Journal: Cell Reports

Article Title: TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions

doi: 10.1016/j.celrep.2019.08.050

Figure Lengend Snippet: TL1A Enhances the Activation of MAIT Cells Suboptimally Stimulated with IL-12 and IL-18 CD8 + T cells were enriched from healthy peripheral blood mononuclear cells (PBMCs) and stimulated overnight with different combinations of cytokines: IL-12 at 2 ng/mL, IL-18 at 50 ng/mL, IL-15 at 25 ng/mL, and TL1A from 0.01 to 100 ng/mL as indicated. (A–C) Proportions of CD8 + MAIT/CD161 + or CD161 − cells producing IFN-γ (A), TNF-α (B), or CD69 (C) following overnight stimulation with suboptimal concentrations of IL-12 and IL-18, plus varying concentrations of TL1A. (D) Representative histograms showing the expression of IFN-γ, TNF-α, GrB, and CD69 by MAIT cells after stimulation with different combinations of cytokines. (E–H) Frequency of MAIT cells expressing IFN-γ (E), TNF-α (F), GrB (G), and CD69 (H) upon stimulation with the indicated cytokines. Data were acquired from seven donors in 2–3 experiments. Error bars represent means ± SEM. Differences among conditions were analyzed by Friedman tests with Dunn’s multiple comparison tests. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. See also Figure S1 .

Article Snippet: Anti-human CD161 (clone 191B8) PE-Vio770 , Miltenyi , Cat# 130-113-594; RRID: AB_2751134.

Techniques: Activation Assay, Expressing, Comparison

Journal: Cell Reports

Article Title: TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions

doi: 10.1016/j.celrep.2019.08.050

Figure Lengend Snippet: TCR-Mediated Activation of MAIT Cells Leads to the Expression of Tissue-Repair-Associated Molecules and Accelerates Wound Healing (A–C) Gene set enrichment summary plots for stimulated sorted MAIT cell-versus-unstimulated cell-ranked genes. Depicted are the individual plots for TCR-stimulated versus UT in (A), TC-stimulated versus UT in (B), and C versus unstimulated in (C). Non-significant for C versus UT, normalized enrichment score (NES) = 1.63; p < 0.0002 for TCR versus UT, NES = 1.57; and p < 0.0002 for TC versus UT. Data were acquired from three donors in one experiment. (D) Flow cytometry analysis of the expression of TNF-α, furin, and CCL3 by CD161 ++ /MAIT CD8 + T cells in response to fixed E. coli presented by THP1 cells in the presence or absence of an anti-MR1 (αMR1) blocking antibody at the 72-h time point. (E) Statistical analysis of the expression of the effector molecules shown in (D). (F) Caco2 cells were grown to confluency and scratched with a WoundMaker device to perform in vitro wound-healing assays. Cells were supplemented with different supernatants collected from 72-h cocultures of enriched CD8 T cells with E. coli -loaded THP1 cells in the presence or absence of αMR1, as indicated. The open wound areas were quantified as percentages of the initial wound size in the Caco2 cultures. Data points are mean ± SEM and were acquired from five biological replicates in two experiments. (G) Representative pictures of the closure of the wounds in Caco2 cultures treated as in (F) were assessed with time-lapse imaging over a time course of 36 h. Data were acquired from seven donors in three experiments. Differences among conditions were analyzed by two-way ANOVA. ns, not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.001. Scale bars, 250 μm. See also Figure S5 and .

Article Snippet: Anti-human CD161 (clone 191B8) PE-Vio770 , Miltenyi , Cat# 130-113-594; RRID: AB_2751134.

Techniques: Activation Assay, Expressing, Flow Cytometry, Blocking Assay, In Vitro, Imaging

Journal: Cell Reports

Article Title: TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions

doi: 10.1016/j.celrep.2019.08.050

Figure Lengend Snippet: MAIT Cells Can Be Found Close to and within the Colonic Epithelium (A–G) Representative images showing the expression of Va7.2, CD161, CD8, PLZF, CD3, and CD103 in the lamina propria and the epithelium of fixed samples of colonic polyp tissue. Samples were mounted on cytometer chips and iteratively stained with sets of three directly fluorochrome-conjugated antibodies as described in the methods section. Depicted are a merged picture (A) and all the individual stains for Va7.2 (B), CD161(C), CD8 (D), PLZF (E), CD3 (F), and CD103 (G). White arrows mark cells showing co-expression of Va7.2, CD161, PLZF, and CD3 that were defined as MAIT cells here. Note that while CD8 was co-expressed in most of them, CD8− MAITs (arrow + asterisk) could also be found. In contrast, CD103 was rarely co-expressed on MAITs (arrow + diamond). During the iterative staining process dust particles and other detritus can be picked up by the solution flowing over the tissue creating autofluorescent artifacts (1–4). While some of these get washed away after completion of the staining cycle (1, 4), others present during multiple imaging rounds (2, 3). Scale bars, 50μm.

Article Snippet: Anti-human CD161 (clone 191B8) PE-Vio770 , Miltenyi , Cat# 130-113-594; RRID: AB_2751134.

Techniques: Expressing, Cytometry, Staining, Imaging

Journal: Cell Reports

Article Title: TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions

doi: 10.1016/j.celrep.2019.08.050

Figure Lengend Snippet:

Article Snippet: Anti-human CD161 (clone 191B8) PE-Vio770 , Miltenyi , Cat# 130-113-594; RRID: AB_2751134.

Techniques: Virus, Recombinant, Reverse Transcription, Activation Assay, Staining, Software

Journal: Nature Communications

Article Title: High-dimensional profiling reveals Tc17 cell enrichment in active Crohn’s disease and identifies a potentially targetable signature

doi: 10.1038/s41467-022-31229-z

Figure Lengend Snippet: Flow cytometry analysis of PMA/Ionomycin stimulated cells indicating percentage of γδ T cells (γδTCR+), NKT cells (CD56+ TCR γδ- Vα7.2-), MAIT cells (CD161 hi Vα7.2+ or CD161 mid Vα7.2+) and conventional T cells (TCR γδ- CD56- Vα7.2-) of CD8+ T cells ( A ) and of Tc17 cells ( B ). Comparison of indicated frequencies in PBMC as well as in the intestinal tissue of HD ( A n = 18, B n = 12), non-inflamed mucosa from CD patients (normal mucosa, A n = 28, B n = 15) and inflamed mucosa from CD patients (inflamed mucosa, A n = 16, B n = 12). C TSNE analysis of conventional and unconventional T cells calculated based on the lineage markers described above from peripheral blood and intestinal biopsies ( n = 10) of 7 different CD patients with active disease. IL-17 production by unconventional (orange dots) and conventional (red dots) T cells is shown. D Frequency of CD56+ cells of Vα24Jα18 (Clone 6B11)+ CD8+ iNKT cells ( n = 5). E Percentage of MAIT cells (CD161 hi Vα7.2+ and CD161 mid Vα7.2+), iNKT cells (Vα24Jα18+), γδ T cells (γδTCR+) and conventional T cells (defined as Vα7.2-, Vα24Jα18-, γδTCR- and αβ TCR+) of IL-17+ CD8+ CD3+ cells were examined in PBMCs of HD ( n = 5), CD patients ( n = 6) and in intestinal biopsies of CD patients (n biopsies = 6). Broad and narrow conventional T cell frequencies were determined taking into account only the iNKT (Vα24Jα18-) vs. both the iNKT and the CD56+ NKT definition (Vα24Jα18-, CD56-), respectively. Data in A, B, D, E are presented as mean + /− SEM. Statistical tests used were two-sided. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: The following antibodies were used for flow cytometry experiments: CD8 BV650 (RPA-T8, BioLegend), PD-1 BV786 (EH12.1, BD Biosciences), PD-1 BV421 (EH12.2H7, BioLegend), PD-1 PerCP-eFluor710 (eBioJ105, eBioscience), CD26 FITC (2A6, eBioscience), CD6 PE (BL-CD6, BioLegend), CD27 PE-Dazzle594 (M-T271, BioLegend), CD39 PerCP-eFluor710 (eBioA1, eBioscience), CD69 PE-Cy7 (FN50, eBioscience), CD161 APC (191B8, Miltenyi), IFN-γ APC-eFluor780 (4 S.B3, eBioscience), Fixable viability dye BV510 (eBioscience), Fixable viability dye APC-eFluor780 (eBioscience), IL-17 BV605 (BL168, BioLegend), IL-17F BV786 (O33-782, BD Biosciences), RORγt PE (# 600380, R&D Systems), CD56 BV650 (5.1H11, BioLegend), TCRαβ AlexaFluor488 (IP26, BioLegend), TCRαβ BV421 (IP26, BioLegend), TCRγδ PE (5A6.E9, Life Technologies), CD8 PE-Dazzle594 (RPA-T8, BioLegend), TCR Vα7.2 PE-Cy7 (3C10, BioLegend), TCR Vα7.2 FITC (3C10, BioLegend), TCR Vα24Jα18 PE-Cy7 (6B11, Invitrogen), CD3 AlexaFluor700 (SK7, BioLegend), IL-17 PE (eBio64DEC17, eBioscience), TNF PE-Cy7 (MAb11, BioLegend), pSTAT3 Alexa 647 (4/P-STAT3, BD Biosciences), CD3 PerCP (SK7, BD Biosciences), CD8 Krome Orange (B9.11, Beckman Coulter), CD45RA PeCy7 (HI100, BD Biosciences), Isotype IgG2a κ Alexa 647 (MOPC-173, BD Biosciences), CD4 BV786 (L200, BD Biosciences), CD4 BV421 (RPA-T4, BioLegend).

Techniques: Flow Cytometry, Comparison

Journal: Nature Communications

Article Title: High-dimensional profiling reveals Tc17 cell enrichment in active Crohn’s disease and identifies a potentially targetable signature

doi: 10.1038/s41467-022-31229-z

Figure Lengend Snippet: A CD8+ T cell transcriptome data and corresponding clinical data from 31 untreated CD patients were extracted from E-MTAB-331 and patients were stratified into two groups based on hierarchical clustering on phenotypic Tc17 signature markers CD6, CD69, CD27, CD39, PD-1, CD26, CD161. Kaplan-Meier-plot depicts flare-free survival. Log rank test was used to determine statistical significance. B Time dependent ROC curve for prediction of flare free survival at 200 days of follow up using a GSVA score based on phenotypic Tc17 signature markers CD6, CD69, CD27, CD39, PD-1, CD26, CD161 and CD8+ T cell transcriptome data and corresponding clinical data from 35 untreated CD patients that were extracted from . C CD6 expression in group 1 and 2 indicated in arbitrary units, n = 35 patients extracted from E-MTAB-331. Data are presented as mean +/− SEM. Statistical tests used were two-sided. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: The following antibodies were used for flow cytometry experiments: CD8 BV650 (RPA-T8, BioLegend), PD-1 BV786 (EH12.1, BD Biosciences), PD-1 BV421 (EH12.2H7, BioLegend), PD-1 PerCP-eFluor710 (eBioJ105, eBioscience), CD26 FITC (2A6, eBioscience), CD6 PE (BL-CD6, BioLegend), CD27 PE-Dazzle594 (M-T271, BioLegend), CD39 PerCP-eFluor710 (eBioA1, eBioscience), CD69 PE-Cy7 (FN50, eBioscience), CD161 APC (191B8, Miltenyi), IFN-γ APC-eFluor780 (4 S.B3, eBioscience), Fixable viability dye BV510 (eBioscience), Fixable viability dye APC-eFluor780 (eBioscience), IL-17 BV605 (BL168, BioLegend), IL-17F BV786 (O33-782, BD Biosciences), RORγt PE (# 600380, R&D Systems), CD56 BV650 (5.1H11, BioLegend), TCRαβ AlexaFluor488 (IP26, BioLegend), TCRαβ BV421 (IP26, BioLegend), TCRγδ PE (5A6.E9, Life Technologies), CD8 PE-Dazzle594 (RPA-T8, BioLegend), TCR Vα7.2 PE-Cy7 (3C10, BioLegend), TCR Vα7.2 FITC (3C10, BioLegend), TCR Vα24Jα18 PE-Cy7 (6B11, Invitrogen), CD3 AlexaFluor700 (SK7, BioLegend), IL-17 PE (eBio64DEC17, eBioscience), TNF PE-Cy7 (MAb11, BioLegend), pSTAT3 Alexa 647 (4/P-STAT3, BD Biosciences), CD3 PerCP (SK7, BD Biosciences), CD8 Krome Orange (B9.11, Beckman Coulter), CD45RA PeCy7 (HI100, BD Biosciences), Isotype IgG2a κ Alexa 647 (MOPC-173, BD Biosciences), CD4 BV786 (L200, BD Biosciences), CD4 BV421 (RPA-T4, BioLegend).

Techniques: Expressing

Journal: Nature Communications

Article Title: High-dimensional profiling reveals Tc17 cell enrichment in active Crohn’s disease and identifies a potentially targetable signature

doi: 10.1038/s41467-022-31229-z

Figure Lengend Snippet: A CD26 hi CD161+ CD8+ T cells contain Tc17 cells and express higher CD6 as illustrated by original plots and overlay histograms of live CD8+ and CD26 hi CD161+ CD8+ T cells from PBMC and intestinal tissue of a CD patient analyzed for IL-17A and CD6. B Reduction in CD26 hi CD161+ CD8+ T cells in presence of anti-CD6 (Itolizumab) treatment during culture of PBMCs overnight (o/n), or for three or seven days (d3, d7) (plots gated on CD8+ T cells). C Cell numbers in anti-CD6 vs. control-treated assays ( n = 4 for each condition). D Percentage of CD26 hi CD161+ CD8+ T cells in presence of RPMI, Itolizumab ( n = 8) or UMCD6 ( n = 5). E Percentage of IL-17A+ CD8+ T cells in presence of RPMI, Itolizumab ( n = 14) or UMCD6 ( n = 13). F Proportion of IFN-γ and TNF mono- and coproduction by IL-17A+ CD8+ CD3+ T cells in presence of RPMI or Itolizumab ( n = 8). G IL-17A production of CD8+ T cells from intestinal biopsies after five-hour stimulation in presence of RPMI or UMCD6. Depicted are samples from a CD patient with clinical and endoscopic remission and a patient with clinical and endoscopic activity. D , E Data after 5 h PMA/Ionomycin stimulation; ( B – E ) assays performed on HD samples. Data in ( C ) and ( F ) are presented as mean +/− SEM. Statistical tests used were two-sided. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: The following antibodies were used for flow cytometry experiments: CD8 BV650 (RPA-T8, BioLegend), PD-1 BV786 (EH12.1, BD Biosciences), PD-1 BV421 (EH12.2H7, BioLegend), PD-1 PerCP-eFluor710 (eBioJ105, eBioscience), CD26 FITC (2A6, eBioscience), CD6 PE (BL-CD6, BioLegend), CD27 PE-Dazzle594 (M-T271, BioLegend), CD39 PerCP-eFluor710 (eBioA1, eBioscience), CD69 PE-Cy7 (FN50, eBioscience), CD161 APC (191B8, Miltenyi), IFN-γ APC-eFluor780 (4 S.B3, eBioscience), Fixable viability dye BV510 (eBioscience), Fixable viability dye APC-eFluor780 (eBioscience), IL-17 BV605 (BL168, BioLegend), IL-17F BV786 (O33-782, BD Biosciences), RORγt PE (# 600380, R&D Systems), CD56 BV650 (5.1H11, BioLegend), TCRαβ AlexaFluor488 (IP26, BioLegend), TCRαβ BV421 (IP26, BioLegend), TCRγδ PE (5A6.E9, Life Technologies), CD8 PE-Dazzle594 (RPA-T8, BioLegend), TCR Vα7.2 PE-Cy7 (3C10, BioLegend), TCR Vα7.2 FITC (3C10, BioLegend), TCR Vα24Jα18 PE-Cy7 (6B11, Invitrogen), CD3 AlexaFluor700 (SK7, BioLegend), IL-17 PE (eBio64DEC17, eBioscience), TNF PE-Cy7 (MAb11, BioLegend), pSTAT3 Alexa 647 (4/P-STAT3, BD Biosciences), CD3 PerCP (SK7, BD Biosciences), CD8 Krome Orange (B9.11, Beckman Coulter), CD45RA PeCy7 (HI100, BD Biosciences), Isotype IgG2a κ Alexa 647 (MOPC-173, BD Biosciences), CD4 BV786 (L200, BD Biosciences), CD4 BV421 (RPA-T4, BioLegend).

Techniques: Control, Activity Assay

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: αβ T-cell receptors from multiple sclerosis brain lesions show MAIT cell–related features

doi: 10.1212/NXI.0000000000000107

Figure Lengend Snippet: Double fluorescence immunohistochemistry identifies brain-infiltrating mucosal-associated invariant T (MAIT) cells in multiple sclerosis (MS) lesions of patient A. Nuclei are stained with 4',6-diamidino-2-phenylindole (white). Green and red dyes were used. Double-positive cells are therefore shown in yellow. Scale bars 20 µm. (A) T cells expressing the T-cell receptor Vα7.2 (red) and Vβ1 chains (green). This combination (see ) was identified by single-cell PCR. (B) Most Vα7.2 + (green) T cells belong to the CD8 + (red) T-cell subset. (C) MAIT cells expressing Vα7.2 (red) and CD161 (green) in the parenchyma of MS brain. (D) The vast majority of CD161 + (green) cells in MS CNS coexpress CD8α (red).

Article Snippet: To isolate MAIT cells from PBMCs in 2013 and 2014, T cells were negatively isolated using the pan T-cell isolation kit (Miltenyi Biotec) and subsequently stained with the FITC-labeled mouse anti-human Vα7.2 (1:20, 3C10, BioLegend) and the allophycocyanin-labeled mouse anti-human CD161 (1:10, 191B8, Miltenyi Biotec) antibodies at 4°C for 30 minutes.

Techniques: Fluorescence, Immunohistochemistry, Staining, Expressing

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: αβ T-cell receptors from multiple sclerosis brain lesions show MAIT cell–related features

doi: 10.1212/NXI.0000000000000107

Figure Lengend Snippet: Analysis of the T-cell receptor (TCR) Vα7.2 repertoire of patient A by pyrosequencing shows oligoclonal T-cell expansions in different samples. Each pie chart represents the total number of nucleotide sequences found in the Vα7.2 repertoire of a certain compartment and each sector represents one distinct T-cell clone. We compared (A) samples from CNS tissue from the biopsy taken in 1996, (B) Vα7.2 + CD161 + mucosal-associated invariant T (MAIT) cells, (C) Vα7.2 + CD161 − cells, (D) CD8 + cells, and (E) CD4 + T cells from peripheral blood taken in 2013 and 2014. For each population we list the designation of the Jα elements and their relative percentage. Clones carrying the MAIT canonical TCR α chain (CAXXDSNYQLIW) are marked in bright blue, and clones containing the noncanonical α chains characterized by Jα12 (CAXXDSSYKLIF) and Jα20 (CAVXXDYKLSF) are marked in dark blue and light blue, respectively. (F) Percentages of identical complementarity determining region 3α amino acid sequences within the TCR Vα7.2 + repertoire between the different samples defined above (A–E). The numbers are percentages indicating how often a particular sequence detected in one sample was also found in another sample. The greatest overlap was between Vα7.2 + CD161 + and CD8 + T cells from peripheral blood, but there was also significant overlap between the CNS sample and the Vα7.2 + CD161 + sample from 2013 to 2014, as highlighted by the red and yellow colors. PBMC = peripheral blood mononuclear cell.

Article Snippet: To isolate MAIT cells from PBMCs in 2013 and 2014, T cells were negatively isolated using the pan T-cell isolation kit (Miltenyi Biotec) and subsequently stained with the FITC-labeled mouse anti-human Vα7.2 (1:20, 3C10, BioLegend) and the allophycocyanin-labeled mouse anti-human CD161 (1:10, 191B8, Miltenyi Biotec) antibodies at 4°C for 30 minutes.

Techniques: Clone Assay, Sequencing