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.
Supplementary Figure 1. Characterization of basophils after reconstitution of SCID mice
Supplementary figure legends Supplementary Figure 1. Characterization of after reconstitution of SCID mice with CD4 + CD62L + T cells. (A-C) SCID mice (n = 6 / group) were reconstituted with 2 x 1 6 CD4
of whole cell cultures in U-bottomed wells of a 96-well plate are shown. 2
Supplementary online material Supplementary figure legends Supplementary Figure 1 Exposure to T reg cells causes loss of T resp cells in co-cultures. T resp cells were stimulated with CD3+CD28 alone or
D CD8 T cell number (x10 6 )
IFNγ Supplemental Figure 1. CD T cell number (x1 6 ) 18 15 1 9 6 3 CD CD T cells CD6L C CD5 CD T cells CD6L D CD8 T cell number (x1 6 ) 1 8 6 E CD CD8 T cells CD6L F Log(1)CFU/g Feces 1 8 6 p
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Supplemental Figure 1. Furin is efficiently deleted in CD4 + and CD8 + T cells. a, Western blot for furin and actin proteins in CD4cre-fur f/f and fur f/f Th1 cells. Wild-type and furin-deficient CD4 +
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Supplementary Figure 1
Supplementary Figure 1 Identification of IFN-γ-producing CD8 + and CD4 + T cells with naive phenotype by alternative gating and sample-processing strategies. a. Contour 5% probability plots show definition
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doi:1.138/nature1554 a TNF-α + in CD4 + cells [%] 1 GF SPF 6 b IL-1 + in CD4 + cells [%] 5 4 3 2 1 Supplementary Figure 1. Effect of microbiota on cytokine profiles of T cells in GALT. Frequencies of TNF-α
<10. IL-1β IL-6 TNF + _ TGF-β + IL-23
3 ns 25 ns 2 IL-17 (pg/ml) 15 1 ns ns 5 IL-1β IL-6 TNF
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Cover Page The handle http://hdl.handle.net/1887/23854 holds various files of this Leiden University dissertation. Author: Marel, Sander van der Title: Gene and cell therapy based treatment strategies
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Peli1 negatively regulates T-cell activation and prevents autoimmunity Mikyoung Chang 1,*, Wei Jin 1,5,*, Jae-Hoon Chang 1, Yi-chuan Xiao 1, George Brittain 1, Jiayi Yu 1, Xiaofei Zhou 1, Yi-Hong Wang
Supplementary Figure 1. Example of gating strategy
Supplementary Figure 1. Example of gating strategy Legend Supplementary Figure 1: First, gating is performed to include only single cells (singlets) (A) and CD3+ cells (B). After gating on the lymphocyte
T H 1, T H 2 and T H 17 polarization of naïve CD4 + mouse T cells
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SUPPLEMENTARY FIGURE 1
SUPPLEMENTARY FIGURE 1 A LN Cell count (1 ) 1 3 1 CD+ 1 1 CDL lo CD hi 1 CD+FoxP3+ 1 1 1 7 3 3 3 % of cells 9 7 7 % of cells CD+ 3 1 % of cells CDL lo CD hi 1 1 % of CD+ cells CD+FoxP3+ 3 1 % of CD+ T
Pearson r = P (one-tailed) = n = 9
8F4-Specific Lysis, % 1 UPN1 UPN3 8 UPN7 6 Pearson r =.69 UPN2 UPN5 P (one-tailed) =.192 4 UPN8 n = 9 2 UPN9 UPN4 UPN6 5 1 15 2 25 8 8F4, % Max MFI Supplementary Figure S1. AML samples UPN1-UPN9 show variable
Supplementary Fig. 1 p38 MAPK negatively regulates DC differentiation. (a) Western blot analysis of p38 isoform expression in BM cells, immature DCs
Supplementary Fig. 1 p38 MAPK negatively regulates DC differentiation. (a) Western blot analysis of p38 isoform expression in BM cells, immature DCs (idcs) and mature DCs (mdcs). A myeloma cell line expressing
BD CBA on the BD Accuri C6: Bringing Multiplexed Cytokine Detection to the Benchtop
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Supplementary Information
Supplementary Information Distinct bone marrow-derived and tissue resident macrophage lineages proliferate at key stages during inflammation. 1 Luke C. Davies, 1 Marcela Rosas, 2 Stephen J. Jenkins, 1
Suppl Video: Tumor cells (green) and monocytes (white) are seeded on a confluent endothelial
Supplementary Information Häuselmann et al. Monocyte induction of E-selectin-mediated endothelial activation releases VE-cadherin junctions to promote tumor cell extravasation in the metastasis cascade
Supplementary Materials for
www.sciencesignaling.org/cgi/content/full/6/305/ra106/dc1 Supplementary Materials for Controlling Long-Term Signaling: Receptor Dynamics Determine Attenuation and Refractory Behavior of the TGF-β Pathway
Supplementary Figure 1. Ex vivo IFNγ production by Tregs. Nature Medicine doi: /nm % CD127. Empty SSC 98.79% CD25 CD45RA.
SSC CD25 1.8% CD127 Empty 98.79% FSC CD45RA CD45RA Foxp3 %IFNγ + cells 4 3 2 1 + IL-12 P =.3 IFNγ p=.9 %IL-4+ cells 3 2 1 IL-4 P =.4 c %IL-1 + cells IFNγ 4 3 2 1 Control Foxp3 IL-1 P =.41.64 4.76 MS 2.96
Supplemental Table I.
Supplemental Table I Male / Mean ± SEM n Mean ± SEM n Body weight, g 29.2±0.4 17 29.7±0.5 17 Total cholesterol, mg/dl 534.0±30.8 17 561.6±26.1 17 HDL-cholesterol, mg/dl 9.6±0.8 17 10.1±0.7 17 Triglycerides,
Supplementary Figure 1
Supplementary Figure 1 Expression of apoptosis-related genes in tumor T reg cells. (a) Identification of FOXP3 T reg cells by FACS. CD45 + cells were gated as enriched lymphoid cell populations with low-granularity.
Krishnamoorthy et al.,
Krishnamoorthy et al., c d e ND ND Supplementary Figure 1 RSV-induces inflammation even in the asence of allergen. Tolerized pups were either infected with RSV or not. The mice were sacrificed a week following
Type of file: PDF Title of file for HTML: Supplementary Information Description: Supplementary Figures
Type of file: PDF Title of file for HTML: Supplementary Information Description: Supplementary Figures Type of file: MOV Title of file for HTML: Supplementary Movie 1 Description: NLRP3 is moving along
Supplementary Figure 1
d f a IL7 b IL GATA RORγt h HDM IL IL7 PBS Ilra R7 PBS HDM Ilra R7 HDM Foxp Foxp Ilra R7 HDM HDM Ilra R7 HDM. 9..79. CD + FOXP + T reg cell CD + FOXP T conv cell PBS Ilra R7 PBS HDM Ilra R7 HDM CD + FOXP
Supplementary Information:
Supplementary Information: Follicular regulatory T cells with Bcl6 expression suppress germinal center reactions by Yeonseok Chung, Shinya Tanaka, Fuliang Chu, Roza Nurieva, Gustavo J. Martinez, Seema
Control GST GST-RAP. α2-mg. 170 kda. b-actin. 42 kda LRP-1
% of max Supplementary Figure 1 Control GST GST-RP 17 kda α2-mg 42 kda b-actin Gate: CD11c+ (DCs) Gate: F4/8+ (Mfs) IgG Cd11cCre + Lrp1 fl/fl LRP-1 Supplementary figure 1. () MDCs were pretreated with
Canberra, Australia). CD11c-DTR-OVA-GFP (B6.CD11c-OVA), B6.luc + and. Cancer Research Center, Germany). B6 or BALB/c.FoxP3-DTR-GFP mice were
Supplemental Materials and Methods Mice Female C57BL/6 (B6, I-E null, H-2 b ), BALB/c (H-2 d ) + ), FVB/N (H-2 q, I-E null, CD45.1 + ), and B6D2F1 (H-2 b/d ) mice were purchased from the Animal Resources
Figure S1. PMVs from THP-1 cells expose phosphatidylserine and carry actin. A) Flow
SUPPLEMENTARY DATA Supplementary Figure Legends Figure S1. PMVs from THP-1 cells expose phosphatidylserine and carry actin. A) Flow cytometry analysis of PMVs labelled with annexin-v-pe (Guava technologies)
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DOI: 1.138/ncb3355 a S1A8 + cells/ total.1.8.6.4.2 b S1A8/?-Actin c % T-cell proliferation 3 25 2 15 1 5 T cells Supplementary Figure 1 Inter-tumoral heterogeneity of MDSC accumulation in mammary tumor
Nature Immunology: doi: /ni Supplementary Figure 1
Supplementary Figure 1 Fatty acid oxidation is emphasized in 1 macrophages compared with that in macrophages. Gene expression of mitochondrial OXPHOS (Atp5j, Cox4i1, Uqcrc1/2, Ndufs1, Sdhb) and β-oxidation
effect on the upregulation of these cell surface markers. The mean peak fluorescence intensity
SUPPLEMENTARY FIGURE 1 Supplementary Figure 1 ASIC1 disruption or blockade does not effect in vitro and in vivo antigen-presenting cell activation. (a) Flow cytometric analysis of cell surface molecules
Rapamycin-treated human endothelial cells preferentially activate allogeneic regulatory T cells
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Tbk1-TKO! DN cells (%)! 15! 10!
a! T Cells! TKO! B Cells! TKO! b! CD4! 8.9 85.2 3.4 2.88 CD8! Tbk1-TKO! 1.1 84.8 2.51 2.54 c! DN cells (%)! 4 3 2 1 DP cells (%)! 9 8 7 6 CD4 + SP cells (%)! 5 4 3 2 1 5 TKO! TKO! TKO! TKO! 15 1 5 CD8
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doi: 1.138/nature775 4 O.D. (595-655) 3 1 -ζ no antibody isotype ctrl Plated Soluble 1F6 397 7H11 Supplementary Figure 1 Soluble and plated anti- Abs induce -! signalling. B3Z cells stably expressing!
Supplementary Figure 1. Double-staining immunofluorescence analysis of invasive colon and breast cancers. Specimens from invasive ductal breast
Supplementary Figure 1. Double-staining immunofluorescence analysis of invasive colon and breast cancers. Specimens from invasive ductal breast carcinoma (a) and colon adenocarcinoma (b) were staining
Contrasting roles for all-trans retinoic acid in TGF- mediated induction of Foxp3 and Il10 genes in developing regulatory T cells
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Reviewers' comments: Reviewer #1 (Remarks to the Author):
Reviewers' comments: Reviewer #1 (Remarks to the Author): The manuscript by Sun et al., provide data showing that short-chain fatty acids produced by intestinal microbiota act through GPR43 to induced
Nature Immunology: doi: /ni Supplementary Figure 1. Id2 and Id3 define polyclonal T H 1 and T FH cell subsets.
Supplementary Figure 1 Id2 and Id3 define polyclonal T H 1 and T FH cell subsets. Id2 YFP/+ (a) or Id3 GFP/+ (b) mice were analyzed 7 days after LCMV infection. T H 1 (SLAM + CXCR5 or CXCR5 PD-1 ), T FH
Nature Medicine: doi: /nm.2109
HIV 1 Infects Multipotent Progenitor Cells Causing Cell Death and Establishing Latent Cellular Reservoirs Christoph C. Carter, Adewunmi Onafuwa Nuga, Lucy A. M c Namara, James Riddell IV, Dale Bixby, Michael
for six pairs of mice. (b) Representative FACS analysis of absolute number of T cells (CD4 + and
SUPPLEMENTARY DATA Supplementary Figure 1: Peripheral lymphoid organs of SMAR1 -/- mice have an effector memory phenotype. (a) Lymphocytes collected from MLNs and Peyer s patches (PPs) of WT and SMAR1