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_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_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

Distribution of observed increase in PCR positive cases as a function of baseline.

<p>Individuals with a low antibody titer baseline (0–1) are in blue; those with a higher baseline (2–4) are in red.</p

Model adequacy to the data.

<p>Distribution of “observed” paired serology as predicted by the model (color bars) and as observed in the data (black point) as a function of season (2008, Spring 2009, Autumn 2009) and subtype (H1N1, H3N2 and B). In each panel, individuals are sorted by baseline AT levels on the y-axis. For a given baseline, the grey bar indicates the expected proportion of individuals with post AT level equal to baseline AT level; the yellow bar indicates the proportion with a 2 fold rise (2f.r.); the red bar indicates the proportion with a 4 fold rise or more (4f.r.+); the green bar indicates the proportion with a decay. The black thin lines give the 95% CI. The legend gives the mean [95% CI]. <b>A</b>: H1N1, 2008. <b>B</b>: H3N2, 2008. <b>C</b>: B, 2008. <b>D</b>: H1N1, Spring 2009. <b>E</b>: H3N2, Spring 2009. <b>F</b>: B, Spring 2009. <b>G</b>: H1N1pdm09, Autumn 2009.</p

Increases in antibody titers.

<p><b>A</b>: Posterior distribution of the percentage of subjects with a 4 fold rise or more in AT (pink) and with a 2 fold rise or more in AT (blue) for the different subtypes and the different seasons (2008 (08), Spring 2009 (S09), Autumn 2009 (A09)). <b>B</b>: Posterior distribution of the percentage of subjects with a 2 fold rise in AT among those with a rise in AT. Boxplots give percentiles 2.5%, 25%, 50%, 75%, 97.5% of the distribution.</p

Less technical description of the statistical method.

<p>This figure illustrates the description of the method that is made in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003061#ppat-1003061-box001" target="_blank">Box 1</a>.</p

Fit of the model to data on replicate measurements.

<p>Observed (red point) and expected (mean: blue point/95% CI: blue bar) number of pairs (observed AT level, replicate AT level). Pairs are sorted by panel according to the number of dilution difference between the observed and the replicate measurement.</p

Performance of the method to reconstruct the true distribution of paired serology.

<p>Eighty datasets are simulated with known parameters (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003061#s4" target="_blank">Methods</a>). <b>A</b>: Estimated percentage of subjects with an increase in antibody titers as a function of the true percentage in the simulated dataset. <b>B</b>: Estimated percentage of subjects with a decay in antibody titers as a function of the true percentage in the simulated dataset. <b>C</b>: Estimated probabilities characterizing jointly baseline AT level and the change in AT level during the epidemic _ similar to those presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003061#ppat-1003061-g001" target="_blank">Figure 1</a> _ as a function of the true probability in the simulated dataset.</p