Vgamma9Vdelta2 T cell-mediated recognition of human solid tumors. Potential for immunotherapy of hepatocellular and colorectal carcinomas.

INTRODUCTION: Vgamma9Vdelta2 T lymphocytes are reported to participate in the anti-tumor immune surveillance in human. They are known to recognize phosphoantigens and molecules expressed on cells undergoing neoplasic transformation. In this study, we investigated phenotype and anti-tumor cytotoxicity of ex vivo expanded Vgamma9Vdelta2 T cells in view of adoptive immunotherapy. MATERIALS AND METHODS: Experiments were performed with peripheral blood samples from eleven patients [six colorectal carcinoma (CRC), four hepatocellular carcinoma (HCC), one sarcoma] and sixteen healthy donors. RESULTS/DISCUSSION: Ex vivo expansion of Vgamma9Vdelta2 T cells could be achieved by a single dose of phosphoantigen, either bromohydrin pyrophosphate or zoledronate, and supported by exogenous IL-2. After 2 weeks, expanded Vgamma9Vdelta2 T lymphocytes acquired the effector memory phenotype CD45RA(-)CD45RO(high)CD27(-). They expressed NKG2D and CD161 and the proinflammatory CXCR3 and CCR5 chemokine receptors. Vgamma9Vdelta2 T cells displayed a strong lytic activity toward a broad panel of tumor cell lines or primary cultures. Interestingly, HCC and CRC primary cells could be lysed by autologous Vgamma9Vdelta2 T cells whereas autologous normal cells were not sensitive to the lysis. mAbs blocking assays demonstrated that TCR was the most important receptor involved in the lysis of tumor cells. However, NKG2D receptor could deliver a costimulatory signal enhancing the lysis of HCC and CRC tumors expressing MICA/B. Treatment of tumor cells by the mevalonate pathway inhibitor, zoledronate, enhanced the killing of both HCC and CRC. Expansion index of Vgamma9Vdelta2 T cells was in similar levels in healthy donors or in cancer patients and total expansion was suitable for adoptive immunotherapy. CONCLUSION: These results provide a rationale for the clinical evaluation of Vgamma9Vdelta2 T lymphocytes in HCC and CRC.

In vitro antitumor lymphocyte generation using dendritic cells and innate immunity mechanisms as tumor cell treatments.

Dendritic cells play a central role in the initiation and regulation of acquired and innate immunity, playing an important role in immunosurveillance and antitumor reaction. This reaction is mediated by effector cells and soluble factors. We chose to investigate four dendritic cell loading methods by mimicking innate immunity mechanisms and using whole tumor cell treatments in order to stimulate lymphocytes: sodium hypochlorite, TNFalpha and IFNgamma and IgG opsonization. These methods were compared in an HLA.A2 model of healthy donors and with the M74 melanoma cell line. Treated tumor cell-loaded DC were able to increase proliferation of lymphocytes. Moreover, a CTL population was stimulated, as shown by their specific cytotoxicity against tumor cells (with w6/32 antibody assays), against MelanA/MART-1 loaded T2 cells and using MelanA/MART-1 tetramer. IgG opsonization seemed to be less efficient than other tumor cell treatments. These loaded DC, or the obtained effector cells, could be interesting for therapeutic applications in antitumor cell therapy.

Ex vivo expansion of antitumor cytotoxic lymphocytes with tumor-associated antigen-loaded dendritic cells.

Cell therapy with lymphocytes is an attractive approach for cancer immunotherapy. Methods to generate ex vivo effector cells directed against whole autologous tumor antigens are under investigation. Our procedure involved stimulation of autologous lymphocytes with antigen-pulsed dendritic cells (DC). Experimental conditions were established with DC, matured with TNFa, LPS and CD40L, from healthy donors and the M74 melanoma cell line. DC were pulsed with either irradiated, apoptotic or necrotic tumor cells or fused with tumor cells. Increase of lymphocyte cytotoxicity and IFNy production were repeatedly observed with tumor cell-loaded DC. Stimulation of tumor-associated antigen-specific lymphocytes was clearly shown. MelanA-MART1 (dominant melanoma-associated antigen) tetramer staining revealed a high frequency of specific T cells. Lymphocytes were able to efficiently lyse MelanA-MART1-pulsed T2 target and MelanA-expressing target cells (M74) after CD56+ cells depletion. We confirmed with other tumor cell lines that this DC-mediated procedure induced activation of cytolytic lymphocytes.

Dendritic cells are defective in breast cancer patients: a potential role for polyamine in this immunodeficiency.

INTRODUCTION: Dendritic cells (DCs) are antigen-presenting cells that are currently employed in cancer clinical trials. However, it is not clear whether their ability to induce tumour-specific immune responses when they are isolated from cancer patients is reduced relative to their ability in vivo. We determined the phenotype and functional activity of DCs from cancer patients and investigated the effect of putrescine, a polyamine molecule that is released in large amounts by cancer cells and has been implicated in metastatic invasion, on DCs. METHODS: The IL-4/GM-CSF (granulocyte-macrophage colony-stimulating factor) procedure for culturing blood monocyte-derived DCs was applied to cells from healthy donors and patients (17 with breast, 7 with colorectal and 10 with renal cell carcinoma). The same peroxide-treated tumour cells (M74 cell line) were used for DC pulsing. We investigated the effects of stimulation of autologous lymphocytes by DCs pulsed with treated tumour cells (DC-Tu), and cytolytic activity of T cells was determined in the same target cells. RESULTS: Certain differences were observed between donors and breast cancer patients. The yield of DCs was dramatically weaker, and expression of MHC class II was lower and the percentage of HLA-DR-Lin- cells higher in patients. Whatever combination of maturating agents was used, expression of markers of mature DCs was significantly lower in patients. Also, DCs from patients exhibited reduced ability to stimulate cytotoxic T lymphocytes. After DC-Tu stimulation, specific cytolytic activity was enhanced by up to 40% when DCs were from donors but only up to 10% when they were from patients. IFN-gamma production was repeatedly found to be enhanced in donors but not in patients. By adding putrescine to DCs from donors, it was possible to enhance the HLA-DR-Lin- cell percentage and to reduce the final cytolytic activity of lymphocytes after DC-Tu stimulation, mimicking defective DC function. These putrescine-induced deficiencies were reversed by treating DCs with all-trans retinoic acid. CONCLUSION: These data are consistent with blockade of antigen-presenting cells at an early stage of differentiation in patients with breast cancer. Putrescine released in the microenvironmement of DCs could be involved in this blockade. Use of all-trans retinoic acid treatment to reverse this blockade and favour ex vivo expansion of antigen-specific T lymphocytes is of real interest.

Cytotoxic effector cells with antitumor activity can be amplified ex vivo from biopsies or blood of patients with renal cell carcinoma for cell therapy use.

Adoptive immunotherapy with antitumor effector cells is an attractive therapeutic approach in metastatic renal cell carcinoma (RCC). The aim of the work was to enhance in vitro activation of lymphocytes with optimal cytotoxic activity against tumor cells. We evaluated a procedure based on the use of dendritic cells (DCs) loaded with irradiated tumor cells (DC-Tu) to stimulate lymphocytes. Experimental conditions were established with cells from healthy donors and melanoma cell lines. Procedures were then applied to cells from RCC patients. A total of 30 tumor biopsies, 14 proximal lymph nodes, and 17 peripheral blood samples from 30 patients were used. When lymphocytes were stimulated in vitro with DC-Tu, they responded to tumor cells with an increased cytolytic activity for all the assays with donor cells (n=18). For RCC patients, DC-Tu stimulation improved the final cytotoxic activity in only half of the assays (16/31). When significantly enhanced (>10%, n=8), responder cells resulted in a final 43% cytotoxicity against autologous RCC cells. Mechanism of lysis was at least in part class I mediated. Effector cells have no lytic activity against normal renal cells. Percentage of cells with regulatory T-cell phenotype was not found to be enhanced in the DC-Tu stimulated lymphocytes. Individual differences were observed in the characteristics of DCs generated from RCC patients in contrast to that observed in donors and could explain why lymphocyte stimulation was not improved by DC-Tu in half of the RCC assays. T-cell spreading was suitable for a therapeutic use (>10(9) cells) irrespective of the procedure (with or without DC-Tu stimulation) or the tissular origin of lymphocytes from patients. Data show that precursors of selective antitumor effector cells are present in patients with RCC and can be amplified in vitro either with or without DC-Tu stimulation. One of these populations could be chosen for an adoptive transfer immunotherapy.

[Antitumor immunity and cellular cancer therapies]. / Immunité anti-tumorale et thérapies cellulaires du cancer.

The identification of tumor specific antigens has provided important advance in tumor immunology. It is now established that specific cytotoxic T lymphocytes (CTL) and natural killer cells infiltrate tumor tissues and are effector cells able to control tumor growth. However, such a natural antitumor immunity has limited effects in cancer patients. Failure of host defenses against tumor is consecutive to several mechanisms which are becoming targets to design new immunotherapeutic approaches. CTL are critical components of the immune response to human tumors and induction of strong CTL responses is the goal of most current vaccine strategies. Effectiveness of cytokine therapy, cancer vaccines and injection of cells improving cellular immunity have been established in tumor grafted murine models. Clinical trials are underway. To day, interest is particularly focused on cell therapy: injected cells are either "ready to use" effector cells (lymphocytes) or antigen presenting cells able to induce a protective immune reaction in vivo (dendritic cells). The challenge ahead lie in the careful optimization of the most promising strategies in clinical situation.

Constitutive expression of TGF-bêta1, interleukin-6 and interleukin-8 by tumor cells as a major component of immune escape in human ovarian carcinoma.

Tumors could use several mechanisms to coexist with the host's immune system or to protect themselves from an immune response. Thus, insufficient expression of cell surface molecules on tumor cells, which are important for T cell recognition or activation, could lead to induction of a state of tolerance. Tumor cells could also produce cytokines that would inhibit the immune response and allow tumor progression. Here, we studied, in vitro, the cell surface expression of immunologically important molecules in seven ovarian carcinoma (OVCA) cell lines and the constitutive expression of cytokines. All OVCA cell lines expressed MHC class I molecules, ICAM-1 and LFA-3 adhesion molecules, necessary to induce a specific cytotoxic T-cell response, as well as the CD40 costimulatory molecules. Conversely, the lack of the dominant costimulatory molecules, CD80 (B7.1) and CD86 (B7.2) could be a possible explanation of poor immunogenicity of OVCA tumors. Immunosuppressive TGF-beta1 was detected at the mRNA level in all cell lines but was weakly secreted in supernatants. By contrast, IL-10 was never found. Most of them constitutively produced IL-8 and IL-6, two cytokines known as tumor promoting factors whereas the proinflammatory cytokines TNF-alpha, IL-1beta and GM-CSF were rarely produced. Data from this study could be useful for designing new strategies of immunotherapy to improve immunogenicity and/or limit protumor cytokine production.