Lenalidomide Decreases PD-1 Expression, Depletes Regulatory T-Cells and Improves Cellular Response to a Multiple Myeloma/Dendritic Cell Fusion Vaccine In Vitro

We have developed a cancer vaccine for multiple myeloma in which patient derived tumor cells are fused with dendritic cells (DCs) such that a broad array of tumor antigens are presented in the context of DC mediated costimulation. In clinical studies, we have demonstrated that vaccination results in the induction of anti-tumor immunity and disease response in a subset of patients. A fundamental challenge limiting the efficacy of cellular immunotherapy is the immunosuppressive milieu that characterizes patients with myeloma. We have previously reported that the PD-1/PDL-1 pathway plays an important role in suppressing T cell immunity in patients with myeloma, PD-1 expression is upregulated on T cells isolated from patients with multiple myeloma, and PD-1 blockade is associated with enhancement of T-cell response to the vaccine. Lenalidomide is a potent anti-myeloma agent whose activity may be linked, in part, to its immunomodulatory properties. We hypothesized that lenalidomide would augment the capacity to elicit anti-myeloma immunity. In our current study, we examined the effect of lenalidomide on T-cell activation and polarization, PD-1 signaling, and vaccine-induced responses in vitro.

Peripheral blood mononuclear cells were cultured in media containing IL-2 with and without 1μM lenalidomide. The expression of cell surface molecules and intracellular cytokines was assessed using flow cytometry. Exposure of unstimulated T cells to lenalidomide resulted in a decrease in the percentage of CD4+ T cells expressing PD-1 (from 8.0% to 5.6%, p=0.04) and a 2 fold increase in T-cell proliferation as measured by incorporation of tritiated thymidine. We then examined the effect of lenalidomide on T cell activation by ligation of the costimulatory complex using antibodies directed against CD3 and anti CD28. Most notably, the upregulation of PD-1 by CD3/CD28 ligation was markedly decreased in the presence of lenalidomide as measured in CD4+ cells (from 26% to 15%, p<0.0001) and in CD8+ cells (from 16% to 10% p<0.01). Ligation of CD3/CD28 in the presence of lenalidomide resulted in greater degree of Th1 polarization as manifested by a 2 fold increase in the percentage of CD8+ T cells expressing IFNγ (p=0.02) and a decrease in the percentage of regulatory T-cells (CD4+CD25+FoxP3+) from 6.88% to 3.13% (p=0.02). In addition, the percentage of NK cells (CD3-CD56+) expressing IFNγ following CD3/CD28 ligation was 5-fold greater (p=0.03) in the presence of lenalidomide. Lastly, we studied the effect of lenalidomide on T-cells stimulated in vitro by the DC/myeloma fusion vaccine. DC/myeloma fusions were generated as previously described. Fusion mediated stimulation of autologous T cells in the presence of lenalidomide resulted in an increase in the percentage CD4+ and CD8+ T cells expressing IFNγ, (5.35% to 8.79%, p=0.06; and 6.37% to 9.85%, p=0.03, respectively). The proportion of regulatory T-cells decreased from 9.57% to 4.43% in the presence of lenalidomide (p<0.01). As with non-specific stimulation, PD-1 expression on CD4+ cells in the presence of lenalidomide decreased from 24% to 19%. In concert with these findings, exposure to lenalidomide resulted in increased cytotoxic T lymphocyte mediated lysis of autologous tumor targets (from 25% to 36%).

In vitro exposure to lenalidomide results in enhanced T-cell activation in response to direct ligation of the co-stimulatory complex and stimulation by the DC/myeloma fusion vaccine. Exposure to lenalidomide suppresses T cell expression of PD-1 and expansion of regulatory T cells, 2 critical pathways responsible for tumor mediated immune suppression. To our knowledge, this is the first demonstration of an interaction between lenalidomide and the PD-1/PDL-1 pathway. These findings support the development of cellular immunotherapy in conjunction with lenalidomide, including its use with the DC/myeloma fusion vaccine.

No relevant conflicts of interest to declare.