A Longitudinal Study of Immune Cells in Severe COVID-19 Patients

Abstract

Little is known about the time-dependent immune responses in severe COVID-19. Data of 15 consecutive patients were sequentially recorded from intensive care unit admission. Lymphocyte subsets and total monocyte and subsets counts were monitored as well as the expression of HLA-DR. For 5 patients, SARS-CoV-2-specific T-cell polyfunctionality was assessed against Spike and Nucleoprotein SARS-CoV-2 peptides. Non-specific inflammation markers were increased in all patients. Median monocyte HLA-DR expression was below the 8,000 AB/C threshold defining acquired immunodepression. A "V" trend curve for lymphopenia, monocyte numbers, and HLA-DR expression was observed with a nadir between days 11 and 14 after symptoms' onset. Intermediate CD14⁺⁺CD16⁺ monocytes increased early with a reduction in classic CD14⁺⁺CD16^- monocytes. Polyfunctional SARS-Cov-2-specific CD4 T-cells were present and functional, whereas virus-specific CD8 T-cells were less frequent and not efficient. We report a temporal variation of both innate and adaptive immunity in severe COVID-19 patients, helpful in guiding therapeutic decisions (e.g. anti-inflammatory vs. immunostimulatory ones). We describe a defect in virus-specific CD8 T-cells, a potential biomarker of clinical severity. These combined data also provide helpful knowledge for vaccine design.

Clinical trial registration: https://clinicaltrials.gov/, identifier NCT04386395.

Keywords: SARS-CoV-2; antigen-specific polyfunctional T-cells; immunity; intensive care unit; monocyte HLA-DR.

Figure 1

Leukocyte absolute number variations according to the delay from the onset of symptoms. HLA-DR: Human Leucocyte Antigen-DR expressed as events per cell (AB/C); *p < 0.05.

Figure 2

Time evolution of HLA-DR expression according to the delay from the onset of symptoms for 3 monocyte subtypes. Blue: intermediate (CD14⁺⁺CD16⁺) monocytes; green: classical (CD14⁺⁺CD16^-) monocytes; red: non-classical (CD14⁺CD16⁺⁺) monocytes. HLA-DR: Human Leucocyte Antigen-DR expressed as events per cell (AB/C). *Kruskall-Wallis test comparing the median values of HLA-DR expression of monocytes subtypes during all stay. Log: Log₁₀. At left normal expected values for HLA-DR expression in total monocytes.

Figure 3

Polyfunctional Index (PI) of SARS-CoV-2–specific CD4 and CD8 T-cells. The PI of antigen-specific CD4 and CD8 T-cells from 5 patients was calculated as previously described (18, 20) for each antigen stimulation. Data are expressed after subtraction of “background polyfunctionality” of medium-stimulated cells (Δ PI). Dotted lines correspond to PI positivity threshold as determined at 3SD of 30 negative controls measures for CD4 and CD8 T-cells. Bars are shown as mean ± SEM. LT CD4: CD4 T-cells, LT CD8: CD8 T-cells.

Figure 4

SARS-CoV-2–specific-T-cells numbers and functions. Peripheral antigen-specific CD4 and CD8 T-cells from 5 patients were identified by intracellular staining of IFN-κ, TNF-α and IL-2 by flow-cytometry as described (see Material and Methods and Suppl. Methods sections). Shown are individual data from stimulation with SARS-CoV-2 Nucleoprotein and Spike Glycoprotein peptides pools as well as by multiple immunodominant microbial peptides (CEFX pool) for CD4 and CD8 T-cells and for each cytokine capability (monofunctional, bifunctional or trifunctional). Basal cytokine production of medium-stimulated cells was subtracted for each antigen-stimulated cell subtype and a 0.01% positivity threshold was defined (dotted lines). Bars are shown as mean ± SEM. LT CD4: CD4 T-cells, LT CD8: CD8 T-cells.