SOCS1 in graft CD8+ T cells ameliorates intestinal aGVHD by modulating chemokine-driven monocyte-to-M1 macrophage polarization Free

Acute graft-versus-host disease (aGVHD), particularly involving the gastrointestinal tract (GI-GVHD), remains a major complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Suppressor of Cytokine Signaling 1 (SOCS1), a negative regulator of JAK/STAT signaling, inhibits cytokine-driven T cell activation, but its role in GVHD pathogenesis remains poorly characterized.

To investigate the immunological impact of SOCS1 loss in T cells during GVHD, we utilized a murine aGVHD model with T cell-specific Socs1 knockout (LckCre-Socs1^fl/fl). We combined single-cell RNA sequencing (scRNA-seq), bulk RNA-seq, ATAC-seq, CUT&Tag, and HD spatial transcriptomics with functional assays to characterize cellular and molecular changes. At steady state, although the overall CD8⁺ T cell proportion remained unchanged between control and knockout (cKO) groups, scRNA-seq revealed a significant shift in CD8⁺ T cell composition, with reduced naïve and increased effector/memory subsets in cKO mice. Splenic CD8⁺ T cells from cKO mice exhibited higher expression of Granzyme B, Perforin, TNF-α, and CD107a, indicating a hyperactivated phenotype.

Bulk transcriptomic and chromatin accessibility profiling revealed upregulation of chemokine signaling genes, including Ccl5, Ccr5, and Cxcr3, in Socs1-deficient CD8⁺ T cells, with increased chromatin accessibility in associated regulatory regions. CUT&Tag and chromatin interaction analyses demonstrated direct occupancy of enhancers upstream of the Ccl5 promoter by STAT1/STAT2, enhancing promoter-enhancer looping and gene activation. These results indicate that SOCS1 restricts inflammatory gene expression via inhibition of STAT-mediated chromatin remodeling.

Importantly, SOCS1 loss reshaped the innate immune landscape. Trajectory analysis of single-cell RNA-seq from monocyte-derived macrophages (MDMs) in spleens showed that cKO-derived MDMs preferentially committed toward a pro-inflammatory MHChi phenotype, driven by interferon-stimulated gene programs and elevated MHC-II expression. Simultaneously, M2-associated gene signatures, such as Mrc1, Tnfsf13, and Tgfb1, were suppressed. Flow cytometry confirmed expansion of iNOS⁺ F4/80^lowCD11b^hi macrophages in cKO mice, characteristic of M1 polarization. CD8⁺ T cells from cKO mice secreted more CCL5, which induced migration and polarization of CCR1⁺ monocytes into M1 macrophages in vitro. Analysis of clinical aGVHD datasets further confirmed increased CCR1 expression and CD8⁺ T–monocyte interactions in patients, implicating the CCL5 pathway in enhancing inflammatory CD8⁺ T cell–macrophage crosstalk.

To assess tissue-level responses to SOCS1 loss, we established lethal GVHD models with WT or Socs1-deficient donor T cells. Compared to controls, recipients receiving cKO T cells exhibited more severe GI pathology, including reduced survival, higher CD8⁺ T cell infiltration into intestinal crypts, and destruction of crypt architecture. Spatial transcriptomics showed CD8⁺ T cells in cKO recipients clustered dominantly in the crypts, expressing high levels of cytotoxic mediators (Gzmb, Tnf, Ifng) and tissue retention molecules (Itgae, Tox), along with decreased expression of epithelial regeneration genes (e.g., Mki67, Top2a). Concurrently, the M1-to-M2 transition seen in WT crypt macrophages was impaired in the cKO group, with persistent pro-inflammatory M1 profiles.

Pharmacologic intervention using Ruxolitinib (JAK1/2 inhibitor) or Maraviroc (CCR5 antagonist) alleviated intestinal inflammation in cKO GVHD models. Ruxolitinib effectively reduced both CD8⁺ T cell infiltration and M1 macrophage burden, preserving crypt structure and supporting mucosal healing more robustly than Maraviroc alone. These findings confirm that SOCS1 regulates GVHD severity by modulating both CD8⁺ T cell activation and downstream macrophage polarization via the JAK/STAT–CCL5 axis.

In clinical datasets, high SOCS1 expression in graft-derived CD8⁺ T cells correlated inversely with expression of JAK/STAT targets and chemokine pathway components, and was associated with a reduced risk of GVHD. These data indicate that SOCS1 is a key regulator of immune homeostasis following allo-HSCT, and suggest its potential use as a predictive biomarker and therapeutic target for GVHD prevention.