Recently, we reported that tumor spheres formed by breast cancer cells were visibly smaller in size in a Th1 enriched microenvironment, differentiation of granulocytic CD14−/HLA-DR−/CD11b+/CD33+ and monocytic CD14+/HLA-DR−/CD11b+/CD33+ MDSC populations was reduced with further reduction and attenuation of their suppressive activity in the presence of aATC . In this study, we investigated the mechanism(s) of aATC mediated inhibition of MDSC in the presence or absence of Th1 microenvironment. We show significantly decreased differentiation and accumulation of MDSC in the presence of aATC or aATC and Th1 cytokines. The decreased percentage of MDSC was paralleled by significantly lower levels of IL-6, COX2, PGE2, ARG1 in the presence of aATC or aATC and Th1 cytokines. While, levels of IFN-γ, IL-2, TNF-α, IL-12 and chemokines CXCL9 and CXCL10 were higher in the presence of aATC or aATC and Th1 cytokines.
Consistent with other studies that inflammation is associated with the expansion of MDSC [21, 22], our data also show that increased numbers of MDSC were accompanied by increased levels of proinflammatory cytokines IL-6 and IL-1β/IL-1Ra ratio. A delayed accumulation of MDSC and reduced primary and metastatic tumor progression was reported in mice that have reduced inflammation due to IL-1 receptor-deficiency [23, 24]. On the other hand, excessive inflammation in IL-1R antagonist-deficient mice promoted the accumulation of MDSC and produced MDSC with enhanced suppressive activity [23, 24]. Relevance of increased levels of TNF-α in the presence of Th1 cytokines or Th1 cytokines + aATC in the context of MDSC is not clear. TNF has been shown to play a crucial role in the differentiation of myeloid cells [25, 26]. However, binding of TNF to TNFR-1 and TNFR-2 activates distinct signaling pathways [27–30]. Depending on TNF signaling pathway it may favor tumor growth and differentiation of MDSC or may induce immune responses .
In addition to cytokines, bioactive lipid mediators, such as PGE2 and COX2 produced by many tumors are known to induce the inflammatory and immune suppressive tumor microenvironment [10, 32–34]. Kalinski et al. showed that PGE2 can modulate the Th1 responses by impairing IL-12, and IFN-γ expression [35–37]. PGE2 and COX2 amplify ARG1 levels in MDSC and suppress the adaptive immune response in part through ARG1 production that enhances the L-arginine catabolism and thus depletion of L-arginine [13, 19, 38, 39]. Catabolism of L-arginine is essential for the suppressive activity of MDSC, which serves as a substrate for two enzymes, oxide synthase (iNOS) and arginase 1 (ARG1). MDSCs express high levels of both ARG1 and iNOS and both these enzymes play roles in the inhibition of T-cell function [13, 19, 20]. Depletion of L-arginine in the tumor microenvironment leads to the inhibition T cell proliferation by decreasing expression of the CD3ζ chains . and induction of T cell apoptosis . Collectively, these studies show a strong association between expansion of MDSCs and inflammation mediated by the arachidonic acid cascade. Consistent with these findings, our data suggest a strong correlation between increased accumulation of MDSC and high levels of COX2/PGE2/ARG1 expression.
Analysis of chemokines showed significantly reduced levels of CCL3/MIP1α, CCL4/MIP-1β, CCL5/RANTES, CXCL9/MIG and CXCL10/IP-10 in the supernatants from control culture conditions (without aATC) which increased dramatically when either aATC or aATC and Th1 cytokines were added to the co-cultures. Co-cultures with reduced chemokine levels contained a significantly higher percentage of MDSC and significantly higher levels of COX2 and PGE2. PGE2 has been shown to inhibit mRNA and protein expression of chemokines including CCL3/MIP1α, CCL4/MIP-1β, CXCL10/IP-10 in activated monocytes and macrophages [41–44]. COX2 and PGE2 were reported to deregulate the chemokine production of DCs, abrogating the CXCL9/MIG, CXCL10/IP-10 and CCL5/RANTES-mediated ability of DC to attract naive, effector, and memory T and NK cells [42, 44–46].
Activated T cells express a variety of surface markers, including CD25, CD71, CD95, CD137, HLA-DR, and secrete Th1 cytokines IL-2 and IFN-γ . We analyzed CD71, CD62L and IFN-γ as T cell activation and functional markers and NKG2D as NK or NKT cell activation marker to assess the suppressive activity of MDSC on T cells and NK cells. In the presence of MDSC isolated from control (without aATC), the expression of all the T cell activation markers were markedly downregulated whereas MDSC isolated from aATC containing co-cultures showed attenuated inhibition of T cells activation markers. Study by Ochoa et al. showed restoration of IFN-γ production and T-cell proliferation after MDSC depletion . MDSC can abrogate the expression of L-selectin (CD62L) on both CD4+ and CD8+ T cells, subverting the homing of these cells to the tumor site leading towards a dominant immunosuppressive microenvironment .