PU.1 Expression Delineates Heterogeneity in Primary Th2 Cells Hua-Chen Chang, Shangming Zhang, Vivian T. Thieu, Roger B. Slee, Heather A. Bruns, R. Nicholas Laribee, Michael J. Klemsz, Mark H. Kaplan  Immunity  Volume 22, Issue 6, Pages 693-703 (June 2005) DOI: 10.1016/j.immuni.2005.03.016 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 PU.1 Expression in Th2 Cells (A) RNA was isolated from Th1 or Th2 cultures generated from mice of the indicated genotypes following stimulation with or without anti-CD3 for the indicated number of hours and was probed for PU.1 expression. Northern blots were probed with TCRα as a loading control. The data are representative of five experiments. (B) RNA was isolated daily from freshly isolated naive CD4+ cells (day 0) and from differentiating Th1 and Th2 cultures over 5 days. PU.1 (top) and GATA-3 (bottom) expression was determined by real-time PCR with samples being normalized to the expression of β2-microglobulin mRNA. Error bars indicate mean ± SEM. The data are representative of two experiments. (C) Cytoplasmic or nuclear extracts (100 or 200 μg) from Th1 or Th2 cultures were immunoblotted with anti-PU.1. Blots were reprobed with PARP as a nuclear loading control. The data are representative of three experiments. (D) Cytoplasmic or nuclear extracts (500 μg) from Th1 or Th2 cells were incubated with a biotinylated PU.1 binding oligonucleotide and complexes were precipitated with streptavidin-agarose. Precipitates were immunoblotted with anti-PU.1. Extract (200 μg) used for the DNA binding assay was immunoblotted in parallel (bottom panel). The data are representative of two experiments. Immunity 2005 22, 693-703DOI: (10.1016/j.immuni.2005.03.016) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 PU.1 Expression Represses Th2 Cytokine Production (A) Schematic of retroviral vectors used for infection of primary T cells (B) EGFP+ cells from differentiating Th2 populations transduced with control or PU.1-expressing retrovirus on the second day of differentiation and cultured for an additional three days after infection were restimulated with anti-CD3 and treated with Monensin for the last 2 hr of a 5 hr incubation before intracellular staining of cytokine expression. Analysis was performed on 5000 EGFP+ events. The data are representative of two experiments. (C) Th2 cultures infected as in (B) were sorted after 5 days in culture to isolate EGFP (+) and (−) populations and restimulated with anti-CD3. Supernatants were collected after 24 hr and analyzed for cytokine levels by ELISA. The asterisk indicates a significant difference from Vector EGFP (+) cells, p < 0.05. Results are means of duplicate samples and representative of more than 10 experiments. (D) CD4+ T cells were cultured under Th2 conditions for 6 days and rested overnight in IL-2. Cells were then cultured with Th2 cytokines and infected with control or PU.1-expressing retroviruses. EGFP (+) and (−) cells were sorted by flow cytometry, and after a total of 11 days in culture, cells were restimulated with anti-CD3. Supernatants were collected after 24 hours and analyzed for cytokine levels by ELISA. The asterisk indicates a significant difference from Vector EGFP (+) cells, p < 0.05. Results are an average of two experiments. (E) Schematic of PU.1 domain structure and mutants used for analysis of function. (F) Expression of retroviral PU.1 and PU.1 mutants in primary T cells. Extracts (50 μg) from differentiated EGFP+ Th2 cells were immunoblotted for PU.1 expression and GAPDH as a control. Sizes of PU.1 and PU.1 mutants are shown at the right side of the panel. In lanes with multiple translation products, a dot indicates full-length bands. (G) EGFP+ cells were prepared as in (B) and analyzed as in (C) following infection with PU.1 or PU.1 mutant-expressing retroviruses. The asterisk indicates a significant difference from Vector EGFP + cells, p < 0.05. The “#” symbol indicates a significant difference from PU.1 EGFP+ cells, p < 0.05. Results represent the average of three experiments. All ELISA results are expressed as mean ± SEM. Immunity 2005 22, 693-703DOI: (10.1016/j.immuni.2005.03.016) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Retroviral PU.1 Expression Does Not Result in a Th1 Phenotype (A) FACS analysis of IL-12Rβ2 and IL-18Rα. Top, staining of Th1 and Th2 cells. Bottom, staining of Th2 cells transduced with control RV or PU.1-expressing RV. (B) Th1 cells, or Th2 cells transduced with control or PU.1-expressing RV, were sorted, cultured and stimulated as in Fig. 2C. Levels of IFN-γ were determined by ELISA. Error bars indicate mean ± SEM. The data are representative of three experiments. Immunity 2005 22, 693-703DOI: (10.1016/j.immuni.2005.03.016) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 PU.1 Is Expressed in IL-4-Low Th2 Cells (A) Wild-type Th2 cells were restimulated with anti-CD3 after 6 days in culture and analyzed as in Figure 2B. Populations were divided into IL-4-high and -low (right side of panel, top and bottom, respectively) and analyzed for IL-5 and IL-10 staining. (B) Wild-type Th2 cells were stimulated with anti-CD3 and purified based on their IL-4 secretion levels. Cells were rested overnight and restimulated with anti-CD3 for 24, 48 or 72 hours. Cytokine levels were analyzed by ELISA. The data are representative of three experiments. (C) RNA was purified from cells isolated as in (B) and subjected to sequential Northern analysis using cDNA probes for PU.1, Th2 cytokines/receptors and TCRα, as a control. The data are representative of two experiments. (D) Total protein extracts (400 μg) from wild-type and Stat6−/− Th2 cultures separated as in (B) were immunoblotted with anti-PU.1 or anti-GAPDH, as a control. (E) Supernatants from Vector or PU.1 transduced cells (EGFP [+] or [−]) generated as in Figure 2C were analyzed for CCL22 levels by ELISA. The data are representative of three experiments. (F) Purified CD4+ T cells from CD2:Stat6VT mice were stimulated with anti-CD3 and analyzed as in Figure 2B. Results are representative of four experiments. (G) Purified CD4+ T cells from CD2:Stat6VT mice were stimulated with anti-CD3 overnight and separated into IL-4-high and IL-4-low populations. PU.1 expression was determined by real-time PCR following normalization of signal to β2-microglobulin expression. Error bars indicate mean ± SD. The data are representative of two experiments. All ELISA results are expressed as mean ± SEM. Immunity 2005 22, 693-703DOI: (10.1016/j.immuni.2005.03.016) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Inhibition of PU.1 Expression Increases Th2 Cytokine Production (A) Nuclear extracts (50 μg) from M12 cells transduced with control or two different PU.1-specific RNAi retroviruses and isolated by cell sorting were immunoblotted with anti-PU.1 or anti-PARP as a control. The ratio of PU.1/PARP and percent decrease in PU.1 expression compared to control RNAi are indicated below. (B) Differentiating Th2 cultures from PU.1+/− mice were transduced with retrovirus expressing a control or PU.1-specific shRNA two days after initial activation. Four days after transduction, cells were stimulated with anti-CD3 and analyzed as in Figure 2B. The data are representative of three experiments. (C) Average fold induction of cytokine production of PU.1 RNAi treated cells versus control cells analyzed in (B). Fold inductions of total IL-4-, IL-5-, or IL-10-cytokine-positive cells or double cytokine-positive (IL-4/IL-5-positive, or IL-4/IL-10 positive) cells, are shown. The data are representative of three experiments. The asterisk indicates a significant difference from control RNAi values, p < 0.05. Error bars indicate mean ± SEM. Immunity 2005 22, 693-703DOI: (10.1016/j.immuni.2005.03.016) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 PU.1 Antagonizes GATA-3 Function (A) Schematics of retroviral vectors used in primary T cell transductions. (B) Stat6−/− Th2 cultures were transduced with control (vector or hCD4 RV), GATA-3-expressing, and PU.1-expressing retroviruses. Populations that were hCD4 and EGFP-double positive (hCD4 RV + Vector RV, GATA-3 + Vector RV, or GATA-3 + PU.1 RV) were isolated by flow cytometry. Purified cells were stimulated with anti-CD3 and cytokine levels were tested by ELISA. Data represent mean ± SEM. The asterisk indicates a significant difference from Vector RV + hCD4 RV transduced cells, p < 0.05. (C) GATA-3 and PU.1, PU.1 mutant or empty retroviral vectors were transfected into Phoenix cells. GATA-3 was immunoprecipitated from total cell extracts and precipitates were immunoblotted for PU.1. The expression of GATA-3 and PU.1 or PU.1 mutants was detected in total cell extracts by immunoblot. (D) Densitometry analysis of immunoblots of PU.1 expression in IL-4-low Th2 cells compared to other cells with transduced PU.1, and RAW cells. (E) PU.1 was immunoprecipitated from PU.1 transduced EL4 cell extracts. Precipitates were immunoblotted for GATA-3 and PU.1. (F) PU.1 was immunoprecipitated from primary Th2 cells. Precipitates were immunoblotted for GATA-3 and PU.1. The amount of GATA-3 and PU.1 present in extracts before immunoprecipitation (input) is shown below. Immunity 2005 22, 693-703DOI: (10.1016/j.immuni.2005.03.016) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 PU.1 Antagonizes GATA-3 DNA Binding (A) Biotinylated DNA used for GATA-3 DNA affinity purification assay. (B) Total cell extracts from EL4-Vector and EL4-PU.1 (200, 400, or 800 μg) were incubated with biotinylated oligonucleotides and levels of DNA bound GATA-3 were assessed by Western (top). Total cell extracts were immunoblotted for GATA-3, followed by reprobing for PU.1. Ratio of DNA bound GATA-3 to total GATA-3 is indicated below. (C) Total cell extracts (50 or 100 μg) from IL-4-low and IL-4-high Th2 cells were tested for GATA-3 DNA binding levels (top) and total levels of GATA-3 expression. Ratio of DNA bound GATA-3 to total GATA-3 is indicated below. (D) Th2 cells were separated in IL-4-high and IL-4-low populations, stimulated for 3 hr, and fixed. Cells were lysed, chromatin sheared, and fragments subjected to chromatin immunoprecipitation with or without anti-GATA-3. DNA was purified from control and GATA-3 ChIP and used as the template in a PCR reaction for IL-4 promoter and 3′ enhancer (Hypersensitivity Site VA). As a control, 4-fold dilutions of 1% of the ChIP input were used in PCR reactions with the same primer sets. (E) Model of the IL-4 locus in IL-4-high and -low cells indicates that GATA-3 binds the IL-4 gene enhancer in IL-4 high cells but that PU.1 interferes with GATA-3 binding in IL-4-low cells. Cytokine profiles of IL-4-high and IL-4-low cells are also indicated. Immunity 2005 22, 693-703DOI: (10.1016/j.immuni.2005.03.016) Copyright © 2005 Elsevier Inc. Terms and Conditions