Cytolytic antitumor effector cells in long-term cultures of human tumor-infiltrating lymphocytes in recombinant interleukin 2

Summary Lymphocytes infiltrating human solid tumors (TIL) and autologous peripheral blood lymphocytes (A-PBL) were cultured with 1000 units/ml of recombinant interleukin 2 (rIL2) in long-term cultures. TIL isolated from 26 primary squamous cell carcinomas of the head and neck expanded better (P<0.01) and achieved higher total lytic units of activity against fresh tumor cell targets (P<0.05) than A-PBL. TIL obtained from primary hepatocellular carcinomas (n=7) showed a higher degree of expansion than those from metastatic liver tumors (n=7). Further, TIL from metastatic tumors of the head and neck, liver, and ovary were delayed up to 50 days in their proliferative response to rIL2. Long-term mass cultures in rIL2 of TIL, A-PBL, or normal PBL were serially monitored for cytotoxicity with different cultured and fresh tumor cell targets and for phenotypic markers of the predominating cell populations. Antitumor cytotoxicity was found in cultures enriched in CD3 + Leu19 + and/or CD3-Leu19 + cells. Two-color sorting of such cultures followed by cytotoxicity assays confirmed that the human antitumor effectors expressed either the CD3 + Leu19 + or CD3-Leu19 + phenotype. CD3 + Leu19- cells had little or no antitumor cytotoxicity. The two types of Leu19 + effector cells were present in low numbers in fresh TIL, A-PBL, or normal PBL; in contrast, in some rIL2-expanded long-term cultures, they represented a majority of proliferating cells. This study identifies for the first time two types of antitumor effector cells in rIL2 cultures of human TIL, one of which may represent activated natural killer cells on the basis of the absence of the CD3 and expression of the Leu19 antigen. These antitumor effector cells mediate non-MHC-restricted cytotoxicity of fresh or cultured tumor cell targets of different histologic types.     

Comparison of recombinant-interleukin-2-activated peripheral blood and tumor-infiltrating lymphocytes of patients with epithelial ovarian carcinoma: Cytotoxicity,growth kinetics and phenotype

Summary We have compared the growth and tumordirected cytotoxic efficacy of recombinant-interleukin-2-(rIL-2)-activated peripheral blood (PBL) and tumor-infiltrating lymphocytes (TIL) from patients with epithelial ovarian carcinoma. These studies demonstrated that TIL and PBL displayed similar levels of cytotoxicity and a broad range of target cell killing, as exemplified by their reactivity against autologous and allogeneic ovarian tumors as well as against tumor cell lines. No specificity of autologous tumor cell killing was manifested by TIL. Even though TIL of some patients showed higher proliferative activity (especially at the later times in rIL-2 culture) this was not a general phenomenon. In fact, in one case TIL did not proliferate at all, and in the other case the PBL proliferated more actively. While the cultures were composed primarily of CD3⁺ lymphocytes, the major cytotoxic cells displayed the CD56⁺ and CD16⁺ phenotype. Addition of OKT3 mAb to rIL-2 cultures resulted in an increased proliferative index, but showed only a minor effect on the cytotoxic potential of cultured lymphocytes. The therapeutic potential of rIL-2-activated TIL and PBL is discussed.Recipient of the Florence Maude Thomas Cancer Research Professorship     

High expression of adhesion molecules/activation markers with little interleukin-2, interferon γ, and tumor necrosis factor β gene activation in fresh tumor-infiltrating lymphocytes from lung adenocarcinoma

Tumor-infiltrating lymphocytes (TIL) were derived from primary breast tumors, metastatic lymph nodes and malignant pleural effusions from 34 patients with breast cancer. TIL were cultured for approximately 30 days and studied for phenotype, cytotoxicity, and the ability to secrete cytokines in response to autologous tumor stimulation. Tumor specimens were obtained from two different sites in 7 patients, resulting in 41 samples from which 38 TIL cultures were established. In addition to screening 38 bulk TIL cultures, TIL from 21 patients were separated into CD4⁺ and CD8⁺ subsets and extensively studied. Three CD4⁺ TIL were found specifically to secrete granulocyte macrophage-colony-stimulating factor and tumor necrosis factor when stimulated by autologous tumor and not by a large panel of stimulators (24–34) consisting of autologous normal cells, allogeneic breast or melanoma tumors and EBV-B cells. This cytokine release was found to be MHC-class-II-restricted, as it was inhibited by the anti-HLA-DR antibody L243. These 3 patients' EBV-B cells, when pulsed with tumor lysates, were unable to act as antigen-presenting cells and induce cytokine secretion by their respective CD4⁺ TIL. These findings demonstrate that MHC-class-II-restricted CD4⁺ T cells recognising tumor-associated antigens can be detected in some breast cancer patients.     

Semiquantitative analysis of Th1 and Th2 cytokine expression in CD3+, CD4+, and CD8+renal-cell-carcinoma-infiltrating lymphocytes

The mRNA expression of Th1 and Th2 cytokines was compared in freshly isolated CD3⁺ tumor-infiltrating lymphocytes (CD3⁺ TIL) and in autologous CD3⁺ peripheral blood lymphocytes (CD3⁺ PBL) obtained simultaneously from 20 patients with renal cell carcinomas (RCC). In addition cytokine expression was compared in CD4⁺ TIL and CD8⁺ TIL from another group of 20 patients with RCC. TIL were isolated from mechanically disaggregated tumor material and PBL from peripheral blood by gradient centrifugation and subsequent selection with anti-CD3, anti-CD4 or anti-CD8 magnetic beads. In these pure lymphocyte preparations the constitutive expression of interleukin-1 (IL-1), IL-2, IL-10, interferon γ (IFN), and tumor necrosis factor α (TNF) was determined by using a polymerase-chain-reaction-assisted mRNA amplification assay. In the CD3⁺ TIL, levels of mRNA for IFN, IL-10, IL-1 and TNF were significantly higher than in the autologous CD3⁺ PBL whereas IL-2 expression was rather low and did not differ in the two populations. Comparison of cytokine mRNA expression in CD4⁺ TIL and simultaneously obtained CD8⁺ TIL revealed a significantly higher expression of IFN in the CD8⁺ cells. These data reflect an in vivo activation of RCC-infiltrating lymphocytes at the mRNA level with respect to the Th1 as well as the Th2 immune response. Th1 activation seems to be most evident in the CD8⁺ TIL. Received: 14 January 1999 / Accepted: 30 April 1999     

Lymphokine production by human melanoma tumor-infiltrating lymphocytes

Summary Lymphokine production by human melanoma tumor-infiltrating lymphocytes (TIL) was studied. Uncultured TIL produced interferon (IFN), but not interleukin-2 (IL-2) or IL-4, in response to anti-CD3 mAb or IL-2. In bulk cultures, IL-2-activated TIL displaying autologous tumor-specific cytotoxicity (CTL-TIL) produced IFN in culture with medium alone, whereas IL-2-activated noncytotoxic TIL did not. Addition of anti-CD3 mAb or autologous tumor cells up-regulated IFN production in IL-2-activated TIL from 10 of 12 or 6 of 12 cases respectively. Those from 4 of 12 cases (2 CTL-TIL and 2 noncytotoxic TIL) produced IL-2 in culture with medium alone. At the clonal level, 5 (4 CD4⁺ and 1 CD8⁺) of 7 autologous tumor-specific CTL clones derived from TIL and 3 (2 CD4⁺ and 1 CD8⁺) of 7 noncytotoxic TIL clones produced IFN in culture with medium alone, which was up-regulated by adding anti-CD3 mAb. Two IFN-producing CTL clones tested produced IL-2 in 4 ×-concentrated supernatants from a 3.5-h culture with medium alone. Furthermore, 2 IFN-producing CTL clones tested expressed mRNA for both IFN and IL-2. IL-2 production and its mRNA expression were up- or down-regulated, respectively, by adding anti-CD3 mAb or autologous tumor cells. IL-4 production was not observed in culture either with medium alone or with IL-2 in any of the cells described above. Anti-CD3 mAb was required for IL-4 production in 3 of 12 IL-2-activated TIL, 2 of 6 CTL clones, and none of 5 noncytotoxic TIL clones. In summary, IFN production was characteristic of melanoma TIL. Some autologous tumor-specific CTL in TIL are suggested to be productive of IL-2 and IFN under unstimulated conditions, both being required for self-activation in an autocrine loop.This work was supported in part by grant CA-47891 from the National Cancer Institute     

In search of specific cytotoxic T lymphocytes infiltrating or accompanying human ovarian carcinoma

Summary Lymphocytes infiltrating human ovarian carcinoma obtained directly from the tumour mass (tumour-infiltrating lymphocytes, TIL) or from the carcinomatous ascites (tumour-associated lymphocytes, TAL) were expanded in vitro in long-term cultures with interleukin-2 and tested for their specific cytolytic activity. Killing of the autologous tumour was detected only in a proportion of the patients, less frequently in TIL compared to TAL. In fact two out of ten TIL and four out of nine TAL cultures tested showed significant levels of lysis against the autologous tumour. This cytotoxic activity was not restricted to the autologous tumour, as other tumour cell lines, including non-ovarian ones, were lysed as well. The cultures that were not cytotoxic against the autologous tumour were in most cases able to lyse other tumour cell lines of ovarian or other histology. Cloning of TIL from one patient was performed: of 22 clones tested, 4 displayed higher cytotoxicity against the autologous tumour compared to the uncloned population and 3 out of these 4 did not kill an irrelevant carcinoma cell line. In order to stimulate the expansion of putative specific effectors we performed mixed lymphocyte/tumour cultures (MLTC) with autologous or allogeneic tumour cells. No stimulation of cytotoxicity against the autologous tumour was detected after MLTC in nine different TAL populations, using autologous or allogeneic tumours as stimulators. On the contrary, peripheral blood lymphocytes from two patients after MLTC with the autologous tumour showed increased killing of the autologous and decreased killing of an allogeneic target. In conclusion TIL and TAL from ovarian carcinoma expanded in vitro with interleukin-2 usually have non-MHC-restricted cytotoxicity and variable degrees of reactivity against the autologous tumour. A preferential killing for the autologous tumour was not observed even after MLTC. These results do not exclude the existence of tumour-specific cytotoxic T lymphocytes in ovarian carcinoma; nevertheless they suggest that putative specific effectors have very low frequency and that culture techniques for expanding their growth more selectively are still to be optimized.     

Characterization of interleukin-2-initiated versus OKT3-initiated human tumor-infiltrating lymphocytes from glioblastoma multiforme: Growth characteristics,cytolytic activity,and cell phenotype

Summary Outgrowth of tumor-infiltrating lymphocytes (TIL) from the human primary brain tumor glioblastoma multiforme was achieved by OKT3 initiation (10 ng/ml), followed by sustained expansion by interleukin-2 (IL-2; 200 U/ml). Tumor-infiltrating lymphocyte (TIL) initiation by this process was performed in parallel with the standard IL-2-only method. Of ten tumors, seven yielded TIL in response to OKT3/IL-2, whereas only three of these seven grew after initiation with IL-2 alone. On the basis of cell doubling times, at least 60 doublings, resulting in (hypothetically) up to 10²³ TIL from as few as 2 × 10⁵ cells in tumor suspensions, could be achieved using OKT3/IL-2. OKT3-initiated TIL proliferated in culture for as long as 288 days, although senescence of some cultures occurred at as early as 73 days. Significant heterogeneity of lymphocytes infiltrating the fresh tumors and heterogeneity of resultant TIL phenotype and function were apparent, yet several common trends were noted. In all cases after OKT3 initiation, significant net growth was not apparent until approximately 14 days. In contrast, in the three samples that grew in response to IL-2 alone, log-phase growth was always observed earlier. During the early phase of the cultures, all TIL expressed some killing activity toward a broad spectrum of tumors, including the autologous tumor. No consistent preference of TIL for lysis of autologous tumor was observed. Glioblastoma multiforme TIL cultures contained a mixture of CD8⁺ and CD4⁺ cells, with few CD16⁺ or NKH-1⁺. Of the six TIL examined in detail for population phenotype in relationship to time in culture, four eventually became exclusively CD4⁺. Further analysis of these CD4⁺ TIL indicated that all were of the helper-inducer class, 4B4⁺ and 2H4^–. Concurrent with the decline in CD8⁺ cells, a decline in the cytolytic activity of these TIL cultures occurred. Furthermore, in two TIL that remained CD8⁺, a decline in the cytolytic activity also occurred. Therefore, loss of killing activity was not merely a reflection of the major cell phenotype changes. These results indicate that the OKT3/IL-2 process provides an alternative to IL-2 alone for TIL initiation and growth, as well as providing a novel system for further analysis of tumorderived lymphocyte and accessory cell functional potential.This work was supported by a grant from the Dunn Foundation, Houston, Texas     

T-Cell tropism of simian T-cell leukaemia virus type 1 and cytokine profiles in relation to proviral load and immunological changes during chronic infection of naturally infected mandrills (Mandrillus sphinx)

Background Although a wide variety of non‐human primates are susceptible to simian T‐cell leukaemia virus type 1 (STLV‐1), little is known about the virological or molecular determinants of natural STLV‐1 infection. Methods We determined STLV‐1 virus tropism in vivo and its relation to the immune response by evaluating cytokine production and T‐cell subsets in naturally infected and uninfected mandrills. Results With real‐time PCR methods, we found that STLV‐1 in mandrills infects both CD4⁺ and CD8⁺ T cells; however, proviral loads were significantly higher (P = 0.01) in CD4⁺ than in CD8⁺ cells (mean STLV‐1 copies number per 100 cells (± SD) was 7.8 ± 8 in CD4⁺ T cells and 3.9 ± 4.5 in CD8⁺ T cells). After culture, STLV‐1 provirus was detected in enriched CD4⁺ but not in enriched CD8⁺ T cells. After 6 months of culture, STLV‐1‐transformed cell lines expressing CD3⁺, CD4⁺ and HLADR⁺ were established, and STLV‐1 proteins and tax/rex mRNA were detected. In STLV‐1 infected monkeys, there was a correlation between high proviral load and elevated levels of interleukin (IL)‐2, IL‐6, IL‐10, interferon‐γ and tumour necrosis factor‐α. The two monkeys with the highest STLV‐1 proviral load had activated CD4⁺HLADR⁺ and CD8⁺HLADR⁺ T‐cell subsets and a high percentage of CD25⁺ in CD4⁺ and CD8⁺ T cells. Conclusions Our study provides the first cellular, immunological and virological characterization of natural STLV‐1 infection in mandrills and shows that they are an appropriate animal model for further physiopathological studies of the natural history of human T‐cell leukaemia viruses.     

Specific immune recognition of autologous tumor by lymphocytes infiltrating colon carcinomas: Analysis by cytokine secretion

Summary Tumor-infiltrating lymphocytes (TIL) were grown in the presence of interleukin-2 from 19 colon carcinoma specimens, including 1 primary lesion and 18 metastatic lesions. These cultures showed a median proliferation of 606-fold (range 13-fold to 28 000-fold) over 49 culture days (range 26–76 days). By phenotype, mature cultures were 69%–99% CD3⁺ (mean 93%) and contained mixed populations of CD4⁺ and CD8⁺ cells (CD4>CD8 in 10 of 19 cultures). Fresh cryopreserved colon tumors were not lysed by autologous TIL in short-term⁵¹Cr-release assays, and were poorly lysed by lymphokine-activated killer cells. Ten TIL cultures were assayed for cytokine secretion in response to autologous and allogeneic tumors during a 6- to 24-h coincubation. Culture supernatants were tested by ELISA for the presence of granulocyte/macrophage-colony-stimulating factor, interferon , and tumor necrosis factor . Of 10 TIL, 4 secreted at least two of these cytokines specifically in response to autologous and/or HLA-matched fresh allogeneic colon carcinomas, but not to melanomas or HLA-unmatched colon carcinomas. Cytokine secretion was mediated by both CD4⁺ and CD8⁺ TIL, and could be inhibited by mAb directed against the appropriate class of MHC antigen. These data provide evidence for specific, MHC-restricted immune recognition of human colon carcinomas by T lymphocytes.     

Tumor-infiltrating lymphocytes from nonrenal urological malignancies

Summary Tumor-infiltrating lymphocytes (TIL) were isolated from 15 of 20 surgical specimens of transitional cell carcinoma of the urinary bladder, prostate cancer, testicular cancer, Wilms tumor and adrenal cancer. Expansion was carried out in four different culture conditions, each containing 1000 U/ml interleukin-2: RPMI medium with or without 20% (by volume) of lymphokine-activated killer cell (LAK) supernatant and AIM V medium with or without 20% LAK supernatant. The resultant cell populations were then assayed for cytotoxicity against a variety of autologous and allogeneic tumor targets and phenotypic analysis was performed with fluorescein-labeled monoclonal antibodies. TIL growth was unrelated to the initial percentage of lymphocytes or tumor cells present in the enzymatically dispersed specimens or whether fresh or cryopreserved tissue was utilized. Better growth was seen in AIM V than in RPMI medium (P = 0.013); the beneficial effect of the addition of LAK supernatant to RPMI was indicated (P = 0.065), and the addition of LAK supernatant to AIM V did not improve the ability to culture TIL (P = 0.5) from these cancers. TIL in long-term culture were predominantly CD3⁺. The ratio of CD4⁺/CD8⁺ cells varied with time in culture and culture medium, but most cultures eventually became CD4⁺. Cells bearing B cell, natural killer cell, and macrophage markers disappeared early in culture. Overall 14/15 TIL samples were lytic against one of the autologous and allogeneic targets tested, but specific lysis against the autologous tumor from which it was derived was seen in only one TIL culture originating from a bladder cancer. Our results suggest that TIL can be expanded to therapeutic levels from a variety of urological malignancies and that their potential role in future therapy should be further explored.     

Tumor mRNA-loaded dendritic cells elicit tumor-specific CD8(+) cytotoxic T cells in patients with malignant glioma

In this study, we demonstrate that tumor mRNA–loaded dendritic cells can elicit a specific CD8⁺ cytotoxic T-lymphocyte (CTL) response against autologous tumor cells in patients with malignant glioma. CTLs from three patients expressed strong cytolytic activity against autologous glioma cells, did not lyse autologous lymphoblasts or EBV-transformed cell lines, and were variably cytotoxic against the NK-sensitive cell line K-562. Also, DCs-pulsed normal brain mRNA failed to induce cytolytic activity against autologous glioma cells, suggesting the lack of autoimmune response. Two patients' CD8⁺ T cells expressed a modest cytotoxicity against autologous glioma cells. CD8⁺ T cells isolated during these ineffective primings secreted large amounts of IL-10 and smaller amounts of IFN- as detected by ELISA. Type 2 bias in the CD8⁺ T-cell response accounts for the lack of cytotoxic effector function from these patients. Cytotoxicity against autologous glioma cells could be significantly inhibited by anti-HLA class I antibody. These data demonstrate that tumor mRNA–loaded DC can be an effective tool in inducing glioma-specific CD8⁺ CTLs able to kill autologous glioma cells in vitro. However, high levels of tumor-specific tolerance in some patients may account for a significant barrier to therapeutic vaccination. These results may have important implications for the treatment of malignant glioma patients with immunotherapy. DCs transfected with total tumor RNA may represent a method for inducing immune responses against the entire repertoire of glioma antigens.     

Culture of tumour-infiltrating lymphocytes from melanoma and colon carcinoma: Removal of tumour cells does not affect tumour-specificity

The therapeutic potential of adoptive therapy using tumour-infiltrating lymphocytes (TIL) has been demonstrated in a number of clinical trials. However, freshly isolated tumour-infiltrating lymphocytes (TIL) are often impaired in their proliferative and cytotoxic responses, which limits their use in immunotherapy. Several hypotheses with regard to the poor effector function of TIL have been postulated, including the production of immunosuppressive factors by tumour cells. In a previous paper we reported the efficient expansion of immunoreactive TIL from a variety of solid tumours by stimulation with a combination of monoclonal antibodies (mAbs) against CD3 and CD28. In the present study we analysed whether this protocol would be improved by the removal of tumour cells at the start of the culture. We tested a highly immunogenic tumour, melanoma, and a poorly immunogenic tumour, colon carcinoma. Removal of tumour cells highly improved anti-CD3/CD28 stimulated expansion of TIL from colon carcinoma, resulting in a significantly higher percentage of potentially tumour-specific CD8-positive T-cells and a reduced CD4/CD8 ratio compared to expansion in the presence of tumour cells. In contrast, expansion and CD4/CD8 ratio of melanoma-derived TIL was not significantly influenced by the removal of autologous tumour cells. CD3/CD28-stimulated melanoma TIL cultured in the absence of tumour cells showed specific lysis of autologous tumour cells comparable to melanoma TIL cultured in high-dose IL2. However, no cytotoxicity could be detected in colon TIL irrespective of the culture conditions used. On the other hand, 3/8 colon carcinoma TIL cultures and 9/12 melanoma-derived TIL cultures showed IFN secretion upon stimulation with autologous tumour cells. We conclude that stimulation of TIL with a combination of mAbs to CD3 and CD28 in the absence of tumour cells induces efficient expansion of potentially tumour-specific cells from a highly and a poorly immunogenic tumour. Removal of tumour cells does not have a negative influence on the generation of tumour-specific T cells, while cell yield improves. Therefore, for large-scale cultures this protocol can efficiently induce the outgrowth of tumour-specific TIL, at the same time providing a useful source of autologous tumour cells that can be stored and used to direct or test antitumour specificity.     

Efficient expansion of tumor-infiltrating lymphocytes from solid tumors by stimulation with combined CD3 and CD28 monoclonal antibodies

Combined CD3 and CD28 monoclonal antibodies (mAb) may initiate efficient activation and expansion of tumor-infiltrating lymphocytes (TIL). In this study we compared phenotypical and functional characteristics of TIL from a group of 17 solid human tumors, stimulated either by high-dose recombinant interleukin 2 (rIL-2, 1000 IU/ml) or by a combination of anti-CD3 and anti-CD28 monoclonal antibodies in the presence of low-dose rIL-2 (10 IU/ml). Compared to activation with high-dose rIL-2, stimulation of TIL with CD3/CD28 mAb induced significantly stronger proliferation and yielded higher levels of cell recovery on day 14. Following the CD3/CD28 protocol, expansion of an almost pure population of CD3⁺ cells was obtained. Whereas CD4⁺ cells dominated in the first week of culturing, within 4 weeks the CD8⁺ population increased to over 90%. The specific capacity to kill autologous tumor cells was not increased as compared to the high-dose rIL-2 protocol, but all cultures showed high cytotoxic T cell activity as measured in a CD3-mAb-mediated redirected kill assay. These studies show that combined CD3 and CD28 mAb are superior to rIL-2 with respect to the initiation of expansion of CD8⁺ cytolytic TIL from solid tumors. Stimulation with specific tumor antigens at a later stage of culturing may further augment the expansion of tumor-specific cytolytic T cells.     

Killer cells induced by stimulation with allogeneic tumor cells and subsequent culture with recombinant interleukin-2

Summary Peripheral blood lymphocytes were cultured for 5 days with allogeneic tumor cells (allogeneic mixed lymphocyte/tumor cell culture), and subsequently cultured with recombinant interleukin-2 for 12 days. These cultured cells were found to be cytotoxic to autologous tumor cells. Results of two-color analysis using monoclonal antibodies to cell markers showed that more than 80% of their cultured cells were CD3⁺ cells, and CD4⁺ cells showed a higher distribution than CD8⁺ cells. However, CD8⁺ cells had a much higher killing activity with autologous tumor than did CD4⁺ cells, when estimated by an elimination study using monoclonal antibodies to T cell phenotypes and complement. The cold-target inhibition test showed that the cytotoxicity of these cells for autologous tumor cells was inhibited by unlabeled autologous tumor cells but not by unlabeled stimulator cells. Furthermore, about 40% of the cytotoxicity was suppressed by blocking of HLA class I antigen with a monoclonal antibody on autologous tumor cells. Thus, cytotoxic activity of lymphocytes to autologous tumor restricted by target cell HLA class I antigen is possibly induced by allogeneic tumor-stimulation.     

Co-Stimulation through 4-1BB/CD137 Improves the Expansion and Function of CD8+ Melanoma Tumor-Infiltrating Lymphocytes for Adoptive T-Cell Therapy

Adoptive T-cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) can induce tumor regression in up to 50% or more of patients with unresectable metastatic melanoma. However, current methods to expand melanoma TIL, especially the “rapid expansion protocol” (REP) were not designed to enhance the generation of optimal effector-memory CD8⁺ T cells for infusion. One approach to this problem is to manipulate specific co-stimulatory signaling pathways to enhance CD8⁺ effector-memory T-cell expansion. In this study, we determined the effects of activating the TNF-R family member 4-1BB/CD137, specifically induced in activated CD8⁺ T cells, on the yield, phenotype, and functional activity of expanded CD8⁺ T cells during the REP. We found that CD8⁺ TIL up-regulate 4-1BB expression early during the REP after initial TCR stimulation, but neither the PBMC feeder cells in the REP or the activated TIL expressed 4-1BB ligand. However, addition of an exogenous agonistic anti-4-1BB IgG₄ (BMS 663513) to the REP significantly enhanced the frequency and total yield of CD8⁺ T cells as well as their maintenance of CD28 and increased their anti-tumor CTL activity. Gene expression analysis found an increase in bcl-2 and survivin expression induced by 4-1BB that was associated with an enhanced survival capability of CD8⁺ post-REP TIL when re-cultured in the absence or presence of cytokines. Our findings suggest that adding an agonistic anti-4-1BB antibody during the time of TIL REP initiation produces a CD8⁺ T cell population capable of improved effector function and survival. This may greatly improve TIL persistence and anti-tumor activity in vivo after adoptive transfer into patients.     

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.     

Tumour-infiltrating lymphocytes mediate lysis of autologous squamous cell carcinomas of the head and neck

Tumour-infiltrating lymphocytes (TIL) and tumours from six patients with squamous cell carcinomas of the head and neck (SCCHN) were investigated. The six tumours all expressed major histocompatibility complex (MHC) class I antigens both in vivo and as tumor cell lines grown in vitro. In addition, the cancer cells either overexpressed the tumour-suppressor gene product p53 or harboured human papilloma virus 16/18 (HPV). The TIL were expanded in vitro in the presence of interleukin-2, immobilised anti-CD3 mAb and soluble anti-CD28 mAb. Expanded TIL cultures contained both CD4+and CD8+T cells, but generally contained few CD56+CD3-cells of the natural killer (NK) phenotype. CD8+T cells dominated the individual TIL cultures from five of the six patients and showed significant autologous tumour cell lysis. In TIL cultures derived from four of these tumour-reactive TIL cultures, killing could be partially blocked by an anti-MHC class I mAb. TIL cultures reacting with autologous tumour cells also showed strong TCR/CD3-redirected cytotoxicity when assayed against hybridoma cells expressing anti-TCR/CD3 mAb as well as natural-killer(NK)-like activity. A number of TIL cultures devoid of autologous tumour cell lysis were capable of lysing the natural-killer(NK)-sensitive K562 cell line suggesting that the SCCHN cells themselves are resistant to NK-like lysis. In conclusion, TIL cultures from head and neck carcinomas contain T cells which, upon expansion in vitro, can lyse autologous tumour cells in a MHC-class-I-restricted fashion. Thus, the results of the present study document that carcinomas of the head and neck in some patients are infiltrated by cytotoxic T cell precursors potentially capable of rejecting the autologous tumour.     

Phenotypic and functional analysis of lymphocytes infiltrating paediatric tumours,with a characterization of the tumour phenotype

Summary Tumour-infiltrating lymphocytes (TIL) of paediatric tumours obtained from 37 lesions of different histo-type (12 osteosarcomas, 5 Wilms' tumours, 7 soft-tissue sarcomas, 5 neuroblastomas and 8 miscellaneous) were studied to establish their potential for therapy. Fresh isolated TIL were cultured for the first 2 weeks with low doses of interleukin-2 (IL-2) (20 Cetus U/ml) to select for tumour-specific lymphocytes potentially present in the neoplastic lesion, followed by culture with high doses of IL-2 (1000 Cetus U/ml) to achieve TIL expansion. TIL were grown with more than 10-fold expansion in only 9 cases (mean expansion: 58-fold, range 13.5–346). In 17 cases no viable cells were obtained. After 30 days of culture with IL-2 the proliferative ability of TIL declined sharply in the majority of cases and TIL became refractory to any further stimulus, including addition of IL-4, tumour necrosis factor (TNF) or interferon , and activation with OKT3 in solid phase. In 20 out of 37 cases TIL were available for phenotypic and functional analysis. TIL after long-term culture were predominantly CD3⁺ but 2 cases of osteosarcoma showed a predominance of CD3⁺TcR / cells. The CD4/CD8 ratio was more than 1 in 10 cases, without correlation with tumour histology, site of lesion or TIL growth. The number of CD16⁺ and CD25⁺ lymphocytes decreased progressively during culture, the latter concomitantly with a reduction of TIL growth rate. The lytic pattern of TIL against allogeneic and autologous tumour (Auto-Tu) cells was variable, but specific lysis of Auto-Tu was seen in only one case (Wilms' tumour) after culture with TNF and irradiated Auto-Tu cells. The immunohistochemical analysis of tumour lesions revealed a limited lymphocyte infiltrate, a low expression of histocompatibility leukocyte antigens (HLA) class I and of the adhesion molecules ICAM1, LFA3, and a significant production of transforming growth factor (TGF). These data indicate that TIL obtained from paediatric patients are difficult to expand at levels required for immunotherapy and lack a significant number of tumour-specific T lymphocytes. A low expression of immunomodulatory molecules on tumour cells or the production of suppressive factors may prevent activation and expansion of TIL in paediatric tumours.