Identifi cation of Novel Cell Surface Markers on Mouse and Human Th17 Cells Christine Goetz, Alberto Guerra, and Jody Bonnevier | R&D Systems, Inc., Minneapolis, MN 55413

ABSTRACT RESULTS Th17 cells are a subset of CD4+ T helper cells that play a crucial role in FIGURE 1 Distribution of Markers Identifi ed in our Screen FIGURE 5 Novel Surface Markers on Human Th17 Cells protection against extracellular bacteria, but may also be involved in auto- Human Surface Antibodies Mouse Surface Antibodies Lymphocytes immune disease progression. Consequently, basic and clinical research on Immune478 Cells 236 Angiogenesis CD43 Myeloid Plexin D1 Metastasis NTB-A CD200 Th17 cells has increased tremendously in the last several years, becoming CD82(-) LAIR1 64 High414 Negative/ 41 High 195 Negative/ Immune Cells CD99 Platelets one of the most prominent and active areas of research in immunology. The Low Low Inflammation CCRL2 RBCs CD49f Th17 subset is defi ned by the ability to produce cytokines, such as IL-17 and IL-1 RAcP TRA-1-85 37 Known 27 Novel 4 13 Novel 28 Known IL-18 R Overlap D IL-22, and also by the expression of cell surface markers, such as the IL-23 BTLA 26 Co-stain with Autoimmunity/ receptor. Much research eff ort in the area of Th17 cell biology is the search IL-17, IL-22, Cancer Th17 Signaling RORJt Signaling/ for surface molecules that can be used to consistently characterize Th17 Adhesion Carrier CD97 cells in diff erent immune environments, which may ultimately lead to CD53 TfR FIGURE 2 Representative Flow Data in Human Th17 Cells Development DEP-1 potential therapies for autoimmunity. To date, only a few surface molecules Notch-1 Axon ALCAM Actin Growth Development Isotype Control Negative Mild Expression High Expression DLL3 DNAM-1 A33 have been found that are exclusively expressed by Th17 cells, making the Sema7A (+) CD48 107.2 0.4% 8.9% 107.2 1.0% 12.7% 107.2 1.8% 48.9% 107.2 3.6% 95.8% Sema4D (-) IGF-II R use of surface markers for this cell type a challenge. Our objective was to 106 106 106 106 LRP-6 (Wnt) 105 105 105 105 identify new surface markers to distinguish Th17 cells from other subsets 104 104 104 104

Antibody X Antibody X Antibody X Antibody X PBMCs were purifi ed as described above and stimulated for 5 days to induce Th17 diff er- of CD4+ helper T cells by fl ow cytometry. In order to accomplish this goal, 103 103 103 103 entiation. The resulting Th17 cells were stained with APC-, Fluorescein-, or PE-conjugated 102 102 102 102

2.6% 88.1% 2.5% 83.8% 1.9% 47.5% 0.0% 0.6% Mouse Anti-Human CD4 Monoclonal Antibody (Catalog # FAB3791A, # FAB3791F, or # FAB3791P, 101 101 101 101 we used a high-throughput fl ow cytometer to screen over 700 surface 1 2 3 4 5 6 7.2 1 2 3 4 5 6 7.2 1 2 3 4 5 6 7.2 1 2 3 4 5 6 7.2 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 respectively) and the surface markers shown. Cells expressing 26/27 of these novel markers also CD4-APC CD4-APC CD4-APC CD4-APC antibodies on mouse and human Th17 cells. We identifi ed 26 novel surface TM + expressed IL-22, IL-17, and RORt. The four novel markers that overlap with mouse Th17 cells are PBMCs were isolated, CD4-purifi ed using the MagCellect Human Naïve CD4 Isolation shown in blue. These results are representative of three and two experiments, respectively. markers on human Th17 cells that also co-express IL-22, IL-17, and RORt, Kit (Catalog # MAGH115), and stimulated for 5 days to induce Th17 diff erentiation. The resulting Th17 cells were then stained with APC-conjugated Mouse Anti-Human CD4 Monoclonal Antibody confi rming these markers were on functional Th17 cells. Interestingly, four (Catalog # FAB3791A) and various human surface markers. These data show the various levels of FIGURE 6 Novel Surface Markers on Mouse Th17 Cells of these markers (BTLA, CD200, CD99, and IL-18 R) overlapped with novel staining with the surface markers ranging from negative to high expression. These results are representative of three experiments. Th17 surface markers identifi ed on mouse Th17 cells. Further study of these Lymphocytes markers will enable the development of therapies for autoimmunity and B7-2 Myeloid FIGURE 3 Examples of Known Human and Mouse Th17 Markers Identifi ed in our Screen CD6 CD200 cancer research. CD200 R3 107.2 1.5% 20.3% 107.2 1.8% 50.2% 107.2 2.1% 38.9% CXCR7 Immune Cells CD97v2 Platelets 106 106 106 CD155 CD151(+) 105 105 105 Inflammation

OBJECTIVES Chain-PE CD99

Human 104 J 104 104 IL-18 RD CCR4-PE IL-1R1-PE 103 103 103 BTLA Common Common GOAL: To identify novel cell surface markers on mouse and human Th17 cells. 102 102 102 16.4% 61.9% 5.6% 42.4% 5.2% 53.8% Autoimmunity/ 101 101 101 101 102 103 104 105 106 107.2 101 102 103 104 105 106 107.2 101 102 103 104 105 106 107.2 Cancer Th17 Signaling SIGNIFICANCE: Identify unique markers on Th17 cells to enable further CD4-APC CD4-APC CD4-APC 107.2 45.2% 26.8% 107.2 28.4% 8.3% 107.2 69.8% 26.7% research on Th17 cells that can include applications such as autoimmune 6 6 6 10 10 10 Signaling/ disease treatment and prevention. 105 105 105 Adhesion Mouse 104 104 104 SLAM/CD150 CCR6-PE CD26-PE LAG-3-PE 103 103 103 Development Development 102 102 102 Frizzled-4 14.5% 13.5% 41.8% 21.4% 1.3% 2.2% 101 101 101 METHODS 1 2 3 4 5 6 7.2 1 2 3 4 5 6 7.2 1 2 3 4 5 6 7.2 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 CD4-APC CD4-APC CD4-APC Th17 DIFFERENTIATION PBMCs were purifi ed as described above, and both human cells and mouse splenocytes were Mouse splenocytes were stimulated for 5 days to induce Th17 diff erentiation. The resulting Th17 stimulated for 5 days to induce Th17 diff erentiation. The resulting Th17 cells were stained with cells were co-stained with APC-, Fluorescein-, or PE-conjugated Rat Anti-Mouse CD4 Monoclonal Isolate lymphocytes from human leukopack by density Isolate splenocytes from C56Bl/6 mice an APC-conjugated Mouse Anti-Human CD4 Monoclonal Antibody (Catalog # FAB3791A) or an Antibody (Catalog # FAB554A, # FAB554F, or # FAB554P, respectively) and the surface markers gradient separation APC-conjugated Rat Anti-Mouse CD4 Monoclonal Antibody (Catalog # FAB554A) and the surface shown. The four novel markers that overlap with human Th17 cells are shown in blue. These markers shown. These data validate that known surface markers associated with Th17 cells were results are representative of three experiments. identifi ed in our screen. These results are representative of three experiments. Isolate CD4+ T cells using MagCellect™ Kit

Stimulate CD4+ T cells 2 diff erent ways: FIGURE 4 CD200, IL-18R, BTLA, and CD99 are Novel Surface Markers on Mouse and Human Th17 Cells SUMMARY 5 day diff erentiation Overnight activation Human Mouse BALANCING THE TREG/Th17 RATIO. TGF-E1 TGF- IL-23 E 107.2 107.2 107.2 107.2 IL-2 PMA 11.5% 59.0% 2.9% 68.0% 7.6% 63.8% 21.7% 30.2% IL-23 LPS 106 106 106 106 Anti-CD3 + + CD4 IL-6 CD4 + IL-6 105 105 105 105 CD4 Anti-CD28 4 4 4 4 CD4 Ionomycin 10 10 10 10 Th IL-1E CD200-PE CD200-PE CD200-PE CD200-PE TCR 103 103 103 103 + IL-2 + IL-6 IL-6 R LRRC32/GARP (FoxP3 feedback loop) CD200 (nin SLE) IL-23 R TGF-E RII 102 102 102 102 BTLA (nin RA) CCR4 1 1.4% 28.2% 1 1.9% 27.2% 1 7.6% 21.0% 1 34.6% 13.4% ( in MS; block for tumor IL-17A 10 10 10 10 GITR p 101 102 103 104 105 106 107.2 101 102 103 104 105 106 107.2 101 102 103 104 105 106 107.2 101 102 103 104 105 106 107.2 Treg Th17 CCR6 immunotherapy) IL-18 RD(nin Lupus) Th17 IL-22-CFS IL-17-PerCP RORJt-APC CD4-APC IL-17F CD25/IL-2 R (T cell activation; FoxP3 RORJt ROR t D CD99(T cell activation) J 107.2 107.2 107.2 107.2 IL-21 7.7% 57.6% 3.5% 62.6% 11.4% 55.4% 22.5% 21.4% pin MS) IFN-J 106 106 106 106 TNF-D IL-22 Tolerance Inflammation 105 105 105 105 (Cancer) (Autoimmunity) -PE -PE -PE -APC D D D 104 104 104 D 104 IL-18 R IL-18 R IL-18 R IL-18 103 103 103 R IL-18 103 102 102 102 102 The intricate balance of immune cells prevents the development of 2.2% 32.5% 3.0% 30.9% 10.1% 23.1% 36.1% 20.0% 101 101 101 101 101 102 103 104 105 106 107.2 101 102 103 104 105 106 107.2 101 102 103 104 105 106 107.2 101 102 103 104 105 106 107.2 autoimmunity. T regulatory cells (Tregs) and Th17 cells derive from CD4+ SCREENING OF R&D SYSTEMS CELL SURFACE ANTIBODIES IL-22-CFS IL-17-PerCP RORJt-APC CD4-PE 107.2 28.2% 47.5% 107.2 5.1% 71.1% 107.2 0.3% 74.2% 107.2 7.0% 25.2% precursors in the presence of TGF-1. The key cytokine that skews these cells 106 106 106 106 toward the Th17 lineage is IL-6, while IL-2 promotes Treg diff erentiation. IL-6 Th17 cells co-stained with CD4 105 105 105 105 104 104 104 104 BTLA-PE

BTLA-APC BTLA-APC BTLA-APC signaling induces STAT3, which upregulates RORt, the master regulator ~500 human cell surface antibodies (~240 mouse) added to cells from master 103 103 103 103 102 102 102 102 of Th17 cells. IL-2 signaling induces STAT5, which thereby induces FoxP3, 2.0% 22.3% 4.7% 19.2% 1.2% 24.3% 49.7% 18.1% plate for staining 101 101 101 101 101 102 103 104 105 106 107.2 101 102 103 104 105 106 107.2 101 102 103 104 105 106 107.2 101 102 103 104 105 106 107.2 IL-17-PerCP RORJt-PE IL-22-CFS CD4-APC leading to Treg diff erentiation. FoxP3 has been shown to be antagonistic Th17 cells analyzed for presence of various surface markers on Accuri C6 107.2 1.9% 91.6% 107.2 91.9% 3.5% 107.2 80.7% 13.5% 107.2 49.2% 37.2% towards Th17 cells, whereas IL-6 blocks Treg diff erentiation. When there is a Flow Cytometer (3 repeats) 106 106 106 106 105 105 105 105 skewing of the ratio towards the Th17 lineage, infl ammatory cytokines are

104 104 104 104 CD99-PE List of novel/known markers Negative markers CD99-CFS CD99-CFS CD99-CFS 103 103 103 103 produced, which sets the stage for autoimmune disease progression. The 102 102 102 102 immunosuppressive function of Tregs helps prevent the development of 1.1% 5.4% 3.8% 0.8% 3.7% 2.1% 8.8% 4.9% Confi rm novel/known markers are on functional 101 101 101 101 101 102 103 104 105 106 107.2 101 102 103 104 105 106 107.2 101 102 103 104 105 106 107.2 101 102 103 104 105 106 107.2 Th17 cells (IL-17, IL-22, and RORt co-stains) IL-17-PerCP RORJt-PE IL-22-APC CD4-APC autoimmunity, but this state of tolerance can hinder the immune system PBMCs were purifi ed as described above, and both human cells and mouse splenocytes were Establish collaborations with researchers stimulated for 5 days to induce Th17 diff erentiation. The resulting Th17 cells were stained with an from destroying cancer cells. Representative surface markers for Tregs and APC- or PE-conjugated Mouse Anti-Human CD4 Monoclonal Antibody (Catalog # FAB3791A or Th17 cells illustrate these points. Thus, understanding the correct balance of FAB3791P, respectively) or an APC- or PE-conjugated Rat Anti-Mouse CD4 Monoclonal Antibody (Catalog # FAB554A or # FAB554P, respectively) and the surface markers shown. In order to Tregs and Th17 cells in autoimmunity and cancer is critical for developing identify if these markers were expressed on functional human Th17 cells, a separate experiment immunotherapies for these patients in the clinic. was carried out to examine CD200, IL-18 R, BTLA, and CD99 levels in conjunction with IL-22, IL-17, and RORt. These data are representative of three and two experiments, respectively. RnDSystems.com PS_12.12_Th17_MidwinterImm. For research use only. Not for use in diagnostic procedures.