Supplemental Information. CD4 + CD25 + Foxp3 + Regulatory T Cells Promote. Th17 Cells In Vitro and Enhance Host Resistance

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1 Immunity, Volume 34 Supplemental Information D4 + D Regulatory T ells Promote Th17 ells In Vitro and Enhance Host Resistance in Mouse andida albicans Th17 ell Infection Model Pushpa Pandiyan, Heather R. onti, Lixin Zheng, lanna. Peterson, Douglas R. Mathern, Nydiaris Hernández-Santos, Mira Edgerton, Sarah L. Gaffen, and Michael J. Lenardo

2 Supplementary figures Fig.S1 Tresp +Tcon Tresp +Treg FS D45.1 Fig. S1. Gating of Tresp cells based on congenic marker D45.1 in co-cultures. D45.1 Tresp and were stimulated alone or in co-cultures with D45.2 Tcon or Treg cells at a ratio 1:1 under Th17 cell conditions for 3 days. Representative flow cytometric dot plots of FS and D45.1 staining in the indicated cultures. Fig.S2 P=.7 P=.1 D3-D28 restimulation Tresp +Tcon Tresp +Treg n.s Tresp Tresp + Tcon Tresp + Treg Tresp Tresp + Tresp + Tresp + 5% D25+T-eff 5% Treg 1% D25+T-eff + 4% Treg IFN-γ Fig. S2. Treg cells enhance Th17 cell cytokines in co-cultures in vitro. ) ells stimulated as in (Fig. 1), were re-stimulated with PM-Ionomycin and were stained for at d6 or d7 and the data from three independent experiments are plotted. ) Flow cytometric contour plots of intracellular and expression of Tresp cells, 96 hours after stimulation under Th17 cell conditions with Tcon cells or Treg cells as indicated (gated on live Thy1.1+ Tresp cells). efore staining, the cells were re-stimulated with D3-D28 antibodies for 4 hours. ) D4+D25-D44- (D45.2) naïve Tresp cells were stimulated for 3 days under Th17 cell conditions without or with congenic D45.1 D4+D25+ pre-activated T effector cells (T-eff) or fresh D45.1+D4+D25+ Treg cell or D45.1+ D4+D25- Tcon cells. T-eff cells were pre-activated separately using D3-D28 and then co-cultured with naïve Tresp cells at 1:1 ratio. In the mixed culture where both Tcon and Treg cells were used, the Tresp:Treg:Tcon ratios were 5:4:1. Thus, Treg cells were mixed 2% D25+ Tcon cells. The analysis for and IFN-γ expression was performed by gating on D45.2 Tresp cells.

3 Fig.S2 ** % positive cells ** Tresp Tresp+Tcon Tresp +Treg P<.5 IL-17F IL-22 IFN-γ Fig. S2. D) Treg cells enhance Th17 cell cytokines in co-cultures at late time points in vitro. ells stimulated as in (S2), were re-stimulated with PM-Ionomycin and were stained for IL-17F, IL-22, or IFN-γ at d6 or d7 (late time points) and the data showing the percentage of respective cytokine positive cells are plotted. Fig.S3 In Th17 co-cultures Gated on D45.2 D45.2 only + IL D45.2 Tcon D45.2 Treg d d3 d4 d6 d Fig. S3. ) and expression in D45.2 population (Tcon or Treg cell) in co-cultures on day 3. D45.2 Tcon or Treg cells were stimulated alone with IL-2 or in co-cultures with D45.1 Tresp at a ratio 1:1 under Th17 cell conditions for 3 days. Tcon or Treg cells were gated using D45.2 co-staining to detect intracellular and. ) fraction of Treg cells lose expression and express transiently in Th17 cell co-cultures. D45.2 gfp+ Treg cells were co-cultured with D45.1D44-D25- Tresp cells under Th17 cell conditions at ratio of 1:1 and expression was measured by staining for in Treg cells. In Th17 cell co-cultures, Treg cells were gated using D45.2 co-staining.

4 Fig.S TGF-β ng/ml.1ng/ml.25ng/ml.5ng/ml Serum free media + Th17 cell milieu (2ng/ml TGF-β) ng/ml 2ng/ml 4ng/ml 8ng/ml Treg E WT Il2rb -/- serum (pg/ml) Treg supe. +Treg +Treg in transwell F ell count D25 d Fig. S4. Th17 cell cultures in saturating concentrations of TGF-β. Naive Tresp cells were stimulated in Th17 cell conditions without or with different concentrations of TGF-β (), or with supernatants (supe.) from Treg cultures or Treg cells in co-cultures or transwells (), in normal media or in serum free media (). and expression were quantified using intracellular staining on day 3. IL-2 suppresses production. (D) Naive Tresp cells were stimulated in Th17 cell conditions without or with Treg cells. IL-2 (1 U/ml) was added at the beginning of cultures. was quantified using ELIS of the supernatants collected from indicated cultures on day 4. (E) ELIS quantification of in the serum of 4 week old WT litter mate or Il2rb -/- mice. (F) Treg cells slightly downmodulate D25 expression in Tresp cells cultured under Th17 cell conditions. Flow cytometry histograms showing D25 expression of Tresp cells that were stimulated in Th17 cell conditions for indicated time-points (d3 or 6) in the absence (top) or the presence of Treg cells (bottom) Mean fluorescence intensities of D25 are indicated in the parentheses Treg D (ng/ml) (94) (76) (193) (144) d6 Tresp +Treg +- +IL-2 +Treg

5 Fig.S4 G WT Tresp Tresp Tresp +Treg Il2 -/- Tresp Tresp Tresp +Treg IFN-γ H IL-17F d6 I d6 WT Tresp Il2 -/- Tresp No exogenous TGF-β in cultures WT Tresp Il2 -/- Tresp Tresp +Treg Tresp+Treg Tresp +Treg+ αtgf-β Tresp +Treg+ αtgf-β ell count ell count Fig. S4. (G) IL-17F and IFN-γ staining of WT (top) or Il2 -/- (bottom) Tresp cells, without (left column) or with Treg cells stimulated for 3 days. Treg cells up-regulates in Il2 -/- Tresp cells on day 6. (H) WT or Il2 -/- Tresp cells were stimulated in Th17 cell conditions with or without Treg cells for 6 days. (I) WT or Il2 -/- Tresp cells were stimulated in Th17 cell conditions as in () without exogenous TGF-β. α-tgf-β antibody (25 µg/ml) added to block TGF-β. Frequencies of + Tresp cells were analyzed by intracellular staining and flow cytometry following re-stimulation with PM-ionomycin for 4 hours. Flow cytometry histograms show expression of Tresp cells.

6 Fig.S5 P =.1 P =.1 andida + Isotype Pa andida + P61 Pa a D andida+ andida+ % + ** P =.1 % IL-2+ E Sham + PS % IL-17F+ Sham + PS andida + Isotype ** P =.5 andida + P61 % IFN-γ+ Sham + PS andida + P61 andida + Isotype Fig.S5. locking D25 render mice more susceptible to. albicans infection. WT-57L/6 mice received.5mg of isotype antibody or P61 antibody by IP injection followed by oral infection with.albicans. Immunosuppressed mice received cortisone injection. () Weight of the mice in each group was monitored on d3 after infection. () Fungal olony Forming Units (FU)/gm of tongue tissue plated in 1 fold serial dilutions and assessed in triplicate (log scale). The black circle indicates PS+Sham group with zero values outside the log scale. Results represent the mean +/- SEM in () and (). () On day 5 after infection, tongues were harvested and stained with PS to detect. albicans (a), stained pinkish red in color. (Pa) denotes papillae of the tongue. Microscopic images of the slides viewed at 2X magnification. (D) Four days after infection, cells were analyzed for the expression of cytokines in LN by flow cytometry (gated on D4 + cells). The frequencies of cytokine expressing cells are plotted. Each data point represents data from each mouse. (E) ells were processed as in (S5D). Flow cytometric analyses of the expression of and in LN (gated on D4 + cells). These data are from 2 independent experiments showing similar results. andida + Isotype andida + P61.7 <

7 Fig.S6 Sham+ andida+ +PS Tresp +Tcon Tresp +Treg +PS Tresp +Tcon Tresp +Treg FS + D45.1 Tcon recipients Treg recipients d2 d3 d5 d cells in the LN (gated on non Tresp) d D PS+ Tresp + cells in the tongue (d7) Treg recipients Tcon recipients 11.1 Tresp 5.6 andida+ Tresp (Treg) Thy1.1 Fig. S6. ) Treg cells downsize Tresp cells only in a delayed manner in. albicans infected mice. () ells from SPLN (upper panel) and LN (lower panel) were isolated 7 days after the infection as in Fig 6. Flow cytometric contour plots show frequency of D45.1 Tresp cells recovered in the recipients from each group. Mice infected with andida show 1-2 fold increase in the frequency of D45.1+ Tresp cells in LN. These results represent data from 2 independent experiments. () Enrichment of expressing cells in mice reconstituted with Treg cells. Flow cytometric analyses show the percentage of expressing cells among cells excluding D45.1+ Tresp cells in LN isolated on indicated days after the infection.data are pooled from two independent experiments () Histological immunostaining showing expressing cells (arrows) in the tongue in Treg recipients on day 7 after infection (4X magnification). (D) Thy1.1 cells were stimulated for 96 hours under Th17 cell conditions with Treg cells (Tresp+Treg), as indicated. Thy1.1 Tresps were washed and injected in to Thy scid mice, that were subsequently infected with sham control (PS) or andida. Thy1.1 Tresp cells were separated from Treg cells in co-cultures using magnetic sorting before injection. Flow cytometric contour plots of Thy1.1 and intracellular of the Tresp cells are shown (gated on live Thy1.1+ Tresp cells).

8 Fig.S7 PS Tcon + Tresp Treg + Tresp Th17 ID Th ID 15 *** p < % weight *** *** ****** *** % weight 9 w w1 w2 w3 w4 w5 w6 w7 w8 w9 7 5 (days post ID induction) (weeks post ID induction) Th17 cell ID (d42) PS+PS Th17(Tresp+Tcon) Th17(Tresp+Treg) Fig. S7. Delayed suppression of Th17 cells during Th17-ID by Treg cells in vivo. () Delayed suppression of Th17-ID by Treg cells in vivo. Thy scid mice (n = 1) were reconstituted with cells from Thy1.1 T resp + Thy1.2 Tcon or Thy1.1 Tresp+Treg cell co-cultures in which Tresp and Tcon or Treg cells were cultured at 1:1 ratio in Th17 polarizing conditions for 4 days. Three control mice received PS (PS). Mean body weight of the groups of mice normalized to untreated mice represented as percentage of weight loss. () Early and strong amelioration of Th-ID by D4 + D25 + Treg cells. Thy1.2,.-17 scid mice (n=1) received 8 x 1 5 fresh Thy1.1, D25 D45R high D4 + congenic cells co-transferred with Thy1.2 D4 + D25 D45R high D4 + Tcon (D4+ D45R high + Tcon) or 8 x 1 5 fresh Thy1.2 D4 + D25 D45R high D4 + Treg cells (D4 + D45R high +Treg). ontrol mice received PS in both the injections (PS+PS) (n=5). The weight of the mice was monitored for 9 weeks after ID induction. () Th17 cell-id was induced as in. H&E staining of colon sections prepared on day 42 after Th17 cell ID induction. These results represent two independent experiments showing similar results.

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