Fig 2: ADNP is associated with genes involved in lymphocyte differentiation(A) Average ChIP-seq signal over all ADNP peaks in Th2 cells. Data are representative of 3 biological replicates.(B) Pie chart displaying distribution of 7,291 ADNP peaks across genomic features. Promoter includes peaks between 1-kb upstream and 1-kb downstream of the transcription start site (TSS). Downstream includes peaks up to 1-kb downstream transcription termination site (TTS).(C) KEGG pathway analysis of the genes associated to ADNP peaks. All pathways shown were enriched (p < 0.05).(D) Venn diagram showing the overlap between ADNP ChIP-seq peaks in Th2 and naive T cells. Peak list was generated using two biological replicates.(E) KEGG pathway analysis of the genes associated with ADNP gained peaks (peaks present in Th2 cells but not in naive T cells). All pathways shown were enriched (p < 0.05).(F) Top two enriched motifs of ADNP peaks in naive T cells and Th2 cells. Enrichment was assessed using a one-sided cumulative binomial distribution in HOMER.(G) Venn diagram showing the overlap between ADNP and CTCF ChIP-seq peaks in Th2 cells. Peak list was generated using two biological replicates.(H) Representative binding profiles of ADNP in wild-type naive T cells and Cd4Cre Th2 cells and CTCF in Cd4Cre or Cd4CreAdnpfl/fl Th2 cells at the Runx3 locus. Data are representative of 3 biological replicates.(I) Average ChIP-seq signal over all CTCF peaks in Cd4Cre or Cd4CreAdnpfl/fl Th2 cells. Data are representative of 2 biological replicates.(J) Representative binding profiles of ADNP in wild-type naive T cells and Cd4Cre Th2 cells; and CTCF in Cd4Cre or Cd4CreAdnpfl/fl Th2 cells at the type 2 cytokine locus. Black arrows indicate CTCF-binding sites (CBSs). Data are representative of 3 biological replicates.(K) Predicted CTCF motifs within the ADNP ChIP-seq peaks at the Il4 and Il13 promoter regions (magenta and purple rectangles in J). Prediction was performed using Jaspar transcription factor database.See also Figure S4 and Tables S3 and S4.

Fig 3: The reduction of CD25+ Nrp1+ expression resulting from AhR activation is dependent on IL-2. (A) CD4+ T cells isolated from NOD mice splenocytes were co-cultured with 1000 ng/ml, 100 ng/ml, 10 ng/ml or 0 ng/ml of anti-IL-2. All cells were also co-cultured with IL-12, anti-IL4, and CD3/CD28 Dynabeads. AhR was activated with 100 nM Cl-BBQ. Following incubation at 37°C for 4 days, cells were stained, and Nrp1 expression was analyzed on CD4+CD25+Foxp3- cells. A two-way ANOVA was performed. * indicates p-value <0.05. ** indicates p-value <0.01. n=4 mice/condition. (B) CFSE-labeled cells from B6 mice were injected into F1 hosts and treated i.p. with anti-IL-2 or isotype control and 15μg/kg TCDD or vehicle. Nrp1 expression on proliferating donor (CFSE dilute) CD4+CD25+ cells were analyzed on day 2.A one-way ANOVA was performed. **** indicates p-value ≤0.0001 n=5 mice/group. Each data point represents an individual mouse.

Fig 4: Increased expression of macrophage activation markers in Dgka -/- BMDM following LPS or IL4 stimulation. (A) WT and Dgka-/- BMDMs were treated with 500ng/mL LPS or 2ng/mL IL-4 for 24h and assessed by qPCR for changes in Nos2 and Arg1 expression, respectively. (B) Immunoblot depicting protein expression of iNOS in lysates of LPS treated WT and Dgka-/- BMDMs (left) and arginase expression of IL-4 treated BMDMs. Quantification of band intensity normalized to PBS treated WT mice (not shown) are indicated in text below bands. (C) Summary plot depicting quantification of immunoblot band intensity of two independent experiments as in (B) for n = 6 per group. (D) WT and Dgka-/- BMDMs treated with LPS and IL-4 were assessed by qPCR for expression of SOCS1 and SOCS3. In (A–D) samples were normalized to the PBS treated WT control. (E) immunoblot of WT and Dgka-/- BMDM whole cell lysates with anti-DGKα antibody. Each data point represents an individual mouse for n = 3 per group. Error bars: SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant; Two-way ANOVA with Tukey post-hoc test.

Fig 5: Knockdown of Dgka from J774 murine macrophage cell line shows increased amplification of macrophage activation markers following LPS or IL4 stimulation. (A) J774 murine macrophages were stimulated with 500ng/mL LPS or 2ng/mL IL-4 following control or Dgka siRNA transfection and assessed by qPCR for Nos2 and Arg1, respectively. Samples were normalized to the PBS treated control siRNA transfected cells. (B) Immunoblot analysis of whole cell J774 lysates probing for iNOS in LPS-treated cells and Arginase in IL4-treated cells. Relative band intensities normalized to PBS-treated control are indicated as text below the immunoblots. Representative data shown. (C) Summary plot depicting quantification of immunoblot band intensity from two independent experiments as in (B). (D) J774 macrophages transfected with either control siRNA or Dgka siRNA were treated with LPS for 24h, and the protein expression of phospho-PKC substrates is shown by immunoblot. Relative band intensities normalized to PBS treated control are indicated below the immunoblots. (E) Summary plot depicting quantification of immunoblot band intensity of two independent experiments as in (D). Error bars: SD. *p < 0.05, **p < 0.01, ****p < 0.0001; Two-way ANOVA with Tukey post-hoc test.