Fig 2: Anti-CD137 mAb Results in Disorganization of B Cell Follicle Architecture in the SpleenFour-week-old WT C57BL/6 male mice were inoculated with 103 FFU of CHIKV. At 2 dpi, 400 µg of agonistic anti-CD137 or isotype control mAb was administered by an i.p. route. Spleens were harvested at 6 dpi (A–D), 7 dpi (E and F), and 14 dpi (I–M) for imaging. (A–D) FDCs (green) were stained for CD21/35; IgD+ B cells (red); and T cell zone (turquoise), CCL21. (B and D) Insets of the respective dotted boxes. (E and F) FDCs (green) were stained for CD21/35; IgD+ B cells (red); and CD4+ T cells (snow). White scale bars indicate 50 µm. Yellow arrows indicate IgD+ B cells surrounding FDCs, and white arrows indicate IgD+ B cells at the CCL21+ T cell zone border. (G and H) The number of CD45-CD21/35+CD54+ FDCs in the spleen at 7 and 14 dpi was analyzed by flow cytometry. (G) Representative flow cytometry dot plots of FDCs are shown. (I) B cells (blue) were stained for B220; FDCs (green), CD21/35; and GC B cells (snow), GL7. White scale bars indicate 500 µm or 100 µm (insets). Each symbol represents an individual FDC-containing follicle (J), GC B cell (K), or spleen (L), and bars indicate mean values. Quantification was performed for FDC area per FDC-containing-follicle (J), distance between the closest GC B cells (K), and GC B cells greater than 10 µm from an FDC (L). (M) Left and middle: FDCs (blue) were stained for CD21/35; VCAM-1 (red); merge (white). (M) Right: GC B cells (white) were stained for GL7; VCAM-1+ FDCs (red). White scale bars indicate 200 µm or 50 µm (insets). The images are representative of 3 spleens per group from 2 independent experiments (Mann-Whitney test: ***p < 0.001; ****p < 0.0001; n.s., not significant).

Fig 3: Anti-CD137 mAb Treatment Dampens T-Cell-Dependent Antibody Responses(A–F) Four-week-old C57BL/6 male mice were injected by i.p. route with 5 µg of NP-KLH (A) or 10 µg of NP-Ficoll (F). At 2 dpi, 400 µg of agonistic anti-CD137 or isotype control mAb was administered by an i.p. route. (B–E) After immunization with NP-KLH, the number of PNA+CD95+ GC B cells (B) in the spleen at 14 days postimmunization was analyzed by flow cytometry. At 30 days postimmunization, antigen-specific MBCs were harvested from a separate set of animals and profiled (C). At day 130, bone marrow was harvested to profile antigen-specific LLPCs (D). Serum was collected at 7, 14, 21, 28, 35, 56, 76, 96, and 130 days postimmunization, and anti-NP IgG (E) was measured.(G–K) After immunization with NP-Ficoll, spleens were harvested at day 3 and 7 to assess antigen-specific IgM+ plasmablasts (G). At 30 days postimmunization, antigen-specific IgM+ MBCs were harvested and profiled (H). Bone marrow was harvested at day 130 to assess antigen-specific IgG+ LLPCs (I). Serum was collected at 7, 14, 21, 28, 35, 56, and 130 days postimmunization, and anti-NP IgM (J) and IgG (K) were measured. Symbols represent individual mice, and bars indicate median values, except for serum antibody titers where bars indicate mean values. The IgM and IgG ELISA binding data were analyzed by subtracting the optical density (OD) of naive serum from the OD of serum from immunized animals. Dotted lines represent the baseline number of plasmablasts that secrete NP-binding natural IgM in naive control mice (G) or limit of detection (E, J, and K). Data are pooled from 3 independent experiments (Mann-Whitney test, except for serum antibody titers where two-way ANOVA with Sidak post-test was used: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n.s., not significant).

Fig 4: Cell-Intrinsic CD137 Signaling in CD4+ or CD8+ T Cells Is Required for Anti-CD137 mAb-Mediated Inhibition of the GC ReactionFour-week-old CD137-/- (A) or TCRßd-/- (D) mice were irradiated and then received bone marrow cells from WT or CD137-/- mice. After 8 weeks, recipient mice were inoculated with 103 FFU of CHIKV. At 2 dpi, 400 µg of agonistic anti-CD137 or isotype control mAb was administered by an i.p. route. (B) Immune cell reconstitution was confirmed in recipient mice. The number of PNA+CD95+ GC B cells (C, E, and G) in the spleen at 14 dpi was determined. (F and G) Four-week-old TCRßd-/- mice were administered 7.1 × 106 WT CD4+ T cells + WT CD8+ T cells, WT CD4+ T cells + CD137-/- CD8+ T cells, CD137-/- CD4+ T cells + WT CD8+ T cells, or CD137-/- CD4+ T cells + CD137-/- CD8+ T cells by an i.v. route. Five days later, recipient mice were inoculated with 103 FFU of CHIKV. At 2 dpi, 400 µg of agonistic anti-CD137 or isotype control mAb was administered by an i.p. route. Symbols represent individual mice, and bars indicate median values. Data are pooled from 3 to 4 independent experiments (Mann-Whitney test: ***p < 0.001; ****p < 0.0001; n.s., not significant).

Fig 5: Effect of Anti-CD137 mAb Treatment on Antigen-Specific B Cell Populations when Administered before Viral Vaccine BoostingNine-week-old WT C57BL/6J female mice were injected by an intramuscular (i.m.) route with recombinant influenza A virus H5 HA protein. A total of 400 µg of agonistic anti-CD137 or isotype control mAb was administered by an i.p. route at either 2 days postimmunization (A) or 27 days postimmunization (E) before boosting with H5 HA at 28 days after initial immunization. Spleens were harvested 7 days after the booster immunization, and the numbers of GC B cells (B and F) and antigen-specific MBCs (C and G) were analyzed by flow cytometry. (D and H) The number of antigen-specific plasmablasts was analyzed by ELISpot. Symbols represent individual mice, and bars indicate median values. Data are pooled from 2 independent experiments (Mann-Whitney test: ***p < 0.001; n.s., not significant).