Clonal analysis and in situ characterization of lymphocytes infiltrating human breast carcinomas

Summary T lymphocytes were isolated from tumor biopsies in 13 patients with breast carcinomas. Immunohistology with monoclonal antibodies confirmed the presence of mononuclear cell infiltrates composed primarily of T lymphocytes in all tumors studied. While the proportion of T lymphocytes expressing the T4 or the T8 surface marker varied from tumor to tumor as determined by morphometric analysis, T8+ cells were more numerous than T4+ cells in 8/12 breast tumors studied. Relatively few T cells (<10% in 11/12 tumors) were in an activated state as judged by the surface expression of HLA-DR antigens or the receptor for interleukin-2 (IL-2). In 1 case 20% of the infiltrating mononuclear cells were expressing the IL-2 receptor. The tumor infiltrating lymphocytes (TIL) recovered from 10 tumors were cloned in a microculture system that permits proliferation of nearly 100% of normal peripheral blood T lymphocytes (PBL-T). In contrast to normal and autologous PBL-T, frequencies of proliferating T lymphocyte precursors (PTL-P) were depressed (<0.01) in 7/10 TIL preparations indicating a decreased responsiveness of TIL to phytohemagglutinin at the single-cell level. The frequency of PTL-P was noticeably higher in 2 cases (0.03 and 0.09) and close to normal in 1 case (0.39).A total of 170 clones were expanded in vitro and analyzed for different functional capabilities. Most of these clones expressed the T4+/T8-phenotype (73%) and strikingly 53% of these T4+/T8– clones were cytolytic in a lectin-dependent assay, a functional subset which is uncommon among normal PBL-T. Some clones (10%) lysed allogeneic breast tumor cells (MCF7). Only 15% of the clones displayed natural killer activity. Among the cytolytic clones, 17 of 31 tested were also IL-2 producers irrespective of the T4 or T8 phenotype. Our results show that human mammary carcinomas contain many infiltrating T cells with cytolytic potential. Interestingly, among the proliferating cytolytic T cell clones (56% of the microcultures), many expressed the T4+/T8– phenotype. These findings may indicate that the in situ cytolytic reaction (against unknown antigens) is associated preferentially with class II antigens.Fogarty International Fellow of NIH, 1984–1985; on leave from Dept of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pa, USA     

Characterization of the CD4+ and CD8+ tumor infiltrating lymphocytes propagated with bispecific monoclonal antibodies

To study the CD4+ and CD8+ tumor infiltrating lymphocytes (TIL) in the antitumor response, we propagated these subsets directly from tumor tissues with anti-CD3:anti-CD8 (CD3,8) and anti-CD3:anti-CD4 (CD3,4) bispecific mAb (BSMAB). CD3,8 BSMAB cause selective cytolysis of CD8+ lymphocytes by bridging the CD8 molecules of target lymphocytes to the CD3 molecular complex of cytolytic T lymphocytes with concurrent activation and proliferation of residual CD3+CD4+ T lymphocytes. Similarly, CD3,4 BSMAB cause selective lysis of CD4+ lymphocytes whereas concurrently activating the residual CD3+CD8+ T cells. Small tumor fragments from four malignant melanoma and three renal cell carcinoma patients were cultured in medium containing CD3,8 + IL-2, CD3,4 + IL-2, or IL-2 alone. CD3,8 led to selective propagation of the CD4+ TIL whereas CD3,4 led to selective propagation of the CD8+ TIL from each of the tumors. The phenotypes of the TIL subset cultures were generally stable when assayed over a 1 to 3 months period and after further expansion with anti-CD3 mAb or lectins. Specific 51Cr release of labeled target cells that were bridged to the CD3 molecular complexes of TIL suggested that both CD4+ and CD8+ TIL cultures have the capacity of mediating cytolysis via their Ti/CD3 TCR complexes. In addition, both CD4+ and CD8+ TIL cultures from most patients caused substantial (greater than 20%) lysis of the NK-sensitive K562 cell line. The majority of CD4+ but not CD8+ TIL cultures also produced substantial lysis of the NK-resistant Daudi cell line. Lysis of the autologous tumor by the TIL subsets was assessed in two patients with malignant melanoma. The CD8+ TIL from one tumor demonstrated cytotoxic activity against the autologous tumor but negligible lysis of allogeneic melanoma targets. In conclusion, immunocompetent CD4+ and CD8+ TIL subsets can be isolated and expanded directly from small tumor fragments of malignant melanoma and renal cell carcinoma using BSMAB. The resultant TIL subsets can be further expanded for detailed studies or for adoptive immunotherapy.     

Role of human circulating and tumor-infiltrating lymphocytes in cancer defense and treatment

We have analyzed the anticancer efficacy of various subsets of human circulating and tumor-infiltrating lymphocytes (TIL). These studies showed that circulating natural killer (NK) cells mediate the most potent oncolytic activity against a variety of tumor targets, after enrichment or stimulation with interleukin-2 (IL-2). Interestingly, NK cell oncolytic activity was directed also against tumor targets frequently designated as 'NK-resistant'. This indicates that NK cells display a broader spectrum of killing than is commonly recognized. TIL did not display any tumoricidal activity when unstimulated, but acquired cytotoxic potential after activation with IL-2. Comparative studies of TIL and circulating lymphocytes from patients with ovarian cancer demonstrated that these two groups of lymphocytes manifested similar levels of cytotoxicity and the same spectrum of target cell killing. No specificity in autologous tumor cell killing was displayed by TIL; instead, TIL were effective against autologous as well as allogeneic tumor targets. The lack of TIL tumor specificity was not detected only in ovarian tumors, but was manifested also in renal- and squamous-cell cancers. Characterization studies demonstrated that the primary oncolytic cells in the periphery and among TIL are NK cells. T lymphocytes displayed some, but rather negligible cytotoxic activity. In contrast, when IL-2-activated NK and T cells were analyzed for lytic activity against normal hematopoietic cells, T cells displayed high levels of bone marrow killing. The anti-bone marrow lytic activity of IL-2-activated T lymphocytes may be harmful after therapy with conventionally prepared lymphokine-activated killers. In light of these observations, new directions to adoptive immunotherapy are discussed.     

Phenotypic characterization of murine tumor-infiltrating T lymphocytes.

Tumor infiltrating lymphocytes (TIL) can be isolated from solid tumors and selectively expanded in long term culture with IL-2 and autologous irradiated tumor. Such long term cultured cells express anti-tumor activity in vitro, mediate the regression of established tumor in murine models of cancer, and have been used for the treatment of cancer in humans. We have characterized freshly isolated mouse Thy-1+ TIL populations, as well as long term TIL cultures, from several different C57BL/6 (B6) tumors. Freshly isolated Thy-1+ TIL include both CD4+ and CD8+ cells, as well as cells bearing NK markers. These cells are predominantly TCR alpha beta+, with a smaller population of TCR gamma delta+ cells. The TCR alpha beta+ cells expressed a broad distribution of V beta phenotypes that was statistically different from that expressed in normal B6 splenic Thy-1+ cells or CD8+ cells, presumably reflecting in vivo selection in the host anti-tumor response. NK cells are present in these tumors at a greater frequency than noted in splenic T cells. Cultured TIL populations rapidly became exclusively Thy-1+/CD8+/CD4- and TCR alpha beta+/gamma delta-. Individual long term TIL populations initially expressed multiple V beta products, but rapidly restricted their V beta expression, frequently expressing a single dominant V beta. The identity of this dominant V beta varied among different TIL lines, but the overall representation of V beta phenotypes in these cultures was statistically different from that seen in Thy-1+ or CD8+ splenocytes. No statistical difference was noted between lines derived from antigenically distinct tumors. The selection of tumor specific T cells in vitro is therefore not reflected in any simple predominance of V beta usage. The complexity of TCR usage in the anti-tumor response may result from the involvement of multiple alpha- and beta-chain regions in the response to a single antigenic determinant, or may reflect multiple antigenic determinants expressed on a single syngeneic tumor.     

Randomized trial of adoptive transfer of melanoma tumor-infiltrating lymphocytes as adjuvant therapy for stage III melanoma

The aim of this study was to demonstrate the interest of using tumor-infiltrating lymphocytes (TIL) as adjuvant therapy for stage III (regional lymph nodes) melanoma. After lymph node excision, patients without any detectable metastases were randomly assigned to receive either TIL plus interleukin-2 (IL-2) for 2 months, or IL-2 only. The primary endpoint was determination of the duration of the relapse-free interval. Eighty-eight patients determined as eligible for treatment were enrolled in the study. After a median follow-up of 46.9 months, for the study population the analysis did not show a significant extension of the relapse-free interval or overall survival. However, a significant interaction ( P<0.001) was found between the treatment and the number of invaded lymph nodes. In the group with only one invaded lymph node, the estimated relapse rate was significantly lower ( P(adjusted)=0.0285) and the overall survival was increased ( P(adjusted)=0.039) in the TIL+IL-2 arm compared with the IL-2 only arm. No differences between the two arms, either as regards the duration of disease-free survival or overall survival, were noted in the group with more than one invaded lymph node whatever the number of invaded lymph nodes. Treatment was compatible with normal daily activity. This study demonstrates for the first time that the efficiency of TIL in stage III melanoma (AJCC) is directly related to the number of invaded lymph nodes, indicating that tumor burden might be a crucial factor in the efficacy and/or in vitro expansion of T cells specific for autologous tumor antigen, a finding which could be of value in future vaccine development for the treatment of melanoma.     

CD8+ tumor-infiltrating lymphocytes are primed for Fas-mediated activation-induced cell death but are not apoptotic in situ

Induction of Fas-mediated activation-induced cell death in antitumor T cells has been hypothesized to permit tumor escape from immune destruction. Several laboratories have proposed that expression of Fas ligand (L) by tumor is the basis for this form of T cell tolerance. In this study, we characterized murine tumor-infiltrating lymphocytes (TIL) for activation status, cell cycle status, level of apoptosis, cytokine secretion, and proliferative capacity. TILs express multiple activation markers (circa CD69, CD95L, CD122, and LFA-1) and contain IL-2 and IFN-gamma mRNAs, but are neither cycling nor apoptotic in situ. In addition, TIL are dramatically suppressed in proliferative response and do not secrete IL-2 and IFN-gamma. However, upon purification and activation in vitro, TIL secrete high levels of IL-2 and IFN-gamma, enter S phase, and then die by Fas-mediated apoptosis. Activation by injection of anti-TCR Ab or IL-2 into tumor-bearing mice induced TIL entrance into S phase preceding apoptosis, showing that TIL have functional TCR-mediated signal transduction in situ. Our data demonstrate that TIL, not tumor, express both Fas and FasL, are arrested in G(1), do not secrete cytokine in situ, and, upon activation in vitro and in vivo, rapidly die by activation-induced cell death.     

Generation of lymphokine-activated killer (LAK) cells from tumor-infiltrating lymphocytes

Culture of tumor-infiltrating lymphocytes (TIL) containing about 20% BMC2 tumor cells with recombinant human interleukin 2 (rIL-2) resulted in the diminish of tumor cells and the growth of lymphocytes. These IL-2-activated lymphocytes showed a strong cytotoxic activity against not only syngeneic tumor cells but also allogeneic tumor cells. Such broad-reactive killer cells, termed lymphokine-activated killer (LAK) cells, are also inducible from spleen cells by in vitro activation with IL-2. However, LAK cells generated from TIL (TIL-LAK) showed higher cytotoxic activity against BMC2 than LAK cells generated from spleen cells (S-LAK). Furthermore, it was demonstrated that TIL-LAK cells revealed marginal cytotoxic activity against normal Con A blasts and YAC-1 cells as opposed to S-LAK. Flow cytometric analysis of TIL-LAK indicated that TIL-LAK cells mainly consisted of Thy 1.2+, Ly 2+, asialo GM1+ cells. TIL-LAK cells displayed not only in vitro cytotoxicity but also in vivo anti-tumor activity. Furthermore, it was also confirmed that TIL-LAK cells could be induced in autochthonous mouse tumor systems and human gastric tumor systems.     

Long-term survival of adoptively transferred tumor-infiltrating lymphocytes in mice

We examined the long term survival of adoptively transferred, cultured tumor-infiltrating lymphocytes (TIL) in sublethally irradiated nontumor-bearing mice. TIL were produced by culturing Thy-1-enriched single cell suspensions from a variety of murine tumors in exogenous IL-2 along with irradiated tumor cells and splenocytes. TIL were adoptively transferred i.v. to mice and such animals demonstrated a statistically significant degree of protection from a subsequent i.v. tumor challenge. This protection lasted up to 6 wk and occurred in the absence of exogenous IL-2 administration in vivo. Using congenic B6.PL Thy-1 alpha/CY mice we demonstrated directly that TIL can survive in vivo for up to 6 wk and that the adoptively transferred TIL were the source of the relative immunity to subsequent tumor challenge. We conclude that TIL contain cells with substantial functional longevity in vivo even in the absence of exogenous IL-2. These studies are relevant to ongoing clinical trials of TIL as a therapy for patients with advanced cancer.     

Long-term follow-up of patients treated by adoptive transfer of melanoma tumor-infiltrating lymphocytes as adjuvant therapy for stage III melanoma

The first analysis of our clinical trial on interest of using tumor-infiltrating lymphocytes (TIL) as adjuvant therapy for stage III (regional lymph nodes) melanoma was published in 2002 [5]. The aim of this paper is to update clinical results of 7 years of follow-up after the last treated patient. In the trial conducted between December 1993 and January 1999, patients without any detectable metastases after lymph node excision were randomly assigned to receive either TIL plus interleukin-2 (IL-2) for 2 months, or IL-2 only. The duration of the relapse-free interval was the primary objective. Eighty-eight patients were enrolled in the study. Currently, the last analysis performed in June 2006, after a median follow-up of 114.8 months, did not show change of non-significant extension of the relapse-free interval or overall survival. However, this second analysis strengthens our first hypothesis about the relationship between number of invaded lymph nodes and TIL treatment effectiveness. In the group with only one invaded lymph node, the estimated relapse rate was significantly lower (P _adjusted = 0.0219) and the overall survival was increased (P _adjusted = 0.0125) in the TIL+IL-2 arm compared with the IL-2 only arm. No differences between the two arms, either with regard to the duration of disease-free survival (P _adjusted = 0.38) or overall survival (P _adjusted = 0.43), were noted in the group with more than one invaded lymph node, whatever the number of invaded lymph nodes. Treatment was compatible with normal daily activity. This study, with a very long follow up (median of almost 10 years), postulates for the first time relationship between TIL efficiency in stage III melanoma (AJCC) and number of invaded lymph nodes, indicating that tumor burden might be a crucial factor in the production of an effective in vitro expansion of T cells specific for autologous tumor antigen, a finding which could be of value in future vaccine development for the treatment of melanoma.     

IL-4-transduced tumor cell vaccine induces immunoregulatory type 2 CD8 T lymphocytes that cure lung metastases upon adoptive transfer.

Vaccinations with tumor cells engineered to produce IL-4 prolonged survival and cured 30% of mice bearing pulmonary metastases, an effect abrogated by in vivo depletion of T cells. Vaccination induced type 2 T cell polarization in both CD4 and CD8 T lymphocyte subsets. We focused on the antitumor activity exerted by type 2 CD8+ T cells (Tc2) activated by IL-4 tumor cell vaccination. Tc2 lymphocytes lacked in vitro tumor cytotoxicity, but released IL-4 upon stimulation with tumor cells, as shown by limiting dilution analysis of the frequencies of tumor-specific pCTL and of CD8 cells producing the cytokine. In vivo fresh purified CD8+ T lymphocytes from IL-4-vaccinated mice eliminated 80-100% of lung metastases when transferred into tumor-bearing mice. CD8+ lymphocytes from IL-4-vaccinated IFN-gamma knockout (KO), but not from IL-4 KO, mice cured lung metastases, thus indicating that IL-4 produced by Tc2 cells was instrumental for tumor rejection. The antitumor effect of adoptively transferred Tc2 lymphocytes needed host CD8 T cells and AsGM1 leukocyte populations, and partially granulocytes. These data indicate that Tc2 CD8+ T cells exert immunoregulatory functions and induce tumor rejection through the cooperation of bystander lymphoid effector cells. Tumor eradication is thus not restricted to a type 1 response, but can also be mediated by a type 2 biased T cell response.     

Cellular characteristics of peripheral blood lymphocytes and tumour-infiltrating lymphocytes in patients with gynaecological tumours

 Immunotherapy of gynaecological cancer with tumour-infiltrating lymphocytes (TIL) or peripheral blood lymphocytes (PBL) has become a valid treatment modality with varying degrees of success in obtaining an antitumour response. TIL consist of lymphocytes, mainly T cells and minor populations of natural killer cells or B cells. Conventional cytogenetic studies of tumour cells from patients with breast and ovarian cancer have shown multiple chromosomal abnormalities including chromosomes 7 and 12. This study was designed to analyse the surface further, as well as investigate the intracellular, characteristics of TIL by multicolour flow cytometry and the cytogenetic features by fluorescence in situ hybridization. Tumour cell, peripheral blood and TIL samples from 25 patients (15 ovarian tumours, 8 breast cancers, 1 uterine sarcoma, 1 cervical carcinoma) were analysed for their phenotype, the expression of major cytokines [interleukin-2 (IL-2), IL-4 and interferon γ (IFNγ)], their proliferation rate, their cytotoxic ability and for the presence of numerical aberrations of chromosomes 7 and 12. All the tumour cells showed a high frequency of numerical aberration in chromosomes 7 and 12, especially trisomies or tetrasomies and combined aberrations. Trisomies of both chromosomes also occured at a low percentage in TIL and PBL. Received: 20 June 1996 / Accepted: 4 January 1997     

The induction of murine tumor infiltrating lymphocytes (TIL) by interleukin-2 or T cell growth factor

Mice were injected in the foot pad with either 5×10⁵ syngeneic plasmacytoma (MOPC104E) or fibrosarcoma cells (Meth A). Lymph nodes containing tumor cells were harvested 14 days later and cultured. In the presence of recombinant interleukin-2 (r-IL-2) predominantly tumor cells proliferated. Culture with T cell growth factor (TCGF) resulted in the growth of lymphoid cells. Concanavalin A (Con A) had only a modest effect on elimination of tumor cells in the culture. Tumor-infiltrating lymphocytes (TIL) prepared from the lymph nodes showed specific tumor-neutralizing activity when grown in the presence of TCGF. In vitro examination revealed that Meth A cells could not be lysed by TIL, while TIL from MOPC tumors showed tumor specific activity. This study may explain negative results in human trials with TIL induced by IL-2 alone.Abbreviations r recombinant - IL-2 interleukin-2 - TCGF T cell growth factor - TIL tumor infiltrating lymphocytes - Con A concanavalin A - HBSS Hanks' balanced salt solution     

Interleukin-2 expanded lymphocytes from lymph node and tumor biopsies of human renal cell carcinoma, breast and ovarian cancer

Adoptive immunotherapy with immune effector cells has proved to be potent for treatment of tumors, however neither the attendant criteria for potential clinical efficacy of the injected cells, nor the method to prepare these cells are presently well established. Our procedure of collecting lymphocytes from biological samples, was based on the use of low IL-2 concentrations (90 to 150 IU/ml) and on the stringent separation of lymphocytes from tumor cells at the very early stages of their outgrowth in culture. When lymphocytes were derived from tumor biopsies (TIL), we observed differences depending on the histological type of tumor. In renal cell carcinoma, natural killer cells were expanded in 4/11 biopsies contrary to what was observed in breast cancer (92 +/- 5% of T lymphocytes from 9 biopsies). The outgrowth of lymphocytes from breast tumors was slower and lower than from renal carcinomas. The autologous tumor cell line was more difficult to obtain from breast carcinoma (23%) than from renal cell carcinoma (61%) biopsies. For ovarian cancer, short-term culture of tumor cells could be obtained for half of the tumor-invaded biological samples. Eight of the 23 tumor-derived cultures contained more than 40% CD8 T. TIL were consistently cytolytic each time they could be evaluated. For ascitic and pleural fluids, data were of similar range. In ascitic-derived cultures, tumor cells and antigen-presenting cells are present and can be supposed to rechallenge T cells with tumor antigens. Lymphocytes derived from lymph nodes could be expanded to a larger number than TIL. However, only 1/18 of these cultures contained more than 40% CD8 T. The presence of few tumor cells in this culture was in favor of significant specific and non-specific cytotoxicity in RCC lymph node cultures and higher percentages of CD8 T in breast cancer lymph nodes. Correlations could not be established between CD8 T percentages and specific in vitro cytotoxicity in our polyclonal populations. Our conclusion is that phenotypic and functional quality of lymphocytes is of interest when the T cells are derived 1) from tumors (RCC, breast or ovarian cancer) and isolated very early to avoid inhibitor factors secreted from tumor cells or 2) from lymph nodes and ascitic and pleural fluids when very few tumor cells are co-cultivated with lymphocytes at initial steps of culture. Final expansion to a number of lymphocytes suitable for therapy (> 109) could be attained in a second step of the procedure by the use of 1,000 IU/ml IL-2 each time it was assayed with 50.106 lymphocytes. In view of these data it appears that phenotypic and functional changes occur during culture depending on the presence of a particular ratio of tumor antigens. This could be artificially reproduced.     

Murine lymphocytes exhibit heterogeneity in their proliferative responsiveness to calcium ionophore.

The calcium ionophore, A23187, when used alone was found to induce proliferation of murine T cells, at concentrations of 0.5-1 mM. This response required the presence of syngeneic splenic adherant cells (SAC) as a source of accessory cells. Interestingly, only CD4+ T cells but not CD8+ T cells or B cells responded to the calcium ionophore by proliferation. The inability of CD8+ T cells or B cells to respond was not related to decreased elevation in the intracellular ionized calcium [Ca2+]i concentration induced by the ionophore, because activated CD4+ T, CD8+ T and B cells all exhibited similar elevation in [Ca2+]i. The inability of CD8+ T cells to respond to calcium ionophore was probably due to insufficient production of autocrine growth factors, such as IL-2, inasmuch as the addition of exogenous IL-2 could completely restore the CD8+ T cell responsiveness. Also, exogenous rIL-1 could partially restore purified T cell response to calcium ionophore, whereas, rIL-6 failed to do so. IL-2, but not IL-4, acted as an autocrine growth factor for T cells responding to the calcium ionophore in the presence of SAC, since, antibodies against IL-2 or IL-2 receptor (IL-2R) but not against IL-4, could inhibit the T cell proliferation. Furthermore, exogenous rIL-2 but not rIL-4 supported the proliferation of T cells to calcium ionophore in the absence of accessory cells. Our results suggest that murine lymphocytes exhibit heterogeneity in their proliferative responsiveness to calcium ionophore and that this may not depend on the early activation signal such as the elevation in [Ca2+]i) induced by the ionophore but may depend on subsequent signals which regulate endogenous growth factor production.     

Lymphocytes infiltrating human ovarian tumors. I. Role of Leu-19 (NKH1)-positive recombinant IL-2-activated cultures of lymphocytes infiltrating human ovarian tumors

Tumor-infiltrating lymphocytes (TIL) were obtained from human ovarian tumors, expanded in the presence of IL-2 in culture and studied for cytotoxicity against fresh autologous and allogeneic ovarian carcinoma (CA) targets. TIL from ovarian tumors grew well in long term cultures, achieving from 8- to 682-fold expansion. TIL cultured with IL-2 were cytotoxic against both autologous and allogeneic fresh ovarian CA targets, and no specificity for autologous tumor could be demonstrated in any of the cultures. In all fresh TIL preparations, CD3+ lymphocytes were the major cell type and contained a high proportion (up to 51%) of activated (IL-2R+) cells as determined by two-color flow cytometry. Sorting of bulk TIL cultures followed by cytotoxicity assays identified the Leu-19+ cells, both CD3+ and CD3-, as effectors of cytotoxicity against autologous and allogeneic tumor cell targets. Cold target inhibition assays showed that allogeneic targets (both ovarian CA and a sarcoma) competed effectively with autologous ovarian CA targets for Leu-19+ effectors in TIL cultures. mAb to Leu-19 or Leu-2a did not block lysis of autologous targets by sorted effectors. OKT3 antibody augmented lysis of autologous targets by CD3+Leu-19- effectors only. These results show that non-MHC-restricted Leu-19+ effectors in cultures of TIL with 1000 U/ml of rIL-2 mediate lysis of autologous and allogeneic tumor cells. The CD3+Leu-19- cells, the main population in these cultures, do not mediate tumor lysis. To determine the phenotype of antitumor effectors in IL-2 cultures of TIL, cell sorting followed by functional assays are necessary.     

Flow cytometry assessment of residual melanoma cells in tumor-infiltrating lymphocyte cultures

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) is in development for the treatment of metastatic melanoma. In phase II clinical trials, patients with metastatic melanoma that received TIL after preconditioning had a 50-70% clinical response rate. The current approach to generate TIL is to culture melanoma enzyme digests in the presence of IL-2 for a 10- to 20-day period followed by 2 weeks of rapid expansion (REP). Prior to administration, cell therapies are characterized and tested for purity. TIL are characterized by CD3 surface marker expression, and purity is assessed by the amount of tumor remaining in culture. Evaluating TIL purity has traditionally been done by immunohistochemistry, which is often considered semiquantitative. To generate a quantitative assay, we used multiparameter flow cytometry to evaluate the presence of viable tumor cells by staining TIL populations with a viability dye and an antibody cocktail that detects intracellular tumor-antigens gp100, Mart-1, tyrosinase, S100, and surface tumor-antigen melanoma chondroitin sulfate proteoglycan (MCSP), and CD3 on T cells. Tumors were identified by gating on the viable CD3(-) population. Antigens in tumors were initially optimized with individual antibodies using both immunohistochemistry and flow cytometry. When eight different tumor cell lines were spiked into an activated T cell culture, flow cytometry was able to distinguish lymphocytes from tumors in all samples tested. Most importantly, the assay was able to detect melanoma cells in all enzyme digests (9/9) from patient samples. After IL-2-induced TIL expansion, there was a significant decrease in tumor cells; tumor cells were detected in only 2 of 12 samples. In eight IL-2-induced TIL samples that were further expanded in REP, no tumor cells were detected. We have demonstrated that flow cytometry is an alternative to immunohistochemistry for defining the purity of a TIL population.     

Histopathological analysis of metastatic melanoma deposits in patients receiving adoptive immunotherapy with tumor-infiltrating lymphocytes

Tumor-infiltrating lymphocytes (TIL) from a wide range of human and murine tumors can be expanded in vitro using interleukin-2 (IL-2). These TIL are cytolytic T lymphocytes with in vivo and in vitro antitumor activity in mice and in humans. TIL from human melanoma can recognize autologous tumor in an MHC-restricted fashion, localize in vivo after¹¹¹In labeling, and mediate regression of large metastatic deposits. Although studied extensively in vitro, less is known in vivo about TIL activity associated with tumor regression. This study was undertaken, in association with a study of TIL localization, to investigate mechanisms of TIL action by evaluating histopathological changes that occur at the tumor site during TIL administration. A total of 106 pre- and post-treatment pathological specimens from 25 patients enrolled in phase II TIL treatment and¹¹¹In-TIL imaging protocols were examined blindly by a single pathologist. Histological subtype, lymphocytic infiltration, melanin content, vascularity, and necrosis were documented for each tumor specimen. Average baseline and post-treatment parameters were compared. Any significant changes were evaluated for correlation with clinical response and¹¹¹In-TIL localization to tumor. Melanin content and vascularity of the tumor did not change as a result of therapy or correlate with either response or TIL localization. However, both increased lymphocytic infiltration and tumor necrosis were present after TIL administration (P=0.044 and 0.032 respectively). Furthermore, increases in lymphocytic infiltration correlated with tumor imaging using¹¹¹In-TIL, and with the percentage of¹¹¹In-labeled injectate present per gram of tumor specimen (P=0.036 and 0.0041 respectively). This suggests that TIL either account for the increased lymphocytes directly, or localize to tumor and recruit endogenous lymphocytes. We were unable to demonstrate any pretreatment histopathological predictors of response or variables that significantly correlated with subsequent clinical response, although peak and average values of necrosis were higher in responding patients compared to non-responding patients.     

Functional properties of tumor-infiltrating and blood lymphocytes in patients with solid tumors: effects of tumor cells and their supernatants on proliferative responses of lymphocytes

Tumor-infiltrating lymphocytes (TIL) were obtained from 22 humans with solid tumors. In three cases only, one colon and two lung carcinomas, TIL which contained from 3 to 10% of T cells expressing the interleukin 2 receptor (IL 2R) were obtained, and these proliferated in the presence of exogenous IL 2. In most TIL preparations, however, the T lymphocytes did not express the IL 2R and failed to proliferate in response to IL 2. In contrast, TIL were able to proliferate in response to irradiated allogeneic spleen cells in mixed lymphocyte culture. Proliferative responses of autologous PBL were not inhibited by the addition of TIL. In most tumors, the TIL showed no response or had significantly lower (p less than 0.01) responses to PHA, Con A, and the phorbol ester TPA than did autologous peripheral blood lymphocytes (PBL). A limiting-dilution microculture system which allows clonal growth of every T cell was used to demonstrate decreased responses of the TIL to PHA at a single-cell level. In contrast to normal PBL-T with proliferating frequencies from 0.46 to 1.0, those for T cells in three TIL preparations were zero, 0.005, and 0.01. Normal PBL exposed in vitro to tumor cells or their supernatants lost the ability to respond to mitogens and to clone normally (e.g., proliferating frequency of 0.147 vs 0.863 in control). The TIL isolated from solid tumors resemble normal PBL exposed in vitro to tumor cells or their supernatants in terms of decreased responses to mitogens and poor clonogenicity in the PHA-dependent microculture system. It is possible that tumor cells may inhibit certain functions of the TIL in human solid tumors.