Fig 2: Mammaglobin specific Treg in BM expresses S1P1. a Dot plot depicting frequencies of mammaglobin specific Treg in PB and BM of a breast cancer patient. b Cumulative data of mammaglobin specific Treg frequencies in PB and BM of breast cancer patients (n = 10). c Graph showing MFI of S1P1 expression on bulk and antigen-specific Tcon and Treg in PB and BM and d Graph showing percentage of S1P1 positive BM Tcon and Treg from all DR04 and DR07 patients analyzed (n = 5). Data distribution is represented by mean with SEM in b–d. Paired t test was used for statistical analysis in b–d. Lines connect data from individual patients

Fig 3: Antigen-specific TCR stimulation induces S1P1 expression on BM Treg. a Histogram of S1P1 expression on a patient’s BM Treg stimulated with anti-CD3 anti-CD28 beads (n = 3). b Histogram depicting S1P1 expression on a patient’s BM Treg stimulated with purified primary APC alone (n = 2) or primary APC with SEB (n = 5). c Histogram depicting S1P1 expression on a patient’s BM Treg stimulated with in vitro differentiated BMDC alone or BMDC with SEB (n = 4) three days after activation. Red line in the histograms depict S1P1 expression on unstimulated Treg alone. Green line in the histograms refer to S1P1 expression on TCR stimulated Treg. Blue line (only in b and c) refers to S1P1 expression on Treg that were cocultured with APC or BMDC alone without the polyclonal SEB stimulus. Solid grey histogram represents S1P1 isotype control. d Cumulative data from three different stimulation settings. Paired t test was used (when n is > or =4) for statistical analysis

Fig 4: S1PR1 regulates the C3-PPIL1 binding complex and inflammasome activation to induce cell migration(A–C) S1PR1 mutants, arginine 120 to alanine (R120A), and tryptophan 269 to leucine (W269L) were expressed with FLAG tag in B16 cell line and analyzed by western blotting, compared with wild-type (WT) S1PR1 and vector controls (A and B). In contrast, their relative S1PR1 mRNA levels were analyzed by qRT-PCR (C). (D and E) The effects of S1PR1 mutants on C3 protein expression and cell migration in comparison with WT-S1PR1 or vector control were analyzed by western blotting (D) and Boyden chambers (E), respectively.(F and G) S1PR1 mutants and WT-S1PR1 or vector B16 cells were lysed, and C3 was pulled down or subjected to co-IP to determine the effects of C3-PPIL1 association in the mutants’ cells. To detect PPIL1 binding in the C3 pull-down lysates, western blotting was performed (F) and quantified (G).(H–J) CRISPR-Cas9-mediated PPIL1 knockout (PPIL1 KO) in S1PR1 overexpression (S1PR1 OE) B16 cells was analyzed by western blotting and quantified, using S1PR1 OE as control, without affecting S1PR1 expression (H). In addition, the Effects of PPIL1 KO in S1PR1 OE cells on cell migration and NLRP3 inflammasome genes compared with vector or S1PR1 OE controls were measured in Boyden chambers (I) and analyzed by western blotting (J), respectively.(K) Effects of PPIL1 KO in response to JTE-013 + DPO treatment for 24 h on NLPR3 protein expression were analyzed by western blotting and quantified, compared with vector or JTE-013 + DPO controls. Data represent at least two independent studies in triplicates. Data are means of ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 5: JTE-013 and DPO drug combination increases B16 cell migration in response to elevated intracellular S1P/S1PR1/C3 signaling(A) Migration of B16 cells in the absence/presence of JTE-013 (1 µM) for 24 h, DPO (5 mM) for 8 h, or JTE + DPO combination for 24 h (JTE-013 pre-treatment alone for 16 h and then with DPO addition for 8 h), was measured in Boyden chambers. Representative images of migrated cells (left panels), and optical density readings of transwell inserts at 630 nm, after staining with crystal violet solution (0.5%) (lower left graph) are shown. In similar studies, the effects of these treatments on B16 cell viability were measured by MTT cell proliferation assay (upper left graph). Scale bars, 100 µm.(B) Effects of JTE-013/DPO (alone or in combination) on complement C3b-a and -ß chain expression were measured in B16 cells using western blotting (upper panel). Actin was used as a loading control. Western blotting was quantified relative to actin levels (lower graph).(C) Effects of JTE/DPO (alone or in combination) on intracellular C3a expression and secretion in B16 cells were measured by western blotting (left panel) and ELISA (right panel), respectively, using anti-C3a-antibody.(D) Intracellular levels of S1P, dhS1P, Sph, and dhSph in B16 cells treated with vehicle or JTE-013/DPO combination were measured by lipidomics as we described.37,38(E–H) Migration of B16 cells transfected with shRNA scrambled (Scr) or shRNA C3 knockdown (shC3) plasmids, confirmed by western blotting and quantified, without significantly affecting S1PR1 expression (E and F) was measured in Boyden chambers in response to JTE013/DPO treatment (G and H). Scale bars, 100 µm.(I and J) Migration of B16 cells transfected for ectopic expression of wild-type (WT)-C3b-a'2 (vector-only transfected cells were used as controls), confirmed by western blotting (I) was measured in Boyden chambers with/without JTE-013/DPO exposure (J).(K–N) Effects of shRNA-mediated knockdown of S1P lyase (shSGPL1) compared with shRNA scrambled (Scr) transfected B16 cells, confirmed using qRT-PCR (K), on cell migration (L) or C3b expression (M and N) were measured using Boyden chambers and western blotting, respectively.(O and P) Effects of exogenous S1P with BSA- or HDL-S1P + JTE-013 combination, compared with endogenous S1P with DPO + JTE-013 combination on migration (O) and invasion (P) assays using B16 cells. Data represent at least two independent studies in triplicates. Data are means of ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.