CD4+ T cells from Sfpi1^lck-/- mice are more sensitive in response to stimuli. CD4+ T cells isolated from Sfpi1^lck-/- and wild-type control mice were stimulated with the indicated concentrations of plate-bound anti-CD3ε and a constant concentration of soluble anti-CD28 (0.5μg/ml) for 2 days. A, Supernatants were collected for determining the levels of IL-2 production using ELISA. B, The expression levels (MFI) of CD25 were evaluated using flow cytometry. Values presented are mean ± SD of 3 mice from each group. *, Significant difference between wild-type control and Sfpi1^lck-/- samples (p< 0.05). Results shown are representative of 2 independent experiments.

Intracellular staining of cytokine production in wild type and PU.1-deficient Th2 cells. Th2 cells were differentiated with anti-CD3 at 0.125 μg/ml (A) or 2 μg/ml (B) were restimulated with plate-bound anti-CD3ε (4 μg/ml) and treated with monensin for the last 2 h of a 5-hour incubation. Intracellular cytokine production was determined by staining cells with fluorochromes-conjugated antibodies including IL-4, IL-5, and IL-10. Results were presented as mean ± SD of 3 mice from each group. *, Significant difference between wild-type control and Sfpi1^lck-/- samples (p< 0.05). Results shown were a representative from 5 independent experiments.

PU.1 and GATA-3 alter TCR expression in Th1 and Th2 cells. CD4+ T cells isolated from both Sfpi1^lck-/- and wild-type control mice were differentiated into Th1 and Th2 cells. A, Expression levels of Gata3 and Tb×21 were determined by real time PCR and presented as mean relative gene expression ± SD of 2 duplicates. B, TCRβ surface expression on wild type Th1 and Th2 cells was evaluated using FACS. C, Expression levels of Tcra in wild type Th1 and Th2 cultures were determined by real time PCR and presented as mean relative gene expression ± SD of 2 duplicates. D, After 5-6 days of differentiation, Th1 cells from wild-type mice were transduced with retroviruses encoding GATA-3 and human CD4 (hCD4) or hCD4 alone. Three days after transduction, expression of TCRβ and CD3ε was evaluated on 5000 hCD4-positive cells by flow cytometry. E, Wild type CD4 T cells differentiated under Th2 conditions were transduced with control or PU.1-expressing bicistronic EGFP retrovirus. EGFP-positive cells were sorted and expression of Tcra was examined using real-time PCR.

T cell development in the absence of PU.1. A, Thymocytes or splenocytes were sorted into the indicated populations and genomic DNA was analyzed for the presence of the WT (+), floxed (fl) or deleted (Δ) allele. Results are representative of two experiments. B, Thymocytes from Sfpi1^lck-/- and wild-type control mice were sorted into double-negative subpopulations and RNA was analyzed for expression of Sfpi1. Results are averages ± SD of two independent experiments. C, Flow cytometry analysis of cells in thymus and spleens from Sfpi1^lck-/- and wild-type control mice. The CD4+ or CD8+ T cell percentage is presented as mean ± SD of 6 mice from spleens and 3 mice from thymus. CD4+ T cell development was normal in Sfpi1^lck-/- mice as shown by similar (p>0.05) percentage of cells staining with anti-CD4 and/or anti-CD8.

Cytokine production from wild type and PU.1-deficient Th2 cultures. Total CD4+ T cells isolated from both Sfpi1^lck-/- and wild-type control mice were differentiated into Th2 cells in the presence of either 0.125 or 2 μg/ml of anti-CD3ε. A, The numbers of differentiated cells were determined by hemocytometer with trypan blue staining. B, Sfpi1 mRNA was determined in wild type Th2 cells differentiated with the anti-CD3 dose indicated using qPCR. C, Differentiated Th2 cells were restimulated with plate-bound anti-CD3ε (2 μg/ml) for 24 h. The supernatant was collected for cytokine ELISA. Data presented were mean ± SD of 3 mice from each group. *, Significant difference between wild-type control and Sfpi1^lck-/- samples (p< 0.05). Results shown were representative from 5 independent experiments. NS, not significant.

TCR expression on wild type and PU.1-deficient CD4+ T cells. A-C, CD4+ splenocytes (A), CD4 single-positive thymocytes (B) or CD4+ splenocytes gated on naïve (CD62L^hi CD44^lo) or memory (CD62L^lo CD44^hi) populations (C) isolated from Sfpi1^lck-/- and wild-type mice were evaluated for the surface expression of CD3ε and TCRβ as indicated. The expression levels are presented as mean MFI ± SD of 3 mice from each group. D, CD4 T cells from isolated from Sfpi1^lck-/- and wild-type splenocytes were stimulated with PMA (10 or 50 ng/ml as indicated) and ionomycin (500 ng/ml) for 48 hours before IL-2 concentration in supernatants was determined using ELISA. E, CD4+CD62L+ (naïve) T cells were isolated using magnetic selection. Total protein extracts were subjected to western blotting analysis for GATA-3 with GAPDH as a loading control. The western blot band intensity was analyzed by densitometry and normalized to GAPDH as the ratio of GATA-3 and GAPDH. Data were presented as the mean ratio ± SD of 3 mice from each group. NS, not significantly different (p> 0.05). F, Isolated total CD4+ T cells were pooled from 3 wild type and 3 Sfpi1^lck-/- mice for ChIP assay to evaluate the binding of GATA-3 to the Tcr Eβ region. The DNA amplified by each primer set using realtime PCR is presented as the mean percent of input ± SD of 2 duplicates. G, CD4 T cells isolated from Sfpi1^lck-/- and wild-type splenocytes were transfected with Gata3-specific siRNA and stimulated with anti-CD3 (1 μg/ml) for 48 hours before determination of IL-2 concentrations in supernatants using ELISA. Inset, immunoblot of GATA-3 and GAPDH in control and Gata3 siRNA transfected cells. Numbers indicated the relative level of GATA-3 protein. Representative of two experiments. H, DNA affinity precipitation assay was performed with total protein extracts from differentiated Th2 cells from both wild-type and Sfpi1^lck-/- mice. Results shown are a representative from 3 independent experiments. I, ChIP assay for GATA-3 binding to the regions of Tcr Eβ and Tcr Eα, and to the Il4 Va regions (24) was carried out with chromatin from Th2 cultures differentiated from wild-type and Sfpi1^lck-/- mice. Data is average of 2 independent experiments. For all panels * indicates a significant difference between wild-type control and Sfpi1^lck-/- samples (p< 0.05).

Functional heterogeneity of TCR high and low populations. A, Cell populations expressing high (MFI:100) and low (MFI:25) levels of TCRβ were collected by flow sorting from wild-type mice. B, TCR-low and TCR-high cells were activated with the indicated concentrations of plate-bound anti-CD3ε and a constant concentration of soluble anti-CD28 (0.5μg/ml) for 2 days before the activation marker CD69 was evaluated by flow cytometry. C, The production of IL-2 cytokine after 2 days of stimulation was evaluated using ELISA. Values are presented as the mean ± SD of 2 independent experiments. D, Total protein extracts from TCR-high and TCR-low populations after sorting were subjected to western blot analysis for GATA-3 protein levels with GAPDH as a loading control. E, Gene expression of Sfpi1 from TCR-low and TCR-high populations after sorting of total CD4 T or Th2 cells were analyzed by real time PCR and presented as mean relative gene expression ± SD of 2 duplicates. Data is a representative from 2 independent experiments. F, Th2 cells sorted as in (E) were stimulated for 24 hours with anti-CD3 before IL-4 concentration in supernatant was determined using ELISA. Data is a representative from 2 independent experiments.