HLA-Cw6 Alloreactive CTL Recognizing HLA-CW6+ Myeloma Cells but Not Non-Myeloma HLA-Cw6+ Cells Are Peptide Dependent.

We investigated the generation of myeloma-specific cytotoxic T lymphocytes (CTL) from unrelated normal donors non-HLA matched with the myeloma cell line SBN. The aim was to obtain alloreactive CTL specific of peptides to identify genes preferentially expressed by to myeloma cells. After coculture of SBN with PBL of unrelated donor, the T-cell line obtained was cloned and each CTL was assessed against SBN and SB-EBV (B-EBV cell line obtained by infecting B cells of SB patient with EBV) simultaneously. Only the CTL recognizing SBN but not SB-EBV were kept for further study. Among more than 200 clones screened, we isolated two different CTL CD8+ recognizing SBN only (60% of specific lysis at effector: target ratio 5). Their cytotoxicity was blocked by antibodies against HLA-I molecules and more precisely by mAb against HLA-B/Cw molecules. Both CTL recognized also other MM cell lines but only those matched with SBN for HLA-Cw0602 (XG6, BCN) suggesting an HLA-Cw6 recognition. Furthermore, antiserum directed against HLA-Cw6 abrogated the recognition of SBN, XG6 and BCN. Both CTL were not cytotoxic against BC-EBV nor against 3 other B-EBV cell lines derived from HLA-Cw0602 donors. This unreactivity was not restricted to B-EBV cell lines since PBL from 2 normal HLA-Cw0602 donors were also not recognized (cytotoxicity, TNF secretion). We directly measured cell surface HLA-Cw6 expression of both B-EBV and myeloma cells with a scFv directed against HLA-Cw6 and we showed that HLA-Cw6 was more expressed by B-EBV cells as compared to myeloma cells (2fold). So, the lack of recognition of B-EBV cells was not related to HLA-Cw6 expression level. We also looked for KIR expression by the CTL since KIR molecules could have blocked B-EBV recognition that expressed more HLA-Cw6 molecules as compared to myeloma cells. No KIR molecules (p58.1, p58.2, CD94) were found to be expressed by the CTL.

To investigate whether the recognition of myeloma cells was peptide dependent, we performed two experiments i.e., acid elution and cold target competition. Acid elution (glycine 0.3M, pH=2.5) abrogated myeloma recognition and cold target competition showed that B-EBV cells were not recognized by the CTL. These data suggest that the CTL recognize a peptide or a set of peptides restricted to myeloma cells or that the CTL recognition is activated by tumor cells only. Anyway, such alloreactive CTL could be helpful for GVL reaction in allotransplanted patients with myeloma.