Anti human cd8 clone rpa t8

Tissue samples were collected and transported in RPMI-1640 (Sigma, cat# R0883-500ML). Single cell suspensions were produced by enzymatic digestion using liberase with subsequent cellular disaggregation using a Miltenyi gentleMACS Octo Dissociator. Lymphocytes were isolated from single cell suspension by gradient centrifugation on Ficoll Paque Plus (GE Healthcare, cat# 17-1440-03) and stored in liquid nitrogen. Blood samples were collected in BD Vacutainer EDTA blood collection tubes (BD cat# 367525), PBMC’s were then isolated by gradient centrifugation on Ficoll Paque (GE Healthcare, cat# 17-1440-03) and stored in liquid nitrogen.
FC receptors were blocked with Human Fc Receptor Binding Inhibitor (Thermo) before staining. Non-viable cells were stained using the eBioscience Fixable Viability Dye eFluor 780 (Thermo). Cells were stained in BD Brilliant stain buffer (BD cat# 563794) with the following monoclonal antibodies: anti-human CD3 (clone SK7, BD cat# 565511), anti-human CD4 (clone SK3, BD cat# 566003), anti-human CD8 (clone RPA-T8, BD cat# 564804). Data was acquired on a BD Symphony flow cytometer and analyzed in FlowJo. Cells were gated for size, single cells, live cells, CD3+CD8+ T cells.

NOD-scid IL2Rgamma^null (NSG) mice were engrafted with either 5 × 10⁶ MEL 526 MEL tumor cells or 5 × 10⁶ autologous primary melanoma tumor cells. On day 12, either 10 ×10⁶ sorted CD8⁺BTLA⁺ or sorted CD8⁺BTLA⁻ were adoptively transferred into tumor-bearing mice (n= 5 to 8 per group). Recombinant human IL-2 (Proleukin, Prometheus) was administered intraperitoneally at a concentration of 6 ×10⁵ I.U. immediately after TIL transfer and daily for three days. Tumor size was measured every other day. Mice were sacrificed when tumors exceeded 15 mm diameter. Peripheral blood was collected every other day, lysed with ACK lysis buffer, then stained for AQUA (Invitrogen), anti-human CD45 (clone HI30, BD Pharmingen™), and anti-human CD8 (clone RPA-T8, BD Pharmingen™).