OTUD1 deficiency subverts T-cell fate, inhibits Th1 and Th17 differentiation, promotes Treg differentiation, and restricts proinflammatory factor secretion. (A) UMAP (uniform manifold approximation and projection) displays cell populations in the spleen of aGVHD mice receiving WT or OTUD1-deficient cells by sc-RNA sequencing (sc-RNA seq) (n = 2 for WT, n = 2 for OTUD1-KO). Cluster 1 (C1): CD8+ T, C2: CD4+ T, C8/C9: proliferating T (MKi67hi T), C6/C11/C0/C19/C5/C10: neutrophil, C16: megakaryocyte (MK), C4: MP, C18/C7: monocyte, C12: M1, C22: M2, C3: macrophage, C14/C20: DC, C15: basophil, C17: B cell, C21: GC-B cell, C13: erythrocyte. (B) Cell proportion of clusters in aGVHD mice receiving WT or OTUD1-deficient cells. (C) The proportion of CD4+ T cells and CD8+ T cells in T cells of aGVHD mice receiving WT or OTUD1-deficient cells are shown. (D) GSEA analysis of T-cell differential genes implicated by sc-RNA seq shows the KEGG signaling pathways. (E) UMAP displays cell populations of T-cell reclusters in aGVHD mice receiving WT or OTUD1-deficient cells by sc-RNA seq (C0/C3/C6/C9: proliferating T (MKi67hi T), C7/C8: activated CD3+ T (aCD3+ T), C5: activated CD4+ T (aCD4+ T) and activated CD8+ T (aCD8+ T), C2: aCD8+ T, C4: helper CD4+ T cell (Th1), C1: cytotoxic CD8+ T cell (Tc1)). (F) Cell proportion of T-cell reclusters in aGVHD mice receiving WT or OTUD1-deficient cells. (G-H) Pseudotime analysis reveals the ordering of T-cell clusters along the pseudotime trajectory. (I-J) GSEA analysis showed inflammatory response (I) and TGF-β signaling pathway (J) enrichments of OTUD1-deficient T cells. (K-L) The percentage of Th1 (IFNγ+CD4+ T cells), Th17 (IFNγ+CD4+ T cells), Th2 (IL-4+CD4+ T cells) (K), and Treg (CD25+Foxp3+CD4+ T cells) (L) in spleen of aGVHD mice receiving WT or OTUD1-deficient cells are analyzed by flow cytometry (n = 10 for WT, n = 16 for OTUD1-KO). Data in panels K-L are represented as mean ± SD; ∗∗P < .01, ∗∗∗P < .001 (2-tailed unpaired Student t test). DC, dendritic cell; MP, myeloid progenitors.
Figure 3.

OTUD1 deficiency subverts T-cell fate, inhibits Th1 and Th17 differentiation, promotes Treg differentiation, and restricts proinflammatory factor secretion. (A) UMAP (uniform manifold approximation and projection) displays cell populations in the spleen of aGVHD mice receiving WT or OTUD1-deficient cells by sc-RNA sequencing (sc-RNA seq) (n = 2 for WT, n = 2 for OTUD1-KO). Cluster 1 (C1): CD8+ T, C2: CD4+ T, C8/C9: proliferating T (MKi67hi T), C6/C11/C0/C19/C5/C10: neutrophil, C16: megakaryocyte (MK), C4: MP, C18/C7: monocyte, C12: M1, C22: M2, C3: macrophage, C14/C20: DC, C15: basophil, C17: B cell, C21: GC-B cell, C13: erythrocyte. (B) Cell proportion of clusters in aGVHD mice receiving WT or OTUD1-deficient cells. (C) The proportion of CD4+ T cells and CD8+ T cells in T cells of aGVHD mice receiving WT or OTUD1-deficient cells are shown. (D) GSEA analysis of T-cell differential genes implicated by sc-RNA seq shows the KEGG signaling pathways. (E) UMAP displays cell populations of T-cell reclusters in aGVHD mice receiving WT or OTUD1-deficient cells by sc-RNA seq (C0/C3/C6/C9: proliferating T (MKi67hi T), C7/C8: activated CD3+ T (aCD3+ T), C5: activated CD4+ T (aCD4+ T) and activated CD8+ T (aCD8+ T), C2: aCD8+ T, C4: helper CD4+ T cell (Th1), C1: cytotoxic CD8+ T cell (Tc1)). (F) Cell proportion of T-cell reclusters in aGVHD mice receiving WT or OTUD1-deficient cells. (G-H) Pseudotime analysis reveals the ordering of T-cell clusters along the pseudotime trajectory. (I-J) GSEA analysis showed inflammatory response (I) and TGF-β signaling pathway (J) enrichments of OTUD1-deficient T cells. (K-L) The percentage of Th1 (IFNγ+CD4+ T cells), Th17 (IFNγ+CD4+ T cells), Th2 (IL-4+CD4+ T cells) (K), and Treg (CD25+Foxp3+CD4+ T cells) (L) in spleen of aGVHD mice receiving WT or OTUD1-deficient cells are analyzed by flow cytometry (n = 10 for WT, n = 16 for OTUD1-KO). Data in panels K-L are represented as mean ± SD; ∗∗P < .01, ∗∗∗P < .001 (2-tailed unpaired Student t test). DC, dendritic cell; MP, myeloid progenitors.

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