5 week-old male C57BL/6J mice (Jackson Labs) were used for microarray analysis within 1 week of arrival. Ccr10-Gfp (N. Xiong, Penn State) and Il4-Gfp reporter (M. Mohrs, Trudeau Institute) mice have been described previously. Axin2 (H. Birchmeier, MDC Berlin), Cenpk (SOLT, IGTC), and Sox5 (V. Lefebvre, Cleveland Clinic) LacZ reporter mice have been published. Ctsl−/− mice were provided by Kenneth Rock (UMMS, Worcester, MA) and Cd74−/ − mice were provided by Eric Huseby (UMMS, Worcester, MA). Mice were housed in a specific pathogen free rodent barrier facility. Experiments were approved by the UMMS IACUC (Worcester, MA).
Sample preparation for microarray analysis
Pooled thymocytes from 4–30 mice were enriched for γδ T cells by depletion of CD8+ cells using magnetic beads and an autoMACS, stained, and sorted directly into Trizol (Invitorgen) using a FACSAria (~2–3 × 104 cells, >99% pure). Independent triplicates were sorted unless noted otherwise (complete sorting details at Immgen.org). As antibodies to Vγ4 are not available, fetal immV4 cells were sorted by gating on TCRδ+ cells that were Vγ1.1−Vγ2−Vγ3−Vγ5− (estimated purity ~95%). Approximate frequencies of TCRδ chains associated with sorted TCRγ subsets are as follows: V2 (Vγ2+, 50% Vδ4+, 40% Vδ5+, all Vδ6.3−, ~45% of total γδ cells); V1 (Vγ1.1+, diverse Vδs including 25% Vδ4+, 15% Vδ5+, others at lower frequencies, all Vδ6.3−, 30% of total γδ cells); V6 (Vγ1.1+, 100% Vδ6.3+, ~8% of total γδ cells); V5 (Vγ5+, 40% Vδ5+ and various others at lower frequencies, ~5% of total γδ cells); and fetal V3 and V4 thymocytes co-express Vδ1.
Data analysis and visualization
RNA processing and microarray analysis using the Affymetrix MoGene 1.0 ST array was performed at the ImmGen processing center (SOP at Immgen.org). Data analysis was performed using GenePattern (Genepattern.org) analysis modules. Raw data were RMA normalized with quantile normalization and background correction (ExpressionFileCreator). The ConsolidateProbeSets module (Scott Davis, Harvard Medical School, Boston, MA) was used to consolidate multiple probe sets into a single mean probe set value for each gene. Identification of differentially regulated genes was performed using Multiplot. Unless otherwise indicated, genes were considered differentially regulated if they differed in expression by more than 2-fold, had a coefficient of variation (cv) among replicates of less than 0.5, had a t-test P value of less than 0.05, and had a mean expression value (MEV) of greater than 120 in at least one subset in the comparison. Heatmaps were generated by hierarchical clustering (HierarchicalClustering module) of data based on gene (row) and subset (column) using the Pearson correlation for distance measurement. Data were log transformed and clustered using pair-wise complete linkage. Data were row centered prior to visualization using the HeatmapViewer module. Principal component analysis was performed using the PopulationDistances PCA program (Scott Davis, Harvard Medical School, Boston, MA). Where indicated, the PCA program was used to identify the 15% most differentially expressed genes among subsets by filtering based on a variation of ANOVA analysis using the geometric standard deviation of populations to weight genes that vary in multiple populations. Data were log transformed, gene and subset normalized, and filtered for genes that had a MEV>120 prior to visualization. Euclidian distance and Pearson’s correlation coefficients were calculated using the “dist” and “cor” commands in R to generate distance and correlation matrixes for a given set of genes and subsets. Heatmaps of Euclidian distance and Pearson’s correlation coefficients were generated by hierarchical clustering (HierarchicalClustering module, Pearson’s correlation for row and column, complete-linkage). Data were visualized using a global color scheme in the HeatmapViewer module. Pathway analysis was performed using Ingenuity software (Ingenuity.com) and by manual inspection. Some functional classifications were performed using AmiGO (Amigo.geneontology.org) and KEGG pathways (Genome.kp.kegg).
The following cell surface antibodies were purchased from BD Biosciences: CD25 (PC61), CD27 (LG.3A10), Vγ2 (UC3-10A6), Vγ3 (536), Vδ6.3 (8F4H7B7), Ly49C/I (5E6), CCR6 (140706), Thy1.2 (53-2.1), CD9 (KMC8), streptavidin APC; eBioscience: CD3 (145-2C11), CD4 (RM4-5), CD8 (53-6.7), CD44 (IM7), CD122 (5H4), CD127 (IL-7Rα A7R34), CD24 (HSA, M1/69), c-Kit (2B8), TCRδ (GL3), NKG2D (CX5), CD62L (MEL-15), PD1 (J43), CD73 (TY/11.8), CD199 (CCR9, CW-1.2), CD197 (CCR7, 4B12), CD183 (CXCR3, 174), CD184 (CXCR4, 2B11), CD244.2 (2B4, 244F4), NKG2ACE (20D5), GITR (DTA-1), CD48 (HM48-1), CD28 (37.51), streptavidin PE-Cy7; or other vendors: IL-6R (D7715A7, BioLegend), CD119 (IFNγrα, RDI/Fitzgerald Industries). Vγ1.1 Ab was purified by Bio-XCell and biotinylated using the FluoReporter Mini-Biotin-XX Labeling Kit (Invitrogen). Vδ1 (17D1) antibody was provided by A.Hayday (King’s College, London). Intracellular proteins (IL-17, 17B7, eBioscience; IFN-γ, XMG1.2, BD Biosciences) were detected using the Cytofix/Cytoperm kit (BD Biosciences). Intranuclear staining was performed using the FOXP3 staining kit (eBioscience) for the following antibodies: TOX (TXRX10), EOMES (Dan11mag), RORγt (AFKJS-9), GATA-3 (L50-823), from eBioscience; BLK (cat# 3262), LEF-1 (C12A5), from Cell Signaling; Ki-67 from BD Biosciences; PLZF (D-9) and SMO (N-19), from Santa Cruz. FDG (Invitrogen) staining was performed according to standard protocols. Data were acquired on a BD LSRII cytometer and analyzed using FlowJo (Treestar). When indicated, data were concatenated from multiple independent samples in FlowJo for visualization in histograms.
Ex vivo stimulation
Ex vivo stimulations were performed by culturing total cells (2×106/well) with PMA (10ng/ml) and Ionomycin (1ug/ml) for 4 hours at 37°C, with Golgi Stop and Golgi Plug (BD Biosciences) added after 1 hour, according to the manufacturers’ protocols.
Mtb infection and analysis
WT Mtb bacterial strains were used to infect 4–10 male 6–8 week old C57BL/6 mice (Jackson Labs) with 2×103 CFU of Mtb by the aerosol route. Two weeks post infection, mice were sacrificed and cells were isolated from the spleen and lungs (enzymatic digestion) and stained immediately, or after culture with PMA and Ionomycin.
For the identification of differentially expressed genes, t-test P values were generated using Multiplot (Genepattern). For statistical analysis of flow cytometry data, Prism (GraphPad Software) was used. Data were tested for normality using F tests and then analyzed using unpaired two-tailed t-tests. Pathway analysis was performed using Ingenuity Pathway Analysis and statistical significance was determined using the program’s built-in Fisher’s exact test.