Calcineurin inhibition rescues alloantigen-specific central memory T cell subsets and promotes chronic GVHD

Calcineurin inhibitors (CNIs) constitute the backbone of modern acute graft-versus-host disease (aGVHD) prophylaxis regimens but have limited efficacy in the prevention and treatment of chronic GVHD (cGVHD). We investigated the effect of CNIs on immune tolerance after stem cell transplantation with discovery-based single cell gene expression and T-cell receptor (TCR) assays of clonal immunity in tandem with traditional protein-based approaches and preclinical modeling. While CNIs suppress the clonal expansion in CD8+ T-cells, alloreactive CD4+ T-cell clusters preferentially expanded during GVHD. Moreover, CNIs mediated reversible dose-dependent suppression of T cell activation and all stages of donor T-cell exhaustion but favored the differentiation to central memory T-cells. Critically, CNIs promoted the expansion of both polyclonal and TCR-specific alloreactive central memory CD4+ T-cells with high self-renewal capacity that mediated cGVHD following drug withdrawal. In contrast to post-transplant cyclophosphamide (PT-Cy), CSA was ineffective in eliminating IL-17A-secreting alloreactive T cell clones that mediate cGVHD. Collectively, we show that although CNIs attenuate aGVHD, they paradoxically rescue alloantigen-specific central memory T-cells, especially within the CD4+ compartment in lymphoid and GVHD target tissues, thus predisposing to cGVHD. These data provide further evidence to caution against CNI-based immune suppression without concurrent approaches that eliminate alloreactive T-cell clones. B6D2F1 recipients were transplanted with B6 BM (5x10^6) + B6 T cells (2x10^6) + TEa TCR transgenic T cells (1x10^3) and treated daily with saline or CSA (5 or 50 mg/kg/kg). Spleens were taken on day 7, pooled from every 2 mice (n = 6 mice per group) and barcoded with hashtag Total Seq C. Donor CD4+ and CD8+ T cells were sort purified and combined per group for single cell RNA sequencing. Please note that we sequenced for mRNA and TCR; There are 3 samples and each sample contains CD4 T and CD8 T from 3 "animals (pooled from every two)". CD4 and CD8 T cells have different metadata column names so were not merged.