Lenalidomide overcomes the immunosuppression of regulatory CD8+CD28- T-cells.

Although lenalidomide and pomalidomide are well-established treatment options in patients with multiple myeloma, their immune-modulating effects are not fully understood. While CD8+CD28- regulatory T-cells in patients with hematologic disorders display a known immune-escape mechanism, we show that lenalidomide can overcome the immunosuppressive impact of CD8+CD28- T-cells. We analyzed in vitro the antigen-specific T-cell responses of healthy donors and patients with multiple myeloma with or without the addition of autologous CD8+CD28- T-cells in the absence and presence of lenalidomide. We found that lenalidomide enhances the antigen-specific secretion of IFN-γ and Granzyme B despite the addition of CD8+CD28- T-cells. Furthermore, we showed that lenalidomide inhibits the IL-6 secretion of mononuclear cells, triggered by CD8+CD28- T-cells. The addition of IL-6 counteracts the action of lenalidomide based stimulation of IFN-γ secretion and induction of T-cell maturation but not the secretion of Granzyme B. Surprisingly, pomalidomide failed to induce IL-6 suppression and displayed immunostimulating effects only after a prolonged incubation time. Analysis of the IL-6 modulating cereblon-binding protein KPNA2 showed the similar degradation capacity of lenalidomide and pomalidomide without explaining the divergent effects. In conclusion, we showed that IL-6 and lenalidomide, but not pomalidomide, are opponents in a myeloma-antigen specific T-cell model.

Lenalidomide enhances myeloma-specific T-cell responses in vivo and in vitro.

Immunomodulation is an important part of lenalidomide's mode of action. We analyzed the impact of lenalidomide on T cells from patients with multiple myeloma during lenalidomide therapy in vivo and in patients with lenalidomide-refractory disease in vitro Patients enrolled in the German Speaking Myeloma Multicenter Group (GMMG) MM5 trial received a consolidation therapy with two cycles of lenalidomide after autologous stem cell transplantation (ASCT). Half of the study population continued treatment with lenalidomide maintenance therapy for 2 y, while the other patients received lenalidomide maintenance therapy until complete remission. We analyzed 58 patients with (n = 30) or without (n = 28) lenalidomide therapy and 12 patients refractory to lenalidomide with regards to their anti-myeloma-specific T-cell responses displayed by IFNγ, Granzyme B, and Perforin secretion. The immunophenotype of T-cells was investigated by flow cytometry. Significantly, more myeloma-specific T-cell responses were observed in patients during lenalidomide therapy, compared to patients without treatment. Furthermore, we found on T-cells from patients treated with lenalidomide a decreased CD45RA expression, indicating a maturated immunophenotype and a decreased expression of CD57, indicating functional T cells. An improved myeloma-specific T-cell response was observed in 6 out of 12 heavily pretreated patients (refractory to lenalidomide) after in vitro incubation with lenalidomide. Complementary to the results in vivo, lenalidomide decreased CD45RA expression on T cells in vitro.

Cytotoxicity of tumor antigen specific human T cells is unimpaired by arginine depletion.

Tumor-growth is often associated with the expansion of myeloid derived suppressor cells that lead to local or systemic arginine depletion via the enzyme arginase. It is generally assumed that this arginine deficiency induces a global shut-down of T cell activation with ensuing tumor immune escape. While the impact of arginine depletion on polyclonal T cell proliferation and cytokine secretion is well documented, its influence on chemotaxis, cytotoxicity and antigen specific activation of human T cells has not been demonstrated so far. We show here that chemotaxis and early calcium signaling of human T cells are unimpaired in the absence of arginine. We then analyzed CD8(+) T cell activation in a tumor peptide as well as a viral peptide antigen specific system: (i) CD8(+) T cells with specificity against the MART-1aa26-35*A27L tumor antigen expanded with in vitro generated dendritic cells, and (ii) clonal CMV pp65aa495-503 specific T cells and T cells retrovirally transduced with a CMV pp65aa495-503 specific T cell receptor were analyzed. Our data demonstrate that human CD8(+) T cell antigen specific cytotoxicity and perforin secretion are completely preserved in the absence of arginine, while antigen specific proliferation as well as IFN-γ and granzyme B secretion are severely compromised. These novel results highlight the complexity of antigen specific T cell activation and demonstrate that human T cells can preserve important activation-induced effector functions in the context of arginine deficiency.