Cd3 7d6

Clear cell RCC patient tissue specimens were prepared by mechanical disruption using a razor blade followed by treatment with 280 U/mL Collagenase Type 3 (Worthington Biochemical) and 4 ug/mL DNase I (Sigma) at 37°C for 1 hour with periodic vortexing. Digested tissues were passed through 70 um filters. Resulting cells were resuspended in 44% Percoll – 66% Percoll gradient (Sigma) and centrifuged for 30 minutes at 1,900 g with no brake. Mononuclear cells were collected and immediately stained for flow cytometry analysis following Fc blocking (company) and live/dead staining (company). The antibodies used for flow cytometry were: HLA-DR (L243, Biolegend #307618), CD14 (HCD14; Biolegend #325608), CD45 (2D1; eBioscience 11–9459-42), CD16 (3G8; Biolegend 302008), CD56 (HCD56; Biolegend 318318), and CD3 (7D6; Invitrogen MHCD0317). Tumor-associated macrophages (TAM) were identified as CD45⁺CD3^–Lin^-HLA-DR⁺CD14⁺CD16⁺. Data are reported as the percent of all cells.

CD19 (HIB19), CD38 (HIT2) and CD20 (2H7) came from Biolegend, CD27 (CLB-27/1) and CD3 (7D6) were purchased from Invitrogen. Influenza stocks were freshly grown in eggs, harvested and purified using PEG virus precipitation (BioVision Research Products, CA) according to the manufacturer's instructions and stored at −80°C. Viral hemagglutination activity units (HAU) was used to quantify virus as measured by incubating serial dilutions of virus with a 1:1 ratio of 0.5% turkey red blood cells (Lampire Biological Laboratories). 1 HAU represents the minimum viral amount capable of causing red blood cell agglutination. Recombinant HA was produced as described previously (54 (link)) (H1N1 strains) or obtained from BEI Resources (H5N1 A/Vietnam/1203/2004).