Multifunctional cytokine production marks influenza A virus-specific CD4 T cells with high expression of survival molecules

Cytokine production by memory T cells is a key mechanism of T cell mediated protection. However, we have limited understanding of the persistence of cytokine producing T cells during memory cell maintenance and secondary responses. We interrogated antigen-specific CD4 T cells using a mouse influenza A virus infection model. Although CD4 T cells detected using MHCII tetramers declined in lymphoid and non-lymphoid organs, we found similar numbers of cytokine⁺ CD4 T cells at days 9 and 30 in the lymphoid organs. CD4 T cells with the capacity to produce cytokines expressed higher levels of pro-survival molecules, CD127 and Bcl2, than non-cytokine⁺ cells. Transcriptomic analysis revealed a heterogeneous population of memory CD4 T cells with three clusters of cytokine⁺ cells. These clusters match flow cytometry data and reveal an enhanced survival signature in cells capable of producing multiple cytokines. Following re-infection, multifunctional T cells expressed low levels of the proliferation marker, Ki67, whereas cells that only produce the anti-viral cytokine, interferon-γ, were more likely to be Ki67⁺. Despite this, multifunctional memory T cells formed a substantial fraction of the secondary memory pool. Together these data indicate that survival rather than proliferation may dictate which populations persist within the memory pool.