Anti-Hemagglutinin B Cell Receptor Heavy Chain Knockin Mouse: A New Model for Studying Memory B cells

Memory B cells, along with plasma cells, are clonally long-lived B cells that have responded to antigen and can participate in a faster, bigger and more specific secondary immune response. Memory B cells can either differentiate into antibody forming cells (AFCs) or re-enter the germinal center (GC) following re-activation. Studying memory B cells in wild-type animals is challenging due to their low frequency and thus many groups use antigen-specific B cell receptor (BCR) heavy chain (IgH) knockin mice, which have an enriched precursor frequency of B cells of a known antigen-specificity. While this approach has provided many insights into memory B cell development, phenotype and function, most research has been conducted with non-physiologic antigens so neither the role of infection-induced inflammation in shaping the memory B cell compartment nor the protectiveness of memory B cells in a secondary immune response can be evaluated.;To better understand memory B cells induced under physiologic conditions, we have developed an anti-hemagglutinin (anti-HA) BCR IgH knockin mouse that has an enhanced precursor frequency of flu (PR8)-specific B cells. This mouse model enables tracking, enumeration and isolation of virus-specific B cells. Importantly, we have generated both a IgH only knockin mouse (the H210 mouse) and a IgH knockin and light chain (IgL) knockin mouse (the H210 VK8R mouse). The latter has the rearranged IgL Vkappa8-J5.;The H210 and H210 Vk8R mouse models were validated through a combination of genetic approaches, an allelic exclusion mating strategy and serum anti-PR8 Ab measurements at both the baseline level and post-PR8 infection. These models have subsequently been used in a transfer system to phenotype memory B cells generated by attenuated PR8-gp33 infection. We have shown that H210 and H210 Vk8R B cells participate in GCs and undergo class-switch recombination (CSR). Memory H210 B cells preferentially use the rearranged IgL Vk14-Jk1 and we observed somatic hypermutation (SHM) of the H210 IgH in memory H210 Vk8R B cells. Furthermore, H210 and H210 Vk8R B cell kinetics following infection mirror earlier findings: production of sera anti-PR8 IgM occurs early in the response with sera anti-PR8 IgG levels increasing over time, splenic B cells are robustly activated by intranasal flu infection and there is a delayed expansion of flu-specific B cells in the lungs due to a delayed onset of GCs in this tissue. We have found that splenic CD80 and PD-L2 expression patterns differ from those seen with hapten-carrier immunization. Memory H210 Vk8R B cells, but not memory H210 B cells, upregulate CD80 and PD-L2 in both the spleens and lungs. The spleens appear to be a permissive environment for CD80, but not PD-L2, expression on memory H210 B cells.;Although H210 B cells are specific for PR8-derived HA they can expand in response to at least one other H1N1 virus, Mel/35. They do not expand significantly in response to the recombinant H3NI virus, J1. Restricting the light chain repertoire to Vk8R prevents H210 B cells from expanding to Mel/35, indicating that differential light chain pairing likely enables H210 B cell expansion to this virus. To this end, we found that the H210 IgH pairs with a more diverse and more mutated IgL repertoire in response to Mel/35.;Finally, we have compared expansion and activation of naive H210 Vk8R B cells to memory H210 Vk8R B cells in the response to PR8-gp33 infection. Memory H210 VK8R B cells expand more rapidly and participate more in GCs in the spleens, but not in the draining lymph nodes, relative to naive H210 Vk8R B cells. Additionally, memory H210 Vk8R B cells mediate more rapid production of PR8-specific IgG than naive H210 Vk8R B cells. Memory H210 B cells expand significantly less than both naive and memory H210 Vk8R B cells in response to PR8-gp33; they are also unable to expand in response to Mel/35, in contrast to naive H210 B cells.;We believe that the H210 and H210 Vk8R mouse models can be used in complementary studies to better understand flu-specific memory B cells generated in response to PR8 and at least one heterologous virus.