In this study we used TEPITOPE, a new epitope prediction software, to identify sequence segments on the MAGE-3 protein with promiscuous binding to histocompatibility leukocyte antigen (HLA)-DR molecules. Synthetic peptides corresponding to the identified sequences were synthesized and used to propagate CD4+ T cells from the blood of a healthy donor. CD4+ T cells strongly recognized MAGE-3281–295 and, to a lesser extent, MAGE-3141–155 and MAGE-3146–160. Moreover, CD4+ T cells proliferated in the presence of recombinant MAGE-3 after processing and presentation by autologous antigen presenting cells, demonstrating that the MAGE-3 epitopes recognized are naturally processed. CD4+ T cells, mostly of the T helper 1 type, showed specific lytic activity against HLA-DR11/MAGE-3–positive melanoma cells. Cold target inhibition experiments demonstrated indeed that the CD4+ T cells recognized MAGE-3281–295 in association with HLA-DR11 on melanoma cells. This is the first evidence that a tumor-specific shared antigen forms CD4+ T cell epitopes. Furthermore, we validated the use of algorithms for the prediction of promiscuous CD4+ T cell epitopes, thus opening the possibility of wide application to other tumor-associated antigens. These results have direct implications for cancer immunotherapy in the design of peptide-based vaccines with tumor-specific CD4+ T cell epitopes.
Intra‐arterial transplantation of mesoangioblasts proved safe and partially efficacious in preclinical models of muscular dystrophy. We now report the first‐in‐human, exploratory, non‐randomized open‐label phase I–IIa clinical trial of intra‐arterial HLA‐matched donor cell transplantation in 5 Duchenne patients. We administered escalating doses of donor‐derived mesoangioblasts in limb arteries under immunosuppressive therapy (tacrolimus). Four consecutive infusions were performed at 2‐month intervals, preceded and followed by clinical, laboratory, and muscular MRI analyses. Two months after the last infusion, a muscle biopsy was performed. Safety was the primary endpoint. The study was relatively safe: One patient developed a thalamic stroke with no clinical consequences and whose correlation with mesoangioblast infusion remained unclear. MRI documented the progression of the disease in 4/5 patients. Functional measures were transiently stabilized in 2/3 ambulant patients, but no functional improvements were observed. Low level of donor DNA was detected in muscle biopsies of 4/5 patients and donor‐derived dystrophin in 1. Intra‐arterial transplantation of donor mesoangioblasts in human proved to be feasible and relatively safe. Future implementation of the protocol, together with a younger age of patients, will be needed to approach efficacy.
Neoplastic cells express tumor-associated antigens, but tumor rejection seldom occurs in vivo. The absence of an effective immune response may be explained by the inability of tumor cells to deliver co-stimulatory signals. Indeed, transfection of either B7-1 or B7-2 co-stimulatory molecules into mouse tumor cells enhances antitumor immune responses. In this study, we stably transfected human melanoma cells with the cDNA encoding the B7-2 molecule to evaluate in vitro: (i) the induction of anti-melanoma cytotoxic T lymphocytes (CTL) by stimulation of CD8+ T cells, purified from healthy donors and a melanoma patient, with B7-2 transfected allogeneic HLA-matched melanoma cells; (ii) the tumor specificity and the HLA restriction of the induced CTL; and (iii) the feasibility to propagate long-term antimelanoma CTL lines. We found that B7-2 transfected, but not untransfected or mock-transfected, melanoma cells activated MHC-class I-restricted, melanoma-specific CD8+ CTL from healthy donors. More importantly, CD8+ tumor-associated lymphocytes, purified from a tumor-invaded lymph node of a melanoma patient and stimulated with B7-2-transfected melanoma cells, acquired a strong reactivity toward the autologous tumor. CTL lines with specific cytolytic activity could be propagated in long-term culture. These results indicate that: (i) the expression of the B7-2 molecule into human melanoma cells makes them immunogenic and able to act as antigen-presenting cells and (ii) purified CD8+ cells, stimulated with B7-2+ allogeneic HLA-matched melanoma cells, preferentially recognize melanoma-specific rather than allogeneic antigens. This study may have clinical implications for passive and/or active immunotherapy in melanoma patients.
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