CT7 (MAGE-C1)-Specific Cellular Immune Responses in the Bone Marrow Microenvironment of Multiple Myeloma Patients.

In previous studies we demonstrated that CT7 (MAGE-C1), a member of the type I MAGE family of Cancer-Testis antigens, was expressed in 82% of primary multiple myeloma specimens. Higher percentages of CT7-expressing cells were correlated with abnormally elevated plasma cell proliferation in these patients, and CT7 was co-expressed in proliferating myeloma cells. Therefore, a tumor vaccine targeting CT7 may preferentially eliminate proliferating myeloma cells, resulting in long-term cures. The type I MAGE were originally identified because they elicited cellular immune responses in melanoma patients whose tumors expressed these antigens. These data suggest the hypothesis that CT7 expression in myeloma cells may similarly elicit T cell immunity in myeloma patients. To test this hypothesis, we examined lymphocytes from myeloma patient bone marrow, the tumor microenvironment. Lymphocytes were isolated from myeloma patient bone marrow aspirates by magnetic bead depletion of CD138 plasma cells followed by plating to remove adherent monocyte. Bone marrow lymphocytes were then expanded by two rounds of stimulation with anti-CD3, anti-CD28 monoclonal antibody (mAb)-conjugated beads and 50 IU/ml recombinant human (rhu) IL-2. Autologous myeloid dendritic cells (mDC) served as antigen-presenting cells for this analysis; they were prepared from peripheral blood monocytes by cultivation with rhu-IL-4 and GM-CSF, then matured with a cytokine cocktail consisting of TNFα , rhu-IL-1β , rhu IL-6, and prostaglandin E2. Myeloid DC were then transduced with in vitro transcribed CT7 mRNA or positive control antigen flu matrix protein (MP) mRNA by electroporation. Negative control mDC were also mock electroporated without mRNA. The dendritic cells were then co-cultured with the expanded bone marrow lymphocytes at 1:1 and 1:20 ratios in IFNγ ELISpot assays. Positive control stimulation with the T cell superantigen Staphylococcal enterotoxin B (SEB) served to assess overall responsiveness of the polyclonal lymphocyte population. Results from four patients are shown in Table 1. Two of four patients, both diagnosed with stage IIIa disease and both expressing CT7 mRNA by RT-PCR, exhibited increases in IFNγ-secreting lymphocytes when co-incubated with CT7-transduced mDC compared to mock electroporated mDC. In contrast, one stage I patient who did not express CT7 mRNA and one stage IIIb, CT7+ patient did not show any increase in IFNγ-secreting cells in response to CT7-transduced mDC stimulation. These data indicated that CT7 expression in myeloma cells can provoke cellular immunity, and suggests that CT7-specific vaccines may boost this immunity. These ongoing studies will also characterize the antigenic T cell epitopes of CT7. Table 1. CT7-specific bone marrow lymphocytes Patient Stage CT7 mRNA expression Mock CT7 Flu MP SEB IFNγ-secreting cells/50,000 bone marrow lymphocytes 1 IIIa + 28.0 ± 7.1 57.7 ± 5.9 nd 288 ± 42.1 2 IIIa + 6.3 ± 2.3 27.7 ± 10.1 17.7 ± 5.3 90.0 ± 50.3 3 Ia − 6.0 ± 1.0 8.5 ± 0.5 nd 91.5 ± 54.5 4 IIIb + 1.5 ± 0.5 4.5 ± 0.5 nd 72.0 ± 43.0