Analysis of Immune Profiles Related to Disease Severity in COVID-19 by Flow Cytometry

Coronavirus Disease 2019 (COVID-19) caused by a new type of coronavirus named Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) was first reported in Wuhan in China's Hubei province at late 2019 (1, 2). The World Health Organization (WHO) declared the viral disease pandemic on 11 March 2020. So far, more than 768 million confirmed cases and 6.9 million deaths have been reported during the pandemic over three years (3). Although SARS-CoV-2 infections may be asymptomatic, symptoms such as fever, dry cough, fatigue, and sputum production or respiratory clinical manifestations ranging from shortness of breath (dyspnea) to pneumonia, pulmonary edema and acute respiratory distress syndrome (ARDS) may present in infected individuals (1, 4-6).

Acute respiratory distress syndrome rapidly develops, especially in elderly individuals and those with secondary underlying diseases such as hypertension, diabetes, chronic obstructive pulmonary disease, and cardiovascular disease. These individuals may develop septic shock, difficult-to-treat metabolic acidosis, and coagulation dysfunctions. The mortality and morbidity associated with COVID-19 are attributed to the abnormal host's immune response, cytokine storm, excessive inflammation, and concomitant comorbidities of infected individuals. Endothelial damage, organ dysfunctions, and lung damage develop due to increased and excessive innate and adaptive immune responses. As the disease progresses, irregularities occur in the functions and distribution of lymphocyte subtypes (1, 7, 8). Cytokine storm characterized by an abnormal host inflammatory response with severe lymphopenia accompanied by high levels of proinflammatory cytokines has been defined as a process that worsens COVID-19 prognosis (9, 10).

Identifying lymphocyte subsets and abnormalities in cytokine levels in COVID-19 patients is essential to gain new insights and data on the immunity mechanisms to virus infection. However, immunological values show heterogeneity due to age, regional differences, and genetic characteristics between cases (11). It is crucial to address these doubts by gathering regional data on potential disparities in peripheral blood lymphocyte subset changes and cytokine levels concerning the survival of patients with different degrees of COVID-19 severity. This study aimed to determine the distribution of lymphocyte subtypes and levels of peripheral blood cytokines in patients hospitalized for mild, moderate, and severe COVID-19 infection at a single center in Turkey. The study also aimed to evaluate the relationship between disease severity, changes in T lymphocytes and cytokine levels, and their impact on prognosis.

Age, hemoglobin and hematocrit values, white blood cell and lymphocyte counts, lymphocyte and monocyte percentages, D-dimer, INR, and CRP values were statistically different between the groups. Hemoglobin values were significantly lower in the severe group than in the others. Also, lymphocyte counts decreased significantly as the disease severity increased. Comparative analyses of demographic features and laboratory parameters are summarized in Table 1. Md represents the middle of values when arranged in increasing order. IQR represents the difference between the first quartile (Q1) and third quartile (Q3) of the data.

According to flow cytometry analysis, alterations in CD3⁺ T cells between the groups were statistically significant. In moderate and severe cases, CD3⁺ cell populations significantly decreased compared to the control and mild groups. No significant changes were found in the CD4⁺/CD8⁺ cell ratio. The CD56⁺ NK cell counts were significantly lower in the patients. Distributions of lymphocyte subsets are summarized in Table 2. In cytokine analysis, IL-2, IL-17A, IL-4, IL-6, TNF-α, IP-10, IFN-λ1 (IL-29), IFN-λ2/3 (IL-28A/B), IFN-β, IL-10, and IFN-γ levels were significantly increased in patients compared to the controls. Severe cases had lower IFN-γ median and interquartile range levels than the other groups. Peripheral blood levels of the tested cytokines are presented in Table 3.

The patients were also divided into death and survival groups. Laboratory parameters are compared in Table 4. According to the results of binomial logistic regression analysis, a decrease in CD56⁺ NK cell percentage [odds ratio (OR) = 0.576, 95% confidence interval (CI) = 0.376 - 0.882, P = 0.011] and lymphocyte count (OR = 0.02, 95% CI = 0.001 - 0.368, P = 0.001) and an increase in CRP (OR = 1.012, 95% CI = 1.002 - 1.023, P = 0.038) and IL-10 (OR = 1.068, 95% CI = 1.000 -1.141, P = 0.049) levels were determined as independent predictors for mortality. The ROC curve analysis was performed with independent predictor variables. When the cutoff value for CD56⁺ NK cell was 2.1, the mortality was predicted with 69% sensitivity and 57% specificity. When the cutoff value for IL10 was 0.75, the mortality was predicted with 65% sensitivity and 64% specificity (Figure 1).

Figure 1.

ROC curve analysis of percentages of CD56⁺ NK cell and IL-10 levels among the deceased and survivors in moderate and severe groups

The main pathogenesis of COVID-19 is severe pneumonia, immune response dysfunctions associated with acute infections, coagulation disorders, and cardiac damage (10, 13). One study indicated that symptomatic patients exhibited significantly higher cell-mediated and humoral adaptive immune responses to the virus than asymptomatic individuals (14). High levels of proinflammatory cytokines such as IL-2, IL-7, IL-10, G-CSF, IP10, MCP1, MIP1α, and TNFα increase the disease severity (1, 10, 13). Also, COVID-19 progresses to severe disease at a higher rate, especially in elderly patients with leukocytosis, lymphopenia, or accompanying comorbidities (15). Besides, TNF-α, alanine aminotransferase, lactate dehydrogenase, CRP, myoglobin, cardiac troponin, D-dimer, IL-1β, IL-2, IL-6, IL-7, IL-8, IL- 10, IL-12, IL-18, and IFN-γ levels were reported to be higher, and platelet, CD4⁺, and CD8⁺ T lymphocyte counts were reported to be lower in severe cases (16-20). Similarly, acute-phase response in SARS-CoV-2 patients is associated with decreased CD4⁺ and CD8⁺ T lymphocyte counts (13, 21, 22). In our study, D-dimer, INR, and CRP values increased as the disease severity increased, which is consistent with the literature.

In this study, similar to other studies, hemoglobin, and hematocrit values were lower in COVID-19 patients than in healthy individuals (23). On the contrary, studies report no significant difference in hemoglobin and hematocrit values between moderate and severe cases (24, 25). The cellular response is the most effective immune response against viruses. Neutrophils, which represent most of the leukocytes in the blood, have a particular role in infection control and disease exacerbation. Absolute neutrophil count increases in the first few days of hospitalization in COVID-19 infection (26-28).

Studies show that CD4⁺ and CD8⁺ cell counts decreased in infected patients who died from SARS-CoV-2. However, this decrease was directly proportional to the increased disease severity without changing the CD4⁺/CD8⁺ T lymphocyte ratio (17, 19, 29). In our study, total T lymphocyte counts, and percentages were lower in the patient groups than in the control group, without any change in the CD4⁺/CD8⁺ ratio. Also, CD56⁺ NK cell percentages were lower in the patient groups compared to the control group. That decrease was directly proportional to the increase in disease severity and was found to be an independent predictor of mortality. In contrast, few studies reported that NK cell counts were higher in dead cases or that NK cell percentages did not differ significantly between mild, moderate, and severe cases (29, 30). In our study, decreased lymphocyte counts and increased CRP and IL-10 levels were other independent mortality predictors.

Han et al. detected increased IFN-γ, TNF-α, IL-2, IL-4, IL-6, and IL-10 levels in 52 patients diagnosed with COVID-19 (31). Liu et al. evaluated cytokine levels during clinical follow-up in 17 moderate and 13 severe cases. They detected higher levels of IL-2, IL-6, IL-10, and IFN-γ in severe cases (13). Huang et al. found increased IL-6, TNF-α, IL-1β, IL-18, IL-12/IL-23p40, IL-10, Tim-3, and IL-8 levels and decreased IFN-γ levels compared to the healthy group (19). In this study, IL-2, IL-17A, IL-4, IL-6, TNF-α, IP-10, IFN-λ1 (IL-29), IFN-λ2/3 (IL-28A/B) IFN-β, IL-10, and IFN-γ levels were statistically significantly higher in the patient groups than in the control group. Some studies associated COVID-19 with Cytokine Storm Syndrome (CSS), characterized by an abnormal immune response and increased proinflammatory cytokines, resulting in multiple organ failure and death, and suggested that immune-modulating treatments such as corticosteroid, IL-6 receptor, and JAK1/2 inhibition may reduce disease severity (32). However, there is still no consensus on this issue because studies report that cytokines were not detected at such high levels in COVID-19 patients as in other CSS diseases (such as hemophagocytic lymphohistiocytosis and autoinflammatory diseases) (33). Considering all these, it should be emphasized that the inflammatory nature of COVID-19 is unique and that more studies should be conducted on the subject to prove appropriate biomarkers and identify personalized treatments.