Fig 2: 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 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: Feedback control of STAT1 desensitization. (A) Single cell analyses of STAT1 responses in presence of transcriptional inhibitors. Reporter cells stimulated with two 1 h pulses of 100 ng/ml IFN-γ (control, left), pre-treated with 5 μg/ml of ActD for 2h before the first pulse (middle) or treated with 5 μg/ml of ActD between the first and second pulse (right). Shown are individual nuclear STAT1-tagRFP trajectories (color-coded according to treatment protocol) as well as ensemble average (in black) for 50, 39 and 68 cells for control, ActD pre-treatment and ActD treatment between pulses, from two replicates, respectively. STAT1-tagRFP fluorescence shown in arbitrary units (a.u.), time in minutes (mins). (B) Characteristics of single cell STAT1 trajectories presented in (B) From the left: distributions of the overall AUC, peak amplitude (in response to first pulse), and time to peak (in response to first pulse) under different treatment conditions. Individual cell data depicted with circles (with mean ± SD per condition) and color-coded according to treatment protocol. Kruskal-Wallis one-way ANOVA with Dunn’s multiple comparisons test was used to assess differences between groups (*p < 0.05, **p < 0.01, ****p < 0.0001, ns, not significant). (C) Phosphorylation pattern of STAT1 (Y701) during pulsatile treatment of iBMDMs. Wild type iBMDMs either untreated (ctr) or stimulated with one or two 1h pulses of 100 ng/ml IFN-γ at 6h interval. In addition, 100 ng/ml of IL4 was used in the second pulse applied at 6h interval following a pulse of IFN-γ. Samples analyzed at 10 mins, 5h, 7h, 7h and 10mins, and 10h after the start of the experiment. B-actin included as a loading control. Schematic diagram represents pulsing protocol and measurement time-points (in red circles). Molecular weight (MW) is shown in kilo Dalton (kDa). Data are representative of two replicates.