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

Abstract

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.

Lung influenza virus specific memory CD4 T cell location and optimal cytokine production are dependent on interactions with lung antigen-presenting cells

“…To investigate the consequences of the anti-MHCII treatment, we used flow cytometry to identify IAV-specific CD4 T cells that recognise the immunodominant nucleoprotein (NP) peptide, 311-325, or by gating on EYFP+ CD4 T cells in infected TRACE mice, Figure 5A-B and gating in Supplementary Figure 5. Our prior study demonstrated similar kinetics and differentiation into cytokine producing cells between CD4 T cells identified via these two methods 29 .…”

“…To first provide an overview of the lung immune response, we combined precision cut lung slices (PCLS) with tissue clearing and confocal microscopy. To enable us to identify IAV-specific CD4 T cells, we took advantage of a reporter mouse, TRACE, that we have developed and previously characterised 28, 29 . Activated T cells in these triple transgenic mice express rtTA under the control of part of the interleukin 2 promoter.…”

“…This leads to the irreversible labelling of activated T cells by expression of the fluorescent protein EYFP from the Rosa locus, Figure 2A. Infection with IAV induces a robust population of EYFP+ CD4 T cells when reporter mice are fed doxycycline in their diet during the initial infection 29 . Our prior studies have shown that EYFP+ cells are found in lung and secondary lymphoid organs following IAV infection 29 .…”

“…Infection with IAV induces a robust population of EYFP+ CD4 T cells when reporter mice are fed doxycycline in their diet during the initial infection 29 . Our prior studies have shown that EYFP+ cells are found in lung and secondary lymphoid organs following IAV infection 29 . This model greatly increases the number of responding T cells we can identify by both flow cytometry and immunofluorescence in comparison to other methodologies such as TCR transgenic cells or MHC tetramers.…”

“…CD4 T cells are known to persist in the lung following IAV infection 4, 5 and we have characterised the number, phenotype and cytokine production of IAV specific CD4 T cells in previous studies 28, 29, 32, 33 . Similar to the MHCII+ cells, IAV-specific CD4 T cells were found in the lung parenchyma, at airways, and in immune clusters at primary and memory time points, Figures 2 and 4A.…”