Programmed cell death-1 receptor deficiency enhances CD30+ T regulatory cell function in Melanoma [Murine NanoString CosMx SMI]

Regulatory T cells (Tregs) are vital for immune suppression rendering their function through multiple pathways including the expression of co-inhibitory receptors. The role of co-receptors namely PD1 in Treg function remains controversial. We demonstrate that PD1 is a master checkpoint in Tregs controlling several aspects of Treg biology in tumor immunity. Our data demonstrates that PD1 controls the expression and function of a unique co-inhibitory receptor network that dictates Treg function. We found that CD30 plays a central role within this network in driving Treg suppressive function within the tumor microenvironment (TME). Mechanistically, PD1 deficiency unleashed enhanced STAT5 signaling in Tregs, which drove CD30 expression. Our work highlights a new role for PD1 as a checkpoint that negatively controls CD30 expression in Tregs thereby dampening their function. Understanding the landscape changes that PD1 renders to Treg function can enable the use of combination therapies for better treatment outcomes in cancer. Tumors were harvested from B16 tumor from B6 Rag1-/- mice when tumor size reached 800mm3. Tumors were fixed with 10% neutral-buffered formalin, embedded in paraffin and mounted onto slides before being subjected to CoxMx SMI analysis. CosMxTM technology [CosMxTM Mouse Universal Cell Characterization RNA Panel (1000-plex); Nanostring, USA] was applied to 4 FFPE samples: 2 tumors with WT Teff and WT Tregs, 2 tumors with WT Teff and PD1KO Tregs with a mean of 29 fields of view (FOVs) per sample. The following cell surface markers were used for morphology visualization: PanCK, CD68, CD229/B2M, CD45 antibodies and DAPI.