Gérard Degiovanni

A tyrosinase peptide presented by HLA-B35 is recognized on a human melanoma by autologous cytotoxic T lymphocytes.

We previously described different cytotoxic T lymphocyte (CTL) clones isolated from the blood lymphocytes of a melanoma patient after in vitro stimulation with autologous tumor cells. These CTL clones recognized at least 2 distinct antigens on the melanoma cells. Here, we show that one of them consists of a peptide derived from tyrosinase and presented by HLA‐B35. The peptide is 9 amino acids long and has the sequence LPSSADVEF. It can be presented by the 2 major B35 allelic subtypes, B*3501 and B*3503. As HLA‐B35 is one of the most frequent HLA‐B specificities, being present in about 20% of Caucasian individuals, it may be a useful target for peptide‐based immunotherapy of melanoma. Int. J. Cancer 83:755–759, 1999.

In vitro production of granular T cells and mast cells by use of different conditioned media. Ultrastructural and functional analysis.

Normal mouse spleen cells were cultured in different conditioned media (CM). Mixed lymphocyte culture supernatant (MLC SN) was shown to promote the proliferation of cytotoxic, Thy‐l+, Lyt‐1+, Lyt‐2+, asialo‐GM‐1 +, weakly adherent cells with numerous vacuoles and lysosomc‐like cytoplasmic granules. In contrast, the Con A SN induced the proliferation of non‐cylotoxic, Thy‐1−. Lyt−1−, Lyt‐2−, asialo‐GM‐I, non‐adherent cells with numerous cytoplasmic granules. The ultrastructural morphology of these cells and the cytochemical characteristics of their granules enable us to identify them as mast celts. The different effects of both CM could be related to their T‐cell growth factor (TCGF) content. When the amount of TCGF of these two CM was determined (by assaying growth‐stimulating activity for T‐cell colonies), it appeared that the MLC SN contained larger amounts of TCGF (han the Con A SN used in these experiments.

Direct in vitro inactivation of murine alloreactive cytotoxic-T-lymphocyte precursors by syngeneic nonspecific cytotoxic-T-cell populations.

Nonspecific cytotoxic T-cell populations, derived from murine fetal calf serum (FCS)-precultured cells expanded in interleukin 2 (IL-2)-containing supernatant (operationally defined as FCS-CM-expanded cells), were investigated for their inactivating properties on syngeneic lymphoid cell populations containing alloreactive cytotoxic-T-lymphocyte precursors (CTL-P). CTL-P were detected and quantitated in a limiting dilution mixed leukocyte microculture (micro-MLC) system supplemented with IL-2. The data show a dramatic decrease in relevant CTL-P frequency in populations of fresh or Day 2 in vitro-alloactivated peripheral blood leukocytes (PBL) after coculturing them for 24 hr with syngeneic mitomycin C-treated populations of FCS-CM-expanded cells. On the contrary, no decrease in CTL-P frequency was observed when Day 7, instead of fresh or Day 2, in vitro-alloactivated PBL were used as responding cells. Throughout these experiments, it was clearly shown that a decrease or an absence of CTL response in the micro-MLC was neither due to a lack of IL-2 nor to a premature destruction of the stimulating cells by the inhibiting FCS-CM-expanded cells still present in the culture. FCS-CM-expanded cells can destroy (in a 3-hr 51Cr-release assay) Day 2 alloreactive PBL populations, and this raises the possibility that the direct inactivation of CTL-P by FCS-CM-expanded cells could result from their cytolytic activities.

Suppression of Cytolytic T‐Lymphocyte Responses through Inactivation of Non‐T Accessory Cells

The suppressive properties of nonspecific cytotoxic T lymphocyte (CTL) populations, derived from murine fetal calf serum (FCS)‐precultured cells expanded in interleukin 2 (operationally defined as FCS‐CM‐expanded cells), were investigated on CTL responses generated by syngeneic alloreactive lymphoid cells. Our results suggest that the addition of FCS‐CM‐expanded cell populations can inhibit the CTL response by elimination of the bone marrow‐derived macrophage (BM Mø) population used as non‐T accessory cells. Indeed, in the culture conditions used, removal of IL‐2 by the FCS‐CM‐expanded cells as well as a direct inactivating effect on the CTL precursors (CTL‐P) could be excluded as a reason for inhibition. On the other hand, we were able to show that the BM Mø population was very sensitive to the cytolytic activity exhibited by the inhibiting cells in a 3 h 51Cr‐release assay and that the suppressor effect observed could be partially circumvented by a second addition of BM Mø on the second day after the initiation of the culture.