Thierry Hercend

A natural cytotoxic T cell response in a spontaneously regressing human melanoma targets a neoantigen resulting from a somatic point mutation.

We have studied a case of human primary melanoma displaying the classical signs of a spontaneous regression in order to characterize potentially efficient anti‐tumor T cell responses. In a previous series of experiments a unique TCR Vβ16+ T cell was shown to be highly expanded at the tumor site. The corresponding clone was isolated in vitro and found to be a CD8+ cytotoxic T lymphocyte with a strong and selective cytolytic activity against the autologous tumor cell line. Here, we demonstrate that this predominant Vβ16+ tumor‐infiltrating lymphocyte recognizes a peptide encoded by a novel unconventional myosin class I gene. This peptide includes a mutation due to a single nucleotide substitution. The resulting Glu  →  Lys replacement at position 911 of the coding sequence is critical to generate the recognized T cell epitope. These experiments demonstrate the existence of a natural tumor‐specific cytolytic T cell response in a primary regressing human melanoma lesion.

A MAGE-6-encoded peptide is recognized by expanded lymphocytes infiltrating a spontaneously regressing human primary melanoma lesion.

In recent years, experiments based on the in vitro stimulation of either autologous peripheral blood lymphocytes or tumor‐infiltrating lymphocytes with melanoma cells have shown that distinct members of the large MAGE gene family encode tumor‐associated antigenic peptides. However, little is still known about natural anti‐MAGE responses in vivo. We have studied a case of spontaneously regressing human melanoma, hypothesizing that in this unique situation, the host immune system had developed an efficient cytotoxic T lymphocyte (CTL) response against the cancer cells. Amongst the dense tumor infiltrate, certain clonal populations of T cells were shown to be amplified, thereby suggesting that an antigen‐driven selection had occurred at the tumor site. One of the expanded tumor‐infiltrating lymphocytes was shown to be a Vβ13+ CD8+ CTL displaying a strong and selective cytotoxic activity against the autologous melanoma cells. Here we show that this cytotoxic T cell clone recognizes a MAGE‐6‐encoded peptide. MAGE‐6 is therefore the fourth gene of the MAGE family shown to encode antigenic peptide recognized by T cells. Together, these data provide further evidence that T cell responses against MAGE antigens may naturally develop in vivo.

Analysis of T-cell immune response in renal cell carcinoma: Polarization to type 1-like differentiation pattern, clonal T-cell expansion and tumor-specific cytotoxicity

We assessed the naturally occurring T‐cell immune response in primary renal cell carcinoma (RCC) tumors from 12 unselected patients. A predominance of CD3+ T‐cell receptor (TCR)α/β+ T cells was observed in tumor‐infiltrating lymphocytes (TILs), in contrast with peripheral blood lymphopenia found in some patients. Activation antigen expression on TILs revealed an imbalance in the activation status, with a significant percentage of CD69+ and HLA‐DR+ and a low percentage of CD25+ and CD71+ TILs. The lymphocyte activation gene‐3 (LAG‐3) was detected in some TIL subpopulations and especially in one patient in whom TILs were predominantly TCRα/β+CD8+DR+LAG‐3+. In addition, we found that RCC TILs are polarized to a global type 1‐like (Th1/Tc1) differentiation pattern (strong secretion of interferon‐γ and interleukin‐2 (IL‐2) following CD3/TCR cross‐linking) but are under the influence of the down‐modulatory cytokines IL‐6 (secreted by tumor cells) and IL‐10, within the tumor microenvironment. In 3 of 5 patients, clonal T‐cell expansion at the tumor site was found for several Vβ specificities, suggesting that in situ stimulation of specific clonotypes in response to potential tumor antigens is a frequent event in RCC. Furthermore, in one patient, selective intratumor amplification of a Vβ1 subpopulation (5% of TCR α/β+ cells) corresponding to 2 distinct Vβ1‐Jβ1.6 and Vβ1‐Jβ2.3 tumor‐specific MHC class I‐restricted cytotoxic T lymphocytes supports the view that discrete T‐cell subsets contribute readily to in situ immunosurveillance. Int. J. Cancer 72:431–440, 1997.

Immunotherapy with interleukin-2 (IL2) and lymphokine-activated natural killer cells: Improvement of clinical responses in metastatic renal cell carcinoma patients previously treated with IL2

Treatment with interleukin-2 (IL2) induces clinical responses in 15-30% of metastatic renal cell carcinoma (MRCC) patients, with mainly partial responses. In order to improve clinical response, we decided to treat partial response patients from a previous IL2 treatment with a second course of IL2 associated with lymphokine-activated natural killer (LANAK) cells. 10 patients who underwent PR after an IL2 protocol (24 x 10(6) U/m2/day, 2 days a week for 5 weeks, either alone or with interferon-gamma) subsequently received a combination of high-dose IL2 (16-20 x 10(6) U/m2/day, 2 days a week) and LANAK cell infusions. Four complete responses were obtained, and 2 additional patients whose tumour mass was further reduced achieved complete response following surgery. These results support the view that initial responses obtained with primary IL2 courses can be improved by complementary treatments. The potential role of cellular immunotherapy and, more particularly, of LANAK cells as an effective procedure to further reduce tumour burden in patients responsive to IL2 will have to be assessed in randomised studies.

In situ T-cell responses in a primary regressive melanoma and subsequent metastases: a comparative analysis.

In an earlier study of the immune response in a patient with a cutaneous primary regressive melanoma, a T‐cell‐receptor diversity analysis demonstrated in situ amplification of certain lymphocytes. Two of them could be cloned and characterized as CD8+ HLA‐class‐I‐restricted CTL with strong selective anti‐tumor activity. Following a disease‐free period of 3 years, the patient developed a gastric metastasis and subsequently (after an additional year) a metastasis in one axillary lymph node. Melanoma cell lines derived from the 2 secondary lesions have been established here. It was found that these metastatic cells have maintained expression of both HLA‐class‐I molecules and the peptidic antigen(s) recognized by the 2 clones amplified at the primary site. However, the corresponding T lymphocytes were either undetectable or poorly represented both in the gastric and in the axillary lesions. These results suggest that substantial alterations in the quality of T‐cell infiltrates occurred during melanoma progression, despite an apparent stability in presentation of tumor‐associated antigen(s) which initially triggered a positive rejection response. Int. J. Cancer 72:241–247, 1997.

MHC-dependent cytolysis of autologous tumor cells by lymphocytes infiltrating urothelial carcinomas.

Tumor‐infiltrating lymphocytes (TIL) were grown from 23 urothelial carcinomas. Phenotyping analysis showed that the TIL cultures were mainly CD3+. Although CD4+ and CD8+ T‐cell sub‐sets were grown in culture, CD4+ T‐cell sub‐sets predominated over CD8+ T cells. Immunohistochemical studies performed on 5 tumor specimens confirmed this observation, and indicated that CD4+ T cells surrounded the tumor islets, whereas CD8+ T lymphocytes were localized among the tumor cells. Five short‐term carcinoma cell lines established from these urothelial tumors were used as target cells in cytolysis assays in order to investigate the functional anti‐tumor activity of autologous TIL. TIL from 4/5 tumors were lytic and 3 TIL lines displayed MHC‐class‐I‐dependent cytotoxicity directed against autologous tumor cells. CD4+ T‐cell‐depletion experiments performed on TIL line 07 confirmed that CD8+ MHC‐class‐I‐dependent CTL were the predominant effectors. Finally, experiments performed on 6 allogeneic urothelial‐cancer cell lines matched for HLA‐class‐I molecules showed that TIL07 exhibited selective lytic activity toward tumor 07. These data indicate that CD8+ MHC‐class‐I‐dependent CTL present in urothelial carcinomas are functional and may participate in the anti‐tumor immune response. Int. J. Cancer 71:585‐594, 1997.

Immunorestorative Capacity of Tuftsin after Long‐Term Administration to Aging Mice

Many attempts have been made to restore the depressed immune functions of old animals. Newborn thymus and adult bone marrow have been shown to reconstitute the antibody and mitogenic response of long-lived hybrid mice for long periods of time.’ However, results obtained with thymic extracts have been less encouraging, leading to limited and transient stimulation of the immune functions of aged mice;2s3 nevertheless, these results support the fact that primary age-related changes involve mainly regulatory T-cell reactions. Immunostimulating drugs such as naturally occurring or synthetic compounds have been utilized infrequently. In our hands, bestatin, an antibiotic, was able to restore some T-cell functions and to increase the life expectancy of treated animak4 We reported in the same study that the incidence of spontaneous tumors arising in old mice is significantly reduced following bestatin administration. Many studies have established that tuftsin, a physiological tetrapeptide, preferentially stimulates macrophage functions and that it has significant tumoricidal activity.’-’ The present work was designed to see whether tuftsin given over a long period of time would display an inhibitory effect on spontaneous development of tumors associated with aging in C57BL6 mice, and in that case what cellular immune antitumor mechanisms could be implicated. Results indicate that in this experimental model, tuftsin exhibits an antitumor activity associated with an increase in the cytostatic capacity of macrophages as well as in cytolytic T-cell activity.

A T chronic lymphocytic leukemia with large granular lymphocytes. Phenotype and functions of leukemic cells under in vitro treatment by differentiation inducers.

We reported the morphologic, phenotypic and functional characteristics of leukemic cells with natural killer (NK) properties in a case of T chronic lymphocytic leukemia with large granular lymphocytes. These cells were cytologically and cytochemically characterized as phosphatase acid positive large granular lymphocytes (LGL), and presented parallel tubular arrays at the ultra structural level. They displayed a CD2, CD3, CD8, CD11, Leu 7, and Leu 11 positive phenotype while they lacked B cell markers including surface immunoglobulins. In addition, they expressed human leukocyte antigens (HLA) Class I, but no Class II antigens. These phenotypic studies were also performed after cells were cultured in vitro with 12–0‐tetradecanoyl phorbol 13‐acetate, γ interferon, 5‐azacytidine, sodium butyrate, phytohemagglutinin, and interleukin 2 (IL2). The cell surface markers underwent several significant changes. Among them we noted a higher percentage of labeled cells with anti‐CD6 and CD7 monoclonal antibodies (moAbs), and a positivity with an anti‐CD19 (B4) moAb. The leukemic LGL spontaneously developed a NK activity on K 562 tumor cells, which was not affected under the various T and B cells growth factors because they became more sensitive to IL2; but they were also stimulated by a 50‐kilodalton (KD) B cell growth factor (BCGF) factor devoid of any T cell proliferation activity. Together these results gave a better characterization of azurophilic granules containing T chronic lymphocytic leukemia, and enabled the documentation of the differentiation of LGL with NK activity.