Tolerogenic Dendritic Cells Induce Apoptosis-Independent T Cell Hyporesponsiveness of SARS-CoV-2-Specific T Cells in an Antigen-Specific Manner

Abstract

Although the global pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still ongoing, there are currently no specific and highly efficient drugs for COVID-19 available, particularly in severe cases. Recent findings demonstrate that severe COVID-19 disease that requires hospitalization is associated with the hyperactivation of CD4⁺ and CD8⁺ T cell subsets. In this study, we aimed to counteract this high inflammatory state by inducing T-cell hyporesponsiveness in a SARS-CoV-2-specific manner using tolerogenic dendritic cells (tolDC). In vitro-activated SARS-CoV-2-specific T cells were isolated and stimulated with SARS-CoV-2 peptide-loaded monocyte-derived tolDC or with SARS-CoV-2 peptide-loaded conventional (conv) DC. We demonstrate a significant decrease in the number of interferon (IFN)-γ spot-forming cells when SARS-CoV-2-specific T cells were stimulated with tolDC as compared to stimulation with convDC. Importantly, this IFN-γ downmodulation in SARS-CoV-2-specific T cells was antigen-specific, since T cells retain their capacity to respond to an unrelated antigen and are not mediated by T cell deletion. Altogether, we have demonstrated that SARS-CoV-2 peptide-pulsed tolDC induces SARS-CoV-2-specific T cell hyporesponsiveness in an antigen-specific manner as compared to stimulation with SARS-CoV-2-specific convDC. These observations underline the clinical potential of tolDC to correct the immunological imbalance in the critically ill.

Keywords: T cell apoptosis; antigen specificity; severe COVID-19; tolerance; tolerogenic dendritic cells.

Figure 1

TolDC display a semimature phenotype and reduced T cell stimulatory capacity. (A) The percentage of cells expressing the markers HLA-DR, CD209 and CD14 and the percentage of cells that are CD209^negCD14^pos. TolDC are indicated in white dots; convDC are indicated in black dots (n = 6). (B) Expression levels of HLA-DR, CD83, CD80 and CD86 shown as MFI values of tolDC (white bars) or convDC (black bars) (n = 6). (C) Representative histogram overlay of CD83, CD80 and CD86 expression levels by tolDC (red), convDC (black) and isotype control (blue). (D) Concentration in pg/mL of IFN-γ secretion by PBL stimulated with convDC (black dots) and tolDC (white dots) (n = 6). Significance (p < 0.05) is indicated by *.

Figure 2

TolDC induce SARS-CoV-2-specific T cell hyporesponsiveness. (A) Gating strategy for the sorting of CD3⁺CD4⁺CD71⁺CD98⁺ and CD3⁺CD8⁺CD71⁺CD98⁺ cells. Cells were sorted from left to right as lymphocytes (FSC-A, SSC-A), single cells (FCS-A, FSC-H), viable cells (Fixable Near-IR Dead Cell Stain) and subsequent surface marker selection, with the sort gates indicated in red, (B) Representative figure of an FACS-sorted CD3⁺CD71⁺CD98⁺ T cell population indicating the purity of the sort. (C) Representative figure of the IFN-γ ELISpot wells from SARS-CoV-2-specific T cells stimulated with tolDC or with convDC, or SARS-CoV-2-specific T cells alone and rechallenged with SARS-CoV-2 peptides. (D) SFU counts normalized to SFU/100.000 cells in cocultures of SARS-CoV-2-specific T cells stimulated with tolDC (white bars) or with convDC (black bars) or SARS-CoV-2-specific T cells alone (grey bars). A p-value < 0.05 is indicated by * (n = 6). (E) Antigen-specific T cell stimulatory capacity in PBL stimulated with tolDC (white) or convDC (black) in the presence SARS-CoV-2 peptides and restimulated in an ELISpot assay with either SARS-CoV-2 peptides or CMV peptides. A p-value < 0.05 is indicated by * (n = 5). Abbreviations used: spot forming units (SFU), tolerogenic dendritic cells (tolDC), conventional dendritic cells (convDC), Interferon-gamma (IFN-γ).

Figure 3

The induction of T cell hyporesponsiveness by tolDC is T cell apoptosis-independent (n = 6). (A) Gating Strategy for the flow cytometric follow-up of T cell apoptosis. Lymphocytes are selected based on light scatter properties (FSC/SSC), and, subsequently, CD3⁺CD4⁺ T cells are gated. Next, a quadrant gating is used to differentiate between viable cells (SytoxAADvanced⁻Annexin-V⁻), early apoptotic cells (SytoxAADvanced⁻Annexin-V⁺) and late apoptotic cells (SytoxAADvanced⁺Annexin-V⁺). The expression levels of caspase 3/7 are assessed via MFI histograms. (B) Follow-up of viable (white), early apoptotic (black) and late apoptotic (grey) cells in T cell–DC cocultures as compared to T cells alone by means of flow cytometry. (C) Mean fluorescence intensity of intracellular caspase 3/7 levels in T cell–DC cocultures at different timepoints, indicated as the median and interquartile range. Significance is indicated by * (p < 0.05) and ** (p < 0.01).