A) Human peripheral blood T cells from two different volunteers were stimulated with syngeneic APC and SEE superantigen for 36 hours. Cell lysates were prepared and sequentially immunoblotted for SLP-2 and actin (as a loading control). Results are representative of three independent experiments. B) Human tonsil cells were prepared and fractionated through Percoll gradient. Whole cell lysates from the four fractions were immunoblotted for SLP-2 and actin. B cell markers for naïve and activated/memory B cells were used to determine the percentage of naïve and activated memory B cells in each fraction (shown in the table). Results are representative of two independent experiments.

A) Jurkat T cells were stimulated with APC and SEE superantigen for the indicated times. Next, whole cell lysates were prepared and used for immunoprecipitation (ip) of SLP2 with cross-linked anti-SLP-2 antibodies. Pre-immune serum immunoprecipitation (lane ‘C’) was used as a negative control. The resulting ip were sequentially immunoblotted for CD3-ε, lck, phospho-ZAP-70, ZAP-70, phospho-LAT, LAT, phospho-PLC-γ1, PLC-γ1, CD45, Ras-GAP, caspase 3, and SLP-2. B) E6.1 Jurkat T cells pretreated with cytochalasin D (+CD) (10 μM) for 30 minutes were stimulated with APC and SEE for the indicated times. Whole cell lysates were prepared and used for immunoprecipitation of SLP-2 and sequentially immunoblotted for SLP-2 and actin. C) Inhibition of actin polymerization with cytochalasin down-regulated the IL-2 response of Jurkat T cells to APC and SEE stimulation. Supernatants from 24 hour cultures of T cells pre-treated with the indicated concentrations of cytochalasin D and stimulated with APC and different concentrations of SEE superantigen were used to measure IL-2 production by ELISA (mean ± s.d.). Results in this figure are representative of at least 4 separate experiments.

A) Jurkat T cells were transfected with a doxycycline-inducible SLP-2-gfp cDNA. Leakiness of the transfected cDNA was apparent as shown by FACS (WTSLP2gfp (-D)). Further over-expression of SLP-2 was induced by addition of doxycycline (1 μg/ml) to the culture for 18 hours (WTSLP2gfp (+D)). Non-transfected parental E6.1 Jurkat T cells (which express lower levels of SLP-2) were used as controls (Parental). Subsequently, these T cells were stimulated with APC and SEE superantigen for 24 hours. Cultures supernatants at that time were collected and used to measure IL-2 production by ELISA (mean ± s.d.). Over-expression of SLP-2-gfp was confirmed by FACS. B) Down-regulation of SLP-2 levels in Jurkat T cells was achieved by RNA interference as detailed in the Material and Methods section. The response of these T cells to APC and SEE was measured by IL-2 production. Down-regulation of SLP-2 expression after siRNA was confirmed by Western blotting.

A) Whole cell lysates from the indicated organs were sequentially immunoblotted for SLP-2 and GAPDH. Equal loading of protein per lane was further confirmed by spectroscopy. Results are representative of four independent experiments. B) Lymph node samples from human non-specific B cell adenitis (top row) or T cell adenitis (middle row), and normal human thymus (bottom rows) were stained for SLP-2 using a C-terminus specific rabbit anti-human SLP-2 antisera or with appropriate controls (CD20_bright for B cells, TdT for developing thymocytes), and biotinylated horse anti-rabbit secondary antibody and avidin (Vector Labs, Burlingame CA). High levels of SLP-2 expression were detected in the germinal centres (B cell area) of lymph nodes of non-specific B cell adenitis, in the paracortical (T cell) area of non-specific T cell adenitis, and in the cortex of the thymus. Much lower expression of SLP-2 was seen in cells outside these areas. The profile of intracellular localization of SLP-2 was predominantly associated to the periphery of the cell and some aggregates in the central area of the cytoplasm. C) Peripheral blood cells from a normal volunteer in whom expression of SLP-2 could be detected in resting peripheral blood mononuclear cells (PBMC) were used to fractionate these cells into T cells (T), B cells (B), and monocytes (M). Whole cell lysates from these subsets were prepared and sequentially immunoblotted for SLP-2 and GAPDH. Densitometric readings for SLP-2 normalized for GAPDH are shown.

A) E6.1 Jurkat T cells were activated with APC and SEE superantigen for the indicated times. Membrane lipid rafts and the detergent-soluble fraction were isolated by sucrose gradient centrifugation and immunoblotted for SLP-2, ERK-1/-2 (as control for the quality of the detergent-soluble fraction), and GM-1 (as a control for the quality of lipid rafts). B) The SLP-2 signal (mean ± s.d.) for lipid raft fractions from T cells activated for the indicated times in three different experiments was quantified by densitometry. C) Lipid rafts from blast PBMC activated with APC and SEE superantigen for 5 and 15 minutes were used to prepare membrane lipid rafts and detergent-soluble fraction by sucrose gradient centrifugation, and immunoblotted for SLP-2, ERK-1/-2 (as control for the quality of the detergent-soluble fraction), and GM-1 (as a control for the quality of lipid rafts).

A) Jurkat T cells were nucleofected with two different sets of siRNA for SLP-2 or control siRNA and used for stimulation with APC and SEE for the indicated times. Cell lysates were prepared and immunoblotted for dually phosphorylated, active ERK-1/-2 (pERKs), total ERK-1/-2 (tERKs), phospho-PLC-γ1, total PLC-γ1, and SLP-2. B) Signals for activated ERK-1/-2 and total ERK were quantified for each group at the indicated time points for three independent experiments and plotted as normalized densitometric units (mean ± s.d.) for active ERK-1/-2. *: p<0.05, **: p<0.01.

A) Peripheral blood lymphocytes from a normal volunteer were isolated and used as resting cells, or after three days of ex vivo activation with PMA and ionomycin and 48hr of resting. Cells were nucleofected with SLP-2 siRNA or gfp control and used 24 hours later for stimulation with autologous APC and SEE. IL-2 production after 24 hours was assessed by ELISA (mean ± s.d.). Additional control of cells nucleofected without any DNA was used to rule out non-specific effect of nucleofection. Inlet figure shows Western blot for SLP-2 to confirm knockdown of SLP-2, and actin as a loading control in the two groups tested. Similar results were obtained in 4 separate experiments. ***: p<0.001. B) Down-regulation of SLP-2 does not affect the IL-2 response to mitogenic stimulation with PMA and ionomycin. Peripheral blood lymphocytes from the experiment shown in figure 7A were used after three days of ex vivo activation and 48hr of resting. Cells were nucleofected with SLP-2 siRNA and used 24 hours later for stimulation with PMA and ionomycin. IL-2 production after 24 hours was assessed by ELISA (mean ± s.d.). Cells nucleofected without any DNA or with non-sense siRNA were used as controls. The effect of SLP-2 siRNA on SLP-2 levels are shown in the right inlet panel of figure 7A. C) Resting and blast human peripheral blood T cells were stimulated with APC and SEE for the indicated times. Cell lysates were prepared and immunoblotted for dually phosphorylated, active ERK-1/-2 (pERKs), total ERK-1/-2 (tERKs), and SLP-2.