Linear regression model of CD8⁺ T cell mediated target killing as a function of polyfunctionality based on three variance-optimal effector molecules.

<p>Linear regression model of CD8⁺ T cell mediated target killing as a function of polyfunctionality based on three variance-optimal effector molecules.</p

Linear regression model of CD8⁺ T cell mediated target killing as a function of polyfunctionality based on five effector molecules.

<p>Linear regression model of CD8⁺ T cell mediated target killing as a function of polyfunctionality based on five effector molecules.</p

Polyfunctionality of HIV-specific CD8⁺ T cells is associated with T cell mediated target killing.

<p><i>In vitro</i> cultured human HIV-specific (KK10) CD8⁺ T cell clones were incubated with KK10-loaded target cells (HLA-B*2705⁺ lymphoblastoid cell lines). To measure T cell killing a standard chromium release assay was employed using ⁵¹Cr charged target cells loaded with a serial dilution of KK10-antigen [10^-6M—10^-13M]. Non-⁵¹Cr charged target cells with an identical serial dilution of KK10-antigen were employed to analyse T cell polyfunctionality by multiparametric flow cytometry (CD107a, IFN-γ, TNF-α, IL-2, MIP-1β). <b>A)</b> Scatter plots showing the association between T cell killing (⁵¹Cr release / max ⁵¹Cr release) and T cell polyfunctionality quantified as the polyfunctionality index (<i>q</i> = 1). <b>B)</b> Non-linear regression for all T cell clones identifies the optimal q-value as 1.24.</p

The principles of single-cell polyfunctionality analysis for modelling associated variables, such as T cell efficacy.

<p>3 (n) bimodial effector molecules (A, B and C) were measured at the single-cell level identifying 2³ = 8 distinct combinatorial cell subsets, which can be stratified according to the number of simultaneous functions at the single-cell level (i). <b>A)</b> “Classical” polyfunctionality analysis generally assess only cells positive for a defined minimal number of simultaneous effector molecules (i.e. 3 simultaneous functions). <b>B)</b> Contrarily, the polyfunctionality index considers all 8 functional subsets and ingeniously parameterizes the influence of individual (φ_A>φ_B>φ_C) as well as combined (<i>q</i>) functionalities. <b>C)</b> Polyfunctionality assessed with the polyfunctionality index algorithm is therefore modulable, contrary to “classic” polyfunctional analysis, enabling a more optimal fit (green line) of model variables. <b>D)</b> Using regression analysis it is feasible to obtain proper parameter estimates (φ_A, φ_B, φ_C and q), which have biological significance. Indeed, interpretation of such parameters enables an objective evaluation of the influence of individual as well as combinatorial functions on the predictive capacity of polyfunctionality with regards to a desired model variable, such as T cell efficacy.</p

Redundancy of effector molecules with regards to the association between HIV-specific CD8⁺ T cell polyfunctionality and T cell mediated target killing.

<p><i>In vitro</i> cultured human HIV-specific (KK10) CD8⁺ T cell clones were incubated with a serial dilution of KK10-antigen [10^-6M—10^-13M] loaded target cells. T cell polyfunctionality was analysed by multiparametric flow cytometry (CD107a, IFN-γ, TNF-α, IL-2, MIP-1β). <b>A)</b> Stacked bar diagram indicates the probability distribution of T cells expressing 5, 4, 3, 2 or 1 simultaneous effector molecules, given that they express one particular effector molecule. <b>B)</b> Variance Inflation Factor (VIF) analysis of the 5 effector molecules shows the lowest VIF for each molecule after iterative retraction of the effector molecule with the highest VIF>5 (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128714#pone.0128714.t003" target="_blank">Table 3</a>).</p

Formulae and application of polyfunctionality algorithms in establishing their relationship with T cell efficacy.

<p>NA = Not Applicable, PI = Polyfunctionality Index</p><p>* For complete description of the parameters, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128714#sec006" target="_blank">materials and methods</a> section.</p><p>** These adjustments are required in the event that the PI does not range [0, 1].</p><p>Formulae and application of polyfunctionality algorithms in establishing their relationship with T cell efficacy.</p

Thermoresponsive Polymer Nanoparticles Co-deliver RSV F Trimers with a TLR-7/8 Adjuvant

Structure-based vaccine design has been used to develop immunogens that display conserved neutralization sites on pathogens such as HIV-1, respiratory syncytial virus (RSV), and influenza. Improving the immunogenicity of these designed immunogens with adjuvants will require formulations that do not alter protein antigenicity. Here, we show that nanoparticle-forming thermoresponsive polymers (TRP) allow for co-delivery of RSV fusion (F) protein trimers with Toll-like receptor 7 and 8 agonists (TLR-7/8a) to enhance protective immunity. Although primary amine conjugation of TLR-7/8a to F trimers severely disrupted the recognition of critical neutralizing epitopes, F trimers site-selectively coupled to TRP nanoparticles retained appropriate antigenicity and elicited high titers of prefusion-specific, T_H1 isotype anti-RSV F antibodies following vaccination. Moreover, coupling F trimers to TRP delivering TLR-7/8a resulted in ∼3-fold higher binding and neutralizing antibody titers than soluble F trimers admixed with TLR-7/8a and conferred protection from intranasal RSV challenge. Overall, these data show that TRP nanoparticles may provide a broadly applicable platform for eliciting neutralizing antibodies to structure-dependent epitopes on RSV, influenza, HIV-1, or other pathogens