Immu-36. pd-1ko hematopoietic stem cells rapidly differentiate into glioma-infiltrating effector t-cells

Abstract INTRODUCTION We previously published that hematopoietic stem cell combination therapy (HSC+aPD1) extends survival in murine CNS malignancies, including glioma. To better understand lymphoid-specific mechanisms, we analyzed various lymphoid tissues in a murine glioma model. We hypothesize early PD-1 blockade in HSCs influences later differentiation trajectories. Transferred HSCs differentiate into T-cells within the thymus and then migrate to the intracranial tumor microenvironment as anticipated. Indeed, blocking PD-1/PD-L1 signaling during HSC differentiation significantly impacts subsequent T-cell maturation. Remarkably, TILs derived from HSCPD1KO cells were biased towards CD4+ Treg or CD8+ effector lineages, whereas HSCPD1+/+ progeny leaned toward immature/naïve cell states. METHODS HSCs were prepared from constitutive GFP-expressing (CD45.2+) and PD1KO (CD45.2+) mice and transferred into CD45.1+ hosts. Sublethal 5Gy total body irradiation was administered day 5 post-intracranial tumor implantation. The next day, 1e6 HSCs from a 1:1-GFP:PD1KO cocktail were injected intravenously. Descriptive statistics are reported as mean ± SD. Transferred HSCs engraft, expand, differentiate, tended and migrate to form approximately 25% of the TIL compartment in only 15 days. RESULTS We found that, 56.2% ± 20.3 of CD3+ TILs display an immature CD4+CD8+ (DP) phenotype in early disease, and 43.8% ± 17.4 of these DP-TILs are HSC-derived. DP-TILs were virtually absent in advanced disease 29 days after treatment, but HSC-derived TILs most commonly fell within the single-positive (SP) TIL subsets, comprising 9.8% ± 5.6 of SP-CD8+ TILs. Notably, HSCPD1KO-derived cells were 4x more abundant amongst SP-CD8 TILs when compared to HSCGFP-derived cells (6.5% vs 1.6%, p < 0.05). SP-CD8 thymocytes were similarly enriched for HSCPD1KO progeny (41.1% vs 24.7%, p < 0.05) suggesting that PD-1 blockade impacts thymopoiesis directly. CONCLUSION PD-1 signaling modulates thymic output. Blocking the PD-1/PD-L1 axis during thymopoiesis increases the abundance of HSC-derived CD8+ effectors at the tumor site. HSC+aPD1 therapy is uniquely positioned to leverage thymus-dependent anti-tumor immunity.