Mucosal CD8+ T cell responses induced by an MCMV based vaccine vector confer protection against influenza challenge

Cytomegalovirus (CMV) is a ubiquitous β-herpesvirus that establishes life-long latent infection in a high percentage of the population worldwide. CMV induces the strongest and most durable CD8+ T cell response known in human clinical medicine. Due to its unique properties, the virus represents a promising candidate vaccine vector for the induction of persistent cellular immunity. To take advantage of this, we constructed a recombinant murine CMV (MCMV) expressing an MHC-I restricted epitope from influenza A virus (IAV) H1N1 within the immediate early 2 (ie2) gene. Only mice that were immunized intranasally (i.n.) were capable of controlling IAV infection, despite the greater potency of the intraperitoneally (i.p.) vaccination in inducing a systemic IAV-specific CD8+ T cell response. The protective capacity of the i.n. immunization was associated with its ability to induce IAV-specific tissue-resident memory CD8+ T (CD8TRM) cells in the lungs. Our data demonstrate that the protective effect exerted by the i.n. immunization was critically mediated by antigen-specific CD8+ T cells. CD8TRM cells promoted the induction of IFNγ and chemokines that facilitate the recruitment of antigen-specific CD8+ T cells to the lungs. Overall, our results showed that locally applied MCMV vectors could induce mucosal immunity at sites of entry, providing superior immune protection against respiratory infections.


Introduction
Respiratory infections caused by influenza viruses usually are associated with mild-to-moderate disease symptoms but are linked with high morbidity and mortality in susceptible populations like the elderly, young children, patients with co-morbidities and immunocompromised patients. Influenza virus causes seasonal epidemics, with typically 3 to 5 million cases of severe illness worldwide, according to WHO reports [1], and influenza type A viruses (IAV) cause the more severe disease form. Vaccines against influenza are based on the induction of adaptive immunity that targets the projected yearly epidemics. While most vaccines are based on inactivated IAV formulations inducing anti-IAV IgG responses, live attenuated influenza vaccines (LAIV) are also used as formulations for i.n. administration. This is based on the assumption that the induction of local immunity may provide superior immune protection [2,3]. However, it remains unclear whether this protection depends on local IgA responses, on cytotoxic T cell responses, or on their combined antiviral activity. Of note, functional T cell responses were shown to substantially contribute to antiviral IAV immunity [4][5][6]. In particular, cytotoxic influenza-specific CD8 + T lymphocytes (CTLs) promote viral clearance indirectly by secretion of pro-inflammatory cytokines such as IFNγ [7] and directly by perforin/ Fas-mediated killing of infected epithelial cells in the bronchoalveolar space [8]. However, it remained unclear if T cell responses alone may control IAV, or if Ig responses were the crucial contributor to LAIV-mediated immune protection. We considered that this question could be addressed by developing a vaccine formulation that optimizes T cell responses against IAV while excluding the humoral ones.
CMV infection induces sustained functional T cell responses that are stronger in the longterm than the immune response to any other infectious pathogen [9]. Experiments in the mouse model have shown that defined CMV epitope-specific CD8 + T cells accumulate in tissues and blood and are maintained at stable high levels during mouse CMV (MCMV) latency [10]. This phenomenon was termed ''Memory Inflation" [11]. While some MCMV derived peptides, as the ones derived from the IE3 ( 416 RALEYKNL 423 ) and M139 proteins ( 419 TVYGFCLL 426 ) induce inflationary responses, other peptides, such as the M45-derived ( 985 HGIRNASFI 993 ), induce conventional CD8 + T cell response [12]. Antigen-experienced CD8 + T cells are subdivided into CD62Leffector memory (CD8T EM ) and CD62L + central memory CD8 + T cells (CD8T CM ). The antigen-specific CD8 + T cells during latent infection bear predominantly a CD8T EM phenotype and localize in secondary lymphoid or non-lymphoid organs [13]. They may provide immune protection against diverse viral targets [14][15][16][17][18], but also against bacterial [19] or tumor antigens [20,21]. The exceptionally long-lasting cellular immunity to CMV antigens has raised the interest in CMV as a potential new vaccine vector [14]. Many studies have demonstrated of optimal design of such CMV-based vaccines display efficient protection against virus infection such as rhesus macaque CMV (RhCMV) based Ebola [22] and SIV [23] vaccines and MCMV based Ebola virus vaccines [15]. Erkes, D.A. and Qiu, Z. et al. demonstrated that CMV based vaccine vectors provide protection in suppressing tumors [24,25].
Both CD8T EM and CD8T CM subsets recirculate between the blood, the lymphoid organs, and the peripheral tissues. A special subset of memory CD8 + T cells (CD8T RM ) resides in nonlymphoid tissues such as lungs, the female reproductive tract (FRT), the skin, the brain or the small intestine [26][27][28][29]. These cells lose the capacity of recirculating, maintain themselves at the site of infection, and their phenotype and transcriptional profile differ from classical memory T cells [30]. The well-characterized CD8T RM cells express C-type lectin CD69 [26] and the integrin αEβ7, also known as CD103 [30]. They provide rapid and superior protection against pathogens at the site of infection [26,30,31]. A recent publication argued that a vaccine formulation adjuvanted by IL-1β enhances the immune control of IAV by improving mucosal T cell responses [32], but IL-1β improved both humoral and cellular responses in their study. Hence, the contribution of CD8T RM to IAV immune control remains unclear.
CD8T RM are found in the salivary glands of MCMV-infected animals [33], but not in their lungs [34]. We showed that i.n. infection with MCMV induces inflationary CD8 + T cell responses, but also that memory inflation is more pronounced in relative and absolute terms upon i.p. infection [35]. The i.n. administration of an MCMV vaccine vector induced CD8T RM responses in the lungs [29] and only i.n. immunization restricted the replication of respiratory syncytial virus (RSV) upon challenge [29], indicating that CD8T RM elicited by i.n. administration of MCMV vectors might provide immune protection against respiratory virus infections, yet this evidence remains correlative. Upon antigen encounter, CD8T RM cells quickly reactivate at the mucosal site and secrete cytokines and chemokines or support the release of inflammatory mediators by other immune cells [28,36,37]. Lung airway CD8T RM cells provide protection against respiratory virus infection through IFNγ and help to recruit circulating memory CD8 + T cells to the site of infection in an IFNγ-dependent way [36]. Therefore, to understand if CD8T RM cell may provide immune control of respiratory infections may help to refine strategies for tissue-targeted vaccine design.
In this study, we constructed an MCMV vector expressing the MHC-I restricted peptide recombinant virus retained its capacity to replicate in host cells, virus replication was assessed by multi-step growth kinetics assays in NIH-3T3 cells in vitro and by ex vivo quantification of virus titers in livers, lungs and spleens 5 days post-infection (dpi) and in salivary glands 21 dpi. MCMV IVL showed identical replication properties as the MCMV WT , both in vitro ( Fig 1B) and in vivo (Fig 1C), indicating that the insertion of the IVL 533-541 epitope does not impair virus replication and spread.

Intranasal immunization with MCMV IVL induces a lower magnitude of CD8 + T cell response compared with intraperitoneal immunization
We have shown that mucosal infection with MCMV by the i.n. route induces memory inflation, although to a lower extent than upon the i.p. infection route [35]. To define if this pattern would hold true for the artificially incorporated influenza epitope as well, we compared the magnitude of the CD8 + T cell responses to MCMV IVL and MCMV WT induced via the i.n. and i.p. route, respectively. S1 Fig shows the gating strategy of flow cytometry analysis. The kinetics of antigen-specific CD8 + T cell responses in peripheral blood was determined by IVL-tetramer staining. While we did not observe striking difference at early times post immunization, i.p. immunization induced an overall higher magnitude of inflationary CD8 + T cell response during latency (Fig 2A and 2B). This pattern was observed both in relative terms (Fig 2A) and in absolute cell counts (Fig 2B). We next analyzed the IVL-specific CD8 + T cell responses in spleens, lungs and mediastinal lymph nodes (mLNs) at times of latency (>3months post infection (p.i)). Similarly, i.p. immunization induced higher levels of IVL-specific CD8 + T cells than the i.n. immunization in the spleen and lungs, both in relative (Fig 2C and 2D) and in absolute terms (Fig 2F and 2G). There were no significant differences in the mLNs (Fig 2E and  2H). In sum, mucosal (i.n.) immunization with MCMV IVL induces a systemic inflationary IVL-specific CD8 + T cell response, whereas the overall magnitude of the IAV-specific CD8 + T cell response is less pronounced compared to that induced in i.p. immunized mice.

Intranasal immunization with MCMV IVL facilitates the elimination of IAV is dependent on CD8 + T cell
To test whether immunization with MCMV IVL protects against IAV infection, latently immunized BALB/c mice were i.n. challenged with IAV. IAV titers in the lungs were quantified 5 dpi. Viral loads in mice that were immunized with MCMV WT via either the i.p. or the i.n. route were comparable to those detected in mock-immunized mice ( Fig 3A). In contrast, mice immunized with MCMV IVL via the i.n. route showed significantly lower IAV titers than in any other group. Interestingly, i.p. MCMV IVL immunization also resulted in reduced IAV loads, but to a lower extent than the i.n. immunization ( Fig 3A). Similarly, animals immunized with MCMV WT suffered the most severe weight loss whilst i.n. immunization of MCMV IVL led to the least pronounced body weight loss. I.p. immunization with MCMV IVL displayed an intermediate level ( Fig 3B). Numerous studies have reported that CD8 + T cells play an important role in protecting against influenza infection [39,40] and it was reasonable to assume that our vector provided immune protection by eliciting CD8 + T cell responses. To formally prove that efficient immune control of IAV observed in the MCMV IVL (i.n.) immunized group depends on CD8 + T cells, we depleted these cells by systemic treatment of mice with a depleting anti-CD8α antibody (depletion efficiency is shown in S2A Fig) one day prior to IAV challenge and quantified viral titers in lungs 6 dpi. While the virus titer was below the detection limit in mice that were i.n. immunized with MCMV IVL and received isotype control antibodies, CD8 + T cell depletion indeed resulted in a significant increase of the IAV titer to levels comparable to the groups that were i.p. immunized with MCMV IVL and to control mice immunized with MCMV WT by i.n. route ( Fig 3C). CD8 + T cell depletion also slightly increased virus titers in both control groups-MCMV IVL (i.p.) and MCMV WT (i.n.), but not as pronounced as in the MCMV IVL i.n. immunized group (Fig 3C). Similar to Fig 3B, animals of all experimental groups showed a comparable body weight loss post-challenge, whereas i.n. MCMV IVL immunized mice showed a faster recovery than the i.p. immunized mice (Fig 3D). Of note, this difference disappeared in the groups lacking CD8 + T cells ( Fig 3E). Together, these data demonstrate that IAV-specific CD8 + T cells induced by the mucosal (i.n.) administration of MCMV IVL confer protection against IAV in the lungs of vaccinated mice. We further compared the lung pathology upon IAV challenge by histology. A moderate perivascular inflammation was observed in the lungs of most mice (stars) and to a lesser degree surrounding the bronchioles (arrows) (Fig 3F). The CD8 + T cell depleted group showed more severe pathology than isotype-treated controls, but the difference was not very pronounced ( Fig 3G). Taken together, these data imply that intranasal immunization with the MCMV IVL vector can limit IAV growth in the lungs by inducing IAV-specific CD8 + T cell responses, whereas the clinical outcome is only moderately improved.

Intranasal immunization with MCMV IVL induces antigen-specific tissueresident memory CD8 + T (CD8T RM ) cells in the lungs
We assumed that intranasal MCMV IVL immunization may control the IAV replication by inducing CD8T RM cell responses in the lungs. In order to test this hypothesis, we identified the CD8T RM cell compartment by staining cells with the CD69 [26] and the CD103 [30] marker at times of MCMV latency (> 3 months p.i.). It is known that resident memory T cells reside in the mucosal tissue layer and are non-migratory [31]. In this study, intravenous (i.v.) injection of a fluorescent anti-CD45 antibody prior sampling allows for the discrimination of circulating leukocytes (fluorescence-positive) from emigrated or tissue-resident leukocytes (fluorescencenegative). The CD69 + CD103 + cells from lungs were virtually absent from the CD45-labelled fraction ( Fig 4A). Barely CD8T RM could be detected in the spleen and blood regardless of the route of immunization ( Fig 4B). CD8T RM (CD69 + CD103 + ) cells were solely induced in the lungs and the frequency was significantly higher when i.n. immunized with MCMV IVL than via the i.p. route ( Fig 4B). Approximately forty percent of these lung CD8T RM cells were IVLtetramer + which is significantly higher than the i.p. immunization group ( Fig 4C). Similar results were observed when gated reversibly ( Fig 4D) and when analyze these tetramer + CD8T RM cell numbers ( Fig 4E). CD8T RM cells were also induced in the group that was i.n. immunized with MCMV WT , but not IVL-specific (S3A Fig In summary, i.n. immunization with MCMV IVL induces an accumulation of IAV-specific CD8T RM response in the lungs. Moreover, antigen-specific CD8 + T cell responses induced via the mucosal route skew towards an effector memory phenotype.

Pulmonary CD8T RM cells improve viral clearance and the production of CD8 + T cell-recruiting chemokines during IAV infection
Resident memory T cells reside in the epithelial barrier of mucosal tissue [31] that is in close proximity to the airways. Hence, they reactivate rapidly to a virus challenge at the site of infection upon encountering cognate antigens [29,31]. To define the relevance of lung CD8T RM cells in protection against IAV challenge, we specifically depleted the airways CD8 + T cells by i.n. administration of αCD8 antibodies one day before challenge (Fig 5A). Upon depletion, tetramer + CD8T RM cell number reduced significantly while the circulating CD8 + T cell number did not change a lot in the lungs (S2B Fig). The local depletion did not affect the CD8 + T cell  An earlier day was chosen (4 days post-challenge) to assess whether CD8T RM cell could rapidly confer protection. The IAV titers in the lungs of i.n. MCMV IVL immunized mice were quantified. Targeted depletion of pulmonary CD8T RM cells was associated with a significantly higher viral burden during IAV infection ( Fig 5B). These data indicate that CD8T RM cells induced by i.n. immunization with MCMV IVL promote the clearance of IAV.
Influenza virus infection can induce a vigorous cytokine storm in airways and lungs, which promotes the recruitment of inflammatory cell. IFNγ as a pivotal antiviral cytokine is expressed early after influenza virus infection [41]. It has been demonstrated that CD8T RM cells activate rapidly when they re-encounter the cognate antigen and provide protection by secreting cytokines such as IFNγ and granzyme B [42,43]. Morabito et al. showed that intranasal immunization with an MCMV-based vaccine vector induced CD8T RM cells and IFNγ was secreted at the very early time upon challenge during RSV infection [29].
Therefore, we measured the production of IFNγ in the bronchoalveolar lavage fluid (BALF) early upon challenge. IFNγ levels were generally low on day 2 post-challenge and no difference could be observed between groups regardless of airway CD8 + T cells depletion (Fig 5C). On day 4, the IFNγ level was significantly increased compared to the level on day 2, but more IFNγ was induced in the control group than in the one lacking CD8T RM cell in the lungs ( Fig  5C). IFNγ was also induced in the MCMV IVL i.p. immunization group and extremely low level of IFNγ could be detected in the MCMV WT control group (S5A Fig). These data suggest that primary cognate antigen immunization is needed for the rapid IFNγ secretion and that resident CD8 + T cells may not be the major IFNγ producer. In contrast to these effects, depletion of lung airway CD8 + T cells increased the concentration of IL-6 as compared to the group that was intranasally immunized with MCMV IVL and treated with isotype control antibodies ( Fig  5D). Similarly, a higher concentration of IL-6 was also detected in the i.p. immunization group, whereas the MCMV WT control group displayed the highest IL-6 levels ( It has been demonstrated that T RM cells help to recruit immune cells to the infection site through the induction of chemokines such as CCL3 and CXCL9 in the female reproductive tract (FRT), and CCL4 in the lungs, either by direct chemokine expression or by their induction in nearby cells, such as epithelial cells [28,29].
To determine whether i.n. immunization with MCMV IVL induced inflammatory chemokines expression upon IAV challenge, a series of inflammatory chemokines were measured in the BALF on day 2 ( Fig 5E) and day 4 ( Fig 5F) upon IAV challenge. Airway depletion of CD8 + T cells reduced CCL3, CCL4, CCL5 levels on day 2. On day 4, CCL3 and CCL4 levels were significantly higher in the MCMV IVL i.n. group than in the MCMV IVL i.p. and in the MCMV WT i.n. immunization groups. Airway CD8 + T cell depletion reduced the level of CCL3 and CCL4 to values in the i.p. MCMV IVL immunization group (Fig 5F). CXCL9 levels were comparable between the MCMV IVL i.n. and i.p. immunization groups, but dramatically lower in the MCMV WT immunization group (Fig 5F), which was consistent with the low IFNγ level in the BALF, as IFNγ is known as an inducer of CXCL9, which then acts as a T cell-attracting chemokine. Together, these data indicate that CD8T RM cells induced by i.n. immunization with MCMV IVL promote the induction of the pro-inflammatory chemokines CCL3, CCL4, CCL5 and CXCL9, along with a reduction of IL-6 in the lungs.

CD8T RM cells facilitate the expansion of CD8 + T cells in the lungs
Since i.n. immunization induced stronger chemokine responses in comparison to the i.p. immunization route, we decided to define whether CD8T RM cells induced by MCMV IVL promoted the accumulation of CD8 + T cells in the lungs. We first analyzed the total IVL-specific and CD8 + T cell numbers (CD45 + plus CD45 -) in the MCMV IVL i.n. immunization group. IVL-specific and CD8 + T cell numbers increased from day 2 to day 4 post-challenge, but only in mice that were not depleted for airway CD8 + T cells (Fig 6A and 6B). Both IVL-specific and total CD8 + T cell counts increased significantly in the BALF of i.n. immunized mice by day 4 post IAV challenge (Fig 6C and 6D), However, in the mice which CD8 + T cells were depleted prior to challenge, very few IVL-tetramer + CD8 + T cells (Fig 6C, filled dots) and CD8 + T cells (Fig 6D, filled dots) could be detected in the BALF, both on day 2 and at day 4 post-challenge. These data indicate that CD8 + T cells accumulate in the lungs and migrate to the lung tissue and bronchoalveolar space upon IAV challenge. In addition, IVL-specific CD8 + T cell counts in the lung tissue and BAL were slightly higher in the MCMV IVL i.n. group than in the i.p. immunized group (S6A and S6B Fig). This differs from results prior to IAV challenge, where significantly larger amounts of IVL-specific CD8 + T cells were detected in the i.p. group ( Fig  2G).
CD8 + T cells in the lung tissue were further analyzed by in vivo labeling with anti-CD45 antibodies in the presence or absence of airway CD8 + T cells. The IVL-specific CD8 + T cell population failed to expand upon airway CD8 + T cell depletion, with significantly lower numbers in CD45subset on day 4. However, IAV-specific CD8 + T cell counts showed an increasing trend both in the CD45 + and in the CD45subsets on day 4 post-challenge (Fig 6E). Airway CD8 + T cell depletion prevented also the expansion of total CD8 + T cells counts on day 4 ( Fig 6F). Interestingly, in contrast to IVL-specific CD8 + T cells, only the CD45fraction of the total CD8 pool expanded on day 4 (Fig 6F), suggesting that bystander CD8 + T cells were also accumulated within the lungs. The depletion effects were more pronounced later upon antibody administration. Hence, direct depletion of incoming cells appears an unlikely scenario. Together with the increased chemokines in the BAL, these data indicate that the increase of IVL-specific CD8 + T cells upon challenge is not due to in situ proliferation or differentiation but most probably by recruiting CD8 + T cells from circulating system.
We also checked whether CD8T RM cells expanded upon IAV challenge that may contribute to the accumulation of CD8 + T cells. We found that the number of CD8T RM cells in the lungs did not expand from day 2 to day 4; if anything, their frequency decreased (Fig 7A and 7B). Likewise, IVL-Tetramer + CD8T RM cell counts were also slightly reduced from day 2 to day 4 post-challenge (Fig 7C), although IVL-Tetramer + CD8 + T cell counts increased at the same time ( Fig 6A). It seems that the effect of the i.n. depletion was local, since the frequencies (S6C and S6D Fig) and counts (S6E and S6F Fig) of IVL-specific CD8 + T cells in the blood and spleen did not significantly differ upon αCD8 or isotype-control antibody i.n. administration. In addition, CD4 + T cell numbers showed no difference upon IAV challenge when airway CD8 + T cells were depleted compared with the control group (S7 Fig), suggesting that CD8T RM cell do not promote CD4 + T cells trafficking into the lungs. Therefore, our data indicated that CD8T RM cells confer protection by recruiting circulating CD8 + T cells upon IAV challenge.

Discussion
Influenza-specific CD8 + T cells are known to contribute to virus elimination, as the clearance of influenza virus is delayed in T cell-deficient mice [5,44]. However, previous evidence did not clarify whether vaccines solely inducing influenza-specific CD8 + T cell responses improve immune protection. To avoid confounding humoral immune responses and focus on the potential of optimally primed CD8 + T cells in protecting against influenza, we generated a new MCMV based vaccine vector. CMV vaccine vectors expressing exogenous antigenic peptides fused to a CMV gene induce CD8 + T cell responses of unparalleled strength [14,15,21,29]. We show here that robust CD8 + T cell responses against a single MHC-I restricted epitope derived from the HA protein of IAV, promote the clearance of IAV from lungs, but only upon i.n. immunization. While some pathology was observed even in immunized mice, arguing that the protection was not complete, depletion assays confirmed that CD8 + T cells are crucial for the immune protection observed in our model. Morabito et al. showed that the volume of MCMV inoculum affects the magnitude of T cell responses [29]. Hence, a larger inoculum volume could have resulted in even stronger lung CD8 + T cell responses and protection. Remarkably, immunization with the same virus dose by the i.p. route induced even higher magnitude of CD8 + T cell responses, but conferred poor protection.
This conundrum was resolved once we noticed that only i.n. immunization induces T RM responses in the lung. CD8T RM cells act as sentinels in the host and form the first line of defense, providing rapid and effective protection to fight against pathogens invasion [27,29,31,45]. Prior studies have revealed that direct delivery of vaccines to the target tissue is necessary for the generation of T RM cells [29,46] and that sustained lung CD8T RM responses in MCMV-infected mice are generated by immunoproteasome-independent antigenic stimulation [47], akin to the CD8 expansions in memory inflation [21], arguing that their induction may share similar or overlapping mechanisms. Some prior studies have claimed that skin-resident CD8T RM cells may confer protection in an antigen-unspecific manner [48], whereas others argued that only the antigen-specific CD8T RM cells respond to cognate antigens [49]. MCMV WT induced robust CD8T RM responses in our model, but these were not specific for IAV, and did not provide any protection against IAV in our study. Site-specific anti-CD8α antibody administration depleted CD8T RM , and increased IAV titers in immunized mice, indicating that CD8T RM cells facilitated IAV elimination. Thus, the protection against IAV challenge requires antigen-specific CD8T RM cells in our model. However, the weight loss was not different in the early days upon challenge and T RM depletion affected chemokine expression and T cell influx at later time points.
Former studies have described that the inflationary MCMV-specific CD8 + T cells induced by intraperitoneal infection display an effector phenotype (KLRG1 + CD62L -CD127 -) [50,51]. Mucosal immunization of MCMV IVL affects not only the magnitude but also the quality of CD8 + T cells skewing cells towards an effector memory phenotype. Since KLRG1 is assumed a marker for terminally differentiated cells which usually are short-lived [52], the low expression of KLRG1 may contribute to a longer life-span of these cells. Additionally, recent work has demonstrated that KLRG1 -CD8 + T EM cells traffic and migrate more rapidly to non-lymphoid tissues than the KLRG1 + CD8 + T EFF cells, which mostly remain in the circulation [53,54]. Accordingly, this may give us a clue that CD8T EM cells induced by mucosal immunization may rapidly migrate to the lungs and exert immune functionality there. Nonetheless, further studies need to be done to prove this hypothesis.
It has been assumed that CD8T RM cells have poor proliferative capacity upon challenge. Previous work has demonstrated that airway CD8 + T cells fail to expand in vivo upon intratracheal transfer [36] and that CD8T RM cells induced by MCMV infection display a limited proliferative capacity in salivary glands [55]. However, this is in contrast to two recent studies demonstrating that CD8T RM cells in the skin [49] and FRT [56] maintain the capacity of in situ proliferation upon cognate antigen stimulation. Such stimulation differentiates circulating effector memory CD8 + T cells into CD8T RM cells without displacing the pre-existing CD8T RM population [49]. In our study, CD8 + T cells accumulated in the lungs upon IAV challenge, but the CD8T RM population did not expand and the number of antigen-specific CD8T RM cells even displayed a reduction trend. This appears unrelated to apoptosis, because CD8T RM cells showed less caspase3 expression than circulating CD8 + T cells upon challenge. It is possible that CD8T RM cells downregulated CD103 from the cell surface upon activation, and this intriguing question needs to be addressed in future studies. Therefore, our data argued that either lung CD8T RM in general or CD8T RM induced by MCMV i.n. immunization in particular, may behave differently from CD8T RM in other organs. This distinction, however, goes beyond the scope of our current work and remains to be addressed in future studies.
Early upon IAV challenge, not only the IVL-specific CD8 + T cells but also the bystander CD8 + T cells in the lung tissue increased significantly, indicating that the accumulation was not due to in situ proliferation but probably due to recruitment from circulating system. In addition, IAV challenge expanded IVL-specific CD8 + T cell counts in the blood and spleen of i.n. immunized mice to levels observed in the i.p. immunized controls, although the levels were significantly lower in the i.n. immunization group before IAV challenge. Overall, these results indicated that i.n. immunization facilitated CD8 + T cell responses upon challenge, both locally in the lungs and systemically in the blood and spleen. It is unclear if this apparent alarming function of T RM cells is exclusive to the lung tissue.
We have shown in this study that concentration of CCL3, CCL4 and CXCL9 in the BALF of the MCMV IVL i.n. immunization group is significantly higher than in MCMV IVL i.p. or MCMV WT i.n. immunized mice. Intravital CD45 labeling showed that CD8 + T cells accumulating in the lungs are sequestered from the bloodstream, but not CD8T RM , arguing that circulating antigen-specific cells were attracted into the lungs under the presence of mucosa-resident CD8 + T cells. This is in line with the work of Schenkel et al. showing a rapid local induction of chemokines CXCL9 and CCL3/4 in the FRT upon re-infection, and recruitment of memory CD8 + T cells from the periphery [28]. Depletion of mucosal CD8 + T cells depressed chemokine levels in the BALF to levels seen in the i.p. immunization group. This, together with the high levels of IFNγ in the MCMV IVL i.n. immunization group and extremely low IFNγ in MCMV WT i.n. immunization points to a putative model where antigen-specific re-stimulation induces IFNγ, which drives chemokine responses that recruit CD8 + T cells from the bloodstream to the lungs. We observed a surprising lower level of IL-6 upon challenge in mice that controlled influenza, because IL-6 is known as a cytokine that is involved in controlling virus infection [57][58][59][60][61]. It is not clear if IL-6 reduction was a consequence of lower virus titers or of negative regulation by T RM . McMaster et al. showed a reduced IL-6 production accompanied with lower virus titer in the appearance of lung airway CD8T RM cells [36] and this was similar to a report by Lee et al. in a clinical study in human patients [62].
In summary, our data demonstrate that CD8T RM cells promote the induction of chemokines, which help to drive the recruitment of IVL-specific CD8 + T cells and facilitates the elimination of IAV. Furthermore, the optimal induction of CD8T RM cells in the lungs by the MCMV vector can be only achieved after i.n. vaccination. Therefore, immunization with an MCMV vector at the local site provided CD8 + T cell-based protection against IAV infection. Our results, therefore, demonstrate that CD8 + T cell induction, and CD8T RM in particular, contribute to vaccination outcomes in influenza infection independently of humoral immune responses, and the selection of the adequate immunization route plays a critical role in terms for promoting superior protective efficacy.

Ethics statement
Mice were housed and handled in agreement with good animal practice as defined by EU directive EU 2010/63 and ETS 123 and the national animal welfare body ''Die Gesellschaft für Versuchstierkunde /Society of Laboratory Animals (GV-SOLAS)". Animal experiments were performed in accordance with the German animal protection law and were approved by the responsible state office (Lower Saxony State Office of Consumer Protection and Food Safety) under permit number: 33.9-42502-04-14/1709.

Mice
BALB/c mice were purchased from Janvier (Le Genest Saint Isle, France) and housed in the animal facility of the HZI Braunschweig under SPF conditions according to FELASA recommendations [63].
Recombinant MCMV was generated by the ''en passant mutagenesis", essentially as described previously [68,69]. In brief, we generated a construct containing an antibiotic resistance cassette coupled with the insertion sequence and the restriction site Sce-I. This construct was flanked by sequences homologous to the target region of insertion within the MCMV BAC genome. Then, the fragment containing the insertion sequences was integrated into the MCMV BAC genome by homologous recombination. In a second step, Sce-I was induced to linearize the BAC followed by a second round of induced homologous recombination to recircularize it and select for clones that discarded the antibiotic selection marker but retained the inserted sequence.
The PR8M variant of Influenza A/PR/8/34 was obtained from the strain collection at the Institute of Molecular Virology, Muenster, Germany. Virus stocks from chorioallantoic fluid of embryonated chicken eggs were generated as previously described [70].

Virus in vitro infection
NIH-3T3 cell monolayers were infected with MCMV WT and MCMV IVL at an MOI of 0.1, incubated at 37˚C for 1h, upon which the inoculum was removed, cells were washed with PBS, and supplied with fresh medium. Cells were incubated for 6 days; the supernatant was harvested every day and stored at -80˚C until titration.

Infectious virus quantification (MCMV)
MCMV virus from organ homogenates was titrated on MEFs with centrifugal enhancement as described previously [17].

Infectious virus quantification (IAV)
Mice were sacrificed by CO 2 inhalation, whole lungs were excised and mechanically homogenized using a tissue homogenizer. Tissue homogenates were spun down and supernatants were stored at -70˚C. Lung virus titers were determined by using the focus-forming assay (FFA), as described before [70] with minor modifications. Briefly, MDCK cells were cultured in MEM, supplemented with 10% FCS, 1% penicillin/streptomycin. Supernatants of lung tissue homogenates were serially diluted in DMEM, supplemented with 0.1% BSA and N-acetylated trypsin (NAT; 2.5 μg/ml) and added to the MDCK cell monolayers. After 1h, cells were overlaid with DMEM supplemented with 1% Avicel, 0.1% BSA and NAT (2.5 μg/ml). After 24h cells were fixed with 4% PFA and incubated with quenching solution (PBS, 0.5% Triton X-100, 20 mM Glycin). Cells were then treated with blocking buffer (PBS, 1% BSA, 0.5% Tween20). Focus forming spots were identified using primary polyclonal goat anti-H1N1 IgG (Virostat), secondary polyclonal rabbit anti-goat IgG conjugated with horseradish peroxidase (KPL) and TrueBlue™ peroxidase substrate (KPL). Viral titers were calculated as FFU per ml of lung tissue homogenate.

Isolation of lymphocytes from blood and organs
Blood, spleen and mLNs were prepared as described previously [35]. Lungs were perfused by injecting 5-10 ml PBS into the right heart ventricle. The lungs were cut into small pieces, resuspended in 1 ml RPMI1640 (0.5% FCS), and digested with 1 ml of RPMI1640 with DNase I (Sigma-Aldrich Chemie) and Collagenase I (ROCKLAND™) in a shaker at 37˚C for 30 min. Digested tissue was passed through cell strainers and single cell suspensions were washed with RPMI1640, centrifuged at 500x g for 10 min. Subsequently, the cells were resuspended in 7 ml of 40% Easycoll solution (Biochrom), overlayed onto 6 ml of 70% Easycoll solution in a 15 ml Falcon and centrifuged at 25 min at 1000x g at room temperature. The interface layer was transferred to a 5 ml tube, washed, and resuspended in RPMI1640 (10% FCS).

Peptide stimulation
T cells were stimulated with peptides (1 μg/ml) in 85 μl RPMI 1640 for 1h at 37˚C, supplemented with brefeldin A (10 μg/ml in 15 μl RPMI 1640) and incubated for additional 5h at 37˚C. Cells incubated without any peptide in the same condition were used as negative controls. Cytokine responses were detected by intracellular cytokine staining.

Cell surface staining, intracellular cytokine staining for flow cytometry
Blood cells and lymphocytes from spleen, lung and mLNs were stained with IVL 533-541 -tetramer-PE and surface antibodies for 30 min, washed with FACS buffer and analyzed. For intracellular cytokine stainings, the cells were first stained with cell surface antibodies for 30 min, washed and fixed with 100 μl IC fixation buffer (eBioscience) for 5 min at 4˚C. Following this, cells were permeabilized for 3 min with 100 μl permeabilization buffer (eBioscience) and incubated with anti-IFNγ antibody for 30 min. Afterwards, cells were washed with FACS buffer and acquired using an LSR-Fortessa flow cytometer (BD Bioscience).

Collection of bronchoalveolar lavage fluid (BALF)
Mice were sacrificed by CO 2 inhalation, the chest cavity was opened and skin and muscle around the neck were gently removed to expose the trachea. A catheter was inserted and the lungs were carefully flushed with 1 mL PBS via the trachea. The BALF was transferred into a 1.5 ml tube and stored on ice. The BALF was centrifuged at 500x g at 4˚C for 10 min. The supernatant was aliquoted and stored at -80˚C until further analysis.

Cytokine and chemokine quantification
Mouse IFNγ enzyme-linked immunosorbent assay (ELISA) MAX™ kits (BioLegend) and the bead-based immunoassay LEGENDplex™ Mouse Inflammation Panel (13-plex, BioLegend) were used to quantify IFNγ and other cytokine levels in the BALF according to the manufacturer's instructions. The bead-based immunoassay LEGENDplex Mouse Pro-inflammation Chemokine Panel (13-plex, BioLegend) was used to quantify multiple chemokine levels in the BALF.

Histopathology
Lungs were harvested from BALB/c mice that were latently infected with MCMV WT and MCMV IVL and challenged with IAV during latency. Lungs were fixed in 4% formalin, paraffin embedded, sliced and hematoxylin and eosin (H&E) stained according to standard laboratory procedures.

Statistics
One-way ANOVA analysis was used to compare multiple groups at single time points. Twoway ANOVA analysis was used to compare different groups at multiple time points. Comparisons between two groups were performed using Mann-Whitney U test (two-tailed). Statistical analysis was performed using GraphPad Prism 7. BALB/c mice were immunized with 2 x 10 5 PFU MCMV IVL via the i.p. or i.n. route. During latency (> 3 months p.i), anti-CD45 antibodies were injected intravenously 3-5 min before mice euthanasia. Leukocytes from blood, spleen and lungs were stained with cell surface markers CD3, CD4, CD8, CD11a, KLRG1, CD62L, IVL-tetramer and analyzed by flow cytometry. T EFF cells are defined as KLRG1 + CD62L -, T EM as KLRG1 -CD62Land T CM as KLRG1 -CD62L + . (A) The percentages of each phenotype subset among CD45tetramer + CD8 + T cells in the lungs and spleen. (B) The percentages of each phenotype subset among CD45 + tetramer + CD8 + T cells and tetramer + CD8T RM cells in the lungs, spleen and blood. (C) The percentages of each phenotype subset among tetramer + CD8T RM cells in the lungs. Two independent experiments were performed and pooled data are shown, n = 5. Each symbol represents one mouse. Group means +/-SEM are shown. Significance was assessed by One-way ANOVA and Two-way ANOVA test. During latency (> 3 months p.i), MCMV IVL (i.n.) immunized mice were administered with 10 μg αCD8 or 10 μg IgG2b antibody (i.n.). MCMV IVL (i.p.) and MCMV WT (i.n.) immunized mice were administered with 10 μg IgG2b antibody (i.n.). One day later, animals were challenged with IAV (PR8) (i.n., 1100 FFU). On day 2 and day 4 post-challenge, BALF was harvested and measured cytokines production by bio-plexing. The concentration of (A) IFNγ and (B) IL-6 in the BALF on day 4 post-challenge. Two independent experiments were performed and pooled data are shown. Each symbol represents one mouse, n = 5-7. Group means +/-SEM are shown. (C) Cytokine concentrations in the BALF in different immunization group on day 2 and day 4 post-challenge. Bars indicate means, error bars are SEM. Two independent experiments were performed and pooled data are shown. Each symbol represents one mouse, n = 5-7. Significance was assessed by One-way ANOVA test. � P <0.05, ��� P <0.001.