Spontaneous CD4 and CD8 Memory T Cell Responses Against MUC1 and Carcinoembryonic Antigen in Bone Marrow of Multiple Myeloma Patients.

Abstract

Introduction: The bone marrow (BM) is a site of induction of tumour antigen specific T cell responses in many malignancies. We have demonstrated in the BM of myeloma patients high frequencies of spontaneously generated CD8 memory T cells with specificity for the myeloma-associated antigen MUC1, which were not detectable in the peripheral blood (PB). Besides MUC1, carcinoembryonic antigen was recently identified as a tumour-associated antigen in a patient with multiple myeloma. Up to now, spontaneous CD4 T cell responses against myeloma-associated antigens have not been reported. We undertook this study to evaluate to what extent spontaneous CD4 T cell responses against myeloma antigens occur during myeloma progression and if MUC1 or carcinoembryonic antigen represent immunogenic targets of spontaneous CD4 and CD8 T cell responses.Methods: Altogether, 78 patients with multiple myeloma were included into the study. Presence of functionally competent antigen specific T cells was evaluated by ex vivo short term (40 h) IFN-γ Elispot analyses. CD4 T cell responses against MUC1 were assessed by stimulation of purified CD4 T cell fractions with antigen pulsed, autologous dendritic cells (DCs) pulsed with two synthetic 100 meric polypeptides (pp1-100ss and (137–157)5 tr) that can be processed and presented via multiple HLA-II alleles. CD4- or CD8 T cell reactivity against carcinoembryonic antigen was assessed on purified CD4- and CD8 T cell fractions by pulsing DCs with highly purified CEA derived from culture supernatants of an epithelial carcinoma cell line. CD8 responses against MUC1 were analyzed by stimulation of HLA-A2+ patients derived purified T cells with DCs loaded with HLA-A2 restricted MUC1-derived nonameric peptide LLLLTVLTV. As negative control antigen for MUC1 polypeptides and CEA human IgG was used for pulsing DCs at identical concentrations while HLA-A2-restricted peptide SLYNTVATL derived from HIV was used as control antigen for LLLLTVLTV. Test antigen specific reactivity was defined by significantly increased numbers of IFN-γ spots in triplicate test wells compared to control wells (p<0.05, students T test).Results: 8 out of 19 tested patients (42%) contained MUC1 specific CD8 T cells in their bone marrow, while MUC1 specific CD4 T cells were detected in the BM of 30% of the cases (3/10). Interestingly, in peripheral blood (PB) CD8 reactivity against MUC1 was detectable in only 1 out of 10 patients while CD4 reactivity in PB was not detectable at all (0/10). CEA was specifically recognized by BM CD8 T cells from 5 out of 30 patients (17%) and by BM CD4 T cells from 5 out of 18 patients (28%). CEA was not recognized by CD4 and CD8 T cells in the PB of the same patients (0/13).Conclusion: Spontaneous T helper responses against tumour-associated antigens occur in the BM at similar levels as antigen specific CD8 T cells responses while they are virtually undetectable in the PB. Compared to CEA, MUC1 induces CD8 T cell responses in a much higher proportion of myeloma patients. Nevertheless, our data suggest that CEA may trigger spontaneous T cell responses against multiple myeloma in a considerable number of patients. Thus, systematic functional analyses of this potential tumour antigen in multiple myeloma appears to be justified.