Journal: Frontiers in Immunology
Article Title: Dependence on Autophagy for Autoreactive Memory B Cells in the Development of Pristane-Induced Lupus
doi: 10.3389/fimmu.2021.701066
Figure Lengend Snippet: Elevated autophagy in autoantigen-specific memory B cells. Splenocytes from MRL or MRL- lpr mice (4-month-old) were stained with PE-conjugated antibodies to CD11b, Gr-1, IgM, IgD and CD138, PE-Cy7-anti-CD19, Pacific Blue anti-CD38, APC-anti-IgG, and Alexa Fluor 488-conjugated dsDNA or RNP. DUMP – (IgM – IgD – CD11b – Gr-1 – CD138 – ) CD19 + B cells were gated. (A) dsDNA-specific memory B cells (DUMP – CD19 + CD38 + dsDNA + IgG + ) or (B) RNP-specific memory B cells (DUMP – CD19 + CD38 + RNP + IgG + ) were analyzed by flow cytometry. (C) The frequency of dsDNA-specific memory B cells and RNP-specific memory B cells, as well as dsDNA-specific germinal center (GC) B cells (DUMP – CD19 + CD38 – dsDNA + IgG + ) and RNP-specific GC B cells (DUMP – CD19 + CD38 – RNP + IgG + ), in the spleen of each mouse was plotted. Data are presented as mean ± s.e.m. ** P < 0.01, *** P < 0.001, **** P < 0.0001, (n=8 for MRL and 11 for MRL- lpr ). (D) RNP-specific memory B cells (DUMP – CD19 + CD38 + RNP + IgG + ) and RNP-negative memory B cells (DUMP – CD19 + CD38 + RNP – IgG + ) were sorted from MRL- lpr mice and stimulated with LPS in vitro for 3 days, the number of ASCs producing RNP-specific antibodies were measured by ELISPOT. Data are presented as mean ± s.e.m. Experiments were performed twice in triplicates using cells from a pool of 3-4 mice. **** P < 0.0001, determined by two-tailed Student’s t -test. (E) Splenocytes from pooled MRL or MRL- lpr mice were stained as in (A) except that both Alexa Fluor 488-conjugated dsDNA and RNP were included for staining. Sorted dsDNA/RNP/Sm-specific memory B cells, and B220 + IgM low IgD + CD23 + IgG − naïve B cells were used for real-time RT-PCR analysis of indicated autophagy-related genes. Data are presented as mean ± s.e.m. Experiments were performed three times using cells from a pool of 10-15 mice. * P < 0.05, ** P < 0.01, determined by two-tailed Student’s t -test. (F) Western blot analysis of LC3 processing in Memory (DUMP - CD19 + IgG + CD38 + ) and naïve B cells isolated from pooled MRL and MRL- lpr mice. Data are representative of two independent experiments.
Article Snippet: From eBioscience: PerCP-Cy5.5-anti-B220 (45-0452-82), PE-anti-IgM (12-5890-83), PE-anti-CD23 (12-0232-82), Biotin-anti-mouse-IgD (13-5993-85), Streptavidin-PE (12-4317-87), PE-Cy7-streptavidin (25-4317-82).
Techniques: Staining, Flow Cytometry, In Vitro, Enzyme-linked Immunospot, Two Tailed Test, Quantitative RT-PCR, Western Blot, Isolation
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