Combined Ablation of PD-1 and Pparγ Synergistically Promotes M1-like Macrophage Polarization and Proinflammatory Differentiation

PD-1 is a crucial checkpoint receptor in cancer immunotherapy. In T cells, the PD-1 inhibitory function is mediated by SHP-2 recruitment. Specific deletion of SHP-2 in myeloid cells, but not in T-cells, enhances anti-tumor immunity. In tumor-associated macrophages (TAMs), the PD-1:SHP-2 axis regulates macrophage differentiation, and ablation of this axis in the context of cancer, reverses polarization from anti-inflammatory M2-like to pro-inflammatory M1-like phenotype, with enhanced signature of NF-κB signaling. PPARγ, a ligand-dependent nuclear transcription factor, promotes M2 polarization and suppresses anti-tumor immunity by inhibiting NF-κB and decreasing pro-inflammatory cytokine production. We sought to investigate the crosstalk between PD-1- and PPARγ-mediated fate commitment in macrophages. To this end, we used mice with conditional targeting of the Pdcd1 allele (Pdcd1^fl/fl) or the Pparγ (PPARγ^fl/fl) allele and crossed them with LysMCre to selectively delete Pdcd1 alone (Pdcd1^fl/flLysMCre, named PD-1 KO), or Pdcd1 and Pparγ in combination (Pdcd1^fl/fl/PPARγ^fl/fl/LysMCre, named DKO) in myeloid cells. Bone marrow derived macrophages (BMDM) were generated from WT, PD-1 KO and DKO mice and were stimulated with the TLR1/2 agonist PAM. Subsequently, IL-6 and TNF-α secretion was quantified by ELISA. To further assess if the polarization potential of macrophages was altered, we also analyzed expression of surface markers in macrophages following culture under classic M1 polarizing conditions with LPS and IFNγ, or classic M2 polarizing conditions with IL-4. At 24 hours of culture with PAM, PD-1 KO BMDM showed the highest pro-inflammatory response compared to WT and DKO BMDM, evidenced by robust TNF-α and IL-6 production. At 48 hours, PAM yielded significantly higher TNF-α production in DKO BMDM than in PD-1 KO BMDM, suggesting a more pronounced pro-inflammatory response with the additional PPARγ ablation, after prolonged stimulation. Under M1 polarizing conditions, MHC-II expression was highest in PD-1 KO macrophages, was not significantly altered by PPARγ deletion, and displayed lowest expression in WT macrophages, suggesting that PD-1 is likely the primary driver for macrophage fate commitment away from an activated phenotype within a pro-inflammatory microenvironment. Analysis of surface markers following M2 polarizing culture revealed that PPARγ and PD-1 synergistically altered macrophage differentiation. Specifically, at 24 hours of M2 polarizing culture, CD86 and MHC-II expression levels were highest in DKO macrophages, intermediate in PD-1 KO, and lowest in WT macrophages. Expression of PD-L1, a marker typically induced under pro-inflammatory conditions, was highest in DKO macrophages, with lower expression in PD-1 KO and lowest levels in WT macrophages. Conversely, expression of CD206, a classic M2 marker, was lowest in DKO, with low level expression in PD-1 KO and highest expression in WT macrophages. These results indicate that PD-1 blockade induced a shift in macrophage polarization towards an M1-like pro-inflammatory phenotype under M2 conditions, which was further enhanced by PPARγ ablation. Notably, PPARγ ablation mediated this additive effect at a later time point, suggesting that PPARγ may be a crucial regulator of macrophage differentiation in an immunosuppressive microenvironment in a time-dependent manner. The demonstrably higher MHC-II and CD86 expression in DKO BMDM signify an enhanced antigen presentation and co-stimulatory capacity of macrophages and suggest that the joint blockade of PD-1 and PPARγ could improve anti-tumor responses by synergistically augmenting T-cell activation. These findings denote the context-dependent roles of PD-1 and PPARγ in macrophage polarization and provide evidence that they synergistically maintain the M2 immunosuppressive macrophage phenotype. Our findings reveal that PPARγ blockade can enhance myeloid cell immune responses beyond PD-1 ablation and suggest PPARγ as a potential therapeutic target to improve the outcome of checkpoint immunotherapy in cancer.