Easysep

Human tonsil was obtained tissue procurement lab in the University of Michigan. Study was approved by the local Institutional Review Board. Human peripheral blood samples were obtained from healthy volunteers by venipuncture or cytopheresis. Human umbilical cord blood was collected by C.S. Mott Children's Hospital (University of Michigan) in blood collection tubes (Becton Dickinson Biosciences). Mononuclear cells were collected by Ficoll-Hypaque or Lymphoprep density gradient centrifugation. Peripheral blood naïve T cells were isolated by RossetteSep (Stemcell Technologies) followed by CD45RA⁺ or CD45RO⁺ microbeads (Myltenyi) column selection. Naïve T cells from umbilical cord blood and human tonsil were isolated by first depleting CD14⁺ monocytes followed by positive selection of CD4⁺ T cells using CD14⁺ monocyte selection EasySep and CD4⁺ T cell selection EasySep (Stemcell Technologies). CD4⁺CD45RA⁺CD31^+/− naïve T cells were sorted from single-cell suspensions by high-speed cell sorter (FACSAria, Becton Dickinson Immunocytometry Systems). Neutrophils were isolated as described from adult blood (18 (link)). Cellular purity was measured by flow cytometry (LSRII, BD). Surface and cytokine profiles were measured through surface and intracellular staining, respectively, and analyzed by LSRII, DIVA and FloJo software (19 (link)–21 ).

PBMC were isolated as described. CD4⁺ memory T cells were enriched from healthy human donor PBMC in 2% PHS/1.5mM EDTA/ Dulbeco’s PBS through cell sorting with dextran-coated magnetic particles using bispecific tetrameric antibody complexes (EasySep™, Stemcell) to CD8, CD14, CD16, CD19, CD20, CD36, CD45RA, CD56, CD123, TCRyS according to the manufacturers’ instructions. In selected experiments, naive CD4⁺ T cells were isolated (EasySep™, Stemcell) from either human peripheral or cord blood-derived PBMC. All T cell cultures were performed in RPMI supplemented with 1 mM HEPES (Life Technologies), 2mM glutamine (Sigma), streptomycin (100 IU/mL)/Penicillin 100 μg/mL, and 5% PHS AB (Gibco) at T 37 °C, and CO₂ 5%. For co-culture studies T cells were cultured with moDCs for a duration of 12–14 days. IL-2 (30 IU/mL) was added on day 3 to the culture system to maintain cultures.

Analyses of blood and splenocytes were performed as previously described [25 (link), 26 (link)]. For recovery of lymphocytes from tumors, tumor masses were placed in digestion media (1x HBSS [Cellgro], 0.1 mg/ml Collagenase A [Worthington], 60 U/ml DNase I [Roche]) [19 (link)] and minced using the gentleMACS™ Octo Dissociator using C Tubes (Miltenyi Biotec). Minced tumors were incubated at 37°C for 30 minutes with continuous rotation, minced again as above, washed twice with T cell media (RPMI 1640 [Cellgro] with L-glutamine + 10% FBS + 1% PenStrep and 5 × 10⁻⁵ M β-mercaptoethanol [Omnipur, Calbiochem]), and filtered through a 70 µm nylon filter. In most experiments, lymphocytes were then either directly assessed by flow cytometry or tested for their ability to produce cytokines upon stimulation. In some experiments (Fig. 6 A–C, and Supplemental Fig 3A), lymphocytes were enriched from tumor homogenates by spinning through a 40%/80% Percoll (GE Lifesciences) gradient for 25 minutes at 600 × g and collecting cells at the interface. For Supplemental Fig. 3ACD8⁺ T cells were further enriched with a biotin selection kit and magnetic beads (EasySep, StemCell Technologies) and biotinylated antibodies specific for NK1.1 (clone PK136), TER-119 (clone TER-119), CD4 (clone GK1.5), and CD19 (clone 6D5), all from Biolegend. Enriched cells were ~80% CD8⁺.