Daan Pieren

104 Chapter 4 -40 -20 0 20 40 60 80 100 Induction of CD69 in subset (%) (Relative to %CD69 + cells at day 0) 1 3 Time (days) TIGIT Helios + + + - - + - - 1 3 1 3 1 3 ** ns * ns *** ns *** ns *** *** -20 0 20 40 60 80 100 Induction of CD25 in subset (%) (Relative to %CD69 + cells at day 0) 1 3 Time (days) TIGIT Helios + + + - - + - - 1 3 1 3 1 3 *** **** *** 0.07 *** *** *** *** *** B C 0 20 40 60 80 % subset of CD8 + T cells ns *** ns ns *** ns ns ns 0 1 3 Time (days) TIGIT Helios + + + - - + - - 0 1 3 0 1 3 0 1 3 A 26.1 17.7 47.4 8.77 TIGIT (PE-CF594) Helios (PE-Cy7) 0 20 40 60 80 100 % Proliferation of subset (Relative to control) *** ns *** *** *** *** TIGIT Helios + + + - - + - - 0 20 40 60 80 0 20 40 60 80 100 % TIGIT + Helios + cells in CD8 + T cells (T=3, 0.005 aCD3) % Proliferation of CD8 + T cells (Relative to control) -0.741 <0.001 r p D TIGIT - Helios - TIGIT - Helios + TIGIT + Helios + TIGIT + Helios - 9.57 54.5 88.2 84.6 E CellTrace Violet (BV421) Figure 1. Co-expression of TIGIT and Helios defines functionally senescent T cells ( A ) Representative flow-cytometry plot with expression of TIGIT and Helios by CD8 + T cells and the frequency of TIGIT/Helios cell subsets among CD8 + T cells over time after stimulation with anti-CD3 (day zero, one, and three) are shown. Induction of T-cell activation was assessed by measuring the frequency of ( B ) CD69 + cells and ( C ) CD25 + cells over time within the indicated TIGIT/Helios cell subsets relative to the frequency of CD69 + and CD25 + cells determined at day zero (% CD69 + or CD25 + cells at indicated day minus their % at day 0) (n=13). T-cell proliferation was assessed by dilution of CellTrace after three days of stimulation with anti- CD3. ( D ) Representative histogram shows the proliferation of the indicated TIGIT/Helios cell subsets by dilution of CellTrace (number indicates the frequency of proliferated cells of the subset). Bar graph shows the frequency of proliferated cells within each TIGIT/Helios cell subset