Anti tcrβ

Mouse tumor samples were excised and incubated with 10 ml RPMI supplemented with 10 μg/ml DNase I (Sigma Aldrich) and 25 μg/ml Liberase (Roche) for 10–15 min at 37 °C. Tumors were homogenized by pipetting and filtering through a 100-μm nylon filter. Following the dissociation, 5 × 10⁶ cells were pelleted and resuspended in FACS buffer and blocked with monoclonal antibody to CD16/32 (Trustain fcX, Biolegend) for 10 min before staining. All tumors were stained with anti-CD45 (Biolegend, Clone 30-F11), anti-TCRβ(BD, Clone H57-97), anti-NK1.1 (eBioscience, Clone PK136), anti-CD11b (Biolegend, Clone M1/70), anti-F4/80 (Biolegend, Clone BM8), anti-Ly6C (BD, Clone AL-21), anti-CD11c (Biolegened, Blone N418) and anti-Gr-1 (Biolegend, Clone RB6-8C5). Dead cells were eliminated from analysis by excluding Hoechst⁺ cells. All samples were assayed using a FACSAria cell sorter (BD Biosciences), and channel compensations were performed using single-stained UltraComp eBeads (Affymetrix) or cells. Data were analyzed using FlowJo, and outliers in each group were removed using Prism Outlier Calculator (http://graphpad.com/quickcalcs/Grubbs1.cfm).

8×10⁶ splenocytes from NY8.3 TCR transgenic mice or BDC2.5 TCR transgenic mice were labeled with cell trace violet (CTV) (ThermoFisher) according to the manufactures protocol and i.v. transferred into 8 weeks old recipient mice. Pancreatic and inguinal LNs from recipient mice were harvested 3 days post-transfer and analyzed by flow cytometry following staining with the following antibodies: anti-TCRβ, anti-B220, anti-CD4, anti-CD8, anti-CD44 Biolegend, anti-Vβ8, anti-Vβ4 BD Biosciences. Transferred cells were gated for expression of TCRβ, CTV, CD4 and Vβ4 (BDC2.5) or CD8 and Vβ8 (NY8.3).

Myeloid and lymphoid subsets were isolated from the tumors and quantitatively analyzed as previously described (Chowdhury et al., 2019 (link)) with some modifications. Briefly, after mechanical homogenization, the tumors were digested with collagenase A (1 mg ml−1; Roche) and DNase I (0.5 μg ml−1; Roche) in isolation buffer (RPMI 1640 supplemented with 5% FBS, 1% L-glutamine, 1% penicillin–streptomycin and 10 mM HEPES) for 45 minutes shaking (150 rpm) at 37 °C. Cells were filtered through 100 μm cell strainers, washed in isolation buffer and stained. Myeloid cells were stained immediately, and lymphoid subset underwent a separation gradient using Percoll (67%, 40%), followed by staining. Dead cells were excluded by staining with Ghost Dye cell viability reagent. Extracellular antibodies included: anti-B220 (BD) (1:200), anti-CD19 (Tonbo) (1:200), anti-CD45 (BD and Biolegend) (1:400), anti-CD4 (BD) (1:400), anti-CD8 (Tonbo) (1:400), anti-NK1.1 (BD) (1:300), anti-CD11b (BD) (1:500), anti-CD11c (BD) (1:200), anti-F4/80 (Tonbo) (1:500), and anti-MHC class II (Tonbo) (1:400) antibodies. Intracellular antibodies included: anti-CD3e (BD) (1:400), anti-TCRβ (BD) (1:300) and anti-FOXP3 (Thermo) (1:300). Cells were fixed using the FOXP3/transcription factor staining buffer set (Tonbo) according to the manufacturer’s protocol. Samples were analyzed using a BD LSRFortessa cell analyzer.