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

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_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_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

A human tissue-based functional assay platform to evaluate the immune function impact of small molecule inhibitors that target the immune system

<div><p>While the immune system is essential for the maintenance of the homeostasis, health and survival of humans, aberrant immune responses can lead to chronic inflammatory and autoimmune disorders. Pharmacological modulation of drug targets in the immune system to ameliorate disease also carry a risk of immunosuppression that could lead to adverse outcomes. Therefore, it is important to understand the ‘immune fingerprint’ of novel therapeutics as they relate to current and, clinically used immunological therapies to better understand their potential therapeutic benefit as well as immunosuppressive ability that might lead to adverse events such as infection risks and cancer. Since the mechanistic investigation of pharmacological modulators in a drug discovery setting is largely compound- and mechanism-centric but not comprehensive in terms of immune system impact, we developed a human tissue based functional assay platform to evaluate the impact of pharmacological modulators on a range of innate and adaptive immune functions. Here, we demonstrate that it is possible to generate a qualitative and quantitative immune system impact of pharmacological modulators, which might help better understand and predict the benefit-risk profiles of these compounds in the treatment of immune disorders.</p></div

The impact of small molecule inhibitors on human T cell function.

<p>The production of IL-2 as well as the proliferation of CD4⁺ and CD8⁺ T cells was evaluated in a human <i>in vitro</i> PBMC assay. The production of IL-2 was evaluated 24h following stimulation with α-CD3 and α-CD28 while the proliferation of CD4⁺ and CD8⁺ T cells was evaluated in the same cultures 72h following α-CD3 and α-CD28 stimulation. Small molecule inhibitors suppress α-CD3 and α-CD28 induced IL-2 production (A), CD4⁺ T cell proliferation (B) and CD8⁺ T cell proliferation (C) in human PBMCs. For each compound, the potency (red circles) is plotted along the top X-axis and the percentage maximal inhibition achieved in the assay (black squares) are plotted along the bottom X-axis. The potencies of the evaluated compounds in these assays are displayed as IC₅₀ ovalues. A maximum of 100% inhibition is possible for small molecule inhibitors in these assays. The reported potency and % max inhibition values were generated from a composite of 8–10 point dose response curves from n = 6–8 donors for each compound.</p

Impact of small molecule inhibitors on gene expression profiles in the T cell stimulation assay.

<p>(A) A nanostring gene expression panel was used to evaluate mRNA profiles of PBMCs 24 h following treatment with 1μM of the SM inhibitors in the T cell stimulation assay. The gene expression profiles of unstimulated and compound treated stimulated samples are shown. All data were normalized to housekeeping genes and stimulated DMSO control samples. Hierarchical agglomerative clustering of genes with greater than a 2-fold change (p-value<0.05) is shown. (B) Transcript expression of IL-2 under unstimulated, stimulated, and compound treated conditions. The mRNA expression levels of the cytokine genes IFNγ (C), IL4 (D), IL13 (E), IL17F (F) as well as the transcription factors Tbx21 (G), GATA3 (H), RORc (I) and Foxp3 (J) are depicted as examples from the gene expression dataset. The gene expression profiles in (A) is a composite of PBMCs from n = 3 donors, while the individual gene expression profiles in (B-J) are mean±SEM of mRNA expression from n = 3 PBMC donors.</p

A conceptual framework to develop an ‘immune impact score’ for compounds.

<p>The impact of compounds in immune system-based functional assays, -omics approaches, PK/PD information and chemical informatics (that classify and predict compound-function relationships based on available information) might be integrated with the aim of assigning an ‘immune impact score’ for individual compounds. Further integration of available or modeled efficacy and adverse event data for compounds with the immune impact score might be useful in better understanding and predicting the efficacy, adverse event profile and differentiation of these compounds in a clinical setting. PK/PD–Pharmacokinetics/Pharmacodynamics, AE–adverse events.</p

Impact of SYK/ZAP-70 inhibitor and prednisolone on gene expression profiles in the T cell stimulation assay.

<p>(A) A nanostring gene expression panel was used to evaluate mRNA profiles of PBMCs 3, 6 and 24 h following treatment with three different concentrations of SYK/ZAP-70 inhibitor and prednisolone. The gene expression profiles of unstimulated and compound treated stimulated samples are shown. All data were normalized to housekeeping genes and stimulated DMSO control samples. Hierarchical agglomerative clustering of genes with greater than a 2-fold change (p-value<0.05) is shown. Transcript expression of IL-2 under unstimulated, stimulated, and stimulated plus SYK/ZAP-70 inhibitor or prednisolone treated conditions at the 3h (B), 6h (C) and 24h (D) time points in the T cell stimulation assay. The gene expression profiles in (A) is a composite of PBMCs from n = 3 donors, while the individual gene expression profiles in (B-D) are mean±SEM of mRNA expression from n = 3 PBMC donors.</p

The effect of small molecule inhibitors on reactive oxygen species (ROS) production by human phagocytes.

<p>Ten minutes following stimulation with opsonized bacteria, the production of ROS in neutrophils and monocytes from human whole blood was evaluated using flow cytometry. Small molecule inhibitors inhibit ROS production neutrophils (A) and monocytes (B) in human whole blood following stimulation with opsonized bacteria. For each compound, the potency (red circles) and the percentage maximal inhibition achieved in the assay (black squares) are plotted along the X-axis. The potencies of the evaluated compounds in these assays are displayed as IC₅₀ values. A maximum of 100% inhibition is possible for small molecule inhibitors in these assays. The reported potency and % max inhibition values were generated from a composite of 8–10 point dose response curves from n = 6–8 donors for each compound. The positive control used in this assay (Diphenyliodonium chloride [DPI], an NADPH oxidase inhibitor), can inhibit ROS production from neutrophils (C) or monocytes (D) in a dose dependent manner. A BTK inhibitor consistently inhibited ~50% neutrophil ROS production (E) across multiple donors evaluated in this assay. Each point in the dose response curve indicates mean±SEM of % inhibition at that dose from n = 6–8 donors.</p