Rbc lysis

Spleens were removed from vaccinated or control BALB/c mice and splenocytes were isolated by disaggregation through a metallic mesh. After RBC lysis (Biolegend, San Diego, CA, USA), resuspension and counting, the cells were ready for analysis. The IFN-γ secreting cells were assessed using the ELISPOT mouse IFN-γ kit (R&D Systems) according to the manufacturer’s protocol. The cells were cultured for 18 h at 5 ×10⁵ cells per well with 2 μg/mL of a peptide pool consisting mainly of 15-mers (overlapping by 11 amino acids) covering the immunodominant sequence domains of the SARS-CoV-2 Spike protein (PepTivator® SARS-CoV-2 Prot_S; Miltenyi Biotec, Bergisch, Gladbach, Germany). The number of spots was determined using an automatic ELISPOT reader and image analysis software (CTL-ImmunoSpot® S6 Micro Analyzer, Cellular Technology Limited (CTL), Cleveland, OH, USA).

Single-cell suspensions were prepared and stained according to standard protocols for flow cytometry including RBC lysis (Biolegend) following manufacturer recommendations. Cells were incubated with anti-CD16/32 to block nonspecific binding and then stained for 1 hr at room-temperature with appropriate dilutions of various combinations of fluorochrome conjugated antibodies. For intracellular staining, cells were fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences). Cells were stained with intracellular mixture of fluorochrome conjugated antibodies for 30 minutes. The stained cells were acquired on a BD fortessa, Canto (BD Biosciences), or Cytek Aurora and the data was analyzed using FlowJo software (version 7.6; Tree Star). Doublets were excluded based upon forward and side scatter and dead cells were excluded by using BV510 Ghost dye. All flow cytometry plots shown and data quantitated from flow cytometry were gated upon doublet exclusion (FSC SSC) and viable (BV510 ghost -) CD45+ cells. Gating schemes found in (Figure S10). All antibodies used including dilutions are found in Supplementary Table 1.

For flow cytometry on intratumoral immune cell populations, tumors were processed as described previously (54 (link)). Briefly, tumors were chopped using a sterile scalpel until 2–3 mm in size, then placed in digestion media containing collagenase I (0.05% wt/vol; MilliporeSigma), DNase type IV (30 U/mL; MilliporeSigma), and hyaluronidase type V (0.01% wt/vol; MilliporeSigma). Mechanical dissociation using the gentleMACS Octo Dissociator (Miltenyi Biotec) was performed followed by a 40-minute incubation at 37°C. Tumor samples were mechanically dissociated again, then passed through a 70 μm filter. RBC lysis (BioLegend) was performed on tumor cell suspension following the manufacturer’s recommendations. For the ATX inhibitor plus anti–PD-1 flow studies, 344SQ tumor samples were stained with a 34-color panel and acquired using a Cytek Aurora. Antibodies and dilutions are listed in Supplemental Table 2. Additional details can be found in Supplemental Methods.

Lungs were digested in RPMI media with 5% FBS, 1 mg/mL collagenase, and 0.02 mg/mL DNase I for 60 minutes at 37°C. A single-cell suspension was generated by straining these digestions through a 70-μm filter. RBC lysis (BioLegend, San Diego, Calif) was performed, according to the manufacturer's instructions. Cells were restimulated with 10 ng/mL phorbol 12-myristate 13-acetate and 1 μmol/L ionomycin in the presence of 0.07% monensin in Iscove modified Dulbecco medium supplemented with 10% FBS, 0.01 mmol/L nonessential amino acids, penicillin/streptomycin, and 1 mmol/L sodium pyruvate for 6 hours at 37°C. Cells were stained for viability and cell-surface proteins, fixed/permeabilized, and stained for intracellular antigens. All samples were run on a BD LSR II Flow Cytometer (BD Biosciences, San Jose, Calif) and analyzed with FlowJo software (Version 10; FlowJo, Ashland, Ore). Total innate lymphoid cells (ILCs) were defined as Lin⁻CD45⁺CD25⁺CD127⁺ cells, where Lin includes CD3, CD5, CD45R (B220), CD11b, Gr-1 (Ly-6G/C), 7-4, and Ter-119. ILC2s were defined as ILCs that expressed IL-5, IL-13, or both. T cells were defined as CD45⁺CD3⁺ cells. Mean fluorescence intensity (MFI) was determined as the geometric mean.