Disease-duration based comparison of subsets of immune cells in SARS CoV-2 infected patients presenting with mild or severe symptoms identifies prognostic markers for severity

Abstract

Introduction: Infection with SARS-CoV-2 leads to a spectrum of symptoms. Understanding the basis for severity remains crucial for better management and therapy development. So far, older age, associated-comorbidities, and IL-6 have been associated with severity/mortality.

Materials and methodology: As a primary step, we analyzed the frequency and functional profile of innate immune cells (NK cells/dendritic cells/monocytes) and adaptive immunity-driving lymphocytes (B cells/T cells/follicular T helper cells) by flow cytometry. Sixty cases of SARS CoV-2 infection (25 severe, 35 mild) and ten healthy subjects without SARS CoV-2 IgG were included. Disease-duration based analysis of immune profile was explored for early events differentiating the two disease forms. Neutralizing antibody titers were determined by PRNT.

Results and conclusion: Disease severity was found to be associated with impaired maturation of mDCs and hyperactivation of NK, follicular T helper cells, and CD8 T cells. Lower IL-21 receptor expression on memory B cells indicated an imbalance in IL-21/IL-21 R ratio. Lower BCMA positive plasmablast cells in severe cases did suggest a probable absence of long-term humoral immunity. Multivariate analysis revealed a progressive association of PD-1+CD4 T cells with PRNT₅₀ titers. Thus, in addition to identifying probable prognostic markers for severity, our study emphasizes the definite need for in-depth viral antigen-specific functional analyses in a larger patient cohort and with multiple sampling.

Keywords: PRNT50; SARS CoV-2; adaptive immune cells; disease severity; innate immune cells.

Figure 1

Gating strategy for the enumeration of immune cells in different study groups. Gating strategy used to distinguish different lymphocyte populations by polychromatic flow cytometry: The PBMCs from SARS CoV‐2 infected cases were stained with given number of panels of fluorochrome‐labeled antibodies to assess the frequency and functional profile of T cells (CD4 and CD8 T cells), follicular T helper cells (T_FH), B cells, Dendritic cells, and monocytes. List of the antibodies with their clones and fluorochrome dyes is provided as Supporting Information Table. Flow cytometry data were analyzed by using Cytexpert Version 2.4. (A) CD4 and CD8 T cells‐Lymphocyte populations were identified based on forward and side scatter properties of PBMCs. The CD3 positive population was further discriminated to identify CD4 and CD8 T cells. Then CD8 T cells were analyzed for HLA DR & CD38 expression whereas the CD4 T cell population was analyzed for CD40L and IL‐2 expression. (B) T_FH cells‐follicular T cells were identified based on PD‐1 and CXCR5 expression on CD4 T cells as mentioned above. The T_FH population was further inspected for ICOS and IL‐21 expression. (c) NK cells‐Natural killer cells were identified as CD3‐CD56+CD16+/− (total NK cell population comprised cytotoxic NK cells (CD3‐CD56+CD16+) and Regulatory NK cells (CD3‐CD56+CD16‐). (D) Dendritic cells‐lymphomono gate was identified by using forward and side scatter properties of the cells. The cells were checked for Lineage cocktail (CD3, CD19, CD14, CD56, and CD20) positivity. Lineage cocktail negative cells were selected and checked for HLA DR expression. Thus, Lin‐ HLA DR+CD11c+CD1C+ cells were considered as myeloid dendritic cells (mDC) whereas Lin‐ HLA DR+CD11c‐CD123+ cells were considered as plasmacytoid dendritic cells (pDC). (E) Monocytes‐monocyte gate was recognized by FS & SS contour plot. Further, the CD3 negative cells from this population were screened for CD14 expression to identify the monocyte population. The monocyte population was further analyzed for CD16 expression to discriminate classical and nonclassical monocytes. (F) B cells and Plasmablast cells‐B cells from lymphocytes were identified with the help of CD19 expression. For further differentiation, expression of CD27 (memory B cells), IgD/IgM (class‐switched memory B cells [CSMB] and unswitched memory B cells [USMB]), and CD138 (plasmablast cells) was considered

Figure 2

Innate immune response in relation to COVID‐19 disease severity: Vertical scatter plots denote the comparisons of frequencies of innate immune cells and their subpopulations among different study groups: (A) Frequencies of dendritic cell subsets. (B) Monocyte frequencies. (C) NK cell population. (D) Activated NK cell (Mann–Whitney U test; Error bars‐median and IQR). IQR, interquartile range

Figure 3

Adaptive immune response in relation to COVID‐19 disease severity (T cells): Vertical scatter plots denote the comparisons of frequencies of adaptive immune cells and their subpopulation among different study groups: (A) CD4 and CD8 frequencies (B) CD4/CD8 ratio. (C) CD8 T cell compartment with HLA DR and CD38 expression. (D) Frequency of CD40L positive CD4 T cells. (E) Follicular T cells (T_FH) cells and T_FH cells with ICOS and IL‐21 expression. (F) CXCR5+CD4+ T cells and PD‐1+CD4+T cells. (Mann–Whitney U test; Error bars, median and IQR). IQR, interquartile range

Figure 4

Adaptive immune response in relation to COVID‐19 disease severity (B cells and plasmablasts): Vertical scatter plots denote the comparisons of frequencies of B cells and plasmablast populations and their subpopulations among different study groups: (A) B cells, (B) Memory B cells, (C) Class switched and unswitched memory B cells, (D) Plasmablasts cells & BCMA+plasmablast cells, (E) SARS CoV‐2 PRNT50 titers in mild (median: 53.05; IQR: 22.49–294.8) and severe cases (median: 571.1; IQR: 50.31–2247) within 15 days of disease onset. (F) Association of SARS CoV‐2 PRNT₅₀ titers (15 days of disease onset) with PD‐1+CD4 T cells. (G) Association of SARS CoV‐2 PRNT₅₀ titers (15 days of disease onset) with memory B cells. (Mann–Whitney U test; Error bars, median and IQR). IQR, interquartile range

Figure 5

Plasma Th1/Th2 Cytokine levels in SARS Cov‐2 Infection: (A) Th1 cytokine profile (IFN‐γ, TNF‐α, and IL‐2). (B) Th2 cytokine profile (IL‐10, IL‐6, and IL‐4; Mann–Whitney U test; Error bars, median and IQR). IQR, interquartile range

Figure 6

Immune profiles in SD (A–C) and MD (D) patients at first and last sampling: Reduction in (A) Il‐21R positive memory B cells, (B] NK cell frequency, and (C) IFN‐γ positive NK cells. In the MD group (D) CD86 expression by myeloid DCs increased (Wilcoxon signed‐rank test).

Figure 7

Schematic presentation of the major findings: Multiparametric flow cytometry was used to enumerate immune cells from innate ((NK cells, DCs, and monocytes and adaptive (T cells and B cells) immunity arms in COVID‐19 patients presenting with mild or severe disease. The findings pointed out major defects in the maturation (CD80 and CD86 expression) of myeloid dendritic cells and reduction in plasmacytoid dendritic cell frequency, emphasizing compromised antigen presentation. Enhanced frequency of nonclassical monocytes could be an important event in maintaining vascular homeostasis in the inflammatory cytokine milieu formed due to the infection. Activated NK and CD8 T cell subsets observed in severe cases may enhance lung tissue injury and disease severity. Likewise, activation of T_FH, and CD4 T cell compartments in these cases may lead to a transient higher humoral response observed during the acute phase of infection. However, whether these antibodies are protective or aid in disease severity remains unclear. Lower IL‐21 receptor expression on B cells may lead to impaired proliferation and differentiation of B cells. Moreover, plasmablast cells showed a comparatively lower BCMA expression, a long term survival marker for plasma cells. Collectively, long term immunity is likely to be hampered, another observation being reported recently. The role of crucial immune cells identified in this study needs to be confirmed by functional assays.