Evaluation of the role of innate lymphoid cells following viral vector vaccination

Abstract

Over the past decade, studies in our laboratory have established that i) compared to systemic route of delivery, mucosal delivery of recombinant fowlpox virus (rFPV) prime can induce excellent high avidity mucosal/systemic HIV-specific CD8+ T cell immunity, ii) this was associated with IL-4/IL-13 cytokine milieu and iii) transient inhibition of IL-13 at the vaccination site can recruit unique lung dendritic cell (DC) subsets, responsible for the induction of high quality CD8+ T cell immunity. Therefore, to understand which cells at the vaccination site was the predominant source of IL-13 was assessed by evaluating the different cells at the vaccination site, specifically innate lymphoid cells (ILC) following rFPV vaccination with and without transient inhibition of IL-13. These studies for the first time revealed that ILC2 were the main source of IL-13 at the vaccination site (24 h post vaccination) responsible for inducing high quality T and B cells immune responses reported previously. Intranasal vaccination induced ST2/IL-33R+ ILC2 in lung, whilst intramuscular vaccination exclusively induced IL-25R+ ILC2 in muscle. Moreover, adjuvants that transiently inhibited IL-13 at the vaccination site significantly influenced the IFN-γ expression by ILC1/ILC3 indicating that ILC2-derived IL-13 at the vaccination site also modulated ILC1/ILC3 function/activity.

As intranasal and intramuscular vaccinations induced different ILC2 subsets, two rFPV vaccines co-expressing adjuvants that transiently sequestered IL-25 and IL-33 at the vaccination site (rFPV-IL-12BP and rFPV-IL-33BP) were used to further evaluate ILC development following vaccination. Unlike IL-13 inhibitor vaccination conditions, intranasal delivery of IL-25BP adjuvanted vaccine induced not only ST2/IL-33R+ ILC2 but also IL-25R+ and TSLPR+ ILC2 subsets that were able to express IL-13. Moreover TSLPR+ ILC2 subset was also able to express IL-4. Interestingly, intranasal delivery of IL-25BP also induced significantly elevated number of NKp46+/- ILC1/ILC3 expressing IL-17A compared to IFN-γ, unlike the unadjuvanted or IL-13 inhibitor conditions. Taken together, these inhibitor studies indicated that IL-25 play a fundamental role in early ILC development than IL-33, suggesting that there is a hierarchical regulation of ILC development, where IL-25 is most likely the master regulator of ILC.

Data also revealed that ILC and their cytokine expression profiles were vastly different during permanent verses transient blockage of IL-13, and STAT6 at the vaccination site. STAT6-/- mice given the FPV-HIV vaccine showed elevated ST2/IL-33R+ ILC2-driven IL-13 expression whilst reduced IFN-γ expression by both NKp46+/- ILC1/ILC3, unlike transient blockage of STAT6 which showed the opposing effect. When IL-13-/- mice were vaccinated with FPV-HIV significantly elevated lung lineage- ST2/IL-33R+ ILC2s were detected compared to BALB/c mice given the FPV-HIV-IL-13Rα2 adjuvanted vaccine (transient inhibition of IL-13), and their NKp46+/- ILC1/ILC3-driven IFN-γ expression was significantly lower compared to transient inhibition of STAT6. In previous studies when IL-13 was inhibited, no or low antibody differentiation has been reported, unlike STAT6 inhibition. Thus, current data further corroborated that ST2/IL-33R+ ILC2-derived IL-13 play a crucial role in modulating downstream B cell immune outcomes. Specifically, co-regulation of ST2/IL-33R+ ILC2-derived IL-13 and NKp46+/- ILC1/ILC3-derived IFN-γ may play an important role in modulating antibody differentiation process in a STAT6 independent manner via the IL-13Rα2 pathway.

When trying to understand the molecular mechanism involved in this process, data revealed that the expression of IL-13Rα2, type I and II IL-4Rs on ST2/IL-33R+ ILC2 and NKp46- ILC1/ILC3 were co-regulated 24 h post intranasal rFPV vaccination. Inhibition of STAT6 signalling significantly impacted the IL-13Rα2 expression on both ST2/IL-33R+ ILC2 and NKp46- ILC1/ILC3, unlike IL-13 inhibition, suggesting that under STAT6 inhibition conditions, IL-13 could signal via IL-13Rα2 pathway. As elevated number of ST2/IL-33R+ ILC2 expressing IL-13Rα2 were detected in BALB/c mice given the FPV-HIV-IL-4R antagonist vaccine, this also indicated an autocrine regulation of IL-13 at the ILC2 via IL-13Rα2. The Il-4/IL-13 receptor expression profile on NKp46+ ILC1/ILC3 and NKp46- ILC1/ILC3 were vastly different, suggesting that these cells may play different roles in downstream immune outcomes.

Collectively, findings in this thesis demonstrated that i) ILC activity is significantly modulated by route of vaccine delivery and vaccine adjuvants early as 24 h post vaccination, ii) When designing vaccines against chronic pathogens, understanding the fundamental roles of ILC at the vaccination site may help design better vaccines in the future, iii) IL-25 regulated initial development/differentiation of all ILCs and IL-33 most likely only play a role in ILC2 homing to the lung mucosae, iv) Post viral vector vaccination ILC-derived IL-13 and IFN-γ balance was crucial for shaping the downstream immune outcomes, v) The IL-13 regulation at the ILC level occurred via an STAT6 independent pathway, most likely IL-13Rα2 (due to low IL-13 conditions). In conclusion, this work has provided unique insights into ILC function and activity during viral vector vaccination, that could be used to tailor vaccine vectors to induce effective immune outcomes against target pathogens (for example TB verses an HIV vaccine).