key: cord-103105-iqjksoim
authors: Marinaik, Chandranaik B.; Kingstad-Bakke, Brock; Lee, Woojong; Hatta, Masato; Sonsalla, Michelle; Larsen, Autumn; Neldner, Brandon; Gasper, David J.; Kedl, Ross M.; Kawaoka, Yoshihiro; Suresh, M.
title: Programming Multifaceted Pulmonary T-Cell Immunity by Combination Adjuvants
date: 2020-07-10
journal: bioRxiv
DOI: 10.1101/2020.07.10.197459
sha: 
doc_id: 103105
cord_uid: iqjksoim

Induction of protective mucosal T-cell memory remains a formidable challenge to vaccinologists. Using a novel adjuvant strategy that elicits unusually potent CD8 and CD4 T-cell responses, we have defined the tenets of vaccine-induced pulmonary T-cell immunity. An acrylic acid-based adjuvant (ADJ), in combination with TLR agonists glucopyranosyl lipid adjuvant (GLA) or CpG promoted mucosal imprinting but engaged distinct transcription programs to drive different degrees of terminal differentiation and disparate polarization of TH1/TC1/TH17/TC17 effector/memory T cells. Combination of ADJ with GLA, but not CpG, dampened TCR signaling, mitigated terminal differentiation of effectors and enhanced the development of CD4 and CD8 TRM that protected against H1N1 and H5N1 influenza viruses. Mechanistically, vaccine-elicited CD4 T cells played a vital role in optimal programming of CD8 TRM and anti-viral immunity. Taken together, these findings provide new insights into vaccine-induced multi-faceted mucosal T-cell immunity with significant implications in the development of vaccines against respiratory pathogens. One Sentence Summary Adjuvants Induce Multipronged T-Cell Immunity in the Respiratory Tract.

Viral mucosal infections such as influenza cause considerable morbidity, and even mortality in very young and geriatric patients (1). Protection afforded against influenza A virus (IAV) by antibodies is typically virus type/subtype specific; however, T cells are believed to provide broad heterosubtypic immunity (2) (3) (4) (5) . IAV infection elicits strong effector CD8 and CD4 T-cell responses in the lungs leading to the development of protective lung and airway-resident memory T cells (3, 6) . However, influenza-specific mucosal memory T cells exhibit attrition and T-cell-based protection wanes in a span of 3-6 months (7, 8) . Therefore, unlike systemic viral infections that typically engender enduring immunity (9, 10) , mucosal viral infections fail to program durable T-cell immunity in the respiratory tract (RT). While engagement of multiple innate receptors early in the response might be key to long-lived immunological memory following systemic infections (11, 12) , there is a lack of understanding of why mucosal infections lead to shorter duration of cellular immunity. There is a general paucity of adjuvants that induce strong T-cell responses, and we have limited knowledge of mucosal T-cell responses to adjuvanted subunit vaccines, especially in the RT. These knowledge gaps pose daunting constraints in the development of immunization strategies targeted at the establishment of durable protective T-cell immunity in the RT (13) (14) (15) (16) .

Adjuplex (ADJ) is a polyacrylic acid-based (carbomer) adjuvant that is a component of some current veterinary vaccines and also known to induce neutralizing antibodies against HIV and malaria (17) (18) (19) (20) . Here, we report that ADJ, in combination with Toll-like receptor (TLR) 4/9

agonists, elicits unexpectedly potent and functionally diverse CD8 and CD4 T-cell responses to a subunit viral protein in the RT. Studies with this adjuvant system provided the means to differentially program distinct patterns of effector and memory T-cell differentiation in the RT.

Further, these studies provided the first glimpse of the evolution of T-cell responses to adjuvanted vaccines in the lungs to define the quantitative, phenotypic and functional attributes of mucosal effector/memory CD8 and CD4 T cells that are associated with effective viral control in the lungs, and protection against H1N1 and H5N1 influenza infections. Collectively, these findings provide novel insights into the immunological apparatus underlying the generation and establishment of protective and durable T-cell immunity in the RT in response to adjuvanted subunit vaccines.

(P<0.05) than in the GLA group. Interestingly, comparison of ADJ, GLA and ADJ+GLA groups suggested that GLA limited the development of CX3CR1 HI CD8 T cells.

As another surrogate marker for effector differentiation, we quantified granzyme B levels in CD8 T cells directly ex vivo (Fig. 1C) . The percentages of granzyme B HI CD8 T cells among NP366specific CD8 T cells in ADJ, CpG and ADJ+CpG groups were significantly (P<0.05) higher than in GLA or ADJ+GLA groups. Clearly, ADJ and CpG promoted granzyme B expression, but GLA antagonized the granzyme B-enhancing effects of ADJ.

Studies to determine the transcriptional basis for the disparate differentiation of effector CD8 T cells in different adjuvant groups showed that the expressions of T-bet, IRF-4 and BATF were substantially greater in ADJ and ADJ+CpG groups, compared to GLA and ADJ+GLA groups ( Fig. 1D) . While ADJ appeared to be the primary driver of T-bet, IRF-4 and BATF expression, GLA effectively negated this effect in ADJ+GLA mice (Fig. 1D) . The levels of EOMES did not differ between adjuvants, but analysis of T-bet and EOMES co-expression showed that a higher percentage of CD8 T cells co-expressed T-bet and EOMES (T-bet HI EOMES HI ) in the CpG and ADJ+CpG groups (Fig. S1C) . By contrast, a greater proportion of CD8 T cells in GLA and ADJ+GLA groups expressed EOMES, but not T-bet (T-bet LO EOMES HI ) (Fig. S1C) . Taken together, terminal differentiation of effector CD8 T cells in ADJ and/or CpG was linked to high levels of T-bet, IRF-4 and BATF.

Next, we assessed expression of CD103 and CD69 to ask whether adjuvants affected mucosal imprinting of CD8 T cells in the RT. The majority of NP366-specific CD8 T cells in lungs and BAL expressed CD69 but not CD103 in all groups. The percentages of CD103 HI CD69 HI CD8 T cells in ADJ, ADJ+CpG and ADJ+GLA groups were higher than in CpG and GLA groups, which suggested that ADJ was a potent inducer of CD103 (Fig. 1E) . Altogether, Fig. 1 shows that ADJ and/or CpG promoted different facets of CD8 T-cell terminal differentiation.

Remarkably however, when combined with ADJ, GLA selectively antagonized ADJ-driven terminal differentiation program without affecting mucosal imprinting of CD8 T cells. Thus, ADJ-driven CD8 T-cell differentiation program can be augmented or antagonized by TLR agonists CpG and GLA respectively.

Next, we characterized NP-specific CD4 T-cell responses to various adjuvants following mucosal immunization. At day 8 PV, high perecentages of NP311-specific CD4 T cells were detected in lungs and airways of all groups of mice ( Fig. 2A) . The percentages and total numbers of NP311-specific CD4 T cells in lungs and airways were comparable between ADJ, CpG, GLA and ADJ+CpG groups. However, the total numbers of NP311-specific CD4 T cells in the lungs and airways of ADJ+GLA group were significantly higher than in other groups ( Fig. 2A) .

Phenotypically, ADJ and CpG promoted the expression of terminal differentiation markers CX3CR1 and KLRG-1, respectively (Fig. 2B) . By contrast, expressions of CX3CR1 and KLRG-1 were lowest in the GLA group (Fig. 2B) and GLA tempered ADJ-induced expression of CX3CR1 in ADJ+GLA group. NP311-specific CD4 T cells from ADJ and/or CpG groups contained greater levels of T-bet, as compared to other groups (Fig. 2C) , but EOMES levels were not different between groups. GLA with or without ADJ induced the lowest levels of T-bet, which resulted in greater percentages of T-bet LO EOMES HI CD4 T cells in GLA and ADJ+GLA groups (Fig. 2D) . Thus, ADJ and CpG promoted terminal differentiation of CD4 T cells by

inducing T-bet expression, as compared to GLA or ADJ+GLA groups. Analysis of mucosal imprinting markers CD103 and CD69 showed that ADJ-containing adjuvants elicited higher percentages of CD103 HI and CD103 HI CD69 HI CD4 T cells in lungs (Fig. 2E) . Thus, in contrast to ADJ and CpG, combining ADJ with GLA promoted the development of less differentiated mucosally-imprinted CD4 T cells in the lungs and airways.

We then asked whether adjuvants regulated functional programming of effector CD8 and CD4 T cells into TC1/TC17 or TH1/TH17 subsets respectively, in lungs. NP366-specific IFN--producing TC1 CD8 T cells were induced in all groups and the percentages of such cells among CD8 T cells were generally higher in the ADJ+GLA group (Fig. 3A) . Interestingly however, IL-17-producing NP366-specific TC17 CD8 T cells were strongly induced only in the GLA and ADJ+GLA groups. To further elucidate the relative dominance of TC1 versus TC17 in different adjuvant groups, we calculated the relative proportions of these cells among total cytokine-producing (IL-17+IFN- producing cells) peptide-stimulated NP366-specific CD8 T cells (Fig. 3B) ; ~80-88% of NP366-specific cytokine-producing CD8 T cells produced IFN- in the CpG and ADJ+CpG groups, and only 65%, 56% and 36% of such cells produced IFN- in ADJ, GLA and ADJ+GLA groups, respectively. Reciprocally, while only a relatively small fraction (12-20%) of NP366specific cytokine-producing CD8 T cells produced IL-17 or IL-17+IFN- in CpG and ADJ+CpG  groups, 40-57% of NP366-specific CD8 T cells produced IL-17 or IL-17+IFN- in GLA and   ADJ+GLA groups. Thus, CpG and ADJ+CpG promoted functional polarization of TC1 cells, and ADJ, GLA and ADJ+GLA drove a balanced differentiation of TC1 and TC17 cells. Evaluation of the ability of NP366-specific CD8 T cells to co-produce IFN-, TNF- and IL-2 ( Fig. 3C) showed that all adjuvants induced polyfunctional CD8 T cells, but a significantly higher percentages of NP366-specific CD8 T cells in the GLA group were polyfunctional, as compared to other groups (Fig. 3C) .

NP311-specific TH1 and TH17 CD4 T cells were induced to varying levels by different adjuvants (Fig. 3D) . ADJ promoted TH17 polarization of effector CD4 T cells but CpG promoted TH1 differentiation and negated the TH17 skewing effects of ADJ in the ADJ+CpG group. TH17 differentiation dominated over the TH1 development in GLA and ADJ+GLA groups (Fig. 3E) . In summary, while CpG and ADJ+CpG promoted the development of TH1 effector cells, ADJ, GLA and ADJ+GLA favored the differentiation of TH17 cells (Fig. 3E) . Polyfunctionality among NP311-specific CD4 T cells was largely comparable between groups (Fig. 3F) .

Antigenic stimulation and the inflammatory milieu govern effector differentiation during infections (25) (26) (27) . In order to determine whether adjuvants differed in terms of antigenic stimulation in draining lymph nodes (DLNs) and lungs, early after vaccination (day 2 and 5), we adoptively transferred 5x10 4 TCR transgenic OT-I CD8 T cells that express GFP under the control of Nur77 promoter; Nur77 expression faithfully reports specific TCR signaling in T cells (28) . Subsequently, mice were vaccinated with chicken ovalbumin (OVA) mixed with different   adjuvants, and GFP expression by OT-I CD8 T cells was assessed at days 2 and 5 PV. OT-I CD8 T cells expressed readily detectable levels of GFP in DLNs and lungs at different days PV (Fig.   4A) . Overall, GFP levels were not significantly different for OT-I CD8 T cells (P<0.05) in DLNs between various groups (except between GLA and ADJ+GLA) at day 5 PV (Fig. 4A) . OT-I CD8 T cells were not detectable in lungs until day 5 PV; at day 5 PV, significantly (P<0.05) higher levels of GFP were detected in OT-I CD8 T cells from the lungs of ADJ mice, compared to CpG, GLA and ADJ+GLA groups (Fig. 4A) . Adoptive transfer of 5x10 4 TCR transgenic CD8 T cells was technically essential to assess T-cell signaling early after vaccination (Fig. 4A ), but transfer of such unphysiologically high numbers of T cells might affect their differentiation (29) .

Therefore, for assessment of TCR signaling at day 8 PV, we adoptively transferred 10 3 Nur77-GFP OT-I TCR transgenic CD8 T cells prior to vaccination. The pattern of GFP fluorescence in donor OT-I CD8 T cells in DLNs and lungs of vaccinated mice at day 8 PV, is shown in Fig. S2 .

On the 8 th day PV, OT-I CD8 T cells in the DLNs of ADJ mice expressed higher levels of GFP, compared to other groups, but the differences did not reach statistical significance. By contrast, on day 8 PV, GFP levels in OT-I CD8 T cells from lungs of ADJ mice were significantly higher (P<0.05) than in OT-I CD8 T cells from lungs of CpG, GLA and ADJ+GLA mice (Fig. 4A) .

Collectively, a greater percentage of OT-I CD8 T cells in the lungs of ADJ group showed evidence of active TCR signaling in the lungs at days 5 and 8 after vaccination, and, notably, this effect of ADJ was dampened by GLA but not CpG. Enhanced TCR signaling in ADJ group (and to a lesser extent in CpG group) was consistent with elevation of IRF-4 and BATF (Fig. 1D) , whose expressions are known to be driven by TCR signaling (30) .

Transcription factor KLF2 plays a key role in regulating T-cell trafficking, and TCR signaling downregulates KLF2 expression (31, 32) . Using KLF2-GFP reporter mice (32), we assessed whether high TCR signaling in ADJ-vaccinated mice led to KLF2 downregulation in polyclonal NP366-specific CD8 T cells in the DLNs and lungs, at day 8 PV. In all groups, NP366-specific CD8 T cells downregulated KLF2 expression in lungs, relative to KLF2 levels in their respective lymph nodes (Fig. 4B) . In lungs of ADJ, CpG and ADJ+CpG groups, NP366-specific CD8 T cells expressed lower levels of KLF2 than in CD8 T cells from GLA and ADJ+GLA groups ( Fig. 4B) . These data suggested that ADJ and/or CpG might enhance TCR signaling-induced KLF2 downregulation in lungs, as compared to ADJ+GLA.

During influenza virus infection in mice, TCR signaling drives PD-1 expression in lungs (33) . Therefore, we investigated whether PD-1 expression was linked to varying levels of TCR signaling induced by different adjuvants. At day 8 PV, higher percentages of NP366-specific CD8 T cells in ADJ mice expressed PD-1, as compared to those in CpG and GLA mice (Fig.   4C) . Interestingly, addition of GLA but not CpG to ADJ significantly reduced ADJ-driven PD-1 expression on NP366-specific CD8 T cells (Fig. 4C) . To elucidate the possible relationship between the frequency of NP366-specific CD8 T cells and their PD-1 expression levels in the lungs, we calculated correlation co-efficient between the two parameters (Fig. 4D) . Strikingly, there was a significant linear inverse correlation between PD-1 expression and the frequency of NP366-specific CD8 T cells in lungs of mice vaccinated with ADJ, CpG and GLA adjuvants.

These findings suggested that TCR signaling-induced PD-1 expression might limit the accumulation of CD8 T cells (clonal burst size) in the lungs. In summary ( Fig. 1 and 4) ,

terminal differentiation of effector CD8 T cells in ADJ and ADJ+CpG groups was associated with enhanced TCR signaling in the lungs. Reciprocally, GLA might protect effector CD8 T cells from ADJ-driven terminal differentiation, by limiting TCR signaling in the lungs.

To explore whether TCR signaling in CD8 T cells in ADJ+GLA mice is governed by the abundance of antigen-presenting cells in lungs, first we quantified innate immune cells including DCs in lungs at day 5 and 8 PV (Fig. S3A) . ADJ+GLA and ADJ+CpG increased the infiltration of neutrophils in lungs at day 5 and 8 respectively. Only at day 5 but not at day 8 PV, lungs of ADJ, ADJ+CpG and ADJ+GLA contained higher numbers of monocytes and monocyte-derived DCs, than in CpG and GLA mice. There were no differences between the groups in the numbers of CD103 +ve DCs or alveolar macrophages on either days after vaccination. We deterimined the abundance and type of antigen-processing cells in lungs by vaccinating mice with DQ-OVA, which emits green/red fluorescence upon degradation by proteases (Fig. S3B) . As compared to CpG and GLA groups, lungs of ADJ and ADJ+CpG (and ADJ+GLA to a slightly lesser degree) contained significantly higher numbers of DQ-OVA-bearing monocyte-derived DCs, monocytes and CD103 +ve DCs at day 5 PV, but not at day 8 PV. These data suggested that dampened TCR signaling in ADJ+GLA group, as compared to augmented signaling in ADJ and ADJ+CpG groups ( Fig. 4A -D) cannot be simply explained by reduced abundance of specific antigenbearing cells in the lungs.

To determine whether early inflammatory response influenced the phenotypic and functional differentiation of effector T cells, we quantified cytokine expression in the lungs. At 24 ( Fig.   S4A ) and 48 hours (Fig. S4B ) PV, the levels of cytokines/chemokines IL-1, IL-1, IL-6, KC, RANTES, G-CSF and GM-CSF were higher in lungs of GLA and/or ADJ+GLA mice. However, the levels of IFN-, IFN-, IL-10, IL-12p40, IL-12p70, MIP1, MIP1, MCP, TGF-1 and TNF in lungs were largely comparable between groups, except for the ADJ group (Fig. S4) .

Thus, terminal differentiation of effector CD8 T cells in ADJ, CpG and ADJ+CpG mice was not associated with excessive inflammation in the lungs, relative to other groups. Notably however, TC17 and TH17 cell development in GLA and ADJ+GLA groups was associated with elevated IL-1 in the lungs. Further, development of TH1 effectors and enhanced T-bet induction ( Fig. 1 and Fig. 2 ) in CpG and ADJ+CpG groups was not associated with elevated levels of IL-12p70 in the lungs. Thus, the differences in accumulation and terminal differentiation of effector T cells in the lungs of vaccinated mice cannot be explained by the degree of early inflammation.

At 100 days PV, we quantified NP366-specific memory CD8 T cells in lungs, airways and spleen. All adjuvants elicited robust CD8 T-cell memory in the RT (Fig. 5) . Notably, both frequencies and total numbers of NP366-specific memory CD8 T cells in lungs, airways and spleen of ADJ+GLA group were significantly (P<0.05) higher than in other groups (Fig. 5A) .

Intravascular staining showed that 60-80% of NP366-specific memory CD8 T cells in the lungs localized to the non-vascular compartment in ADJ, CpG, GLA and ADJ+GLA groups; the percentages of non-vascular memory CD8 T cells were slightly reduced in the ADJ+CpG group ( Fig. 5B) . The percentages of CD103 +ve CD69 +ve lung resident memory (TRM) cells among NP366-specific CD8 T cells were comparable for various adjuvants (Fig. 5C) . However, lungs of ADJ+GLA group contained significantly (P<0.05) greater numbers of both non-vascular and vascular CD103 +ve NP366-specific CD8 T cells, as compared to other groups (Fig. 5D) . Thus, ADJ+GLA was the most effective adjuvant that elicited high numbers of CD103 +ve TRM CD8 T cells in the airways and the non-vascular compartment of the lungs.

At 100 days after vaccination, all adjuvants induced strong CD4 T-cell memory and the percentages of NP311-specific memory CD4 T cells ranged from 1.5-4% in the lungs (Fig. 5E) .

The percentages of memory CD4 T cells in lungs of ADJ+GLA group were consistently higher than in other groups (Fig. 5E) . Regardless of adjuvants 60-80% of memory CD4 T cells localized to the non-vascular compartment in the lungs (Fig. 5F) . Likewise, the percentages (15-20%) of lung CD69 +ve TRM-like CD4 T cells were comparable for various adjuvants.

We determined whether polarization of TH1 versus TH17 was maintained in memory CD4 T cells of vaccinated mice. At 100 days after vaccination, IFN- and/or IL-17-producing NP-specific memory CD4 T cells were detectable in the lungs of vaccinated mice (Fig. 5G) . Fig. 5H illustrates that the percentages of NP-specific cytokine-producing CD4 T cells that produce IFN- and/or IL-17 differed amongst various groups. IFN--producing CD4 T cells were only dominant (~60%) in the CpG group, but IL-17-producing CD4 T cells formed the dominant subset (~75%) in the GLA and ADJ+GLA groups. About 50-60% of NP-specific memory CD4

T cells produced IL-17 in the ADJ and ADJ+CpG groups. Therefore, functional programming in effector cells is largely preserved in memory T cells.

Mice were vaccinated twice with NP protein formulated in various adjuvants. At day 100 after the booster vaccination, we investigated whether NP-specific T-cell memory protected against respiratory challenge with the virulent PR8/H1N1 influenza A virus (IAV). On the 6 th day after challenge, viral burden was high in lungs of mice that were unvaccinated or vaccinated with NP alone (without adjuvants) (Fig. 6A) . Compared to the unvaccinated and NP-only groups, other groups exhibited varying degrees of protection. The ADJ+GLA vaccine provided the most effective protection, followed by GLA and ADJ+CpG vaccines ( Fig. 6A and S5A ). Although relatively less effective, ADJ and CpG vaccines still reduced viral titers by >90%. Kinetically, at 100 days PV, viral burden was reduced in the lungs of all vaccinated mice within 2-4 days after PR8/H1N1 challenge (Fig. S5B) , but clear differences in viral control among adjuvants emerged beween days 4 and 6 postchallenge ( Fig. 6A and Fig. S5B ). Protection against IAV afforded by various adjuvant groups was durable and was maintained for at least until day 180 PV (Fig.   S5C ).

To elucidate correlates of protection afforded by various adjuvanted vaccines, we quantified recall CD8 and CD4 T-cell responses in the lungs at day 6 after PR8/H1N1 challenge.

Interestingly, despite varying levels of protection afforded by various vaccines (Fig. 6A) , the numbers and extra-vascular localization of NP366-specific CD8 T cells and NP311-specific CD4 T cells in the lungs were comparable between the groups (Fig. S5D) . The percentages of NP366specific IFN--producing CD8 T cells were also comparable for all groups of mice (Fig. 6B) . In striking contrast, percentages of NP366-specific IL-17-producing TC17 cells were considerably higher in the lungs of ADJ+GLA and GLA groups (Fig. 6B) . The percentages of NP311-specific IFN--producing CD4 T cells in the CpG and ADJ+CpG groups were significantly higher than in other groups (Fig. 6C) . In addition, lungs of GLA and ADJ+GLA mice contained higher percentages of IL-17-producing NP311-specific TH17 CD4 T cells, than in other groups. In this adjuvant system, all adjuvants afforded considerable protection. However, differences in viral control between groups appeared to associate with disparate levels of TC17 and/or TH17 cells, but not TC1 or TH1 cells. For example, better viral control by GLA and ADJ+GLA groups was associated with increased percentages of IL-17-producing NP366-specific TC17 and NP311specific TH17 cells ( Fig. 6B and 6C ). CpG and ADJ+CpG groups also differed in the percentages of NP311-specific TH17 cells but not TH1 or TC1 cells. These data suggest that stimulation of TC17/TH17 cells in parallel with TC1/TH1 cells might constitute a correlate of enhanced immunity conferred by ADJ and GLA, as compared to ADJ and CpG groups. To test this inference, we assessed the importance of IL-17A in mediating protective immunity to IAV in mice vaccinated with NP formulated in ADJ+GLA. At 180 days after vaccination, ADJ+GLAvaccinated mice were treated with isotype control antibodies or anti-IL-17A antibodies, just prior to viral challenge. Data in Fig. 6D show that treatment with anti-IL-17A antibodies did not affect the accumulation of NP366-specific CD8 T cells or NP311-specific CD4 T cells in lungs following viral challenge. In mice treated with isotype control antibodies but not in anti-IL-17Atreated mice, lung viral titers were significantly lower than in unvaccinated control mice (Fig.   6D ). These data suggested that IL-17A might have contributed to viral control in lungs in mice vaccinated with ADJ+GLA. Although IL-17 production is known to be protective against certain fungal and bacterial infections, it is also linked to immune pathology (34, 35) . In order to evaluate whether vaccine-induced protective immunity in ADJ+GLA mice was associated with lung pathology, we analyzed histopathological changes in lungs after viral challenge (Fig. S6) .

With the exception of the ADJ group, moderate necrotizing bronchiolitis was present in all mice, and was most severe in the CPG where it progressed to early-stage bronchiolitis obliterans and organizing pneumonia. Very mild extension to the surrounding alveoli was present in the GLA and AJ GLA group. Thus, we did not find any evidence of augmented lung pathology in ADJ+GLA mice following viral challenge.

Next we assessed whether NP-based adjuvanted vaccines conferred heterosubtypic immunity against a highly lethal infection with H5N1 avian influenza virus at 50 days PV. In the unvaccinated and NP-vaccinated group, 100% of mice lost significant weight and succumbed to H5N1 infection (Fig. 6E) . By contrast, 100% of ADJ+GLA and ADJ+CpG mice lost little weight and survived H5N1 challenge, while other groups showed excellent protection ranging from 70-90% (Fig. 6E) .

In order to determine whether CD4 T cells regulate the quality of CD8 T-cell memory and protective immunity induced by the ADJ+GLA vaccine, we depleted CD4 T cells, only at the time of prime and boost vaccination. At 80 days PV, we examined CD4 and CD8 T-cell memory in the RT (Fig. 7) . NP311-specific memory CD4 T cells were only detected in lungs and airways of non-depleted mice (Fig. 7A) . CD4 T-cell depletion had no adverse effect on the numbers of NP366-specific memory CD8 T cells in the RT (Fig. 7B) . Among TRM markers, only the expression of CD103, but not CD69 or CD49a was significantly reduced by CD4 T-cell depletion (Fig. 7C) . Coincident with impaired CD103 expression, memory CD8 T cells in CD4 T-cell-depleted mice poorly localized to the lung parenchyma (Fig. 7D) . Functionally, the percentages of NP366-specific IFN--producing CD8 T cells were significantly (P<0.05) increased in the lungs of CD4 T cell-depleted mice, with no effect on IL-17-producing CD8 T cells ( Fig. 7E and 7F) . In summary, loss of CD4 T cells impaired CD103 expression and extravascular localization of memory CD8 T cells but increased the percentages of NP366-specific memory TC1 cells in the lungs.

To assess whether depletion of CD4 T cells affected protective immunity, we challenged undepleted and CD4 T-cell-depleted vaccinated mice with the PR8/H1N1 virus. On the 6 th day after challenge, we assessed recall CD8 T-cell responses and viral control in the lungs. The percentages of NP366-specific CD8 T cells in lungs of CD4 T-cell-depleted mice were higher than in un-depleted mice (Fig. 7G) , and the majority of these effector cells localized to the nonvascular compartment (Fig. 7H) . NP311-specific CD4 T cells were only detected in the lungs of un-depleted mice (Fig. 7I) . CD4 T-cell depletion had no effect on the percentages of CD69 +ve CD8 T cells, but the percentages of CD49a +ve cells were significantly (P<0.05) increased in CD4 T-cell-depleted mice. A small percentage of NP366-specific CD8 T cells in the lungs of undepleted mice expressed CD103, and this fraction was significantly (P<0.05) reduced by CD4 T-cell depletion (Fig. 7J) . In the CD4 T-cell-depleted group, the percentages of CXCR3 +ve NP366-specific CD8 T cells were significantly reduced, but the percentages of NP366-specific CD8 T cells that expressed elevated levels of CX3CR1, T-bet and EOMES were higher in CD4 T-cell-depleted group than in undepleted group (Fig. 7K) . Increased accumulation of CX3CR1 Hi cells and reduced expression of CXCR3 on CD8 T cells in CD4 T-cell-depleted mice is consistent with elevated expression of T-bet (23, 36) . Significantly, the percentages of IFN-producing and granzyme B +ve CD8 T cells were higher, but there was a concurrent reduction in IL-17-producing CD8 T cells in the lungs of CD4 T-cell-depleted mice (Fig. 7L-M) . Strikingly, >90% of NP366-specific CD8 T cells produced IFN- in the CD4 T-cell-depleted group as opposed to ~56% in undepleted mice (Fig. 7N) . Undepleted mice effectively controlled viral replication in the lungs (Fig. 7O ; >99% reduction in lung viral titers). Thus, surprisingly, despite markedly increased development of IFN--producing granzyme B +ve TC1 CD8 T cells, CD4 Tcell-depleted mice showed poor control of influenza virus in the lungs (Fig. 7O) and also exhibited exaggerated weight loss (Fig. 7P) . Taken together data in In order to dissect whether impaired viral control in CD4 T cell-depleted mice was due to defective programming of CD8 T cells and/or due to loss of CD4 T cell-dependent viral control, we depleted CD4 or CD8 T cells just prior to influenza virus challenge (Fig. S7) . As shown in 

Mucosal viral infections such as influenza fail to induce durable T-cell immunity and therefore, studies of T-cell responses to IAV have failed to provide clues about how to induce durable Tcell immunity in the RT (7, 37) . Here, we report an adjuvant system comprised of a polyacrylic acid-based adjuvant ADJ and TLR agonists that elicits surprisingly potent, durable and functionally diverse mucosal T-cell immunity to disparate strains of IAV.

As a mucosal adjuvant, ADJ afforded protection against IAV in mice (21) . Here, we define ways to broaden T-cell immunity and enhance the protective efficacy of ADJ by combining with TLR4 and TLR9 agonists, GLA and CpG respectively. This adjuvant system's ability to elicit impressive numbers of antigen-specific CD8 and CD4 T cells enabled us to perform in-depth characterization of vaccine-elicited effector and memory T cells directly ex vivo, without the need for tetramer enrichment. Following IAV infection, activated CD8 T cells migrate from DLNs to the lungs, undergo another round of antigenic stimulation and differentiate into effector cells (6) . Likewise, antigen-specific CD8 T cells in all adjuvant groups experienced varying levels of TCR signaling in the lungs. Significantly, adjuvants differed in terms of the degree of effector differentiation, for both CD8 and CD4 T cells. ADJ and/or CpG-adjuvanted vaccines drove terminal differentiation into CX3CR1 HI KLRG-1 HI effector cells; as in IAV-infected mice (3, 38) , the pathway to terminal differentiation is attributed at least in part to higher TCR signaling in the lungs, leading to induction of transcription factors T-bet, IRF-4 and BATF (30, 39, 40) . Notably, high TCR signaling also induced PD-1 expression in ADJ, CpG and ADJ+CpG groups, and likely limited the accumulation of CD8 T cells in lungs. PD-1 might restrain RT inflammation (33) , but it would be worthwhile determining whether PD-1 limits vaccine-induced memory and protective immunity. It is noteworthy that despite the presence of similar numbers of antigen-bearing cells in lungs of ADJ, ADJ+CpG and ADJ+GLA mice, effector CD8 T cells in ADJ+GLA mice displayed substantially lower levels of TCR signaling in lungs. It is possible that GLA-induced TLR4 stimulation antagonized antigen-triggered TCR signaling in ADJ+GLA mice (41) . By dampening TCR signaling, GLA might have mitigated terminal differentiation of effectors and promoted the development of TRMs in ADJ+GLA mice. High levels of inflammation and IL-12 early in the response have been linked to T-bet induction and terminal differentiation of CD8 T cells in spleen (25, 27) , but the rules that govern T cell differentiation in lungs versus spleen are likely different and worthy of further exploration.

We find that ADJ enhances CD103 expression in responding CD4 and CD8 T cells. TCR signaling, IL-10 and exposure to TGF- promote CD103 expression and mucosal imprinting in T cells (3, 42) . However, we find that at 24 and 48 hours after vaccination, the levels of TGF1 or IL-10 in lungs did not explain differences in CD103 expression. ADJ promotes crosspresentation of antigen to CD8 T cells (21) and hence, ADJ-induced increase in the number of antigen-bearing cells in lungs likely enhances TCR signaling and CD103 expression on effector CD8 T cells. Interestingly, GLA inhibited TCR signaling in ADJ+GLA mice without abrogating the CD103-inducing effects of ADJ. It is possible that the residual TCR signaling in ADJ+GLA mice is sufficient to induce CD103 or other mechanisms including IFN production by CD4 T cells might have contributed to CD103 expression on CD8 T cells (43) . In summary, we infer that the magnitude of TCR signaling in lungs is a key factor that controls accumulation, mucosal imprinting and effector/memory differentiation.

A salient feature of ADJ-based adjuvants is the diverse functional programming of effector and memory T cells. For CD8 T cells, all adjuvants induced comparable levels of IL-12 and elicited a strong TC1 response. However, GLA, by virtue of its ability to induce IL-1 and IL-6, also enabled a significant TC17/TH17 response, and induction of TH17 cells by GLA is consistent with published work (44) . Importantly, from a vaccination perspective, we have discovered the means to tailor an adjuvant based on pathogen-specific correlates of protection. For example, ADJ formulated with CpG elicits strong TC1/TH1 memory, which protects against viruses and protozoan pathogens (e.g. leishmania). Alternatively, ADJ formulated with GLA stimulates balanced differentiation of TC1/TH1 and TH17 cells, which is protective against fungi, tuberculosis and other bacterial pathogens (45, 46) .

The hallmark of effective adjuvants is their ability to elicit protective immunity. Effective T-cellbased protection against IAV requires a critical number of TRMs in the airways and the lung parenchyma (3, 36) . In this study, all adjuvants elicited readily detectable CD8 and CD4 TRMs in the RT. ADJ+GLA induced the largest number of TRMs and vascular memory CD8/CD4 T cells in the lungs, which is likely a sequel to less terminal differentiation and larger clonal burst size during the effector phase (47) . TRMs are known to reside primarily in the tissue parenchyma and in the DLNs, but not as circulating cells (48) . We find that lungs of ADJ+GLA mice contained CD103 +ve memory CD8 T cells in the vasculature, which are likely similar to circulating skinresident CD103 +ve memory T cells in humans (49) . Parabiosis studies are needed to elucidate whether vascular CD103 +ve memory CD8 T cells in ADJ+GLA mice are circulating cells or lung vasculature-resident memory T cells. The numbers of memory T cells in lungs of other adjuvant groups were comparable, but the differential polarity (TH1 vs. TH17) programmed by each during the effector phase was preserved in memory T cells; CpG and ADJ+CpG displayed TH1 dominance and ADJ, GLA and ADJ+GLA showed skewed TH17 differentiation. Upon challenge with the PR8/H1N1 IAV, all vaccinated groups afforded significant protection in the lungs.

Interestingly, the extent of protection varied between the groups; ADJ+GLA provided the most effective protection, and the descending order of adjuvants in terms of protection is GLA ≥ ADJ+CpG > CpG ≥ ADJ. Upon challenge, all vaccinated groups mounted a strong recall response, and the accumulations of NP366-specific CD8 T cells and NP311-specific CD4 T cells in lungs were comparable. The percentages of IFN-producing NP366-specific CD8 T cells were similar between the groups and the percentages of IFN-producing NP311-specific CD4 T cells showed no correlation with viral control. However, interestingly, differences in viral control tend to associate with the combined percentages of IL-17-producing CD8 and CD4 T cells. Further, blocking IL-17A modestly affected IAV control in mice vaccinated with ADJ+GLA. These data are consistent with a report that TH17 cells can provide some degree of protection against IAV (50) . In addition to IL-17A, TC17/TH17 cells also produce cytokines such as IL-17F, IL-22 and GM-CSF, whose role in IAV control is unknown. We postulate that adjuvants (ADJ+GLA) that stimulate TC17/TH17 memory provide an additional layer of immune defense, that augments other mechanisms of CD8/CD4 T cell immunity, leading to enhanced protection. It is also possible that TC17/TH17 programming, and not IL-17-mediated antiviral functions per se, might be important in engendering protective immunity, because TH17 programming is associated with stem cell-like functionally plastic memory T cells (51) . It is likely that a battery of redundant mechanisms including but limited to IL-17, IFN- and MHC I/MHC II-restricted cytotoxicity orchestrate vaccine-induced protective immunity to influenza A virus (52) (53) (54) (55) .

Our investigations into the CD4 T cells' role in programming vaccinal immunity to IAV provided further insights into the mechanisms of protection in ADJ+GLA-vaccinated mice.

Depletion of CD4 T cells during vaccination precluded priming of NP311-specific CD4 T cells, but had no adverse effect on IFN-or IL-17-producing NP366-specific memory CD8 T cells in lungs. Importantly, however, CD4 T cell depletion reduced CD103 expression and the number of non-vascular CD8 TRMs in the lungs, as reported before (43) . Upon IAV challenge, despite 

Request for further information, resources and reagents should be directed to the Lead Contact:

M. Suresh (sureshm@vetmed.wisc.edu).

No unique or new materials or reagents were developed in this study. All materials or reagents used in this manuscript are available commercially or were obtained from other researchers.

All data generated in this study are presented in Figures For intracellular cytokine staining, 1x10 6 cells were stimulated for 5 hours at 37C in the presence of human recombinant IL-2 (10 U/well), and brefeldin A (1 μl/ml, GolgiPlug, BD Biosciences), with one of the following peptides: SIINFEKL, NP366 or NP311 (thinkpeptides ® , ProImmune Ltd. Oxford, UK) at 0.1ug/ml. After stimulation, cells were stained for surface markers, and then processed with Cytofix/Cytoperm kit (BD Biosciences, Franklin Lakes, NJ). To stain for transcription factors, cells were first stained for cell surface molecules, fixed, permeabilized and subsequently stained for transcription factors using the transcription factors staining kit (eBioscience). All samples were acquired on a LSRFortessa (BD Biosciences) flow cytometer.

Data were analyzed with FlowJo software (TreeStar, Ashland, OR). 

Statistical analyses were performed using GraphPad software (La Jolla, CA). All comparisons were made using an one-way ANOVA test with Tukey corrected multiple comparisons or Students t test where p<0.05 = *, p<0.005 = **, p<0.0005 = *** were considered significantly different among groups. In some experiments (Fig. 4) , we used two-way ANOVA, Students t test and simple regression analysis. In Fig. 6 , we used non-linear regression for analyzing weight loss data. Data are presented as mean ± SEM for biological replicates. Viral titers were log transformed prior to analysis. No data or outliers were excluded from analyses.

Funding: This study was supported by a PHS grant from the National Institutes of Health (grant# U01AI124299 and R21 AI149793 ) and funds from the John E. Butler Professorship to M. Suresh. Woojong Lee was supported by a pre-doctoral fellowship from the American Heart Association. DJG's contribution was supported by National Institutes of Health Training Grant (grant# T32OD010423). 

Calculated proportions of IFN- and/or IL-17-producing cells among cytokine-producing peptide-stimulated IFN-+IL-17 NP366-specific CD8 T cells. (G-P) At day 80 after booster vaccination, non-depleted and CD4 T cell-depleted mice were challenged intranasally with PR8/H1N1 influenza A virus

Percentages of NP366-specific tetramer-binding cells among CD8 T cells in lungs. (H) Percentages of NP366-specific tetramer-binding CD8 T cells in vascular and nonvascular lung compartment. (I) Percentages of NP311-specific tetramer-binding cells among CD4 T cells in lungs. (J) Expression of tissue residency markers on NP366-specific tetramer-binding CD8 T cells. (K) Chemokine receptor and transcription factor expression in NP366-specific CD8 T cells in lungs. (L) Granzyme B expression by NP366-specific CD8 T cells directly ex vivo. (M) Percentages of IFN- and IL-17 producing NP366-specific CD8 T cells. (N) Relative proportions of IFN- and/or IL-17 producing cells among total IFN- plus IL-17-producing peptide

Body weight, measured as a percentage of starting body weight prior to challenge. Data are pooled from two independent experiments. *, **, and *** indicate significance at

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We thank Dr. Jameson for providing the KLF2-GFP mice and Advanced Bioadjuvants for sproviding Adjuplex. Thanks to Amulya Suresh for peparing the graphic abstract. We wish to acknowledge sincere appreciation for the efforts of the veterinary and animal care staff at UW-Madison.

The authors declare no competing interests REAGENT