Resource Availability
Any further information and requests for resources and/or reagents should be directed and will be fulfilled by the senior investigator, Ross Kedl ([email protected])
Materials availability
No unique reagents were generated in this study. All materials used are available upon request.
Data and code availability
Data generated and reported in this paper will be shared upon request by the lead contact. This paper did not generate any original code. Any additional information required to reanalyze data reported in this manuscript is available by the lead contact upon request. Single-cell RNAseq data have been deposited at GEO and are publicly available as of the date of publication. Accession numbers are listed in the key resources table. Flow cytometry data has been deposited at the Harvard Dataverse and are publicly available as of the date of publication. DOIs are listed in the key resources table.
Mice
C57BL/6 mice were obtained from The Jackson Laboratory and bred in our own facility at the University of Colorado Anschutz Medical Campus to provide recipient mice for transfer studies. 129 mice were obtained from Taconic Biosciences. 129 males were bred with B6 females to produce B6x129 F1 mice used for adoptive transfer studies of mixed background donor cells. All recipient mice used in experiments were born in the vivarium on Anschutz Medical Campus. Opa1fl/fl mice were obtained from the Hiromi Sesaki Lab and crossed with CD45.1/1 OT1+ dLck-Cre+ mice for use in these studies. Mfn1fl/fl (Strain# 026401) and Mfn2 fl/fl (strain# 026525) mice were cryorecovered from the Jackson Laboratory and subsequently crossed with CD45.1/1 OT1+ dLck-Cre+ mice upon receipt. For all floxxed strains (Opa1, Mfn1, Mfn2, Mfn dKO) WT mice are sex-matched OT1+ dLck-Cre(-) littermates. While the Opa1fl/fl mice were maintained on a mixed B6/129 background, B6/129 F1 sex-matched mice were used as recipients for experiments until the Opa1fl/fl mouse was backcrossed to B6 11 times. Experiments then proceeded using B6 recipients. Strain of donor and recipient mice is noted in the figure legends. Unless otherwise stated, data was generated using B6 donor and recipient mice. All experiments were conducted in accordance with protocols approved by the University of Colorado Denver Institutional Animal Care and Use Committee.
Isolation of cells
For isolation of splenic T cells, spleens were harvested from mice at indicated timepoints and crushed through a 70um filter to generate single cell suspensions. Cell counts were obtained using a Vi-cell automated cell counter (Beckman Coulter) to determine total number of viable cells. Representative population size was determined by flow cytometry and used to calculate total CD8 T cells within the spleen.
For isolating CD8 T cells <72 hours after LM.Ova.ActA infection or vaccination, spleens were minced in a 6 well plate in 2 mL of Clicks media (Irvine Scientific) containing 500 ug/mL Collagenase D (Roche) and 50ug/mL DNase I (Worthington) per spleen. Following a 30-minute incubation at 37C, 2 mLs of 0.1 M EDTA (VWR) was added to each well followed by another 5-minute incubation at 37C. Samples were then pipetted through 70um filters and washed with 25mL of HBSS (Gibco) containing 5mM EDTA. To prepared samples for positive enrichment using LS columns (BioLegend), each spleen was suspended in 1mL complete RPMI + 20% 24G2 supernatant for 10 minutes at RT. Following centrifugation (500g, 3 minutes), media was replaced with 500uL of complete RPMI + 20% 24G2 and 10uL of CD45.1-APC (A20, BioLegend), and incubated 20 minutes at 4C while rotating. After washing cells and resuspending in 450uL of RPMI, 5uL of anti-APC magnetic beads (BioLegend) were added per spleen and samples were incubated for 15 minutes at 4C while rotating. Cells were then washed and added to magnetic columns (LS columns, Miltenyi Biotec) through a mesh filter. For each solution run through magnetic columns, wait until column has stopped dripping before moving onto the next solution. First, 3-4mL 70% ethanol, followed by 3-5 mLs of complete RPMI. Add the cells (~500uL) to the column through a mesh filter. Once cells have been loaded onto the column, fill the column to the top with complete RPMI (~7mL). To evacuate cells of interest from the column, place 15mL conical tube under the column and add 5mL of complete RPMI to column to the top. Immediately remove column from the magnet and plunge media through the column and into the 15 mL tube. The cell suspensions were then washed in complete RPMI and counted on a Vi-Cell cell counter (Beckman Coulter) to determine total viable cell number. Blood was collected into tubes containing HBSS with 5mM EDTA and lysed in ACK buffer, washed with complete media, and stained for flow cytometry.
Western Blots
Protein was isolated from CD8 T cells using RIPA buffer (Thermo Scientific) with Halt Protease Inhibitor Cocktail (Thermo Scientific) while on ice and with agitation from a vortex and subsequent centrifugation at max speed (20817 rcf) for 20 minutes at 4oC. Prior to isolation, CD8 T cells were washed 3 times with PBS during which the cells were spun for 3 minutes at 500 G. Protein loading solutions were prepared using 6x reducing Laemmli SDS sample buffer (Thermo Scientific) followed by heating for 10 minutes at 70oC. Protein solutions were loaded into a 7.5% Mini-PROTEAN TGX Precast Protein Gel (Bio-Rad). The gel was processed with up to 120 V in Tris/Glycine/SDS buffer (Bio-Rad). The transfer utilized the Trans-Blot Turbo Transfer System (Bio-Rad) and 0.2 mm PVDF in pre-packaged Trans-Blot Turbo Transfer Packs (Bio-Rad) with 25 V and 2500 mA for 7 minutes. The blot was then blocked with 5% w/v milk in TBST for 1 hr at room temperature with gentle agitation and then incubated with the primary antibody at 1:1000 in TBST at 4oC overnight on a rocker. The blot was subsequently incubated in secondary antibody conjugated to horse-radish peroxidase (Bio-Rad) for 1 hr at room temperature with gentle agitation. The blot was visualized with a luminol – peroxide solution (Thermo Scientific) on a G:BOX imager (Syngene). Antibodies used: Opa1 (D6U6N; Cell Signaling), goat anti-mouse IgG (H/L):HRP (Bio-Rad), goat anti-rabbit IgG (H/L):HRP (Bio-Rad), b Tubulin (D-10;Santa Cruz).
Immunizations and infections
All immunizations and infections were administered via i.v. injection. Vaccinations included 40ug polyinosinic-polycytidylic acid (polyIC; GE), InVivoMAb 40ug aCD40 (FGK4.5; BioXCell), and 150ug Ova protein (Sigma) in a total volume of 200uL. Mice were infected with 2000 CFUs of virulent LM expressing whole OVA (LM.Ova), 106 CFUs of attenuated non-replicating LM expressing OVA (LM.Ova.ActA) or 5 x 106 plaque forming units (PFUs) per mouse of Vaccinia virus expressing SIINFEKL (VV.Siin). For secondary challenges with virulent LM.Ova, four days after challenge, spleens, blood, and liver were harvested from euthanized mice. Livers were massed and homogenized in 5mL of 0.02% NP40 solution using sterile tissue homogenizers (Miltenyi). Liver homogenates were plated at multiple dilutions for 48 hours at 37C on BHI agar plates with 5ug/mL erythromycin (Sigma) before bacteria counts were taken.
Flow cytometry
Cells were incubated with aCD16/32 (clone 24G2; hybridoma supernatant) and plated on U-bottom 96-well plates at <= 4 x 106 cells/well in complete RPMI. Where indicated, cells were stained with Kb-SIINFEKL tetramer conjugated to either APC or PE (NIH tetramer core) at 37C for 30 minutes in complete RPMI with 20% 24G2. For viability and surface staining, cells were washed with media and stained in media for 10 minutes at 37C with Fixable Viability Dye 780 (Tonbo) and surface antibodies for CD19 ( 1D3; BD Biosciences), CD8 (53-6.7; BioLegend), CD44 (IM7; Tonbo), CD45.1 (A20; BioLegend), CD45.2 (104; BioLegend), Klrg1 (2F1/KLRG!; BioLegend), CD127 (A7R34; BioLegend), CD98 (RL388; BioLegend), and CD71 (C2F2; BD Biosciences). After wash with RPMI, cells were fixed and permeabilized for 30 minutes at RT in FoxP3/Transcription Factor 1x Fix/Perm solution (Tonbo), followed by wash with 1x Flow Cytometry Perm Buffer (Tonbo) and intracellular staining for 30 minutes at RT with intracellular antibodies including GzmB (QA16A02; BioLegend), Tcf1 (C63D9; Cell Signaling), T-Bet (4B10; BioLegend), Eomes (Dan11mag; Invitrogen), and Irf4 (Irf4.3e4; BioLegend). For intracellular cytokine staining, splenocytes were plated at 3 x 106 cells per well and incubated for 6 hours at 37C in complete RPMI with 1:200 aCD107a APC (1D4B; BioLegend), 0.5 ug/mL SIINFEKL, and 3 ug/mL Brefeldin A (AdipoGen). After incubation, cells were stained, fixed, and permeabilized as stated above and stained intracellularly with Tnfa (MP6-XT22; BioLegend) and Ifng (XMG1.2; BioLegend) antibodies for 30 minutes at 37C. Following intracellular staining, cells were washed twice and resuspended in 1x Flow cytometry perm buffer (Tonbo). Flow cytometry data was acquired using a four-laser (405, 488, 561, 638nm) Cytoflex S (Beckman Coulter) and analyzed using FlowJo software (BD Biosciences).
Lymphopenia-induced proliferation
Mice were sub-lethally irradiated (cesium; 600 rads). The following day, mice received 105 CTV-labeled WT and Opa1-/- OT1 T cells. At various timepoints following cell transfer, mice were euthanized, splenocytes were isolated, and samples were analyzed via flow cytometry.
Quantification and Statistical analysis
GraphPad Prism (version 10.1.0, GraphPad) was used for all statistical analyses. Number of experimental replicates, mouse strain, and n-values, and statistical tests used are reported in the figure legends. Unless otherwise noted, data presented are means +/- SEM. Significance was defined using paired and unpaired Student’s t-test or analysis of variance (ANOVA). Significance was denoted as follows: ns, not significant (p > 0.05) or significant (p < 0.05).
Single-cell RNA sequencing sample preparation
A physiologically relevant number (500) of congenically marked (CD45.1) OT1 T cells were adoptively transferred into a naïve B6 recipient (CD45.2). Recipient mice were infected intravenously (i.v.) with LM.Ova.ActA as described previously. On day 5, T cells were isolated and pooled from 4 mice prior to cell sorting of CD45.1+ CD8+ CD19- cells. Naïve cells were isolated using Biolegend CD8 isolation kit. Naïve and d5 cells for each genotype were pooled at a 1:1 ratio and roughly 10,000 cells were loaded onto Single Cell A chips (10X Genomics) and partitioned into Gel Bead In-Emulsions in a Chromium Controller (10X Genomics). Single-cell RNA libraries were prepared according to the 10X Genomics Chromium Single Cell 5’ Reagent Kits v3.1 User Guide and sequenced on a NovaSeq 6000 (Illumina).
scRNAseq mapping
scRNAseq reads were aligned to mm10 oligo sequences and collapsed into unique molecular identifier (UMI) counts using the 10X Genomic Cloud Cell Ranger Count software (version 7.1.0). The sample had an appropriate number of genes detected (>1000), a high percentage of reads mapped to the genome (>70%), and a sufficient number of cells detected (>1000).
scRNAseq dataset statistical analysis
The Seurat package (version 4.3.0) in R (version 4.0.4) was used to process, analyze, and visualize the scRNAseq dataset. Cell read output from Cell Ranger was loaded into Seurat. UMIs with gene counts less than 200, fewer than 1000 reads, or mitochondrial gene content of greater that 20% were filtered out. Four mice per group were used for this single cell experiment. Gene count data was normalized using Seurat’s NormalizeData function. Top variable genes, principle components analysis (PCA), and uniform manifold approximation and projection for dimension reduction (UMAP) were calculated by the functions: FindVariableGenes, RunPCA, and RunUMAP, respectively. Only the top 2000 genes were considered in the PCA calculation and only the top 20 PCs were used in UMAP. Shared nearest-neighbor (SNN) identification and clustering were performed using the functions FindNeighbors and FindClusters using the top 20 PCs with resolution set to 0.15 and k set to 20. Myc targets module score was calculated using the AddModuleScore function with the gene sets Hallmark_Myc_targets_v1 and Hallmark_Myc_targets_v2.
cNMF: Identification of Gene Expression Programs.
The count matrix was used for conducting non-negative matrix factorization (NMF) through the cNMF method53. This process enabled us to infer both identity and activity programs, along with their respective contributions in each cell. Cells were assigned to the GEP with the highest GEP score and this assignment was added to the metadata of the Seurat object. To determine the genes associated with each program, we plotted the gene ranks (ranging from most associated to least associated) against the gene_spectra_score output from the cNMF analysis. This ranked gene expression was utilized in GSEA using the fGSEA (version 1.24.0) package in R. Both positive and negative normalized enrichment scores (NES) were obtained and scaled NES were graphed for all gene sets with an adjusted p value > 0.05.
scRNA analysis packages used.
The R packages Seurat (version 4.3.0), SCpubR (version 2.0.2), ggplot (version 3.4.4), and pheatmap (version 1.0.12) were all used for the graphing of scRNA data.