Source: Dissertation Abstracts International, Volume: 74-11(E), Section: B.

Adviser: Mark Shlomchik.

Memory B cells (MBCs) protect against host re-infection. The different functions of MBCs in protecting against reinfection have been attributed to their B cell receptor isotype. However, we found the expression of CD80 and/or PD-L2, which are differentially expressed on MBCs and not naive B cells, separate memory B cells into three distinct subsets and best correlates to specific functions of MBCs after secondary immunization, rather than their isotype. We developed a series of adoptive transfers in mice to generate MBCs, identify and sort subsets of MBCs and then evaluate the function of subsets of MBCs 3.5 days, 10.5 days and 4 weeks or more post-secondary immunization. We took advantage of transgenic knock-in mice with increased precursor frequencies for the 4-hydroxy-3-nitrophenyl (NP) and thus evaluated responses in immunized mice with NP conjugated to carrier proteins chicken gamma globulin or ovalbumin and the adjuvant alum.

We found that 3.5 days post-secondary immunization all MBCs were able to differentiate into antibody forming cells (AFCs), but naive B cells were not able to generate AFCs. The subset of CD80+ PD-L2 + MBCs from both IgG1neg (IgM+) and IgMneg (IgG1+) isotypes made the most AFCs. Our results also show that naive T cell help was sufficient to help MBCs function. T cells were required for isotype switching of MBCs and expansion of AFCs, but not for differentiation into AFCs. These results indicate that the early burst of antibody-mediated protection is generated by CD80 + PD-L2+ MBCs and not limited to IgG1+ MBCs.

At 10.5 days post-secondary immunization CD80- PD-L2 - MBCs produced just as many germinal center (GC) B cells as naive B cells. CD80- PD-L2+ MBCs of IgG1neg (IgM+) and IgMneg (IgG1+) isotypes were able to generate small numbers of GC B cells. However, CD80 + PD-L2+ MBCs were not able to generate GCs. Thus, MBCs that generate GCs are not defined by IgM isotype, since CD80+ PD-L2+ MBCs from IgG1neg (IgM +) MBCs were not able to generate new GCs.

Interestingly, at 4 weeks post-secondary immunization CD80- PD-L2- MBCs with the help of memory-like T cells were not able to generate new MBCs. However, CD80- PD-L2- MBCs that had naive T cell help were able to generate new MBCs. These MBCs had all subset phenotypes, but the CD80- PD-L2 - subset represented the highest proportion of MBCs. We conclude that CD80- PD-L2- MBCs have the capacity to self-renew, which was impeded by memory-like T cell help.

Overall, the work described here advances our knowledge of the function of MBC subsets in a recall response. These results suggest that IgG1 neg MBCs have diverse functional heterogeneity and that dividing MBCs into subsets according to CD80 and PD-L2 expression provides a better indicator of function of MBCs in recall responses than isotype expression. Understanding which subset is most protective after different infections and then determining how to generate these subsets has implications for the design of better vaccines.