Suppression of Cytotoxic T Cell Functions and Decreased Levels of Tissue Resident Memory T cell During H5N1 infection

Seasonal influenza virus infections cause mild illness in healthy adults, as timely viral clearance is mediated by the functions of cytotoxic T cells. However, avian H5N1 influenza virus infections can result in prolonged and fatal illness across all age groups, which has been attributed to the overt and uncontrolled activation of host immune responses. Here we investigate how excessive innate immune responses to H5N1 impair subsequent adaptive T cell responses in the lungs. Using recombinant H1N1 and H5N1 strains sharing 6 internal genes, we demonstrate that H5N1 (2:6) infection in mice causes higher stimulation and increased migration of lung dendritic cells to the draining lymph nodes, resulting in higher numbers of virus specific T cells in the lungs. Despite robust T cell responses in the lungs, H5N1 (2:6) infected mice showed inefficient and delayed viral clearance as compared to H1N1 infected mice. In addition, we observed higher levels of inhibitory signals including increased PD1 and IL-10 expression by cytotoxic T cells in H5N1 (2:6) infected mice, suggesting that delayed viral clearance of H5N1 (2:6) was due to suppression of T cell functions in vivo. Importantly, H5N1 (2:6) infected mice displayed decreased numbers of tissue resident memory T cells as compared to H1N1 infected mice; however, despite decreased number of tissue resident memory T cells, H5N1 (2:6) were protected against a heterologous challenge from H3N2 virus (X31). Taken together, our study provides mechanistic insight for the prolonged viral replication and protracted illness observed in H5N1 infected patients. Importance Influenza viruses cause upper respiratory tract infections in humans. In healthy adults, seasonal influenza virus infections result in mild disease. Occasionally, influenza viruses endemic in domestic birds can cause severe and fatal disease even in healthy individuals. In avian influenza virus infected patients, the host immune system is activated in an uncontrolled manner and is unable to control infection in a timely fashion. In this study, we investigated why the immune system fails to effectively control a modified form of avian influenza virus. Our studies show that T cell functions important for clearing virally infected cells are impaired by higher negative regulatory signals during modified avian influenza virus infection. In addition, memory T cell numbers were decreased in modified avian influenza virus infected mice. Our studies provide a possible mechanism for the severe and prolonged disease associated with avian influenza virus infections in humans.


Abstract 24 25
Seasonal influenza virus infections cause mild illness in healthy adults, as timely viral 26 clearance is mediated by the functions of cytotoxic T cells. However, avian H5N1 influenza 27 virus infections can result in prolonged and fatal illness across all age groups, which has been 28 attributed to the overt and uncontrolled activation of host immune responses. Here we 29 investigate how excessive innate immune responses to H5N1 impair subsequent adaptive T 30 cell responses in the lungs. Using recombinant H1N1 and H5N1 strains sharing 6 internal 31 genes, we demonstrate that H5N1 (2:6) infection in mice causes higher stimulation and 32 increased migration of lung dendritic cells to the draining lymph nodes, resulting in higher 33 numbers of virus specific T cells in the lungs. Despite robust T cell responses in the lungs, 34 H5N1 (2:6) infected mice showed inefficient and delayed viral clearance as compared to H1N1 35 infected mice. In addition, we observed higher levels of inhibitory signals including increased 36 PD1 and IL-10 expression by cytotoxic T cells in H5N1 (2:6) infected mice, suggesting that 37 delayed viral clearance of H5N1 (2:6) was due to suppression of T cell functions in vivo. 38 Importantly, H5N1 (2:6) infected mice displayed decreased numbers of tissue resident memory 39 T cells as compared to H1N1 infected mice; however, despite decreased number of tissue 40 resident memory T cells, H5N1 (2:6) were protected against a heterologous challenge from 41 H3N2 virus (X31). Taken together, our study provides mechanistic insight for the prolonged 42 viral replication and protracted illness observed in H5N1 infected patients. self-limiting in healthy adults; however, seasonal infections can be severe in young children 66 and the elderly (2, 3). In addition to humans, influenza viruses can infect a variety of zoonotic 67 species including domestic poultry, pigs, horses, seals and waterfowl (4-6). Occasionally, 68 influenza virus strains circulating in zoonotic reservoirs can cross the species barrier and 69 cause infections in humans. Unlike seasonal H1N1 and H3N2 strains, infections with avian 70 influenza viruses such as H5N1 and H7N9 are often severe in all age groups and cause 71 extensive alveolar damage, vascular leakage, and increased infiltration of inflammatory cells in 72 the lungs. The virulent nature of avian influenza viruses has been attributed to both viral and 73 host determinants; while the viral determinants of virulence are well defined, the contribution of 74 host responses to disease severity remain to be elucidated. 75 The H5N1 strain of avian influenza virus was first detected in humans during a domestic 76 poultry outbreak in Hong Kong in 1997 (7, 8). Despite considerable efforts for containment, 77 H5N1 strains have spread globally and are now endemic in domestic poultry on several 78 continents. Over the past 20 years, H5N1 viruses from infected domestic poultry have crossed 79 the species barrier, causing severe and often fatal infections in humans with mortality rates as 80 high as 60% (9). Many of the viral components critical for the enhanced virulence of H5N1 81 have been identified through the generation of recombinant and/or reassortant viruses (10) (11, 82 12). Prior studies have shown that the multibasic cleavage site (MBS) in the viral 83 hemagglutinin of H5N1 facilitates higher viral replication and mediates extrapulmonary spread 84 (13-15). In addition, our group has recently demonstrated that the endothelial cell tropism of 85 H5N1 contributes to barrier disruption, microvascular leakage, and subsequent mortality (12). 86 Moreover, polymorphisms that increase viral replication have been identified in the viral 87 polymerase subunits of H5N1 strains (16)(17)(18)(19)(20). Together, these studies have helped to define 88 the viral components that are responsible for the enhanced virulence of H5N1. 89 Apart from viral determinants, overt and uncontrolled activation of the innate immune 90 responses also contribute to the disease severity associated with H5N1 infection (21,22). 91 Histological analyses of lungs from fatal H5N1 cases demonstrate severe immunopathology, 92 as evidenced by excessive infiltration of immune cells into the lungs and higher numbers of 93 viral antigen positive cells in the lungs (23, 24). In corroboration with these studies, H5N1 94 viruses have been shown to induce higher DC activation and increase cytokine production as 95 compared to H1N1 viruses (25). Moreover, studies with H5N1 strains in animal models 96 demonstrate hyperactivation of resident immune cells in the lungs and a consequent upsurge 97 in cytokine levels (26,27). As such, these heightened proinflammatory responses result in the 98 excessive recruitment of neutrophils and inflammatory monocytes into the lungs, correlating 99 with severe disease (24). Despite robust activation of innate immune responses against H5N1 100 infection, higher and prolonged virus replication can be detected in the lungs of infected 101 individuals, suggesting a possible dysregulation of adaptive immune responses(28). 102 We have previously demonstrated that appropriate activation of respiratory DC is required for 103 effective T cell responses against a mouse adapted H1N1 strain (29). Here, we sought to 104 determine if excessive activation of innate immune cells during avian H5N1 infection impairs 105 subsequent adaptive T cell responses. In order to investigate the immune responses against 106 H5N1 in comparison to a mouse adapted H1N1 strain, we generated a closely matched 107 recombinant H5N1 virus carrying the 6 internal genes of H1N1 (H5N1 (2:6)). Our studies 108 demonstrated that H5N1 (2:6) infection in mice induced higher lung DC activation and 109 promoted increased migration of lung DC to the draining lymph nodes, resulting in increased 110 numbers of virus specific CD8+ and CD4+ T cells in the lungs as compared to H1N1 infected 111 mice. Despite higher numbers of virus specific T cells, we observed delayed clearance of 112 H5N1 from the lungs, which correlated with higher PD-1 expression and increased production 113 of the anti-inflammatory cytokine IL-10 by T cells in H5N1 infected mice. Importantly, we 114 observed lowered numbers of virus specific tissue resident memory T cells in H5N1 infected 115 mice as compared to H1N1 infected mice. Taken together, our study demonstrates that 116 hyperactivation of innate immune cells during H5N1 infection impairs cytotoxic T cell functions 117 as well as subsequent generation of influenza virus specific tissue resident memory T cells. 118

H5N1 infection induces higher activation of innate immune cells 121
To establish if infection with a low pathogenic H5N1 virus results in higher activation of innate 122 immune cells, we infected C57BL/6 mice with a recombinant H5N1-GFP 123 mice infected with H5N1-GFP as compared to H1N1-GFP ( Figure 1A-B). In addition, we 129 observed higher upregulation of CD86 on inflammatory DC and inflammatory monocytes from 130 H5N1-GFP infected mice as compared to H1N1-GFP infected mice, demonstrating that H5N1 131 infection results in higher activation of innate immune cells ( Figure 1C-D). 132 Next, to determine if the HA and NA of H5N1 virus are sufficient to induce higher activation of 133 innate immune cells, we generated a 2:6 reassortant virus carrying the HA and NA from H5N1 134 with the 6 internal genes of PR8 (H5N1 (2:6)) and compared it to the parental strain in 135 subsequent studies. In this way, we can minimize the differences in viral replication between 136 H5N1 and H1N1, as well as monitor T cell responses against the same epitopes in the internal 137 viral genes. To confirm higher activation of innate immune cells by the H5N1 (2:6) reassortant 138 strain, C57BL/6 mice were infected with H5N1 (2:6) or H1N1 and the levels of CD86 were 139 analyzed by flow cytometry on day 2 post-infection (pi). In mice infected with H5N1 (2:6), we 140 observed higher expression of CD86 on both types of lung resident DC (CD103+ DC and 141 CD11b+ DC) as compared to H1N1 infected mice ( Figure 1E-F). In addition, we observed 142 increased expression of IFNβ and interferon stimulated genes (ISG) in the lungs of H5N1 (2:6) 143 infected mice on day 4 pi as compared to H1N1 infected mice ( Figure 1G). Together, these 144 results demonstrate that the HA and NA of H5N1 can induce higher innate immune responses 145 in the lungs. in the CD11b+ DC subset in both groups. Next, to determine the levels of lung DC migration to 155 the MLN, we labeled cells in the respiratory tract by instilling CFSE dye on day 2 pi and 156 measured the levels of CFSE positive lung DC in the MLN after 16h. As compared to H1N1 157 infected mice, we observed increased accumulation of CFSE+ lung DC in the MLN of H5N1 158 (2:6) infected mice ( Figure 2C-E). In addition, we observed increased numbers of total lung DC 159 in the MLN of H5N1 (2:6) infected mice as compared to H1N1 infected mice ( Figure 2E). 160 These data demonstrate that H5N1 (2:6) infection induces higher activation of lung DC, 161 resulting in increased migration and accumulation of lung DC in the MLN. 162

Mice infected with H5N1 (2:6) show robust activation of T cell responses but display 163 delayed viral clearance 164
Next, we determined if the higher numbers of DC observed in the MLN of H5N1 (2:6) infected 165 mice resulted in enhanced T cell responses and viral clearance. To evaluate primary T cell 166 responses, C57BL/6 mice were infected with 100 PFU of H5N1 (2:6) or H1N1 and T cell 167 responses were measured on day 8 pi by tetramer staining and by monitoring for cytokine 168 production upon ex vivo stimulation. Using tetramers specific for viral NP or PA, we observed 169 increased frequencies of both NP and PA tetramer positive CD8+ T cells in H5N1 (2:6) 170 infected mice as compared to H1N1 infected mice ( Figure 3A-B). The absolute numbers of 171 virus specific CD8 T cells were also higher in H5N1(2:6) infected mice as compared to H1N1 172 infected mice ( Figure 3C). In addition, ex vivo stimulation with X-31 (H3N2) virus or viral 173 peptides showed increased frequencies of interferon gamma (IFNγ) and granzyme B (GrB) 174 producing CD8+ T cells in H5N1 (2:6) infected mice as compared to H1N1 infected mice 175 ( Figure 3D). Moreover, H5N1 (2:6) infected mice showed increased frequencies of IFNγ and 176 GrB producing CD4+ T cells as compared to H1N1 infected mice ( Figure 3E). These results 177 demonstrate that hyperactivated lung DC promote robust activation of virus specific T cell 178 responses in the lung. 179 In the mouse model of influenza virus, innate immune cells restrict viral replication prior to the 180 establishment of adaptive T cell responses. However, after day 6 pi, T cells primed in the MLN 181 migrate to the lungs and participate in the clearance of virus infected cells. Therefore, we 182 determined if the higher numbers of virus specific T cells observed in H5N1 (2:6) infected mice 183 resulted in efficient viral clearance in the lungs. C57BL/6 mice were infected with 100 PFU of 184 H5N1 (2:6) or H1N1, viral loads in the lungs were measured by plaque assay at various days 185 pi. Prior to and including day 6 pi, we observed similar viral loads in the lungs of both groups of 186 infected mice, suggesting that both viruses replicate to similar levels ( Figure 3F). However, on 187 day 8 and day 9 pi, we observed higher viral loads (~5-10 fold) in the lungs of H5N1 (2:6) 188 infected mice as compared to H1N1 infected mice. These results demonstrate that, despite the 189 presence of more virus specific T cells in the lungs, viral clearance was delayed in H5N1 (2:6) 190 infected mice. 191 To understand the basis for the delayed clearance of H5N1 (2:6) in the lungs, we evaluated 192 the functionality of T cells by in vitro T cell killing assay. In this assay, T cells isolated from 193 H5N1 (2:6) or H1N1 infected mice were co-cultured with CFSE labeled splenocytes pulsed 194 with NP peptide, and the amount of target cell death was determined by quantification of 7-195 AAD positive splenocytes. Interestingly, we observed more splenocyte death in co-cultures 196 containing T cells from H5N1 (2:6) infected mice as compared to co-cultures containing T cells 197 from H1N1 infected mice ( Figure 3G-H). Next, we performed in vivo killing assay with peptide 198 pulsed splenocytes. Splenocytes were labeled with either low CFSE or high CFSE and pulsed 199 with influenza virus NP peptide or control peptide, respectively. Splenocytes were adoptively 200 transferred into mice previously infected with either H5N1(2:6) or H1N1 (day 8pi; Figure  shown to be upregulated in T cells in response to direct activation of TCR. Although the T cells 211 isolated from H5N1 (2:6) infected mice were efficient in killing peptide pulsed splenocytes, we 212 observed delayed viral clearance in the lungs ( Figure 3F-G). Thus, we investigated if the T cell 213 functionality was suppressed in vivo through PD-1/PD-L1 interactions by measuring the 214 expression of PD-1/PD-L1 by flow cytometry. We observed significantly higher levels of PD-1 215 on CD8+ T cells isolated from H5N1 (2:6) infected mice as compared to H1N1 infected mice 216 ( Figure 4A-B). Next, we analyzed different cellular compartments in the lungs for PD-L1 217 expression and observed significantly higher levels of PD-L1 on inflammatory monocytes 218 (CD11b+ Ly6C hi Ly6G-) isolated from H5N1 (2:6) infected mice as compared to H1N1 infected 219 mice ( Figure 4C). In addition, we observed increased numbers of inflammatory monocytes in 220 H5N1(2:6) infected mice group ( Figure 4D). However, the levels of PD-L1 on other cellular 221 compartments in the lungs including inflammatory DC were similar between the two groups. 222 Next, we measured IL-10 production in T cells isolated from infected mice to determine the 223 possibility of IL-10 mediated suppression of T cell functions. T cells isolated from H5N1 (2:6) or 224 H1N1 infected mice on day 8 pi were co-cultured with DC pulsed with MHC-I or MHC-II peptide 225 or infected with X-31 (H3N2) virus, and production of IFNγ and IL-10 in T cells was measured 226 by flow cytometry. We observed increased production of IFNγ and IL-10 in both CD8+ and 227 CD4+ T cells isolated from H5N1 (2:6) infected mice as compared to H1N1 infected mice 228 were infected with 50PFU of H1N1 or H5N1(2:6) virus and subsequently challenged with a 249 heterologous H3N2 strain (X-31), a reassortant strain that share 6 internal genes with H1N1 250 and H5N1(2:6) viruses. We did not observe significant differences in weight loss between 251 H1N1 and H5N1(2:6) infected groups upon lethal challenge with the H3N2 (X-31) strain. These 252 data suggest that the lowered levels of memory T cells in H5N1 (2:6)  Infections with avian H5N1 influenza virus induce higher innate immune responses as 258 compared to human H1N1 viruses (21, 36). However, due to inherent differences in replication 259 levels, it is difficult to discern if this hyperactivation of innate immune responses against H5N1 260 is due to higher viral replication in the lungs. To overcome this caveat, we generated an H5N1 261 strain sharing the 6 internal genes of H1N1 (H5N1 (2:6)), and observed that the HA and NA of 262  despite mounting robust T cell responses, H5N1 (2:6) infected mice showed delayed viral 298 clearance in the lungs as compared to H1N1 infected mice ( Figure 3F). This delayed viral 299 clearance in H5N1 (2:6) infected mice was likely due to active suppression of cytotoxic T cell 300 functions in vivo, as T cells isolated from H5N1 (2:6) infected mice showed efficient cytotoxic 301 activity against NP peptide pulsed splenocytes ( Figure 3G). In corroboration, we observed 302 higher levels of inhibitory signals (PD-1 and IL-10) that likely suppress cytotoxic T cell 303 functions in vivo and delay viral clearance ( Figure 4A and 3F). In our in vivo killing assays, 304 H5N1 (2:6) infected mice showed robust killing of viral peptide loaded splenocytes, suggesting 305 that inhibition of T cells may occur by direct suppression by cell-cell contact rather than by the (2:6) infected mice as compared to H1N1 infected mice ( Figure 4C). In addition, the numbers 318 of inflammatory monocytes were higher in H5N1(2:6) infected mice as compared to H1N1 319 infected mice. It should be noted that PD-L1 expression was observed on others cell types as 320 well, yet there was no significant difference in PD-L1 levels between the two groups (data 321 shown for inflammatory DCs; Figure 4C). In a prior study, anti-PD-L1 treatment of PR8 infected 322 mice showed increased virus specific T cells and decreased viral titers (30); however, anti-PD-323 L1 treatment did not alter disease outcome, suggesting that there may be additional 324 mechanisms for suppression of T cell functions. In agreement, we observed increased 325 expression of the anti-inflammatory cytokine IL-10 in T cells isolated from H5N1 (2:6) infected 326 mice as compared to H1N1 infected mice ( Figure 4C-E). Taken together, our data suggest that 327 higher levels of IL-10 production and PD-1/PD-L1 mediated inhibition likely contribute to 328 suppression of T cell functions and consequently results in delayed clearance of H5N1 (2:6) in 329 the lungs. In conclusion, our study demonstrates that hyperactivation of innate immune cells by H5N1 352 (2:6) dampens T cells responses and delays viral clearance in the lungs. This is likely due to 353 higher expression of the inhibitory molecule PD-1 on T cells as well as higher production of the

Quantitative RT-PCR analysis 394
Total RNA from lung tissue was extracted using Trizol (Life technologies) following the 395 manufacturer's instructions, and cDNA was synthesized with SuperScript II using Oligo dT 396 primers (Roche Diagnostics). Quantitative PCR was performed using previously described 397 gene specific primers in an ABI7300 Real Time PCR system with SYBR Green Master Mix 398 (Invitrogen) (12). 399

Flow cytometric analyses 400
Preparation of lung samples for flow cytometric analysis and T cell assays were performed 401 following techniques previously described by us (29)

DC and T cell assays 422
Bone marrow derived dendritic cells (BMDC) were generated from C57BL/6 mice and T cell re-423 stimulation experiments were performed as previously described (59)

Acknowledgements 477
We are grateful to Dr. Adolfo Garcia-Sastre at the Icahn School of Medicine for providing 478 numerous reagents used in this study. H5N1 reverse genetics plasmids were kindly provided 479 by Dr. John Steel at Emory University. We would like to thank the NIH Tetramer Core Facility 480 at Emory University for providing us with influenza virus specific T cell tetramers. We would 481 also like to thank the staff at the Office of Research Safety and Animal Resource Center at the 482 Pathways Contribute to Lung CD8+ T Cell Impairment and Protect against 620