Fixable viability dye

Two days prior to mRNA immunization, 2 × 10⁶ OT-I or OT-II cells were purified and labeled with 5 µM of CFSE (Invitrogen, Merelbeke, Belgium) and subsequently adoptively transferred via i.v. injection into mice that had been s.c. inoculated with B16 cells. Four days after the mRNA treatment, draining lymph nodes were isolated and OT-I or OT-II cell division was analyzed by flow cytometry. Cells were stained with anti-CD16/CD32 (500× dilution) (BD Biosciences) to block Fc receptors followed by staining with Fixable Viability Dye (1000× dilution) (BD Biosciences), CD8 PE-Cy7 (200× dilution) (eBiosciences), CD3 efluor450 (200× dilution) (eBioscience), anti-CD19 allophycocyanin (APC; 200× dilution) (BD Biosciences), and MHC-I dextramer H-2 Kb/SINFEKL-PE (500× dilution) (Immundex). The experiments were performed on a triple-laser (B-V-R) LSR-II (Becton Dickinson, San Jose, CA, USA), and data were analyzed using the FlowJo software (Treestar, OR). Single cells were gated based on FSC and SSC. Living cells were selected and T cells gated for CD3⁺ CD19⁻ T cells. Within the CD8⁺ T cells or CD4⁺ T cells, OVA-specificity was gated by labeling with MHC-I SINFEKL-PE dextramer. See Supplementary Fig. 4 for the gating strategy.

Corneas from infected mice were dissected and incubated with 3 mg/ml collagenase (C0130; Sigma-Aldrich) in RPMI (Life Technologies), with 1% HEPES (Life Technologies), 1% penicillin-streptomycin (Life Technologies), and 0.5% BSA (Fisher Bioreagents) for 1 h and 15 min at 37◦C. All subsequent steps were at 4⁰C. Cells were recovered following centrifugation and incubated 5 min with anti-mouse CD16/32 Ab (BioLegend) to block Fc receptors, then 20 min with anti-mouse CD45-allophycocyanin, Ly6GBV510, Ly6C-PE-Cy7, CD11b-PETxRed, CCR2-BV421, or F4/80-FITC (BioLegend) and fixable viability dye (BD Biosciences). Cells were washed with FACS buffer and quantified using an ACEA Novocyte flow cytometer and NovoExpress software. The gating strategy identified total cells in infected corneas by forward and side scatter, followed by gating on single cells and live cells were identified using the E780 viability dye (Biolegend). Neutrophils were identified as CD45 + , CD11b + , Ly6G + , CCR2-, and monocytes were CD45 + , CD11b + , Ly6G- CCR2 + . The gating strategy is shown in Fig. S3K.

Freshly peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood samples by lymphoprep (Ficoll-Hypaque, Cedarlane, Burlington, ON, Canada) density gradient centrifugation. For surface antigens, PBMCs were stained with specific monoclonal anti-human antibodies (mAbs) combined in appropriated panels and incubated for 20 min at 4 °C in the dark. A detailed list of mAbs used is reported in Supplementary Table 2. Fixable viability dye (BD Biosciences, Franklin Lakes, NJ, USA) was used to discriminate live and dead cells. For intracellular antigens (transcription factors, cytotoxic molecules and cytokines), PBMCs were fixed and permeabilized with the Foxp3/Transcription factor Staining kit (cat. #: 00-5523-00, eBioscience, Thermo Fisher, Waltham, MA, USA), according to manufacturer’s instructions, and then incubated for 40 min at 4 °C in the dark.
Sample acquisition was performed by LRSFortessa flow cytometer (BD Biosciences, San Jose, CA, USA) with 355-,405-, 488-, 561-, and 640-nm lasers.