Fig 2: Gating strategy for lung cell phenotyping. The fully stained sample was unmixed with AF included in the ID7000 software, then FCS files were exported and opened in FlowJo. Sequential gating was used to identify cell populations. Data clean-up included time gating (A), doublet exclusion (B), viability gating (C), and debris removal (D). CD45 and Sca-1 were used to discriminate 3 populations (E): hematopoietic cells (CD45+; red gate), endothelial cells (ECs) (CD45−/Sca-1+; pink gate), and non-hematopoietic/non-EC cells (CD45−/Sca-1−; yellow gate). A scatter plot (G) and expression of C-kit and Sca-1 defined proangiogenic cells (PACs) (H). CD11c+/CD11b− cells were gated (I) then expression of Siglec-F and absence of CD24 was used to gate alveolar macrophages (J). CD11b+ cells (K) were gated from the remaining cells in plot I. CD24 was expressed on granulocytes (L) then Siglec-F+ eosinophils and Ly-6G+ neutrophils were gated (M). The remaining cells in plot L were selected and MHC II+ cells were gated (N). CD64+/CD24− cells were gated as interstitial macrophages (O). From the EC gate (pink gate in plot (P)], CD90.2 and VWF were used to gate pulmonary EC subsets as alveolar ECs (CD90.2-/VWF-), lymphatic ECs (CD90.2+/VWF-), or blood vessel ECs (CD90.2-/VWF+) (R). From the non-hematopoietic/non-EC subset (yellow gate in plot (Q), α-SMA+ cells were gated (U) then myofibroblasts expressing low or high amounts of collagen-1 were gated (V). Also from the non-hematopoietic/non-EC subset, PDGFRb+/CD146+ pericytes (S), and MUC2+/EPCAM+ epithelial goblet cells (T) were gated.

Fig 3: Improved resolution of fully stained sample with AF included in unmixing. AF was either included or not included in the unmixing of the fully stained sample. By including the AF in the unmixing, the resolution for BUV395 was enhanced, enabling for better detection of the non-hematopoietic/non-EC fraction (CD45-/Sca-1-; yellow gate) (A). Increased resolution for AF532 was also seen, allowing for the detection of eosinophils (B). The absence of AF in the unmixing masked the signals of PE and BV605 which reduced the resolution between these fluorophores and lead to false positives of epithelial goblet cells (C). The poor identification of the non-hematopoietic/non-EC subset in (A) resulted in the loss of events downstream in the gating strategy as seen by a reduced α-SMA + population (D).

Fig 4: Triptolide decreases the production of donor-specific antibodies (DSA) and reduces the amounts of B cells and plasma cells in vivo. Transplant-recipient sera collected at the indicated time points were reacted with donor spleen cells and evaluated for antibody production by flow cytometry. Circulating levels of (A) IgG and (B) IgM are expressed as mean fluorescence intensity ± SD (n = 5). B cells (CD45+, CD19+) and plasma cells (CD45+, CD38+) were detected by flow cytometry in recipient blood and spleen 4 weeks after transplantation. Representative dot plots for frequencies and counts of B cells and plasma cells in recipient blood (C–F) and spleen (G–J) were shown. Data were represented as mean ± SD of at least three independent samples. ∗p < 0.05; ∗∗p < 0.01, Student’s t-test.

Fig 5: Generation and characterization of Foxp3LSL mice.a, Schematics of the targeting construct and the Foxp3LSL allele before and after Flp recombinase-mediated removal of the neo cassette. Homologous regions between the targeting construct and the WT allele are demarcated with dashed gray lines. Gene map is based on RefSeq record NM_001199347.1. pA, bovine growth hormone polyadenylation signal; neo, neomycin resistance gene; PGK, mouse phosphoglycerate kinase 1 promoter; 3xSV40pA, triple-tandem SV40 early polyadenylation signals (STOP cassette); DTA, diphtheria toxin A. b, Genotyping PCR showing the WT (275 bp) and knock-in-specific (350 bp) bands using primers labeled in a. c, d, Flow cytometric analysis of T cells and TCRßhi single positive thymocytes from 3-week-old male Foxp3LSL/y (c) and 8–10-week-old female Foxp3LSL/WT (d) mice. LN, lymph nodes. e, Expression of molecules associated with T cell activation in splenic Foxp3+ Treg and Thy1.1+ Treg “wannabes”, and naïve (CD44loCD62Lhi) and activated (CD44hiCD62Llo) conventional CD4 T cells shown in d. f, Percentages of TCRVß5+, Vß6+ and Vß8+ cells among the indicated splenic CD4 T cell populations from 8–10-week-old female Foxp3LSL/DTR-GFP mice. Cells are gated on the CD44loCD62Lhi subset to avoid potential clonal expansion during activation. One way ANOVA. g, 2×106 CD45.1+Foxp3- conventional CD4 T cells were co-transferred with 2×105 CD45.2+ Treg “wannabes” from 2–3-week-old Foxp3LSL/y mice or Treg cells from 6–8-week-old Foxp3GFP mice into Tcrb–/–Tcrd–/– mice. Recipients were orally gavaged with tamoxifen and injected i.p. with diphtheria toxin (DT) at the indicated time points (top). DT was administered to deplete the few contaminating DTR-expressing Treg cells in the transferred FACS-purified conventional T cells or those induced to express Foxp3 after transfer. Cellularity and proliferation of CD45.1+ responder cells from lymph nodes were analyzed using flow cytometry (bottom). Data are representative of two independent experiments. All error bars denote mean ± s.e.m. ns, non-significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.