Control of nutrient uptake by IRF4 orchestrates innate immune memory - scRNA-seq
Natural Killer (NK) cells are innate cytotoxic lymphocytes with adaptive immune features, including antigen-specificity, clonal expansion, and memory. As such, NK cells share many transcriptional and epigenetic programs with their adaptive CD8+ T cell siblings. Various signals ranging from antigen, co-stimulation, and proinflammatory cytokines are required for optimal NK cell responses in mice and humans during virus infection; however, the integration of these signals remains unclear. In this study, we identified the transcription factor IRF4 as a signal integrator to coordinate the NK cell response during viral infection. Loss of IRF4 was detrimental to the expansion and differentiation of virus-specific NK cells. This defect was partially attributed to the inability of IRF4-deficient NK cells to uptake nutrients required for survival and memory generation. Altogether, these data suggest IRF4 is a signal integrator that acts as a secondary metabolic checkpoint to orchestrate the adaptive response of NK cells during viral infection. Overall design: Spleens were dissociated with glass slides and filtered through 100-µm cell strainer. Flow cytometry and cell sorting were performed on the Cytek Aurora (Cytek Biosciences) and Aria II cytometers (BD Biosciences), respectively. Before cell sorting, NK cells were enriched by incubating whole splenocytes with the following antibodies at 20 µg/ml against CD3e (17A2), CD4 (GK1.5), CD8 (2.43), Ter119 (TER-119), CD19 (1D3), Ly6G (1A8) (BioXCell) followed by magnetic depletion using goat anti-rat beads (QIAGEN). For scRNA-seq enriched splenocytes were then stained with surface markers to identify Ly49H+ NK cells (CD3/TCRb/CD19- NK1.1+ CD49b+ Ly49H+) and sorted based on congenic markers (CD45.1 and CD45.2) to identify WT and Irf4-/- cells with > 95% purity. Ly49H+ NK cells (CD3/TCRb/CD19- NK1.1+ CD49b+ Ly49H+) from either WT or Irf4-/- NK cells were sorted from spleens of MCMV-infected WT:Irf4-/- mBMC mice as described from day 0, 2, 4 and 7 PI, and stained with barcoded antibodies (Total-Seq B, Biolegend). After hash-staining, WT and Irf4-/- Ly49H+ NK cells were mixed at 1:1 ratio for each time point. The single-cell RNA-seq from these pooled FACS-sorted cell suspensions was then performed on Chromium instrument (10X genomics) following the user guide manual for 3' v3.1. In brief, FACS-sorted cells were washed once with PBS containing 1% bovine serum albumin (BSA) and resuspended in PBS containing 1% BSA to a final concentration of 700–1,300 cells per µl. The viability of cells was above 80%, as confirmed with 0.2% (w/v) trypan blue staining (Countess II). Cells were captured in droplets. Following reverse transcription and cell barcoding in droplets, emulsions were broken, and cDNA purified using Dynabeads MyOne SILANE followed by PCR amplification per manual instruction. Samples were multiplexed together on one lane of 10X Chromium (using Hash Tag Oligonucleotides - HTO) following a previously published protocol. Final libraries were sequenced on Illumina NovaSeq S4 platform (R1 – 28 cycles, i7 – 8 cycles, R2 – 90 cycles). The cell-gene count matrix was constructed using the Sequence Quality Control (SEQC) package.