Human and mouse T cell regulation mediated by soluble CD52 interaction with Siglec-10. Esther Bandala-Sanchez, Yuxia Zhang, Simone Reinwald,

Transcription

1 Human and mouse T cell regulation mediated by soluble CD52 interaction with Siglec-1 Esther Bandala-Sanchez, Yuxia Zhang, Simone Reinwald, James A. Dromey, Bo Han Lee, Junyan Qian, Ralph M Böhmer and Leonard C. Harrison. Nature Immunology doi:1.138/ni.261

2 SUPPLEMENTARY TABLES Supplementary Table 1: Characteristics of study subjects Gender M, F Median age (range) Proportion HLA DR3 and/or 4 Pre-clinical T1D 9, 5 11 (7-21) 13/14 T1D 9, 5 29 (5-64) 13/14 Healthy 9, 5 39 (26-52) 13/14 T2D 3, 2 54 (52-59) 3/5 Supplementary Table 2: Agents tested for ability to prevent suppression by GAD65- activated CD52 hi CD4 + T cells. Agent Antibody to IL-1Rα or TGF-bRII, singly or in combination Indomethacin (cyclooxygenase-2 inhibitor) N(G)-monomethyl-L-arginine (nitric oxide synthase inhibitor) 1-methyl-dl-tryptophan (indoleamine 2,3 dioxygenase inhibitor) SCH58261 (adenosine A2A receptor blocker) Concentration Action tested 1 mg/ml Neutralizes IL-1Rα or TGF-bRII 2 mm Blocks prostaglandin E2 production 8 mm Blocks nitric oxide production 2 µm Blocks production of tryptophan metabolites 2 mm Blocks action of adenosine Nature Immunology doi:1.138/ni.261

3 SUPPLEMENTARY FIGURES AND LEGENDS 3 H-thymidine uptake (cpm) a 2, 1, GAD Number of Treg clone cells b Cell number (% of max) CD Supplementary figure 1: CD52 expression distinguishes GAD65-specific CD4 + suppressor T cell clones. a, Proliferation of a GAD65-specific T cell clone (# 1.4) in the presence of an autologous GAD65-specific suppressor clone (# 3.19). A fixed number (25) of GAD65-specific non-suppressor clone cells was co-cultured in 2 µl in round bottom 96-well plates with increasing numbers of an autologous GAD65-specific suppressor clone and irradiated PBMCs (1x1 5 ) as antigen presenting cells, in the presence or absence of GAD65. 3 H-thymidine uptake was measured after 72 h. The result is representative of multiple autologous suppressor and non-suppressor clone pairs as previously described 14. b, Flow cytometric histograms of CD52 expression by autologous GAD65-specific suppressor (solid line) and non-suppressor (dashed line) clones after overnight stimulation by plate-bound anti-cd3 antibody (1µg/ml). Staining by isotype control antibody is depicted in solid grey. The result is representative of clone pairs from 3 healthy individuals. Nature Immunology doi:1.138/ni.261

4 a b CD45RA CD45RO c IFN-γ (spots/well) d IFN-γ (spots/well) CD45RO 23.2 Hi CD25 CD45RA CD45RO Lo Hi+Lo Lo+Lo CD25 hi cell-depleted PBMCs.5 Post-sort memory Hi 1 * Post-sort naïve 1.2 Lo Hi+Lo Lo+Lo Post-sort ath Control-depleted Hi Lo Hi+Lo Lo+Lo Hi+Lo calc CD8 + T cells Post-sort rt REG.5.5 CD Post-sort at REG CD25 Log (base 2) CD52/HPRT mrna Log (base 2) FOXP3/HPRT mrna Log (base 2) CD25/HPRT mrna CD25 Naive rt REG atreg FOXP3 Naive rt REG at REG ath CD52 Naive rt REG atreg ath Memory Memory ath Memory Supplementary figure 2: CD52 hi CD4 + suppressor T cells are distinct from CD4 + CD25 + T REG cells. a, Resting CD4 + T cells were flow-sorted into naïve (CD45RA + CD25 - ), resting (r) T REG (CD45RA + CD25 + ), adaptive (a) T REG (CD45RO + CD25 ++ ), adaptive T helper (ath) (CD45RO + CD25 + ) and memory (CD45RO + CD25 - ) cell subsets 9. b, RNA was extracted from T cell subsets and analysed in triplicate by quantitative RT-PCR (see Methods). Results are expressed as median+interquartile range. c, ELISpot assay (as in Figure 1b) of IFN-γ secretion by tetanus toxoidactivated () CD4 + T cells sorted and re-activated by, after depletion of CD25 + cells from PBMCs. CD25 + cells were depleted (~9%) from PBMCs by anti-cd25 antibody on an AutoMACS column; an isotype-matched antibody was used for the control depletion. d, ELISpot assay of IFN-γ secretion by -activated CD8 + T cells sorted and re-activated by. * P <.1 (ANOVA) for -activated cells; posttest paired comparisons were all significant (P <.5). Nature Immunology doi:1.138/ni.261

5 6 IFN-γ (spots/well) 4 2 CD52 hi CD52 hi CD52 lo CD52 lo CD24 lo CD24 hi CD24 lo CD24 hi Supplementary figure 3: CD24 expression is not a marker of suppression by CD52 hi CD4 + T cells. CFSE-labeled PBMCs were incubated with tetanus toxoid () for 7 days and sorted into CD4 + T cell populations based on CD52 and CD24 expression. Each population (5x1 3 cells) was incubated with sorted CD52 lo responder cells (5x1 3 ), and irradiated PBMCs (2x1 4 ) in triplicate and analysed by IFN-γ ELISpot assay. Nature Immunology doi:1.138/ni.261

6 3H-thymidine uptake (cpm) 3H-thymidine uptake (cpm) 3, 2, 1, 3, 2, 1, Donor 1 Donor 2 CD52 lo CD52 lo hi CD52 CD52 lo hi CD52 CD52 lo Supplementary figure 4: Suppression by CD52 hi CD4 + T cells occurs without cellcell contact. Proliferation by tetanus toxoid ()-activated and sorted CD52 hi and CD52 lo CD4 + T cells combined or separated by a semi-permeable.4 mm transwell then re-activated with. CFSE-labeled PBMCs were incubated with for 7 days and sorted into CD52 hi and CD52 lo CD4 + T cells. Sorted cells (1 5 each) were incubated with irradiated autologous PBMCs (4x1 5 ) and in quadruplicate in 48- well plates. In the presence of the transwell both compartments contained irradiated PBMCs and. 3 H-thymidine uptake by cells in the lower compartment was measured after 48 h. Results shown for two donors. Nature Immunology doi:1.138/ni.261

7 a b 3, 3 H-thymidine uptake (cpm) 2, 1, CD52-Fc (2.5 μg/ml) + CD52-Fc (1 μg/ml) + CD52-Fc (25 μg/ml) + Fc control (25 μg/ml) + 3 H-thymidine uptake (cpm) 4, 2, No antibodies Anti-CD3/CD28 Anti-CD3/CD28 + CD52-Fc (5 μg/ml) Anti-CD3/CD28 + Fc (5 μg/ml) c Concentration (pg/ml) 1, IFN-γ IL-2 IL-1 IL-17 G-CSF TNF-α CD52-Fc + Fc + CD52-Fc + Fc + Supplementary figure 5: Recombinant CD52-Fc suppresses T cell proliferation and cytokine secretion. a, PBMCs (2x1 5 ) were cultured with tetanus toxoid () for 7 days or b, purified CD4 + T cells (2x1 4 ) were cultured with plate-bound anti-cd3 (1 ng/ml) and soluble anti-cd28 (2 ng/ml) antibodies for 48 h, in the presence of CD52-Fc or Fc proteins. 3 H-thymidine uptake was measured over the last 16 h of culture. c, media from PBMCs incubated for 48 h with ± the highest concentration of CD52-Fc or Fc in a were assayed for cytokines by multiplex bead array. Results are representative of six independent experiments , 2, 1, 1, , 1,5 1, 5 CD52-Fc + Fc + Nature Immunology doi:1.138/ni.261

8 1 % of maximum % of Max Anti-CD3 antibody Tetanus toxoid No stimulus <PE-A>: Siglec-1 Siglec-1 Supplementary figure 6: Siglec-1 expression is upregulated on activated human T cells. Flow cytometric histograms of siglec-1 expression on CD4 + T cells. PBMCs were incubated with tetanus toxoid () or soluble anti-cd3 antibody (1µg/ml) for 4 days. Nature Immunology doi:1.138/ni.261