High macrophage PD-L1 expression not responsible for T cell suppression.

Tumors are often comprised of microenvironments (TMEs) with a high proportion of cells and molecules that regulate immunity. Peritoneal cavity (PerC) cell culture reproduces key features of TMEs as lymphocyte proliferation is suppressed by PerC macrophages (Mϕs). We monitored the expression of T cell stimulatory (Class II MHC, B7) and inhibitory (PD-L1) molecules by PerC APCs before and after culture and report here that IFNγ-driven PD-L1 expression increased markedly on PerC Mϕs after TCR ligation, even more so than seen with direct APC activation by LPS. Considering the high APC composition of and pronounced PD-L1 expression by PerC cells, it was surprising that blocking PD-1/PD-L1 interaction by mAb neutralization or genetic ablation did not relieve suppression. This result parallels TME challenges observed in the clinic and validates the need for further study of this culture model to inform strategies to promote anti-tumor immunity.

CD28 ligation increases macrophage suppression of T-cell proliferation.

When compared to spleen or lymph node cells, resident peritoneal cavity cells respond poorly to T-cell activation in vitro. The greater proportional representation of macrophages in this cell source has been shown to actively suppress the T-cell response. Peritoneal macrophages exhibit an immature phenotype (MHC class II(lo), B7(lo)) that reduces their efficacy as antigen-presenting cells. Furthermore, these cells readily express inducible nitric oxide synthase (iNOS), an enzyme that promotes T-cell tolerance by catabolism of the limiting amino acid arginine. Here, we investigate the ability of exogenous T-cell costimulation to recover the peritoneal T-cell response. We show that CD28 ligation failed to recover the peritoneal T-cell response and actually suppressed responses that had been recovered by inhibiting iNOS. As indicated by cytokine ELISpot and neutralizing monoclonal antibody (mAb) treatment, this 'cosuppression' response was due to CD28 ligation increasing the number of interferon (IFN)-γ-secreting cells. Our results illustrate that cellular composition and cytokine milieu influence T-cell costimulation biology.Cellular & Molecular Immunology advance online publication, 23 April 2012; doi:10.1038/cmi.2012.13.

Cytokine treatment of macrophage suppression of T cell activation.

High Mphi:T cell ratios suppress the immune response to the retroviral superantigen Mls by IFNgamma-triggered production of the arg- and trp-consuming enzymes iNOS and IDO. Attempts to reverse suppression by treatment with pro-inflammatory cytokines revealed that IL-6 improved the T cell response to Mls and the pro-hematopoietic cyokines IL-3 and GM-CSF increased suppression. GM-CSF treatment increased Mphi expression of CD80, a ligand for the immune suppressive B7H1 and CTLA-4 receptors. These results illustrate potential strategies for reversing the suppression of cell-mediated immunity characteristic of the high Mphi:T cell ratios found in many tumors.