Antigen delivery to dendritic cells shapes human CD4+ and CD8+ T cell memory responses to Staphylococcus aureus

Abstract

Intracellular persistence of Staphylococcus aureus favors bacterial spread and chronic infections. Here, we provide evidence for the existence of human CD4+ and CD8+ T cell memory against staphylococcal antigens. Notably, the latter could provide a missing link in our understanding of immune control of intracellular S. aureus. The analyses showed that pulsing of monocyte-derived dendritic cells (MoDC) with native staphylococcal protein antigens induced release of Th2-associated cytokines and mediators linked to T regulatory cell development (G-CSF, IL-2 and IL-10) from both CD4+ and CD8+ T cells, thus revealing a state of tolerance predominantly arising from preformed memory T cells. Furthermore, G-CSF was identified as a suppressor of CD8+ T cell-derived IFNγ secretion, thus confirming a tolerogenic role of this cytokine in the regulation of T cell responses to S. aureus. Nevertheless, delivery of in vitro transcribed mRNA-encoded staphylococcal antigens triggered Th1-biased responses, e.g. IFNγ and TNF release from both naïve and memory T cells. Collectively, our data highlight the potential of mRNA-adjuvanted antigen presentation to enable inflammatory responses, thus overriding the existing Th2/Treg-biased memory T cell response to native S. aureus antigens.

Fig 1. Comparison of IFNγ production by T cells stimulated with mRNA- or protein-derived staphylococcal antigens.

(a) CD4⁺ T cells and (b) CD8⁺ T cells in co-culture with MoDC were stimulated with either mRNA-encoded antigens or the corresponding protein antigens, e.g. spa / SpA, mecA / PBP2a and sitC / SitC. Lipofectamine (LF) alone, non-coding mRNA (NC) and a peptide pool from matrix protein 1 (MP1) of H1N1 Influenza virus and Tetanus toxoid (TT) served as controls. The number of IFNγ ELISpot spots after overnight culture is shown as mean values ± SEM of n = 8 donors. p**<0.01, p*< 0.05 (Wilcoxon matched-pairs signed rank test). Experiments were done in duplicates.

Fig 2. mRNA-promoted cytokine response and adjuvant effect.

(a) PBMC were co-cultured with autologous MoDC loaded with either non-coding control mRNA (NC), spa mRNA, SpA protein or the lipofection control (LF). IFNγ was detected after overnight incubation by ELISpot. The individual results of the single donors (n = 7) were connected with lines and displayed as IFNγ spots. Analysis was carried out in technical duplicates. Following testing for normal distribution, paired Student’s t-test was used to calculate significance. (b) Cytokine production by MoDC stimulated with mRNA-encoded staphylococcal antigens or the respective proteins and controls (lipofectamine (LF), non-coding control mRNA (NC), Influenza MP1 peptides (MP1) and tetanus toxoid (TT)) was measured in supernatants after 24 hours. The results of duplicates for TNF and IFNα are shown as mean values ± SEM of n = 4 donors. p≥ 0.05 n.s. (One-way ANOVA) (c) Stimulation of MoDC/CD8⁺ T cell co-cultures with PBP2a or SpA in the presence and absence of NC mRNA after overnight IFNγ ELISPOT analysis. The ELISPOT enzymatic activity relative to the unstimulated control is shown as mean values ± SEM of n = 8 independent donors. Statistical analysis was done using the Wilcoxon matched-pairs signed rank test. Experiments were carried out in duplicates. (d) Upon transfection of MoDC with spa mRNA and co-culture with CD8⁺ T cells, blocking antibodies against IFNα and TNFα alone or in combination were added and IFNγ secretion of duplicates was quantified by ELISPOT. IFNγ enzymatic activity of n = 5 independent donors is normalized to the unstimulated isotype control and displayed as mean ± SEM. Following testing for normal distribution, paired Student’s t-test was used to calculate significance. p**<0.01, p*< 0.05.

Fig 3. T cell cytokine profiles in response to spa mRNA-encoded and SpA protein delivered antigens.

Cytokine secretion profiles in day 5 supernatants of CD4⁺ (left panel) or CD8⁺ T cells (right panel) stimulated with spa mRNA or SpA protein antigens was performed using a multiplex cytokine array: (a) Th1 cytokines (IFNγ, TNF) and (b) Th2 cytokines (IL-5, IL-13). The graphs depict the mean values ± SEM obtained from n = 6 independent donors. Experiments were carried out in duplicates. For Th1 cytokines, one-way ANOVA was used to test significance of multiple conditions; for Th2 cytokines, p values refer to condition with spa mRNA (paired student’s t-test; p**<0.01, p*< 0.05).

Fig 4. Protein-derived antigens activate memory T cells.

Human (a) CD4⁺ or (b) CD8⁺ T cells were isolated from frozen PBMC via magnetic beads. Cell fractions were either purified for (a) CD14^-CD8^- and (b) CD14^-CD8⁺, and CD45RO^-CD45RA⁺ (naïve) or CD45RO⁺CD45RA^- (memory) phenotype. Cytokine secretion profiles after 5 days of MoDC/T cell co-culture stimulated with SpA protein were measured by multiplex cytokine array, done in duplicates. TNF, IFNγ, IL-5 and IL-13 are presented as mean ± SEM of n = 7 or 8 donors, respectively. p value refers to the same condition in naïve T cells. p**<0.01, p*< 0.05 (paired student’s t-test). For testing of multiple conditions, one-way ANOVA was used.

Fig 5. Antigen source-dependent activation of different T cell subsets.

MoDC/T cell co-cultures of n = 8 independent donors were stimulated with spa-encoding mRNA and/or SpA protein and analyzed by IFNγ/IL-13 FluoroSpot demonstrating cytokine secretion by different cell subsets dependent on the antigen delivery. The number of spots is displayed as mean values ± SEM. p*< 0.05, n.s. not significant (Wilcoxon matched-pairs signed rank test). Experiments were done in technical duplicates.

Fig 6. G-CSF-mediated regulation of IFNγ production.

Cytokine secretion profiles of (a) G-CSF, IL-2 and IL-10 in day 5 supernatants of CD4⁺ (left panel) or CD8⁺ T cells (right panel): MoDC/T cell co-cultures stimulated with spa, mecA or sitC mRNA or the corresponding protein antigens (SpA, PBP2a, SitC) were performed using a multiplex cytokine array. The graphs depict the mean values ± SEM obtained by n = 6 independent donors. Paired t-test was used to test for significance. The p-value refers to the mRNA condition of the corresponding protein antigen and cytokine. (b+c) MoDC were transfected with non-coding (NC) or spa mRNA and cultured in the presence of recombinant IL-2, IL-10 or G-CSF with (b) CD4⁺ or (c) CD8⁺ T cells. IFNγ ELISPOT enzymatic activity of n = 8 different donors was normalized to the NC mRNA control. Wilcoxon matched-pairs signed rank test was used to test for significance. If not indicated otherwise, p values refer to the untreated control transfected with spa mRNA. p**<0.01, p*< 0.05. All experiments were done in duplicates.