Mice: C57BL/6, SMARTA LCMV-specific TCR transgenic mice, Perforin (Prf1) deficient, and Cas9-expressing mice were purchased from Jackson Laboratory (Bar Harbor, ME). KLF2-GFP mice were a gift from Sing Sing Way. Cas9 or KLF2-GFP mice were crossed to SMARTA and bred to homozygosity to create Cas9-SMARTA and KLF2-GFP SMARTA mice. Male mice between 8 to 20 weeks of age were routinely utilized in experiments. Mice were housed under barrier conditions and experiments performed under ethical guidelines approved by the Institutional Animal Care and Use Committees of Cincinnati Children’s Hospital Medical Center. Staff performing experimental measures were blinded to genotype and treatment status of experimental groups during sample processing and data acquisition.
Virus and viral vector injections: Mice were infected with the Armstrong strain of LCMV via intraperitoneal injection of 5x104 plaque forming units per mouse. Viral stocks were previously generated in house via propagation on BHK21 cells with titer determined using Vero cells.
SMARTA transfer model: SMARTA mouse spleens were harvested and processed into single cell suspensions (See Flow Cytometry) from which CD4 T cells were enriched by depletion of non-CD4 T cells (Miltenyi Biotec, Germany). 5x105 CD4 T cells were injected retro-orbitally into isotype control and NK cell-depleted mice 1 day prior to LCMV infection. Two to four days following infection, spleens were harvested and subjected to flow cytometry or immunofluorescence. To ensure validity of the model an experiment was performed wherein groups of mice were treated as above but only 50,000 SMARTA were adoptively transferred. 4-7 days post infection the same trend of enrichment was seen in the SMARTA and endogenous T cell pools that recognize the MHC tetramer loaded with LCMV-GP64-78. This ensures that the supraphysiologic doses of SMARTA we used to detect this novel subpopulation behave similarly to a physiologic precursor frequency.
In vivo NK-cell depletion: One day before infection, selective depletion of NK cells10 was achieved through a single intraperitoneal injection of 25 micrograms of mouse anti-NK1.1 monoclonal antibody (PK136) or 25 micrograms of a control mouse IgG2a isotype antibody (C1.18.4) produced by Bio-X-Cell (West Lebanon, NH).
Flow cytometry and in vitro peptide stimulation: Single-cell leukocyte suspensions were prepared from spleens by mechanical homogenization of tissues between frosted glass microscope slides (VWR, Radnor, PA) and filtration through a 70 µm nylon mesh. Following lysis of red blood cells, lymphocytes were plated at 2x106/well in 96-well round-bottom plates and subjected to flow staining. Leukocytes were washed with PBS then stained for dead cells with Zombie UV (BioLegend, San Diego, CA) used at 1:1000 for 5 min at room temperature, washed twice with PBS, and then Fc Receptor blocked with anti CD16/32 (BD BioSciences, San Jose, CA) used at 1:200 in FACS buffer (Phosphate buffer saline + 2% fetal bovine serum + 0.5 mM EDTA) for 5 min at 4C prior to surface staining with the following antibodies: CD162 (Clone 2PH1 used at 1:300), CD8α (Clone 53-6.7 1:200), CD45R (Clone RA3-6B2 used at 1:200), CD4 (Clone GK1.5 used at 1:200), SLAMF6 (Clone 13G3 used at 1:200), Ly6c (HK1.4 used at1:300), CD69 (Clone H1.2F3 used at 1:200), Integrin ß7 (Clone FIB504 used at 1:100), GP64 tetramer (Obtained from the NIH 1:75), CXCR5 (Clone SPRCL5 used at 1:100), S1PR1 (clone MAB7089 used at 1:50), SLAMF1 (Clone TC15-12F12.2 used at 1:100), CD19 (Clone 6D5 used at 1:200), NK1.1 (Clone PK136 used at 1:100), CD49b (Clone DX5 used at 1:100), CD45.1 (Clone A20 used at 1:100), CD62L (Clone MEL-14 used at 1:200). All antibodies were purchased from BioLegend (San Diego, CA), BD Biosciences (San Jose, CA), or ThermoFisher Scientific (Waltham, MA). GP64 loaded MHCII tetramers and S1PR1 stains were used at 37C for 90 min prior to addition of the remaining antibodies in Brilliant Stain buffer and room temperature incubation for 30 minutes. Following staining, cells were washed and fixed with BD fixation buffer (BD Biosciences, San Jose, CA) for 5 minutes at 4oC. For experiments utilizing KLF2-GFP expressing mice flow cytometry was run on the same day as harvest without fixation to preserve GFP signal. Cells were washed twice in FACS and resuspended in 100 microL FACS buffer and run on a Cytek Aurora Spectral flow cytometer. Spectral unmixing was performed in SpectroFlow and flow plots created using FlowJo.
Single cell RNA sequencing: GP66-81:I-Ab+ CD45.1+ cells were sorted from LCMV infected animals, loaded onto the Chromium platform (10 X Genomics) to generate cDNAs carrying cell- and transcript-specific barcodes that were used to construct sequencing libraries using the Chromium Single cell 5’v2 Library & Gel Bead Kit according to the manufacturer instructions. Libraries were sequencing on a single run of Illumina Novaseq 6000 using paired-end reads to reach a read depth of 28-30,000 reads per cell. Raw base call files were de-multiplexed with Cell Ranger 26 v3.0.2 mkfastq (10x Genomics). Reads were aligned to mouse reference genome mm10 and gene expression quantified using Cell Ranger count. Further data analysis was carried out with Seurat 27,28v4.3.0.1 in R v4.2 29. Cells displaying more than 5% mitochondrial gene expression, less than 500 total expressed genes, or less than 1000 RNA counts were excluded from the analysis. Gene expression counts were normalized with the NormalizeData function in Seurat, which uses a logarithmic normalization method where gene counts for each cell are divided by its total counts and natural log-transformed using log1p and multiplied by a scale factor of 10,000. Data was scaled using the ScaleData function in Seurat and mitochondrial score and cell cycle genes were regressed out of the data. Doublets were removed using doubletFinder30. The six samples were integrated together using FindIntegrationAnchors and IntegrateData functions from Seurat. This integrated dataset was used for principal component analysis, variable gene identification, Shared Nearest Neighbor (SNN) clustering analysis, and Uniform Manifold Approximation and Projection (UMAP). Clusters were defined to optimize silhouette scoring. DotPlot of common CD4 T cell defining genes shown in Supplemental Figure 3. Clusters were collapsed for each individual animal for pseudobulk analysis using AggregateExpression and compared using DESeq2.
Tissue processing, sectioning, and immunohistochemistry: Tissues to be analyzed by fluorescence microscopy were placed into 4% formaldehyde solution for 5 hours followed by overnight dehydration in a 30% sucrose (Sigma-Aldrich, St. Louis, MO) solution at 4oC. Samples were washed with phosphate buffered saline prior to embedding within optimal cutting temperature (OCT) media (Sakura Finetek, Maumee, OH) and frozen using a dry ice slurry in 100% ethanol and stored in -20oC. Tissues were sectioned (7-10 µm thick) using a cryostat (Leica CM3050 S) and affixed to positively charged Denville slides (Thomas Scientific, Swedesboro, NJ). Slides were dried at room temperature for 5 to 10 minutes preceding a 10-minute fixation in chilled 100% acetone at -20oC. Slides were subsequently dried at room temperature for 5 to 10 minutes and washed twice in chilled phosphate-buffered saline before being placed in saturation buffer containing 10% normal donkey serum (Sigma-Aldrich, St. Louis, MO) and 0.1% Triton-X (Sigma-Aldrich, St. Louis, MO) in phosphate buffered saline for blocking at room temperature for 45 minutes. Slides were incubated with a primary antibody cocktail containing one or more of the following antibodies overnight at 4oC: 1:100 goat anti-NKp46 (R&D Systems, Minneapolis, MN), 1:200 rat anti-CD3e-AF647 (Clone 17A2 from R&D Systems, Minneapolis, MN), 1:200 rat anti-CD169-AF594 (3D6.112, BioLegend, San Diego, CA) 1:200 anti-CD45R/B220-AF657 (Clone RA3-6B2 from ThermoFisher Scientific, Waltham, MA), and 1:100 chicken anti-GFP (Aves). The following day, slides were washed twice in chilled phosphate buffered saline. Subsequent secondary antibody staining with 1:1000 donkey anti-goat AF555 (ThermoFisher Scientific, Waltham, MA) for 2 hours at room temperature to reveal NKp46 primary staining. Slides were again washed thrice with chilled phosphate buffered saline and additional secondary staining was performed with 1:5000 donkey anti-chicken AF488 (ThermoFisher Scientific, Waltham, MA) for an additional 2 hours at room temperature to reveal GFP primary staining. Slides were again washed twice with chilled phosphate buffered saline, mounted with prolong diamond mounting media (Thermo-Fisher Scientific, Waltham, MA), covered with a coverslip, and allowed to cure overnight prior to imaging.
Confocal microscopy & NK-cell quantification: Confocal imaging was performed using a Laser Scanning Nikon AXR Inverted Confocal Microscope with NIS Elements Confocal software. Z-stacked tissue images were acquired through a 20X objective (Nikon Plan Apo λ) from which a maximum intensity projection was generated prior to cell enumeration. Using tools available in NIS Elements Analysis software and guided by CD169- and CD3-staining, borders around white pulp (CD169 boundary) and T cell zones (CD3 boundary) were drawn. To enumerate KLF2-GFP+ CD3+ T cells, a cell expansion algorithm was used to determine the number of GFP and CD3 positive cells (cells borders determined by DAPI staining). Brightness and contrast for each representative image were adjusted equally across all channels using Photoshop CS6.
Statistics: Experimental results are consistently presented as the mean with individual data point spread. Statistical differences between control and experimental groups were calculated using a one-way analysis of variance (ANOVA) with either the Holm-Šídák multiple comparison test or a Dunnett’s multiple comparisons test. A p-value of less than 0.05 was considered significant. Graphing and statistical analysis were routinely performed using GraphPad Prism (San Diego, CA). Researchers were blinded to groupings and treatment during experimental measurements.