Early Initiation of Calcineurin Inhibitor Induces CD8+ Transitory Exhausted-like and CD4+ Cytotoxic T Cells, Maintaining Responsiveness to PD-1 Blockade and Contributing to the Development of Chronic Gvhd after Post-Transplant Cyclophosphamide

[Introduction] We have previously reported that early initiation of calcineurin inhibitors from day 0 (early-CNI) induces CD8⁺ transitory exhausted-like T cells (transitory-Tex-CNIs) and granzyme B⁺ (GZMB⁺) CD4⁺ T cells after mouse allogeneic hematopoietic cell transplantation (allo-HCT) with post-transplant cyclophosphamide (PTCy), while delayed initiation of CNIs from day 5 post-transplant did not induce these populations (Senjo H: 65^th ASH annual meeting). In human PTCy-based haploidentical peripheral blood stem cell transplantation (Haplo-PTCy-PBSCT), we confirmed that early-CNI induced transitory-Tex-CNIs and CD74⁺GZMB⁺CD4⁺ cells. Although we previously showed that transitory Tex-CNIs contribute to the development of chronic GVHD (cGVHD) after mouse allo-HCT (Senjo H: Blood 2023), the functions of CNI-induced T cells in human, as well as their contribution to cGVHD, remained to be clarified. In the current study, we explored the phenotype and functions of human transitory Tex-CNIs and CD74⁺GZMB⁺ CD4⁺ T cells, using peripheral blood T cells harvested from patients on day 28 after Haplo-PTCy-PBSCT. We also examined the relationship between the abundance of these cells on day 28 and the development of cGVHD. [Methods] In Haplo-PTCy-PBSCT, tacrolimus (TAC) was initiated either on day -1 (early-TAC; n = 20) or day 5 (late-TAC; n = 20) of the transplantation, and blood samples were prospectively collected following institutional review board approval. For xenograft transplantation, transitory-Tex-CNIs and GZMB⁺ CD4⁺ T cells were purified from patients’ peripheral blood on day 28 after Haplo-PTCy-PBSCT with early-TAC. These cells were mixed at a 1:1 ratio and injected into irradiated NOD-SCID-IL2Rγ^-/- (NOG) mice. [Results] By comparing scRNA-seq data from T cells on day 28 after Haplo-PTCy-PBSCT with early-TAC to those with late-TAC, we identified Cx3cr1⁺ Cd16⁺ Cd8⁺ cells with higher expression of Gzmb and Mki67 as transitory-Tex-CNIs. We further found that Cd74⁺ Gzmb⁺ Cd4⁺ T-cell cluster induced by early CNIs specifically expressed Crtam, a marker for CD4⁺ cytotoxic T cells (CD4⁺ CTLs). Therefore, we annotated this population as CD4⁺ CTLs. Importantly, scTCR-seq showed that both clusters had markedly less TCR diversity compared to other clusters, suggesting that these clusters were enriched with alloreactive donor T cells that clonally expanded after allo-HCT. As expected, Flow cytometric analysis confirmed that both CX3CR1⁺CD16⁺CD8⁺ transitory-Tex-CNIs and CRTAM⁺CD74⁺CD4⁺ CTLs were significantly increased in the early-TAC group compared to the late-TAC group on day 28. In an in vitro stimulation assay, we found that transitory-Tex-CNIs and CD4⁺ CTLs purified from patients in the early-TAC group showed higher responsiveness to PD-1 blockade compared to other CD8⁺ and CD4⁺ T cells, resulting in higher upregulation of GZMB and Ki67. In a xenograft model, NOG mice transplanted with both transitory-Tex-CNIs and CD4⁺ CTLs showed significantly lower body wight and reduced tear secretion volume compared to those transplanted with either population alone or with other CD4⁺ and CD8⁺ T cells, indicating that CNI-induced CD4⁺ and CD8⁺ populations are responsible for the development of cGVHD, as demonstrated in murine models (Senjo H: Blood 2023, Wang Y: JCI 2024). In the prospective cohort analysis, we found that the cumulative incidence of moderate to severe cGVHD at one-year post-transplant was significantly higher in patients treated with early-TAC compared to late-TAC (20.0% vs 0.0%, p=0.048). Additionally, the cumulative incidence of immunosuppressant cessation at two years was significantly lower in the early-TAC group compared to the late-TAC group (61.1% vs 92.6%, p=0.047). In early-TAC group, we found that patients who developed cGVHD had significantly higher proportions of CD8⁺ transitory-Tex-CNIs and CD4⁺ CTLs on day 28 compared to those who did not develop cGVHD thereafter (CD8; 20.8% vs 9.0%, p=0.002, CD4; 13.9% vs 2.8%, p=0.001). [Conclusion] We found that human transitory-Tex-CNIs and CD4⁺ CTLs induced by early-CNI maintain the responsiveness to PD-1 blockade and contribute to the development of chronic GVHD after Haplo-PTCy-PBSCT. These populations could be promising biomarkers for predicting the development of cGVHD. Delayed initiation of CNIs is crucial for tolerance induction after PTCy without inducing these T-cell populations.