Spatiotemporal Single-Cell Analysis Reveals T Cell Clonal Dynamics and Phenotypic Plasticity in Human Graft-versus-Host Disease

Shi, L.; Uzuni, A.; Wang, X. K.; Pressler, M.; Harle, D. W.; Chakrabarti, S.; Macedo, R.; Belay, K.; Gordillo, C. A.; Raps, E.; Zhang, J. Y. A.; Nazaret, A.; Fan, J. L.; Jin, Y.; Shen, X.; Fuller, J. S.; Azad, T.; Huang, J.; Chainani, P.; Abrams, J. A.; Del Portillo, A.; Mapara, M. Y.; Alhamar, M.; Sykes, M.; McFaline-Figueroa, J.; Azizi, E.; Reshef, R.

doi:10.1101/2025.05.24.655962

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bioRxiv · 10.1101/2025.05.24.655962

Spatiotemporal Single-Cell Analysis Reveals T Cell Clonal Dynamics and Phenotypic Plasticity in Human Graft-versus-Host Disease

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Abstract

Allogeneic hematopoietic cell transplantation (alloHCT) is curative for various hematologic diseases but often leads to acute graft-versus-host disease (GVHD), a potentially life-threatening complication. We leverage GVHD as a uniquely tractable disease model to dissect complex T-cell-mediated pathology in 27 alloHCT recipients. We integrate pre-transplant identification of alloreactive T-cells with longitudinal tracking across blood and gut, using mixed lymphocyte reaction-based clonal "fingerprinting", TCR clonotyping, single-cell RNA/TCR sequencing, and spatial transcriptomics. Using DecompTCR, a novel computational tool for longitudinal TCR analysis, we uncover clonal expansion programs linked to GVHD severity and TCR features. Multi-omics profiling of gut biopsies reveals enrichment and clonal expansion of CD8 effector and ZNF683(Hobit) resident memory T-cells, cytolytic remodeling of regulatory and unconventional T-cells, and localization of CD8 effector T-cells near intestinal stem cells in crypt loss regions. This framework defines dynamic immune circuit rewiring and phenotypic plasticity with implications for biomarkers and therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/655962v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@178b77dorg.highwire.dtl.DTLVardef@569226org.highwire.dtl.DTLVardef@1951281org.highwire.dtl.DTLVardef@1f1e046_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPersistent expansion of diverse alloreactive T cell clones is a hallmark of severe GVHD C_LIO_LIDecompTCR reveals dynamic clonal expansion programs linked to GVHD severity and clinical outcome C_LIO_LICD8+ T cell clones exhibit phenotypic plasticity in vivo across intestinal tissue compartments in GVHD C_LIO_LIHigh-resolution spatial profiling shows CD8+ effector T cells localize near intestinal stem cell niches and drive epithelial injury in GVHD C_LI

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Shi, L., Uzuni, A., Wang, X. K., Pressler, M., Harle, D. W., Chakrabarti, S., Macedo, R., Belay, K., Gordillo, C. A., Raps, E., Zhang, J. Y. A., Nazaret, A., Fan, J. L., Jin, Y., Shen, X., Fuller, J. S., Azad, T., Huang, J., Chainani, P., Abrams, J. A., Del Portillo, A., Mapara, M. Y., Alhamar, M., Sykes, M., McFaline-Figueroa, J., Azizi, E., Reshef, R.. 2025-05-28. Spatiotemporal Single-Cell Analysis Reveals T Cell Clonal Dynamics and Phenotypic Plasticity in Human Graft-versus-Host Disease. https://doi.org/10.1101/2025.05.24.655962

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