PMC

STAT3 signaling in B cells is required for the GC B cell maintenance upon hRBC immunization. B cell-specific STAT3 KO and control mice were immunized i.p. with 0.2 ml of 50% v/v hRBC. (A) Splenocytes were stained with B220, CD95, and GL7 Abs and cells were gated on B220⁺ population. Representative dot plots are shown. Summarized data are presented as mean ± SEM and pooled from three experiments (n = 8–19 mice per group). (B) Immunohistochemical staining for peanut agglutinin (PNA, brown) of spleens from control and STAT3 KO mice obtained on days 7 and 21 after immunization. Summarized data are presented as mean ± SEM (n=2–5 mice per group). *p<0.05, **p<0.01, ***p<0.001.

STAT3 signaling in B cells is essential for the GC formation and GC B cell maintenance after immunization with OVA with adjuvant CFA. Control and STAT3 KO mice were immunized s.c. with 50 μg OVA in CFA. (A) Splenocytes were stained with B220, CD95, and GL7 Abs and cells were gated on B220⁺ population. Representative dot plots are shown. Summarized data are presented as mean ± SEM and pooled from three experiments (n = 10–17 mice per group). (B) Immunohistochemical staining for the GC marker PNA (brown) of spleens from control and STAT3 KO mice obtained on days 7 and 21 after immunization. Summarized data are presented as mean ± SEM (n=4–6 mice per group). *p<0.05, **p<0.01, ***p<0.001.

Depletion of STAT3 signaling in B cells down-regulates autoAb production. Anti-snRNP Ig Tg control and STAT3 KO mice of C57Bl/6 background (n=5) were immunized by intravenous injection of 2×10⁷ apoptotic thymocytes weekly for 9 weeks. (A) Sera were collected at day 7 after last injection, and autoantibodies anti-snRNP IgG and anti-dsDNA IgG in serum (1:10 dilution in PBS) were measured by ELISA. (B and C) Splenocytes were collected and the frequencies of the GC B cells (B, B220⁺ population) and Tfh cells (C, gated on CD4⁺ population) were analyzed by flow cytometry. Data are representative of two independent experiments. **p<0.01, ***p<0.001.

GC B cell apoptosis is increased in the absence of STAT3 signaling. Control and STAT3 KO mice were immunized with hRBC or OVA in CFA. Spleens were collected at day 21 and a single cell suspension was prepared. (A) Splenocytes were stained with anti-mouse GL7 followed by staining with Vybrant DyeCycle Green. DNA content of GC B cells was analyzed by flow cytometry. Numbers in plot indicate the percent of cells in the phase of S/G2-M. Each symbol represents an individual mouse. (B) GC B cell apoptosis was measured by staining of Annexin V and 7-AAD. Cells were gated on the GC B cells. (C) qRT-PCR for the expression of Bcl6, Mcl-1 and Bcl-xL in the GC B cells sorted from immunized mice. Data are representative of at least three independent experiments. *p<0.05, **p<0.01.

STAT3 signaling in B cells regulates IgG Ab production and plasma cell differentiation. (A) Serum samples from OVA/CFA immunized mice were assessed for OVA IgG and IgG isotype Ab levels were measured by ELISA. Sera were diluted at 1:10,000 or 1:100 (IgG3). (B) Flow cytometric analysis of splenic plasma cells at day 21 after immunization. Representative dot plots and summarized data (n=8–9) are shown. (C) qRT-PCR for the expression of Aicda and Blimp-1 in the sorted GC B cells from immunized mice. (D) Purified B cells from control and KO mice (n=3–4) were stimulated in vitro with LPS (10 μg/ml) for 3 days and then analyzed for CD138 and B220 expression. (E) Purified B cells were stimulated in vitro with LPS+IL-4 (10 ng/ml) and analyzed 4 days later for CD138 and IgG1 expression by flow cytometry. Summarized data on the percentages of plasma cells and IgG1⁺ cells are presented. Data are representative of at least two independent experiments. *p<0.05, **p<0.01, ***p<0.001.

Depletion of STAT3 signaling in B cells protects B6.MRL/lpr lupus-prone mice from developing autoAbs, splenomegaly, and lupus nephritis. (A) AutoAbs of snRNP IgG (1:20 dilution) and dsDNA IgG (1:50 dilution) were measured from 8-mo-old B6. MRL/lpr control and B cell STAT3 KO mice. (B) Representative ANA staining of a control and a STAT3 KO mouse serum (1:20). (C) Representative spleens and the total cellular numbers in the spleens from control and STAT3 KO mice are shown. (D, E, and F) The frequencies of the GC B cells, CD138⁺ plasma cells, and Tfh were analyzed by flow cytometry. The percentages of GC B cells (gated on CD19⁺ population), CD138⁺ cells, and Tfh cells (gated on CD4⁺ population) are shown. (G) Splenocytes from aged control and STAT3 KO mice were stimulated with PMA/Ionomycin and then stained for intracellular IFN-γ. Representative dot plots and summarized data are shown. (H) Splenocytes from 6-mo-old control and STAT3 KO mice were stained for surface markers expression and then stimulated with PMA/Inomycin for intracellular cytokine staining. Representative dot plots and summarized data are shown. Cells were gated on Cd19+ population. (I) A representative image of H&E-stained kidney sections from control and STAT3 KO mice. Scale bars: 200 μm. (J and K) Kidney sections were probed with FITC anti-mouse IgG or anti-mouse C3 to detect IgG (J) and C3 deposition (K) in the glomeruli of aged mice. *p<0.05, **p<0.01, ***p<0.001.