Dynamic assessment of T-lymphocytes and humoral immunity in patients with acute coronary syndrome, with and without COVID-19, depending on the content of CD3⁺CD8⁺T-lymphocytes

Introduction

Acute myocardial infarction (AMI) is one of the leading causes of death worldwide, placing a significant burden on the global economy. Immune cell modulation is a crucial factor in the development of myocardial ischemia and myocardial reperfusion injury. T-cells have been observed to accumulate at the site of myocardial injury and immediately release both pro-inflammatory and anti-inflammatory cytokines. T-cells and macrophages significantly contribute to maintaining cardiac homeostasis and promoting tissue repair [1]. COVID-19 has been shown to damage the immune system, specifically T-lymphocytes [2][3]. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may cause damage to the cardiovascular system, resulting in a variety of complications, including acute myocardial injury. Myocardial injury may result from direct viral invasion or indirect mechanisms of systemic inflammation, immune-mediated response, and dysregulation of the renin-angiotensin system. Myocardial injury affects about 25% of COVID-19 patients, even those without a prior cardiovascular disease, and is associated with increased mortality rates and long-term complications [4][5]. The pathophysiological mechanisms underlying delayed complications remain poorly understood, while symptoms and objective measures of cardiopulmonary health are not always correlated. COVID-19 is anticipated to alter the long-term course of numerous chronic heart conditions that frequently impact individuals at high risk of severe illness [6]. According to Zuin et al. [7], COVID-19 has been shown to increase the risk of AMI [8]. Overproduction of pro-inflammatory cytokines (IL-6 and TNF-α) plays a crucial role in the pathophysiology of SARS-CoV-2 infection, which is associated with systemic inflammation and multiple organ dysfunction syndrome, causing acute injury of the cardiovascular system. An increased risk of myocardial infarction, fulminant myocarditis characterized by a rapid progressive decline in the left ventricular systolic function, arrhythmias, venous thromboembolism, and cardiomyopathy mimicking ST elevation (STE) AMI are the most common cardiovascular complications described in COVID-19 patients. Moreover, SARS-CoV-2 tropism and interaction with the renin-angiotensin system through the angiotensin-converting enzyme 2 receptor may exacerbate the inflammatory response and heart injury. T-cell immune dysfunctions in patients with long COVID have been demonstrated by Savchuk et al. [9][10]. In AMI patients, CD8⁺ T-cells accumulate in the necrotic myocardium. It is currently unclear whether CD8⁺CD28⁺ T-cells, a subset of CD8⁺ T-cells, contribute to myocardial injury. The study by Zhang et al. [11] included 92 AMI patients and 28 healthy controls. Peripheral blood CD8⁺CD28⁺ T-cells were assayed by flow cytometry. Cardiac troponin I plasma levels and left ventricular ejection fraction (LVEF) were also measured. Long-term outcomes were evaluated based on major adverse cardiac and cerebrovascular events over the 12-month follow-up. The findings indicated that AMI patients who presented with high levels of CD8⁺CD28⁺ T-cells had an increased infarction size and aggravated ventricular function. The authors suggested that cytotoxic CD8⁺CD28⁺ T-cell-mediated myocardial necrosis may act as a novel and alternative pathway for AMI. Currently, the T- and B-cell-mediated immunity that plays a role in regulating mechanisms of acute coronary syndrome (ACS), including in post-COVID-19 patients, has not been fully understood, which highlights the relevance of the study.

The aim of the study was to investigate T- and B-cell responses over time in post-COVID-19 and COVID-19 naive patients with ACS, based on CD3⁺CD8⁺ T-cell counts.

Materials and Methods

The study included 65 male patients aged 40–65 years with ACS (unstable angina and AMI) who required coronary artery stenting by coronary angiography. Prior to the study, all patients provided written informed consent (Protocol No. 9 approved by the Ethics Committee of the South Ural State Medical University of the Russian Ministry of Health on September 11, 2006 and Protocol No. 12 approved by the Ethics Committee of the Chelyabinsk City Clinical Hospital No. 1 Chelyabinsk on October 10, 2022).

All the patients had flow cytometry tests for immune cell profiling [12]. Based on the number of CD3⁺CD8⁺ T-lymphocytes, the patients were assigned to one of the three groups with low, normal, or high values of CD3⁺CD8⁺ T-cells, for post-COVID-19 or COVID-19 naive patients separately. T- and B-cell counts were measured on admission and 28 days later.

Standard hematology (25 parameters for white blood cell, red blood cell, and platelet lineages) and hematopoietic lineage profiling were performed using a Medonic M20 Cell Analyzer (Sweden).

The flow cytometry with CD45⁺ pan-leukocyte marker gating (BioLegend, Beckman Coulter, USA) showed CD3⁺ (total T-cells), CD3⁺CD4⁺ (helper T-cells), CD3⁺CD8⁺ (cytotoxic T-cells), CD3⁺CD16⁺CD56⁺ (TNK cells) CD3^-CD16⁺CD56⁺ (natural killer cells), CD3^-CD19⁺CD5⁺ (B-lymphocyte subpopulation), CD3⁺CD4⁺CD25⁺CD127^- (regulatory/suppressor T-cells), CD3⁺CD4⁺CD25⁺ (activated helper T-cells, early activated lymphocytes), CD45⁺CD3⁺HLA-DR (activated T-cells, late activated lymphocytes), and CD27⁺ memory B-cells. The immune status was assessed by flow cytometry (Navios Flow Cytometer, Beckman Coulter, USA) using a standardized technique for separating specific subsets of immune cells [12][13].

The laboratory tests also included neutrophil phagocytosis of 1.7 µm latex particles (phagocytosis activity, phagocytosis intensity, and phagocytic index). The neutrophil activity was morphologically estimated by a spontaneous and induced nitroblue tetrazolium reduction test (using optical microscopes from Olympus, Japan) [14][15][16]. The study used established functional assays to assess phagocytic activity.

Total IgA, IgG, IgM titers, SARS-CoV-2-specific IgM, IgA, IgG titers, C1 inhibitor, and C3a and C5a complement fragments (VectorBest, Russia, Cytokine OOO, Russia) were measured by an enzyme immunoassay using a Multiskan FC Microplate Reader (Thermoscientific, China). In this study, the established enzyme immunoassay methods were used according to the manufacturers' protocols.

Data were processed and analyzed using the Statplus 2005 statistical software. Since the quantitative data were normally distributed, parametric statistics were used [17].

Results

It is noteworthy that higher levels of CD3⁺CD8⁺T-lymphocytes were only found in those patients who had a history of the novel coronavirus infection (COVID-19). All the patients with high CD3⁺CD8⁺ T-cells had AMI, including two patients with ST elevation (STE) and two patients with non-ST elevation (non-STE). Among the COVID-19 naive individuals with low or normal CD3⁺CD8⁺ T-cell counts, patients with unstable angina were more prevalent. The highest GRACE risk scores were observed in the post-COVID-19 patients with normal or elevated CD3⁺CD8⁺ T-cells. The duration of hospitalization was the longest in the patients with a history of COVID-19 who had elevated CD3⁺CD8⁺ T-cells, and the shortest in the COVID-19 naive patients with normal CD3⁺CD8⁺ T lymphocytes. Stent thrombosis was found in 3 (12.5%) post-COVID-19 patients with normal CD3⁺CD8⁺T-cell counts. During the current hospitalization, the patients with elevated CD3⁺CD8⁺ T-lymphocytes received the highest number of stents, with an average of 2. The highest troponin levels were reported for the individuals with normal or elevated CD3⁺CD8⁺ T lymphocytes who had previously suffered from COVID-19 (10.61±2.57 ng/mL and 8.96±6.98 ng/mL, respectively). These groups most frequently required morphine for coronary pain management with 9 (37.5%) patients having normal levels of CD3⁺CD8⁺ T-cells and 3 (75%) patients having elevated CD3⁺CD8⁺ T-cells. Thus, the highest clinical severity was observed in the post-COVID-19 patients with normal or elevated CD3⁺CD8⁺T-cells.

According to Table 1, there were no age differences between the groups. The individuals without a history of COVID-19 were more commonly diagnosed with unstable angina, particularly those with normal CD3⁺CD8⁺ T-cell counts. All the patients (100%) with a history of COVID-19 and elevated CD3⁺CD8⁺ T-cells had STE (50%) or non-STE (50%) AMI. There were no STE AMI patients among those with normal CD8 lymphocytes and no history of COVID-19 infection. Among the STE AMI patients, those with normal or elevated CD3⁺CD8⁺T-cells who had a history of COVID-19 were predominant. There was a greater number of COVID-19 naive patients who had AMI in the past history compared to those with a previous COVID-19 infection. A greater GRACE risk score was found in the post-COVID-19 individuals with high or normal CD8 lymphocyte counts. Among the patients with baseline and follow-up assessments, the largest number of stenting procedures was reported for those with low CD3⁺CD8⁺ T cells and a history of COVID-19.

The post-COVID-19 patients with ACS had more significant changes in clinical signs and immune cell profiles compared to the COVID-19 naive individuals. In particular, they required longer hospitalizations with the highest duration in those with elevated CD3⁺CD8⁺T-cells, followed by patients with normal CD3⁺CD8⁺ T-cells. Additionally, the atherogenic index and troponin values were higher in the ACS patients with a history of COVID-19 infection. The highest troponin levels were observed in the post-COVID-19 individuals with ACS and normal CD3⁺CD8⁺ T-cells. Stent thrombosis and deaths were only reported for the post-COVID-19 patients, more frequently for those with low CD3⁺CD8⁺ T-cells who had the follow-up immune status assessments. As previously noted, individuals with normal or elevated CD3⁺CD8⁺ T-cells had higher mortality rates and incidence of stent thrombosis. However, not all of these individuals had follow-up immune assessments due to a rapid fatal outcome. The ACS patients with normal CD3⁺CD8⁺ T-cells and a history of COVID-19 infection required morphine most frequently for coronary pain management.

Table 1 compares T- and B-cell immunity in the ACS patients.

The table shows that the post-COVID-19 patients with high baseline levels of CD3⁺CD8⁺ T-cells had the smallest percentage of T-cells (CD45⁺CD3⁺CD19^-), which was significantly different from the other groups. However, the group with a history of COVID-19 infection and low CD3⁺CD8⁺ T-cells had the lowest absolute T-cell counts, which were significantly lower compared to the other groups. After 28 days, T-cell values increased significantly (p<0.0001) only in patients with initially low CD3⁺CD8⁺ T-cells. At baseline, this population also had the lowest levels of helper T-cells, which increased significantly at follow-up (p<0.001), as did CD3⁺CD8⁺ T-cells (p<0.0001). The patients with elevated CD3⁺CD8⁺ T-cells experienced a significant increase in the helper/suppressor ratio (p<0.05). The patients with high CD3⁺CD8⁺ T-cells had the lowest CD4/CD8 ratio at baseline, which was significantly different from the other groups.

The individuals with low CD3⁺CD8⁺ T-cells, both with and without a history of COVID-19, exhibited significantly lower levels of NK T-cells compared to the other groups.

There was a trend towards an increase in NK T-cells, except in patients with high CD3⁺CD8⁺ T-cells, who showed a trend towards a decrease in NK T-cells. The post-COVID-19 patients with low or normal baseline CD3⁺CD8⁺ T-cell values experienced a significant increase in natural killer levels. The individuals with high CD3⁺CD8⁺ T-cells had the highest NK T-cell count, which tended to decrease over time compared to the other groups.

A significant decrease in percentages of early activated T-cells (p<0.05) was reported for patients with normal CD3⁺CD8⁺ T-cells and COVID-19 infection in the past medical history. The lowest levels of late-activated T-cells were seen in the individuals with low CD3⁺CD8⁺T-cells and significantly increased over time. The highest late-activated T-cell counts were observed in the post-COVID-19 patients with high CD3⁺CD8⁺ T-cells. Regulatory T-cells and late activated regulatory T-cells significantly (p<0.01) increased in the patients with low baseline CD3⁺CD8⁺ T cell values who had a history of COVID-19, and significantly decreased in the COVID-19 naive patients.

Table 2 presents baseline and follow-up B-cell levels in the ACS patients, both with and without a history of COVID-19 infection.

The lowest B-cell counts (CD45⁺CD3^-CD19⁺) were observed in the patients with low CD3⁺CD8⁺ T-cells who had a history of previous COVID-19 infection, with a statistically significant difference from other groups and a significant increase over time (p<0.05). The lowest IgA values were reported for the patients with elevated CD3⁺CD8⁺T-cells. The highest IgG was observed among the post-COVID-19 patients with low CD3⁺CD8⁺T-cells. The levels decreased statistically significantly over time (p<0.05). The highest levels of the C5a complement fragment were found in the patients with a history of COVID-19 and low CD3⁺CD8⁺ T-cells, and these levels showed a significant increase (p<0.05) at follow-up. B-cells (CD3^-CD19⁺CD5⁺) were lowest in the individuals with low levels of cytotoxic cells and increased statistically significantly (p<0.05) in the post-COVID-19 patients with low CD3⁺CD8⁺ T-cells. The lowest B-cell counts (CD3^-CD19⁺CD5^-) were reported for the patients with low CD3⁺CD8⁺ T-cells.

Discussion

Despite early reperfusion, patients with ST-elevation myocardial infarction (STEMI) may experience extensive myocardial necrosis and significant ventricular dysfunction. The study by Casarotti et al. [18] aimed to assess the role of B-cell subpopulations and related cytokines in the infarcted mass and LVEF obtained by cardiac magnetic resonance imaging (MRI) performed after 30 days of STEMI. This prospective study included 120 STEMI patients who had pharmacological thrombolysis and percutaneous intervention (pharmacoinvasive strategy). Blood samples were collected on days 1 and 30 post STEMI. The values of CD3^-CD19⁺CD5⁺ lymphocytes (cells/mL) on day 1 were correlated with the infarcted mass measured by cardiac MRI on day 30. CD3^-CD19⁺CD5⁺ cells were associated with CD4⁺ T-cells on days 1 and 30, whereas classic CD3^-CD19⁺CD5 lymphocytes on day 30 were related to LVEF. The multiple linear regression analysis demonstrated that high-sensitivity troponin T and IL-6 collected on day 1 and high-density lipoprotein cholesterol levels on day 30 were independent predictors of the infarcted mass. The independent predictors of the LVEF included high-sensitivity troponin T and high-sensitivity C-reactive protein on day 1, and classic CD3^-CD19⁺CD5^- lymphocytes on day 30. Despite early reperfusion, the amount of infarcted mass and ventricular function were associated with inflammatory responses triggered by circulating B-cells.

In the present study, the most affected clinical group was those who had a history of COVID-19 and higher levels of CD3⁺CD8⁺ T-cells. This is consistent with Santos-Zas et al. [19]. In a murine model of AMI, CD8⁺ T-cells are recruited and activated in the ischemic heart tissue and release Granzyme B, which leads to cardiomyocyte apoptosis, adverse ventricular remodeling, and deterioration of myocardial function. Depletion of CD8⁺ T-lymphocytes reduces apoptosis in the ischemic myocardium hampers an inflammatory response, limits myocardial injury, and improves cardiac function. These effects are reproduced in mice with Granzyme B-deficient CD8⁺ T-cells. The protective effect of CD8⁺ T-cell depletion on cardiac function has been confirmed using a porcine ischemia/reperfusion model. Finally, the study demonstrated that elevated blood Granzyme B levels in the patients with AMI were indicative of an increased risk of 1-year death. The authors revealed the deleterious role of CD8⁺ T-cells after acute ischemia and suggested potential therapeutic strategies targeting pathogenic CD8⁺ T-cells in AMI.

Conclusions

In most cases, coronary artery stenting has resulted in statistically significant changes in immune cell profiles of the individuals with low CD3⁺CD8⁺T-cells and a history of COVID-19, with an increase in T-cells (CD45⁺CD3⁺CD19^-), helper T-cells, CD3⁺CD8⁺T-cells, NK T-cells, NK cells, late activated T-cells, B-cells, and a decrease in IgG and C3a complement fragment levels.

Regulatory T-cells and late activated regulatory T-cells significantly (p<0.01) increased in the patients with low baseline CD3⁺CD8⁺ T cell values who had a history of COVID-19, and significantly decreased in the COVID-19 naive patients.

In the post-COVID-19 individuals with normal CD3⁺CD8⁺T-cells, a significant increase in T-cells (CD45⁺CD3⁺ CD19^-), NK cells, late activated regulatory T-cells, and a decrease in percentages of early activated T-cells were observed at follow-up.

The patients with elevated CD3⁺CD8⁺T-cells experienced an increase in IgA/IgM titers and CD4/CD8 ratio values over time.