Long-term surviving influenza infected cells evade CD8+ T cell mediated clearance

Influenza A virus (IAV) is a seasonal pathogen with the potential to cause devastating pandemics. IAV infects multiple epithelial cell subsets in the respiratory tract, eliciting damage to the lungs. Clearance of IAV is primarily dependent on CD8+ T cells, which must balance control of the infection with immunopathology. Using a virus expressing Cre recombinase to permanently label infected cells in a Cre-inducible reporter mouse, we previously discovered infected club cells that survive both lytic virus replication and CD8+ T cell-mediated clearance. In this study, we demonstrate that ciliated epithelial cells, type I and type II alveolar cells can also become survivor cells. Survivor cells are stable in the lung long-term and demonstrate enhanced proliferation compared to uninfected cells. When we investigated how survivor cells evade CD8+ T cell killing we observed that survivor cells upregulated the inhibitory ligand PD-L1, but survivor cells did not use PD-L1 to evade CD8+ T cell killing. Instead our data suggest that survivor cells are not inherently resistant to CD8+ T cell killing, but instead no longer present IAV antigen and cannot be detected by CD8+ T cells. Finally, we evaluate the failure of CD8+ T cells to kill these previously infected cells. This work demonstrates that additional cell types can survive IAV infection and that these cells robustly proliferate and are stable long term. By sparing previously infected cells, the adaptive immune system may be minimizing pathology associated with IAV infection.

Influenza A virus (IAV) is a seasonal pathogen with the potential to cause devastating pandemics. IAV infects multiple epithelial cell subsets in the respiratory tract, eliciting damage to the lungs. Clearance of IAV is primarily dependent on CD8 + T cells, which must balance control of the infection with immunopathology. Using a virus expressing Cre recombinase to permanently label infected cells in a Cre-inducible reporter mouse, we previously discovered infected club cells that survive both lytic virus replication and CD8 + T cell-mediated clearance. In this study, we demonstrate that ciliated epithelial cells, type I and type II alveolar cells can also become survivor cells. Survivor cells are stable in the lung long-term and demonstrate enhanced proliferation compared to uninfected cells. When we investigated how survivor cells evade CD8 + T cell killing we observed that survivor cells upregulated the inhibitory ligand PD-L1, but survivor cells did not use PD-L1 to evade CD8 + T cell killing. Instead our data suggest that survivor cells are not inherently resistant to CD8 + T cell killing, but instead no longer present IAV antigen and cannot be detected by CD8 + T cells. Finally, we evaluate the failure of CD8 + T cells to kill these previously infected cells. This work demonstrates that additional cell types can survive IAV infection and that these cells robustly proliferate and are stable long term. By sparing previously infected cells, the adaptive immune system may be minimizing pathology associated with IAV infection.

Author summary
Influenza A virus is a seasonal respiratory pathogen that can cause severe lung damage and death. We previously made the discovery that cells infected with influenza virus do not have a death sentence. An infected cell can survive both influenza virus infection and the immune response to eliminate the virus, specifically CD8 + T cells which are required for virus clearance. Here, we investigated how an infected cell could survive the CD8 + T cell immune response. We used an influenza virus expressing a recombinant protein that a1111111111 a1111111111 a1111111111 a1111111111 a1111111111

Introduction
Influenza A virus (IAV) is a negative sense segmented RNA virus causing significant morbidity and mortality annually. IAV has a broad tropism in the respiratory tract infecting club cells, ciliated cells, type I and type II alveolar cells among others [1]. IAV drives destruction of infected cells directly through lytic virus replication and indirectly from the innate and adaptive immune response's efforts to eliminate the infection. However, we and others, have previously shown IAV infected pulmonary epithelial cells can survive acute IAV infection [2][3][4]. Survivor cells express a robust interferon simulated gene (ISG) signature, facilitating a transient non-specific antiviral environment in the lung, and protecting against secondary viral infection [2,4]. Despite the extensive damage caused by viral replication and antiviral immune responses, the lung is able to recover through significant remodeling and repair. While the turnover of pulmonary epithelial cells in the steady state is slow, after injury epithelial cells undergo proliferation to prevent vascular leakage and differentiation to restore lung function [5]. In addition, rare pulmonary progenitor cells are also critical for the repair of the upper and lower airway [6][7][8][9].
CD8 + T cells are critical for the control of acute IAV infections. CD8 + T cells use perforin and granzyme as well as FasL and TRAIL to specifically kill IAV infected cells [10][11][12]. There are several mechanisms by which cells can evade CD8 + T cell-mediated control. Cells can downregulate MHC-I, a strategy employed by quiescent stem cells [13], tumor cells, and cells infected with some viruses [14]. The inhibitory ligand, PD-L1, can bind to PD-1 on CD8 + T cells and inhibit T cell effector functions. During IAV infection epithelial cells upregulate PD-L1 resulting in decreased IAV titers [15,16]. CD8 + T cells can also control virus infected cells without destroying the cell. After hepatitis B virus infection, CD8 + T cells secrete IFN-γ and TNF-α to stop viral replication and cure infected hepatocytes [17,18]. It is unclear if survivor epithelial cells are inherently resistant to cytolysis, downregulate MHC-I, or if non-cytolytic control of IAV facilitates their survival and evasion from CD8 + T cell-mediated elimination.
In this study we demonstrate that in addition to club cells, ciliated epithelial cells, type I and type II alveolar cells can survive IAV infection. Surviving cells undergo enhanced proliferation compared to uninfected cells after IAV clearance and survivor cells are stable in the lungs until at least 99 days post infection. When we investigated how the adaptive immune system fails to kill survivor cells, we discovered that survivor cells are not inherently resistant to CD8 + T cell killing, do not employ active mechanisms to dampen the response and demonstrate that they lack IAV antigen. These results suggest that survivor cells rapidly clear the infection to evade killing from the adaptive immune system. This study sheds new light on the biology of cells that survive acute IAV infection.

Multiple epithelial cell types survive acute IAV infection
We generated a H1N1 strain (A/Puerto Rico/8/1934) of IAV expressing Cre recombinase (Cre) (IAV_Cre) downstream of the PA gene. IAV_Cre showed similar replication kinetics to the wt PR8 virus (S1A and S1B Fig) and was cleared between 10 and 12 days post infection (dpi) (S1C Fig). We had previously determined that club cells were the primary cell type surviving IAV infection [2]. However, digestion, processing, sorting and lysing of lung epithelial cells for RNA-seq could bias cell types detected. To determine if a different digestion method could result in a greater breadth of lung epithelial cells recovered, we compared two distinct digestion methods for flow cytometry analysis [2,19]. Transgenic mice containing a Cre-inducible fluorescent reporter protein were infected with IAV_Cre. With a dispase-based lung digestion, 6-fold more epithelial cells and 10-fold more reporter + epithelial cells were recovered from the lung (S1D and S1E Fig). Therefore, we used this digestion method for all experiments analyzing lung epithelial cells by flow cytometry.
We next determined the presence of reporter + cells by microscopy and flow cytometry. Reporter + lung epithelial cells were observed by flow cytometry at multiple time points after IAV_Cre infection (Fig 1A and S1F Fig). There were large numbers of reporter + epithelial cells early after infection (4-8 dpi), which decreased by 12 dpi, likely to due to lytic IAV replication and CD8 + T cell mediated killing ( Fig 1B). The number of reporter + epithelial cells remained stable after clearance, and long-term survivor cells could be detected as late as 99 dpi ( Fig 1B). We observed similar trends for reporter + cells detected by microscopy (Fig 1C and 1D). Reporter + cells were observed both in the large airways and in the lung parenchyma (Fig 1C). At later time points (15-30 dpi) reporter + cells were frequently observed in clusters with other reporter + cells. The percentage and number of reporter + cells with a reporter + neighbor cell increased from 7 to 30 dpi (Fig 1E and S1G-S1H Fig). As the expression of the fluorescent reporter is a permanent heritable change in the DNA, a reporter + cell with a reporter + neighbor may be the result of a parental infected cell undergoing division.
Surviving cells could have been infected during the first wave of replication or later after induction of antiviral immune responses, or a combination thereof. To determine if survivor cells could be generated late during IAV infection we infected Cre-inducible reporter mice with wt PR8, then infected with IAV_Cre at 5 or 7 dpi and quantified reporter + epithelial cells on 10 dpi. We observed detectable levels of reporter + cells when IAV_Cre was delivered at 5 or 7 dpi (Fig 1F), demonstrating that survivor cells can be generated late during the acute phase of IAV infection, after the induction of antiviral immune responses.
We next assessed if other cell types are able to survive IAV infection and evade CD8 + T cell mediated killing, or if survival is a property specific to club cells, as suggested by our previous study [2]. We stained for CD24 (ciliated cells), and podoplanin (type I alveolar cells), to assess cells by flow cytometry (S2 Fig). Total ciliated cells and type I alveolar cells are relatively stable after IAV infection (S2B,E). We observed reporter + cells expressing CD24 or podoplanin at multiple timepoints ( Together these data demonstrate that in addition to club cells diverse pulmonary epithelial cells can survive acute IAV infection and CD8 + T cell-mediated killing.

Survivor cells proliferate after IAV clearance
We observed that the number of survivor cells increased with time (Fig 1B), and are present in clusters after viral clearance (Fig 1E), indicative of cell division. To assess if the survivor cells After IAV clearance the level of BrdU incorporation increased in both wt PR8 and IAV_Cre infected mice when compared to naïve epithelial cells ( Fig 3B). Interestingly, a greater percentage of survivor cells had incorporated BrdU than total lung epithelial cells at both 21 and 27 dpi. These data demonstrate that survivor cells are proliferating after IAV clearance, and are doing so at a higher rate than bystander cells after clearance. These data may explain the longterm stability of the surviving cell populations.

Cellular immune responses do not impact the types of surviving cells
To determine if the immune system exerts bias in the cell subsets that survive IAV infection, we assessed which cells are infected and survive in a primary mouse epithelial cell culture devoid of innate and adaptive immune cells. Tracheal epithelial cells from reporter mice were differentiated at air liquid interface, and infected with IAV_Cre and collected at 1 and 8 dpi, to quantity infected and survivor cell, respectively. In this system, IAV is cleared by 8 dpi (S5D Fig). Using confocal microscopy we demonstrated that the cell types that were initially infected went on the become survivor cells at similar ratios (S5A-S5C Fig, Fig 4A and 4B). These data suggest that in the absence of immune cell killing, a broad array of cell types can survive acute lytic replication.
To determine if these results were consistent in an in vivo model, we assessed reporter + cells in mice treated with CD4 and CD8α depletion antibodies or control antibody. These data demonstrated that while the CD8 + T cell depletion increased the overall number of reporter + Together these data suggest that CD8 + T cells do not fail to survey and kill specific cell types.

Survivor cells do not evade CD8 + T cell killing via PD-L1:PD-1 interactions
To understand how survivor cells circumvent destruction by IAV-specific CD8 + T cells, we assessed our RNA-seq data for molecules known to protect cells from T cell killing [2]. We observed an upregulation of CD247 (PD-L1) mRNA in reporter + cells at 5 dpi (S6A Fig) [2]. PD-L1 is an inhibitory ligand for PD-1, expressed on CD8 + T cells, which suppresses CD8 + T cell signaling and killing of target cells [20]. We confirmed increased surface protein expression of PD-L1 on reporter + cells in the lung on 10 and 13 dpi during CD8 + T cell-mediated clearance ( Fig 5A). Additionally, IAV-specific CD8 + T cells in the lung expressed high levels of PD-1 ( Fig  5B). To determine if survivor cells use PD-L1 to inhibit CD8 + T cell-mediated killing, we treated IAV infected mice with PD-L1 blocking antibody. We chose to block PD-L1 instead of PD-1, to avoid potential interactions between PD-1 and PD-L2, which is expressed on cells other than pulmonary epithelial cells [20]. PD-L1 blocking antibody was given on 2, 5 and 8 dpi and

Survivor cells are not inherently resistant to CD8 + T cell killing
Viruses can suppress the surface expression of MHC-I to evade CD8 + T cell-mediated clearance. We assessed the mRNA expression of MHC-I (β2M) in lung reporterand reporter + epithelial cells after IAV_Cre infection. Greater b2m mRNA levels were observed on reporter + cells, compared to reporterepithelial cells (S6D Fig) [2]. MHC-I (H-2K b ) was also upregulated on the surface of reporter + cells, when compared to total epithelial cells after infection ( Fig  6A). It is possible that survivor cells clear IAV infection and no longer display IAV peptide-MHC-I, thereby rendering them invisible from IAV-specific CD8 + T cells. It is also possible that survivor cells are inherently resistant to cytolysis.
To assess whether antigen-specific CD8 + T cells can kill survivor cells we used a system where IAV_Cre permanently induces a CD8 + T cell antigen. To accomplish this, we utilized the just eGFP death-inducing (JEDI) CD8 + T cell system, and Cre-inducible eGFP-expressing mice [13,21,22]. JEDI T cells recognize eGFP on MHC-I (H-2K d ), and specifically deplete eGFP expressing cells [13,21]. To use JEDI T cells in our system we crossed B10.D2 (H-2K d ) mice to Cre-inducible eGFP expressing mice [21,22]. F1 mice express H-2K d in all cells and will express eGFP in cells infected with IAV_Cre. Reporter + cells will express eGFP even after IAV is cleared, so we can determine if the survivor cells are resistant to cytolysis or lack viral antigen. H-2K d reporter mice were infected with IAV_Cre, and JEDI T cells were transferred and stimulated with Adenovirus expressing eGFP (AD_eGFP) and eGFP 200-208 peptide. Survivor cells were then assessed at 10 dpi (S6E Fig). As a control, AD_eGFP was administered to mice and eGFP expression in the lung was restricted to the endothelial (CD31 + ) compartment and was absent from epithelial cells (S6F Fig). Therefore, AD_eGFP priming will not induce CD8 + T cell-mediated killing of pulmonary epithelial cells. While the addition of JEDI T cells did not alter the number of reportercells in the lung there was a significant decrease in reporter + cells (Fig 6B and 6C). These data demonstrate that survivor cells are susceptible to killing by antigen-specific CD8 + T cells, and that survivor cells are not inherently resistant to cytolysis.
We previously demonstrated that survivor cells do not have infectious virus by 10 dpi [2]. However, antigen can have a long half-life and these cells may still present IAV peptide. To determine if survivor cells lack IAV-antigen, we performed an in vitro proliferation assay. We generated a new reporter virus containing the GP33 epitope from lymphocytic choriomeningitis virus glycoprotein. The new reporter virus infects epithelial cells to a similar level as the IAV_Cre, and induces a robust GP33-specific CD8 + T cell response (S6G and S6H Fig). By incubating in vitro activated GP33-specific P14 TCR-Tg CD8 + T cells with sorted lung CD45 -CD31reporter + cells from IAV_Cre/NA_GP33 infected mice, we can directly assess if the epithelial cells are presenting antigen. P14 CD8 + T cells mixed with 4 dpi reporter + cells undergo division, indicating these epithelial cells express IAV antigen (S6I Fig). In contrast, 10 dpi

Discussion
In this study, we sought to elucidate how epithelial cells that survive the lytic phase of IAV infection are able to avoid CD8 + T cell mediated killing. We observed that multiple epithelial cell types could survive IAV infection, suggesting that survival is not a cell specific mechanism, and that CD8 + T cells are able to survey all areas of the lung. Survivor cells upregulate PD-L1, but do not use PD-L1 to suppress CD8 + T cell killing. Survivor cells are not inherently resistant to CD8 + T cell killing, do upregulate MHC-I, and no longer express IAV antigen by the time they are under CD8 + T cell surveillance.
In our original study, we identified club cells as the primary lung epithelial cell type that survived IAV infection. By incorporating fluorescent microscopy and using an alternative lung digestion method for flow cytometry analysis, we determined that multiple distinct cell types could survive IAV infection. We observed that in addition to club cells, ciliated cells, type I and type II alveolar cells can become survivor cells. We found increased numbers of ciliated epithelial cells and type I alveolar cells long-term compared to acute infection. However, it is difficult to determine if increased presence of these cell types represents a bias in the cells that can survive, increased proliferation, or preferential differentiation into these cell types. In a similar system, Schwemmle and colleagues also observed type I and type II survivor cells [3]. These cells are widespread throughout the lung, requiring CD8 + T cells to survey not only a broad array of cells types, but anatomical locations.
The indelible Cre-inducible reporter system allowed us to follow survivor cells out to 99 dpi, demonstrating the longevity and stability of survivor cells. This may be partially due to the slow turnover of many lung epithelial cells [5] and/or capacity to proliferate. As transient antiviral immunity is mediated by surviving club cells [2,4], we propose that this immunity is lost as survivor cells proliferate and daughter cells lose the ISG expression profile that was acquired during infection. IFN signaling can induce epigenetic changes [23], but it is unclear if these epigenetic changes are heritable in lung epithelial cells.
One interesting finding of our study is increased proliferation of survivor cells compared to bystander cells within the same lung at multiple different time points. Although the benefit of this proliferation is unclear, one potential advantage of increased survivor cell division is that it could aid in the repopulation of the respiratory epithelium. Proliferation of phenotypically altered survivor cells could also alter the pulmonary cytokine and chemokine environment and modulate susceptibility to secondary viral infection [4]. Survivor cells may also proliferate in order to enhance the repair of DNA damage that occurs during viral infection. Some DNA damage repair pathways only function during cell proliferation and DNA repair has been recently demonstrated to help aid in infected cell survival [24]. IAV uses several distinct mechanisms to disrupt cell cycle progression and cell division has been shown to decrease IAV replication [25][26][27]. Survivor cells could initiate cell division as an antiviral mechanism to blunt virus replication and to dilute antigen to prevent killing by the adaptive immune system. Interestingly, we have previously found that cells infected early have increased activation of genes involved in cell cycle [19].
The cellular events that occur for a survivor cell to be generated are still unclear. Survivor cells can be generated early during infection as well as later during the acute phase after type I and III IFN responses have been generated. As small amounts of Cre recombinase are needed to express the fluorescent reporter, infected cells undergoing abortive replication may turn on the reporter. However, these cells are still impacted by IAV replication as demonstrated by altered ISG profiles [2,4]. These cells could still express IAV antigen and be targeted by CD8 + T cells.
The efforts of the immune system to control IAV drive significant pathology within the lung [28,29]. Eliminating the virus without killing infected cells could be one way of ameliorating this affect. CD8 + T cells have been shown to control hepatitis B virus, sindbis virus, lymphocytic choriomeningitis virus, and simian immunodeficiency virus in the absence of cell lysis [17,18,[30][31][32][33]. Another method to curb pathogenic T cell responses in the lungs is though engagement of inhibitory ligands. Airway epithelial cells increased the expression of PD-L1 after IAV infection [15,16]. PD-L1 blockade enhanced CD8 + T cell-mediated clearance of infected cells [16], increased CD8 + T cell numbers and reduced IAV titers [15]. Similar results were observed in PD-1 KO mice during acute IAV infection [34]. Therefore, we hypothesized that this axis may be responsible for protecting surviving cells. However, PD-L1 blockade or systemic blockade of PD-L1 and PD-1 did not result in increased frequency or number of surviving cells. This could be a result of redundancy in inhibitory ligand mediated suppression as others have demonstrated [35,36]. While inhibitory ligands clearly play a role in IAV clearance and pathogenesis they do not appear to be involved in promoting survival of previously infected cells.
Our data demonstrate that survivor cells are not inherently resistant to CD8 + T cell killing. We propose that survivor cells no longer express IAV antigen, thereby rendering the survivor cell invisible to an antigen-specific CD8 + T cell. This is consistent with our in vitro proliferation assay where only reporter + cells from 4, and not 10, dpi could activate IAV antigen-specific CD8 + T cells. There are several mechanisms that could drive this. Cell division could dilute the amount of antigen between two daughter cells, survivor cells could have cleared IAV protein/antigen, and/or survivor cells could have had an abortive infection. Interestingly, while in the presence of JEDI T cells and constitutive antigen there was a substantial decrease in the number of survivor cells, but not all were eliminated. These remaining cells could evade CD8 + T cell cytolysis through loss of antigen processing and presentation pathways and/or multiple redundant inhibitory mechanisms.
Together the data presented herein demonstrate that a broad array of epithelial cells are capable of surviving acute lytic virus replication. As these cells are not resistant to killing, these epithelial cells must rapidly clear the infection to evade CD8 + T cell mediated cytolysis. This antiviral strategy may be critical for preventing immunopathology.

IAV infection of mice, BrdU Treatment and antibody depletion and blockade
For IAV infection, mice were anesthetized using a weight-based dose of ketamine/xylazine delivered intraperitoneally (i.p.). Mice were infected intranasally with 40 plaque forming units (PFUs) of PR8, 100 PFU of IAV_Cre, 500 PFU of IAV_Cre/NA_GP33 or 40 PFU IAV_-NA_GP33. For sequential infections, mice were infected with 40 PFU of PR8 or 100 PFU of IAV_Cre. PR8 infected mice were infected on 5 or 7 dpi with 100,000 PFU of IAV_Cre. During infection, all mice having weight loss exceeding 25% of their starting weight were sacrificed. For cell proliferation experiments, mice were injected i.p. with 1 mg BrdU (Sigma) once, and given BrdU in drinking water at 0.8 mg/mL for 6 days prior to euthanasia. At indicated dpi lungs were harvested and lung cells were processed and surface staining was performed. Cells were fixed and permeabilized with the BD Cyotfix/Cytoperm kit as per the manufacturer's instructions, incubated with 1 mg/mL DNase I (Sigma-Aldrich) for 60 min at 37˚C washed and stained with anti-BrdU (clone MoBU-1) (Life Technologies). For CD4 and CD8 depletion experiments, IAV infected mice were treated on 2, 5, and 8 dpi with 100 μg of control IgG (SF128) or 100 μg of anti-CD4 (GK1.1) and 100 μg of anti-CD8 (2.43). For PD-L1 blockade experiments, IAV infected mice were treated on 2, 5 and 8 dpi with 250 μg of control IgG (SF128), anti-PD-L1 (M1H6) antibody, or anti-PD-L1 and PD-1 (J43) delivered i.p. [37].

JEDI Experiments
B10.D2 mice were lethally irradiated (1100 rad) and were injected with 5 x 10 6 bone marrow cells isolated from JEDI mice. At least 8 weeks later, spleen and lymph nodes (cranial, axillary, brachial, inguinal and mesenteric) were harvested, RBCs were lysed and CD8 + T cells were negatively selected using the mouse CD8 + T cell isolation kit from Stem Cell Technologies following the manufacturer's instructions. 1 x 10 6 -5 x 10 6

Isolation of epithelial cells from the lung
Mice were euthanized and lungs were inflated via intratrachial injection with 2 mL dispase (Corning) and 0.5 mL 1% low melt agarose (Lonza) and were incubated under an ice pack for 2 minutes. Lungs were removed, transferred to 1 mL dispase and incubated at room temperature for 45 minutes. Lungs were minced into small pieces, and transferred to DMEM with DNaseI (Sigma-Aldrich) 95 U/L and rocked for 10 minutes at room temperature. Lungs were homogenized in a GentleMACS dissociator, filtered through 70 μm filter mesh to generate a single cell suspension, RBCs were lysed and cells were stained for flow cytometry as described above.

Isolation of lymphocytes from the lung
Mice were euthanized and lungs were harvested, minced into small pieces and washed twice with harvest buffer (ice-cold RPMI 1640 supplemented with 5% bovine serum, 4 mM L-glutamate and 10 mM HEPES). Lungs were incubated in a solution of RPMI 1640/ 10% bovine serum/ 2 mM MgCl 2 / 2mM CaCl 2 / 10 mM HEPES/ 4 mM L-glutamate medium containing 100 U/mL of collagenase type I (Worthington) for 45 min at 37˚C. Lung pieces were then incubated in a solution of RPMI 1640/ 10% bovine serum/ 10 mM HEPES/ 4 mM L-glutamate medium containing 1.3 mM EDTA (Calbiochem) for 45 min at 37˚C. Single cell suspensions of all tissues were generated using a Gentle MACS dissociator and cells were passed through 70 μm filter mesh, RBC lysed, and stained for flow cytometry as described above.

In vitro proliferation assay
Spleen and lymph nodes (cranial, axillary, brachial, inguinal and mesenteric) were harvested from a CD45.1 P14 TCR-Tg mouse. RBCs were lysed and CD8 + T cells were negatively selected using the mouse CD8 + T cell isolation kit from Stem Cell Technologies following the manufacturer's instructions. 1 x 10 6 P14 T cells were placed in 24 well plates pre-treated with anti-CD3 and anti-CD28 (10 μg/mL of each). P14 T cells were incubated at 37˚C for 48 hours in the presence of 0.0025 μg/mL IL-12 (R&D Systems). After 48 hours, cells were washed and plated at 5 x 10 5 cells/mL in the presence of 10 U/mL recombinant human IL-2 for 24 hours. P14 T cells were labeled with CellTrace Violet (CTV; Invitrogen) per the manufacturer's instructions. P14s were mixed at a 10:1 ratio with sorted CD45 -CD31reporter + cells from the lungs of B6.129(Cg)-Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo /J mice infected with IAV_Cre/ NA_GP33 4 or 10 days prior. After 48 hours, cells were stained for flow cytometry. Cell cultures were maintained in RPMI 1640 supplemented with 10% FCS. 4mM L-glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 U/mL penicillin and streptomycin and 10 mM HEPES.

Plasmid design & virus rescue
In-Fusion primers (Takara Bio, Inc.) were designed for insertion of a PA2A site followed by Cre and the complete 5' vRNA packaging signal (184 nts) into PA of influenza A/Puerto Rico/ 8/1934 (PR8) [2,38]. PA_Cre was then cloned into pDZ rescue system via In-Fusion HD cloning. NA_GP33 was inserted into the stalk of NA at a position amenable to insertion via infusion cloning as described [39,40]. All viruses were rescued via HEK293T transfection and amplified in embryonated chicken eggs as previously described [41]. Rescued viruses were sequence confirmed and titered on Madin-Darby canine kidney (MDCK) cells (ATCC).

Tissue preparation and microscopy
B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J mice previously infected with PR8 or IAV_Cre were euthanized at indicated dpi. Lungs were harvested and fixed in 2% PFA for two hours, immersed overnight in 30% sucrose, inflated and flash frozen in optimum cutting temperature compound (O.C.T.). 7 μm sections were cut from each block with a Leica CM 1860 cryostat and stained prior to imaging on a Leica DM6000B EPI fluorescent microscope (violet LED). Histo-cytometry was performed using Imaris (Bitplane) by creating surfaces for the reporter + cells (tdTomato) and markers (SPC or CC10), and performing a distance transformation to detect double positive cells. Reporter + cells were manually counted in ImageJ. Neighbor cells were defined as two or more reporter + DAPI + cells within the proximity of one DAPI-stained nucleus to one another. Abs used were: CC10 (polyclonal) (Abcam), proSP-C (polyclonal) (Millipore Sigma), goat anti-rabbit (polyclonal) (ThermoFisher Scientific).

Primary mouse pulmonary epithelial cell cultures
Tracheal epithelial cells were collected from the lungs of Cre-inducible reporter mice and plated on 4 μm pore transwell membranes (Corning). Cultures were grown in mTec + for 2 days of proliferation then switched to mTec-serum free media for differentiation. After 21 days of differentiation, the pseudostratified cultures were infected with IAV_Cre then harvested at 1 dpi (to measure actively infected cells) and 8 dpi (to measure survivor cells after the clearance of viral infection). These cultures were then fixed with 2% PFA and antigen retrieval was performed in a citrate buffer. Samples were then stained with antibodies for active infection (HA, PY102), basal stem cells (Krt5, Biolegend, clone Poly19055, cat 905501), ciliated cells (FoxJ1, eBioscience, clone 2A5, cat 14-9965), secretory cells (SSEA-1, Biolegend, clone MC-480, cat 125611) using tdTomato as an endogenous marker of infection. Wholemount membranes were mounted in Prolong Diamond (Life Technologies) then imaged on a SP5 inverted confocal microscope (Leica) and images processed using Fiji.

Statistical analysis
GraphPad Prism (version 6.0h, GraphPad Software, La Jolla, CA) was used to determine statistical significance using as Student unpaired two-tailed t test. A p value of < 0.05 was considered statistically significant.

Ethics statement
Care and use of the animals was in accordance with The guide for the Care and Use of Laboratory Animals from the National Research Council and the USDA Animal Care Resource Guide. All experimental protocols involving the use of mice were approved by the Institutional Animal Care and Use Committee at the University of Minnesota (protocol: 1708-35040A. approved 09/14/2017; expires 09/13/2020). Eggs were obtained from Charles River Laboratories and were grown at 37˚C until 12 days of embryonation.