Fig 2: Btk expression and activation in T cells. a The expression of Btk mRNA normalized to that of actin in different subsets of lymphocytes. b The expression of Btk in rTreg and aTreg cells. c Western blot analysis of BTK protein levels in different cell subsets. d Immunoblot of BTK expression after TCR engagement. e Immunoblot of p-BTK (Y223) in WT and Btk−/− CD4+ T cells at different times after TCR engagement. f Phosflow analysis of p-BTK (Y223) after TCR engagement. The data shown are the means ± SD and are representative of at least three independent experiments with similar results. *P < 0.05. Tn, naïve T cells; Tm, effector/memory T cells; NK, natural killer cells

Fig 3: Intrinsic Btk is responsible for defective T-cell proliferation. a A schematic of the experimental approach for the analysis of the extrinsic and intrinsic roles of Btk. b Analysis of the WT (CD45.1+) and Btk-/- (CD45.1-) ratio in CD4+ and CD8+ T cells in the spleen on different days after adoptive transfer into DBF1 mice gated on H2Kd- donor T cells. c Statistic analysis of the WT:Btk-/- T-cell ratio at different times after transfer. d, e Representative flow cytometry plot (d) and the percentages (e) of EdU incorporation in WT or Btk-/- T cells transferred into DBF1 mice at a 1:1 ratio (gated on CD4+H2Kd- or CD8+H2Kd-) in the spleen. The numbers in the plot are the percentages of total CD45.1+ or CD45.1- T cells that are CD45.1+ EdU+ or CD45.1- EdU+. The data shown are the means ± SD and are representative of at least three independent experiments with similar results. *P < 0.05; **P < 0.01

Fig 4: Btk deficiency ameliorates T-cell activation and proliferation. Flow cytometry a and analysis b of CD69 expression in CD4+ and CD8+ T cells from WT and Btk-/- mice 4 h post anti-CD3/anti-CD28 or PMA/ionomycin stimulation. c Flow cytometry analysis of CD25/CD69 expression 1 day after activation. d, e Apoptosis of CD4+ and CD8+ T cells 1 day after anti-CD3/anti-CD28 stimulation in vitro. f CFSE dilution assay of WT and Btk-/- T cells simulated with anti-CD3/anti-CD28 or PMA/ionomycin. The numbers shown in the plot are the division index (upper) and the percentage of proliferated cells (bottom). g EdU incorporation in WT and Btk-/- T cells stimulated with anti-CD3 plus anti-CD28. h Intracellular cytokine production by differentiated Th1, Th17, and Treg cells from WT and Btk-/- CD4+ T cells. The data shown are the means ± SD and are representative of at least three independent experiments with similar results. *P < 0.05; **P < 0.01

Fig 5: In vitro and in vivo efficacy of RC-1.a XLA cells overexpressing wild-type or C481S mutant BTK were treated with RC-1 for 24 h. Then western blotting was performed to evaluate the degradation of BTK. Duplicates were performed. b RC-1 induced degradation of BTK, pBTK, IKZF1, and IKZF3 in mino cells. Duplicates were performed. c Pharmacokinetics of RC-1 in mice. RC-1 (20 mg kg−1) was injected intraperitoneally in ICR mice (n = 3) and the plasma was analyzed using LC-MS/MS. Data are presented as mean values ± SD (n = 3 mice per group). d Representative western blot (left) and quantification (right) of splenic BTK level in mice (n = 3) treated with 7 once daily i.p. injection of 20 mg kg−1 RC-1 (blue bar, spleens were harvested 24 h after the 7th injection) and DMSO (violet bar). Data are presented as mean values ± SEM (n = 3 biologically independent animals). Asterisks indicate that the differences between samples are statistically significant, using two-tailed, unpaired t-test (*P < 0.05). Source data are provided as a Source Data file.