CD4⁺ T cell purification and in vitro differentiation of Th cells

CD4⁺ T cells were positively selected using biotinylated anti-CD4 (RM4-5), followed by streptavidin microbeads on a MACS column (Miltenyi Biotec, Auburn, CA). Purity, as assessed by flow cytometry, was typically ≥92%. CD4⁺CD62L⁺ cells were isolated by negative selection for CD4 and then by positive selection using anti-CD62L MACS beads (Miltenyi Biotec). Purified T cells were plated in 24-well dishes (1 × 10⁶/well) that were coated with 5 µg/ml anti-CD28 (clone 2.43 rat IgG) and 0.5 µg/ml anti-TCR (H57-H59). The cells were cultured under Th1 (10 U/ml IL-2, 5 ng/ml IL-12, and 3.3 µg/ml anti–IL-4) or Th2 (10 U/ml IL-2, 10 ng/ml IL-4, 0.12 µg/ml anti–IL-12, and 5 µg/ml anti–IFN-γ) skewing conditions in 2 ml RPMI 1640 complete T cell media (RPMI 1640 + l-glutamine, 10% FBS, 50 µM2-ME, 10 mM HEPES, 1 mM sodium pyruvate, and 0.05 mg/ml gentamycin). In some experiments, cells were differentiated using HL-1 complete T cell media (HL-1, ± 1% FBS, 2 mM l-glutamine, 50 µM 2-ME, 10 mM HEPES, 1× MEM nonessential amino acids, and 0.05 mg/ml gentamycin). After 4 d, the cells were further expanded for 3 d under Th1 or Th2 skewing conditions without anti-CD28 and anti-TCR. Recombinant mouse IL-4 and IL-12 were purchased from PeproTech (Rocky Hill, NJ). Low endotoxin-grade anti–IL-12 (C17.8), anti–IFN-γ (XMG1.2), anti-TCR, and anti–IL-4 (11B11) Abs were purchased from eBioscience (San Diego, CA). In some experiments, cells were differentiated 3 or 4 d with anti-TCR and anti-CD28 stimulation in complete media. Secondary stimulations were performed by incubating day 7 cells with PMA (5 ng/ml) and ionomycin (500 ng/ml) for 4 h.

For experiments using OTII mice, the cells (1 × 10⁶) were differentiated in the presence of 10 µM OVA peptide 323–339 (ISQAVHAAHAEINEAGR), a gift from Dr. W. J. Karpus (Northwestern University, Chicago, IL), and T cell-depleted irradiated APCs (2 × 10⁶) for 5 d in 2 ml complete media in a 24-well dish. The cells were then expanded for 2 d in complete media under skewing conditions in the absence of OVA peptide and APCs.

Intracellular cytokine staining and cytokine capture assays

Cells skewed for 7 d were incubated with monensin (eBioscience) or GolgiStop (BD Biosciences, San Jose, CA), 5 ng/ml PMA (Roche, Basel, Switzerland), and 500 ng/ml ionomycin (Roche) for 4 h at 37°C in complete media. Cells skewed for 3 d were incubated with monensin in the presence of plate-bound anti-TCR (0.5 µg/ml) and anti-CD28 (5 µg/ml) for 5 h. The cells were then fixed and permeabilized with the BD Cytofix/Cytoperm fixation/permeabilization kit (BD Biosciences) following the manufacturer’s guidelines. The cells were stained with fluorochrome-conjugated CD4 (RM4-5), IFN-γ (XMG1.2), IL-4 (11B1), IL-10 (Jes5-16E3), and IL-13 (ebio13A) Abs at 4°C for 30 min, washed, resuspended in FACS buffer, and analyzed on a FACSCalibur or FACSCanto (BD Biosciences). With regards to the GATA-3 staining, cells were fixed and permeabilized using the eBioscience FoxP3 Staining buffer kit (eBioscience), and then the cells were incubated with GATA-3 (clone TWAJ) Ab or an isotype control for 30 min, washed, resuspended in FACS buffer, and analyzed on a FACSCanto (BD Biosciences). All Abs for intracellular cytokine staining (ICCS) were obtained from eBioscience or BD Pharmingen (San Diego, CA). IL-5 cytokine capture assays (Miltenyi Biotec) were performed according to the manufacturer’s guidelines.

For experiments using OTII mice, the cells were restimulated in the presence of OVA-pulsed APCs and monensin for 5 h. The cells were then analyzed for IL-4 and IFN-γ as above.

RNA isolation and quantitative RT-PCR

RNA was isolated from day 4 and day 7 skewed cells using TRIzol and reverse transcribed with Reverse Transcriptase III, according to the manufacturer’s protocol (Invitrogen Life Technologies, Carlsbad, CA). Day 7 skewed cells were restimulated prior to RNA isolation. Quantitative RT-PCR (qRT-PCR) was performed using Sybr Green incorporation on an ABI prism 7000 detection system (Applied Biosystems). Target levels were normalized to hypoxanthine phosphoribosyltransferase (HPRT). The relative expression for the qRT-PCR samples is calculated as 2^{−(ΔCTsample)}, where CT is the cycle number it took for the sample to reach the analysis threshold. ΔCT is the CT of the sample for the gene of interest minus the CT of the normalizing gene, HPRT. The primers used for qRT-PCR can be found in Table I.

Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) was performed according to the manufacturer’s protocol (Upstate Biotechnology, Lake Placid, NY). Briefly, 4 × 10⁶ cells were fixed with formaldehyde, and the nuclear lysates were sonicated and subjected to immunoprecipitation overnight at 4°C with anti-acetyl H3 (Millipore, Bedford, MA) or rabbit IgG isotype (The Jackson Laboratory, Bar Harbor, ME). qRT-PCR was performed using Sybr Green incorporation on equal volumes of immunoprecipitated DNA. The relative expression for the ChIP samples is calculated as 2^{(CTinput − CTIP)}, where CT is the cycle number it took for the samples to reach the analysis threshold. The primers used for ChIP are found in Table II. qRT-PCR was performed using primers for the Rad50 promoter as a positive control for the ChIP samples.

Protein isolation and Western blots

The cells were lysed in radioimmunoprecipitation assay buffer, and debris was pelleted by centrifugation at 100,000 rpm for 10 min at 4°C. Day 7 skewed cells were restimulated for 4 h prior to harvesting. The lysates were run on a 10% SDS gel, transferred to polyvinylidene difluoride, and immunoblotted with the following Abs: anti-mouse GATA-3 (clone HG3-31; Santa Cruz Biotechnology, Santa Cruz, CA), anti-mouse β-actin (clone C4; Santa Cruz Biotechnology), and anti-mouse T-bet (clone ebio4b10; eBioscience).

Retrovirus infections

CD4⁺ splenocytes were isolated and stimulated under Th1 and Th2 conditions as above and infected with MigR1 or GATA-3 MigR1 (20) retroviruses at 24 h as described previously (21).

Lck-deficient Th2 skewed cells produce Th2 cytokines IL-10, IL-13, and IL-5

The cytokine IL-4 is genetically linked to the cytokines IL-5 and IL-13 at what is termed the Th2 cytokine gene locus (26), and these cytokines have been shown to be important for Th2-mediated responses. In addition, IL-10, which is located elsewhere in the genome, is also expressed by Th2 cells (2). Therefore, we analyzed IL-5, IL-10, and IL-13 expression by qRT-PCR and flow cytometry in both bulk CD4⁺ cells and CD4⁺CD62L⁺ cells (Fig. 2 and data not shown). We found that the lck^−/− Th2 skewed cells have modestly decreased levels of IL-10 and normal IL-13 expression compared with wt Th2 cells (Fig. 2A). Furthermore, we found that IL-5 expression is relatively normal in lck^−/− Th2 skewed cells compared with wt Th2 cells (Fig. 2B). Overall, these data indicate that the defect in lck^−/− Th2 skewed cells predominately affects IL-4 with minimal impact on the other Th2 cytokines.

The expression of T-bet and GATA-3 is aberrant in lck^−/− Th2 skewed cells

Th1 and Th2 differentiation is dependent on transcription factors T-bet and GATA-3, respectively (reviewed in Refs. 1, 3, 22). Therefore, we analyzed T-bet and GATA-3 mRNA and protein expression in wt and mutant Th2 and Th1 skewed cells at days 3 and 4, a time when the cells are committing to their respective lineages, and at day 7, upon secondary stimulation of fully differentiated cells (Fig. 3A, 3B). T-bet protein expression is present in lck^−/− Th2 skewed cells in the early stages of differentiation, and this correlates with elevated levels of T-bet mRNA (Fig. 3A, 3B). GATA-3 protein is reduced in lck^−/− Th2 skewed cells early in the differentiation process, but mRNA levels are similar to wt; this suggest that the decrease in GATA-3 is occurring posttranscriptionally (Fig. 3A, 3B). These aberrant protein expression patterns are more pronounced in day 7 lck^−/− Th2 skewed cells compared with B6 Th2 cells (Fig. 3B). These data again emphasize that the loss of Lck results in a failure of cells to properly commit to the Th2 lineage.

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FIGURE 3

The expression of T-bet and GATA-3 is aberrant in lck^−/− Th2 skewed cells. The sequences used for qRT-PCR are listed in Table I. A, GATA-3 and T-bet mRNA expression from wt and lck^−/− Th2 cultures. CD4⁺CD62L⁺ T cells were stimulated under Th2 conditions for 4 d, and qRT-PCR was performed for T-bet and GATA-3. The data are graphed as the ratio of the expression of the target gene in lck^−/− Th2 cells relative to B6 Th2 skewed cells. Note that T-bet is overexpressed in these cultures. The graphs illustrate the average of a minimum of three experiments. B, GATA-3 and T-bet protein expression is altered in lck^−/− Th2 cultures. CD4⁺ cells were skewed under Th1 and Th2 conditions for 3 and 7 d, and protein expression was evaluated by immunoblotting. The data are representative of two (day 3) and three (day 7) experiments. C, GATA-3 overexpression normalizes IL-4 cytokine expression in lck^−/− Th2 cells. lck^−/− and B6 cells were infected with GATA-3 or empty vector 24 h after initiation of Th2 skewing and then skewed for an additional 6 d. Infected cells were identified by GFP expression and then analyzed for IL-4 and IFN-γ expression by ICCS. The flow plots show a representative experiment. The graph illustrates the average for the percentage of cells expressing IL-4 in GFP⁺ cells (four experiments). The expression of IL-4 in Lck Th2 vector versus Lck Th2 GATA-3–infected cells is statistically different. The asterisk indicates that p < 0.05 (Student paired t test). Note: IL-4 expression is similar in the wt Th2 and lck^−/− GATA-3–infected cells. D, c-Maf and Runx3 are elevated in lck^−/− Th2 cultures. lck^−/− and B6 CD4⁺CD62L⁺ cells were skewed under Th2 conditions for 7 d, and qRT-PCR was performed for c-Maf and Runx3. The data are graphed as the ratio of the expression of the target gene in lck^−/− Th2 cells relative to B6 Th2 skewed cells. The graph illustrates the average of three experiments.

T-bet can antagonize GATA-3 expression and function (6, 9). Therefore, we hypothesized that T-bet may be inhibiting GATA-3 expression and/or function in lck^−/− Th2 skewed cells and that overexpression of GATA-3 would normalize the Th2 phenotype in lck^−/− cells. CD4⁺ T cells from wt and lck^−/− mice were skewed under Th2 or Th1 conditions and infected at 24 h after initial stimulation with a retrovirus encoding GATA-3 or an empty vector control. We observed that GATA-3 overexpression, unlike the empty vector control, resulted in similar levels of IL-4 expression in wt and lck^−/− Th2 skewed cells (Fig. 3C). In addition, the increased IL-4 expression following infection with GATA-3 retrovirus is not due to gross overexpression of GATA-3. Although GATA-3 expression in mutant cells transduced with the GATA-3 retrovirus (GFP⁺) is slightly higher than wt B6 Th2 cells, the overall expression of GATA-3 in these cells is less than that of wt cells infected with the empty vector (Supplemental Fig. 2). These data suggest that the reduction of GATA-3 expression in lck^−/− Th2 skewed cells may result in decreased IL-4 cytokine levels.

Previous studies have shown that the loss of GATA-3 results in reduced Th2 cytokines (2). Lck^−/− Th2 skewed cells have decreased IL-4 expression but relatively normal levels of other known Th2 cytokines (Figs. 1, ​,2);2); this implies that other transcription factors involved in IL-4 gene regulation are affected. Knockout studies have shown that c-Maf is important for IL-4 expression but not for the production of other Th2 cytokines (27). We found c-Maf mRNA expression is elevated in lck^−/− Th2 skewed cells at days 4 and 7 compared with wt Th2 cells, and this suggests that the IL-4 defect we observe is not due to c-Maf (Fig. 3D and data not shown). We also analyzed the expression of Runx3, which has been shown to interact with T-bet to silence IL-4 expression in Th1 cells by binding to the Il4 silencer or HSIV (28). We found Runx3 mRNA expression to be elevated 2-fold in lck^−/− Th2 skewed cells (Fig. 3D). Significantly, the level of Runx3 expression in lck^−/− Th2 skewed cells was equivalent to that of wt Th1 cells (data not shown). These data suggest that the decrease in IL-4 expression may be due to a combination of diminished GATA-3 as well as Runx3/T-bet complexes binding to the Il4 silencer.

We thank Sheila Chari, Dr. William Karpus, Mary Quirion, Dr. Steve Reiner, Aki Ueda, Sarah Umetsu, and Dr. Susan Winandy for technological expertise and support.

This work was supported in part by National Institutes of Health Grants R01CA086867 (to P.L.S.) and F31AI077322 (to K.L.K.) and National Institutes of Health, National Cancer Institute Training Grant T32CA09560 (to K.L.K.).