Fig 1: CD16+ monocyte subset exacerbated B-cell activation in SLE. SLE CD16+ monocytes had an enhanced capacity on PB and Breg differentiation but attenuated MB development. Monocyte subsets were cocultured with CD19+ B cells isolated from freshly collected SLE blood or blood bank collected HC blood buffy coat for 3 days in the presence anti-CD40 antibody (3 µg/mL) and M-CSF (50 ng/mL). The expression of CD20 and CD27 as well as CD24 was assessed. (A) Representative histogram plots from HCs and patients with SLE displayed the frequencies of MB (CD27+CD19+) cells in monocyte/B-cell cocultures. Graphs showed the cumulative MB frequencies in cocultures of monocytes/B cells and CD19+ B cells cultured alone from eight healthy individuals and seven patients with SLE. The difference in the percentage increase in MB cells in cocultures of monocytes and B cells compared with CD19+ B cells cultured alone was compared between HCs and SLEs. (B) Representative dot plots from HCs and patients with SLE demonstrated the frequencies of PB (CD20-CD27+CD19+) cells in monocyte/B-cell cocultures. Graphs showed the cumulative frequencies of PBs in cocultures of monocytes/B cells and CD19+ B cells cultured alone from eight healthy individuals and seven patients. Percentage increase in PBs in cocultures of monocytes and B cells compared with CD19+ B cells cultured alone was identified between HCs and patients with SLE. (C) Representative contour plots from HCs and patients with SLE showed the frequencies of Breg (CD24+CD27+CD19+) cells in monocyte/B-cell cocultures. Graphs displayed the cumulative frequency of Bregs in cocultures of monocytes/B cells and CD19+ B cells cultured alone from eight healthy individuals and five patients with SLE. Percentage increase in Bregs in cocultures of monocytes and B cells compared with CD19+ B cells cultured alone was compared between HCs and patients with SLE. MBs, memory B cells; PBs, plasma B cells; Bregs, regulatory B cells. Data were expressed as mean ± SD and analyzed by non-parametric paired t test and Mann–Whitney U test. *P < 0.05, **P < 0.01; NS, no significance.
Fig 2: T-cell reconstitution following autologous hematopoietic stem cell transplantation (autoHSCT).Absolute numbers (cells per milliliter) of (A) total CD3+ T-cells, (B) total, naive (CD27+CD45RO-) and memory (CD45RO+) CD3+CD4+ T-cells, (D) total, naive (CD27+CD45RO-) and memory (CD45RO+) CD3+CD8+ T-cells, and (C) the CD4:CD8 ratio over time for the duration of the study are depicted. Box plots represent the distribution of values for healthy controls (HCs) (N = 17 for CD3+, CD4+, CD8+, and CD4:CD8 ratio, other N = 27, box = 25th to 75th percentile, black line=median, whiskers=min and max values). Absolute numbers shown in the graph are not normalized. (E) Bar graphs show the median percentage of naive (CD27+CD45RO-), central memory (CM, CD27-CD45RO+), effector memory (EM, CD27+CD45RO+), and effector (CD27-CD45RO-) CD4+ and CD8+ T-cells of autoHSCT patients (A–F) and HCs (n = 6) in the indicated colors. For the T-cell subset distribution per patient over time, see Figure 3—figure supplement 1 and Figure 3—source data 1. For the gating strategy, see Figure 3—figure supplement 2.Figure 3—source data 1.T-cell numbers and percentages in blood of patients and healthy controls.
Fig 3: B-cell reconstitution following autologous hematopoietic stem cell transplantation (autoHSCT).(A) Absolute numbers (cells per milliliter) of total CD19+ B-cells, naive (CD19+IgM+CD27-), Ig class-switched memory (CD19+IgM-CD27+), and IgM+ memory (CD19+IgM+CD27+) B-cells in peripheral blood over time. Graphs show the absolute cell counts per milliliter in autoHSCT patients (patients A–F) over the duration of the study. Box plots represent the distribution of values for healthy controls (HCs) (N = 10, box = 25th to 75th percentile, black line=median, whiskers=min and max values). Absolute numbers shown in the graph are not normalized. (B) Bar graphs show the median percentage of naive, Ig class-switched memory, and IgM+ memory B-cells within total CD19+ B-cells of autoHSCT patients (patients A–F) and HCs (N = 10). For the B-cell subset distribution per patient over time, see Figure 6—figure supplement 1 and Figure 6—source data 1. Note the different y-axes in panel A.Figure 6—source data 1.B-cell numbers and percentage in blood of patients and healthy controls.
Fig 4: Activated Dsg3-Specific Memory B Cells Are Detected Exclusively in Patients with Active PV(A) High levels of anti-Dsg3 serum antibody titers are present in PV patients at the time of diagnosis and during relapse following previous treatment with Rituximab (Rtx), but not in healthy controls and patients in remission. Cut-off values were determined based on manufacturer recommendations: dark gray = negative; light gray = indeterminate. Representative data of two individual experimental repeats are shown.(B) Representative Dsg3-specific MBC assay.(C) High frequencies of Dsg3+ MBCs were detected in PV patients at the time of diagnosis and after relapse but were largely undetectable in patients in remission and absent in healthy controls. Reported frequency of Dsg3-specific MBCs is the average of 3—5 replicates of a single experiment.(D) Dsg3-specific MBCs (gated on CD3-CD19+IgD-CD20+ lymphocytes) are readily detected from two patients with high frequencies of Dsg3-specific MBCs (as shown in C).(E) Dsg3-specific MBCs express the classic memory cell marker CD27.(F) Dsg3-specific MBCs expressed the activation marker CD71, similar to activated HA-specific MBCs induced 7 days post-influenza vaccination. Steady-state MBCs found later after the vaccination are quiescent and do not express CD71. A one-way ANOVA was used to analyze these data. ***p = 0.001.
Fig 5: Metformin reduces the secretion of IFN-γ in CD8 + senescent T cells. Representative flow histogram of IFN-γ production by CD8 + senescent T cells (CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57 + KLRG1 +) (a) and non- senescent T cells (CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57-KLRG1-) (e) in the control group and the 20 mM Met treatment group relative to the unstimulated controls from middle-age subjects. Analysis of IFN-γ-secreting CD8 + senescent T cells (b) and MFI of IFN-γ in CD8 + senescent T cells (c) with 20 mM metformin treatment. d Frequency of CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57 + KLRG1 + IFN-γ + T cells in lymphocytes with Met treatment and control. f Percentage and MFI of IFN-γ in CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57-KLRG1- T cells with Met treatment and control. g Representative flow histogram of IFN-γ secretion by CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57 + /CD57- population relative to the unstimulated controls from middle-age donors. h Percentage and MFI of IFN-γ in CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57 + / CD57- population. Expressed as the mean ± SEM. **P < 0.01, ***P < 0.001; Paired t test. Met, metformin
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