CCR2 Regulates Vaccine-Induced Mucosal T-Cell Memory to Influenza A Virus

Abstract

Elicitation of lung tissue-resident memory CD8 T cells (TRMs) is a goal of T-cell based vaccines against respiratory viral pathogens, such as influenza A virus (IAV). Chemokine receptor 2 (CCR2)-dependent monocyte trafficking plays an essential role in the establishment of CD8 TRMs in lungs of IAV-infected mice. Here, we used a combination adjuvant-based subunit vaccine strategy that evokes multifaceted (TC1/TC17/TH1/TH17) IAV nucleoprotein-specific lung TRMs, to determine whether CCR2 and monocyte infiltration are essential for vaccine-induced TRM development and protective immunity to IAV in lungs. Following intranasal vaccination, neutrophils, monocytes, conventional DCs and monocyte-derived dendritic cells internalized and processed vaccine antigen in lungs. We found that Basic Leucine Zipper ATF-Like Transcription Factor 3 (BATF-3)-dependent DCs were essential for eliciting T cell responses, but CCR2 deficiency enhanced the differentiation of CD127HI/KLRG-1LO, OX40+veCD62L+ve and mucosally imprinted CD69+veCD103+ve effector and memory CD8 T cells in lungs and airways of vaccinated mice. Mechanistically, increased development of lung TRMs, induced by CCR2 deficiency was linked to dampened expression of T-bet, but not altered TCF-1 levels or T cell receptor signaling in CD8 T cells. T1/T17 functional programming, parenchymal localization of CD8/CD4 effector and memory T cells, recall T cell responses and protective immunity to a lethal IAV infection were unaffected in CCR2-deficient mice. Taken together, we identified a negative regulatory role for CCR2 and monocyte trafficking in mucosal imprinting and differentiation of vaccine-induced TRMs. Mechanistic insights from this study may aid the development of T-cell-based vaccines against respiratory viral pathogens including IAV and SARS-CoV-2. Importance While antibody-based immunity to influenza A virus (IAV) is type and sub-type specific, lung and airway-resident memory T cells that recognize conserved epitopes in the internal viral proteins are known to provide heterosubtypic immunity. Hence, broadly protective IAV vaccines need to elicit robust T-cell memory in the respiratory tract. We have developed a combination adjuvant-based IAV nucleoprotein vaccine that elicits strong CD4 and CD8 T cell memory in lungs and protects against H1N1 and H5N1 strains of IAV. In this study, we examined the mechanisms that control vaccine-induced protective memory T cells in the respiratory tract. We found that trafficking of monocytes into lungs might limit the development of anti-viral lung-resident memory T cells, following intranasal vaccination. These findings suggested that strategies that limit monocyte infiltration can potentiate vaccine-induced frontline T-cell immunity to respiratory viruses, such as IAV and SARS-CoV-2.

Multimodal profiling of lung granulomas in macaques reveals cellular correlates of tuberculosis control

“…Further supporting a hybrid cell state, we independently confirmed the presence of a subset of granuloma T cells expressing both T-bet and RORα by flow cytometry ( Grant et al., 2022 ). Notably, although Th1 ∗ and ex-Th17 subsets are described primarily as CD4 T cells ( Darrah et al., 2020 ; Gideon et al., 2015 ; Lyadova and Panteleev, 2015 ; Mpande et al., 2018 ), our T1-T17 sub-cluster was characterized by the expression of both CD4 and CD8A/B transcripts ( Figures 3 C, 4 A–4C, S4 D, and S4E), suggesting that this phenotype is not an identity program but context dependent, consistent with findings in other systems ( Lee et al., 2021 ).

Figure 4 Phenotypic Diversity in T1-T17 cells (A) T1-T17 subcluster overlaid on unified T/NK cell cluster (left) and colored by normalized expression values for T1-T17 subcluster-defining genes (bold outlined boxes) and non-enriched canonical Type1 and type 17 genes (right).

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“…Aspects of these data are consistent with recent observations that granulomas established in immune-primed environments—e.g., existing Mtb infection ( Cadena et al., 2018 ) or intravenous or intrabronchial BCG vaccination—are characterized by Th1/17 expression patterns that are associated with protection ( Darrah et al., 2020 ; Dijkman et al., 2019 ); however, we extend these findings, defining appreciable substructure among the T1-T17 subcluster of relevance to control. The CD4 T1-T17 subpopulation (subpopulation 1) is most consistent with published descriptions of Th1/17 cells (e.g., Th1 ∗ or ex-Th17) ( Acosta-Rodriguez et al., 2007 ; Amezcua Vesely et al., 2019 ; Becattini et al., 2015 ; Lee et al., 2021 ; Lindestam Arlehamn et al., 2013 ; Mahnke et al., 2013 ; Nikitina et al., 2018 ). These cells could represent precursors to long-lived tissue memory, which has been shown to play a crucial protective role in autoimmunity, bacterial control, and memory immune responses to pathogens ( Amezcua Vesely et al., 2019 ; Liang et al., 2015 ; van Hamburg and Tas, 2018 ; Wacleche et al., 2016 ), including Mtb infection.…”

Epigenetic variation impacts ancestry-associated differences in the transcriptional response to influenza infection

“…Unexpectedly, BATF, which is not a classical immune-related TF, showed the greatest increase in activity upon infection. Our results thus suggest that BATF likely plays a previously unappreciated role in the macrophage response to flu infection, paralleling its already established role in the induction of effector programs and epigenetic landscape of CD8 + T cells and innate lymphoid cells infected with flu (Wu et al 2022;Lee et al 2021;Scott-Browne et al 2016). Collectively, our results show that transcriptional and epigenetic changes in response to flu infection are highly coordinated and likely driven by the activation of infection-induced TFs involved in the regulation of antiviral responses.…”

Vaccine adjuvants to engage the cross-presentation pathway

“…Specifically, upon subcutaneous vaccination of mice, ADJ elicited effector CD8 T cells that differentiated into a distinct subset of granzyme B-expressing CD27 LO ‘effector-like’ memory CD8 T cells, which provided highly effective immunity to intracellular bacteria L. monocytogenes in spleen and liver ( 222 ). Additionally, we have reported that ADJ, in combination with TLR agonists CpG and GLA, stimulated high numbers of tissue resident memory CD4 and CD8 T cells in the respiratory tract and protected against antigenically distinct strains of influenza viruses ( 218 – 220 ). Recently, Kingstad-Bakke et al.…”

“…Like vaccine adjuvants, various innate immune cells, including monocytes, neutrophils, and conventional dendritic cells, are rapidly recruited within 24-48 hours of intradermal, intraperitoneal, and intranasal vaccination of ADJ-containing vaccines at the injection sites ( 219 , 222 , 230 ). CD8 T cell response induced by mucosal or parenteral administration of vaccine antigens formulated in ADJ was ablated in BATF-3-deficient mice ( 220 , 222 ). These data suggest that stimulation of CD8 T cell responses to subunit vaccine antigens formulated in ADJ requires cross-presentation, presumably by BATF3-dependent conventional migratory DCs.…”