenting cells for 23 to 65 days, selection of T-cell lines expressing a restricted number of Vβ genes occurred. Although in 4/5 TIL cultures this selection involved the Vβ7 gene, no relationship could be established between Vβ gene expression in fresh TIL and that in T-cell lines outgrowing in long-term cultures. Selection in culture of CD3⁺ CD8⁺ T-cell lines with Vβ-gene expression restricted to I or 2 Vβ families did not correlate with the presence or level of AuTu cytotoxicity mediated by these T cells. The results indicate that in TIL cultures random selection of T-cell lines with reactivity not relevant to AuTu may account for poor expression or loss of AuTu cytotoxicity by most TIL cultured long-term in the presence of cytokines and in the absence of specific antigenic stimulation.     

T-cell receptor v-alpha and v-Beta gene usage in interleukin-2-cultured tumor-infiltrating lymphocytes from patients with breast-cancer

Tumor-infiltrating lymphocytes (TIL) are often found in malignant breast tumors, and have been claimed to be of prognostic value. It has been proposed that TIL may represent an enriched population of tumor-specific cytotoxic lymphocytes, reacting with antigenic determinants on the tumor cell surface through the T cell receptor (TCR) complex. We have studied the phenotype, cytotoxicity, and expression of TCR variable (V) alpha and beta chain on in vitro IL-2-cultured TIL isolated from primary malignant breast tumors from 11 patients. 10/11 cultures were dominated by CD4(+) (T-helper) cells. The different TIL cultures exhibited varying levels of cytotoxicity against the natural killer (NK)-sensitive cell line K562 and breast cancer cell line T47D. The level of clonality, as measured by PCR-based analyses of usage of the different V segments was low, as only a few tumors showed patterns of restricted V gene expression. The mean number of V alpha segments per TIL culture was higher than the number of V beta segments per culture. A significant negative correlation was observed between the number of CD4+ cells and the number of V beta segments per culture, and no other correlations between phenotypes and expression of any particular V segments were found. Neither was there any correlation between the expression of specific V alpha/V beta segments and cytotoxicity against allogeneic tumor cells.     

Phenotype and functional activity of tumor-infiltrating lymphocytes isolated from immunogenic and nonimmunogenic rat brain tumors

The purpose of the present study was to define the immunogenicity of two transplantable rat gliomas, designated F98 and D74, and to relate this to the phenotype and functional activity of tumor-infiltrating lymphocytes (TIL). Fischer rats, immunized with irradiated F98 tumor cells and challenged with intracerebral implants of ten F98 cells, had a median survival time of 49 days compared to 36 days for nonimmunized controls. In contrast, no statistically significant increases in survival times were noted in animals similarly immunized and challenged with the D74 tumors. No in vivo protection could be demonstrated in animals immunized and cross-challenged with either F98 or D74 glioma cells. Lymph node lymphocytes and TIL, isolated from animals immunized and challenged with F98 cells, were more cytolytically active than effector cells obtained from D74-immunized animals. Phenotypes of TIL isolated from intracerebral F98 gliomas of immunized rats were 52% OX-8+ and 21% W3/25+ compared to 31% OX-8+ and 19% W3/25+ for D74-immunized animals. Cytolytic activity against glioma targets was mediated by OX-8+ TIL, as determined by cell depletion experiments. Limiting dilution analysis showed that cytolytic T-lymphocyte precursors were present in TIL of F98 gliomas of immunized rats at a frequency of 1/3547 and were specific for F98 targets, while natural killer cell-like activity was low. Our data indicate that the F98 glioma was more immunogenic than the D74 glioma, as evidenced by increased numbers and activity of cytolytic effector cells and their precursors among TIL. This may explain in part the longer survival times observed in immunized animals challenged intracerebrally with the F98 gliomas compared to D74-immunized and -challenged hosts.     

Adoptive T-cell therapy using autologous tumor-infiltrating lymphocytes for metastatic melanoma: current status and future outlook

Immunotherapy using autologous T cells has emerged to be a powerful treatment option for patients with metastatic melanoma. These include the adoptive transfer of autologous tumor-infiltrating lymphocytes (TILs), T cells transduced with high-affinity T cell receptors against major tumor antigens, and T cells transduced with chimeric antigen receptors composed of hybrid immunoglobulin light chains with endodomains of T-cell signaling molecules. Among these and other options for T-cell therapy, TILs together with high-dose interleukin 2 have had the longest clinical history with multiple clinical trials in centers across the world consistently demonstrating durable clinical response rates near 50% or more. A distinct advantage of TIL therapy making it still the T-cell therapy of choice is the broad nature of the T-cell recognition against both defined and undefined tumors antigens against all possible major histocompatibility complex, rather than the single specificity and limited major histocompatibility complex coverage of the newer T cell receptors and chimeric antigen receptor transduction technologies. In the past decade, significant inroads have been made in defining the phenotypes of T cells in TIL-mediating tumor regression. CD8+ T cells are emerging to be critical, although the exact subset of CD8+ T cells exhibiting the highest clinical activity in terms of memory and effector markers is still controversial. We present a model in which both effector-memory and more differentiated effector T cells ultimately may need to cooperate to mediate long-term tumor control in responding patients. Although TIL therapy has shown great potential to treat metastatic melanoma, a number of issues have emerged that need to be addressed to bring it more into the mainstream of melanoma care. First, we have a reached the point where a pivotal phase II or phase III trial is needed in an attempt to gain regulatory approval of TILs as standard of care. Second, improvements in how we expand TILs for therapy are needed that minimize the time the T cells are in culture and improve the memory and effector characteristics of the T cells for longer persistence and enhanced anti-tumor activity in vivo. Third, there is a critical need to identify surrogate and predictive biomarkers to better select suitable patients for TIL therapy to improve response rate and duration. Overall, the outlook for TIL therapy for melanoma is very bright. We predict that TILs will indeed emerge to become an approved treatment in the upcoming years through pivotal clinical trials. Moreover, new approaches combining TILs with targeted signaling pathway drugs, such as mutant B-RAF inhibitors, and synergistic immunomodulatory interventions enhancing T-cell costimulation and preventing negative regulation should further increase therapeutic efficacy and durable complete response rates.     

Interleukin 2 expanded tumor-infiltrating lymphocytes in human renal cell cancer: isolation, characterization, and antitumor activity

We have described a method for the generation, from fresh human renal cell cancers, of lymphoid cells that are capable of exhibiting significant antitumor reactivity when tested in short term 51Cr release assays. Tumor cell suspensions obtained from 37 consecutive fresh human renal cell cancer specimens (35 patients) could be separated by using enzymatic techniques and culturing in medium containing recombinant interleukin 2 (IL-2). The total cell recovery was 1.5 X 10(9) +/- 2.2 (SE) per tumor with a range of 1 X 10(8) to 5 X 10(9) cells. The percentage of tumor cells in the suspension ranged from 6 to 75% with a mean of 39.1 +/- 3.3%. The remaining cells were predominantly lymphocytes. Viability of mononuclear cells was greater than 90%. Activated tumor-infiltrating lymphocytes (TIL) within these tumors expand and by 10 to 14 days after initiation of culture a 5- to 15-fold increase in the number of lymphocytes could be achieved with elimination of all autologous tumor cells. Lymphocytes were recultured in fresh medium containing IL-2 and continued to expand between 2- and 10-fold every 4 to 6 days for an average of 33.7 +/- 4.5 days, resulting in greater than 50,000-fold increase in the total number of lymphocytes. The average number of splits was 4.9 +/- 0.8, with a range of 0 to 21. In 11 of 11 cases tested, TIL exhibited a far better expansion capability in vitro compared to that of peripheral blood lymphocytes obtained from the same patient and grown under identical conditions. The majority of TIL were T cytotoxic/suppressor cells (Leu 2+ Leu 4+). With continued in vitro expansion (up to 50 days) there was a concomitant increase in the helper T (Leu 3+) and pan T populations (Leu 4+) and decrease in Leu 2+ and HLA-DR+ cells. Compared with expanded peripheral blood lymphocytes, these cells demonstrated higher levels of IL-2+ receptors and HLA-DR+ antigens. Renal TIL effectors expanded in IL-2 could lyse almost all autologous tumor targets in 4-h chromium release assays. Allogeneic renal as well as nonrenal targets were equally lysed. TIL lysis of cultured tumor targets K562 and Daudi was significantly better than lysis of autologous, allogeneic-renal, and nonrenal targets. No statistically significant difference in the cytotoxic activity of renal TIL or peripheral blood lymphocyte effectors in killing autologous or allogeneic targets could be demonstrated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of autologous tumor-specific T-helper 2 cells in tumor-infiltrating lymphocytes from a patient with metastatic melanoma

Human autologous tumor-specific T-helper 2 (Th2) cells were investigated in melanoma tumor-infiltrating lymphocytes (TILs). Both a CD4⁺ T-cell line and its 5 potential T-cell clones established from TILs of a patient with metastatic melanoma produced significant levels of IL-4, IL-6, IL-10 and granulocyte-macrophage colony-stimulating factor (GM-CSF) in response to autologous, but not any of 12 allogeneic, melanoma cell lines. They also produced IL-3 and IL-8 but not IL-2, IFN-γ, TNF-α or TNF-β in response to autologous tumor cells. Furthermore, they showed autologous melanoma-specific cytotoxicity only in an 18-hr ⁵¹Cr-release assay. Specific IL-4, IL-6 or IL-10 production by the CD4⁺ M73 T-cell line and its clone was inhibited by anti-class 11 DR (but not anti-class 1) MAb, whereas their specific cytotoxicity was inhibited by anti-class 1 (but not anti-class 11) MAb. Anti-CD3 and -CD4 MAb (but not anti-CD8) abrogated both IL-4, IL6 and IL-10 production and cytotoxicity, while anti-IL-4 antibody did not inhibit cytotoxicity. CD4⁺ potential T-cell clones, but not CD8⁺ clones, that were established from freshly isolated TILs without in vitro sensitization by autologous tumor cells also produced IL-4, IL-6 and IL-10 but not IFN-γ or tumor necrosis factor (TNF)α in an autologous tumor-specific fashion. These Th2 cells were neither reactive to EBV-B cells nor suppressive against CD8⁺ T-cell clones. PMA and PHA stimulated these potential T-cell clones, regardless of their specific lymphokine production, to produce IL-3, IL-4, IL-6, IL-8, IL-10, GM-CSF, TNFα and IFN-γ. Our results demonstrate the presence of autologous tumor-specific Th2 cells at the melanoma sites.     

Telomerase activity and telomere length in lymphocytes from patients with cutaneous T-cell lymphoma.

BACKGROUND: Telomeres shorten with successive cell divisions in normal somatic cells. Telomerase is a ribonucleoprotein enzyme associated with cellular proliferation and plays an important role in maintaining the stability of chromosomes and the length of DNA telomeres. Telomerase activity has been detected in tissues from many human tumors, but is not present in the majority of normal tissues. Thus, measurement of telomerase activity and telomere length may contribute to understanding the mechanism of tumorigenesis and provide useful diagnostic or prognostic information. The aim of this study was to investigate the telomerase activity and telomere length from patients with cutaneous T-cell lymphoma (CTCL). METHODS: Eighteen skin-homing T-cell lines were established from skin biopsies and 10 peripheral blood mononuclear cells (PBMC) were isolated from patients with various stages of CTCL together with 22 PBMC from healthy donors. For each sample an identical amount of cellular protein was measured quantitatively for telomerase activity using the telomerase polymerase chain reaction-enzyme-linked immunosorbent assay based on the telomeric repeat amplification protocol method. Telomere length was assayed using a commercial kit. RESULTS: Eight of ten PBMC and 16 of 18 skin-homing T-cell lines from patients with CTCL showed moderate to strong telomerase activity. Freshly obtained PBMC from healthy donors showed weak levels of telomerase activity. A shorter telomere length was found in cell lines and PBMC from patients with CTCL compared with healthy controls. Four skin-homing T-cell lines going into growth crisis showed sharply reduced telomerase activity. CONCLUSIONS: The results of the current study indicate that both skin-homing T-cells and PBMC from CTCL have high telomerase activity and short telomere length. These changes are similar to the changes observed in the majority of malignant cells including other types of T-cell lymphoma. It is interesting to note that even in the very early stages of CTCL such as parapsoriasis (which is a clinically benign disease) the changes already are present, indicating that a significantly high level of telomerase activity frequently occurs in CTCL and may be an important event in tumorigenesis. Telomerase activity and telomere length are useful markers for CTCL risk assessment.     

T-cell receptor v-Beta usage of tumor-infiltrating lymphocyte lines cloned from human breast-tumor and melanoma

A total of forty-one tumor infiltrating T cell lines (TIL) were cloned, in the presence of interleukin-2, from nine breast tumor and five melanoma specimens with limiting dilution in a microculture system. Nineteen (46%) of the lines/clones reacted to autologous tumor targets. The T cell receptor (TcR) V beta gene usage was analyzed using polymerase chain reaction (PCR) technique and a set of oligonucleotide primers specific for 20 V beta families. T cell lines generated from paired peripheral blood lymphocytes (PBL) under similar condition were used as control. Our data revealed a limited heterogeneity in TcR V beta gene usage with a biased expression of V beta 6 in both breast tumor- and melanoma-derived TIL lines/clones. In contrast, a random pattern of TcR V beta usage was observed in 27 control T cell lines derived from PBL of patients with breast cancer and melanoma. The results lend support to oligoclonal expansion of TIL at tumor sites but fail to directly correlate the preferential expression of V beta 6 with the functional property of the TIL in recognition of tumor antigens.     

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.     

Generation of tumour-specific cytotoxic T-cell clones from histocompatibility leucocyte antigen-identical siblings of patients with melanoma

Lymphodepletion and infusion of autologous expanded tumour-infiltrating lymphocytes is effective therapy for patients with malignant melanoma. Antitumour responses are likely to be mediated by HLA class I- and II-restricted immune responses directed at tumour antigens. We assessed whether the peripheral blood of normal HLA-matched siblings of patients with melanoma could be used to generate lymphocytes with antimelanoma activity for adoptive immunotherapy after allogeneic blood or marrow transplantation. Melanoma cell lines were derived from two donors and were used to stimulate the mononuclear cells of three HLA-identical siblings. CD4(+) clones dominated cultures. Of these, approximately half were directly cytotoxic towards recipient melanoma cells and secreted interferon-gamma in response to tumour stimulation. More than half of the noncytotoxic clones also secreted interferon-gamma after melanoma stimulation. No CD4(+) clones responded to stimulation with recipient haemopoietic cells. The majority of CD8(+) clones directly lysed recipient melanoma, but did not persist in long-term culture in vitro. No crossreactivity with recipient haemopoietic cells was observed. The antigenic target of one CD4(+) clone was determined to be an HLA-DR11-restricted MAGE-3 epitope. Antigenic targets of the remaining clones were not elucidated, but appeared to be restricted through a non-HLA-DR class II molecule. We conclude that the blood of allogeneic HLA-matched sibling donors contains melanoma-reactive lymphocyte precursors directed at tumour-associated antigens. Adoptive immunotherapy with unselected or ex vivo-stimulated donor lymphocytes after allogeneic stem cell transplantation has a rational basis for the treatment of malignant melanoma.     

Identification of colon-tumor-associated antigens by T-cell lines derived from tumor-infiltrating lymphocytes and peripheral-blood lymphocytes from patients immunized with an autologous tumor-cell/bacillus calmette-Guérin vaccine

Tumor immunity developing as a response to an autologous colon-tumor/bacillus Calmette-Guerin (BCG) vaccine appears to be associated with induction of CD4 helper T cells, implied by the observation that vaccine efficacy is associated with major histocompatibility complex class-ll molecule expression on the vaccine tumor cells. Therefore, in an attempt to identify colon-tumor-associated antigens responsible for conferring immunity, we examined and compared the proliferative responses of peripheral-blood lymphocytes (PBL) from patients immunized with the autologous tumor/BCG vaccine to T-cell lines cloned expanded from colon-tumor-infiltrating lymphocytes to 5 antigens isolated on the basis of their reactivity by colon-tumor-reactive human monoclonal antibodies. Enzymatically dissociated colon tumors provided a source for establishment of cloned T-cell lines, tumor cell lines propagated in vitro or in vivo as nude-mouse xenografts and EBV-transformed B-cell lines used as antigen-presenting cells. Of 104 different T-cell lines tested, only 3 proliferated in response to CTAA 28A32-46K, and I to the CTAA28A32-32K antigen. In contrast, PBL from 64% of patients immunized with the autologous colon-tumor/BCG vaccine responded to the CTAA 28A32-32K antigen. This antigen is related to a family of calcium- and phospholipid-binding placental proteins termed annexins. Since proliferative responses developed to this antigen after vaccination in 64% of individuals, this antigen may be an important common colon-tumor-associated rejection antigen.     

Continuous interleukin-2 and tumor-infiltrating lymphocytes as treatment of advanced melanoma. A national biotherapy study group trial

Melanoma metastases were harvested from 82 patients for the purpose of growing and expanding tumor-infiltrating lymphocytes (TIL). Tumor tissue cell suspensions were incubated with interleukin-2 (IL-2), followed by repeated exposure to tumor antigen with or without OKT3 monoclonal antibody (MoAb). Initial growth success was achieved in 56 of 82 cultures (72%). Efforts were made to expand 26 of these 56 cultures for therapeutic TIL; 23 of 26 early cultures (88%) were successfully expanded for in vivo therapy. It took a mean of 78.5 +/- 25.4 days to grow sufficient TIL for treatment. Therapy included cyclophosphamide (1 g/m2) on day 1, followed by a 96-hour continuous infusion of IL-2 (18 x 10(6) IU/m2/d) on days 2 to 5, and approximately 10(11) (mean 1.49 +/- 0.93 x 10(11)) TIL on day 2. Patients who responded received monthly IL-2 as a 96-hour infusion. Median patient age was 45 years of age. Sixty-seven percent of the patients were men. Performance status was 0 to 1 in 77% of patients. Thirty-four percent of the patients had liver metastases. The usual IL-2 toxicities were seen. Response rate for 21 patients was 24% (95% confidence interval, 10% to 49%). One complete response was achieved with cells 98% CD4+; four partial responses were achieved with cells 80%, 94%, 98%, and 98% CD8+, respectively. Four of eight patients who received TIL, which had never been stimulated with OKT3, had tumor response. The authors conclude that a treatment plan for IL-2/TIL is technically difficult, costly, and effective for only a minority of patients. Overall, clinical results are not clearly superior to those obtained with other IL-2 regimens.