Predicted H2-Db restricted PA CD8 epitopes for CA/E3/09.

<p>X31 and CA/E3/09 PA amino acid sequences <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046166#pone.0046166-Guo1" target="_blank">[18]</a> were introduced into NetMHC 3.0 for H2-Db 10 mer CD8 epitope analysis. Listed are the top 10 peptides with higher MHC binding potential. Bolded peptides indicated identical sequences shared between CA/E3/09 and X31 viruses.</p

Detection of cross-reactive NP366 CD8 population after secondary challenge.

<p>Mice were primed with X31 virus (3×10⁵ EID₅₀) and rested for 42 days and then were challenged with a lethal dose of CA/E3/09 virus (3000 PFU). At day 6 after re-infection, co-staining with X31NP366 and CANP366 tetramers were performed on cell samples obtained from spleen (A), MLN (B) and lung (C). Data are representative of three independent experiments.</p

IFN-γ response to five new CD8 peptides after CA/E3/09 infection.

<p>A panel of predicted CD8 peptides excluding the NP366 and PA224 as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046166#pone-0046166-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046166#pone-0046166-t002" target="_blank">2</a> were assessed for their abilities to induce IFN-γ response at day 10 after naïve mice were non-lethally infected with X31 virus (3×10⁵ EID₅₀, filled column) or CA/E3/09 virus (3 PFU, open column) using the ELISPOT methods. Among these peptides, only five (CANP39, CANP41, CANP55, CANP130, CANP296 and CAPA439) were able to induce spots formation after CA/E3/09 infection. Data presented were average values ± SD from 5 mice of each group and representative of at least four independent experiments.</p

Detection of peptide specific CD8 cells after X31 and CA/E3/09 infection.

<p>Mice were non-lethally infected with X31 (3×10⁵ EID₅₀) or CA/E3/09 virus (3 PFU). At day 10 of infections, CD8 antibody and H2-Db restricted X31NP366 and PA224 tetramers were used to detect peptide specific CD8 cells in live tissue cells from lung (A), spleen (B) and MLN (C) pooled from five mice. Data are representative of the results for at least three independent experiments.</p

CANP366 specific CD8 cells revealed by new CD8 tetramer.

<p>Mice were non-lethally infected with X31 (3×10⁵ EID₅₀) or CA/E3/09 virus (3 PFU) or a mixture of X31 and CA/E3/09 virus containing 3×10⁵ EID₅₀ X31 and 3 PFU of CA/E3/09. At day 10 of infections, CD8 antibody and the newly generated H2-Db restricted tetramer specific for CANP366 were used to detect CA/E3/09 virus specific CD8 cells. Lung cell samples from X31 and CA/E3/09 infected mice were individually stained for X31NP366 and CANP366 tetramers (A) or co-stained with both NP366 tetramers (B). In addition, lung cell samples from X31 and CA/E3/09 co-infected mice were co-stained with X31NP366 and CANP366 tetramers (C). Data are representative of the results for two independent experiments.</p

Predicted H2-Db restricted NP CD8 epitopes for CA/E3/09.

<p>X31 and CA/E3/09 NP amino acid sequences <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046166#pone.0046166-Guo1" target="_blank">[18]</a> were introduced into NetMHC 3.0 for H2-Db 9 mer CD8 epitope analysis. Listed are the top 10 peptides with higher MHC binding potential. Bolded peptides indicated identical sequences shared between CA/E3/09 and X31 viruses.</p

Immune responses to the newly identified CD8 peptides after rechallenge.

<p>Mice were primed with X31 virus (3×10⁵ EID₅₀) and rested for 42 days before challenged with a lethal dose of CA/E3/09 virus (3000 PFU). At day 6 after the rechallenge, the IFN-γ responses for identified CA/E3/09 CD8 peptides including CANP399, CANP41, CANP55, CANP130, CANP296 and CAPA439 were measured by ELISPOT assay (A) Data are representative of three independent experiments.</p

IFN-γ responses to NP366 peptides after rechallenge.

<p>Mice were primed with X31 virus (3×10⁵ EID₅₀) and rested for 42 days before challenged with a lethal dose of CA/E3/09 virus (3000 PFU). At day 6 after the rechallenge, the IFN-γ responses for X31NP366 and CANP366 were measured by ELISPOT assay (A). In addition, after in-vitro NP366 peptide or PMAI stimulation, intracellular staining was used to measure IFN-γ production with or without X31 priming (B). At day 6 after the rechallenge, NP366 peptide or PMAI stimulated cell samples were also stained for CD107a before intracellular IFN-γ staining (C). Data are representative of two independent experiments.</p

The Effects of Acute Neutrophil Depletion on Resolution of Acute Influenza Infection, Establishment of Tissue Resident Memory (T_RM), and Heterosubtypic Immunity

<div><p>After disease resolution, a small subset of influenza specific CD8⁺ T cells can remain in the airways of the lung as a tissue resident memory population (T_RM). These cells are critical for protection from subsequent infections with heterosubtypic influenza viruses. Although it is well established that expression of the collagen IV binding integrin alpha 1 is necessary for the retention and maintenance of T_RM cells, other requirements allowing them to localize to the airways and persist are less well understood. We recently demonstrated that inhibition of neutrophils or neutrophil derived chemokine CXCL12 during acute influenza virus infection reduces the effector T cell response and affects the ability of these cells to localize to the airways. We therefore sought to determine whether the defects that occur in the absence of neutrophils would persist throughout resolution of the disease and impact the development of the T_RM population. Interestingly, the early alterations in the CD8⁺ T cell response recover by two weeks post-infection, and mice form a protective population of T_RM cells. Overall, these observations show that acute neutrophil depletion results in a delay in the effector CD8⁺ T cell response, but does not adversely impact the development of T_RM.</p></div

Motility parameters by quadrant.

<p>Motility parameters by quadrant.</p