Clone 6d5

To block Fc receptors, cells were incubated with 0.5 µl of TruStain FcX PLUS in 50 µl FACS of buffer. Thirty minutes later, anti-CD45 APC (1.25 µl; clone 30-F11, Biolegend; 5 µg/ml) and anti-CD19 BV421 (1.5 µl; clone 6D5, Biolegend; 1.5 µg/ml) were added in 50 µl of FACS buffer. Following incubation in darkness for 30 min at room temperature (RT), the cells were washed and resuspended in 300 µl of FACS buffer containing 0.3 µl of Sytox Green viability dye (Biolegend; 5 µM). A BD FACS Aria cell sorter was used to isolate CD45⁺ cells into RPMI + 2% FBS and resuspend them in 50 µl of PBS + 2% FBS. CD19⁺ signal was used to assess bone marrow contamination. In all samples, CD19⁺ cells represented less than 5% of total CD45⁺ cells, indicating minimal bone marrow contamination. Pooled CD45⁺ cell suspensions (n = 2–3 mice per group) were transferred to the MD Anderson Advanced Technology Genomics Core (ATGC, CA016672) for single-cell library preparation. Cell density and viability were determined using a Countess II FL auto-counter. Single cells were captured (~ 6000–9000 cells/sample) using Chromium Controller (10X Genomics), and sequencing was done by Novaseq 6000.

SA1/N and B16-SIY tumor analyses were performed 2 days following the second dose of antibody treatment. Tumors were weighed on a microscale on plastic weigh boats and dissociated with a 0.25mg/mL Liberase TL (Sigma-Aldrich), 0.05mg/mL DNaseI (Roche) from bovine pancreas, and RPMI Medium mixture for 20 min at 37To determine ICOS receptor quantity on primary cells, tumors were processed as described above. Peripheral blood was collected by cardiac puncture with 25-gauge needles and tuberculin syringes and dispersed into EDTA coated blood collection tubes. RBCs were lysed in conical tubes with 1mL of ACK lysis buffer for 3min on ice. Blood was centrifuged at 400xg for 5mins and resuspended in 100uL FACS buffer. Tumors and peripheral blood were transferred to round bottom 96-well plates and Fc blocked as described above. Following Fc block, cells were stained with 1ug/mL DyLight-650 (Life Technologies) labelled ICOS antibody 37A10 or isotype control, in addition to the staining cocktail described above. Cells were stained in 100uL of cocktail for 30mins at 4ºC. Cells were then washed twice with FACS buffer and resuspended 100uL in Fix/Perm buffer (Foxp3/Transcription Factor Staining Kit, eBioscience) and kept at 4ºC for 15mins. Cells were washed twice in Perm buffer and resuspended in 100uL of intracellular staining cocktail containing fluorescent labelled anti-FoxP3 (FJK-16s, 1:100, eBioscience) diluted in Perm buffer. Following a 30min incubation at 4ºC, cells were washed twice in FACS buffer and resuspended in 100uL of FACS buffer supplemented with 0.1% paraformaldehyde. Receptor quantitation was performed using Quantum Simply Cellular beads (Bangs Laboratories) according to manufacturer’s protocol. To determine the number of cells per milligram of tumor, a fixed number of CountBrite beads (Life Technologies) were added to samples and analyzed by flow cytometry.
For TIL analysis of MC38 tumors, huCTLA-4 mice were randomized in groups when tumor reached an average size of 153mm³ and treated with a single dose of clinical grade ipilimumab (Yervoy®, Bristol-Meyers Squibb) or human IgG1 isotype control administered intravenously at 10 mg/kg, or a single dose of ICOS antibody or mouse IgG2a isotype control administered intraperitoneally at 0.25 mg.kg. Mice were sacrificed 72 hours later and tumors excised, dissociated, and processed for flow cytometry analysis. The following antibodies were used: CD45-BB515 (clone 30-F11, BD), CD3-BUV395 (clone 145-2C11, BD), CD4-APCeF780 (clone GK1.5, eBiosciences), CD8-PEeF610 (clone 53–6.7, eBiosciences), FoxP3-PE (clone NRRF-30, eBiosciences), CD25-PerCP/Cy5.5 (clone PC61, Biolegend), CD19-BV605 (clone 6D5, Biolegend), CD49b-APC (clone DX5, Biolegend), ICOS-BV421 (clone C398.4A, Biolegend). Viability was assessed by staining with Fixable Viability Dye eF506 (Biolegend).

Cells were fixed with 2% PFA for 10 min, spun down, resuspended in PBS and stored at 4 °C until analysis. Cells were washed two times with FACS buffer (PBS containing 2% FBS). Labeled with anti-CD3 (APC-Cy7 – Biolegend; Clone 17A2), anti-CD11b (Pacific Blue – Biolegend; Clone M1/70), anti-CD11c (Pe-Cy7 – BD Pharmingen; Clone HL3), and anti-CD19 (PE – Biolegend; Clone 6D5) antibodies for 1 h at room temperature, and washed twice with FACS buffer. They were then permeabilized and probed with TLR3 (APC - Biolegend; Clone 11F8) or a Rat IgG2a isotype (APC – Biolgend; Clone RTK2758) for 30 min at room temperature, and washed twice with FACS buffer. Samples were run on a BD LSRII flow cytometer and analyzed using FlowJo software, gating with the assistance of unstained and isotype stained samples.