STAT3 Inhibition Promotes Potent Th1 Responses By Down Regulating Pdl-1 Expression On Tumor Cells

STAT3 signaling is upregulated in cancer cells and is thought to be a critical mediator of tumor associated immune suppression. We examined the relationship of STAT3 and PDL-1, an important negative regulator of T cell immune responses via interactions with PD-1. The 5'upstream, putative promoter region of PDL-1 gene contains four potential STAT3 binding sites close to transcription start site, suggesting a possible role of STAT3 in transcriptional regulation of PDL-1. We investigated for a possible relationship between STAT3 and PDL-1 in multiple myeloma (MM-RPMI) cell line. Using chromatin immunoprecipitation assay (ChIP assay), we observed direct binding of STAT3 to the PDL-1 promoter region in MM-RPMI cells. Moreover, an enhanced occupancy of STAT3 on the PDL-1 promoter region was also observed when the cells were treated with IL-6 (20 ng/ml). Concurrent with the above observation, IL-6 treated MM-RPMI cells showed an enhanced expression of PDL-1 protein. Treatment of RPMI-MM cells with STAT3 inhibitors AG490 and CDDO-Me for 72 hrs resulted in PDL-1 expression levels of 24.5% and 15.1% respectively, compared to 97.5% for untreated control cells as determined by FACS analysis. Based on these observations, we evaluated the effect of STAT3 inhibition by exposure to small molecule STAT3 inhibitor on response to a tumor vaccine consisting of myeloma cells fused with dendritic cells (DC). DC/myeloma fusion cells were co-cultured with autologous T cells in the presence or absence of small molecule STAT3 inhibitor (CDDO-Me). Treatment of the cocultures with CDDO-Me resulted in an increased in the percentage of CD4/IFNγ (11.57±SEM 1.16; n=3) and CD8/IFNγ (23.37±SEM 2.25; n=3) expressing cells as compared to untreated cocultures (3.1± SEM 0.72; n=3) and (4.43±SEM 0.81; n=3) respectively. Moreover, we also observed a concomitant decrease in both CD4+ and CD8+ T cells expressing IL-10 or IL-4 compared to untreated cocultures. We also noted a decrease in the percentage of immunosuppressive regulatory T cells (CD4+/CD25+/FOXP3+) in treated cocultures (8.33±SEM 0.59; n=3) as compared to untreated cocultures (28.50±SEM 2.0; n=3). These findings indicate a pivotal role of inflammatory cytokine IL-6 and its downstream activator STAT3 in regulating PDL-1 expression. Thus, strategic inhibition of STAT3/IL-6 pathway and therefore down regulation of PDL-1 could enhance the potency of tumor/DC fusion cell based cancer vaccines for immunotherapeutic treatments. Furthermore, the data demonstrate that the availability of novel adjuvants offers the potential for combination therapy that may have profound effect on the potency and durability of responses to cellular immunotherapy.