The expression of checkpoint blockade molecules PD-1, PD-L1, CTLA-4, and foxp3+CD25+CD4+ T cells (Tregs) regulate donor T cell activation and graft-vs-host disease (GvHD) in allogeneic hematopoietic stem cell transplant (allo-HSCT). Detailed kinetics of PD-1-, CTLA-4-, and PD-L1 expression on donor and host cells in GvHD target organs have not been well studied. Using an established GvHD model of allo-HSCT (B6 → CB6F1), we noted transient increases of PD-1- and CTLA-4-expressing donor CD4+ and CD8+ T cells on day 10 post transplant in spleens of allo-HSCT recipients compared with syngeneic HSCT (syn-HSCT) recipients. In contrast, expression of PD-1- and CTLA-4 on donor T cells was persistently increased in bone marrow (BM) of allo-HSCT recipients compared with syn-HSCT recipients. Similar differential patterns of donor T cell immune response were observed in a minor histocompatibility (miHA) mismatched transplant model of GvHD. Despite higher PD-1 and CTLA-4 expression in BM, numbers of foxp3+ T cells and Tregs were much lower in allo-HSCT recipients compared with syn-HSCT recipients. PD-L1-expressing host cells were markedly decreased concomitant with elimination of residual host hematopoietic elements in spleens of allo-HSCT recipients. Allo-HSCT recipients lacking PD-L1 rapidly developed increased serum inflammatory cytokines and lethal acute GvHD compared with wild-type (WT) B6 allo-HSCT recipients. These data suggest that increased expression of checkpoint blockade molecules PD-1 and CTLA-4 on donor T cells is not sufficient to prevent GvHD, and that cooperation between checkpoint blockade signaling by host cells and donor Tregs is necessary to limit GvHD in allo-HSCT recipients.

Background Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment for patients with both malignant and non-malignant hematologic disorders. But life-threatening graft-vs-host diseases (GvHD) caused by alloreactive donor T cells limits its clinical use. Alloreactive T cells are also required for graft-vs-leukemia (GvL) and to fight opportunistic infections. Hence, a method that modulates donor T cells activity to reduce GvHD but to retain GvL effect is highly desirable. The inhibitory receptor programed cell death-1 (PD-1) reduces T cell activation through binding with its ligand PD-L1 or PD-L2. Interaction between PD-1 and PD-L1 induces cardiac allograft tolerance and expression of PD-L1 is upregulated in presence of inflammatory stimuli. Here, we studied the role of PD-L1 expression on hematologic and non-hematologic tissues and PD-1 - PD-L1 binding in the development of GvHD. Methods Wild type C57BL/6 (WT B6), PD-L1 knock out B6 (KO) and PD-L2 KO B6 mice were transplanted with 2 x106 splenic T cells and bone marrow (BM) cells from H-2K B10.BR donors. The average acute GvHD scores were determined by combining the GvHD scores obtained from the histological tissue sections of small intestine, large intestine and liver, and weight-loss, posture, activity, fur texture and skin integrity data following standard published procedures. The activation status of splenic T cells was analyzed by flow cytometry. Serum cytokines were determined by using 26 plex Luminex assay. The requirement of hematopoietic and or non-hematopoietic tissues expressing PD-L1 to reduce GvHD was investigated by generating radiation chimeras using WT B6 mice engrafted with PD-L1 KO BM and vice versa. Two months later radiation chimeras were transplanted again with 2 x106 splenic T cells along with 2 x106 BM cells from congenic na•ve H-2K donors. The role of PD-L1 expressing donor hematopoietic cells on the development of acute GvHD was tested by transplanting B10.BR mice with donor PD-L1 KO B6 and WT B6 splenocytes. Results PD-L1 KO B6 recipients had significantly increased acute GvHD (scores 1.68 ± 0.07) compared with WT B6 GvHD (0.78 ± 0.024, p<0.0005) and B6 PD-L2 KO B6 (0.86 ± 0.14, p<0.0005) within day 8 after transplant. All PD-L1 KO B6 recipients had severe GvHD with >25% weight loss on day 8 after transplant and were sacrificed. The WT B6 and PD-L2 KO B6 recipients survived 75% and 80%, respectively until 34 days of transplantation with similar levels of chronic GvHD. To test whether excessive activation of donor T cells caused severe acute GvHD in PD-L1 KO B6 recipients, we determined the activation status of donor T cells in the spleen. The numbers of donor CD4+ and CD8+ T cells expressing ICOS-1 and PD-1 in the spleen were significantly higher (p<0.005) in PD-L1 KO B6 recipients compared with the WT B6 and PD-L2 KO B6 recipients. Additionally, significantly increased levels of serum inflammatory cytokines (IFN-g and TNF-a) were also detected in the PD-L1 KO B6 recipients compared with the WT B6 recipients on day 8 post transplant (Figure 1). Using WT B6 or PD-L1KO hematopoietic cell radiation chimeras as allo-HSCT recipients, we further confirmed that both allo-HSCT radiation chimeras having PD-L1 expressing hematopoietic (10% survival, open square) and non-hematopoietic cells (10% survival, closed triangle) were required to protect from GvHD (Figure 2). We next investigate whether PD-L1 KO donor cells cause increased GvHD. The B10.BR recipients transplanted with donor PD-L1 KO B6 splenocytes had 70% survival while the same recipients transplanted with WT B6 donor splenocytes had only 20% survival until 100 days post transplant. These data suggest that only PD-L1 expressed by host tissues is required to inhibit the development of GvHD. In summary, our data suggest that PD-L1 expressed by host tissues are required to control GvHD and method(s) that enhance expression of PD-L1 in allo-HSCT recipients may represent a novel strategy to control GvHD. Disclosures: No relevant conflicts of interest to declare.