Fig 2: CD57- and NKG2A-defined NK subsets show phenotypes of trained immunity. (A) Upper panel: representative flow cytometry plots of the percentages of Ki67+ or IFN-γ+ cell populations in NK cells stimulated with IL-12/15/18 for 16 h on day 0 and restimulated with IL-12/18 on day 7; lower panel: PMA+ionomycin restimulated cells on day 7 to obtain positive controls. Data are representative of 3 independent experiments. (B) Upper panel: summary bar graph showing the percentage of IFN-γ+ cells in the CD57− and CD57+ NK subsets in the upper panel of (A); Lower panel: summary bar graph showing the percentage of IFN-γ+ cells in the CD57− and CD57+ NK subsets in the lower panel of (A). (C) Flow cytometry plot of the gating strategy for CD3−CD56+ NK cell subsets. NK cells were stained with NKG2A, CD57, and IFN-γ, and grouped into 4 subsets according to NKG2A and CD57 for IFN-γ expression analysis. (D, E) Summary data showing the percentage of each of the 4 NK cell subsets (D) and the percentage of IFN-γ+ NK cells (E) in the control and preactivated groups on day 7 of in vitro culture. For all experiments, n = 5–7, *P < 0.05, **P < 0.01, and ***P < 0.001 (error bars, mean ± SEM). Data are representative of at least 3 independent experiments.

Fig 3: The percentage and fold change GMFI of pSTAT5 in B (CD20+) and T cells (CD3+) after IL-21, IL-10 + IL4, and IL-2 in sIgAD individuals and healthy controls. The figure shows % of phosphorylated and fold change GMFI of STAT5 as measured ex vivo in PBMCs after 15min stimulation of IL-21 (50 ng/ml), IL-10 (100 ng/ml) + IL4 (100 ng/ml), and IL-2 (50 ng/ml). Analysis of (A) % pSTAT5 positive B cells (CD20+) and (B) fold change GMFI shows a no significant difference in the percentage or fold change GMFI of B cells expressing pSTAT5 after IL-21, IL-10 + IL4, and IL-2 stimulation in sIgAD and HCs. Analysis of (C) % pSTAT5 positive T cells (CD3+) and (D) fold change GMFI shows neither differences between phosphorylation responses in T cells after IL-21, IL-10 + IL4, and IL-2 stimulation in sIgAD and HCs. Significance was calculated in relation to the control group. *p < 0.05, ****p < 0.0001 (as determined by one-way ANOVA).

Fig 4: The percentage and fold change GMFI of pSTAT6 in B (CD20+) and T cells (CD3+) after IL-4 and IL-10 + IL4 in sIgAD individuals and healthy controls. The figure shows % of phosphorylated and fold change GMFI of STAT6 as measured ex vivo in PBMCs after 15-minute stimulation of IL-4 (100 ng/ml) and IL-10 (100 ng/ml) + IL4 (100 ng/ml). Analysis of (A) % pSTAT6 positive B cells (CD20+) and (B) fold change GMFI shows a no significant difference in the percentage or fold change GMFI of B cells expressing pSTAT6 after IL-4 or IL-10 + IL4 stimulation in sIgAD and HCs. Analysis of (C) % pSTAT6 positive T cells (CD3+) and (D) fold change GMFI shows neither differences between phosphorylation responses in T cells after IL-4 or IL-10 + IL4 stimulation in sIgAD and HCs. Significance was calculated in relation to the control group. *p < 0.05, ****p < 0.0001 (as determined by one-way ANOVA).

Fig 5: Characterisation of the peripheral T and NK cell populations in SLE using a single-cell multi-omics approach.( A) Summary of the experimental workflow based on the BD Rhapsody single-cell RNA-sequencing system combining the quantification of mRNA and surface protein targets (AbSeq). Experiment was carried out using samples from three donors: (i) an SLE patient with low expression of the type I interferon (IFN)-induced gene expression signature (IFN low; depicted in blue); (ii) an SLE patient with a previously detected IFN-induced gene expression signature (IFN hi; depicted in red); and (iii) a healthy donor (HD; depicted in green). Cells isolated from each donor were barcoded with oligo-conjugated antibodies and pooled together for cell capture and library preparation. ( B) Gating strategy used for the isolation of the six T and NK cell populations assessed in this study. ( C) Frequency of NK56 br cells, defined as CD3 -CD127 +CD56 hi NK cells, in each of the three donors.