Fig 2: ALOX15 inhibition decreases FOXP3 expression in Tregs and alters their phenotype.Tregs were obtained from healthy volunteers and incubated with an ALOX15 inhibitor (PD146176; 5 µM) on day 0 (D0) or day 3 (D3) or vehicle (0.01% DMSO). (A) The number of FOXP3 positive cells (left panel) and the expression of FOXP3 (right panel), (B) Cellular proliferation, together with the number of (C) FOXP3 and/or T-bet expressing cells and (D) FOXP3 and/or ROR?t expressing cells was determined using flow cytometry and fluorescently labeled antibodies. Results are expressed mean ± s.e.m of % change from vehicle control. n = 10 healthy volunteers from two distinct experiments. (E–H) Mouse Tregs were obtained from WT and Alox15-/- mice. (E) The number of FOXP3 positive cells (left panel) and the expression of FOXP3 (right panel), together with the number of (F) FOXP3 and/or T-bet expressing cells and (G) FOXP3 and/or ROR?t expressing cells and (H) Cellular proliferation was determined using flow cytometry and fluorescently labeled antibodies. Splenocytes were isolated from WT and Alox15-/- mice and (I) the number of FOXP3 positive cells and (J) the expression of FOXP3 was determined using flow cytometry. n = 4 mice per group from two distinct experiments. For A–D *p < 0.05, **p < 0.01, using One sample t test and Unpaired t test with Welch’s correction. For E–J *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; One sample t test. (K) Mouse Tregs were obtained from WT and Alox15-/- mice and the concentrations of ALOX15-derived lipid mediators was determined using LC-MS/MS-based lipid mediator profiles. Results are mean ± sem. n = 3–4 mice per group A–D *p < 0.05 Unpaired t test with Welch’s correction.

Fig 3: Increased ALOX15-derived lipid mediators by Treg precursors leads to FOXP3 upregulation.(A–D) CD4+ T-cells were isolated from healthy volunteers and incubated for 30 min with p38 inhibitor (SB202190) or vehicle (0.01% DMSO), then with TGF-ß+CD3/CD28 for (A, B) 12 h for the expression of (A) p-smad2/3, (B) p-STAT1 and p-STAT3, (C, D) or for 24 h and the expression of (C) ALOX15 and (D) FOXP3 assessed using flow cytometry. Results are mean ± s.e.m. and expressed as percent change from cells incubated with TGF-ß+CD3/CD28 alone; n = 4–6 healthy volunteers; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, One sample t test or Unpaired t test with Welch’s correction. (E) CD4+ T-cells were incubated with a FOXP3 inhibitor peptide (P60 peptide; 100 µM) or vehicle (0.01% DMSO) and cells differentiated to Tregs (see methods for details) for 7 days. ALOX15 expression was determined using flow cytometry. (F) HEK 293 cells were co- transfected with a vector containing the promoter region of ALOX15 tagged with luciferase and either a vector containing a FOXP3 or a control vector and luminescence was determined after 24 h. Results are mean ± s.e.m. n = 7 cell preparations from three independent experiments. For E ***p < 0.0001, One sample t test, For F **p < 0.01, Unpaired t test with Welch’s correction. (G) CD4+ T-cells were isolated from healthy volunteers and incubated with or without the Treg differentiation cocktail for 1 h (37 °C). Incubations were quenched and lipid mediator profiles determined using lipid mediator profiling. Pathway analysis for the differential expression of mediators from the (left panel) DHA and n-3 DPA, and (right panel) EPA and AA bioactive metabolomes in cells incubated with the Treg differentiation cocktail when compared to Th0 cells incubated in media alone. Statistical differences between the normalized concentrations (expressed as the fold change) of the lipid mediators from the cells incubated with Treg cocktail and Th0 cells were determined using a Mann–Whitney test followed by a multiple comparison correction using Benjamini–Hochberg procedure. Results are representative of n = 6 donors from three distinct experiments. (H) Human Tregs were obtained as detailed above, in the presence or absence of ALOX15 inhibitor (5 µM) and with RvD3, RvD5n-3 DPA (1nM) each or vehicle (0.1% EtOH; 37 °C; 7 days). Abundance of FOXP3 positive cells (left panel) and expression of FOXP3 per cell (right panel). Results are mean ± s.e.m. and expressed as percent change from Tregs + ALOX15 inhibitor, n = 4 donors from two independent experiments. *p < 0.05, One sample t test or Unpaired t test with Welch’s correction. (I) Scheme summarizing the proposed ALOX15-initiated Treg differentiation pathway.

Fig 4: Comparisons of functional markers between TNFR2+Tregs and TNFR2-Tregs in MPE. (A) Gating strategy for identifying TNFR2hi, TNFR2low, and TNFR2neg in CD4+Foxp3+T cells. (B) The representative percentages of Ki-67+ cells in TNFR2hiTregs, TNFR2lowTregs, and TNFR2negTregs were determined using FACS. (B) Summary data of Ki-67+ cells frequency in TNFR2hiTregs, TNFR2lowTregs, and TNFR2negTregs (n=16). (D) Correlation between Ki-67 and TNFR2 expressed by Tregs in MPE (n=16). (E) The histogram showing the expression of CTLA-4 and PD-L1 on TNFR2+Tregs (red filled histogram) and TNFR2-Tregs (blue filled). The gray filled histogram depicts the isotype control. (F) The graph summarizes the frequency of CTLA-4+ cells and PD-L1+ cells within each cell type (n=11). (G) TNFR2 expression correlates with CTLA-4 and PD-L1 expression on Tregs in MPE (n=11). The percentages were determined by flow cytometry. Data are expressed as means ± SEM. Flow analysis was gated on live cells. ***, P<0.001, ****, P<0.0001 by one-way ANOVA or Paired Student’s t-test. Correlations were determined by Spearman’s rank correlation coefficients. TNFR2, tumor necrosis factor receptor type II; MPE, malignant pleural effusion.

Fig 5: TEPP-46 Inhibits EAE Development(A and B) C56Bl/6 mice were immunized with MOG35–55 peptide emulsified in complete Freund adjuvant. Mice were treated every other day from day 0 to day +14 post-immunization (red arrows) with vehicle (PBS + 40% cyclodextrin) or TEPP-46 50 mg/kg in vehicle.(A) Mean clinical score (left) and percentage of weight loss (right) in EAE mice treated with vehicle or TEPP-46.(B) Mean day of onset in vehicle and TEPP-46-treated EAE mice. N = 14–16 mice per group (A) or 12–16 mice/group (B) from 3 independent experiments.(C–E) Vehicle- and TEPP-46-treated EAE mice were sacrificed 11 days post-immunization. CNS infiltrates were isolated, stained, and analyzed by flow cytometry as described in the STAR Methods section.(C) Total number of CD45+ cells in the CNS of vehicle- and TEPP-46-treated EAE mice.(D) Percentage of Foxp3+CD25+ cells in the CD4+ T cells population isolated from the CNS of EAE mice.(E) Percentages of IL-17A-, IFN-?-, and GM-CSF-producing and Ki67+ cells in the CNS CD4+ and CD8+ T populations of vehicle and TEPP-46-treated mice. For (C–E), n = 9–11 from 2 independent experiments.(F) Transfer EAE was induced in C57BL/6 mice by injection of MOG35–55-primed CD3+ T cells re-stimulated in vitro as described in STAR Methods section. N = 7–9 mice/group from 2 independent experiments. For panels (A) and (F), data are the mean ± standard error of the mean (SEM). For panels (B–E), data are the mean ± SD. p values were calculated by two-tailed unpaired Student’s t test. #p < 0.01, *p < 0.05, and ****p < 0.0001, compared to vehicle condition (A–E) or CTRL cell condition (F).