American Society of Hematology

Figure 6.

$Figure 6. CD30 signaling enhances PC differentiation upon TD immunization with NP-CGG. (A) Displayed are the percentages of reporter-positive lymphocytes, CD38lowCD95high reporter-positive GC B cells, and NP+ reporter-positive GC B cells in LMP1/CD30//γ1-Cre mice and CAR//γ1-Cre upon NP-CGG immunization at the indicated time points. Gating strategy of reporter-positive lymphocytes, GC B cells, and NP+ GC B cells, as well as graphs compiling values of different experiments including the statistics, is shown in supplemental Figure 10A-C. (B) GCs were clearly visible 14 days after immunization with NP-CGG in LMP1/CD30//γ1-Cre and CAR//γ1-Cre mice. Splenic sections from these mice as well as from unimmunized control (ctrl) mice were stained for GC B cells (PNA; blue) and IgM+ cells (IgM; brown). Slices were analyzed as described in Figure 2B. (C) Left graph shows the percentages of CD23lowCD43+ cells within the fraction of reporter-positive (CAR+, hCD2+) lymphocytes 5 and 14 days postimmunization in LMP1/CD30//γ1-Cre (hCD2+) and CAR//γ1-Cre (CAR+) mice. Right graph shows the percentages of CD138+B220low PBs and PCs in the fraction of reporter-positive (CAR+, hCD2+) lymphocytes. Corresponding gating strategies are shown in supplemental Figure 11A. (D) Upper row: NP-IgM as well as total (NP13) and high-affinity (NP4) NP-specific IgG1-secreting PCs were determined by ELISpot analysis 14 days postimmunization using splenocytes from the 2 genotypes. Lower row: serum titers of the indicated antibodies were measured by ELISA 14 days after immunization with NP-CGG. (E) Splenic sections from LMP1/CD30//γ1-Cre and CAR//γ1-Cre mice 5 and 14 days after immunization with NP-CGG and unimmunized (n.i.) ctrl mice were stained for GC B cells (GL7; green), B cells (B220; red), and PBs (IRF4; cyan). Images of immunofluorescences were obtained with the Leica TCS SP5 II with an 8-kHz resonant scanner and HCX PL APO CS 20× objective and LAS AF software. (F) Percentage of IRF4+ cells in the fraction of reporter-positive centroblasts (CBs) (CXCR4highCD86low) and CCs (CXCR4lowCD86high) was determined 14 days postimmunization. Graph compiles the percentages of IRF4+ cells in CBs and CCs from different experiments. Gating strategy of reporter+ CB and CC as well as IRF4+ cells is shown in supplemental Figure 12B. *P < .05, **P < .01, ***P = .001, ****P < .0001.$

CD30 signaling enhances PC differentiation upon TD immunization with NP-CGG. (A) Displayed are the percentages of reporter-positive lymphocytes, CD38^lowCD95^high reporter-positive GC B cells, and NP⁺ reporter-positive GC B cells in LMP1/CD30//γ1-Cre mice and CAR//γ1-Cre upon NP-CGG immunization at the indicated time points. Gating strategy of reporter-positive lymphocytes, GC B cells, and NP⁺ GC B cells, as well as graphs compiling values of different experiments including the statistics, is shown in supplemental Figure 10A-C. (B) GCs were clearly visible 14 days after immunization with NP-CGG in LMP1/CD30//γ1-Cre and CAR//γ1-Cre mice. Splenic sections from these mice as well as from unimmunized control (ctrl) mice were stained for GC B cells (PNA; blue) and IgM⁺ cells (IgM; brown). Slices were analyzed as described in Figure 2B. (C) Left graph shows the percentages of CD23^lowCD43⁺ cells within the fraction of reporter-positive (CAR⁺, hCD2⁺) lymphocytes 5 and 14 days postimmunization in LMP1/CD30//γ1-Cre (hCD2⁺) and CAR//γ1-Cre (CAR⁺) mice. Right graph shows the percentages of CD138⁺B220^low PBs and PCs in the fraction of reporter-positive (CAR⁺, hCD2⁺) lymphocytes. Corresponding gating strategies are shown in supplemental Figure 11A. (D) Upper row: NP-IgM as well as total (NP13) and high-affinity (NP4) NP-specific IgG1-secreting PCs were determined by ELISpot analysis 14 days postimmunization using splenocytes from the 2 genotypes. Lower row: serum titers of the indicated antibodies were measured by ELISA 14 days after immunization with NP-CGG. (E) Splenic sections from LMP1/CD30//γ1-Cre and CAR//γ1-Cre mice 5 and 14 days after immunization with NP-CGG and unimmunized (n.i.) ctrl mice were stained for GC B cells (GL7; green), B cells (B220; red), and PBs (IRF4; cyan). Images of immunofluorescences were obtained with the Leica TCS SP5 II with an 8-kHz resonant scanner and HCX PL APO CS 20× objective and LAS AF software. (F) Percentage of IRF4⁺ cells in the fraction of reporter-positive centroblasts (CBs) (CXCR4^highCD86^low) and CCs (CXCR4^lowCD86^high) was determined 14 days postimmunization. Graph compiles the percentages of IRF4⁺ cells in CBs and CCs from different experiments. Gating strategy of reporter⁺ CB and CC as well as IRF4⁺ cells is shown in supplemental Figure 12B. *P < .05, **P < .01, ***P = .001, ****P < .0001.

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