Critical requirement for the Wiskott-Aldrich syndrome protein in Th2 effector function by Vanessa Morales-Tirado, Dorothy K. Sojka, Shoshana D. Katzman, Christopher A. Lazarski, Fred D. Finkelman, Joseph F. Urban, and Deborah J. Fowell Blood Volume 115(17):3498-3507 April 29, 2010 ©2010 by American Society of Hematology

Nonredundant role for WASp in IL-4 but not IFNγ production. Nonredundant role for WASp in IL-4 but not IFNγ production. WT and WAS−/− Th1- and Th2-primed OTII TCR Tg+ CD4 T cells were restimulated with plate-bound anti-TCRβ (left panels) or pOVA/APC (WT T-depleted splenocytes; right panels) for 24 hours, and the supernatants collected for cytokine detection by ELISA. Results represent 1 of at least 4 comparable experiments. *P ≤ .01 for differences between WT and WAS−/− T-cell cytokine production, Student t test. Error bars indicate mean and SEM. Vanessa Morales-Tirado et al. Blood 2010;115:3498-3507 ©2010 by American Society of Hematology

Early events in the Th2 differentiation program are WASp-independent. Early events in the Th2 differentiation program are WASp-independent. (A) WT and WAS−/− naive CD4+ T cells (CD4+CD62LhighCD44low) were loaded with CFSE and primed under Th2 (anti-IFNγ mAb and rIL-4) conditions with plate-bound anti-TCRβ and anti-CD28 Ab for 5 days. Cells were harvested at given time points, and mRNA was extracted for qRT-PCR. mRNA was normalized to CD3δ and expressed as fold change over unstimulated naive CD4+ cells. mRNA from WT and WAS−/− Th1 (anti–IL-4 mAb and rIL-12)–primed cells were collected at 48 hours as controls. Data from 1 representative experiment of 3. Results were not statistically different for all cytokine mRNAs tested over 3 independent experiments, by paired Student t test. (B) CFSE-labeled CD4+ T cells were harvested after 48 hours of primary Th2 stimulation and analyzed for phospho-STAT6 by FACS: dot plots are gated on CD4+ T cells, and quadrant numbers equal the percentage of CD4 T cells. Percentage of phospho-STAT6+ cells in negative control (Th1-primed) was less than 5%. (C) At 48 hours, IL-4 production was analyzed by the cytokine secretion assay (CSA) on gated live CD4+ T cells. Quadrant gates were set on “no catch” controls (< 0.5% IL-4+ for WT and WAS−/−); numbers in quadrants equal the percentage of CD4+ T cells. (D) Kinetic analysis of IL-4 production by CD4+ T cells during Th2 differentiation by CSA. (E) IL-4 dose response during initial cell culture; IL-4 effects determined by the induction of IL-4 production by CD4+ T cells by CSA 48 hours after primary stimulation. Results represent one of at least 4 comparable experiments. (F) WT and WAS−/− naive CD4+ T cells were primed under Th1 or Th2 conditions with pOVA/APC for 5 days. Th2 cells were primed a subsequent time under opposing Th1 conditions (anti–IL-4 mAb and rIL-12) with pOVA/APC. At 5 days later, cells were restimulated for 24 hours in the absence of exogenous cytokines with pOVA/APC. IFNγ ELISA analysis of collected supernatants 24 hours after restimulation, nonsignificant by Student t test. Error bars indicate mean and SEM. Vanessa Morales-Tirado et al. Blood 2010;115:3498-3507 ©2010 by American Society of Hematology

Impaired cytokine production by WASp-deficient Th2-primed effectors. Impaired cytokine production by WASp-deficient Th2-primed effectors. WT and WAS−/− naive CD4+ T cells were primed under Th2 conditions with plate-bound anti-TCRβ and anti-CD28 mAbs for 5 days. Cells were harvested and restimulated with plate-bound anti-TCRβ mAb. (A) ELISA from supernatants 24 hours after restimulation. (B) Th2-primed cells were restimulated with anti-TCRβ mAb for 24 hours, followed by intracellular cytokine staining. Numbers in quadrants equal the percentage of Th2-primed cells. (C) Kinetic analysis of CD4+ IL-4+ cells after restimulation using the CSA. *P ≤ .05 for differences between WT and WAS−/− Th2-primed effectors across 3 independent experiments; paired Student t test. (D) Ex vivo memory cells (CD4+CD62LlowCD44high) were isolated from the spleens (SPNs) and LNs of unimmunized mice by FACS and restimulated using plate-bound anti-TCRβ mAb for 24 hours; cytokines in supernatants were assayed by ELISA. Results represent 1 of at least 3 comparable experiments. *P ≤ .05 for differences between WT and WAS−/− Th2-primed effectors across 3 independent experiments; paired Student t test. Error bars indicate mean and SEM. Vanessa Morales-Tirado et al. Blood 2010;115:3498-3507 ©2010 by American Society of Hematology

IL-4 transcriptional enhancement but no IL-4 protein production in the absence of WASp. (A) Th2-primed WT and WAS−/− CD4+ T cells were restimulated with pOVA/APC (WT T-depleted splenocytes). IL-4 transcriptional enhancement but no IL-4 protein production in the absence of WASp. (A) Th2-primed WT and WAS−/− CD4+ T cells were restimulated with pOVA/APC (WT T-depleted splenocytes). Cells were collected at given times after stimulation, and mRNA was extracted for qRT-PCR. mRNA was normalized to CD3δ and expressed as fold change over Th2-primed unstimulated (resting) cells. Results were not statistically different for IL-4 mRNA tested over 3 independent experiments by paired Student t test. (B) Intracellular staining on Th2-primed cells stimulated with pOVA/APC. (C) ELISPOT analysis of Th2-primed cells for frequency of IL-4 secretors 24 hours after restimulation with pOVA/APC. Data are from 1 of 3 comparable experiments. *P ≤ .05; paired Student t test. (D) Fold change in mRNA versus protein in WAS−/− Th2 cells compared with WT Th2 cells. *P = .03, paired Student t test, for relative differences in IL-4 protein in WAS−/− and WT Th2 cells. Differences in absolute IL-4 mRNA between WAS−/− and WT Th2 cells was not significant. Average and SEM of data from 3 independent experiments. (E) Th2-primed WT and WAS−/− CD4+ T cells were restimulated with phorbol myristyl acetate (PMA) and ionomycin: IL-4 mRNA analysis (left) and protein secretion by ELISA (right) 12 hours after restimulation. Data are from 1 of 3 comparable experiments. *P ≤ .05; paired Student t test. Error bars indicate mean and SEM. (F) ERK phosphorylation after pOVA/APC restimulation of Th2-primed WT and WAS−/− CD4+ T cells using an anti–phospho-specific ERK Ab. Specificity of binding of the phospho-specific Ab was confirmed by treatment with the ERK inhibitor U0126 (bottom panel). Representative plots from 1 of 4 experiments. (G) qRT-PCR analysis of ERK transcriptional target gene c-FOS in Th2-primed WT and WAS−/− effectors restimulated with pOVA/APC. Representative data from 1 of 2 similar experiments. (H) Cytokine production after restimulation of WT Th1- and Th2-primed cells in the presence of the ERK inhibitor U0126. IFNγ and IL-4 were measured by ELISA and normalized to effector cell stimulation in the absence of the ERK inhibitor (DMSO vehicle control = 100%). Mean and standard error from 4 independent experiments. *P ≤ .05; Student t test. Error bars indicate mean and SEM. Vanessa Morales-Tirado et al. Blood 2010;115:3498-3507 ©2010 by American Society of Hematology

WT Th2 cells require WASp signaling for IL-4 production. WT Th2 cells require WASp signaling for IL-4 production. WT naive CD4+ T cells were primed under Th2 (A-C) or Th1 (D) conditions with peptide and WT APCs. Cells were retrovirally transduced with DN WASp (WASpΔVCA-IRES-GFP) or empty vector (-IRES-GFP) for 36 hours late during priming (day 4). On day 6, retrovirally transduced cells were sorted based on GFP expression and restimulated. (A) GFP− and GFP+ cells were not activated or activated with anti-TCRβ mAb for 15 minutes. Cells were fixed and stained with phalloidin and analyzed by FACS. (B-C) Cells were restimulated with pOVA/APC for 24 hours. (B) The frequency of cytokine producers was determined using CSA (left panel) and cytokine produced in the supernatant by ELISA (right panel). Mean ± SEM; n = 3. *P ≤ .05 between GFP+ and GFP− cells in 3 independent experiments; paired Student t test. (C) mRNA analysis 24 hours after restimulation. mRNA was normalized to CD3δ and expressed as fold change over naive unstimulated CD4+ T cells. (D) Th1-primed cells were restimulated with pOVA/APC for 24 hours. The frequency of cytokine producers was determined using CSA (left panel) and cytokine produced in the supernatant by ELISA (right panel). Mean ± SEM; n = 3. Data are from 1 of 3 comparable experiments. Vanessa Morales-Tirado et al. Blood 2010;115:3498-3507 ©2010 by American Society of Hematology

IL-4 production from non-αβ T cells in the absence of WASp IL-4 production from non-αβ T cells in the absence of WASp. (A) Left panel shows intracellular staining for IL-4 from CD4−Gr1−FcϵR1α+ cells in peripheral blood after 6 hours of ionomycin stimulation, with brefeldin added for the final 4 hours of culture. IL-4 production from non-αβ T cells in the absence of WASp. (A) Left panel shows intracellular staining for IL-4 from CD4−Gr1−FcϵR1α+ cells in peripheral blood after 6 hours of ionomycin stimulation, with brefeldin added for the final 4 hours of culture. Cells were gated on negative staining for CD19, CD4, and GR-1 from red cell–depleted peripheral blood. Representative plots are from 3 independent experiments. Numbers in quadrants equal the percentage of gated (isotype control for IL-4, 0.0048%). Right panel shows frequency of IL-4 producers within the FcϵR1α+ compartment. Mean ± SEM from 3 independent experiments. (B) Frequency and total number (×104 per mouse) of IL-4 producers within the γδ T-cell compartment from LNs by intracellular staining after 6 hours of PMA/ionomycin stimulation as in panel A. Symbols represent individual mice from 2 independent experiments; P value by Mann-Whitney. Mean isotype control staining of activated cells is 1.460%. (C) Total serum IgE from 6- to 8-week-old mice was measured by ELISA. Symbols represent individual mice from 2 independent experiments. P values by Mann-Whitney. (D) Frequency and number of IL-4–producing γδ T cells from WAS−/− TCR-Cα−/− and WAS+/+ TCR-Cα−/− mice analyzed as in panel B. Symbols represent individual mice from 2 to 3 independent experiments. Vanessa Morales-Tirado et al. Blood 2010;115:3498-3507 ©2010 by American Society of Hematology

WAS−/− CD4+ T cells exert Th1 effector function in vivo. WAS−/− CD4+ T cells exert Th1 effector function in vivo. WT and WAS−/− CD4+ T cells (C57BL/6) were adoptively transferred into Rag-deficient mice. At 24 hours later, mice were infected with 2.0 × 105 L major promastigotes. (A) Progression of disease measured by footpad (lesion) size, mean of 4 to 6 mice per group. *P ≤ .05 (Mann-Whitney) for differences between Rag−/− with no reconstitution and either WT or WAS−/− CD4 reconstitution. (B) Limiting dilution analysis of parasite load in the footpad. *P ≤ .05 by paired Student t test across 3 independent experiments. (C) Draining LN CD4+ T-cell numbers 8 weeks after reconstitution of RAG−/− mice and L major infection. ns indicates not significant; paired Student t test. (D) ELISPOT analysis of antigen-specific IFNγ-producing cells upon 6 hours of restimulation with SLA. ns indicates P = .5 between WT and WAS−/− IFNγ production across 3 independent experiments; paired Student t test. Error bars indicate mean and SEM. (E) CSA for IL-4 and IFNγ production by WT and WAS−/− CD4+ T cells in the L major–infected dermis at 8 weeks. Cytokines were measured after 6 hours of restimulation with SLA. Dot plots were gated on CD4+ T cells. Numbers in quadrants equal the percentage of CD4+ T cells. Data are from 1 experiment representative of at least 3 comparable independent experiments. Vanessa Morales-Tirado et al. Blood 2010;115:3498-3507 ©2010 by American Society of Hematology

WASp-deficient CD4+ T cells fail to support Th2 immune responses to N brasiliensis infection in vivo. WASp-deficient CD4+ T cells fail to support Th2 immune responses to N brasiliensis infection in vivo. WT or WAS−/− CD4+ T cells (BALB/c) were adoptively transferred into TCR Ca−/− mice. After 24 hours, mice were infected with 500 infective N brasiliensis third-stage larvae (Nb). Ten days after infection, small intestines were excised for direct counting of adult worms. Symbols represent individual mice, 6 mice per group. *P ≤ .01 for WT versus WAS−/− CD4-reconstituted; Student t test. (B) Serum IgE levels measured by ELISA 10 days after infection. (C) In vivo cytokine capture for IL-4 in TCR Ca−/− mice reconstituted with WT or WAS−/− CD4+ T cells, 10 days after Nb infection. (D) Serum IL-13 in TCR Ca−/− mice reconstituted with WT or WAS−/− CD4+ T cells, 10 days after Nb infection. (E) CD4 T-cell numbers in mesenteric LNs 10 days after Nb infection; symbols represent individual mice. (F) ELISPOT for cytokine-secreting cells from the lungs and mesenteric LNs of infected mice restimulated ex vivo for 18 hours with Nb antigen. Statistical analysis by Student t test: lung tissue, *P < .008; mesenteric LN, *P < .004 for difference between IL-4 production by WT and WAS−/− cells. Data are from 1 experiment representative of at least 3 independent experiments. (G) Ex vivo ELISPOT for IL-13 with or without addition of blocking anti–MHC class II Ab. (H) mRNA analysis of Th2 cytokine gene expression in WT and WAS−/− CD4+ T cells from the mesenteric LNs of Nb-infected mice at day 10. Transcripts were normalized to HPRT and expressed relative to uninfected CD4+ T cells from the mesenteric LN. (I) Innate cell number in the lung, 10 days after Nb infection. Noninfected lungs contained 306 ± 60 basophils, 1130 ± 248 eosinophils, and 507 ± 140 mast cells. Results are the mean of individual mice from 3 independent experiments. Differences between WT and WAS−/− recipients were not statistically significant. Error bars indicate mean and SEM. Vanessa Morales-Tirado et al. Blood 2010;115:3498-3507 ©2010 by American Society of Hematology