Blk is expressed in thymic and BM progenitor cells. A, Black histograms show representative staining of the i.c. levels of Blk, Lck, and Fyn in gated DN1 (Lin⁻ CD25⁻ CD44⁺), DN2 (Lin⁻ CD25⁺ CD44⁺), DN3 (Lin⁻ CD25⁺ CD44⁻), DN4 (Lin⁻ CD25⁻ CD44⁻) thymocyte populations from TCRα^−/− mice. Lin⁻ is defined as CD4⁻ CD8⁻ CD11b⁻ TCRβ ⁻ TCRγδ⁻ CD19⁻ NK1.1⁻ IA^b− TER-119⁻ Ly6-G/Ly6-C⁻. Dark shaded histograms represent negative staining controls (DP thymocytes for i.c. Blk staining, Lck^−/− thymocytes for i.c. Lck staining, and Fyn^−/− thymocytes for i.c. Fyn staining). Light shaded histograms represent positive staining controls (splenic B cells for i.c. Blk staining and mature CD4⁺ T cells for both i.c. Lck and Fyn staining). B, Black histograms show representative staining of the i.c. levels of Blk in Linv⁻ Sca-1⁺ c-Kit^hi (LSK) progenitors from B6 BM. DP thymocytes (dark shaded histogram) are shown as a negative control. C, Black histograms show representative staining of the i.c. levels of Blk in Lin⁻ Sca-1^lo c-Kit^lo cells or common lymphoid progenitors (CLPs) from B6 BM. DP thymocytes (dark shaded histogram) are shown as a negative control. D, Black histograms show representative staining of the i.c. levels of Blk in lin⁻ CD25⁻ CD44⁺ c-Kit⁺ thymocytes or early thymic progenitors (ETPs) from TCRα^−/− thymus. DP thymocytes (dark shaded histogram) are shown as a negative control.

Effects of Blk-deficiency on γδ T cell development. A. Phenotypic analysis of γδ lineage cells in Blk^+/+ and Blk^−/− mice. Left panel: Dot plots show representative Vγ5 versus CD3 staining profiles on gated DN thymocytes from E17 Blk^+/+ and Blk^−/− fetuses. Right panel: Dot plots show representative TCRγδ versus CD3 staining on total thymocytes and LN cells from 3-wk-old Blk^+/+ and Blk^−/− mice. Numbers represent percentage of cells in each gate. B, Quantification of Vγ5⁺ and Vγ5⁻ fetal thymocytes in E17 Blk^+/+ and Blk^−/− fetuses. Bars represent mean ± SEM of at least 8 thymic lobes per genotype. #p≤0.001. C, Quantification of DN TCRγδ⁺ cells in the thymus and LN of 3-wk-old Blk^+/+ and Blk^−/− mice. Bars represent mean ± SEM of 9 to 10 mice per genotype. *p≤0.05, #p≤0.001. D, Percentage of Vγ1⁺ and Vγ4⁺ cells in gated DN TCRγδ⁺ LN cells from 3-wk-old Blk^+/+ and Blk^−/− mice. Bars represent mean ± SEM of 5 mice per genotype. #p≤0.001. E, Percentage of Vγ1⁺ and Vγ4⁺ cells in gated DN TCRγδ⁺ LN cells from 6-wk-old Blk^+/+ and Blk^−/− mice. Bars represent mean ± SEM of 3 mice per genotype. *p≤0.05.

Effects of Blk-deficiency on γδ T cell effector function. A, Comparison of IL-17 versus IFNγ production and IFNγ versus TNFα production by DN γδTCR⁺ LN cells from γδTCR Tg Blk^−/− and γδTCR Tg Blk^+/+ mice. LN cells from the two genotypes were in vitro stimulated with 1 μg/ml of immobilized anti-CD3 mAb (2C11) or 5 μg/ml of immobilized hamster IgG. 48 h later, cells were harvested and cytokine production was assayed by i.c. flow cytometric analysis. Dot plots show representative i.c. staining for gated DN γδTCR⁺ cells. Numbers in the quadrants represent percentage of cells in that quadrant. Data shown are representative of 6 mice per genotype. B, Histogram showing representative i.c. Blk staining on gated CCR6⁺ (black histogram) and CD27⁺ (dashed dark gray histogram) DN γδTCR⁺ cells in the thymus and LN of Blk^+/+ mice. Total γδ thymocytes (light shaded histogram) are shown as a staining control in the left histogram and B cells (light shaded histogram) are shown as a staining control in the right histogram. C, Dot plots showing representative CD44 versus CCR6 staining on DN γδTCR⁺ thymocytes and LN cells from γδTCR Tg Blk^−/− (n=4) and γδTCR Tg Blk^+/+ (n=6) mice. Numbers represent percentage of cells in each gate. D, Histograms showing representative IL-23R (GFP⁺) staining on DN γδTCR⁺ thymocytes and LN cells from Blk^−/− IL-23R-GFP.KI (n=3) and Blk^+/+ IL-23R-GFP.KI (n=5) mice. Numbers represent percentage of cells in each marker.

Blk is expressed in γδ lineage cells. A, Quantitative real-time RT-PCR analysis of the relative transcript levels of Blk and Lyn in DN γδ T cells purified from γδTCR Tg mice and in CD8⁺ αβ T cells, CD4⁺αβ T cells, and CD19⁺ B cells purified from B6 mice. Data were normalized to Gapdh transcript levels and are presented as fold change over B cells (set to 1). Bars represent mean ± SEM of at least 3 samples of each lymphocyte population. **p≤0.01. B, Black histograms show representative staining of the intracellular (i.c.) levels of Blk in gated populations of thymic and peripheral γδ lineage cells from γδTCR Tg, B6, TCRβ^−/− and Lck^−/−Fyn^−/− mice. DP thymocytes (dark shaded histogram) and splenic B cells (light shaded histogram) are shown as negative and positive controls, respectively. C, Black histograms show representative staining of the i.c. levels of Blk in gated populations of Vγ1⁺ and Vγ4⁺γδ T cells from the LNs of B6 mice. DP thymocytes (dark shaded histogram) are shown as a negative control. D, Black histograms show representative staining of the i.c. levels of Blk in gated populations of Vγ5⁺ and Vγ5⁻ thymocytes from B6 embryonic day 17 (E17) fetuses. DP thymocytes (dark shaded histogram) are shown as a negative control. E, Black histograms show representative staining of the i.c. levels of Blk in gated populations of DN and CD8αα⁺γδTCR⁺ iIELs from B6 mice. DP thymocytes (dark shaded histogram) are shown as a negative control. F, Serial dilutions of cleared lysates from a known number of DN γδ thymocytes were resolved by SDS-PAGE in parallel with serial dilutions of known amounts of recombinant His-tagged Blk or Lck, transferred to a PVDF membrane, and immunoblotted with anti-Blk, Lck or β-actin Abs. Data are representative of four independent experiments. G, Mean number of Blk and Lck molecules per DN γδ thymocytes. #p≤0.001.

Effects of Blk-deficiency on T cell development. A, Cellularity of the thymus and LNs of Blk^+/+ () and Blk^−/− (ν) mice plotted as a function of age. Mean cell number ± SEM from 3 to 10 mice are shown for each age. *p≤0.05, **p≤0.01, #p≤0.001. B, Phenotypic analysis of thymocytes from 3-wk-old Blk^+/+ and Blk^−/− mice. Top panel: Dot plots show representative CD4 versus CD8 staining profiles of total thymocytes. Bottom panel: Dot plots show representative CD44 versus CD25 staining on gated Lin⁻ thymocytes, where Lin⁻ is defined as in Fig. 2A. Numbers in quadrants represent percentage of cells in each quadrant. The mean thymus cell number ± SEM for each genotype are displayed above the two-color plots. C, Effect of Blk-deficiency at 3 wks of age on the proliferative capacity of the four DN subsets. Bars represent the mean percentage of Ki-67⁺ cells ± SEM from at least 3 mice per genotype. #p≤0.001. E, Dot plots show representative CD44 versus CD117 (c-Kit) staining profiles on gated Lin⁻ CD25⁻CD44⁺ (DN1) thymocytes from 3-wk-old Blk^+/+ and Blk^−/− mice. Numbers represent percentages of ETPs. F, Comparison of the number of ETPs in 3-wk-old Blk^+/+ and Blk^−/− mice. The mean number of ETPs ± SEM from 3 mice are shown. *p≤0.05.

Defects in T cell development in Blk^−/− mice are cell intrinsic. A, Degree of chimerism in the thymus of Blk^+/+:CD45.1 and Blk^−/−:CD45.1 mixed BM chimeras. Bars represent the percentages of CD45.2⁺ thymocytes ± SEM in Blk^+/+:CD45.1 chimeras (n=4) and Blk^−/−:CD45.1 chimeras (n=3). *p≤0.05. B, Phenotypic analysis of thymocytes from Blk^+/+:CD45.1 mixed BM chimeras. Two-color plots show expression of CD4 versus CD8 on total and on gated CD45.2⁺ and CD45.1⁺ thymocytes. Numbers in quadrants of the two-color plots represent the percentage of cells in each quadrant. C, Phenotypic analysis of thymocytes from Blk^−/−:CD45.1 mixed BM chimeras. Two-color plots show expression of CD4 versus CD8 on total and on gated CD45.2⁺ and CD45.1⁺ thymocytes. Numbers in quadrants of the two-color plots represent the percentage of cells in each quadrant.

Effects of Blk-deficiency on T cell development in γδTCR Tg mice. A, Phenotypic analysis of 4 to 6-wk-old γδTCR Tg Blk^−/− and γδTCR Tg Blk^+/+ mice. Dot plots show representative CD4 versus CD8 staining profiles on total thymocytes. Numbers in quadrants of the two-color plots represent the percentage of cells in each quadrant. Adjacent dot plots show representative TCRγδ versus CD3 staining on DN thymocytes and LN cells. Numbers represent percentage of cells in each gate. B, Mean number ± SEM of DN (DN TCRγδ⁺; γδ lineage) and DP (αβ lineage) thymocytes in γδTCR Tg Blk^−/− and γδTCR Tg Blk^+/+ mice. Data represent 5 mice per genotype. C, Mean number ± SEM of DN γδ T cells in the LNs of γδTCR Tg Blk^−/− and γδTCR Tg Blk^+/+ mice. Data represent 5 mice per genotype. D, Comparison of CD5 and CD3 surface levels on DN TCRγδ⁺ cells in the thymus and LNs of γδTCR Tg Blk^−/− and γδTCR Tg Blk^+/+ mice. Data are representative of 4 mice per genotype.