Journal: Cell reports

Article Title: JAK1 signaling in dendritic cells promotes peripheral tolerance in autoimmunity through PD-L1-mediated regulatory T cell induction.

doi: 10.1016/j.celrep.2022.110420

Figure Lengend Snippet: Figure 1. Effects of filgotinib on DC responses (A) JAK1 mean fluorescence intensity (MFI) of BM-DCs stimulated with LPS over 72 h and representative histograms; n = 4. (B) JAK1 MFI of BM-DCs stimulated with the indicated TLR agonists for 24 h; n = 4 per group. (C) Cytokine profile of LPS-stimulated, filgotinib-treated BM-DCs after 24 h. Data are represented as log2 fold change compared to vehicle control; n = 4. (D) In vitro T cell proliferation assay with naive CD62L+CD44, CFSE-labeled OT-II CD4+ T cells co-cultured with vehicle- or filgotinib-treated OVA-loaded BM- DCs. Percentage of CFSElow CD4+ T cells and percentage of CD4 T cells in indicated division cycle; n = 4. (E) Representative CFSE profile of data shown in (D). (F) CD80, CD86, and MHC class II MFI of vehicle- or filgotinib-treated, LPS-stimulated BM-DCs after 24 h; n = 4. (G) Representative histograms of data shown in (F). (H) CD80, CD86, and MHC class II MFI of vehicle-, ruxolitinib-, or tofacitinib-treated, LPS-stimulated BM-DCs after 24 h; n = 4. The data are represented as means ± SEMs of biologically independent samples. **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired t test (A, D, and F), 1-way ANOVA (A and H), and 2-way ANOVA (D).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Myelin oligodendrocyte glycoprotein (MOG)35–55 Charitè Berlin NA Incomplete Freund’s adjuvant Sigma-Aldrich Cat# F5506 Mycobacterium tuberculosis H37Ra BD Cat# 231141 Pertussis toxin List Biological Laboratories Cat# 181 recombinant murine GM-CSF R&D Cat# 415-ML-050 recombinant murine IL-4 Miltenyi Biotec Cat# 130-097-761 recombinant murine IFN-ɣ Miltenyi Biotec Cat# 130-105-774 recombinant murine IL-10 R&D Cat# 417-ML-005 Lipopolysaccharide ultra pure InvivoGen Cat# tlrl-pelps Filgotinib TargetMol Cat# T1929 Stattic MedChem Express Cat# HY-13818 Fludarabine MedChem Express Cat# HY-B0069 Ruxolitinib Cayman Chemical Cat# 11609-5 Tofacitinib MedChem Express Cat# HY-40354 OVA323–399 Bachem Cat# H-5532 CD11c MACS Micro Bead System Miltenyi Biotec Cat# 130-125-835 Naive CD4+ T Cell Isolation Kit, mouse Miltenyi Biotec Cat# 130-104-453 CFSE Thermo Fisher Scientific Cat# 65-0850-84 Recombinant Mouse TGF-beta 1 R&D Cat# 7666-MB-005/CF Animal-Free Recombinant Human IL-2 Peprotech Cat# AF-200-02 Recombinant mouse PD-L1 Fc-Fusion protein Kerafast Cat# Pr00112-1.9 Recombinant human IgG1 Fc control protein R&D Cat# 110-HG-100 Adult brain dissociation kit Miltenyi Biotec Cat# 130-107-677 Fixable Viability Dye eFluorTM 780 Thermo Fisher Scientific Cat# 65-0865-14 SYTOXTM AADvancedTM Thermo Fisher Scientific Cat# S10274 FoxP3/Transcription factor staining buffer set Thermo Fisher Scientific Cat# 00-5523-00 eBioscienceTM Intracellular fixation & permeabilization buffer set Thermo Fisher Scientific Cat# 88-8824-00 True-Phos Buffer Set BioLegend Cat# 425401 Ionomycin Sigma Aldrich Cat# I0634 PMA Sigma Aldrich Cat# P1585 Brefeldin A solution Thermo Fisher Scientific Cat# 00-4506-51 High-Capacity cDNA Reverse Transcription Kit Thermo Fisher Scientific Cat# 4368814 TaqManTM Gene Expression Master Mix Thermo Fisher Scientific Cat# 4369016 Luna Master Mix New England BioLabs Cat# M3003E Critical commercial assays LEGENDplexTM Mouse Inflammation Panel BioLegend Cat# 740446 LEGENDplexTM Mouse Th Panel BioLegend Cat# 740741 Experimental models: Organisms/strains Mouse: Jak1tm1a(EUCOMM)Hmgu EUMMCR MGI:4456720 Mouse: ROSA26::FLPe knock in; B6.129S4-Gt(ROSA)26Sortm1(FLP1)Dym/RainJ The Jackson Laboratory Cat# JAX:009086, RRID:IMSR_JAX:009086 Mouse: CD11c cre; B6.Cg-Tg(Itgax-cre)11Reiz/J mice The Jackson Laboratory Cat# JAX:008068, RRID:IMSR_JAX:008068 (Continued on next page) Cell Reports 38, 110420, February 22, 2022 e3

Techniques: Control, In Vitro, Proliferation Assay, Labeling, Cell Culture

Journal: Cell reports

Article Title: JAK1 signaling in dendritic cells promotes peripheral tolerance in autoimmunity through PD-L1-mediated regulatory T cell induction.

doi: 10.1016/j.celrep.2022.110420

Figure Lengend Snippet: Figure 2. Effect of genetic JAK1 deficiency on DC responses (A) Scheme for the conversion to the conditional Jak1flallele by Flp-mediated deletion. CD11c cre-mediated recombination excises loxP-flanked exon 3 of the Jak1 gene. (B) Percentage of splenic cDC1 (CD11c+) and cDC2s (CD11c+CD11b+) of healthy WT and Jak1DDC animals; n = 7 per genotype. (C) qRT-PCR of Jak1 in isolated splenic DCs of WT and Jak1DDC mice; n = 4. (D) JAK1 MFI of differentiated immature WT and JAK1-deficient BM-DCs; n = 4. (E) CD80, CD86, and MHC class II MFI of immature WT and JAK1-deficient BM-DCs; n = 4. (F) Representative histograms of data shown in (E). (G) Cytokine profile of WT and JAK1-deficient BM-DCs after 24 h of LPS stimulation. Data are represented as log2 fold change compared to WT control; n = 4. (H) In vitro T cell proliferation assay with naive CD62L+CD44, CFSE-labeled OT-II CD4+ T-cells co-cultured with WT or JAK1-deficient OVA-loaded BM-DCs. CFSE dilution is represented by the percentage of CFSElow CD4+ T cells and the percentage of CD4+ T cells in the indicated division cycle; n = 4. (I) Representative CFSE profile of data shown (H). (J) CD80, CD86, and MHC class II MFI of WT and JAK1-deficient BM-DCs after 24 h of LPS stimulation; n = 4. (K) Representative histograms of data shown in (J). The data are represented as means ± SEMs of biologically independent samples or animals. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired t test (C, D, E, H, and J) and 2-way ANOVA (H).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Myelin oligodendrocyte glycoprotein (MOG)35–55 Charitè Berlin NA Incomplete Freund’s adjuvant Sigma-Aldrich Cat# F5506 Mycobacterium tuberculosis H37Ra BD Cat# 231141 Pertussis toxin List Biological Laboratories Cat# 181 recombinant murine GM-CSF R&D Cat# 415-ML-050 recombinant murine IL-4 Miltenyi Biotec Cat# 130-097-761 recombinant murine IFN-ɣ Miltenyi Biotec Cat# 130-105-774 recombinant murine IL-10 R&D Cat# 417-ML-005 Lipopolysaccharide ultra pure InvivoGen Cat# tlrl-pelps Filgotinib TargetMol Cat# T1929 Stattic MedChem Express Cat# HY-13818 Fludarabine MedChem Express Cat# HY-B0069 Ruxolitinib Cayman Chemical Cat# 11609-5 Tofacitinib MedChem Express Cat# HY-40354 OVA323–399 Bachem Cat# H-5532 CD11c MACS Micro Bead System Miltenyi Biotec Cat# 130-125-835 Naive CD4+ T Cell Isolation Kit, mouse Miltenyi Biotec Cat# 130-104-453 CFSE Thermo Fisher Scientific Cat# 65-0850-84 Recombinant Mouse TGF-beta 1 R&D Cat# 7666-MB-005/CF Animal-Free Recombinant Human IL-2 Peprotech Cat# AF-200-02 Recombinant mouse PD-L1 Fc-Fusion protein Kerafast Cat# Pr00112-1.9 Recombinant human IgG1 Fc control protein R&D Cat# 110-HG-100 Adult brain dissociation kit Miltenyi Biotec Cat# 130-107-677 Fixable Viability Dye eFluorTM 780 Thermo Fisher Scientific Cat# 65-0865-14 SYTOXTM AADvancedTM Thermo Fisher Scientific Cat# S10274 FoxP3/Transcription factor staining buffer set Thermo Fisher Scientific Cat# 00-5523-00 eBioscienceTM Intracellular fixation & permeabilization buffer set Thermo Fisher Scientific Cat# 88-8824-00 True-Phos Buffer Set BioLegend Cat# 425401 Ionomycin Sigma Aldrich Cat# I0634 PMA Sigma Aldrich Cat# P1585 Brefeldin A solution Thermo Fisher Scientific Cat# 00-4506-51 High-Capacity cDNA Reverse Transcription Kit Thermo Fisher Scientific Cat# 4368814 TaqManTM Gene Expression Master Mix Thermo Fisher Scientific Cat# 4369016 Luna Master Mix New England BioLabs Cat# M3003E Critical commercial assays LEGENDplexTM Mouse Inflammation Panel BioLegend Cat# 740446 LEGENDplexTM Mouse Th Panel BioLegend Cat# 740741 Experimental models: Organisms/strains Mouse: Jak1tm1a(EUCOMM)Hmgu EUMMCR MGI:4456720 Mouse: ROSA26::FLPe knock in; B6.129S4-Gt(ROSA)26Sortm1(FLP1)Dym/RainJ The Jackson Laboratory Cat# JAX:009086, RRID:IMSR_JAX:009086 Mouse: CD11c cre; B6.Cg-Tg(Itgax-cre)11Reiz/J mice The Jackson Laboratory Cat# JAX:008068, RRID:IMSR_JAX:008068 (Continued on next page) Cell Reports 38, 110420, February 22, 2022 e3

Techniques: Quantitative RT-PCR, Isolation, Control, In Vitro, Proliferation Assay, Labeling, Cell Culture

Journal: Cell reports

Article Title: JAK1 signaling in dendritic cells promotes peripheral tolerance in autoimmunity through PD-L1-mediated regulatory T cell induction.

doi: 10.1016/j.celrep.2022.110420

Figure Lengend Snippet: Figure 3. JAK1 deficiency in DCs aggravate EAE by affecting T cells (A) Cytokine profile of supernatants from co-cultures of OT-II CD4+ T cells and LPS-stimulated WT or JAK1-deficient OVA-loaded BM-DCs. The data are rep- resented as log2 fold change compared to WT control; n = 4. (B) Clinical course, cumulative scores, and weights of WT and Jak1DDC mice after EAE induction; n = 13 per genotype. (C) Representative H&E staining of spinal cord sections of WT and Jak1DDC mice 21 days after EAE induction. Scale bars, 500 mm. (D) Representative immunofluorescence images of spinal cord sections stained with DAPI (blue), fluoromyelin (green), and CD3 (pink) of WT and Jak1DDC mice 21 days after EAE induction. Scale bars, 500 mm. (E) CD3+ T cells and IFN-g+CD4+ T cells in the CNSs of WT and Jak1DDC mice 21 days after EAE induction; n = 5 per genotype. (F) Percentage of CD4+ and CD8+ T cells of total CD3+ T cells in lymph nodes of WT and Jak1DDC mice 8 days after EAE induction; n = 6–7 per genotype. (G) Percentage of Ki67+CD3+ and Ki67+CD4+ T cells in lymph nodes of WT and Jak1DDC mice 8 days after EAE induction; n = 6–7 per genotype. (H) Representative fluorescence-activated cell sorting (FACS) plots of data shown in (G). (legend continued on next page)

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Myelin oligodendrocyte glycoprotein (MOG)35–55 Charitè Berlin NA Incomplete Freund’s adjuvant Sigma-Aldrich Cat# F5506 Mycobacterium tuberculosis H37Ra BD Cat# 231141 Pertussis toxin List Biological Laboratories Cat# 181 recombinant murine GM-CSF R&D Cat# 415-ML-050 recombinant murine IL-4 Miltenyi Biotec Cat# 130-097-761 recombinant murine IFN-ɣ Miltenyi Biotec Cat# 130-105-774 recombinant murine IL-10 R&D Cat# 417-ML-005 Lipopolysaccharide ultra pure InvivoGen Cat# tlrl-pelps Filgotinib TargetMol Cat# T1929 Stattic MedChem Express Cat# HY-13818 Fludarabine MedChem Express Cat# HY-B0069 Ruxolitinib Cayman Chemical Cat# 11609-5 Tofacitinib MedChem Express Cat# HY-40354 OVA323–399 Bachem Cat# H-5532 CD11c MACS Micro Bead System Miltenyi Biotec Cat# 130-125-835 Naive CD4+ T Cell Isolation Kit, mouse Miltenyi Biotec Cat# 130-104-453 CFSE Thermo Fisher Scientific Cat# 65-0850-84 Recombinant Mouse TGF-beta 1 R&D Cat# 7666-MB-005/CF Animal-Free Recombinant Human IL-2 Peprotech Cat# AF-200-02 Recombinant mouse PD-L1 Fc-Fusion protein Kerafast Cat# Pr00112-1.9 Recombinant human IgG1 Fc control protein R&D Cat# 110-HG-100 Adult brain dissociation kit Miltenyi Biotec Cat# 130-107-677 Fixable Viability Dye eFluorTM 780 Thermo Fisher Scientific Cat# 65-0865-14 SYTOXTM AADvancedTM Thermo Fisher Scientific Cat# S10274 FoxP3/Transcription factor staining buffer set Thermo Fisher Scientific Cat# 00-5523-00 eBioscienceTM Intracellular fixation & permeabilization buffer set Thermo Fisher Scientific Cat# 88-8824-00 True-Phos Buffer Set BioLegend Cat# 425401 Ionomycin Sigma Aldrich Cat# I0634 PMA Sigma Aldrich Cat# P1585 Brefeldin A solution Thermo Fisher Scientific Cat# 00-4506-51 High-Capacity cDNA Reverse Transcription Kit Thermo Fisher Scientific Cat# 4368814 TaqManTM Gene Expression Master Mix Thermo Fisher Scientific Cat# 4369016 Luna Master Mix New England BioLabs Cat# M3003E Critical commercial assays LEGENDplexTM Mouse Inflammation Panel BioLegend Cat# 740446 LEGENDplexTM Mouse Th Panel BioLegend Cat# 740741 Experimental models: Organisms/strains Mouse: Jak1tm1a(EUCOMM)Hmgu EUMMCR MGI:4456720 Mouse: ROSA26::FLPe knock in; B6.129S4-Gt(ROSA)26Sortm1(FLP1)Dym/RainJ The Jackson Laboratory Cat# JAX:009086, RRID:IMSR_JAX:009086 Mouse: CD11c cre; B6.Cg-Tg(Itgax-cre)11Reiz/J mice The Jackson Laboratory Cat# JAX:008068, RRID:IMSR_JAX:008068 (Continued on next page) Cell Reports 38, 110420, February 22, 2022 e3

Techniques: Control, Staining, FACS

Journal: Cell reports

Article Title: JAK1 signaling in dendritic cells promotes peripheral tolerance in autoimmunity through PD-L1-mediated regulatory T cell induction.

doi: 10.1016/j.celrep.2022.110420

Figure Lengend Snippet: Figure 5. DC-expressed PD-L1 is regulated via IFN-g, JAK1, STAT1 signaling (A) PD-L1 MFI of filgotinib- or vehicle-treated or WT and JAK1-deficient BM-DCs after 24 h LPS stimulation; n = 5 per group. (B) p-STAT1(Ser727), p-STAT1(Tyr701), p-STAT3(Tyr705), PD-L1, and IRF1 kinetics following LPS stimulation of splenic WT and JAK1-deficient DCs analyzed by western blotting. (C) PD-L1 MFI of BM-DCs 24 h after IFN-ɣ stimulation in the presence of an IFN-ɣ-blocking or isotype control antibody; n = 4 per group. (D) PD-L1, p-STAT1(Ser727), and p-STAT3(Tyr705) MFI of BM-DCs after 24 h of LPS stimulation in the presence of an IFN-ɣ-blocking or isotype control antibody; n = 3–4 per group. (E) PD-L1 and p-STAT1(Ser727) MFI of WT and JAK1-deficient BM-DCs after 24 h of LPS stimulation in the presence of an IFN-ɣ-blocking or isotype control antibody; N = 3 per group. (F) PD-L1, p-STAT1(Ser727), and p-STAT3(Tyr705) MFI of BM-DCs after 24 h of LPS stimulation in the presence of a IL10-receptor-blocking or isotype control antibody; n = 3–4 per group. (G) PD-L1 MFI of BM-DCs treated with vehicle or a STAT3 inhibitor (Stattic) after 24 h of LPS stimulation; n = 4 per group. (H) PD-L1 MFI of WT and STAT1-deficient BM-DCs after 24 h of LPS stimulation. n = 4 per group. The data are represented as means ± SEMs of biologically independent samples. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, 1-way ANOVA (C), 2-way ANOVA (A and D–H).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Myelin oligodendrocyte glycoprotein (MOG)35–55 Charitè Berlin NA Incomplete Freund’s adjuvant Sigma-Aldrich Cat# F5506 Mycobacterium tuberculosis H37Ra BD Cat# 231141 Pertussis toxin List Biological Laboratories Cat# 181 recombinant murine GM-CSF R&D Cat# 415-ML-050 recombinant murine IL-4 Miltenyi Biotec Cat# 130-097-761 recombinant murine IFN-ɣ Miltenyi Biotec Cat# 130-105-774 recombinant murine IL-10 R&D Cat# 417-ML-005 Lipopolysaccharide ultra pure InvivoGen Cat# tlrl-pelps Filgotinib TargetMol Cat# T1929 Stattic MedChem Express Cat# HY-13818 Fludarabine MedChem Express Cat# HY-B0069 Ruxolitinib Cayman Chemical Cat# 11609-5 Tofacitinib MedChem Express Cat# HY-40354 OVA323–399 Bachem Cat# H-5532 CD11c MACS Micro Bead System Miltenyi Biotec Cat# 130-125-835 Naive CD4+ T Cell Isolation Kit, mouse Miltenyi Biotec Cat# 130-104-453 CFSE Thermo Fisher Scientific Cat# 65-0850-84 Recombinant Mouse TGF-beta 1 R&D Cat# 7666-MB-005/CF Animal-Free Recombinant Human IL-2 Peprotech Cat# AF-200-02 Recombinant mouse PD-L1 Fc-Fusion protein Kerafast Cat# Pr00112-1.9 Recombinant human IgG1 Fc control protein R&D Cat# 110-HG-100 Adult brain dissociation kit Miltenyi Biotec Cat# 130-107-677 Fixable Viability Dye eFluorTM 780 Thermo Fisher Scientific Cat# 65-0865-14 SYTOXTM AADvancedTM Thermo Fisher Scientific Cat# S10274 FoxP3/Transcription factor staining buffer set Thermo Fisher Scientific Cat# 00-5523-00 eBioscienceTM Intracellular fixation & permeabilization buffer set Thermo Fisher Scientific Cat# 88-8824-00 True-Phos Buffer Set BioLegend Cat# 425401 Ionomycin Sigma Aldrich Cat# I0634 PMA Sigma Aldrich Cat# P1585 Brefeldin A solution Thermo Fisher Scientific Cat# 00-4506-51 High-Capacity cDNA Reverse Transcription Kit Thermo Fisher Scientific Cat# 4368814 TaqManTM Gene Expression Master Mix Thermo Fisher Scientific Cat# 4369016 Luna Master Mix New England BioLabs Cat# M3003E Critical commercial assays LEGENDplexTM Mouse Inflammation Panel BioLegend Cat# 740446 LEGENDplexTM Mouse Th Panel BioLegend Cat# 740741 Experimental models: Organisms/strains Mouse: Jak1tm1a(EUCOMM)Hmgu EUMMCR MGI:4456720 Mouse: ROSA26::FLPe knock in; B6.129S4-Gt(ROSA)26Sortm1(FLP1)Dym/RainJ The Jackson Laboratory Cat# JAX:009086, RRID:IMSR_JAX:009086 Mouse: CD11c cre; B6.Cg-Tg(Itgax-cre)11Reiz/J mice The Jackson Laboratory Cat# JAX:008068, RRID:IMSR_JAX:008068 (Continued on next page) Cell Reports 38, 110420, February 22, 2022 e3

Techniques: Western Blot, Blocking Assay, Control

Journal: Cell reports

Article Title: JAK1 signaling in dendritic cells promotes peripheral tolerance in autoimmunity through PD-L1-mediated regulatory T cell induction.

doi: 10.1016/j.celrep.2022.110420

Figure Lengend Snippet: Figure 7. JAK1 gain-of-function DCs exhibit increased PD-L1 expression, and their adoptive transfer positively affects Treg generation (A) PD-L1 MFI of WT or Jak1spade BM-DCs after 24 h of LPS stimulation; n = 4 per group. (B) PD-L1 MFI of WT or Jak1spade BM-DCs after 24 h of IFN- ɣ stimulation; n = 4 per group. (C) PD-L1 MFI of WT, Jak1spade, or Jak1spade BM-DCs treated with a STAT1 inhibitor (fludarabine) after 24 h of LPS stimulation; n = 4 per group. (D) Representative histograms of data shown in (C). (E) Percentage of splenic FoxP3+ Tregs of EAE-bearing mice receiving either WT or Jak1spade MOG-loaded BM-DCs together with the administration of an aPD-1- blocking antibody or an isotype control 3 days after the first DC transfer (day 11); n = 6–9 per group. (F) Representative FACS plots of data shown in (E) depicting cells in the CD25+ parent gate. The data are represented as means ± SEMs of biologically independent samples or animals. *p < 0.05, ****p < 0.0001, 1-way ANOVA (A–C and E).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Myelin oligodendrocyte glycoprotein (MOG)35–55 Charitè Berlin NA Incomplete Freund’s adjuvant Sigma-Aldrich Cat# F5506 Mycobacterium tuberculosis H37Ra BD Cat# 231141 Pertussis toxin List Biological Laboratories Cat# 181 recombinant murine GM-CSF R&D Cat# 415-ML-050 recombinant murine IL-4 Miltenyi Biotec Cat# 130-097-761 recombinant murine IFN-ɣ Miltenyi Biotec Cat# 130-105-774 recombinant murine IL-10 R&D Cat# 417-ML-005 Lipopolysaccharide ultra pure InvivoGen Cat# tlrl-pelps Filgotinib TargetMol Cat# T1929 Stattic MedChem Express Cat# HY-13818 Fludarabine MedChem Express Cat# HY-B0069 Ruxolitinib Cayman Chemical Cat# 11609-5 Tofacitinib MedChem Express Cat# HY-40354 OVA323–399 Bachem Cat# H-5532 CD11c MACS Micro Bead System Miltenyi Biotec Cat# 130-125-835 Naive CD4+ T Cell Isolation Kit, mouse Miltenyi Biotec Cat# 130-104-453 CFSE Thermo Fisher Scientific Cat# 65-0850-84 Recombinant Mouse TGF-beta 1 R&D Cat# 7666-MB-005/CF Animal-Free Recombinant Human IL-2 Peprotech Cat# AF-200-02 Recombinant mouse PD-L1 Fc-Fusion protein Kerafast Cat# Pr00112-1.9 Recombinant human IgG1 Fc control protein R&D Cat# 110-HG-100 Adult brain dissociation kit Miltenyi Biotec Cat# 130-107-677 Fixable Viability Dye eFluorTM 780 Thermo Fisher Scientific Cat# 65-0865-14 SYTOXTM AADvancedTM Thermo Fisher Scientific Cat# S10274 FoxP3/Transcription factor staining buffer set Thermo Fisher Scientific Cat# 00-5523-00 eBioscienceTM Intracellular fixation & permeabilization buffer set Thermo Fisher Scientific Cat# 88-8824-00 True-Phos Buffer Set BioLegend Cat# 425401 Ionomycin Sigma Aldrich Cat# I0634 PMA Sigma Aldrich Cat# P1585 Brefeldin A solution Thermo Fisher Scientific Cat# 00-4506-51 High-Capacity cDNA Reverse Transcription Kit Thermo Fisher Scientific Cat# 4368814 TaqManTM Gene Expression Master Mix Thermo Fisher Scientific Cat# 4369016 Luna Master Mix New England BioLabs Cat# M3003E Critical commercial assays LEGENDplexTM Mouse Inflammation Panel BioLegend Cat# 740446 LEGENDplexTM Mouse Th Panel BioLegend Cat# 740741 Experimental models: Organisms/strains Mouse: Jak1tm1a(EUCOMM)Hmgu EUMMCR MGI:4456720 Mouse: ROSA26::FLPe knock in; B6.129S4-Gt(ROSA)26Sortm1(FLP1)Dym/RainJ The Jackson Laboratory Cat# JAX:009086, RRID:IMSR_JAX:009086 Mouse: CD11c cre; B6.Cg-Tg(Itgax-cre)11Reiz/J mice The Jackson Laboratory Cat# JAX:008068, RRID:IMSR_JAX:008068 (Continued on next page) Cell Reports 38, 110420, February 22, 2022 e3

Techniques: Expressing, Adoptive Transfer Assay, Blocking Assay, Control