Marilen Benner

CHAPTER 3 78 Supplementary Figure S4. Treg suppression of T cell proliferation. Related to Figure 2. A) CFSE dilution of 6-12wk decidual CD4+ Teff cells stimulated with anti-CD3/28 and co-cultured with or without PD1 dim , PD1 HI or CD25 HI cells and B) CFSE dilution of peripheral blood CD4+ Teff cells (upper panels) and CD8+ Teff cells (bottom panels) stimulated with anti-CD3/28 and co-cultured with or without CD25 HI , PD1 HI or TIGIT+ cells. Cells were cultured for four days in a 1:2, Treg:Teff ratio. Percentages of the undivided Teff cells are shown. C) Fold Change (FC) in percentage undivided peripheral blood Teff cells and D) FC in division index after addition of CD25 HI , PD1 HI or TIGIT+ Treg, compared to CD4+ Teff cells (left panels) and CD8+ Teff cells (right panels) cultured alone. Bars represent median and interquartile range; n=5-7; *P<0.05, Kruskal-Wallis with Dunn’s post-test comparing columns B-D to column A was performed. Values >1 for FC %undivided cells and <1 for FC division index represent suppression of proliferation. Division index reflects the number of divisions per cell as calculated in FlowJO v7.6.5. B A C CD4+ Teff Undivided cells 5.5% Division Index 1.45 CD4+ Teff + CD25 HI Undivided cells 14.6% Division Index 0.89 CD4+ Teff + PD1 HI Undivided cells 16.7% Division Index 0.74 CD4+ Teff +PD1dim Undivided cells 6.7% Division Index 1.39 CD8+ Teff CD4+ Teff Teff Teff + TIGIT+ Teff + PD1 HI Teff +CD25 HI 7.3% 24% 18% 14% 12% 29% 22% 18% Teff + PD1 HI Teff +TIGIT+ Teff Teff + CD25 HI 0 1 2 3 4 F C % U n d iv id e d c e l ls * 0 1 2 3 4 S u p p r e s s io n * * - C D 2 5 H I P D 1 H I T IG IT + 0 .0 0 .5 1 .0 1 .5 F C D iv is io n In d e x * - C D 2 5 H I P D 1 H I T IG IT + 0 .0 0 .5 1 .0 1 .5 S u p p r e s s io n CD8+ Teff CD4+ Teff CD8+ Teff CD4+ Teff D 5.5% 6.7% 17% 15% Supplementary Figure S4. Treg suppression of T cell proliferation. Related to Figure 2. (A) CFSE dilution of 6-12wk ecidual CD4+ Teff cells stimulated with anti-C 3/28 and co-cultured with or without PD1 dim , PD1 HI or CD25 HI cells and (B) CFSE dilution of peripheral blood CD4+ Teff cells (upper panels) and 8+ Teff cells (bottom panels) stimulated ith anti-CD3/28 and co-cultured with or without CD25 HI , PD1 HI or TIGIT+ cells. Cells were cultured for four days in a 1:2, Treg:Teff ratio. Percentages of the undivided Teff cells are shown. (C) Fold Change (FC) in percentage undivided peripheral blood Teff cells and (D) FC in division index after addition of CD25 HI , PD1 HI or TIGIT+ Treg, compared to CD4+ Teff cells (left panels) and CD8+ Teff cells (right panels) cultured alone. Bars represent median and interquartile range; n=5-7; *P<0.05, Kruskal-Wallis with Dunn’s post-test comparing columns B-D to column A was performed. Valu s >1 for FC %undivided cells and <1 for FC division index represent suppression of proliferation. Division index reflects the number of divisions per cell as calculated in FlowJO v7.6.5.