Fig 2: Gating strategy to identify T cell subsets (CD4+, CD8+ and ?dTCR+) and counting beads based on a representative sample.(a) Gating strategy to identify T cell subsets. T lymphocytes were selected based on CD3 expression and side scatter (SSC) characteristics. The CD3+ population was further divided into CD4+/CD8+ T lymphocytes (based on CD4 and CD8 expression) and ?dT positive and negative CD3 cells (based on ?dTCR expression). (b) Gating strategy to identify counting beads. First, two populations of counting beads were selected based on the forward and side scatter characteristics. Second, FITC positive beads were counted for the cell count analyses. Third, the frequency of each bead population was assessed to compare with bead lot characteristics.

Fig 3: Analysis of genetic and epigenetic conservation in mouse and human Treg and CD4+ T cell subsets.(A) Schematic representation of profiled CD4+ T-cell subsets. Abbreviations: naive T cell (Tn); effector T cell (Teff); resting regulatory T cell (rTreg); activated regulatory T cells (aTreg). (B) The indicated human CD4+ T-cell subpopulations were FACS sorted based on CD3, CD4, CD45RO, and CD25 expression from preparations of peripheral blood mononuclear cells (PBMCs) from healthy human donors. Highly purified Treg cell subpopulations were obtained using a FACS Aria II fluorescent cell sorter (Figure 1—figure supplement 1A). Epigenetic profiling was performed using the following 16 cell samples isolated from 7 healthy donors: including 7 aTreg, 4 rTreg, 2 Teff, 2 Tmem, and 1 Tn independently isolated cell populations. See also Figure 1—figure supplement 1A,B. (C) Resting and activated murine CD4+ T-cell subpopulations were FACS sorted from Foxp3DTR-GFP mice injected with PBS or diphtheria toxin (DT), respectively. In Foxp3DTR-GFP mice, Treg cells express diphtheria toxin receptor (DTR). Mice injected with DT underwent punctual Treg cell depletion and consequent transient systemic inflammation, which resulted in activation of rebounding Treg and conventional T cells. A total of 10 mouse cell samples isolated using FACS sorting from DT-treated and DT-untreated Foxp3DTR mice were analyzed: 2 aTreg, 4 rTreg, 2 Teff, and 4 Tn biological replicates. (D, E) Genetic and epigenetic conservation at select loci. Multiple regulatory elements near LRRC32 and YY1 are genetically and epigenetically conserved: YY1 has two epigenetic elements that are not conserved in human; LRRC32 has a regulatory element that is genetically, but not epigenetically conserved. (D) Acetylation at the LRRC32 locus shows multiple conserved genetic elements that illustrate concordant and discordant epigenetic states across species (highlighted regions). The human LRRC32 locus (top) and murine Lrrc32 locus (bottom) feature extensive genetically orthologous elements (lines connecting human and murine genomic coordinates) containing species-specific insertions/deletions (white space). H3K27ac ChIP-seq reads per million (RPM) are shown on y-axis for the indicated species and cell lineages. Orthologous regions with regulatory elements of interest are shown by blue background highlighting and red connecting lines. A genetically conserved element near LRRC32 is epigenetically active in mouse, but not in human (leftmost highlighted region). (E) Two regulatory elements near YY1 are epigenetically active in human but are not genetically conserved in mouse (leftmost and rightmost highlighted regions). (F) Genome-wide fractions of genetically conserved acetylated loci. Loci with high read counts are more frequently genetically conserved (shown) as are regulatory elements more proximal to gene body (Figure 1—figure supplement 1H). (G) Genome-wide quantification of epigenetic conservation. Axes show H3K27ac quantification (reads per million: RPM) of murine (x-axis) and human (y-axis) acetylated loci. Qualitatively, the vast majority of regulatory elements are epigenetically conserved in mouse and human Treg cells, with genome-wide quantitative correlation of r = 0.48 (‘†’ indicates that correlation is computed only for genetically conserved loci; non-conserved loci are shown on axes and by definition cannot be epigenetically conserved). Correlation across mouse biological replicates was r > 0.99 and between human donors r > 0.94, indicating that the observed conservation and lack thereof are reflective of biology and not technical/replicate reproducibility (Figure 1—figure supplement 1K).DOI: http://dx.doi.org/10.7554/eLife.07571.003

Fig 4: MAIT cell frequency and activation in PBMCs and LPLs by flow cytometric analysis.Lamina propria lymphocytes (LPLs) and PBMCs were isolated from patients. (A) Frequency of MAIT cells (live (DAPI-) CD3+CD161hiVa7.2+ cells), as % of total CD3+ lymphocytes, and (B) Frequency of CD3+ T cells, as of total lymphocyte population, in LPLs at day 2 (n = 10) and day 30 (n = 8) p.i., and PBMCs, at day 2 (n = 10), day 7 (n = 10), and day 30 (n = 9) p.i. (C) Frequency of CD38+ cells, and (D) frequency of CD69+ cells, gated on MAIT cells in LPLs at day 2 (n = 9) and day 30 (n = 7) p.i., and PBMCs, at day 2 (n = 9), day 7 (n = 9), and day 30 (n = 8) p.i. Horizontal bar shows median values in graphs. (E) Gating strategy used to identify MAIT cells and representative histograms showing % CD38+ and % CD69+ cells gated on MAIT cells in LPL and PBMCs. Statistical significance of difference between groups was determined using one-way ANOVA with Tukey’s post hoc testing. * denotes p = 0.05, ** denotes p = 0.01, and *** denotes p < 0.001.

Fig 5: MAIT cell localization and frequency in LP by Immunohistochemistry.Cryosections of duodenal biopsies obtained from five cholera patients (n = 5) were stained with antibodies as mentioned in methods and imaged using Leica CM3000 Cryostat. (A) Representative images of duodenal biopsies at day 2 and day 30, shows CD3+ T cells (red arrows), IL-18Ra+ T cells (green arrows), Va 7.2+ cells (magenta arrows), and the merged image shows MAIT cells (yellow arrows indicating CD3+ IL-18Ra+ Va7.2+ cells). (B) Frequency of MAIT cells, as a % of total CD3+ cells in the duodenum at day 2 and day 30 post infection (p.i.), and (C) Frequency of CD3+ cells, as % of total cells in the lamina propria area at day 2 and day 30 p.i. Statistical significance of the difference between day 2 and day 30 was determined using the Wilcoxon signed-ranked test.