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IL-4 production is associated with low-avidity, poorly cytotoxic T cell induction that contributes to viral immune evasion and the failure of T cell-based vaccines. Yet, the precise mechanisms that regulate IL-4 signalling in T cells remain elusive. Mounting evidence indicates that cells can dynamically alter their IL-4/IL-13 receptor signature to modulate downstream immune outcomes upon pathogen encounter. Here, we describe how naïve (CD62L+CD44lo–mid) CD4 and CD8 T cells distinctly engage both STAT6 and STAT3 in response to IL-4. We further show that IL-4R⍺ expression is both time- and IL-4 concentration-dependent. Remarkably, our findings reveal that STAT3 inhibition can ablate IL-4R⍺ and affect transcriptional expression of other Stat and Jak family members. By extension, the loss of STAT3 lead to aberrant STAT6 phosphorylation, revealing an inter-regulatory relationship between the two transcription factors. Moreover, IL-4 stimulation down-regulated TGF-β1 and IFN-γR1 expression on naïve T cells, possibly signifying the broad regulatory implications of IL-4 in conditioning lineage commitment decisions during early infection. Surprisingly, naïve T cells were unresponsive to IL-13 stimulation, unlike dendritic cells. Collectively, these findings could be exploited to inform more efficacious vaccines, as well as design treatments against IL-4/IL-13-associated disease conditions.

We have shown that manipulation of IL-13 and STAT6 signaling at the vaccination site can lead to different innate lymphoid cell (ILC)/dendritic cell (DC) recruitment, resulting in high avidity/poly-functional T cells and effective antibody differentiation. Here we show that permanent versus transient blockage of IL-13 and STAT6 at the vaccination site can lead to unique ILC-derived IL-13 and IFN-γ profiles, and differential IL-13Rα2, type I and II IL-4 receptor regulation on ILC. Specifically, STAT6−/− BALB/c mice given fowl pox virus (FPV) expressing HIV antigens induced elevated ST2/IL-33R+ ILC2-derived IL-13 and reduced NKp46+/− ILC1/ILC3-derived IFN-γ expression, whilst the opposite (reduced IL-13 and elevated IFN-γ expression) was observed during transient inhibition of STAT6 signaling in wild type BALB/c mice given FPV-HIV-IL-4R antagonist vaccination. Interestingly, disruption/inhibition of STAT6 signaling considerably impacted IL-13Rα2 expression by ST2/IL-33R+ ILC2 and NKp46− ILC1/ILC3, unlike direct IL-13 inhibition. Consistently with our previous findings, this further indicated that inhibition of STAT6 most likely promoted IL-13 regulation via IL-13Rα2. Moreover, the elevated ST2/IL-33R+ IL-13Rα2+ lung ILC2, 24 h post FPV-HIV-IL-4R antagonist vaccination was also suggestive of an autocrine regulation of ILC2-derived IL-13 and IL-13Rα2, under certain conditions. Knowing that IL-13 can modulate IFN-γ expression, the elevated expression of IFN-γR on lung ST2/IL-33R+ ILC2 provoked the notion that there could also be inter-regulation of lung ILC2-derived IL-13 and NKp46− ILC1/ILC3-derived IFN-γ via their respective receptors (IFN-γR and IL-13Rα2) at the lung mucosae early stages of vaccination. Intriguingly, under different IL-13 conditions differential regulation of IL-13/IL-13Rα2 on lung DC was also observed. Collectively these findings further substantiated that IL-13 is the master regulator of, not only DC, but also different ILC subsets at early stages of viral vector vaccination, and responsible for shaping the downstream adaptive immune outcomes. Thus, thoughtful selection of vaccine strategies/adjuvants that can manipulate IL-13Rα2, and STAT6 signaling at the ILC/DC level may prove useful in designing more efficacious vaccines against different/chronic pathogens.

In this study, recombinant pox viral vaccination was shown to induce highly elevated IgG2a and low IgG1 antibody expression in mice lacking IL-4 or STAT6, whilst IL-13-/- mice exhibited elevated IgG1, but very low IgG2a. These findings revealed that IL-13 and IL-4 differentially regulated antibody development. To understand this further, when STAT6-/- mice were given a vaccine co-expressing IL-13Rα2 that temporarily sequestered IL-13, significantly reduced IgG2a expression, was detected. These findings for the first time demonstrated that IL-13 regulated IgG2a differentiation utilising an alternative IL-13R signalling pathway independent of STAT6 (IL-13Rα2 pathway). This was further corroborated by the (i) elevated IL-13Rα2 expression detected on STAT6-/- lung MHCII+ CD11c+ cells 24 h post IL-13 inhibitor vaccination and ii) significant up-regulation of IL-13Rα2 expression on spleen and lung derived MHCII+ CD11c+ following inhibition of STAT6 signalling in vitro, or vaccination with IL-4R/STAT6 antagonist in vivo. When T follicular helper (Tfh) cells which regulate antibody differentiation were assessed post vaccination, although no difference in IL-4 expression was observed, greatly reduced IFN-γ expression was detected in IL-13-/- and STAT6-/- mice compared to wild-type. These findings support the notion that the balance of IL-13 level at the vaccination site can differentially regulate T and B-cell immune outcomes.

The efficacy of the intranasally (i.n.) delivered live attenuated influenza vaccine (LAIV) is variable and, in some seasons, suboptimal. In this study, we report that LAIV exhibits cross-protective efficacy in mice, potentially associated with cellular immunity as opposed to antigen-specific antibody responses. However, pre-exposure to the intramuscularly (i.m.) delivered inactivated influenza vaccine (IIV) severely impaired LAIV-induced cross-protection against heterologous challenge, potentially by inhibiting replication of LAIV. Our findings suggest that pre-existing immunity afforded by IIV suppresses cross-protective T cell immunogenicity of LAIV.

Asthma is a global problem that affects millions of individuals. An increased risk of respiratory viral and bacterial infections is one of the complications of asthma. We recently reported that mice with ovalbumin-induced allergic airway disease (AAD) are protected against influenza-Streptococcus pneumoniae co-infection. Here, we describe in detail a protocol on how to induce AAD and influenza-S. pneumoniae co-infection in mice and to evaluate the specific roles of asthma on immunity to viral and bacterial pathogens in the hope of translating findings to benefit asthmatic individuals.

Many of the biological properties of C5a are mediated through activation of its receptor (C5aR1), the expression of which has been demonstrated convincingly on myeloid cells, such as neutrophils, monocytes, and macrophages. In contrast, conflicting results exist regarding C5aR1 expression in dendritic cells (DCs) and lymphoid lineage cells. In this article, we report the generation of a floxed GFP-C5aR1 reporter knock-in mouse. Using this mouse strain, we confirmed strong C5aR1 expression in neutrophils from bone marrow, blood, lung, and spleen, as well as in peritoneal macrophages. Further, we show C5aR1 expression in lung eosinophils, lung- and lamina propria–resident and alveolar macrophages, bone marrow–derived DCs, and lung-resident CD11b+ and monocyte-derived DCs, whereas intestinal and pulmonary CD103+ DCs stained negative. Also, some splenic NKT cells expressed GFP, whereas naive NK cells and B2 cells lacked GFP expression. Finally, we did not observe any C5aR1 expression in naive or activated CD4+ Th cells in vitro or in vivo. Mating the floxed GFP-C5aR1 mouse strain with LysMCre mice, we were able to specifically delete C5aR1 in neutrophils and macrophages, whereas C5aR1 expression was retained in DCs. In summary, our findings suggest that C5aR1 expression in mice is largely restricted to cells of the myeloid lineage. The novel floxed C5aR1 reporter knock-in mouse will prove useful to track C5aR1 expression in experimental models of acute and chronic inflammation and to conditionally delete C5aR1 in immune cells.