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

Influenza A virus (IAV) infection leads to the formation of mucosal memory CD4 T cells that can protect the host. An in-depth understanding of the signals that shape memory cell development is required for more effective vaccine design. We have examined the formation of memory CD4 T cells in the lung following IAV infection of mice, characterising changes to the lung landscape and immune cell composition. IAV-specific CD4 T cells were found throughout the lung at both primary and memory time points. These cells were found near lung airways and in close contact with a range of immune cells including macrophages, dendritic cells, and B cells. Interactions between lung IAV-specific CD4 T cells and MHCII+ cells during the primary immune response were important in shaping the subsequent memory pool. Treatment with an anti-MHCII blocking antibody increased the proportion of memory CD4 T cells found at lung airways but reduced interferon-γ expression by IAV-specific immunodominant memory CD4 T cells. The immunodominant CD4 T cells expressed higher levels of PD1 than other IAV-specific CD4 T cells and PD1+ memory CD4 T cells were located further away from MHCII+ cells than their PD1-low counterparts. This distinction in location was lost in mice treated with anti-MHCII antibody. These data suggest that sustained antigen presentation in the lung impacts on the formation of memory CD4 T cells by regulating their cytokine production and location.

Triphasic production of IFNγ by innate and adaptive lymphocytes following influenza A virus infection.

Interferon gamma (IFNγ) is a potent antiviral cytokine that can be produced by many innate and adaptive immune cells during infection. Currently, our understanding of which cells produce IFNγ and where they are located at different stages of an infection is limited. We have used reporter mice to investigate in vivo expression of Ifnγ mRNA in the lung and secondary lymphoid organs during and following influenza A virus (IAV) infection. We observed a triphasic production of Ifnγ expression. Unconventional T cells and innate lymphoid cells, particularly NK cells, were the dominant producers of early Ifnγ, while CD4 and CD8 T cells were the main producers by day 10 post-infection. Following viral clearance, some memory CD4 and CD8 T cells continued to express Ifnγ in the lungs and draining lymph node. Interestingly, Ifnγ production by lymph node natural killer (NK), NKT, and innate lymphoid type 1 cells also continued to be above naïve levels, suggesting memory-like phenotypes for these cells. Analysis of the localization of Ifnγ+ memory CD4 and CD8 T cells demonstrated that cytokine+ T cells were located near airways and in the lung parenchyma. Following a second IAV challenge, lung IAV-specific CD8 T cells rapidly increased their expression of Ifnγ while CD4 T cells in the draining lymph node increased their Ifnγ response. Together, these data suggest that Ifnγ production fluctuates based on cellular source and location, both of which could impact subsequent immune responses.

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.

The Jeff Mouse Mutant Model for Chronic Otitis Media Manifests Gain-of-Function as Well as Loss-of-Function Effects.

Chronic otitis media (OM) is the most common cause of hearing loss worldwide, yet the underlying genetics and molecular pathology are poorly understood. The mouse mutant Jeff is a single gene mouse model for OM identified from a deafness screen as part of an ENU mutagenesis program at MRC Harwell. Jeff carries a missense mutation in the Fbxo11 gene. Jeff heterozygotes (Fbxo11 Jf/+ ) develop chronic OM at weaning and have reduced hearing. Homozygotes (Fbxo11 Jf/Jf ) display perinatal lethality due to developmental epithelial abnormalities. In order to investigate the role of FBXO11 and the type of mutation responsible for the phenotype of the Jeff mice, a knock-out mouse model was created and compared to Jeff. Surprisingly, the heterozygote knock-outs (Fbxo11 tm2b/+ ) show a much milder phenotype: they do not display any auditory deficit and only some of them have thickened middle ear epithelial lining with no fluid in the ear. In addition, the knock-out homozygote embryos (Fbxo11 tm2b/tm2b ), as well as the compound heterozygotes (Fbxo11 tm2b/Jf ) show only mild abnormalities compared to Jeff homozygotes (Fbxo11 Jf/Jf ). Interestingly, 3 days after intranasal inoculation of the Fbxo11 tm2b/+ mice with non-typeable Haemophilus influenzae (NTHi) a proportion of them have inflamed middle ear mucosa and fluid accumulation in the ear suggesting that the Fbxo11 knock-out mice are predisposed to NTHi induced middle ear inflammation. In conclusion, the finding that the phenotype of the Jeff mutant is much more severe than the knock-out indicates that the mutation in Jeff manifests gain-of-function as well as loss-of-function effects at both embryonic and adult stages.

GREETING AND RESPONSE: PREDICTING PARTICIPATION FROM THE CALL OPENING.

Although researchers have used phone surveys for decades, the lack of an accurate picture of the call opening reduces our ability to train interviewers to succeed. Sample members decide about participation quickly. We predict participation using the earliest moments of the call; to do this, we analyze matched pairs of acceptances and declinations from the Wisconsin Longitudinal Study using a case-control design and conditional logistic regression. We focus on components of the first speaking turns: acoustic-prosodic components and interviewer's actions. The sample member's "hello" is external to the causal processes within the call and may carry information about the propensity to respond. As predicted by Pillet-Shore (2012), we find that when the pitch span of the sample member's "hello" is greater the odds of participation are higher, but in contradiction to her prediction, the (less reliably measured) pitch pattern of the greeting does not predict participation. The structure of actions in the interviewer's first turn has a large impact. The large majority of calls in our analysis begin with either an "efficient" or "canonical" turn. In an efficient first turn, the interviewer delays identifying themselves (and thereby suggesting the purpose of the call) until they are sure they are speaking to the sample member, with the resulting efficiency that they introduce themselves only once. In a canonical turn, the interviewer introduces themselves and asks to speak to the sample member, but risks having to introduce themselves twice if the answerer is not the sample member. The odds of participation are substantially and significantly lower for an efficient turn compared to a canonical turn. It appears that how interviewers handle identification in their first turn has consequences for participation; an analysis of actions could facilitate experiments to design first interviewer turns for different target populations, study designs, and calling technologies.