image_5_Exposure of Human CD8+ T Cells to Type-2 Cytokines Impairs Division and Differentiation and Induces Limited Polarization.jpeg

<p>Effector CD8⁺ T cells generally produce type-1 cytokines and mediators of the perforin/granzyme cytolytic pathway, yet type-2-polarized CD8⁺ cells (Tc2) are detected in type-2 (T2) cytokine-driven diseases such as asthma. It is unclear whether T2 cytokine exposure during activation is sufficient to polarize human CD8⁺ T cells. To address this question, a protocol was developed for high-efficiency activation of human CD8⁺ T cells in which purified single cells or populations were stimulated with plate-bound anti-CD3 and anti-CD11a mAb for up to 8 days in T2 polarizing or neutral conditions, before functional analysis. Activation of CD8⁺ naïve T cells (T_N) in T2 compared with neutral conditions decreased the size of single-cell clones, although early division kinetics were equivalent, indicating an effect on overall division number. Activation of T_N in T2 conditions followed by brief anti-CD3 mAb restimulation favored expression of T2 cytokines, GATA3 and Eomes, and lowered expression of type-1 cytokines, Prf1, Gzmb, T-BET, and Prdm1. However, IL-4 was only weakly expressed, and PMA and ionomycin restimulation favored IFN-γ over IL-4 expression. Activation of T_N in T2 compared with neutral conditions prevented downregulation of costimulatory (CD27, CD28) and lymph-node homing receptors (CCR7) and CD95 acquisition, which typically occur during differentiation into effector phenotypes. CD3 was rapidly and substantially induced after activation in neutral, but not T2 conditions, potentially contributing to greater division and differentiation in neutral conditions. CD8⁺ central memory T cells (T_CM) were less able to enter division upon reactivation in T2 compared with neutral conditions, and were more refractory to modulating IFN-γ and IL-4 production than CD8⁺ T_N. In summary, while activation of T_N in T2 conditions can generate T2 cytokine-biased cells, IL-4 expression is weak, T2 bias is lost upon strong restimulation, differentiation, and division are arrested, and reactivation of T_CM is reduced in T2 conditions. Taken together, this suggests that exposure to T2 cytokines during activation may not be sufficient to generate and retain human Tc2 cells.</p

image_3_Exposure of Human CD8+ T Cells to Type-2 Cytokines Impairs Division and Differentiation and Induces Limited Polarization.jpeg

<p>Effector CD8⁺ T cells generally produce type-1 cytokines and mediators of the perforin/granzyme cytolytic pathway, yet type-2-polarized CD8⁺ cells (Tc2) are detected in type-2 (T2) cytokine-driven diseases such as asthma. It is unclear whether T2 cytokine exposure during activation is sufficient to polarize human CD8⁺ T cells. To address this question, a protocol was developed for high-efficiency activation of human CD8⁺ T cells in which purified single cells or populations were stimulated with plate-bound anti-CD3 and anti-CD11a mAb for up to 8 days in T2 polarizing or neutral conditions, before functional analysis. Activation of CD8⁺ naïve T cells (T_N) in T2 compared with neutral conditions decreased the size of single-cell clones, although early division kinetics were equivalent, indicating an effect on overall division number. Activation of T_N in T2 conditions followed by brief anti-CD3 mAb restimulation favored expression of T2 cytokines, GATA3 and Eomes, and lowered expression of type-1 cytokines, Prf1, Gzmb, T-BET, and Prdm1. However, IL-4 was only weakly expressed, and PMA and ionomycin restimulation favored IFN-γ over IL-4 expression. Activation of T_N in T2 compared with neutral conditions prevented downregulation of costimulatory (CD27, CD28) and lymph-node homing receptors (CCR7) and CD95 acquisition, which typically occur during differentiation into effector phenotypes. CD3 was rapidly and substantially induced after activation in neutral, but not T2 conditions, potentially contributing to greater division and differentiation in neutral conditions. CD8⁺ central memory T cells (T_CM) were less able to enter division upon reactivation in T2 compared with neutral conditions, and were more refractory to modulating IFN-γ and IL-4 production than CD8⁺ T_N. In summary, while activation of T_N in T2 conditions can generate T2 cytokine-biased cells, IL-4 expression is weak, T2 bias is lost upon strong restimulation, differentiation, and division are arrested, and reactivation of T_CM is reduced in T2 conditions. Taken together, this suggests that exposure to T2 cytokines during activation may not be sufficient to generate and retain human Tc2 cells.</p

image_1_Exposure of Human CD8+ T Cells to Type-2 Cytokines Impairs Division and Differentiation and Induces Limited Polarization.tif

<p>Effector CD8⁺ T cells generally produce type-1 cytokines and mediators of the perforin/granzyme cytolytic pathway, yet type-2-polarized CD8⁺ cells (Tc2) are detected in type-2 (T2) cytokine-driven diseases such as asthma. It is unclear whether T2 cytokine exposure during activation is sufficient to polarize human CD8⁺ T cells. To address this question, a protocol was developed for high-efficiency activation of human CD8⁺ T cells in which purified single cells or populations were stimulated with plate-bound anti-CD3 and anti-CD11a mAb for up to 8 days in T2 polarizing or neutral conditions, before functional analysis. Activation of CD8⁺ naïve T cells (T_N) in T2 compared with neutral conditions decreased the size of single-cell clones, although early division kinetics were equivalent, indicating an effect on overall division number. Activation of T_N in T2 conditions followed by brief anti-CD3 mAb restimulation favored expression of T2 cytokines, GATA3 and Eomes, and lowered expression of type-1 cytokines, Prf1, Gzmb, T-BET, and Prdm1. However, IL-4 was only weakly expressed, and PMA and ionomycin restimulation favored IFN-γ over IL-4 expression. Activation of T_N in T2 compared with neutral conditions prevented downregulation of costimulatory (CD27, CD28) and lymph-node homing receptors (CCR7) and CD95 acquisition, which typically occur during differentiation into effector phenotypes. CD3 was rapidly and substantially induced after activation in neutral, but not T2 conditions, potentially contributing to greater division and differentiation in neutral conditions. CD8⁺ central memory T cells (T_CM) were less able to enter division upon reactivation in T2 compared with neutral conditions, and were more refractory to modulating IFN-γ and IL-4 production than CD8⁺ T_N. In summary, while activation of T_N in T2 conditions can generate T2 cytokine-biased cells, IL-4 expression is weak, T2 bias is lost upon strong restimulation, differentiation, and division are arrested, and reactivation of T_CM is reduced in T2 conditions. Taken together, this suggests that exposure to T2 cytokines during activation may not be sufficient to generate and retain human Tc2 cells.</p

image_2_Exposure of Human CD8+ T Cells to Type-2 Cytokines Impairs Division and Differentiation and Induces Limited Polarization.jpg

<p>Effector CD8⁺ T cells generally produce type-1 cytokines and mediators of the perforin/granzyme cytolytic pathway, yet type-2-polarized CD8⁺ cells (Tc2) are detected in type-2 (T2) cytokine-driven diseases such as asthma. It is unclear whether T2 cytokine exposure during activation is sufficient to polarize human CD8⁺ T cells. To address this question, a protocol was developed for high-efficiency activation of human CD8⁺ T cells in which purified single cells or populations were stimulated with plate-bound anti-CD3 and anti-CD11a mAb for up to 8 days in T2 polarizing or neutral conditions, before functional analysis. Activation of CD8⁺ naïve T cells (T_N) in T2 compared with neutral conditions decreased the size of single-cell clones, although early division kinetics were equivalent, indicating an effect on overall division number. Activation of T_N in T2 conditions followed by brief anti-CD3 mAb restimulation favored expression of T2 cytokines, GATA3 and Eomes, and lowered expression of type-1 cytokines, Prf1, Gzmb, T-BET, and Prdm1. However, IL-4 was only weakly expressed, and PMA and ionomycin restimulation favored IFN-γ over IL-4 expression. Activation of T_N in T2 compared with neutral conditions prevented downregulation of costimulatory (CD27, CD28) and lymph-node homing receptors (CCR7) and CD95 acquisition, which typically occur during differentiation into effector phenotypes. CD3 was rapidly and substantially induced after activation in neutral, but not T2 conditions, potentially contributing to greater division and differentiation in neutral conditions. CD8⁺ central memory T cells (T_CM) were less able to enter division upon reactivation in T2 compared with neutral conditions, and were more refractory to modulating IFN-γ and IL-4 production than CD8⁺ T_N. In summary, while activation of T_N in T2 conditions can generate T2 cytokine-biased cells, IL-4 expression is weak, T2 bias is lost upon strong restimulation, differentiation, and division are arrested, and reactivation of T_CM is reduced in T2 conditions. Taken together, this suggests that exposure to T2 cytokines during activation may not be sufficient to generate and retain human Tc2 cells.</p

Innate Immunity and the Inter-exposure Interval Determine the Dynamics of Secondary Influenza Virus Infection and Explain Observed Viral Hierarchies

<div><p>Influenza is an infectious disease that primarily attacks the respiratory system. Innate immunity provides both a very early defense to influenza virus invasion and an effective control of viral growth. Previous modelling studies of virus–innate immune response interactions have focused on infection with a single virus and, while improving our understanding of viral and immune dynamics, have been unable to effectively evaluate the relative feasibility of different hypothesised mechanisms of antiviral immunity. In recent experiments, we have applied consecutive exposures to different virus strains in a ferret model, and demonstrated that viruses differed in their ability to induce a state of temporary immunity or viral interference capable of modifying the infection kinetics of the subsequent exposure. These results imply that virus-induced early immune responses may be responsible for the observed viral hierarchy. Here we introduce and analyse a family of within-host models of re-infection viral kinetics which allow for different viruses to stimulate the innate immune response to different degrees. The proposed models differ in their hypothesised mechanisms of action of the non-specific innate immune response. We compare these alternative models in terms of their abilities to reproduce the re-exposure data. Our results show that 1) a model with viral control mediated solely by a virus-resistant state, as commonly considered in the literature, is not able to reproduce the observed viral hierarchy; 2) the synchronised and desynchronised behaviour of consecutive virus infections is highly dependent upon the interval between primary virus and challenge virus exposures and is consistent with virus-dependent stimulation of the innate immune response. Our study provides the first mechanistic explanation for the recently observed influenza viral hierarchies and demonstrates the importance of understanding the host response to multi-strain viral infections. Re-exposure experiments provide a new paradigm in which to study the immune response to influenza and its role in viral control.</p></div

Re-exposure behaviour of Model R1 for different IFN production rates.

<p>A smaller IFN production for the primary virus for Model R1 does not lead to any qualitative difference, in terms of the dependence of model behaviours for the challenge virus on the IEI, from the case of very large IFN production rate of the first virus. The pattern is also independent of the choice of <i>q</i>₂. The meaning of each colour is explained in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004334#pcbi.1004334.g008" target="_blank">Fig 8</a>. They all exhibit four types of behaviours (seen vertically, separated by dashed lines) and within each type the phase decomposition and their order are preserved.</p

Parameter values for the three models.

<p>The units of <i>V</i>, <i>F</i> and <i>A</i> are denoted as <i>u</i>_<i>v</i>, <i>u</i>_<i>F</i> and <i>u</i>_<i>A</i> respectively. <i>T</i>, <i>I</i>, and <i>R</i> have the same unit of <i>u</i>_<i>T</i>; the number of cells. Time (<i>t</i>) has a unit of days (<i>d</i>). Some units are symbolised, as our study is highly qualitative and thus substantially independent of the choice of units. Such units would need to be transformed for different experimental protocols. “varied” indicates that the parameter was assigned different values for different simulations, with the value/s specified whenever necessary. Other parameters are taken or estimated from the literature (references are provided beside those parameter values), and others chosen such that 1) the viral load during infection experiences at least a three orders of magnitude increase and peaks at around the second day post infection [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004334#pcbi.1004334.ref010" target="_blank">10</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004334#pcbi.1004334.ref017" target="_blank">17</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004334#pcbi.1004334.ref022" target="_blank">22</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004334#pcbi.1004334.ref028" target="_blank">28</a>]; 2) IFN is maximally activated at around 2–4 days post infection [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004334#pcbi.1004334.ref028" target="_blank">28</a>]; and 3) antibodies are observable (i.e. rise above a lower detection threshold limit) later than six days post infection [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004334#pcbi.1004334.ref024" target="_blank">24</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004334#pcbi.1004334.ref035" target="_blank">35</a>].</p

Re-exposure behaviour of Model R3 for different IFN production rates.

<p>Different IFN productions of the first virus for Model R3 lead to qualitatively different patterns of re-exposure simulation. The pattern is independent of the choice of <i>q</i>₂. The meaning of each colour is explained in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004334#pcbi.1004334.g008" target="_blank">Fig 8</a>. In spite of different patterns, the order of phase is preserved for any cases. We assume here that <i>κ</i>₁ = <i>κ</i>₂ = 3.</p

Schematic diagram showing the three major functions of IFN.

<p>The three major functions of IFN labelled by ①, ② and ③ correspond to Models 1–3 defined in the main text. Dashed curves with arrows indicate up-regulation while those with bars indicate down-regulation. Binding of virus to target cells leads to infected cells which then produce new virions. Infected cells produce IFN which hinders viral infection via three different hypothesised mechanisms: Model ① converting target cells to virus-resistant state; Model ② decreasing the viral production rate; and Model ③ inducing killing of infected cells by activation of NK cells. Virus activates B cells which produce antibodies to inactivate free virus.</p

Re-exposure experimental data for primary exposure with influenza B virus followed by challenge with A(H1N1)pdm09.

<p>In contrast to <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004334#pcbi.1004334.g001" target="_blank">Fig 1</a>, changing the IEI does not block the infection of A(H1N1)pdm09 virus for any infection period. It does, however, result in a reduced growth rate and delayed time to peak virus titre for A(H1N1)pdm09 at short IEIs. Missing data points in the curves indicate undetectable levels of viral load and no sample was taken on the day of challenge. Each graph represents the data from a single ferret, so that two ferrets within each interval are shown here. All symbols are the same as those in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004334#pcbi.1004334.g001" target="_blank">Fig 1</a>. Data used from [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004334#pcbi.1004334.ref010" target="_blank">10</a>].</p