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. 2018 Feb 6;71(5):527–542. doi: 10.1016/j.jacc.2017.11.055

Figure 5.

Figure 5

TRAF-STOPs Do Not Impair Classical CD40-Mediated Immune Responses

(A) TRAF-STOPs did not affect immunoglobulin M (IgM), IgG levels in the plasma of 28-week-old Apoe−/− mice that were treated for 6 weeks (n = 11 to 12). (B) IgG2b, IgG2c, and IgG3 ratios were not altered by TRAF-STOPs. Immunoglobulin levels were determined by enzyme-linked immunosorbent assay (ELISA) (n = 11 to 12). (C) Representative images (hematoxylin and eosin–stained sections and immunohistochemical staining of CD21 and Bcl6) of splenic germinal centers demonstrated that TRAF-STOPs did not affect germinal center formation in 28-week-old Apoe−/− mice that were treated for 6 weeks (scale bar = 100 μm) (n = 12 to 15 per group). (D and E) C57Bl6 mice were vaccinated with myelin oligodendrocyte glycoprotein peptide (MOG35-55) and antigen-specific immunoglobulin levels were determined by ELISA (n = 6 to 8 per group). TRAF-STOP treatment did not affect the levels of IgM and IgG, or the IgG1, IgG2b, IgG2c, and IgG3 ratios. Antigen-specific anti-MOG IgG levels at 16 and 28 days after vaccination were not affected by TRAF-STOP treatment (n = 8). (F) Coculture of TRAF-STOP–treated, ovalbumin peptide-loaded dendritic cells (DCs) and CFSE-labeled, OTII transgenic CD4+ T cells demonstrated that T-cell proliferation was not affected. T-cell proliferation was assessed by flow cytometry (n = 6). (G) Jurkat T cells were activated using CD3/CD28 beads. TRAF-STOP treatment did not affect T-cell cytokine expression nor expression of costimulatory molecules. (H) CA46 B-cells were activated using the CD40-agonistic antibody G28.5 and treated using TRAF-STOP 6877002 and 6860766. No effects of TRAF-STOP on B-cell activation could be detected (n = 3 experiments). (I) C57Bl6 mice (n = 8 per group) were treated with the TRAF-STOPs, and peritonitis was induced by intraperitoneal injection of Escherichia coli. Sixteen hours after inoculation the bacterial outgrowth in various tissues was analyzed. TRAF-STOP did not increase the bacterial outgrowth in the peritoneal lavage fluid, lungs, liver and blood. *p < 0.05, **p < 0.01. Abbreviations as in Figures 3 and 4.