Table_1_Immunomodulatory regulator blockade in a viral exacerbation model of severe asthma.docx

Asthmatics are more susceptible to viral infections than healthy individuals and are known to have impaired innate anti-viral defences. Influenza A virus causes significant morbidity and mortality in this population. Immuno-modulatory regulators (IMRs) such as PD-1 are activated on T cells following viral infection as part of normal T cell activation responses, and then subside, but remain elevated in cases of chronic exposure to virus, indicative of T cell exhaustion rather than activation. There is evidence that checkpoint inhibition can enhance anti-viral responses during acute exposure to virus through enhancement of CD8+T cell function. Although elevated PD-1 expression has been described in pulmonary tissues in other chronic lung diseases, the role of IMRs in asthma has been relatively unexplored as the basis for immune dysfunction. We first assessed IMR expression in the peripheral circulation and then quantified changes in IMR expression in lung tissue in response to ex-vivo influenza infection. We found that the PD-1 family members are not significantly altered in the peripheral circulation in individuals with severe asthma but are elevated in pulmonary tissues following ex-vivo influenza infection. We then applied PD-1 Mab inhibitor treatment to bronchial biopsy tissues infected with influenza virus and found that PD-1 inhibition was ineffective in asthmatics, but actually increased infection rates in healthy controls. This study, therefore, suggests that PD-1 therapy would not produce harmful side-effects when applied in people with severe asthma, but could have important, as yet undescribed, negative effects on anti-viral responses in healthy individuals that warrant further investigation.</p

Presentation_2_Immunomodulatory regulator blockade in a viral exacerbation model of severe asthma.pptx

Asthmatics are more susceptible to viral infections than healthy individuals and are known to have impaired innate anti-viral defences. Influenza A virus causes significant morbidity and mortality in this population. Immuno-modulatory regulators (IMRs) such as PD-1 are activated on T cells following viral infection as part of normal T cell activation responses, and then subside, but remain elevated in cases of chronic exposure to virus, indicative of T cell exhaustion rather than activation. There is evidence that checkpoint inhibition can enhance anti-viral responses during acute exposure to virus through enhancement of CD8+T cell function. Although elevated PD-1 expression has been described in pulmonary tissues in other chronic lung diseases, the role of IMRs in asthma has been relatively unexplored as the basis for immune dysfunction. We first assessed IMR expression in the peripheral circulation and then quantified changes in IMR expression in lung tissue in response to ex-vivo influenza infection. We found that the PD-1 family members are not significantly altered in the peripheral circulation in individuals with severe asthma but are elevated in pulmonary tissues following ex-vivo influenza infection. We then applied PD-1 Mab inhibitor treatment to bronchial biopsy tissues infected with influenza virus and found that PD-1 inhibition was ineffective in asthmatics, but actually increased infection rates in healthy controls. This study, therefore, suggests that PD-1 therapy would not produce harmful side-effects when applied in people with severe asthma, but could have important, as yet undescribed, negative effects on anti-viral responses in healthy individuals that warrant further investigation.</p

Presentation_3_Immunomodulatory regulator blockade in a viral exacerbation model of severe asthma.pptx

Asthmatics are more susceptible to viral infections than healthy individuals and are known to have impaired innate anti-viral defences. Influenza A virus causes significant morbidity and mortality in this population. Immuno-modulatory regulators (IMRs) such as PD-1 are activated on T cells following viral infection as part of normal T cell activation responses, and then subside, but remain elevated in cases of chronic exposure to virus, indicative of T cell exhaustion rather than activation. There is evidence that checkpoint inhibition can enhance anti-viral responses during acute exposure to virus through enhancement of CD8+T cell function. Although elevated PD-1 expression has been described in pulmonary tissues in other chronic lung diseases, the role of IMRs in asthma has been relatively unexplored as the basis for immune dysfunction. We first assessed IMR expression in the peripheral circulation and then quantified changes in IMR expression in lung tissue in response to ex-vivo influenza infection. We found that the PD-1 family members are not significantly altered in the peripheral circulation in individuals with severe asthma but are elevated in pulmonary tissues following ex-vivo influenza infection. We then applied PD-1 Mab inhibitor treatment to bronchial biopsy tissues infected with influenza virus and found that PD-1 inhibition was ineffective in asthmatics, but actually increased infection rates in healthy controls. This study, therefore, suggests that PD-1 therapy would not produce harmful side-effects when applied in people with severe asthma, but could have important, as yet undescribed, negative effects on anti-viral responses in healthy individuals that warrant further investigation.</p

Presentation_1_Immunomodulatory regulator blockade in a viral exacerbation model of severe asthma.pptx

Asthmatics are more susceptible to viral infections than healthy individuals and are known to have impaired innate anti-viral defences. Influenza A virus causes significant morbidity and mortality in this population. Immuno-modulatory regulators (IMRs) such as PD-1 are activated on T cells following viral infection as part of normal T cell activation responses, and then subside, but remain elevated in cases of chronic exposure to virus, indicative of T cell exhaustion rather than activation. There is evidence that checkpoint inhibition can enhance anti-viral responses during acute exposure to virus through enhancement of CD8+T cell function. Although elevated PD-1 expression has been described in pulmonary tissues in other chronic lung diseases, the role of IMRs in asthma has been relatively unexplored as the basis for immune dysfunction. We first assessed IMR expression in the peripheral circulation and then quantified changes in IMR expression in lung tissue in response to ex-vivo influenza infection. We found that the PD-1 family members are not significantly altered in the peripheral circulation in individuals with severe asthma but are elevated in pulmonary tissues following ex-vivo influenza infection. We then applied PD-1 Mab inhibitor treatment to bronchial biopsy tissues infected with influenza virus and found that PD-1 inhibition was ineffective in asthmatics, but actually increased infection rates in healthy controls. This study, therefore, suggests that PD-1 therapy would not produce harmful side-effects when applied in people with severe asthma, but could have important, as yet undescribed, negative effects on anti-viral responses in healthy individuals that warrant further investigation.</p

Quantification of infection of human lung explants.

<p>Histograms showing flow cytometry data of viral NP1 (% cells) and PDL1 expression (specific mean fluorescence intensity—SMFI) in epithelial cells <b>(A & C)</b> and macrophages <b>(B & D).</b> Data are expressed as means ±SE of 9 independent experiments. Data analysed using a paired t-test ** p<0.01. **** p<0.0001.</p

Infection of human lung macrophages by X31.

<p>Human lung macrophages were isolated by plate adherence prior to infection with 4000 pfu/ml of H3N2 X31 influenza virus or a UV-irradiated aliquot of virus (UVX31) for 2 h. After washing, media was replaced and the cells incubated for a further 22 h before supernatants and cells were harvested. Cells were analysed for intracellular viral NP1 expression or cell surface expression of HLA-DR and PD-L1 using flow cytometry. Histograms showing flow cytometry data of <b>A)</b> Viral NP1 expression (% cells), <b>B)</b> HLA-DR expression (specific mean fluorescence intensity—SMFI) and <b>C)</b> PDL1 expression (SMFI) from isolated human lung macrophages are expressed as means ±SE of 4 independent experiments. <b>D)</b> Representative histograms demonstrating increase in PDL1 expression in response to influenza infection are shown. Data analysed using a paired t-test * p<0.05, ** p<0.01.</p

Functional effect of PDL1 on T cell activation.

<p>MDM were differentiated in the presence of 2 ng/ml GM-CSF for 12 d prior to infection with 500 pfu/ml of H3N2 X31 influenza virus or a UV-irradiated aliquot of virus (UVX31) for 2 h. Infected MDM were transferred to a coated ELISpot plate and autologous lymphocytes added and incubated for a further 22 h before IFNγ release was measured using ELISpot. <b>A)</b> 2.5 x 10⁵ monocyte-depleted PBMC co-cultured with 5 x 10⁴ autologous MDM not infected (NI) or treated with X31 or UVX31(n = 5). <b>B)</b> 1 x 10⁵ CD8+ T cells co-cultured with 5 x 10⁴ autologous MDM NI or infected with X31 (n = 6) <b>C)</b> 1 x 10⁵ CD8+ T cells co-cultured with 5 x 10⁴ autologous MDM infected with X31 in the presence of 10 μg/ml anti-PDL1 antibody or isotype control (n = 5). Data are expressed as means ±SE of n independent experiments and analysed using a paired t-test * p<0.05, ** p<0.01.</p

Ex vivo infection of human lung explants.

<p>After resting explanted lung tissue overnight, 1 x 10⁶ pfu/ml H3N2 X31 influenza virus or a UV-irradiated aliquot of virus (UVX31) was added for 2 h. After washing, media was replaced and the tissue was incubated for a further 22 h followed by collagenase digestion and flow cytometry analysis <b>A)</b> Gating strategy for identification of CD45+HLADR+ macrophages and CD45-EpCAM+ epithelial cells expressing viral NP1 and PDL1 from lung tissue. <b>B)</b> FACS plots demonstrating increases in influenza infection (NP1) with corresponding increases in PDL1 expression. Plots are representative of nine independent experiments.</p

Regulation of PDL1 mRNA expression by influenza infection and IFNβ.

<p><b>A)</b> PDL1 gene expression was assessed in MDM differentiated in the presence of 2 ng/ml GM-CSF for 12 d before incubation with the indicated doses of rhIFNβ for 24 h using RT-PCR (n = 5). <b>B)</b> MDM were differentiated in the presence of 2 ng/ml GM-CSF for 11 d before transfection with non-specific siRNA or siRNA specific for IFNβ in the presence of GM-CSF for 24 h. MDM were then infected with 500 pfu/ml of H3N2 X31 influenza virus or a UV-irradiated aliquot of virus (UVX31) for 2 h. After washing, media was replaced and the cells incubated for a further 22 h before cells were harvested for RT-PCR analysis of IFNβ and PDL1 mRNA expression (n = 6). PCR data were normalised to β2MG and are expressed as mean fold induction over the non-infected (NI) sample ± SEM of n independent experiments. Data analysed using a paired t-test * p<0.05, ** p<0.01.</p

Effects of influenza infection on MDM cytokine expression.

<p>MDM were differentiated in the presence of 2 ng/ml GM-CSF for 12 d prior to infection with 500 pfu/ml of H3N2 X31 influenza virus or a UV-irradiated aliquot of virus (UVX31) for 2 h. After washing, media was replaced and the cells incubated for a further 22 h before supernatants and cells were harvested. Inflammatory cytokine expression was measured by <b>A)</b> real time PCR (IFNα, IFNβ n = 6) and <b>B)</b> ELISA (IFNβ, TNFα, IL-6, IL-8 n = 6) N.D. = not detected. PCR data were normalised to β2MG and are expressed as mean fold induction over the non-infected (NI) sample ± SEM of 6 independent experiments. ELISA data are expressed as means ±SE of 6 independent experiments. Data analysed using a paired t-test. * p<0.05, ** p<0.01.</p