SRX18329004 GSM6745548_r1 SRP408987 PRJNA903887 SRS15818240 GSM6745548 GSM6745548 RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA Murine cells were isolated from isolated from spleen and 4-6 neonatal tissues were pooled per biological sample. Single cell suspensions from spleen were obtained by manual dissociation and filtration through a 40uM strainer. CD8+ T cells were isolated by positive magnetic separation using anti-CD8a microbeads. 2x105 cells were plated per well in round-bottomed 96 well plates. Cells were incubated in RPMI supplemented with 10% fetal bovine serum, L-glutamine, penicillin-streptomycin, 2-mercaptoethanol (RP-10) and 10 ng/ml recombinant human IL-2 (Thermo Fisher Scientific) for 18-22 hours. For bystander-activated samples, media was additionally supplemented with recombinant murine IL-12p70 (Thermo Fisher Scientific) and recombinant murine IL-18 (Thermo Fisher Scientific) at 1 ng/ml. 1.5 mg/ml Brefeldin A (Millipore Sigma; St. Louis, MO) was added to the cells for the final 4 hours of incubation prior to antibody labeling for flow cytometry analysis. Live Va2+CD8+ cells were sorted into 0.04% BSA in PBS. Cells were counted and 3-8k cells per sample were loaded in the Chromium controller for the formation of gel bead-in-emulsions (GEMs) following the manufacturer's instructions (10X Genomics). The single-cell RNA-seq libraries were prepared using Chromium Single Cell 3′ Reagent Kit v2 following the accompanied protocol and were sequenced on Illumina NextSeq500. NextSeq 500 SRX18329005 GSM6745549_r1 SRP408987 PRJNA903887 SRS15818241 GSM6745549 GSM6745549 RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA Murine cells were isolated from isolated from spleen and 4-6 neonatal tissues were pooled per biological sample. Single cell suspensions from spleen were obtained by manual dissociation and filtration through a 40uM strainer. CD8+ T cells were isolated by positive magnetic separation using anti-CD8a microbeads. 2x105 cells were plated per well in round-bottomed 96 well plates. Cells were incubated in RPMI supplemented with 10% fetal bovine serum, L-glutamine, penicillin-streptomycin, 2-mercaptoethanol (RP-10) and 10 ng/ml recombinant human IL-2 (Thermo Fisher Scientific) for 18-22 hours. For bystander-activated samples, media was additionally supplemented with recombinant murine IL-12p70 (Thermo Fisher Scientific) and recombinant murine IL-18 (Thermo Fisher Scientific) at 1 ng/ml. 1.5 mg/ml Brefeldin A (Millipore Sigma; St. Louis, MO) was added to the cells for the final 4 hours of incubation prior to antibody labeling for flow cytometry analysis. Live Va2+CD8+ cells were sorted into 0.04% BSA in PBS. Cells were counted and 3-8k cells per sample were loaded in the Chromium controller for the formation of gel bead-in-emulsions (GEMs) following the manufacturer's instructions (10X Genomics). The single-cell RNA-seq libraries were prepared using Chromium Single Cell 3′ Reagent Kit v2 following the accompanied protocol and were sequenced on Illumina NextSeq500. NextSeq 500 SRX18329006 GSM6745551_r1 SRP408987 PRJNA903887 SRS15818242 GSM6745551 GSM6745551 RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA Murine cells were isolated from isolated from spleen and 4-6 neonatal tissues were pooled per biological sample. Single cell suspensions from spleen were obtained by manual dissociation and filtration through a 40uM strainer. CD8+ T cells were isolated by positive magnetic separation using anti-CD8a microbeads. 2x105 cells were plated per well in round-bottomed 96 well plates. Cells were incubated in RPMI supplemented with 10% fetal bovine serum, L-glutamine, penicillin-streptomycin, 2-mercaptoethanol (RP-10) and 10 ng/ml recombinant human IL-2 (Thermo Fisher Scientific) for 18-22 hours. For bystander-activated samples, media was additionally supplemented with recombinant murine IL-12p70 (Thermo Fisher Scientific) and recombinant murine IL-18 (Thermo Fisher Scientific) at 1 ng/ml. 1.5 mg/ml Brefeldin A (Millipore Sigma; St. Louis, MO) was added to the cells for the final 4 hours of incubation prior to antibody labeling for flow cytometry analysis. Live Va2+CD8+ cells were sorted into 0.04% BSA in PBS. Cells were counted and 3-8k cells per sample were loaded in the Chromium controller for the formation of gel bead-in-emulsions (GEMs) following the manufacturer's instructions (10X Genomics). The single-cell RNA-seq libraries were prepared using Chromium Single Cell 3′ Reagent Kit v2 following the accompanied protocol and were sequenced on Illumina NextSeq500. NextSeq 500 SRX18329007 GSM6745550_r1 SRP408987 PRJNA903887 SRS15818243 GSM6745550 GSM6745550 RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA Murine cells were isolated from isolated from spleen and 4-6 neonatal tissues were pooled per biological sample. Single cell suspensions from spleen were obtained by manual dissociation and filtration through a 40uM strainer. CD8+ T cells were isolated by positive magnetic separation using anti-CD8a microbeads. 2x105 cells were plated per well in round-bottomed 96 well plates. Cells were incubated in RPMI supplemented with 10% fetal bovine serum, L-glutamine, penicillin-streptomycin, 2-mercaptoethanol (RP-10) and 10 ng/ml recombinant human IL-2 (Thermo Fisher Scientific) for 18-22 hours. For bystander-activated samples, media was additionally supplemented with recombinant murine IL-12p70 (Thermo Fisher Scientific) and recombinant murine IL-18 (Thermo Fisher Scientific) at 1 ng/ml. 1.5 mg/ml Brefeldin A (Millipore Sigma; St. Louis, MO) was added to the cells for the final 4 hours of incubation prior to antibody labeling for flow cytometry analysis. Live Va2+CD8+ cells were sorted into 0.04% BSA in PBS. Cells were counted and 3-8k cells per sample were loaded in the Chromium controller for the formation of gel bead-in-emulsions (GEMs) following the manufacturer's instructions (10X Genomics). The single-cell RNA-seq libraries were prepared using Chromium Single Cell 3′ Reagent Kit v2 following the accompanied protocol and were sequenced on Illumina NextSeq500. NextSeq 500