Heightened Circulating Interferon-Inducible Chemokines, and Activated Pro-Cytolytic Th1-Cell Phenotype Features Covid-19 Aggravation in the Second Week of Illness

Abstract

Covid-19 features a delayed onset of critical illness occurring approximately one week from the beginning of symptoms, which corresponds to the bridging of innate and adaptive immunity. We reasoned that the immune events occurring at the turning point of disease might mark the direction toward pathogenic versus protective inflammatory responses. Subjects with either severe (s; PaO2/FiO2 ratio <200) or mild (m; PaO2/FiO2 ratio>300) Covid-19 were enrolled. A range of chemokines and cytokines as well as reactive oxygen species (ROS) were measured in plasma. Dendritic and NK cell frequency, monocyte and B-/T-cell phenotype and SARS-CoV-2-specific T-cell responses were assessed in PBMC. Twenty mCovid-19 and 20 sCovid-19 individuals were studied. sCovid-19 patients displayed higher non-classical monocytes, plasma chemokines (CXCL8, CXCL9, CXCL10), cytokines (IL-6, IL-10), and ROS versus mCovid-19. sCovid-19 also showed significantly increased activated CD38+HLA-DR+ T-lymphocyte, and granzyme-B+/perforin+ pro-cytolytic T-cells. All Covid-19 patients showed SARS-CoV-2 specific-T-cell response with a predominance of Th1 bi- or trifunctional IFN-γ/IL-2/TNF-α-expressing CD4+, while no difference according to disease severity was observed. Severe Covid-19 features heightened circulating IFN-inducible chemokines and activated pro-cytolytic Th1 cell phenotype in the second week of illness, yet SARS-CoV-2-specific responses are similar to that of mild illness. Altogether, our observations suggest Th1 polarization coupled to higher cytolytic profile in sCovid-19 as correlate of disease pathogenesis and as potential targets to be investigated in the roadmap to therapy and vaccine development.

Keywords: Covid-19; S/M/N protein-reactive T-cells; SARS-CoV-2; immunity; immunopathology.

Figure 1

Frequency and phenotypes of monocyte subsets and levels of plasma chemokines according to the degree of Covid-19 diseases. Peripheral blood mononuclear cells (PBMCs) were isolated and analyzed from blood samples of 40 COVID-19 patients [severe Covid-19 (sCovid-19) n = 20 and mild Covid-19 (mCovid-19) n = 20]. (A, B) No differences were observed in classical monocytes (CD14+CD16−) and in intermediate monocytes (CD14++CD16+) between the two study groups. (C) Non-classical monocytes (CD14+CD16++) were increased in (sCovid-19) compared to (mCovid-19). (D–F) HLA-DR mean fluorescent intensity (MFI) on monocytes is lower in sCovid compared to mCovid patients. (G) CXCL8/IL-8 plasma levels were increased in sCOVID-19 patients in comparison to mCOVID-19 patients, albeit not reaching significance (p  =  .06). (H, I) sCOVID-19 patients showed significantly higher circulating levels of CXCL9/MIG and CXCL10/IP-10 in comparison to mCOVID-19 patients (p  =  0.01 and p = 0.02) (J, K) When mCOVID-19 patients were compared to sCOVID-19 patients, no differences in CCL5/RANTES and CCL2/MCP-1 plasma levels were detected. Graph box and whiskers represent medians and 10^th–90^th range percentile range. Dotted lines indicate the median levels, and solid lines indicate 10^th–90^th range of healthy controls (HC) from archived material. Comparison between groups (sCovid-19 vs mCovid-19) Mann–Whitney test.

Figure 2

Levels of plasma cytokines and oxidative stress according to the degree of Covid-19 diseases. Plasma samples were collected and analyzed from blood samples of 20 COVID-19 patients [severe Covid-19 (sCovid-19) n = 10 and mild Covid-19 (mCovid-19) n = 10]. (A–D) No major differences in Interferon (IFN)-α, IFN-γ, Interleukin (IL)-4 and IL-5 were observed between (mCovid-19) and (sCovid-19). (E, F) We observed increased levels of IL-6 and IL-10 in sCovid-19 compared to m-Covid-19 (p = 0.004 and p = 0.02). (G–I). The levels of IL-12, IL-17A and tumor necrosis factor (TNF)-α were comparable between mCovid-19 and sCovid-19. (J) sCOVID-19 patients showed significantly higher circulating levels of reactive oxygen species (ROS) in comparison to mCOVID-19 patients (p  =  0.032). Graph box and whiskers represent medians and 10^th–90^th percentile range. Dotted lines indicate the median levels, and solid lines indicate 10^th–90^th range of healthy controls (HCs) from archived material. Comparison between groups (sCovid-19 vs mCovid-19) Mann–Whitney test.

Figure 3

T-cell activation, maturation, and cytolysis markers among COVID-19 patients. Peripheral blood mononuclear cells (PBMCs) were isolated and analyzed from blood samples of 40 COVID-19 patients [severe Covid-19 (sCovid-19) n = 20 and mild Covid-19 (mCovid-19) n = 20]. (A, B) HLA-DR+CD38+ expressing both in CD4+ and CD8 T-cells were significantly increase in (sCovid-19) compared to (mCovid-19) (p = 0.06 and p = 0.06). (C, D) We observed no difference in naïve (CCR7+CD45RA+), CM: central memory (CCR7+CD45RA−) and TD: terminally differentiated (CCR7−CD45RA+). sCOVID-19 patients showed significantly lower CD4+ and CD8+ EM: effector memory (CCR7−CD45RA−) (p = 0.03 and p = 0.02). (E) Compared to mCovid-19, sCovid-19 patients tended to have a higher number of CD4+granzyme+ cells (p = 0.001). We fail to observe any modification in CD4+ perforin + cells. The number of CD4+granzyme+perforin+ cells was higher in sCovid-19 patients when compared to mCovid-19 (p = 0.05). (F) No major differences in CD8+ granzyme+ and CD8+ perforin+ were observed between mCovid-19 and sCovid-19. A non-significant tendency towards increased CD8+ granzyme-perforin+ frequency was observed in sCovid-19 patients in comparison to mCovid-19 (p  =  .09). In each graph the columns represent the median values, while the error bars indicate the 10^th–90^th percentile range. Dotted lines indicate the median levels and solid lines indicate 10^th–90^th percentile range of healthy controls (HCs) from archived material. Comparison between groups (sCovid-19 vs mCovid-19) Mann–Whitney test.

Figure 4

T-cell cytokine secretion upon phorbol myristate acetate (PMA)/ionomycin, and Sars-Cov-2 challenge: Severe Covid-19 (sCovid-19) vs mild Covid-19 (mCovid). Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples of unselected 20 COVID-19 patients (n = 10 sCovid-19 and n = 10 mCovid-19). PBMCs were incubated for 16 h with SARS-CoV-2 spike (S)-, membrane (M)-, and nucleocapsid (N)-protein peptide pools and analyzed with flow cytometry. (A, B) Frequencies of interferon-γ (IFN-γ)−, tumor necrosis factor-α (TNF-α)−, interleukin (IL)-2−, IL-4−, and IL-17A producing antigen-specific CD4+ and CD8+ T-cells upon (A) Phorbol myristate acetate (PMA) and ionomycin, (B) Sars-Cov-2 pool. In each graph, the columns represent the median values, while the error bars indicate the 10^th–90^th percentile range. Frequencies were corrected by background subtraction as determined in non-stimulated controls. Dotted lines indicate the median levels of healthy controls (HC) from archived material. No major differences in CD4 and CD8 cytokine productions were observed between mCovid-19 and sCovid-19. Comparison between groups (sCovid-19 vs mCovid-19) Mann–Whitney test. (C, D) Tables represent the total production of different cytokines for all enrolled patients. Green boxes represent mCovid-19 patients, and red boxes represent sCovid-19 patients. (E–P) Composition of bi- and trifunctional CD4+ and CD8+ T-cells after stimulation with Sars-Cov-2 pool. The fraction of positive subsets (above 90^th percentile of healthy controls) of all bi- or trifunctional cells was calculated for each combination of cytokine. Colored lines indicate 90^th and 10^th percentile ranges of healthy controls.