Established GC-dependent and GC-independent MBCs can be BAFF dependent. (A) Transfer and treatment strategy to examine BAFF neutralization on WT MBC responses. WT recipient mice were placed on BrdU drinking water 2 d before receiving SW_HEL donor cells (day −2). WT SW_HEL B cells (CD45.1⁺) were adoptively transferred and challenged with HEL^3X-SRBC i.v. (day 0). Recipients were either killed on days 4 and 21 for spleen and LN analysis or placed on normal water from day 4. Mice either were treated with mouse IgG1κ isotype control or anti-BAFF blocking antibody on day 21 or day 29 or received OT-II CD4⁺ T cells i.v. on day 28 and rechallenged with HEL-OVA s.c. 24 h later (day 29) for analysis of the day 5 recall response (day 35). (B) Flow cytometric analysis of donor-derived (CD45.1⁺, CD45.2⁻) SW_HEL CD38^hi B cells (B220^hi, CD38^hi), with gates showing BrdU-labeled HEL^3X-binding low-affinity (BrdU⁺, HEL^3Xhi) and unlabeled high-affinity (BrdU⁻, HEL^3Xlo) MBC compartments in WT recipients before mAb treatment (days 4 and 21) and 8 d after control or anti-BAFF mAb treatment (day 29). Flow data represent eight concatenated mice from one experiment and are representative of two independent experiments. (C) Enumeration of splenic B cell responses in mice treated with isotype control (black dots) or anti-BAFF antibody (red triangles). From left to right: Total splenic B cells (CD45.1⁺, CD45.2⁺, B220⁺) before (day 21) and after (day 29) treatment, donor-derived (CD45.1⁺, CD45.2⁻) SW_HEL IgG⁺ high-affinity MBCs (IgG1⁺, CD38^hi, HEL^3Xhi), SW_HEL GC-independent unswitched MBCs (BrdU⁺, IgM⁺, CD38^hi), and SW_HEL GC-independent IgG-switched MBCs (BrdU⁺, IgG⁺, CD38^hi) on day 29. (D) Enumeration of LN B cell responses in mice treated with isotype control (black dots) or anti-BAFF (red triangles) antibody. From left to right: Total B cells in inguinal LNs before (day 21) and after (day 29) treatment, SW_HEL IgG⁺ high-affinity MBCs, and SW_HEL GC-independent unswitched MBCs on day 29. Enumerated data in C and D represent eight replicate mice from one experiment and are representative of two independent experiments. Enumerated data were analyzed using an unpaired Student’s t test with Welch’s post hoc correction; *, P < 0.05; **, P < 0.01; ****, P < 0.0001. (E) Flow cytometric analysis of SW_HEL B cell responses in paired inguinal LNs of isotype control–treated recipients and (F) anti-BAFF mAb–treated recipients before HEL-OVA challenge (day 29) and 5 d after rechallenge (day 35), with gates showing GC B cells (B220^hi, CD38^lo), CD38^hi B cells (B220^hi, CD38^hi), and PCs (B220^lo, CD38^int). (G) Flow cytometric analysis of day 5 recall SW_HEL GC and PC responses (arrows from E) of control mAb-treated mice, with gates showing intracellular (i.c.) IgG versus HEL^3X-binding high- and low-affinity compartments. (H) Flow cytometric analysis of day 5 recall SW_HEL GC and PC responses (arrows from F) of anti-BAFF mAb–treated mice, with gates showing IgG (i.c.) versus HEL^3X-binding (i.c.) high- and low-affinity compartments. Flow data in E–H represent five concatenated mice from one experiment and are representative of two independent experiments.