FIGURE 5.

Anti-hCD79A treatment inhibits B cell immune responses. (A) ELISPOT analysis of NP-specific, IgM⁺ ASCs 7 d after immunization with NP₅₉-Ficoll. Twenty-four hours prior to immunization, cCD79A mice received 250-µg i.p. injections of either anti-hCD79A D265A or control hIgG4. (B) Flow cytometric analysis of germinal center B cell induction 5 d after immunization with 0.1% SRBCs in PBS. Twenty-four hours prior to immunization, cCD79A animals received 250-µg i.p. injections of either anti-hCD79A D265A or control hIgG4. On day 5 postimmunization, RBC-lysed splenocytes were stained with Abs recognizing B220, CD95, and GL7. (C) ELISPOT analysis of NP-specific IgM⁺ (left) and IgG⁺ (right) ASCs at 16 d postimmunization with NP-OVA in alum. Twenty-four hours prior to immunization, cCD79A mice received 250-µg i.p. injections of either anti-hCD79A D265A or control hIgG4. For detection of total IgM and IgG anti-NP ASCs, plates were coated with NP₁₉-BSA. For detection of high-affinity IgG anti-NP ASCs, plates were coated with NP₂-BSA. (D) ELISPOT analysis of NP- (left) and tetanus toxoid–specific (right) IgG⁺ ASCs following overnight incubation with anti-hCD79A D265A. NP-specific ASCs were generated by immunizing cCD79A knockin mice with NP-OVA in alum and harvesting spleens 16 d later. RBC-lysed splenocytes (1 × 10⁷) were put into 1-ml cultures containing 25 µg of either anti-hCD79A D265A, anti-mCD20, control hIgG4, or control mIgG2a. After 18-h incubations, cells were washed and loaded onto ELISPOT plates coated with NP₁₉-BSA. n = 3 wells per condition. This experiment was repeated three times. PBMCs were isolated from peripheral blood obtained from subjects receiving Tdap booster vaccines 7–10 days prior. PBMCs (5 × 10⁶) were put into 1-ml cultures containing 25 µg of either anti-hCD79A D265A, anti-hCD20, or control hIgG4. After overnight incubation, cells were washed and loaded onto tetanus toxoid–coated ELISPOT plates. n = 3 subjects whose PBMCs were incubated with anti-hCD79; n = 1 subject whose PBMCs were also incubated with anti-hCD20. Points represent individual counted wells. (E) Effects of serial, weekly treatments with hIgG4 D265A anti-hCD79A in pristane-induced development of autoantibodies (anti-chromatin IgG ELISAs). Pooled sera from aged/diseased SLE1.2.3 mice and wild-type C57BL/6 served as positive and negative controls, respectively. n = 20 male and 20 female cCD79A mice per treatment arm. Error bars show SEM. All data represents at least three independent experiments; representative data are shown. A Student t test was used to evaluate statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.