Cd8 fitc sk1

Viably frozen BM mononuclear cells (N = 31) were first thawed and stained using CD3-PerCP-Cy5.5 (SK7, BD Pharmingen), CD8-FITC (SK1, BD Pharmingen), CD4-BV510 (SK3, BD Pharmingen), TIM3-PE-Cy7 (F38-2E2, Invitrogen), PD1-AlexaF647 (EH12.1, BD Pharmingen), CD45-BV421 (HI30, BD Horizon), CCR7-PE (150503, R&D Systems), and CD45RA-AlexaF700 (HI100, BD Pharmingen) antibodies. Fluorescence was measured with FACSVerse (BD Pharmingen). The proportion of CD3+CD4+/PD1+TIM3+ cells was used in survival analysis similarly as with mIHC data (Supplementary Figure S1).

Immunophenotyping was performed on peripheral blood samples collected longitudinally and at the study endpoint on whole blood and on mononuclear cells isolated from the tissues of humanized mice. Prior to antibody incubation, Ig-binding sites were blocked. The fluorochrome-conjugated antibodies included CD45-V500 (HI30, BD Biosciences, San Jose, USA), CD3-APC-R700 (UCHT1, BD Biosciences, San Jose, USA), CD4-APC-H7 (RPA-T4, BD Biosciences, San Jose, USA), CD8-FITC (SK1, BD Biosciences, San Jose, USA), CD19-PE-Cy7 (SJ25C1, BD Biosciences, San Jose, USA), CD27-PE (M-T271, BD Biosciences, San Jose, USA), CD38-APC (HB7, BD Biosciences, San Jose, USA), CD45RA-Pacific Blue (F8-11-13, Bio-Rad, Hercules, USA), HLA-DR-PerCP (L243, BD Biosciences, San Jose, USA), mouse IgG1k-APC (MOPC-21, BD Biosciences, San Jose, USA), mouse IgG1k- Pacific Blue (MOPC-21, BD Biosciences, San Jose, USA), mouse IgG1k-PE (MOPC-21, BD Biosciences, San Jose, USA) and mouse IgG2ak-PerCP (X39, BD Biosciences, San Jose, USA). After antibody incubation, blood samples were lysed with t 1× BD FACS lysing solution (BD Biosciences, San Jose, USA). Samples were then analyzed on a BD LSRFortessa instrument (BD Biosciences, San Jose, USA).