Napolitani et al Nat. Immunol. 2018 Mass Cytometry data

Clonal analysis of Typhoidal Salmonella specific effector CD4+ T cells in challenged human volunteers reveals immunodominant Serovar specific and cross-reactive T cell responses The characterization of the phenotype and specificity of human T cells responding to bacterial pathogens is hampered by the large number of protein antigens and by the possible presence of cross-reactive responses dependent on the large degree of sequence homology between related species. We took advantage of a human challenge model of enteric fever to provide the first characterization of the pathogen specific repertoire of effector CD4 T cells elicited in response to infection with Salmonella Typhi and Paratyphi A. Performing an unbiased clonal analysis of the specificity of a population of CD38+CCR7-Ki67+ IFN- Mip1 producing effector CD4 T cells released during enteric fever we found that the Salmonella specific effector T cell pool included clones cross-reactive against distantly related Salmonella Serovars as well as clones selectively recognizing the Typhoidal Serovars S. Typhi and S. Paratyphi A. The Typhoidal specific response was dominated by cells specific for two immune-dominant S. Typhi and S. Paratyphi A virulence factors, namely Cytolethal distending toxin (CDTB) and Hemolysin E (HlyE). Furthermore, a single amino acid variation in the sequence of CDTB accounted for a major fraction of clones non-cross-reactive among the two Typhoidal Serovars, providing a unique example of how minor antigenic variation in antigenically complex micro-organism can have a sizable impact in the pathogen specific repertoire of CD4 T cells. s
Conclusion:
Stimulation, Staining, and CyTOF Data Acquisition Cryopreserved cells were thawed and washed with pre-warmed RPMI supplemented with 1-% FBS (GIBCO, Life Technologies), 1X Penicliin/Streptomycin/L-glutamine (GIBCO, Life Technologies), 1% 1M HEPES (GIBCO, Life Technologies) and 1X β-mercaptoethanol (GIBCO, Life Technologies). Cells from each sample were split into two wells, followed by staining with the indicated antibodies (Supplementary Table 1) in 96-well round bottom plates for 30 minutes at 37°C prior to stimulation. Cells were left untreated or stimulated at 37°C for 4 hours with 150 ng/ml phorbol-12-myristate-13-acetate (PMA) (Sigma-Aldrich) and 1 μM ionomycin (Sigma-Aldrich) in the presence of monensin and Brefeldin A (eBioscience). After stimulation, cells were washed twice in cold PBS, followed by incubation on ice with 200 μM cisplatin (Sigma-Aldrich) for 5 minutes. Cells were then washed with CyFACS buffer (PBS + 4% FBS + 0.05% sodium azide) and stained with streptavidin-alpha Galcer with 10 μM free biotin for 30 minutes at room temperature. This was followed by 30 minutes incubation in primary antibody cocktail on ice. Cells were then washed twice in CyFACS and stained with metal-tagged surface antibodies. After 30 minutes, stimulated cells were washed twice with CyFACS, once with PBS, and then fixed in 2% paraformaldehyde (PFA; Electron Microscopy Sciences) at 4°C. The untreated cells were washed twice with CyFACS and incubated in Foxp3 Fixation/Permeabilization buffer (eBioscience) on ice for 30 minutes. Cells were then washed twice in 1 x Permeabilization (perm) Buffer (Biolegend) and stained with biotin anti-human Foxp3 and metal-tagged intracellular antibodies for 30 minutes on ice. After washing twice with perm buffer, cells were incubated on ice with metal-tagged streptavidin for 10 minutes. Cells were then washed twice in perm buffer, once in PBS, and then fixed in 2% PFA at 4°C. The next day, stimulated cells were washed twice with perm buffer and stained with intracellular antibodies on ice. After 30 minutes, all the untreated and stimulated cells were washed twice with perm buffer and once with PBS. Cells were then incubated with cellular barcodes for 30 minutes as previously described 40. Subsequently, cells were washed once with perm buffer and incubated in CyFACS for 10 minutes on ice. Cellular DNA was labeled at room temperature with 250 nM iridium interchelator (Fluidigm) diluted in PBS with 2% PFA. After 20 minutes, cells were washed twice with CyFACS and twice in distilled water prior to acquisition. To accommodate the required numbers of samples, two barcoded batches were prepared for each panel, with each batch having comparable number of samples for each of the time-points. Mass-tag barcoding was used so that all samples could be acquired simultaneously. A uniquely barcoded sample of PBMCs from a healthy donor was also included in batch as an internal control. EQ Four Element Calibration Beads (Fluidigm) were added at a final concentration of 1% prior to sample acquisition. Cells acquisition was performed on CyTOF2 (Fluidigm). Data Analysis: After mass cytometry acquisition, data were exported in flow-cytometry file (FCS) format, normalized41 and events with parameters having zero values were randomized using a uniform distribution of values between minus-one and zero. Each sample containing a unique combination of two metal barcodes was de-convoluted using manual gating in FlowJo to select cells stained with two and only two barcoding channels. Heterogeneity CD4+Ki67+ cells was evaluated with the Phenograph algorithm20 embedded in the Bioconductor CyTOFkit package 42. A total of 6185 cells was analysed. TiSNE analysis of CD4+ T cells stimulated with PMA/Ionomycin was performed using Cytobank 43 on CD4+CD45RA- T cells from volunteers 6, 22 49 and 72, at time points D0, ED and D28, based on the expression of CD40L, CTLA-4, MIP-1, TNF, IFN-, IL-2, GM-CSF, IL-17, IL-22, CD107a, IL-4, IL-8 and IL-10.