Regulatory T Cells

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For more information, please visit our website at www.RnDSystems.com/go/Treg R&D Systems Products for Treg Cell Research PRSRT STD CELL SELECTION KITS* RECOMBINANT ELISAs/ ELISpot KITS* RECOMBINANT ELISAs/ U.S. POSTAGE R&D Systems Tools for Cell Biology Research™ MOLECULE ANTIBODIES ELISpot KITS* & NATURAL PROTEINS ASSAYS MOLECULE ANTIBODIES CELL SELECTION KITS* & NATURAL PROTEINS ASSAYS PAID 4-1BB/TNFRSF9/CD137 H M H M H M IFN-γ H M R B Ca H M R P Ca E F Pr H M R B Ca H M R B Ca 5’-Nucleotidase/CD73 H M H M CR E F P RM CR E F P RM CR E F P Pr USA & Canada IFN-γ R1/CD119 H M H M H M R&D Systems, Inc. R&D SYSTEMS B7-1/CD80 H M R H M R H M 4 4 10 61410 McKinley Place NE, Minneapolis, MN 55413 B7-2/CD86 H M R H M R R IFN-γ R2 H M 70 15 100 Tel: (800) 343-7475 (612) 379-2956 100 B7-H2 H M H M IGSF2/CD101 M Change Service Requested IL-1α/IL-1F1 H M R CR P H M R CR P H M R Fax: (612) 656-4400 60 E-Cadherin H M H M H M H M [email protected] 80 cAMP Ms Ms β H M R Ca 103 H M R Ca 103 80 IL-1 /IL-1F2 CR E F P H P CR E F P RM H M R F P 50 CCL1/I-309/TCA-3 H M H M H M MA104_Treg_NOV 10 IL-1 RI/CD121a H M H M R H 60 CCL4/MIP-1β H M Ca CR H M Ca CR H M 40 60 CCL17/TARC H M H M H M IL-1 RII/CD121b H M H M H H M R B Ca H M R B Ca H M R B Ca 2 30 CCL19/MIP-3β H M H M H M IL-2 H M R Ca E F102 10 40

LAP 40 CR E F P CD4 CR E F P E F 5 CCL20/MIP-3α H M R H M R H M R 20 IL-2 Rβ H M H Number Cell Relative Relative Cell Number Cell Relative CCL22/MDC H M H M H M Number Cell Relative Number Cell Relative Regulatory T Cells H M R B Ca H M R B Ca H M R CR 20 20 CCR2 H M IL-4 CR E F P H M Ca E CR E F P RM E F P 10 1 101 CCR4 H IL-4 Rα H M 10 H M CCR5 H H 0 0 0 0 H M R Ca H M R Ca H M R Ca 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 100 101 102 103 104 CCR6 H M IL-10 CR E F P V H M Ca F CR E F P V E F P 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 CD39 NRP-1 CCR7 H M IL-10 Rα H M H M GITR CTLA-4 100 Printed100 on recyclable paper 10% post consumer waste. IL-10 CCR8 H M R IL-10 Rβ H M 0 H 1 2 3 4 100 101 102 103 104 10 10 10 10 10 R&D Systems is a registered trademark of TECHNE Corporation. CD3 H M H M R IL-35 p35 H M P CD127 CD25 Latent TGF-β1 CD3e H M Integrin αE/CD103 M LRRC32 CD4 H M Ca F H M R H Integrin αEβ7 H Galectin-1 CD5 H M Integrin αL/CD11a H + + ™ CD25/IL-2 Rα H M R H M R H M R H M Integrin αVβ8 H CD4 CD25 Regulatory T Cell Enrichment using MagCellect Cell Selection Kits 4 CD27/TNFRSF7 H M H M M Integrin β2/CD18 H M H R&D Systems offers MagCellect Cell Selection Kits for human, mouse, or rat CD4+CD25+ Treg cell isolation. A. 10 CD27 Ligand/TNFSF7 H M M M Jak1 H M R The MagCellect Kits are designed to isolate CD4+CD25+ Treg cells from a mononuclear cell suspension FoxP3 CD127/IL-7 Rα H M R H M R M Jak3 H IL-35 + + CD28 H M H M LAG-3 H M 80 H H using a two-step100 procedure. CD4 T cells are initially enriched by negative selection. CD25 T cells are then 3 Regulatory T Cell CD30/TNFRSF8 H M H M M LAP (TGF-b1) H M H isolated by positive selection from the CD4+ T cell fraction. The90 typical purity of the recovered CD4+CD25+ 10 70 CD30 Ligand/TNFSF8 H M H M M LRRC32/GARP M H M 80 Treg cells ranges80 between 85-95% for the human kit, 84-94% for the mouse kit, and 75-85% for the rat kit. CD34 R P Ca MAdCAM-1 M 60 M M 70 IL-2 Rβ CD38 H M H M Neuropilin-1/BDCA4 H M R H M R TGF-β1 50 MagCellect KIT CATALOG # 60 KIT CONTENTS 102 CD39/ENTPD1 H M H M OX40/TNFRSF4 H M H M 60 IL-2 Rα/CD25 + + + CD25 IL-2 Rγ CD40/TNFRSF5 H M H M M OX40 Ligand/TNFSF4 H M 40 H M M Human CD4 CD25 Regulatory T Cell Isolation Kit MAGH104 Human CD4 T Cell50 Biotinylated Antibody Cocktail, Streptavidin Ferrofluid, Anti-Human CD25 Ferrofluid (Clone 24238), 10X Buffer, Staining Reagent for CD40 Ligand/TNFSF5 H M H M H M 40 PD-1 H M 30 H M H 40 Human CD4+CD25+ Regulatory T Cells CD44 H Ca H H PDCD6 H M R 30 1 + + + 10 CD45 H M H H M H PD-L1/B7-H1 H M Number Cell Relative 20 H M Mouse CD4 CD25 Number Cell Relative Regulatory T Cell Isolation Kit MAGM208 Mouse CD4 T Number Cell Relative Cell Biotinylated Antibody Cocktail, Streptavidin Ferrofluid, 20 Mouse CD25 Biotinylated20 Antibody (Clone PC61.5), 10X Buffer CD45R/B220 M PD-L2 H M H M 10 10 CD69 H M PRAT4A M Rat CD4+CD25+ Regulatory T Cell Isolation Kit MAGR304 Rat CD4 T Cell Antibody Cocktail, Anti-Mouse IgG Ferrofluid, Streptavidin CD72 M PRAT4B M R 0 0 Ferrofluid, Rat CD250 Biotinylated Antibody, 10X Buffer 100 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 Eﬀector T Cells CD83 H M H M RANK/TNFRSF11A H M 10 H M 10 10 H 10 10 10 10 10 10 10 10 10 10 10 10 100 101 102 103 104 CD109 H H RARa/NR1B1 H IL-12/p35 Assay Principle LAG-3 CD27 CD4 γ Common Chain/IL-2 H M H M c-Rel H M H Rγ RXRa/NR2B1 H ENRICHMENT OF CD4+ T CELLS ENRICHMENT OF CD4+ CD25+ T CELLS B. 104 CREB H M R H M R E-Selectin/CD62E H M R H M H M CTLA-4 H M H M M CD4 Biotinylated CD25 Antibody L-Selectin/CD62L H M R H M R H M R Antibody Cocktail Anti-CD25 Ferro uid or CXCL9/MIG H M H H M H M P-Selectin/CD62P H M H M H M Streptavidin Ferro uid + Streptavidin Ferro uid 3 CXCL10/IP-10/CRG-2 H M CR H H M CR H M SLAM/CD150 H M M or Anti-IgG Ferro uid Enriched Enriched 10 + + + CXCL12/SDF-1 H M H M F RM H M Smad3 H M H CD4 T Cells CD4 CD25 T Cells CXCR3 H M STAT5a, STAT5b H M CXCR4 H M F H TGF-β1 H M Ms H H P H M R Ca P 102 Fas/TNFRSF6/CD95 H M R F H M R F H M TGF-β RI/ALK-5 H M M CD25 FoxP3 H M R H TGF-β RII H M H M H Galectin-1 H M H M M TGF-β RIIb H H GITR/TNFRSF18 H M H M H M TLR4 H M H 101 GITR Ligand/TNFSF18 H M H M H M TLR4/MD-2 Complex H Granzyme A H H TLR7 H Granzyme B H M H M H M M TNF RII/TNFRSF1B H M H M H M 0 HLA-DR H TOR H M R H 10 100 101 102 103 104 HO-1/HMOX1/HSP32 H M R H TRAIL/TNFSF10 H M H M H KEY: Undesired cells CD4+ T Cells CD4+CD25+ T Cells ICAM-1/CD54 H M R H M R H M R CD4 TRANCE/TNFSF11/RANK H M H M M ICOS H M H M L Enrichment of CD4+CD25+ T cells using the MagCellect CD4+CD25+ Regulatory T Cell Isolation Kits. The CD4 Antibody Cocktail is added to a mononuclear cell suspension. Undesired Enrichment of CD4+CD25+ Treg Cells using the MagCellect Kits. CD4+CD25+ Treg IDO H TSLP H M H M H M cells are bound by the antibodies and then captured by MagCellect Ferrofluid magnetic particles, or equivalent. The undesired cells are isolated from the sample by negative selection cells were isolated from (A) human PBMCs using the MagCellect Human CD4+CD25+ IFN-a H M P CR H M R CR F P RM H M TSLP R H M H M using a MagCellect Magnet (Catalog # MAG997), or equivalent, and an enriched CD4+ T cell population is aspirated from the sample solution. MagCellect anti-Human CD25 Ferrofluid or Regulatory T Cell Isolation Kit (Catalog # MAGH104) or (B) mouse splenocytes using the Biotinylated CD25 Antibody and Streptavidin Ferrofluid is then added to the CD4+ T cell solution. CD4+CD25+ T cells are captured by applying the MagCellect Magnet, or any compatible MagCellect Mouse CD4+CD25+ Regulatory T Cell Isolation Kit (Catalog # MAGM208). Total KEY: H: Human M: Mouse R: Rat B: Bovine Ca: Canine CR: Cotton Rat E: Equine F: Feline Ms: Multispecies P: Porcine Pr: Primate + + * ELISpot Kits & Development Modules * Cell Selection & Detection Kits & Reagents RM: Rhesus Macaque V: Viral magnet system. CD4 CD25 Treg cells were detected using Fluorescein-conjugated CD4 and PE-conjugated CD25 antibodies.

For research use only. Not for use in diagnostic procedures. www.RnDSystems.com Regulatory T Cells Flow Cytometry Markers for Treg Cells Treg Cell Multi-Color 104 104 104 104

+ The immune system has regulatory mechanisms that prevent sustained TABLE 1. Characteristics of CD4 Treg Cells SPECIES APC FLUORESCEIN PE PerCP SPECIES APC FLUORESCEIN PE PerCP SPECIES APC FLUORESCEIN PE PerCP Flow Cytometry Kits inflammatory responses and attacks on healthy tissue. Regulatory T cells Naturally Occurring Treg Cells Tr1 Cells Th3 Cells 5’-Nucleotidase/CD73 CTLA-4 LAG-3/CD223 Cell Type + + + –/variable + low/variable 3 3 3 3 (Treg cells) play a role in maintaining immune homeostasis, preventing CD4 CD25 CD4 CD25 CD4 CD25 H • • H • H • • • Kit Contents: (Contents also sold separately) 10 10 10 10 M • • • M • M •  autoimmunity, moderating inflammation, and minimizing collateral Differentiation CD28:B7 signaling b APC-conjugated anti-FoxP3 Factors IL-2 IL-10 TGF- 1 damage to tissue. A primary function of Treg cells is to inhibit the function CD4 FoxP3 LAP (TGF-b1)  PE-conjugated anti-CD25 + low + H • • • • H/M/R • H/M • • • 102 102 102 102

FoxP3 , CD127 , LRRC32/GARP , + + + +  FoxP3 FoxP3 FoxP3 FoxP3 of antigen-presenting cells and effector T cells (Teff cells). Consequently, Associated + + + CTLA-4 , LAP , CTLA-4 , LAP , PerCP-conjugated anti-CD4 or CD39 , GITR , NRP-1 (mouse), low – low + M • • • • GITR/TNFRSF18 NRP-1 reduced Treg cell activity may be associated with human autoimmune Markers CTLA-4+, LAP+ CD45RB , FoxP3 CD45RB , FoxP3 FITC-conjugated anti-CD4 CD25/IL-2 Ra H • • • H • • • • diseases, including rheumatoid arthritis, type I diabetes, multiple sclerosis,  Specifically formulated staining buffers  Induction of cytolysis 1 1 H • • M • • M/R • • 10 10 101 101  Disruption of metabolic activities  Goat IgG-APC isotype control systemic lupus erythematosus, and myasthenia gravis. In addition, Treg Proposed  Cell-cell contact M • • IL-10 OX40/TNFRSF4  Inhibition of dendritic cell cells may play a causative role in aplastic anemia, graft-versus-host disease, Regulatory  Secretion of  Secretion of TGF-b1 CD127/IL-7 Ra H • • H • • • Species (Catalog #): Human (FMC013) Mechanisms maturation IL-10, TGF-b1, IFN-g and transplant rejection. CD4+ Treg cells are traditionally divided into 3  Secretion of IL-10, IL-35, TGF-b1, H • • IL-35/p35 TGF-b1 Mouse (FMC014 100 100 0 0 + + Galectin-1 Rat (FMC015) 10 10 subsets. These include, naturally occurring CD4 CD25 Treg cells that M • H/M • • • • Ms • • • 100 100 101 101 102 102 103 103 104 104 100 100 101 101 102 102 103 103 104 104 develop in the thymus and induced CD4+ Tregs, known as Tr1 and Th3 cells, CD39/ENTPD1 L-Selectin/CD62L TGF-b1, 2, 3 Size: 50 Tests CD4CD4 CD25CD25 H • • • H • Ms • • that develop in the periphery. Although the characteristics of these Detection of Human Treg Cells using Multi-Color Flow Cytometry. Human peripheral blood mononuclear cells (PBMCs) were assessed for FoxP3, CD25, and CD4 expression M • • • M • • subtypes continue to be defined, they typically have different surface KEY: H: Human M: Mouse R: Rat Ms: Multispecies using antibodies and buffers included in the Human Regulatory T Cell Multi-Color Flow Cytometry Kit (Catalog # FMC013). Quadrants were set based on isotype controls. CD101/IGSF2 markers, secreted products, and mechanisms of action (Table 1). Studies M • • • have suggested that the naturally occurring CD4+CD25+ Treg cells, which comprise 5-10% of the total peripheral CD4+ T cells, have a central role in Proteins

4 immune control. 4 104 104 10 20000 10 4 2.1 10 70 15 100 702000 15 100 100 100 60 60 2.0 Suppressive Activities of Treg Cells 1800 80 15000 80 3 1. Secretion of TGF-b1, IL-10, IL-35, and Galectin-1 3 3 3 10 80 80 10 10 103 50 10 50 1.9 • Inhibits the differentiation, proliferation, and activation of Teff cells 10 1600 10 60 60 • Suppresses cytokine production by Teff cells 40 60 40 1.8 60 Ν-formylkynurenine • IL-35 and TGF-b1 induces IL-10 production and regulates FoxP3 expression, 10000 1400 Dendritic Cell (DC) + + Tryptophan promoting the maintenance and expansion of CD4 CD25 Treg cells 2 2 2 30 30 (O.D.) IL-2 1.7 102 10 102 10 40 10 40

10. CTLA-4-dependent Suppression LAP 40 LAP CD4 CD4 40 CD4 5 (RFU) Proliferation 5 • Interacts with B7 (CD80 and CD86) on 201200 20 1.6 Number Cell Relative Relative Cell Number Cell Relative Relative Cell Number Cell Relative Relative Cell Number Cell Relative Number Cell Relative Relative Cell Number Cell Relative Relative Cell Number Cell Relative Number Cell Relative 5000 DCs, triggering indoleamine 2, Active TGF-β1 (cpm/well) Proliferation 3-dioxygenase (IDO) expression (which IDO Eﬀector T Cells (Teﬀ Cells) 20 20 20 20 10 101000 is also induced by IFN-g receptor 1 1.5 α β 1 1 1 1 10 stimulation) Integrin V 8 10 10 10 10 Latent TGF-β1 0 0 0 0 0 0800 0 1.4 0 0 • IDO catabolizes tryptophan, depleting α 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 0 2 1 3 2 4 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 stores needed for Teff cell proliferation, β IL-10, IL-35, 10 10 10 10 10 10 10 10 10 10 10 10 0.01 10 0.1 10 1 10 10 10 100 10 10100.1 10110 101010 10010 100010 10000 10 0.000110 0.001 10 0.01 10 0.1 10 1 1010 10 10 10 10 and producing the pro-apoptotic Galectin-1 CD39 GITR CTLA-4 LRRC32 Concentration (µg/mL) NRP-1 CD39LAP (ng/mL) GITR OX40 (µg/mL) CTLA-4 NRP-1 metabolite Ν-formylkynurenine 0 MHC II 100 2. Membrane-associated LAP-TGF-b1 (Latent TGF-b1) 100 100 100 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 Inhibition0 of T Cell 1Proliferation by LRRC32.2 The induction3 of Human T cell4 prolifera- LAP Suppression of TGF-b1 Activity. The ability of Recombinant Human TGF-b1 OX40 Suppresses OX40 Ligand-induced IL-2 Production. Increasing concentrations IFN-γ R2 Latent TGF-β1 100 101 102 103 104 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 IFN-γ R1 LAG-3 TCR-CD3 • Cell surface LAP-TGF-b1 (latent TGF-b1), tion induced by 2 mg/mL Human CD3ε Monoclonal Antibody (Catalog # MAB100) was (Catalog # 240-B; 1 ng/mL) to inhibit the proliferation of HT2 mouse helper T cells is of Recombinant Mouse OX40 Fc Chimera (Catalog # 1256-OX) inhibit IL-2 production IL-2 complexed with LRRC32, suppressesCD127 the CD25 LAG-3 CD127 CD25 Perforin CD4 LRRC32 proliferation of activated Teff cells inhibited in a dose-dependent manner by Recombinant Human LRRC32 (Catalog # suppressed by increasing concentrations of Recombinant Human LAP (Catalog # 246- induced by Recombinant Mouse OX40 Ligand (Catalog # 1236-OX) in mouse T cell B7 Detection of LAP on CD25+ Mouse Splenocytes. Splenocytes from BALB/c mice were Detection of LAG-3 on Mouse Splenocytes. Mouse splenocytes were treated with PMA/ Detection of CD127 on CD4+ Mouse Splenocytes. Splenocytes from BALB/c mice were 6055-LR). T cell proliferation was measured by 3H-thymidine incorporation. LP) as measured using Resazurin (Catalog # AR002). culture supernatants as measured using the Mouse IL-2 Quantikine® ELISA Kit (Cata- 9. Binding of LAG-3 to MHC II IFN-γ labeled using APC-conjugated Mouse CD25/ IL-2 Ra Monoclonal Antibody (Catalog # calcium ionomycin and then labeled using APC-conjugated Mouse CD4 Monoclonal Anti- labeled using APC-conjugated Mouse CD4 Monoclonal Antibody (Catalog # FAB554A) and log # M2000). • Induces an immunoreceptor CD25/IL-2 Rα tyrosine-based activation motif CTLA-4 IL-2 Rγ 3. High Levels of CD25/IL-2 Ra Expression FAB2438A) and PE-conjugated Human/Mouse LAP Monoclonal Antibody (Catalog # body (Catalog # FAB554A) and PE-conjugated Mouse LAG-3 Polyclonal Antibody (Catalog # PE-conjugated Mouse CD127/IL-7 Ra Antigen Affinity-purified Polyclonal Antibody (Cata- + + (ITAM)-mediated inhibitory + + FAB2463P). Quadrants were set based on isotype controls. FAB3328P). Quadrants were set based on isotype controls. log # FAB747P). CD127, which is present on most mature T cells, is absent on CD4 CD25 • IL-2 maintains CD4 CD25 Treg cell populations 4 104 signaling pathway, blocking the + + Treg cells. Quadrants10 were set based on isotype controls. CD4 CD25 Treg Cell IL-2 Rβ • Depletes local IL-2, inhibiting activation maturation and immunostimulatory NRP-1 capacity of DCs and proliferation of Teff cells 104 104 104 104 Antibodies for IHC & ICC ELISAs ELISpot IFN-γ ATP/ADP 80 70 100 70 15 15 100 100 80 100 Granzyme A/B 350 90 3 CD39 90 10 100 100 10 70 70 60 60 80 80 Unstimulated 80 3500 FoxP3 AMP 80 80 80 Stimulated 3 CD73 3 3 A R 4060 3 10 10 6010 2A 70 80 80 70 10 50 50 3000 Adenosine 10 10 50 60 250 60 102 Perforin 4. Generation of Extracellular Adenosine 60 60 60 10 60

40 40 30 CD25 CD25 60 2500 8. Granzyme A/B Secretion by Cell Surface Expression of CD39/CD73 60 50 40 50 40 • Induces apoptosis in DCs and • Activation of A receptors by Adenosine 2000 2 2 2 2 2A 30 30 40 40 Teff cells in both a 10 10 10 10 blocks the expression of costimulatory 40 40 40 30 40 LAP LAP 30 20 40 40 CD4 LRRC32 CD4 cAMP

perforin-dependent and molecules and growth factor receptors in 5 5 30 (pg/mL) IL-10 1500 1 30 101 10

20 20 Number Cell Relative Number Cell Relative Number Cell Relative Relative Cell Number Cell Relative Relative Cell Number Cell Relative

Galectin-1 Number Cell Relative Number Cell Relative Number Cell Relative Relative Cell Number Cell Relative Relative Cell Number Cell Relative

-independent manner Number Cell Relative Number Cell Relative Relative Cell Number Cell Relative 20 Teff cells, inhibiting Teff cell activation, Number Cell Relative Number Cell Relative 20 Latent TGF-β1 20 20 proliferation, and expansion 20 20 20 10 20 20 20 1000 10 10 10 10 1 1 10 10 101 101 10 10 500 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 100 FoxP3 10 0 1 2 3 4 0 1 0 2 1 3 2 4 3 4 0 1 2 3 4 5. Transfer of Inhibitory cAMP0 through Gap1 Junctions 2 3 4 0 0 10 1 21 2 32 3 43 4 40 100 211 322 433 44 0 10 100 21 101 32 102 43 103 4 0 104 1 2 10 3 10 4 10 10 10 10 0 10 10 10 10 0 1 2 3 4 10 10 10 10 10 10 10 1010 10 1010 10 1010 10 1010 10 1010 1010 101010 1010 101010 10 10 1010 10100 1010 10101 1010 10102 1010 10 1031010 10 1010410 1010 1010 1010 10 10 10 10 10 10 • Inhibits Teff cell proliferation and IL-2 gene expression 24 hr 72 hr 120 hr IL-12/p35 CD39 LAG-3 CD39 GITR CD27GITR CTLA-4 CTLA-4IL-12/p35CD73CD4 NRP-1 NRP-1 LAG-3 CD27 CD4 0 100 100 100 10 + + + + + 0 0 1 1 2 2 3 3 4 04 0 1 1 2 2 3 3 4 Detection4 of CD39 on Mouse Splenocytes. Mouse splenocytes were labeled using APC- Detection of GITR on CD4 Lymphocytes. Human peripheral blood CD4 lymphocytes Detection of CD73 on Mouse CD4 Splenocytes. Mouse CD4 splenocytes were stained FoxP3 and CD4 in Human Tonsil. FoxP3 was detected in human tonsil tissue using Measurement of IL-10 Levels Using the Quantikine ELISA Kit. Human PBMCs were Detection of Granzyme B-secreting CD4 Cells using ELISpot. Mouse splenocytes 7. Induction of Infectious Tolerance 10 10106. Membrane-associated1010 Galectin-11010 1010 1010 1010 1010 1010 1010 10 conjugated Mouse CD39/ENTPD1 Monoclonal Antibody (Catalog # FAB4398A; filled histo- were stimulated with PHA and then labeled using APC-conjugated Human GITR using PE-conjugated Mouse 5’-Nucleotidase/CD73 Monoclonal Antibody (Catalog # + CD127 CD127 CD25 CD25 Human FoxP3 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3240) fol- stimulated with 10 mg/mL PHA for 24, 72, or 120 hours. Aliquots of the cell culture enriched for CD4 cells were assessed for Granzyme B secretion using the Mouse • In mice, membrane-associated LAP-TGF-b1 converts non-Treg cells • Binds to GM1 ganglioside on Teff cells gram) or an APC-conjugated Isotype Control Antibody (Catalog # IC005A; open histo- Monoclonal Antibody (Catalog # FAB689A; filled histogram) or an APC-conjugated Mouse FAB4488P; filled histogram) or a PE-conjugated Isotype Control Antibody (Catalog # + + into functional, FoxP3-expressing Treg cells lowed by staining with NorthernLights™ 557-conjugated Anti-Goat IgG Secondary supernatants were assayed using the Human IL-10 Quantikine ELISA Kit (Catalog # CD4 /Granzyme B ELISpot Kit (Catalog # EL6024). CD4 cells were first enriched by a • Induces cross-linking of associated integrins, triggering TRPC5 channel gram). IgG Isotype Control Antibody (Catalog # IC002A; open histogram). IC003P; open histogram). Antibody (Catalog # NL001; red). CD4 was detected using Human CD4 Monoclonal D1000B). short incubation in ELISpot wells coated with anti-CD4. Following a wash, CD4-en- activation and calcium influx, inhibiting Teff proliferation 1 Antibody (Catalog # MAB379) followed by staining with NorthernLights 493-conju- riched cells were then stimulated overnight in culture media with PMA/Ca2+ ionomy- gated Anti-Mouse IgG Secondary Antibody (Catalog # NL009; green). The nuclei were cin. During the incubation, anti-mouse Granzyme B antibodies capture the secreted

4 counterstained with DAPI (blue). enzyme which is then visualized as blue spots. This illustration represents general pathways suggested in the scientific literature and is not to be considered comprehensive nor definitive. 104 10 www.RnDSystems.com For research use only. Not for use in diagnostic procedures. www.RnDSystems.com 80 80 100 100 90 90 103 103 70 70 80 80 80 80 60 60 70 70

perforin-dependent and molecules and growth factor receptors in 5 5 30 (pg/mL) IL-10 1500 1 30 101 10

20 20 Number Cell Relative Number Cell Relative Number Cell Relative Relative Cell Number Cell Relative Relative Cell Number Cell Relative

Galectin-1 Number Cell Relative Number Cell Relative Number Cell Relative Relative Cell Number Cell Relative Relative Cell Number Cell Relative

perforin-dependent and molecules and growth factor receptors in 5 5 30 (pg/mL) IL-10 1500 1 30 101 10

20 20 Number Cell Relative Number Cell Relative Number Cell Relative Relative Cell Number Cell Relative Relative Cell Number Cell Relative

Galectin-1 Number Cell Relative Number Cell Relative Number Cell Relative Relative Cell Number Cell Relative Relative Cell Number Cell Relative

50 50 60 60 2 2 60 60 10 10 CD25 CD25 40 40 50 50 40 40 30 30 40 40 30 30 101 101 Relative Cell Number Cell Relative Number Cell Relative Number Cell Relative Relative Cell Number Cell Relative 20 20 Number Cell Relative Number Cell Relative 20 20 20 20 10 10 10 10 0 0 0 0 0 0 100 100 0 10 21 32 43 4 0 10 21 32 43 4 0 10 21 32 43 4 10 1010 1010 1010 1010 10 10 1010 1010 1010 1010 10 10 1010 1010 1010 1010 10 100 10100 1 10101 2 10102 3 10103 4 104 IL-12/p35 IL-12/p35 LAG-3 LAG-3 CD27 CD27 CD4 CD4 For more information, please visit our website at www.RnDSystems.com/go/Treg R&D Systems Products for Treg Cell Research PRSRT STD CELL SELECTION KITS* RECOMBINANT ELISAs/ ELISpot KITS* RECOMBINANT ELISAs/ U.S. POSTAGE R&D Systems Tools for Cell Biology Research™ MOLECULE ANTIBODIES ELISpot KITS* & NATURAL PROTEINS ASSAYS MOLECULE ANTIBODIES CELL SELECTION KITS* & NATURAL PROTEINS ASSAYS PAID 4-1BB/TNFRSF9/CD137 H M H M H M IFN-γ H M R B Ca H M R P Ca E F Pr H M R B Ca H M R B Ca 5’-Nucleotidase/CD73 H M H M CR E F P RM CR E F P RM CR E F P Pr USA & Canada IFN-γ R1/CD119 H M H M H M R&D Systems, Inc. R&D SYSTEMS B7-1/CD80 H M R H M R H M 4 4 10 61410 McKinley Place NE, Minneapolis, MN 55413 B7-2/CD86 H M R H M R R IFN-γ R2 H M 70 15 100 Tel: (800) 343-7475 (612) 379-2956 100 B7-H2 H M H M IGSF2/CD101 M Change Service Requested IL-1α/IL-1F1 H M R CR P H M R CR P H M R Fax: (612) 656-4400 60 E-Cadherin H M H M H M H M [email protected] 80 cAMP Ms Ms β H M R Ca 103 H M R Ca 103 80 IL-1 /IL-1F2 CR E F P H P CR E F P RM H M R F P 50 CCL1/I-309/TCA-3 H M H M H M MA104_Treg_NOV 10 IL-1 RI/CD121a H M H M R H 60 CCL4/MIP-1β H M Ca CR H M Ca CR H M 40 60 CCL17/TARC H M H M H M IL-1 RII/CD121b H M H M H H M R B Ca H M R B Ca H M R B Ca 2 30 CCL19/MIP-3β H M H M H M IL-2 H M R Ca E F102 10 40

For research use only. Not for use in diagnostic procedures. www.RnDSystems.com For more information, please visit our website at www.RnDSystems.com/go/Treg R&D Systems Products for Treg Cell Research PRSRT STD CELL SELECTION KITS* RECOMBINANT ELISAs/ ELISpot KITS* RECOMBINANT ELISAs/ U.S. POSTAGE R&D Systems Tools for Cell Biology Research™ MOLECULE ANTIBODIES ELISpot KITS* & NATURAL PROTEINS ASSAYS MOLECULE ANTIBODIES CELL SELECTION KITS* & NATURAL PROTEINS ASSAYS PAID 4-1BB/TNFRSF9/CD137 H M H M H M IFN-γ H M R B Ca H M R P Ca E F Pr H M R B Ca H M R B Ca 5’-Nucleotidase/CD73 H M H M CR E F P RM CR E F P RM CR E F P Pr USA & Canada IFN-γ R1/CD119 H M H M H M R&D Systems, Inc. R&D SYSTEMS B7-1/CD80 H M R H M R H M 4 4 10 61410 McKinley Place NE, Minneapolis, MN 55413 B7-2/CD86 H M R H M R R IFN-γ R2 H M 70 15 100 Tel: (800) 343-7475 (612) 379-2956 100 B7-H2 H M H M IGSF2/CD101 M Change Service Requested IL-1α/IL-1F1 H M R CR P H M R CR P H M R Fax: (612) 656-4400 60 E-Cadherin H M H M H M H M [email protected] 80 cAMP Ms Ms β H M R Ca 103 H M R Ca 103 80 IL-1 /IL-1F2 CR E F P H P CR E F P RM H M R F P 50 CCL1/I-309/TCA-3 H M H M H M MA104_Treg_NOV 10 IL-1 RI/CD121a H M H M R H 60 CCL4/MIP-1β H M Ca CR H M Ca CR H M 40 60 CCL17/TARC H M H M H M IL-1 RII/CD121b H M H M H H M R B Ca H M R B Ca H M R B Ca 2 30 CCL19/MIP-3β H M H M H M IL-2 H M R Ca E F102 10 40

For research use only. Not for use in diagnostic procedures. www.RnDSystems.com