PD-1 deficiency augments bone marrow failure in a minor-histocompatibility antigen mismatch lymphocyte infusion model

Abstract

Although PD-1 blockade has revolutionized cancer immunotherapy, immune-related adverse events (irAEs) present life-threatening complications. Recent reports of aplastic anemia (AA) as irAEs implicate PD-1/PD-L1 as important in preventing immune-mediated destruction of the hematopoietic niche. Infusion of PD-1-deficient (PD-1 knockout [KO]) lymph node (LN) cells into minor-antigen mismatched mice resulted in early mortality, as well as more severe bone marrow (BM) hypoplasia, anemia, and BM microarchitecture disruption in PD-1 KO LN-infused mice relative to mice that received B6 LN cell infusion. Mice that received PD-1 KO LN cells had more CD8⁺ T-cell infiltration of the BM and greater expansion of H60-specific CD8⁺ T cells than did their B6 LN-infused counterparts. In the spleen, CD8⁺ T cells were skewed to an effector memory phenotype, suggesting accelerated differentiation of PD-1 KO T cells. Our data suggest that PD-1 dysregulation has a role in murine BM failure and vigilance in irAE monitoring may be desirable to treat early AA and related cytopenias.

Figure 1

PD-1 KO LN infusion induces severe BM failure. (A) Bone marrow (BM) nucleated cell number in TBI (n=2), B6 LN-induced BM failure (n=4), and PD-1 KO LN-induced BM failure (n=8). (B) Neutrophils (NE), white blood cells (WBC), red blood cells (RBC), and platelets (PLT) in the peripheral blood at day 8. (C) Representative H&E staining of the sternums of TBI, B6 LN-induced, and PD-1 KO LN-induced BM failure mice at day 8. Top row: 100× original magnification, with 50-μm scale bars (white bar). Bottom row: 200× original magnification. Capped lined indicate a p-value obtained through unpaired t test. *, p < 0.05; **, p < 0.01.

Figure 2

PD-1 KO LN-infused mice have increased T cell BM infiltration and antigen-specific T cell expansion. (A) Representative flow plots and statistical analyses of CD4⁺ and CD8⁺ T cells in the BM of PD-1 KO LN-infused mice. CD4⁺ and CD8⁺ T cells were gated on all live BM cells. (B) Representative flow plots and summarized results of H60-specific T cells in the BM of BM failure mice. H60 tetramer-positive cells were gated on live BM cells. (C) Representative flow plots and summarized results of H60-specific T cells in the spleens of BM failure mice. H60 tetramer-positive cells were gated on live splenocytes. Capped lines indicate a p-value obtained through unpaired t-test, while brackets indicate a p-value obtained through multiple comparisons. *, p < 0.05; **, p < 0.01.

Figure 3

CD8⁺ T cells are terminally differentiated in the BM, while PD-1 KO T cells have accelerated terminal differentiation in the spleen. (A) Representative flow plots and statistical analyses of CD8⁺ T cell differentiation subsets in the BM of TBI (dark grey), B6 LN-induced BM failure (black), and PD-1 KO LN-induced (light grey) BM failure mice. Cells shown in the flow plots were gated on all live CD8⁺ T cells. (B) Representative flow plots and statistical analyses of CD8⁺ T cell differentiation subsets in the spleen. (C) Representative ELISpot wells (Right) showing IFN-γ secretion by splenocytes isolated from TBI only, B6 LN-infused, and PD-1 KO LN-infused mice. Cells were rested overnight at 37°C, 5% CO₂ prior to ELISpot development and analysis. Quantification of IFN-γ-positive spots per 10⁵ splenocytes plated (Left). Capped lines indicate a p-value obtained through unpaired t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.