Computational protocol: Association of Decreased Percentage of Vδ2+Vγ9+ γδ T Cells With Disease Severity in Multiple Sclerosis

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[…] Peripheral blood mononuclear cells were collected by density gradient centrifugation using Lymphoprep tubes (AXIS-SHIELD Poc AS, Oslo, Norway) containing Ficoll-Paque (GE Healthcare, Little Chalfont, UK) and then suspended in RPMI-1640 medium (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (Gibco, Waltham, MA, USA). Immunophenotyping was performed using the antibodies shown in Tables S1 and S2 in Supplementary Material. For surface staining, cell suspensions were incubated with an optimal concentration of antibodies for 20 min at 4°C in the dark. For intracellular staining, cell suspensions were incubated with 25 ng/ml of phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA) and 1 µg/ml of ionomycin (Sigma-Aldrich) in the presence of 10 µg/ml of brefeldin A (Sigma-Aldrich) for 4 h at 37°C. Stained cells were analyzed on a FACSVerse flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).γδ T cells (CD3+TCRγδ+TCRαβ−) were characterized by surface staining with anti-Vδ1, Vδ2, and Vγ9 antibodies, and then cytokine production was determined by intracellular cytokine staining for IL-17A and IFN-γ (Figures S1A,B in Supplementary Material). αβ T cells were characterized as CD4+ or CD8+ T cells, and subsequently as naïve T (Tnaïve, CCR7+CD45RA+), central memory T (Tcm, CCR7+CD45RA−), effector memory T (Tem, CCR7−CD45RA−), effector T (Teff, CCR7+CD45RA−), or activated T (HLA-DR+) cells by surface staining (Figure S2A in Supplementary Material). Regulatory CD4+ T (Treg) cells were defined as CD25+CD127low/−. In addition, CD4+CD25+CD127low/− T cells expressing FoxP3 were measured in HCs (Figure S3 in Supplementary Material), because FoxP3 expression in CD4+CD25+ T cells has been reported to be tightly linked to the suppressive functions of Treg cells (, ). We found that FoxP3+ cells were 77.7 ± 7.9% of CD4+CD25+CD127low/− T cells, and that the percentage of CD25+CD127low/− T cells (defined as Tregs) had a significant positive correlation with CD25+FoxP3+ T cells in CD4+ T cells (r = 0.6396, p = 0.0138) (Figure S4 in Supplementary Material). These data are consistent with previous studies (, ).For intracellular cytokine staining of αβ T cells after in vitro stimulation with PMA and ionomycin, IL-17A, IFN-γ, IL-4, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were measured in CD4+ T cells, while IL-17A and IFN-γ were measured in CD8+ T cells (Figure S2B in Supplementary Material). B cells (CD19+CD3−) were characterized by surface staining as class-switched memory (CS+ memory, CD27+IgD−), non-class-switched memory (CS− memory, CD27+IgD+), naïve B (CD27−IgD−), and transitional B (CD24+CD38+) cells and plasmablasts (CD38highCD20−) (Figure S5 in Supplementary Material). Appropriate isotype controls were used in each experiment. The data were analyzed using FlowJo software (TreeStar, San Carlos, CA, USA). [...] Fisher’s exact test was used to compare categorical variables, and the Wilcoxon rank sum test was used to analyze continuous scales. Correlations among continuous scales were calculated using Spearman’s rank correlation coefficient. Uncorrected p values (puncorr) were multiplied by the number of comparisons to calculate the corrected p values (pcorr), as indicated in the footnote of the tables (Bonferroni–Dunn’s correction). Statistical analysis was performed using JMP Pro 12.2.0 software (SAS Institute, Cary, NC, USA). A p-value <0.05 was considered statistically significant. […]

Pipeline specifications

Software tools FlowJo, JMP Pro
Applications Miscellaneous, Flow cytometry
Organisms Homo sapiens
Diseases Multiple Sclerosis
Chemicals Ionomycin