Impaired immune cell cytotoxicity in severe COVID-19 is IL-6 dependent

Abstract

BACKGROUNDCoronavirus disease 19 (COVID-19) is an emerging infectious disease caused by SARS-CoV-2. Antiviral immune response is crucial to achieve pathogen clearance; however, in some patients an excessive and aberrant host immune response can lead to an acute respiratory distress syndrome. The comprehension of the mechanisms that regulate pathogen elimination, immunity, and pathology is essential to better characterize disease progression and widen the spectrum of therapeutic options.METHODSWe performed a flow cytometric characterization of immune cell subsets from 30 patients with COVID-19 and correlated these data with clinical outcomes.RESULTSPatients with COVID-19 showed decreased numbers of circulating T, B, and NK cells and exhibited a skewing of CD8+ T cells toward a terminally differentiated/senescent phenotype. In agreement, CD4+ T and CD8+ T, but also NK cells, displayed reduced antiviral cytokine production capability. Moreover, a reduced cytotoxic potential was identified in patients with COVID-19, particularly in those who required intensive care. The latter group of patients also showed increased serum IL-6 levels that inversely correlated to the frequency of granzyme A-expressing NK cells. Off-label treatment with tocilizumab restored the cytotoxic potential of NK cells.CONCLUSIONThe association between IL-6 serum levels and the impairment of cytotoxic activity suggests the possibility that targeting this cytokine may restore antiviral mechanisms.FUNDINGThis study was supported by funds from the Department of Experimental and Clinical Medicine of University of Florence (the ex-60% fund and the "Excellence Departments 2018-2022 Project") derived from Ministero dell'Istruzione, dell'Università e della Ricerca (Italy).

Keywords: Cellular immune response; Cytokines; Immunology; Infectious disease; NK cells.

Figure 1. Absolute numbers of circulating WBCs in patients with COVID-19.

(A) Absolute numbers of total WBCs, neutrophils (N), lymphocytes (L), monocytes (M), eosinophils (E), basophils (B), and platelets (P) in healthy controls (black dots) and COVID-19 patients (gray dots). (B) Absolute numbers of CD3⁺ T cells, CD19⁺ B cells, and CD56⁺ NK cells in healthy controls (black dots) and COVID-19 patients (gray dots). (C) Absolute numbers of CD3⁺CD4⁺ Th cells, CD3⁺CD8⁺ cytotoxic T cells (CTL), and CD3⁺CD56⁺ NKT cells in healthy controls (black dots), and COVID-19 patients (gray dots). (D) Ratio of CD4/CD8 absolute numbers cells in healthy controls (black dots) and COVID-19 patients (gray dots). Red lines represent mean values for each population. Data obtained from 19 healthy controls and 27 COVID-19 patients. *P < 0.05; **P < 0.01; ***P < 0.001; calculated with Student’s t test.

Figure 2. Frequency of CD4⁺ and CD8⁺ T cell subsets in patients with COVID-19.

(A) Frequency of naive (CD45RA⁺CCR7⁺), central memory (CD45RA^–CCR7⁺), effector memory (CD45RA^–CCR7^–), TEMRA (CD45RA⁺CCR7^–), and HLA-DR⁺ cells among CD4⁺ T cells in healthy controls (black dots) and COVID-19 patients (gray dots). (B) Frequency of naive (CD45RA⁺CCR7⁺), central memory (CD45RA^–CCR7⁺), effector memory (CD45RA^–CCR7^–), TEMRA (CD45RA⁺CCR7^–), senescent (CD57⁺), and HLA-DR⁺ cells among CD8⁺ T cells in healthy controls (black dots) and COVID-19 patients (gray dots). Red lines represent mean values for each population. Data obtained from 8 healthy controls and 21 COVID-19 patients. *P < 0.05; **P < 0.01; ***P < 0.001; calculated with Student’s t test.

Figure 3. Functional characterization of CD4⁺ and CD8⁺ T cells and NK cells in patients with COVID-19.

(A) Frequency of IL-2–secreting cells among CD4⁺ and CD8⁺ T cells after in vitro polyclonal stimulation in healthy donors (black dots) and COVID-19 patients (gray dots). Frequency of IFN-γ–secreting cells (B) and TNF-α– secreting cells (C) among CD4⁺ and CD8⁺ T cells and NK cells after in vitro polyclonal stimulation in healthy donors (black dots) and COVID-19 patients (gray dots). Frequency of perforin-expressing cells (D) and granzyme A–expressing cells (E) among CD4⁺ and CD8⁺ T cells and NK cells in healthy donors (black dots) and COVID-19 patients (gray dots). Data obtained from 30 healthy controls and 30 COVID-19 patients. Red lines represent mean values for each population. *P < 0.05; ***P < 0.001; calculated with Student’s t test.

Figure 4. Absolute numbers of immune cell subsets in non-ICU versus ICU hospitalized patients with COVID-19.

(A) Absolute numbers of total WBCs, neutrophils (N), lymphocytes (L), monocytes (M), eosinophils (E), basophils (B), and platelets (P) in non-ICU (black dots) and ICU (gray dots) COVID-19 patients. (B) Absolute numbers of CD3⁺ T cells, CD19⁺ B cells, and CD56⁺ NK cells in non-ICU (black dots) and ICU (gray dots) COVID-19 patients. (C) Absolute numbers of CD3⁺CD4⁺ Th cells, CD3⁺CD8⁺ cytotoxic T cells (CTL), and CD3⁺CD56⁺ NKT cells in non-ICU (black dots) and ICU (gray dots) COVID-19 patients. (D) Frequency of naive (CD45RA⁺CCR7⁺), central memory (CD45RA^–CCR7⁺), effector memory (CD45RA^–CCR7^–), TEMRA (CD45RA⁺CCR7^–), and HLA-DR⁺ cells among CD4⁺ T cells in non-ICU (black dots) and ICU (gray dots) COVID-19 patients. (E) Frequency of naive (CD45RA⁺CCR7⁺), central memory (CD45RA^–CCR7⁺), effector memory (CD45RA^–CCR7^–), TEMRA (CD45RA⁺CCR7^–), senescent (CD57⁺), and HLA-DR⁺ cells among CD8⁺ T cells in non-ICU (black dots) and ICU (gray dots) COVID-19 patients. Data in A–C obtained from 16 non-ICU and 11 ICU COVID-19 patients. Data in D and E obtained from 10 non-ICU and 11 ICU COVID-19 patients. Red lines represent mean values for each population. *P < 0.05; calculated with Student’s t test.

Figure 5. Immune cell functional characterization in non-ICU versus ICU hospitalized COVID-19 patients.

(A) Frequency of IL-2–producing cells among CD4⁺ and CD8⁺ T cells following in vitro polyclonal stimulation in non-ICU (black dots) and ICU hospitalized patients (gray dots). Frequency of IFN-γ⁺ (B) and TNF-α⁺ (C) cells among CD4⁺ and CD8⁺ T cells and NK cells after in vitro polyclonal stimulation in non-ICU (black dots) and ICU hospitalized patients (gray dots). Frequency of perforin-expressing cells (D) and granzyme A–expressing cells (E) among CD4⁺ and CD8⁺ T cells and NK cells in non-ICU (black dots) and ICU hospitalized patients (gray dots). Data obtained from 18 non-ICU and 12 ICU COVID-19 patients. Red lines represent mean values for each population. **P < 0.01; calculated with Student’s t test.

Figure 6. Biological effects of tocilizumab administration in patients with COVID-19.

(A) IL-6 serum levels in non-ICU and ICU COVID-19 patients. Red lines represent mean values for each population. *P < 0.05; calculated with Student’s t test. (B) Correlation between serum IL-6 levels and percentage of granzyme A⁺ cells among NK cells in non-ICU (black dots) and ICU (gray dots) COVID-19 patients, calculated with Pearson correlation coefficient. Red line represents the trend line. Data presented in A and B obtained from 30 COVID-19 patients. Evaluation of (C) serum CRP, (D) absolute lymphocyte counts, (E) perforin⁺ cells among CD8⁺ T cells, (F) granzyme A⁺ cells among CD8⁺ T cells, (G) perforin⁺ cells among NK cells, (H) granzyme A⁺ cells among NK cells, and (I) PaO₂/FiO₂ ratio in 5 selected ICU COVID-19 patients before and after (72 hours) tocilizumab treatment. Each colored line represents the same patient in all plots. *P < 0.05; **P < 0.01; calculated with Student’s t test.