1. IL-17 Gene Ablation Does Not Impact Treg-Mediated Suppression of Graft-Versus-Host Disease after Bone Marrow Transplantation 
Lucrezia Colonna, Mareike Florek, Dennis B. Leveson-Gower, Emanuela I. Sega, Jeanette Baker, Aaron T. Smith, Robert S. Negrin 
Biology of Blood and Marrow Transplantation 
Volume 19, Issue 11, Pages (November 2013)
DOI: /j.bbmt
Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions

2. Figure 1
IL-17 KO Tregs have mildly enhanced suppressive ability on the proliferation of allogeneic T cells in vitro. Tregs from WT or IL-17 KO donors were compared for their suppressive ability in an MLR in vitro. Irradiated total splenocytes from Balb/c mice (d/d haplotype) were used as stimulators (S) and CD4+ and CD8+ WT C57BL/6 mice (b/b haplotype) were used as responders (R) at a fixed 2:1 S/R ratio. At the same time, Treg isolated from either WT or IL-17 KO donors (both on a C57BL/6 background, b/b haplotype) were added at 1:1, 2:1, and 10:1 Tcon/Treg ratios. After 4 days of co-incubation, the cells were pulsed with 1 μCi [3H]-thymidine per well for 16 to 18 hours and Tcon proliferation was quantified by measuring [3H]-thymidine incorporation. The bar graphs are representative of 2 different MLRs combined. Probability values are as indicated, representing differences in suppressive function of the 2 populations of Treg.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions

3. Figure 2
WT and IL-17 KO Treg have a comparable GVHD suppressive ability in vivo. (A) Survival curves of recipient mice transplanted with BM alone (BM), BM on day 0 + Tcon on day 2 (Tcon group), and BM cotransplanted with either WT or IL-17 KO Treg, followed by Tcon transplantation 2 days later (Treg group). Both 1:1 and 4:1 Tcon/Treg ratios are shown. Data are representative of 2 independent experiments (BM n = 9, Tcon n = 10; WT Tcon/Treg 1:1 n = 6; WT Tcon/Treg 4:1 n = 7; IL-17 KO Tcon/Treg 1:1 n = 7; IL-17 KO Treg 4:1 n = 7). Statistical differences between survival curves were calculated with a log rank test. For the 1:1 Tcon/Treg ratio: Tcon vs. WT Treg P = .05; Tcon vs. IL-17 KO Treg P = .02; BM vs. Tcon P = .0002; WT Treg vs. IL-17 KO Treg P = NS; BM vs. WT Treg P = .04; BM vs. IL-17 KO Treg P = .08. For the 4:1 Tcon/Treg ratio: BM vs. Tcon P = .0002; Tcon vs. WT Treg P = .06; Tcon vs. IL-17 KO Treg P = NS; WT Treg vs. IL-17KO Treg P = NS; BM vs. WT Treg P = .04; BM vs. IL-17 KO Treg P = .08. (B) Weight curves of the same animals as in (A). For consistency, the last measurement of each diseased animal was kept in the analysis until the last mouse in each given group expired Statistical differences between groups were calculated with a 2-tailed unpaired Student t-test. For the 1:1 Tcon/Treg ratio: BM vs. Tcon P < .0001; Tcon vs. WT Treg P < .0001; Tcon vs. IL-17 KO Treg P < .0001; BM vs. WT Treg P < .0001; BM vs. IL-17 KO Treg P < For the 4:1 Tcon/Treg ratio: BM vs. Tcon P < .0001; Tcon vs. WT Treg P = .13; Tcon vs. IL-17 KO Treg P = .03; WT Treg vs. IL-17 KO Treg P = .34; BM vs. WT Treg P < .0001; BM vs. IL-17 KO Treg P < (C) In vivo BLI images of representative animals showing the luminescent signal (in photons per second) derived from the allogeneic luc+ Tcon during acute GVHD, in the presence or absence of WT and IL-17-KO Tregs. Both 1:1 and 4:1 Tcon/Treg ratios are shown. (D) Quantification of the in vivo BLI data from luc+ Tcon as a measure of ongoing GVHD. Shown are 1:1 and 4:1 Tcon/Treg ratios. Data are representative of 2 independent experiments (BM n = 9; Tcon n = 10; WT Tcon/Treg 1:1 n = 6; IL-17 Tcon/Treg 1:1 n = 7; WT Tcon/Treg 4:1 n = 7; IL-17 KO Tcon/Treg 4:1 n = 7). Statistical differences between curves were calculated with a 2-tailed Student t-test. For the 1:1 Tcon/Treg ratio: BM vs. Tcon P = .05; WT Treg vs. IL-17 KO Treg P = .84; Tcon vs. WT Treg P = .19; Tcon vs. IL-17 KO Treg P = .14; BM vs. WT Treg P = .19; BM vs. IL-17 KO Treg P = .12. For the 4:1 Tcon/Treg ratio: BM vs. Tcon P = .05; WT Treg vs. IL-17 KO Treg P = .8; Tcon vs. WT Treg P = .72; Tcon vs. IL-17 KO Treg P = .54; BM vs. WT Treg P = .08; BM vs. IL-17 KO Treg P = .09.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions

4. Figure 3
IL-1R signaling blockade does not affect the GVHD suppressive capacity of Treg in vivo. (A) Survival curves of recipient mice transplanted with BM alone (BM), BM on day 0 + Tcon on day 2 (Tcon group), and BM cotransplanted with either WT or IL-1R KO Treg, followed by Tcon transplantation 2 days later (Treg group). Both 1:1 and 4:1 Tcon/Treg ratios are shown. Data are representative of 2 independent experiments (BM n = 10; Tcon n = 10; WT Tcon/Treg 1:1 n = 7; IL-1R KO Tcon/Treg 1:1 n = 7; WT Tcon/Treg 4:1 n = 6; IL-1R KO Tcon/Treg 4:1 n = 9). Statistical differences between curves were calculated by the log rank test. For the 1:1 Tcon/Treg ratio: BM vs. Tcon P = .0002; Tcon vs. WT Treg P = .008; Tcon vs. IL-1R KO Treg P = .004; WT Treg vs. IL-17KO Treg P = .96. For the 4:1 Tcon/Treg ratio: BM vs. Tcon P = .0002; Tcon vs. WT Treg P = .03; Tcon vs. IL-1R KO Treg P = .04; WT Treg vs. IL-17KO Treg P = .88. (B) Weight curves of the same animals as in (A). For consistency, the last weight measurement of each diseased animal was kept in the analysis until the last mouse in the group expired Statistical differences between curves were calculated with a 2-tailed Student t-test. For the 1:1 Tcon/Treg ratio: BM vs. Tcon P < ; Tcon vs. WT Treg P < .0001; Tcon vs. IL-1R KO Treg P < .0001; WT Treg vs. IL-1R KO Treg P = .43; BM vs. WT Treg P < .0001; BM vs. IL-1R KO Treg P < For the 4:1 Tcon/Treg ratio: BM vs. Tcon P < .0001; Tcon vs. WT Treg P = .001; Tcon vs. IL-1R KO Treg P = .19; WT Treg vs. IL-1R KO Treg P = .04; BM vs. WT Treg P < .0001; BM vs. IL-1R KO Treg P < (C) In vivo BLI images of representative animals during acute GVHD in the presence or absence of WT and IL-1R KO Treg. Both 1:1 and 4:1 Tcon/Treg ratios are shown. (D) Quantification of the in vivo BLI data derived from luc+ Tcon as a measure of ongoing GVHD. Both Tcon/Treg ratios of 1:1 and 4:1 are shown. Data are representative of 2 independent experiments (BM n = 10; Tcon n = 10; WT Tcon/Treg 1:1 n = 7; IL-1R KO Tcon/Treg 1:1 n = 7; WT Tcon/Treg 4:1 n = 6; IL-1R KO Tcon/Treg 4:1 n = 9). Probability values were calculated with a 2-tailed unpaired Student t-test. For the 1:1 Tcon/Treg ratio: BM vs. Tcon P = .02; Tcon vs. WT Treg P = .05; Tcon vs. IL-1R KO Treg P = .06; WT Treg vs. IL-1R KO Treg P = .72; BM vs. WT Treg P = .1; BM vs. IL-1R KO Treg P = .11. For the 4:1 Tcon/Treg ratio: BM vs. Tcon P = .02; Tcon vs. WT Treg P = .27; Tcon vs. IL-1R KO Treg P = .26; WT Treg vs. IL-1R KO Treg P = .99; BM vs. WT Treg P = .08; BM vs. IL-1R KO Treg P = .07.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions

5. Figure 4
WT Treg do not produce IL-17 after GVHD induction in vivo. (A) TCD-BM cells and Treg were cotransplanted on day 0 after lethal irradiation, followed by Tcon 2 days later (d +2). The animals were sacrificed 7 and 14 days after BM transplantation (5 and 12 days post-GVHD induction), and lymphoid organs were subjected to intracellular IL-17 and Foxp3 staining for FACS analysis. Treg were gated as live CD4+CD45.1−Thy1.2+H-2b+, whereas Tcon were identified as CD4+CD45.1+Thy1.1+H-2b+. Donor BM cells and residual recipient hematopoietic cells (H-2d+) were gated out as CD45.1−Thy.1.1+H-2b+ and CD45.1−Thy1.1−H-2d+ cells, respectively. (B) Quantification of the intracellular Foxp3 staining data presented in (A). Live CD4+CD45.1−Thy1.2+H-2b+ Treg were reisolated 7 and 14 days after in vivo transfer and reanalyzed for intracellular Foxp3 expression. Data are representative of 2 independent experiments. Day 7: n = 7 in all groups. Day 14: spleen n = 7; mLN = mesenteric lymph nodes: n = 5; pLN = peripheral lymph nodes: n = 9. Statistical differences between groups were calculated with a 2-tail unpaired Student t-test. Day 7: spleen vs. mLNs P = .002; mLNs vs. pLNs P = NS; spleen vs. pLNs P = NS. Day 14: spleen vs. mLNs P = NS; mLNs vs. pLNs P < .0001; spleen vs. pLNs P = (C) Quantification of the Treg intracellular IL-17 staining data presented in (A). CD4+CD45.1−Thy1.2+H-2b+ transferred Treg were re -isolated 7 and 14 days after in vivo transfer and analyzed for intracellular IL-17 production. IL-17 production by both Foxp3+ and Foxp3- reisolated Treg is shown. Statistical differences between groups were calculated with a 2-tail unpaired Student t-test. Day 7: spleen vs. mLNs P = NS; mLNs vs. pLNs P = NS; spleen vs. pLNs P = NS. Day 14: spleen vs. mLNs P = .04; mLNs vs. pLNs P = .1; spleen vs. pLNs P = NS.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions

6. Supplemental Figure 1
Comparable expression of Treg markers on WT and IL-17 KO Tregs. Freshly isolated Tregs from WT and IL-17 KO donors were compared for the expression of the Treg markers CD25 and intracellular Foxp3 by FACS analysis. Foxp3 and CD25 expression were comparable in WT and IL-17KO Tregs and the purity of the transferred Tregs was confirmed to be >98% for both strains.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions

7. Supplemental Figure 2
IL-17 production by CD4+ Tcons. Quantification of the Tcon intracellular IL-17 staining data presented in Figure 4A. CD4+CD45.1+Thy1.1+H-2b+ transferred Tcons were reisolated 5 and 12 days after in vivo transfer and analyzed for intracellular IL-17 production. Data are representative of 2 independent experiments. Day 7: n = 7 in all groups. Day 14: spleen n = 9; mLN = mesenteric lymph nodes: n = 4; pLN = peripheral lymph nodes: n = 4. Statistical differences between groups were calculated with a 2-tail unpaired Student t-test. Day 7 vs. day 14 spleen: P = NS; day 7 vs. day 14 mLNs: P = .005; day 7 vs. day 14 pLNs: P =
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions