Daan Pieren

116 Chapter 4 A TIGIT Helios CD27 CD28 viSNE2 viSNE1 Lymphocytes Single cells Live CD3 + T cells FSC-A FSC-H Live/Dead CD3 (FITC) SSC-A FSC-A CD8 + T cells CD8 (BUV395) CD4 (PerCP-Cy5.5) CD27 (BV510) CD28 (BV711) TIGIT (PE-CF594) Helios (PE-Cy7) CD27/CD28 differentiation stage TIGIT/Helios gating B Supplementary Figure 2. Gating strategy towards CD8 + T cells and their separation into TIGIT/Helios subsets and CD27/CD28 subsets. ( A ) Fingerprint color dot plots generated by viSNE indicate expression of each of the indicated markers within the CD8 + T-cell population of a representative donor as measured by flow cytometry. ( B ) Gating strategy towards stratification of CD8 + T cells into TIGIT/Helios cell or CD27/CD28 subsets as performed in healthy individuals using flow cytometry. CD8 + T cells identified by gating of total lymphocytes, single cells, and live CD3 + T cells. Additionally, CD8 + T cells were gated into early (CD27 + CD28 + ), intermediate (CD27 + CD28 - ), and late-differentiated (CD27 - CD28 - ) cells based on their expression of CD27 and CD28. Total CD8 + T cells as well as CD8 + T cells in one of the three differentiation stages were also separated into TIGIT/Helios cell subsets.