Cd4 rm4 5

The following antibodies were used in this study: CD4 (RM4–5; Biolegend), CD8⍺ (53–6.7; Biolegend), Thy1.1 (OX-7; Biolegend), CD44 (1M7; Biolegend), CD69 (H1.2F3; Biolegend), CD103 (2E7; Biolegend), V⍺2 (B20.1; Biolegend), IFNγ (XMG1.2; Biolegend), TNF⍺ (MP6-XT22; Biolegend), IL-2 (JES6–5H4; Biolegend). MHC-II tetramers were obtained from the National Institutes of Health tetramer core facility. Tetramer staining was performed for 1 hour at 37° C and a tetramer loaded with a human CLIP peptide was used as a negative staining control. All other staining was performed for 15–30 minutes at 4° C. Intravascular labeling was performed as described previously (25 (link)). Briefly, 3 μg of anti-V⍺2 was injected i.v. in 200 μl of PBS, and tissues were harvested 3 minutes later. Data were acquired using a LSRII Flow Cytometer (BD) in the OHSU Flow Cytometry Core Facility. Flow cytometry data were analyzed using FlowJo software (BD) version 9.9.6 or 10.5.3.

To prepare single cell suspension, ventral and dorsal sheets of the ears were separated from the cartilage and incubated for 90 min in CO2 incubator at 37°C in 1 mL volume of RPMI 1640 (Sigma-Aldrich, #R7388) containing 0.25 mg ml⁻¹ Liberase TL (Roche Diagnostics, #5401020001). The digested ears were passed through a 3 mL syringe to make single-cell suspension. The cells were filtered through 70 μm nylon mesh and washed in FACS buffer at 1500 rpm for 5 minutes. Cells were suspended in FACS buffer for further analysis. For surface staining the following antibodies were used at 1:100 dilutions in FACS buffer according to the manufacture’s specifications. CD45 (30-F11, eBiosciences), CD3 (17A2, eBiosciences), CD90.2 (53–2.1, eBiosciences), βTCR (H57–597, eBiosciences), CD4 (RM4–5, Biolegend), CD8 (YTS5167.7, eBiosciences), CD11b (M1/70, eBiosciences), Ly6G (1A8, eBiosciences), and Ly6C (AL-21, BD Pharmingen). For counting the cells AccuCount Fluorecent particles (Spherotech) were used. The stained cells were run on BD FACSymphony^™A3 (BD Biosciences) and the acquired data were analyzed using FlowJo software (Tree Star).

Excised tumors were minced into pieces, and then dissociated by passing through a 70 μm cell strainer to obtain single cell suspension. Spleens were processed by mechanical dissociation, followed by lysis of red blood cells (Cat#A10492-01, Gibco). Ficoll-Paque PLUS (Cat#17-1440-03, GE Healthcare Life Sciences) was added to the bottom of the conical tubes containing single cell suspension in RPMI-1640. Density gradient centrifugation was performed to purify immune cells. Rare sample with inadequate number of TILs is excluded from further processing. Flow cytometry antibodies include: CD3 (17A2, BD Biosciences), CD4 (RM4-5, Biolegend), CD8 (53-6.7, Biolegend), CD366 (RMT3-23, Biolegend), CD279 (29F.1A12, Biolegend), CD16/32 (93, eBioscience), MHC-class II (M5/114.15.2, eBioscience), CD86 (GL1, eBioscience), tetramer recognizing HLA-A*0201-restricted EGFR 854L.ILDFGLAKL (NIH tetramer core), tetramer recognizing H-2D^b-restricted HPV16 E7 epitope RAHYNIVTF (NIH tetramer core), and viability dye (Cat#65-0865-14, eBioscience). All data were analyzed using FlowJo.

Antibodies used for flow cytometry included B220 (RA3-6B2, Biolegend), CD3 (clone 145-2C11, BD Biosciences), CD4 (RM4-5, Biolegend), CD8 (53-6.7, Biolegend), CD11b (M1/70, eBioscience), CD11c (N418, eBioscience), CD38 (90, eBioscience), CD44 (IM7, eBioscience), F4/80 (BM8, eBioscience), GL7 (GL7, BD Biosciences), Gr-1 (RB6, eBioscience), IgM (eB121-15F9, eBioscience), IL-17A (TC11-18H10, BD Biosciences), IL-17F (18F10, eBioscience), and IL-17RA (PAJ-17R, eBioscience).

Antibodies against the following proteins were purchased from BD Biosciences: CD4 (RM4-5), CD69 (H1.2F3), CD90.1 (OX-7), phospho-STAT5^Y694 (C71E5), and CD90.2 (53-2.1). Antibodies against the following proteins were purchased from eBioscience: CD44 (IM7), CD98 (RL388), ICOS (7E.17G9), IRF4 (3E4), Ki-67 (SolA15), CD39 (24DMS1), KLRG1 (2F1), and FoxP3 (FJK-16s). Antibodies against the following proteins were purchased from BioLegend: CD4 (RM4-5), CD45 (30-F11), CD62L (MEL-14), CTLA-4 (UC10-4F10-11), PD-1 (29F.1A12), CD25 (PC61), and Bcl2 (BCL/10C4). Normal rabbit IgG (2729) and anti-phospho-S6^S240/244 (5364) were purchased from Cell Signaling Technology. Goat anti-rabbit-Alexa Fluor 647 secondary antibody was purchased from Invitrogen. Fc Block (2.4G2) and anti-CD28 (37.51) were purchased from Bio X Cell. Stimulatory anti-CD3 (2C11) was purified from hybridoma supernatants prepared in-house. Fixable viability dye eFluor780 was purchased from eBioscience. MitoTracker Deep Red dye was purchased from Invitrogen. Flow cytometry experiments were performed on a FACSCalibur, LSR II, or FACSCelesta (BD Biosciences), and analyzed using FlowJo software (Treestar, v.10.3) or FCS Express (De Novo Software, v.6). Cell sorting was performed on a FACSAria II or FACSAria Fusion (BD Biosciences).