Generation of antigen-specific memory CD4 T cells by heterologous immunization enhances the magnitude of the germinal center response upon influenza infection

Current influenza vaccine strategies have yet to overcome significant obstacles, including rapid antigenic drift of seasonal influenza viruses, in generating efficacious long-term humoral immunity. Due to the necessity of germinal center formation in generating long-lived high affinity antibodies, the germinal center has increasingly become a target for the development of novel or improvement of less-efficacious vaccines. However, there remains a major gap in current influenza research to effectively target T follicular helper cells during vaccination to alter the germinal center reaction. In this study, we used a heterologous infection or immunization priming strategy to seed an antigen-specific memory CD4+ T cell pool prior to influenza infection in mice to evaluate the effect of recalled memory T follicular helper cells in increased help to influenza-specific primary B cells and enhanced generation of neutralizing antibodies. We found that heterologous priming with intranasal infection with acute lymphocytic choriomeningitis virus (LCMV) or intramuscular immunization with adjuvanted recombinant LCMV glycoprotein induced increased antigen-specific effector CD4+ T and B cellular responses following infection with a recombinant influenza strain that expresses LCMV glycoprotein. Heterologously primed mice had increased expansion of secondary Th1 and Tfh cell subsets, including increased CD4+ TRM cells in the lung. However, the early enhancement of the germinal center cellular response following influenza infection did not impact influenza-specific antibody generation or B cell repertoires compared to primary influenza infection. Overall, our study suggests that while heterologous infection or immunization priming of CD4+ T cells is able to enhance the early germinal center reaction, further studies to understand how to target the germinal center and CD4+ T cells specifically to increase long-lived antiviral humoral immunity are needed. T follicular helper (Tfh) cells are specialized CD4+ T cells that provide help to B cells and are required to form germinal centers within secondary lymphoid organs during an immune response. Germinal centers are necessary for generating high affinity virus-specific antibodies necessary to clear influenza infections, though current vaccines fail to generate long-lived antibodies that universally recognize different influenza strains. We used a "heterologous priming" strategy in mice using a non-influenza viral infection or viral protein subunit vaccination to form memory CD4+ Tfh cells (in previously na & iuml;ve mice) that can be rapidly recalled into secondary Tfh cells following influenza infection and ideally enhance the germinal center reaction and formation of high affinity antibodies to influenza better than primary Tfh cells. Our study showed that heterologous priming induced an increase in both CD4+ T and B cells early following influenza infection, suggesting we could successfully target enhancement of the germinal center. Despite the enhancement of the early germinal center cellular response, we did not see an increase in influenza-specific antiviral antibodies. Thus, while Tfh cells are critical for the generation of high affinity antibodies, other strategies to target expansion of Tfh cells during influenza vaccination will need to be developed.