Fig 2: Altered frequency and function of dCD2+CD4+T cells in RS patients. (A) Frequency of CD2 expressing cells in gated CD4+ T cells from decidual immune cells from NN and RS as determined by flow cytometric analysis (n=8, Treatment groups were compared via Student’s t-test). (B) Quantitation of flow cytometric analysis of Th1(TNF-α and IFN-γ), Th2 (IL-4), Treg (TGF-β1) and Th17 (IL-17A)- type cytokine expression of dCD2+CD4+T cells (n=8, Treatment groups were compared via Student’s t-test). (C) Cytokine expression on dCD2+CD4+T cells from NN and RS was assessed by flow cytometric analysis (n=8, Treatment groups were compared via Student’s t-test). A representative dot plot is also shown. Data represent the mean ± SEM. The data points represent the biological replicates obtained from three independent experiments. **P<0.01, ***P<0.001. NN, normal pregnancy with normal sleep; RS, patient of RPL with sleep disturbance.

Fig 3: Schematic diagram of dysfunctional dCD2+CD4+T cells regulated by Rev-erbα-GFPT1-CD58 axis of DSCs in RS. Our integrated bioinformatics, proteomic, functional and model studies elucidated that GFPT1, which regulated by the circadian gene Rev-erbα, increased CD58 expression via GPI modification, leading to the disorder of dCD2+CD4+T cells and consequently, resulting in miscarriage. In addition, anti-CD2 antibody clearly decreased the risks of abortion of SD. This schematic clarified important implications for understanding the etiology of RPL and developing targeted therapy of RPL, especially those with sleep problems. RS, patient of RPL with sleep disturbance; SD, mice with sleep disturbance.

Fig 4: Rev-erbα-GFPT1-CD58-CD2 axis played an important role in maternal-fetal tolerance by regulating cytokine profile of dCD4+T cells. (A) Quantitation of flow cytometric analysis of TNF-α, IFN-γ, IL-4, TGF-β1 and IL-17A expression on dCD2+CD4+T cells in the coculture system of decidual immune cells and DSC with or without CD58 overexpression, in the presence or absence of anti-CD2 antibody (n=5, Treatment groups were compared via Student’s t-test). (B) Cytokine expression on dCD2+CD4+T cells in the coculture system of decidual immune cells and DSC with or without GFPT1 overexpression, in the presence or absence of anti-CD2 antibody (n=5, Treatment groups were compared via Student’s t-test). (C) Cytokine expression on dCD2+CD4+T cells in the coculture system of decidual immune cells and DSCs with or without Rev-erbα knockdown, or DSCs with Rev-erbα and GFPT1 knockdown. Images are representative of three individual experiments. Data represent the mean ± SEM (n=5, Treatment groups were compared via Student’s t-test). The data points represent the biological replicates obtained from three independent experiments. *p<0.05, **p<0.01, ***p<0.001.

Fig 5: Eif4g2 deletion specifically ablates the IL-7 response(A and B) Single-cell transcriptomic landscape of thymocytes. UMAP visualization of (A) all thymic cells and (B) the subtypes of T cells, color-coded by cell type.(C) Heatmap showing the expression pattern of 15 core signaling component genes from seven selected KEGG pathways across the cell clusters identified in (A and B).(D–F) Functional response of CD4+CD8lo transitional cells to IL-7. Relative mRNA level of (D) Runx3 and (E) Bcl2 following 10 ng/ml IL-7 stimulation (n = 3, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).(F) Western blot analysis of key signaling pathway activation with or without IL-7 stimulation.(G) Quantification of cell death in peripheral naive T cells under IL-7 stimulation ex vivo (n = 3, ∗∗∗∗p < 0.0001).(H–J) TCR signaling evaluation in DP cells under stimulation with anti-TCRβ/CD2 ex vivo, including (H) representative plots of CD69 expression examination, (I) frequency of CD69+ cells and (J) cell death level (n = 4, ns p > 0.5). Data are representative of at least two independent experiments. Bar graphs show mean ± SEM and unpaired Students’t test was used to perform the statistical analysis. See also Figures S3–S5.