Figure 3. MPER⁺ Ab and splenic B-cell responses in the MHC H-2^d responder haplotype require cognate CD4 T-cell help.

A. WT or CD154 knockout (CD40L^-/-) mice on BALB/c (H-2^d) or C57BL/6 (H-2^b) backgrounds were immunized with either control (50 μg TNP-KLH plus Alum) or experimental (JRFL/TLR4/9-MPER peptide-liposome boost) regimens, as described in Materials and Methods and Fig. 1, respectively. All control and experimentally immunized groups were measured for both MPER⁺ and TNP⁺ Ab responses, as revealed by α-IgG-AP detection of plates coated with the 2F5 nominal epitope peptide SP62 or TNP-BSA, respectively (and as described further in the Materials and Methods section). B. CD154-dependent H-2^d-restriction of MPER-specific IgG+ B-cells in immunized mice. Shown are graphical representations of TNP or MPER-specific IgG1⁺ B-cells/10⁶ total (singlet, live, kappa+ gated) splenic B-cells from BALB/c (H-2^d) and C57BL/6 (H-2^b) mice (on either CD154-sufficient or deficient backgrounds), immunized with control (TNP-KLH) or experimental (JRFL prime/TLR-MPER peptide-Liposome boost) regimens. For all data shown, splenocytes were taken 10d after 5^th immunizations and ≥2 mice/immunization group is shown. MPER-specific B-cells were measured by 2F5 nominal MPER epitope (SP62) tetramers, as previously described (28,44), whereas TNP-specific cells were enumerated by TNP-Fluorescein-AECM-FICOLL as described in the Materials and Methods. Similar differences in total and MPER-specific IgG1+ splenic B-cells were also seen between immunized BALB/c (H-2^d) and BALB.B (H-2^b) congenic MHC strains on CD154-sufficient backgrounds (data not shown).