Proteins from SARS-CoV-2 reduce T cell proliferation: A mirror image of sepsis

Abstract

Increased cytokine levels, acute phase reactants and immune checkpoint expression changes have been described in patients with Coronavirus Disease 2019 (COVID-19). Here, we have reported a monocyte polarization towards a low HLA-DR and high PD-L1 expression after long exposure to proteins from SARS-CoV-2. Moreover, CD86 expression was also reduced over SARS-CoV-2 proteins exposure. Additionally, T-cells proliferation was significantly reduced after stimulation with these proteins. Eventually, patients with long-term SARS-CoV-2 infection also exhibited a significant blockade of T-cells proliferation.

Keywords: COVID19; Immune checkpoints; Immunology; Infectious disease; Inflammation; PD-L1/PD-1; Pathology; Sepsis; T cell exhaustion; T cell proliferation; Virology.

Figure 1

Proteins from SARS-CoV-2 polarize human monocytes towards an HLA-DR^lowPD-L1^high phenotype. (A) Experimental design. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were cultured for 7 days and stimulated once or every 48 h with a cocktail of proteins from the virus including S glycoprotein (250 ng/mL), N protein (250 ng/mL) and the Papain-like protease (P, 25 nM). (B) Viability of CD14⁺ cells were measured by 7-AAD staining of dead cells cultured following the experimental design (A) is shown. (C) Mean Fluorescence Intensity (MFI) expressions of HLA-DR on CD14⁺ cells are shown (left panel) (∗, p < 0.05, one-way ANOVA test), n = 5. A representative histogram overlay is shown, fluorescent minus one (FMO, dotted line), unstimulated control (empty), S + N + P (solid black), S + N + P multiple stimulations (solid red), right panel. (D) MFI expressions of CD86 on CD14⁺ cells are shown (left panel) (∗, p < 0.05, one-way ANOVA test), n = 5. A representative overlay is shown, fluorescent minus one (FMO, dotted line), unstimulated control (empty), S + N + P (solid black), S + N + P multiple stimulations (solid red), right panel. (E) MFI expressions of PD-L1 on CD14⁺ cells are shown (left panel) (∗, p < 0.05, one-way ANOVA test), n = 5. A representative overlay is shown, fluorescent minus one (FMO, dotted line), unstimulated control (empty), S + N + P (solid black), S + N + P multiple stimulations (solid red), right panel. (F and G, left panels) Expressions of PD-1 on CD4⁺ (F) and CD8⁺ (G) cells are shown. Viability of CD4⁺ (F) and CD8⁺ (G) cells are shown (right panels).

Figure 2

Long exposure to SARS-CoV-2 proteins reduces the T lymphocytes proliferative ability in vitro.PBMCs from healthy volunteers were labelled with CFSE, then cells were exposed or not (open circle) to S, N and P proteins for 5 days (solid black square) or multiple stimulations (Figure 1A) until 5 days (solid red triangle). Cells were also stimulated or not (open triangle) with Pokeweed (PWD, 2.5 μg/mL). (A) Percentage of proliferative CD4⁺ populations (CFSE^dim), measured by flow cytometry, are shown (left panel) (∗, p < 0.05, Mann-Whitney t-test; ∗∗, p < 0.01, one-way ANOVA test), n = 5. A representative histogram overlay is shown (right panel). (B) Percentage of proliferative CD8⁺ populations, measured by flow cytometry, are shown (left panel) (∗, p < 0.05, Mann-Whitney t test; ∗∗, p < 0.01, one-way ANOVA test), n = 5. A representative histogram overlay is shown (right panel).

Figure 3

T-cells proliferation is recovered by PD-L1/PD-1 axis blockage. PBMCs from healthy volunteers were labelled with CFSE, then cells were exposed or not (open circle) to S, N and P proteins for 5 days (solid black square). Anti-PD-L1 (solid black triangle) or anti-PD-1 (solid grey inverted triangle) were added. All cultures were also stimulated with Pokeweed (PWD, 2.5 μg/mL). Percentage of proliferative (CFSE^dim) CD4⁺ (A) and CD8⁺ (B) populations, measured by flow cytometry, are shown (∗, p < 0.05, paired t-test), n = 5. (C) An array analysis is shown for indicated cytokines, that were analyzed in the supernatant by Cytometric Bead Array (CBA) after stimulation with S, N and P proteins from SARS-CoV-2 (S + N + P) or not (control) of PBMCs from healthy volunteers for 5 days; n = 5.

Figure 4

SARS-CoV-2 proteins induce cytokine production. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were stimulated with SARS-CoV-2 proteins S glycoprotein (250 ng/mL), N protein (250 ng/mL) and the Papain-like protease (P, 25 nM). Levels of IL-2, IL-6, IL-10, IL-17, TNFα and INFγ in cell culture supernatants after 24, 72, 120 and 168 h are shown, n = 5.

Figure 5

Monocytes from COVID-19 patients have decreased HLA-DR and increased PD-L1 expression. (A) HLA-DR (left) and PD-L1 (right) expression on CD14⁺ cells from COVID-19 patients (n = 25) and HVs (n = 6). (B) COVID-19 patients were classified according their admission time. Their HLA-DR (left) and PD-L1 (right) expression on CD14⁺ cells are shown. (C) Spearman correlation between sample collection time collection from patient admission time and the expression of HLA-DR (left) and PD-L1 (right). ∗, p < 0.05; ∗∗, p < 0.01 Mann-Whitney t-test for (A) and (B) and Spearman correlation test in (C); MFI, Mean Fluorescence Intensity; R = Spearman r; vs, versus.

Figure 6

Patients exposed to SARS-CoV-2 exhibit a low rate of T-cells proliferation. Four groups of COVID-19 patients were recruited according to their length of hospital stay: 0–2 days (n = 5), 5–7 days (n = 5), 8–19 days (n = 6), or more than 20 days (n = 9). PBMCs were isolated from these and from healthy volunteers (HVs, n = 6), labelled with CFSE and stimulated with Pokeweed (PWD, 2.5 μg/mL, solid black circle) or anti CD3/CD28 beads (0.5 μL/well, open black circle) for 5 days. Percentage of proliferative CD4⁺ (A) and CD8⁺ (B) populations, measured by flow cytometry, are shown (∗, p < 0.05; ∗∗, p < 0.01 Mann-Whitney t-test). The RT-PCR status for SARS-CoV-2 infection at moment of blood recruitment for each group of patients are also shown.