Journal: The Journal of Clinical Investigation

Article Title: CD38 expression by neonatal human naive CD4 + T cells shapes their distinct metabolic and tolerogenic properties

doi: 10.1172/JCI200062

Figure Lengend Snippet: ( A and B ) CD25 + percentages from CB and AB naive CD4 + T cells after 96 hours of anti-CD3/CD28 stimulation under No Cytokine or IL-2 + TGF-β conditions. ( C – E ) Marker expression on CD25 + fraction by flow cytometry. ( F and G ) ELISA of cytokines in supernatant. ( H ) Allogeneic MLR measuring suppressive capacity of CB and AB No Cytokine and IL-2 + TGF-β-stimulated cells. ( I ) Allogeneic MLR assay for CD25 + versus CD25 neg iTreg-stimulated cells. ( J – L ) FOXP3 CNS2 methylation was assessed by bisulfide sequencing in CD4 + CD25 + CD127 lo cells from iTreg cultures and endogenous blood Tregs (CD4 + FOXP3 + CD25 + CD127 lo ). Two separate experiments were analyzed together with adjustment for batch effects. ( J ) Heatmap, ( K ) violin plot, and ( L ) graphs of CpG island methylation ratios. ( M ) FOXP3 stability assessed through 2 rounds of stimulation and rest. CD25 + and FOXP3 MFI on CD25 + cells after second rest. Data points represent individual donors. ( H and I ) N = 5/group. ( A – E ) One of 3 representative experiments. ( H ) One of 2 representative experiments. ( F , G , I , and M ) Performed once. ( J – L ) Combined data from 2 experiments. ( B – G and M ) Two-way ANOVA with multiple comparisons used. ( I ) One-way ANOVA with multiple comparisons used. ( L ) Linear mixed model (included batch correction). * P ≤ 0.05, ** P ≤ 0.02, *** P ≤ 0.001, **** P < 0.0001. Horizontal bars and column heights in panels B – G and M depict mean values. In H and I , mean value plus standard deviation is shown.

Article Snippet: In , other protocols for cell activation were used: Human T-Activator CD3/CD28 Dynabeads and plate-coating with anti-CD3/CD28 antibodies (UCSF Antibody Core clone: OKT3 2 μg/mL; Miltenyi Biotec 130-093-375, clone: 15E8, 4 μg/mL) overnight at 4°C or for 4 hours at 37°C.

Techniques: Marker, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Mlr Assay, Methylation, Sequencing, Standard Deviation