CPM (x 10-3 ) Tregs +Teffs. Tregs alone ICOS CLTA-4
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1 A 2,5 B 4 Number of cells (x 1-6 ) 2, 1,5 1, 5 CPM (x 1-3 ) Days of culture 1/1 1/2 1/4 1/8 1/16 1/32 Treg/Teff ratio C alone alone alone alone CD25 FoxP3 GITR CD44 ICOS CLTA-4 CD127 CD13 Supplemental Figure 1. In vitro Treg expansion and phenotype of expanded boosted. (A) In vitro expansion of purified HA- stimulated by HA-pulsed DCs. Viable cell numbers were determined after trypan blue staining. (B) The in vitro suppressive activity of HA- was determined after 3 weeks of culture. HA-Teffs (1 5 cells) stimulated by DCs (2 x 1 4 cells) and anti-cd3 mab (1 µg/ml of 2C11) were co-cultured with various numbers of in vitro expanded HA- for 68 hours in flat bottom 96 well plates. Proliferation was measured by thymidine DNA incorporation during the last 12 hours. (C) Ins-HA mice were injected with -labeled Thy in vitro expanded HA- alone or co-transferred with pre-activated HA-Teffs. Representative expression out of 3 independent experiments of the indicated markers on donor (CD4 + Thy ) in PLNs 7 days after cell injection. Horizontal dashed lines delineate positive staining.
2 <.1 + Teffs Thy FoxP3 CD4 Supplemental figure 2. High numbers of divided islet-specific in the pancreas of mice co-transferred with islet-specific Teffs and. Ins-HA mice were injected with -labeled Thy-1.1+ expanded HA- alone () or with pre-activated HA-Teffs ( ). Representative dot plot of CD4 vs Thy-1.1 among total lymphocytes (left) and vs FOXP3 within CD4+Thy-1.1+ cells (right) in the pancreatic islets 7 days after transfer. Data were representative of 3 independent experiments with 2 to 3 mice per group.
3 A 5% 3% f-teffs a-teffs Relative cell number IL-2 18% IFN-γ 2% B Diabetes incidence (%) f- (n=17) f-+ f-teffs (n=7) f-+ a-teffs (n=17) Days after first injection Supplemental Figure 3. Pre-activated, but not freshly-purified HA-Teffs, were strongly re-activated in PLNs of ins-ha mice and induced long-term protection from diabetes challenge when combined with freshly purified. (A) Four days after injection of -labeled Thy freshly purified HA-Teffs (f-teffs) or pre-activated HA-Teffs (a-teffs), ins-ha mice were sacrificed for analyses of profile, IL-2 and IFN-γ production among CD4 + Thy cells in PLNs. Representative data out of 4 experiments. (B) Ins-HA mice were transferred with freshly purified HA- alone or co-injected with freshly purified HA- Teffs or pre-activated HA-Teffs. Three weeks later (arrow), mice were challenged with pre-activated HA-Teffs, to test their sensitivity to diabetes. Data were pooled from 5 independent experiments.
4 A B 15 2x1 6 2x1 6 FoxP3 Donor (X1-3 ) 1 5 2x1 6 2x1 6 Supplemental Figure 4. Numbers of boosted and non-boosted 3 weeks after transfer. Ins-HA mice were transferred with 2 x 1 6 -labeled expanded Thy HA- alone, or with 2 x 1 6 labeled expanded Thy HA- and 2 x 1 6 pre-activated HA-Teffs. Mice were sacrificed either at day 19 or were challenged with pre-activated HA-Teffs at day 19 and sacrificed 4 days after for flow cytometry analyses in PLNs. (A) Representative profile of FoxP3 staining vs dilution among donor- (CD4 + Thy FoxP3 + ) at day 19. (B) Absolute number of donor (CD4 + Thy FoxP3 + cells) before (, ) and after (, ) the challenge with HA-Teffs. Each symbol represents an individual mouse from 3 independent experiments.
5 Divided donor (x 1-3 ) *** ** alone + IL-2 +/+ Teffs + IL-2 -/- Teffs Supplemental Figure 5. IL-2 deficient Teffs strongly boost freshly purified. Ins-HA mice were transferred with labeled Thy freshly purified HA- alone ( alone) or co-injected with IL-2 +/+ or IL-2 -/- pre-activated HA-Teffs. Divided HA-Treg numbers ( dim CD4 + Thy cells) were quantified in PLNs 1 days later. Each dot represents individual mice from at least 2 independent experiments. ***p<.1; **p<.1.
6 A Time after IL-2 or PBS administration B 3h 12h 24h 48h 1 PBS PBS Low IL-2 High IL-2 Count % p-stat-5 + among Low IL-2 High IL-2 P-STAT Hours after IL-2 or PBS administration Supplemental Figure 6. Persistence of p-stat-5 signal after IL-2 administration. Percentage of p-stat- 5+ cells among FoxP3+ cells were determined by flow cytometry various time after one i.p. injection of low (25, IU) or high (25, IU) dose of human IL-2 in BALB/c mice. (A) Representative STAT-5 staining among (CD4 + FoxP3 + ). Bars delimitate positive staining. (B) Percentage of p-stat-5+ cells among (CD4 + FoxP3 + ).
7 alone + Teffs Nrp1 Sca-1 Supplemental Figure 7. HA- boosted in vivo acquired phenotypic modifications as in vitro. Ins-HA mice were transferred with -labeled Thy expanded HA- alone or were co-transferred with pre-activated HA-Teffs. Expression of Sca-1 and Nrp1 on donor (CD4 + Thy FoxP3 + ) was assessed 1 days later. Representative data of 2 independent experiments.
8 Supplemental Figure 8. Boosted upregulated the expression of molecules of the alternative NF-kB pathway. Graphic representation of the NF-kB signaling pathway downstream of the lymphotoxin-ß receptor. Genes are represented as nodes, and the biological relationship between two nodes is represented as an edge (line). The node color indicates the degree of up- (red) or down- (green) regulation of gene expression in boosted versus non-boosted generated from the transcriptome data and analyzed using the Ingenuity software.
9 Divided donor (x 1-3 ) ** * P<.7 +anti-tnf Supplemental Figure 9. The Teff Treg boost was inhibited by an anti-tnf-a mab. Four to 6 week-old NOD mice were transferred with freshly isolated -labelled CD45.2+ BDC2.5- alone or with pre-activated CD45.1+ BDC2.5-Teffs with or without an anti-tnf-α mab treatment. Absolute numbers of divided donor ( dim CD4 + CD FoxP3 + cells) were quantified in PLNs 5-6 days after transfer. Each dot represents individual mice pooled from 3 independent experiments. *p<.5, **p<.1.
10 Teffs Inflammatory factors Chronic inflammation or auto-immune disease + Boost Suppression Inflammation Homeostatic equilibrium and tolerance Dynamic equilibrium Progressive resolution 5 Chronic inflammation or auto-immune disease Supplemental Figure 1. A new model of regulation in inflammation and auto-immunity. At the steady state (1), rare auto-reactive Teffs and co-exist in lymphoid tissues, generating a homeostatic Teff/Treg equilibrium and tolerance. During sustained inflammation (2), Teffs are strongly activated. These cells thus produce or induce factors favoring a strong Teff Treg boost (3), leading to subsequent Treg expansion. This expansion strengthens Treg-mediated suppression exerted on Teffs (4). This inhibition of Teff activation probably reduces Treg boost factors, eventually leading to a progressive resolution and a return to the basal homeostatic Teff/Treg equilibrium. In some conditions, the response may progress toward chronic inflammation or autoimmune disease (5). This may be due to either ineffective Treg-mediated suppression or a defect of the Teff Treg boost.
11 Ingenuity Canonical Pathways -Log(P-value) Ratio Molecules Glycolysis/Gluconeogenesis 3,99E+ 4,96E-2 TPI1, ACSS1, PFKL, ENO3, PFKP, HK2, GALK1 Fructose and Mannose Metabolism 3,92E+ 3,6E-2 TPI1, PFKL, PFKP, HK2, GALK1 Galactose Metabolism 2,8E+ 3,57E-2 PFKL, PFKP, HK2, GALK1 CD4 Signaling 2,67E+ 7,35E-2 NFKBIA, FOS, ICAM1, TRAF5, LTA CCR5 Signaling in Macrophages 2,64E+ 5,81E-2 CCL4, FOS, CCL5, CCL3, GNG12 CD27 Signaling in Lymphocytes 2,47E+ 8,16E-2 NFKBIA, FOS, TRAF5, CD7 2,12E+ 7,2E-2 LTB, NFKBIA, TRAF5, LTA Antigen Presentation Pathway 2,8E+ 7,69E-2 CALR, HLA-DQB2, HLA-DRA Activation of IRF by Cytosolic Pattern Recognition Receptors 1,86E+ 5,48E-2 ZBP1, NFKBIA, ISG15 (includes EG:9636), LTA Chemokine Signaling 1,69E+ 5,19E-2 CCL4, CXCR4, FOS, CCL5 p53 Signaling 1,43E+ 4,6E-2 SERPINE2, GADD45G, GADD45B, E2F1 Glycosphingolipid Biosynthesis - Neolactoseries 1,4E+ 2,99E-2 ST8SIA4, ST3GAL6 Eicosanoid Signaling 1,32E+ 3,57E-2 PTGER2, ALOX5AP, AKR1C3 Glucocorticoid Receptor Signaling 1,21E+ 2,54E-2 ADRB2, NFKBIA, FOS, ICAM1, CCL5, CCL3, ANXA1 4-1BB Signaling in T Lymphocytes 1,11E+ 5,41E-2 NFKBIA, TNFRSF9 Aryl Hydrocarbon Receptor Signaling 8,53E-1 2,58E-2 FOS, MCM7, TGM2, E2F1 IL-6 Signaling 8,48E-1 3,12E-2 NFKBIA, FOS, IL6R CXCR4 Signaling 8,5E-1 2,44E-2 CXCR4, FOS, EGR1, GNG12 IL-2 Signaling 7,47E-1 3,57E-2 IL2RB, FOS G-Protein Coupled Receptor Signaling 5,E-1 1,86E-2 ADRB2, P2RY1, NFKBIA, DUSP4 4,9E-1 2,8E-2 NFKBIA, TRAF5, LTA IL-8 Signaling 3,79E-1 2,19E-2 ICAM1, MMP9, PLD4, GNG12 Role of NFAT in Regulation of the Immune Response 3,79E-1 1,61E-2 NFKBIA, FOS, GNG12 Cell Cycle: G2/M DNA Damage Checkpoint Regulation 3,62E-1 2,33E-2 CDC25B Supplemental Table 1. Top canonical pathways differently used by boosted. Gene array data were processed using Ingenuity pathway analysis software. The table shows the canonical pathways, identified from the Ingenuity canonical pathways library, that were most significant to the data set according to the following 2 criteria: 1) Fischer s exact test was used to calculate a p-value determining the probability that the association between the genes in the dataset and the canonical pathway is explained by chance alone ( Log(P-value) ). 2) A ratio of the number of genes from the data set that map to the pathway divided by the total number of genes that map to the canonical pathway is displayed ( Ratio ). Molecules show the genes from the data set included in the corresponding canonical pathway
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