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.     

Enhancement of in vitro tumor-infiltrating lymphocyte cytotoxicity by heteroconjugated antibodies.

Tumor-infiltrating lymphocytes (TIL) were obtained from a mouse melanoma cell line (CL 62) transfected with the gene for the human melanoma Ag p97. TIL were cultured with anti-CD3 antibody and IL-2 for up to 38 days. Flow cytometry identified these TIL as Thy-1.2 + ve/CD4-ve/CD8 + ve cells. A heteroconjugated antibody 500A2 x 96.5, specific for both the CD3 Ag on TIL and the p97 Ag on CL 62 melanoma cells, was prepared using N-succinimidyl-3-(2-pyridyldithio)-propionate as a linking agent. TIL alone demonstrated low levels of cytotoxicity against autologous CL 62 tumor and also against the parental K1735 tumor and an allogeneic murine melanoma (B16). The addition of 500A2 x 96.5 heteroconjugated antibody enhanced TIL-mediated lysis of CL 62 tumor, but not of the K1735 or B16 tumors. This enhanced cytotoxicity was elicited at E:T ratios as low as 0.4:1, and in TIL cultured for 7 to 38 days. These results suggest that hetero-conjugated antibody may enhance the anti-tumor effect of TIL in vivo.     

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.     

Specific antitumor activity of tumor-infiltrating lymphocytes expanded first in a culture with both anti-CD3 monoclonal antibody and activated B cells and then in a culture with interleukin-2

In order to expand tumor-infiltrating lymphocytes (TIL) efficiently and in order to use them for immunotherapy, we utilized lipopolysaccharide-activated B cells (LPS blasts) as costimulatory-signal-providing cells in an in vitro culture system. TIL, prepared from subcutaneously inoculated B16 melanoma, failed to expand when cultured with anti-CD3 monoclonal antibody (mAb) alone followed by a low dose of interleukin(IL)-2. In contrast, such TIL did expand efficiently in culture with both anti-CD3 mAb and LPS blasts followed by culture with IL-2. These findings suggest that the presence of LPS blasts in the initial culture was essential for the cell expansion. The expansion of TIL was partially blocked by the addition of CTLA4 Ig, which is an inhibitor of costimulatory molecules such as CD80 and CD86, and was almost blocked by the addition of anti-(Fc receptor γII)mAb. These findings thus indicate that such molecules, in conjunction with the receptor on the LPS blasts, participate in the efficient expansion of TIL. The B16-derived TIL, which expanded in our culture system, were predominantly CD8+T cells and showed a higher level of cytolytic activity against B16 melanoma than either lymphokine-activated killer cells or TIL cultured with a high dose of IL-2. In addition, the in vitro expanded B16-derived TIL produced interferon γ, but not IL-4, in response to B16 melanoma. What is more important, the adoptive transfer of such TIL had a significant antitumor effect against pulmonary metastasis in B16 melanoma, even without the concurrent administration of IL-2. Collectively, our results thus indicate the therapeutic efficacy of the protocol presented here for antitumor immunotherapy with TIL.     

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.     

Preferential in situ CD4+CD56+ T cell activation and expansion within human glioblastoma

Recent evidence suggests that suppression of the cellular immune response is often attributable to populations of functionally distinct T cells that act to down-regulate Ag-specific effector T cells. Using flow cytometry, we evaluated tumor-infiltrating lymphocytes (TIL) from patients undergoing neurosurgical resection of glioblastoma multiforme (GBM), metastatic lung carcinoma, and meningioma for markers known to be expressed on immunoregulatory T cells. Ex vivo phenotypic characteristics, cellular proliferation, and cytokine expression patterns were compared between T cell subsets found in the PBMC and within TIL from fresh tumor samples. Interestingly, nearly half of all T cells infiltrating GBM specimens were CD56(+) T cells, while much smaller percentages of similar cells were identified within metastatic lung tumors and meningiomas. CD56(+) T cells identified within GBM were not canonical, or "invariant," NKT cells, as they demonstrated diverse TCR expression, a primarily CD4 single-positive phenotype, and lack of CD1d reactivity. The percentage of CD56(+) T cells exhibiting evidence of proliferation within GBM was 3- to 4-fold higher than the proportion of proliferating CD56(-) T cells from these lesions. In addition, direct ex vivo analysis of cytokine expression by TIL from GBM demonstrated significant numbers of IL-4/IL-13 positive cells, cytokines that are integral in the cell-mediated repression of tumor immunity in experimental models. We propose that GBM has a unique capacity to recruit and activate CD4(+)CD56(+) T cells, a population that has not been previously described within human tumors.     

Specific antitumor activity of tumor-infiltrating lymphocytes expanded first in a culture with both anti-CD3 monoclonal antibody and activated B cells and then in a culture with interleukin-2

In order to expand tumor-infiltrating lymphocytes (TIL) efficiently and in order to use them for immunotherapy, we utilized lipopolysaccharide-activated B cells (LPS blasts) as costimulatory-signal-providing cells in an in vitro culture system. TIL, prepared from subcutaneously inoculated B16 melanoma, failed to expand when cultured with anti-CD3 monoclonal antibody (mAb) alone followed by a low dose of interleukin(IL)-2. In contrast, such TIL did expand efficiently in culture with both anti-CD3 mAb and LPS blasts followed by culture with IL-2. These findings suggest that the presence of LPS blasts in the initial culture was essential for the cell expansion. The expansion of TIL was partially blocked by the addition of CTLA4 Ig, which is an inhibitor of costimulatory molecules such as CD80 and CD86, and was almost blocked by the addition of anti-(Fc receptor II)mAb. These findings thus indicate that such molecules, in conjunction with the receptor on the LPS blasts, participate in the efficient expansion of TIL. The B16-derived TIL, which expanded in our culture system, were predominantly CD8+T cells and showed a higher level of cytolytic activity against B16 melanoma than either lymphokine-activated killer cells or TIL cultured with a high dose of IL-2. In addition, the in vitro expanded B16-derived TIL produced interferon , but not IL-4, in response to B16 melanoma. What is more important, the adoptive transfer of such TIL had a significant antitumor effect against pulmonary metastasis in B16 melanoma, even without the concurrent administration of IL-2. Collectively, our results thus indicate the therapeutic efficacy of the protocol presented here for antitumor immunotherapy with TIL.This work was supported in part by a grant from the Ministry of Education, Science and Culture     

Bimodal ex vivo expansion of T cells from patients with head and neck squamous cell carcinoma: a prerequisite for adoptive cell transfer

Background aimsAdoptive transfer of tumor-infiltrating lymphocytes (TIL) has proven effective in metastatic melanoma and should therefore be explored in other types of cancer. The aim of this study was to examine the feasibility of potentially expanding clinically relevant quantities of tumor-specific T-cell cultures from TIL from patients with head and neck squamous cell carcinoma (HNSCC) using a more rapid expansion procedure compared with previous HNSCC studies.MethodsIn a two-step expansion process, initially TIL bulk cultures were established from primary and recurrent HNSCC tumors in high-dose interleukin (IL)-2. Secondly, selected bulk cultures were rapidly expanded using anti-CD3 antibody, feeder cells and high-dose IL-2. T-cell subsets were phenotypically characterized using flow cytometry. T-cell receptor (TCR) clonotype mapping was applied to examine clonotype dynamics during culture. Interferon (INF)-γ detection by Elispot and Cr⁵¹ release assay determined the specificity and functional capacity of selected TIL pre- and post-rapid expansion.ResultsTIL bulk cultures were expanded in 80% of the patients included, showing tumor specificity in 60% of the patients. Rapid expansions generated up to 3500-fold expansion of selected TIL cultures within 17 days. The cultures mainly consisted of T-effector memory cells, with varying distributions of CD8⁺ and CD4⁺ subtypes both among cultures and patients. TCR clonotype mapping demonstrated oligoclonal expanded cultures, ranging from approximately 10 to 30 T-cell clonotypes. TIL from large-scale rapid expansions maintained functional capacity, and contained tumor-specific T cells.ConclusionThe procedure is feasible for expansion of TIL from HNSCC, ensuring clinically relevant expansion folds within 7 weeks. The cell culture kinetics and phenotypes of the TIL resemble previously published results on TIL from melanoma, setting the stage for clinical testing of this promising treatment strategy for patients with HNSCC.     

CD8-independent tumor cell recognition is a property of the T cell receptor and not the T cell

The CD8 coreceptor enhances T cell function by stabilizing the TCR/peptide/MHC complex and/or increasing T cell avidity via interactions with the intracellular kinases Lck and LAT. We previously reported a CD4(+) T cell (TIL 1383I), which recognizes the tumor-associated Ag tyrosinase in the context of HLA-A2. To determine whether CD8 independent tumor cell recognition is a property of the TCR, we used retroviral transduction to express the TIL 1383I TCR in the CD8(-) murine lymphoma, 58 alpha(-)/beta(-). Immunofluorescent staining of TCR-transduced cells with human TCR V beta subfamily-specific and mouse CD3-specific Abs confirmed surface expression of the transferred TCR and coexpression of mouse CD3. Transduced effector cells secreted significant amounts of IL-2 following Ag presentation by tyrosinase peptide-pulsed T2 cells as well as stimulation with HLA-A2(+) melanoma lines compared with T2 cells alone or HLA-A2(-) melanoma cells. Further analysis of TCR-transduced clones demonstrated a correlation between T cell avidity and cell surface expression of the TCR. Therefore, the TIL 1383I TCR has sufficient affinity to mediate recognition of the physiologic levels of Ag expressed by tumor cells in the absence of CD8 expression.     

Tumor-specific cytolysis by lymphocytes infiltrating human melanomas

Tumor infiltrating lymphocytes (TIL) were grown in IL-2 from single cell tumor suspensions of 14 human melanomas resected from 12 patients. As a function of time in culture, 4 of 14 TIL cultures eventually expressed highly specific cytolytic activity against fresh autologous melanoma targets in short term chromium release assays, failing to lyse multiple allogeneic tumors or autologous normal cells. These highly specific TIL were identified as CTL by phenotype (CD3+/CD4-/CD8+/Leu7-) and by function (lysis inhibited by antibodies directed against CD3 and MHC class I molecules). Cell separation experiments using immunomagnetic beads identified a highly tumor-specific CTL subpopulation within a nonspecific TIL culture, suggesting that the lytic activity of tumor-specific CTL may be diluted by the nonspecific killer activity present in heterogeneous TIL cultures. These studies provide evidence for specific MHC-restricted human immune responses against autologous tumor in cancer-bearing patients, and may be of importance to ongoing clinical trials using TIL in the immunotherapy of advanced malignancies.     

Modulation by IL-2 of CD70 and CD27 expression on CD8+ T cells: importance for the therapeutic effectiveness of cell transfer immunotherapy

Proper T cell function relies on the integration of signals delivered by Ag, cytokine, and costimulatory receptors. In this study, the interactions between IL-2, CD27, and its ligand CD70 and their effects on human T cell function were examined. Unstimulated CD8(+) T cells expressed relatively low levels of CD70 and high levels of CD27. Incubation in vitro with high doses of IL-2 (3,000 IU/ml) or administration of IL-2 in vivo resulted in substantial up-regulation of CD70 expression and the concomitant loss of cell surface CD27 expression on CD8(+) cells. Withdrawal of IL-2 from activated CD8(+) T cells that had been maintained in IL-2 resulted in a reversal of the expression of these two markers, whereas reciprocal changes were seen following treatment of PBMCs with IL-2. The proliferation observed in cells stimulated with IL-2 primarily occurred in a subset of the CD70(+)CD8(+) T cells that up-regulated IL-2 receptor expression but did not occur in CD70(-)CD8(+) T cells. Blocking CD70 resulted in a significant reduction of T cell proliferation induced by high-dose IL-2, indicating that the interaction of CD70 with CD27 played a direct role in T cell activation mediated by IL-2. Finally, studies conducted on tumor-infiltrating lymphocyte (TIL) samples that were administered to melanoma patients indicated that the size of the pool of CD27(+)CD8(+) T cells in bulk TILs was highly associated (p = 0.004) with the ability of these TILs to mediate tumor regression following adoptive transfer.     

Increased tumor-specific CTL activity in human tumor-infiltrating lymphocytes stimulated with autologous tumor lines

Two long-term tumor-infiltrating lymphocyte (TIL) lines and their autologous tumor lines have been established from solid tumors derived from different patients with metastatic melanoma. In 4-hr 51Cr release assays, each TIL culture lysed only the autologous cryopreserved fresh or established melanoma line, but failed to lyse other melanoma tumors or K562 cells. Repeated stimulation of TIL with the autologous melanoma lines resulted in significant increases in anti-tumor CTL activity with no apparent loss in specificity. Stimulated cells have retained cytotoxic activity for up to 5 months in culture. Tumor cell CTL activity for both long-term TIL lines is inhibited by several mAbs, including those against CD3, CD8, and class I MHC molecules, indicating that the effector cells are class I-restricted CD8+, CTL. Furthermore, recognition of Ag on one of the established melanoma lines by TIL is restricted by HLA A-2. The availability of autologous tumor lines may prove clinically useful for the selective stimulation and expansion of cells with anti-tumor activity within a heterogeneous TIL population.     

Ex vivo enrichment of circulating anti-tumor T cells from both cutaneous and ocular melanoma patients: clinical implications for adoptive cell transfer therapy

Tumor-infiltrating lymphocytes (TILs) have been successfully used for adoptive cell transfer (ACT) immunotherapy; however, due to their scarce availability, this therapy is possible for a limited fraction of cutaneous melanoma patients. We assessed whether an effective protocol for ex vivo T-cell expansion from peripheral blood mononuclear cells (PBMCs), suitable for ACT of both cutaneous and ocular melanoma patients, could be identified. PBMCs from both cutaneous and ocular melanoma patients were stimulated in vitro with autologous, irradiated melanoma cells (mixed lymphocyte tumor cell culture; MLTCs) in the presence of IL-2 and IL-15 followed by the rapid expansion protocol (REP). The functional activity of these T lymphocytes was characterized and compared with that of TILs. In addition, the immune infiltration in vivo of ocular melanoma lesions was analyzed. An efficient in vitro MLTC expansion of melanoma reactive T cells was achieved from all PBMC's samples obtained in 7 cutaneous and ocular metastatic melanoma patients. Large numbers of melanoma-specific T cells could be obtained when the REP protocol was applied to these MLTCs. Most MLTCs were enriched in non-terminally differentiated T(EM) cells homogeneously expressing co-stimulatory molecules (e.g., NKG2D, CD28, CD134, CD137). A similar pattern of anti-tumor activity, in association with a more variable expression of co-stimulatory molecules, was detected on short-term in vitro cultured TILs isolated from the same patients. In these ocular melanoma patients, we observed an immune infiltrate with suppressive characteristics and a low rate of ex vivo growing TILs (28.5% of our cases). Our MLTC protocol overcomes this limitation, allowing the isolation of T lymphocytes with effector functions even in these patients. Thus, anti-tumor circulating PBMC-derived T cells could be efficiently isolated from melanoma patients by our novel ex vivo enrichment protocol. This protocol appears suitable for ACT studies of cutaneous and ocular melanoma patients.     

Treatment with tumour infiltrating lymphocytes and interleukin-2 in patients with metastatic melanoma: A pilot study

Tumour infiltrating lymphocytes (TIL) were isolated and expanded from biopsy samples of 4 patients with metastatic melanoma. The patients were treated with autologous expanded TIL and continuous or bolus infusion of Interleukin 2 (IL-2) at a dose of 18 × 10⁶ International Units/m²/day for 5 days starting 36–48 hours after administration of cyclophosphamide at a dose of 1 g/m². The number of TIL infused ranged from 10¹⁰ to 5,56 × 10¹⁰ cells. Two patients had stable disease (SD) lasting for 2 1/2 and 4 months respectively and they died 24 and 13 months after therapy. One patient died during therapy due to a pseudomonas septicaemia and another patient developed progressive disease (PD). He died 3 months after the start of therapy. The side effects were substantial but most of them were reversible upon cessation of the treatment.The majority of the expanded TIL of all patients were of the CD8+ phenotype. Cutaneous metastases from two patients, removed after treatment with IL-2 and TIL, showed moderate lymphocytic infiltration also mainly of CD8+ T cells.The treatment with IL-2 and TIL is feasible, but further investigations should continue in an attempt to improve the efficacy of the therapy, to reduce toxicity and to diminish the costs and labour of the culture methods.     

Specific release of granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-alpha, and IFN-gamma by human tumor-infiltrating lymphocytes after autologous tumor stimulation

Tumor-infiltrating lymphocytes (TIL) have been cultured from a variety of human tumors, and some melanoma TIL have demonstrated specific, MHC-restricted recognition of autologous tumor in short term lysis assays. The current study investigates cytokine release by TIL as an indicator of specific tumor recognition. We have identified two of four melanoma and one of seven breast carcinoma TIL cultures that specifically release granulocyte-macrophage-CSF, TNF-alpha, and IFN-gamma after autologous tumor stimulation. The other cultures either do not secrete cytokine or secrete cytokine in a nonspecific fashion. The amount of specific cytokine released is directly related to the number of TIL and stimulating tumor cells. Studies of TIL, from two melanoma patients, separated into CD4+ and CD8+ populations revealed that CD8+ cells were responsible for virtually all of the specific cytokine secretion, although both populations released cytokines when activated by immobilized anti-CD3 antibody. Specific cytokine release by CD8+ TIL was inhibited by anti-MHC class I mAb. Specific cytokine release was also detected from a CD4+ breast cancer TIL culture, and this was inhibited by anti-MHC class II mAb. The clinical significance of this specific mode of immune antitumor reactivity is currently under investigation.     

Incubation of antigen-sensitized T lymphocytes activated with bryostatin 1 + ionomycin in IL-7 + IL-15 increases yield of cells capable of inducing regression of melanoma metastases compared to culture in IL-2

Regression of established tumors can be induced by adoptive immunotherapy (AIT) with tumor draining lymph node (DLN) lymphocytes activated with bryostatin and ionomycin (B/I). We hypothesized that B/I-activated T cells cultured in IL-7 + IL-15 might proliferate and survive in culture better than cells cultured in IL-2, and that these cells would have equal or greater anti-tumor activity in vivo. Tumor antigen-sensitized DLN lymphocytes from either wild-type or T cell receptor transgenic mice were harvested, activated with B/I, and expanded in culture with either IL-2, IL-7 + IL-15 or a regimen of alternating cytokines. Cell yields, proliferation, apoptosis, phenotypes, and in vitro responses to tumor antigen were compared for cells grown in different cytokines. These T cells were also tested for anti-tumor activity against melanoma lung metastases established by prior i.v. injection of B16 melanoma cells. IL-7 + IL-15 or alternating cytokines resulted in much faster and prolonged proliferation and much less apopotosis of B/I-activated T cells than culturing the same cells in IL-2. This resulted in approximately tenfold greater yields of viable cells. Culture in IL-7 + IL-15 yielded higher proportions of CD8+ T cells and a higher proportion of cells with a central memory phenotype. Despite this, T cells grown in IL-7 + IL-15 had higher IFN-γ release responses to tumor antigen than cells grown in IL-2. Adoptive transfer of B/I-activated T cells grown in IL-7 + IL-15 or the alternating regimen had equal or greater efficacy on a “per-cell” basis against melanoma metastases. Activation of tumor antigen-sensitized T cells with B/I and culture in IL-7 + IL-15 is a promising modification of standard regimens for production of T cells for use in adoptive immunotherapy of cancer.     

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.     

Cytokine production by peripheral lymphocytes in melanoma

BACKGROUND: The differentiation of T cells towards a T helper 1 (Th1) or Th2 phenotype based on their profile of cytokine production, is of great relevance in the regulation of immune responses. We have determined by flow cytometry, the expression of selected Th1 and Th2 cytokines by activated T cells in whole blood samples (WB) from normal donors and from patients with different clinical stages of melanoma in different clinical stages. METHODS: WB samples from 6 normal donors and 19 patients with melanoma were activated over 4 hours with PMA + ionomycin in presence or absence of a protein secretion inhibitor. Following surface staining (CD3-Cy5+CD8-FITC), fixation and permeabilization, cells were stained with PE-labelled antibodies against Th1 cytokines (IL-2, IFN-gamma, TNF-alpha) and Th2 cytokines (IL-4, IL-10). RESULTS: The most relevant results were related to IFN-gamma and IL-10 production. The percentage of IFN-gamma producer cells was significantly lower in melanoma patients, independent of the stage, than in controls. IL-10 production was significantly increased in melanoma patients with respect to normal donors. CONCLUSIONS: Our data support the notion that the pattern of cytokines produced by lymphocytes from melanoma patients may help to explain the impairment in their T cell immune response. More extensive studies regarding the pattern of cytokines, not only in peripheral blood, but also in tumour tissue and sentinel lymph nodes, are needed to confirm these data.