premixed magnetic microsphere kits  (R&D Systems)

Journal: Nature immunology

Article Title: Altered differentiation is central to HIV-specific CD4 + T cell dysfunction in progressive disease

doi: 10.1038/s41590-019-0418-x

Figure Lengend Snippet: (a) Representative RNA flow FISH plots from an CP of CXCL13 and IL21 mRNA in CD69 + CD40L + CD4 + T cells after a 15h stimulation with a Gag peptide pool. Numbers represent the frequency of mRNA + cells among (red) CD69 + CD40L + and (grey) total CD4 + T cells (n= 6 CPs and 5 ECs) (b,c) Quantification of (a). Comparison of frequencies of CXCL13 or IL21 mRNA + CD4 + T cells between CPs (n=6) and ECs (n=5) obtained as in a after Gag or SEB stimulation. Bars represent median frequencies +/−interquartile range (two-tailed Mann-Whitney test, **p<0.01). (d,e) RNA flow FISH analysis of co-expression of CXCL13 and IL21 mRNA by Gag-specific CD4 + T cells with ( d ) representative plots from CP and EC people and (e) related statistical analysis by permutation test (10000 permutations) in SPICE (n= 6 CPs and 5 ECs). Pie slices represent median frequency of CXCL13/IL21 mRNA + subpopulations. (f) Representative histograms from CP and EC people of CXCR5 expression on CXCL13 or IL21 mRNA + HIV-specific CD4 + T cells overlaid on total HIV-specific CD4 + T cells (grey). Numbers represent the percentages of CXCR5 + cells within each cytokine + population (n= 6 CPs and 5 ECs). (g) Statistical analysis from (f) on 6 CPs and 5 ECs of the ratios between the CXCR5 mem to the CXCR5 neg subsets of frequencies of cytokine or chemokine mRNA + HIV-specific CD4 + T cells ( CXCL13 , IL21 , IL2 and IFNγ ) as assessed by RNA flow cytometry. Bars represent median frequencies +/−interquartile range (two-tailed Mann-Whitney test). (h,i) Co-expression patterns of PD-1 and TIGIT (surface protein Ab labeling) on CXCL13 mRNA + or IL21 mRNA + Gag-specific CD4 + T cells identified by RNA flow FISH (n= 6 CPs and 5 ECs). ( h ) Representative examples of CP and EC people; cytokine/chemokine mRNA + HIV-specific CD4 + T cells (red dots for CPs, blue dots for ECs) are overlaid on total HIV-specific CD4 + T cell subpopulations (grey dots). Numbers represent the percentages of CXCL13 RNA + or IL21 mRNA + HIV-specific CD4 + T cells located in each quadrant. (i) SPICE analysis of PD-1 and TIGIT expression from (h) on CXCL13 mRNA + or IL21 mRNA + HIV-specific CD4 + T cells. (*p<0.05, **p<0.01 by permutation test, 10000 permutations). Slices represent median frequency of PD-1 and TIGIT subpopulations. (j) Comparison of p24-specific antibodies levels in plasma between CPs and ECs as assessed by ELISA. (two-tailed Mann-Whitney test (n= 12 CPs and 13). Bars represent medians +/− interquartile range. (k,l,m) Correlation between p24-specific antibodies levels measured by ELISA and frequencies of k ) total HIV-specific CD4 + T cells; l) CXCR5 mem HIV-specific CD4 + T cells and m ) CXCR5 neg HIV-specific CD4 + T cells assessed by the CD69/CD40L AIM assay after a 9h stimulation with a Gag peptide pool (two-tailed Spearman test, n=12 CPs and 13 ECs).

Article Snippet: Measurements were performed in duplicates using the Human High Sensitivity Cytokine Premixed Kit B (R&D Systems) for IL-2, IFN-γ, TNF-α, IL-17A, IL-22 and IL-31 or for IL-17F on a Bio-plex 200 array system (Bio-Rad Laboratories) per manufacturer’ s instructions.

Techniques: Comparison, Two Tailed Test, MANN-WHITNEY, Expressing, Flow Cytometry, Labeling, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Nature immunology

Article Title: Altered differentiation is central to HIV-specific CD4 + T cell dysfunction in progressive disease

doi: 10.1038/s41590-019-0418-x

Figure Lengend Snippet: (a,b). Representative flow cytometry plots depicting detection of IL22 and IL17F in CD69 + CD40L + CD4 + T cells in one EC after stimulation with a Gag peptide pool by delayed ICS for cytokine protein and RNA flow FISH for cytokine mRNA, respectively. Numbers represent frequencies of mRNA + HIV-specific CD4 + T cells among (blue) CD69 + CD40L + and (grey) total CD4 + T cells (n= 8 CPs and 8 ECs). (c,d) Statistical comparison from (a) of frequencies of cytokine mRNA + antigen-specific CD4 + T cells between CPs and EC after (c) a Gag peptide pool (n= 8 CPs and 8 ECs) or (d) SEB stimulation (n=6 CPs and 8 ECs) (two-tailed Mann-Whitney test). (e) Statistical comparison of IL-22 and IL-17F protein levels detected by Luminex beads array in the supernatant of CD8-depleted PBMCs 48h after stimulation with a Gag peptide pool (n= 8 CPs and 8 ECs). (c,d,e) Bars represent median with +/−interquartile range. *p<0.05, **p<0.01 by two-tailed Mann-Whitney test. (f) Representative flow cytometry plots of co-expression patterns of IL22 mRNA and IL17F mRNA in HIV-specific CD4 + T cells in CP and EC people (n=6 CPs and ^ ECs). (g) Statistical analysis from (f) by SPICE (*p<0.05 by permutation test, 10000 permutations). Slices represent median frequency of IL22 / IL17F mRNA + subpopulations of HIV-specific CD4 + T cells (n= 6 CPs and 6 ECs). (h,i) Comparison of the frequencies of CCR6 and CXCR3 expression on IL22 mRNA + and IL17F mRNA + HIV-specific CD4 + T cells compared to total HIV-specific CD4 + T cells in CPs (n=6) and ECs (n=6) by two-tailed Mann-Whitney test and by two-tailed Wilcoxon matched-pairs signed-ranked test. Only p ˂0.05 are displayed for clarity. Bars represent median with interquartile range. (j,k) Correlation of ( j ) IL22 and ( k ) IL-17 F mRNA levels assessed by qRT-PCR on sorted HIV Gag-specific CD4 + T cells and CD4 + T cell activation measured by HLA-DR and CD38 co-expression (two-tailed Spearman test; n= 10 CPs, 6 VCs, 12 ECs). ( l,m ) Statistical comparisons of microbial translocation in plasma in cohorts of CP, EC, and uninfected control donors (UD) ( l ) quantitation of bacterial RNA reads in plasma (transcripts per million, TPM); ( m ) bacterial RNA species diversity (Shannon entropy score) (Kruskal Wallis test; n= 10 CPs, 8 ECs and 6 UDs). Bars represent median +/−interquartile range. ( n,o ) Correlation between bacterial RNA species diversity in plasma and frequencies of ( n ) HIV-specific IL22 + CD4 + T cells and ( o ) HIV-specific IL17 + CD4 + T cells (two-tailed Spearman test; n=10 CPs and 8 ECs). ( p,q ) Correlation between abundance of Proteobacteria translocation and frequencies of ( p ) HIV-specific IL22 + CD4 + T cells and ( q ) HIV-specific IL17 + CD4 + T cells (two-tailed Spearman test; n=10 CPs and 8 ECs).

Article Snippet: Measurements were performed in duplicates using the Human High Sensitivity Cytokine Premixed Kit B (R&D Systems) for IL-2, IFN-γ, TNF-α, IL-17A, IL-22 and IL-31 or for IL-17F on a Bio-plex 200 array system (Bio-Rad Laboratories) per manufacturer’ s instructions.

Techniques: Flow Cytometry, Comparison, Two Tailed Test, MANN-WHITNEY, Luminex, Expressing, Quantitative RT-PCR, Activation Assay, Translocation Assay, Clinical Proteomics, Control, Quantitation Assay