Control of nutrient uptake by IRF4 orchestrates innate immune memory - ChIP-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: Splenic WT NK cells (NK1.1+ CD49b+) were expanded ex vivo for two weeks in complete IMDM media (10% FBS, 1% Pen/Strep, 1X BME, 1X MEM-NAA, 1X HEPES) in the presence of 50ng/ml of human IL-15 (Miltenyi Cat#130-095-766). 10 million IL-15 expanded NK cells were then stimulated with anti-NK1.1 + cytokines (IFNa+IL-12/18+IL-2/15) or left unstimulated in 5ng/ml of hIL-15 overnight in a 6-well plate. Upon stimulation, cells were then washed with 1X PBS and crosslinked by slowly adding 16% formaldehyde to a final concentration of 1% formaldehyde, agitated, and incubated for 10 mins at RT. Glycine was then added to quench the sample, and incubated at RT 5 min, and on ice for 15 minutes. Cells were then centrifuged, and 600 ul of cell lysis buffer (50mM HEPES-KOH, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100) was added into cell pellets, and incubated for 4 minutes on ice, spun at 600 xg for 10 minutes at 4C, and supernatant was discarded. 600 ul of nuclei wash buffer (10mM Tris-Cl pH 8.0, 200mM NaCl, 1mM EDTA, 0.5 mM EGTA) was then added to each sample and resuspended gently, spun at 600xg for 10 minutes at 4C, and supernatant was discarded. Cell pellets were then resuspended with nuclei lysis buffer and incubated for 5 minutes on ice, and sonicated for 8 cycles, 30s on and 30s off high at 4C. Upon sonication, Triton-X 100 was added to a final concentration of 1%. Nuclei were centrifuged for 20 minutes at 14000 RPM at 4C, and supernatant was transferred into a new tube where 10% of samples were then aliquoted into a new tube as 'input'. 25 ul of Protein A (ThermoScientific), was added into each sample and agitated for 30mins at 4C on a rotator. Moreover, protein A beads were blocked with ice cold RIPA buffer by adding BSA to a final concentration of 0.1 % and agitated for 3 mins at 4C on a rotator. Upon incubation, immunoprecipitated samples were centrifuged for 30s for 4C at 2400 xg and supernatant was transferred into new tubes and combined with blocked protein A beads. Anti-IRF4 (Clone D9P5H, Cell Signaling Cat# 15106S) was added gently to prevent bead shearing and incubated on a rotator overnight at 4C. Samples were then centrifuged at 2400 xg for 1 min at 4C, and supernatant was removed gently. Beads were then washed twice with RIPA buffer, four times with ChIP wash buffer (100mM Tris-Cl pH 8.5, 500mM LiCl, 1% NP-40, 1% NaDeoxycholate, twice again with RIPA buffer, and twice with ChIP TE buffer (10mM Tris pH 7.5, 1mM EDTA, 50mM NaCl). Beads were centrifuged at 5000 RPM for 1 min at 4C between all washes and supernatant was removed after the last wash. 100ul of decrosslinking buffer (TE buffer, 0.5% SDS buffer, and 1X Proteinase K) was added into every sample and incubated for 2 hours at 55C. After incubation, samples were vortexed, and then transferred to 65C and incubated overnight. Samples were then centrifuged at 2400 xg for 1min at RT, transferred into new tubes, and purified using Qiagen PCR purification kit (Qiagen). Immunoprecipitated DNA was quantified by PicoGreen and the size was evaluated by Agilent BioAnalyzer. Illumina sequencing libraries were prepared using the KAPA HTP Library Preparation Kit (Kapa Biosystems KK8234) according to the manufacturer's instructions with up to 5 ng input DNA and 8-12 cycles of PCR. Barcoded libraries were run on the NovaSeq 6000 in a PE100 run, using the NovaSeq 6000 S4 Reagent Kit (200 Cycles) (Illumina). An average of 29 million paired reads were generated per sample.