Features of T cell immunity depending on the content of natural killer cells in patients with acute coronary syndrome following COVID-19

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Features of the T-cell link of immunity in patients with acute coronary syndrome, who had and did not have COVID-19, depending on the content of natural killers

Introduction
Inflammation in the myocardium in COVID-19 can lead to myocarditis, acute coronary syndrome (ACS), rapid deterioration and sudden death [7]. Direct myocardial injury and cytokine storm, leading to the destabilization of pre-existing plaques and the accelerated formation of new plaques, are the mechanisms that provoke ACS in COVID-19 [10].
In the work of L.N. Khusainova et al. [4] showed that in patients with ACS, the number of T-lymphocytes is reduced, and the number of lymphocytes that affect apoptosis is increased. According to O.K. Lebedeva et al. [2], the development of acute heart failure in patients with acute myocardial infarction (AMI) is associated with a decrease in the number of T and NK cells (natural killer cells).
The available literature lacks data on T- and NK-cell immunity in patients with ACS who have undergone COVID-19, which determines the relevance of the study.
The aim of the study was to study the T-cell immunity depending on the number of natural killers in patients with acute coronary syndrome who underwent COVID-19 in comparison with these parameters in patients without COVID-19.
Research objectives:
1. To identify clinical differences in the course of the disease in patients with ACS who underwent COVID-19 and did not have it, depending on the number of NK cells.
2. To determine indicators of T-cell immune status in patients with ACS who had a history of COVID-19 and did not have a new coronavirus infection, depending on the indicators of NK-cell immunity.
3. To identify differences in the content of the main populations of T-lymphocytes in the above categories of patients.
Material and methods. We examined 65 men with unstable angina and acute myocardial infarction (acute coronary syndrome) from 40 to 65 years old who had previously had COVID-19 and 20 people with ACS who had not undergone COVID-19. All persons also had hypertension, and they required stenting of the coronary arteries within the next 3 days after admission to the hospital. Coronary angiography was performed on an Innova JE device, drug-eluting stents from Xience Alpine were implanted. All patients signed an informed consent (protocol of the Ethics Committee of the South Ural State Medical University of the Ministry of Health of Russia No. 9 dated September 11, 2006 and the protocol of the ethical committee of the GAUZ OTKZ City Clinical Hospital No. 1 of Chelyabinsk No. 12 dated October 10, 2022).
From the immunological parameters by flow cytometry on the Navios cytofluorimeter (BeckmanCoulter, USA), according to the standardized technology for assessing the lymphocytic link of immunity [1], the following were determined: ), CD45+ CD3+ CD8+ (cytotoxic T-lymphocytes), CD45+CD3-CD19+ (B-lymphocytes), CD45+CD3+CD16+CD56+ (TNK cells), CD45+CD3-CD16+CD56+ (natural killer cells), CD45+ CD3+CD4+CD25+CD127- (T-regulatory cells), CD45+ CD3+CD4+CD25+ (T-lymphocytes - early activation), CD45+CD3+HLA-DR (T-lymphocytes - late activation).
StatPlus 2009 Professional software was used to statistically process the material. The following were calculated: arithmetic mean (M), arithmetic mean error (m), Student's t-test was determined for independent samples.
Research results.
All patients (who separately had COVID-19 and who did not have it, but were vaccinated against a new coronavirus infection) were divided into 3 groups depending on the number of NK cells: with reduced NK cell counts, normal and elevated [1]. The general clinical characteristics of these individuals are illustrated in Table 1.

Table 1. General characteristics of patients with acute coronary syndrome who had COVID-19 and did not have it, depending on the content of NK cells

Index	sick with COVID-19			not sick with COVID-19
	1 NK cells are reduced (n=15) M±m, р	2 NK cells are normal (n=31) M±m, р	3 NK cells are raised (n=19) M±m, р	4 NK cells are reduced (n=9) M±m, р	5 NK cells are normal (n=11) M±m
Age, years	57,33±1,59	56,19±1,13	56,63±1,6	54,78±2,53	54,06±2,06
Unstable angina, absolute number of patients, %	6 (40%)	14 (45,16%)	7 (36,84%)	5 (55,56%)	9 (81,82%)
STEMI, absolute number of patients and %	5 (33,33%)	12 (38, 71%)	6 (31,58%)	3 (33,33%)	1 (9,09%)
NSTEMI, absolute number of patients and %	4 (26,67%)	5 (16,13%)	6 (31,58%)	1 (11,11%)	1 (9,09%)
AMI                in the past, absolute number of patients, %	3 (21,43%)	7 (22,58%)	6 (31,58%)	3 (33,33%)	1 (9,09%)
Grace risk, points	112,55±12,43	119,58±9,77	122,75±7,74 Р3-5=0,049	113,67±11,90	101,70±9,74
Duration         of hospitalization, days	10,93±0,89 Р1-5=0,023	9,77±0,43 Р2-5=0,003	10,16±0,61	9,44±0,89	8,54±0,49
Triglycerides, mmol/l	2,29± 0,27 Р1-4=0,049	1,84±0,27	1,80±0,27	1,58±0,26	1,57±0,22
Troponin I, ng/ml	8,99±2,82 Р1-5=0,028	9,23±1,96 Р2-5=0,019	9,97±2,68	6,22±3,44	1,85±1,60
IgM to COVID-19, positivity coefficient	8,21±1,71 Р1-4= 0,003	7,82±1,30 Р2-3=0,033 Р2-4= 0,005 Р2-5=0,010	11,90±1,79 Р3-4=0,0002 Р3-5=0,0003	1,21±0,44	2,46±0,72
IgG to COVID-19, positivity coefficient	20,89±1,30 Р1-4= 0,001	18,12±1,47 Р2-4=0,052	20,88±1,44 Р3-4=0,0002 Р3-5=0,0011	9,95±2,55	13,43±3,26

Note: AMI acute myocardial infarction, - STEMI - acute myocardial infarction with ST segment elevation, NSTEMI  - acute myocardial infarction without ST segment elevation

According to Table 1, in patients with ACS without COVID-19, patients with unstable angina prevailed, and in the group who underwent COVID-19, patients with AMI prevailed, mainly with AMI with nonST - in the group with normal NK cells. In terms of the number of stents installed, the leaders were patients with a normal and elevated number of NK cells who recovered from COVID-19. Stent thrombosis was recorded in those who recovered from COVID-19: 3 (9.68%) individuals with normal NK cells and 1 (6.67%) with reduced NK cells. In the same groups of patients, deaths were more common: 2 (10.53%) with elevated NK cells, 3 (9.67%) with normal NK cells, and 1 (6.67%) with reduced NK cells. There were no deaths among individuals who did not have a history of a new coronavirus infection. More than 30% of patients with ACS with a history of COVID-19 were used to relieve retrosternal pain, and only 10% of patients with ACS without COVID-19 with normal NK cells were used at the Emergency Medical Care stage. There was a statistically significant difference between Jg M and Jg G in individuals with COVID-19 compared with ACS patients without it, but vaccinated. The maximum numbers of Jg M were observed in patients with COVID-19, with elevated NK cells.
The duration of hospital stay is higher in patients with COVID-19 and ACS (p<0.05). In addition, they had higher blood troponin I (greater with elevated and normal NK cells), atherogenic quotient, and Grace risk (with elevated NK cells) compared to these parameters in patients without COVID-19. It should be noted that in the group of patients without a history of COVID-19, none of the patients had elevated NK cells.
Table 2 presents data on T-lymphocytes, T-NK-lymphocytes, NK-lymphocytes, as well as T-regulatory cells.

Table 2. Comparison of indicators of T-cell immunity in persons with acute coronary syndrome, sick and not sick with covid-19, depending on the number of NK cells

Index           NK cells	sick with COVID-19			not sick with COVID-19
	1 NK cells are reduced (n=15) M±m, р	2 NK cells are normal (n=31) M±m, р	3 NK cells are raised (n=19) M±m, р	4 NK cells are reduced (n=9) M±m, р	5 NK cells are normal (n=11) M±m
T-lymphocytes (CD45+CD3+ CD19-), %	66,81±2,11 Р1-2=0,006 р1-3=0,008 р1-4=0,040 р1-5=0,026	73,29±1,43	74,97±2,42	73,24±2,52	73,82±2,52
T-lymphocytes (CD45+CD3+ CD19-), 106 cells/l	1866,21±157,84 Р1-3=0,001 р1-4=0,01	1637,97±129,72 р2-4=0,049	945,47±117,68 р3-5= 0,001	1215,44±179,24 р4-5= 0,004	1931,00±163,76
T-helpers (CD45+CD3+ CD4+), %	40,22±2,13 Р1-2=0,0019 р1-3=0,001 р1-4=0,006 р1-5= 0,003	48,79±1,76	50,83±2,31	50,09±2,78	50,26±2,13
T-helpers (CD45+CD3+ CD4+), 106 cells/l	1116,05±102,77 Р1-3=0,001 р1-4=0,049	1067,29±87,66 р2-3= 0,0014 р2-5=0,049	631,27±80,25 р3-5= 0,001	838,56±127,59 р4-5=0,007	1324,20±125,73
T-cytotoxic (CD45+CD3+ CD8+), 106 cells/l	674,95±76,27 Р1-2=0,020 р1-3= 0,001 р1-4= 0,005	504,77±42,93	334,87±65,85	347,44±60,84	549,20±48,07
Immunoregulatory index (Tx/Tc)	1,95±0,26 р1-3=0,049	2,42±0,23	2,75±0,43	2,66±0,43	2,60±0,39
T-NK lymphocytes (CD45+CD3+CD 16+ CD 56+), %	8,42±1,29 р1-4=0,019	6,77±0,93	6,00±1,16	4,00±1,01 р4-5=0,092	6,30±1,29
T-NK lymphocytes (CD45+CD3+CD 16+ CD 56+), 106 cells/l	242,24±56,86 р1-2=0,044 р1-3=0,009 р1-4=0,021	145,13±26,48 р2-3=0,049 р2-4=0,049	78,00±17,92 р3-5=0,018	62,22±16,11 р4-5=0,017	164,400±39,708
T-lymphocytes CD45+CD3+CD4+CD25+ (early activation), %	7,84±0,84	8,37±0,52	7,89±0,53	8,84±0,66 р4-5=0,016	6,85±0,55
T-lymphocytes CD45+CD3+CD4+CD25+ (early activation), 106 cells/l	79,05±11,81 р1-3=0,033	86,35±8,41 р2-3= 0,0051	50,93±7,37 р3-4= 0,049  р3-5=0,002	73,56±12,02	90,60±11,04
T-lymphocytes CD45+CD3+CD4+HLA-DR+ (late activation), %	6,39±0,95	5,78±0,45 р2-4=0,015	6,33±0,63 р3,4=0,07	8,11±1,13 р4,5=0,032	4,75±1,25
T-lymphocytes CD45+CD3+CD4+HLA-DR+ (late activation), 106 cells/l	65,21±10,42 р1-3=0,034	56,00±4,53 р2-3=0,029	40,87±6,12 р3-4= 0,035	65,44±13,36	67,00±21,56
T-regulatory cells (CD 45+CD3+CD4+CD25+CD127-), %	2,39±0,25 р1-2=0,049 р1-3=0,002 р1-4= 0,001	3,22±0,35 р2-4=0,014 р2-5=0,049	3,85±0,42 р3-5= 0,003	4,98±0,78 р4-5=0,002	2,13±0,34
T-regulatory cells (CD 45+CD3+CD4+CD25+CD127-), 106 cells/l	24,05±2,01 р1-2=0,049 р1-4=0,014	34,03±4,55 р2-3=0,074	23,80±3,33 р3-4= 0,034	38,11±7,94	27,70±4,68
T-regulatory cells late activation (CD 45+CD3+CD4+CD25+CD127-HLA-DR+), %	6,39±0,95 р1-2=0,049 р1-4=0,014	5,78±0,45 р2-3=0,074	6,33±0,63 р3-4= 0,034	8,11±1,13	4,75±1,25
T-regulatory cells late activation (CD45+CD3+CD4+CD25+CD127-HLA-DR+) 106 cells/l	0,92±0,14 р1-3=0,049 р1-4= 0,0012	1,04±0,14   р2-4=0,002	1,37±0,23	2,02±0,36	0,62±0,11

According to the indicators presented in Table 2, individuals with ACS and COVID-19 with elevated NK cells have a minimal number of T-lymphocytes, T-helpers, T-cytotoxic lymphocytes, early-activated T-lymphocytes, late-activated T-lymphocytes, as well as T-regulatory cells in absolute units compared to other groups. The lowest immunoregulatory index and, at the same time, the maximum number of T-NK-lymphocytes were observed in persons who had undergone COVID-19 and had reduced NK cells. The minimum number of T-NK lymphocytes was recorded in patients with low NK cells who did not have COVID-19. Minimal T-lymphocytes (CD45+CD3+CD4+HLA-DR+) of late activation were found in people who recovered from COVID-19 with elevated and normal NK cells.
The lowest number of late activation regulatory T cells was observed in patients who did not have COVID-19, but were vaccinated, and had a normal content of NK cells.
Discussion of the results
According to the studies of Lebedeva O.K. et al. [2], in patients with acute myocardial infarction, lower CD16(+) T and NK cells were recorded.
Other authors [3] note that in 63.6% of persons with ACS with implanted stents, an increase in NK-lymphocytes was noted, which could be due to an increase in the activity of anti-transplant immunity.
According to Liu Y. et al. [8], a pathological autoreactive immune response is responsible for plaque rupture and the subsequent onset of ACS. Regulatory T cells are required to suppress the pathological autoreactive immune response and maintain immune homeostasis. In patients with ACS, a decrease in the number and suppressive function of T-regulatory cells was revealed. This is also observed in our patients who underwent COVID-19 and had elevated NK cells. According to Tian X. et al. [11], in patients with ACS, compared with patients with stable angina and the control group, impaired formation of T-regulatory cells is closely associated with hyperactivity of the sympathetic system.
Gang H. et al. [6] noted that T-cytotoxic cells from patients with AMI showed increased cytotoxicity compared to the control group. Dysregulation of cytotoxic T-lymphocytes in patients with ACS and COVID-19 was noted by Shafeghat M. et al. [9].
According to Liu P.P. [7], in COVID-19, lymphopenia is potentially associated with a decrease in the number of T-helpers and cytotoxic T-cells, which leads to an imbalance in the immune response and a delay in virus clearance. Inflammation in the vascular system can lead to microangiopathy with thrombosis, ACS, and sudden death.
In our studies, we have shown that not everything is so simple with patients who have undergone ACS and have certain indicators associated with the determination of NK cells. Thus, patients who have undergone COVID-19 have 3 phenotypes of disorders (lower NK cell count, normal and increased), and those who have not undergone 2 phenotypes (lower NK cell count and normal). At the same time, in these groups, both disturbances in other compartments of lymphocytes and the severity of clinical and laboratory changes manifested themselves somewhat differently. The most severe condition and severity of immune disorders were found in patients who had undergone COVID-19. The study also allows us to more clearly define the groups of patients with ACS who need additional immunocorrection.
Conclusions
1. In patients with acute coronary syndrome and COVID-19, predominantly with normal and elevated levels of NK cells, in comparison with ACS patients without COVID-19, a more severe course of the disease was observed - patients with acute myocardial infarction prevailed, they had a higher level mortality, increased duration of treatment, and more frequent stent thrombosis.
2. Individuals with ACS and COVID-19 with increased NK cells showed a maximum decrease in the T-cell link of immunity: T-lymphocytes of general, T-lymphocytes-helpers, T-cytotoxic lymphocytes, T-lymphocytes of early activation, T-regulatory cells in absolute numbers compared to other groups.

About the authors

Eleonora A. Safronova

South Ural State Medical University

Author for correspondence.
Email: safronovaeleonora68@gmail.com

PhD (Medicine), Associate Professor, Department of Polyclinic Therapy and Clinical Pharmacology

Russian Federation, Chelyabinsk

Liana V. Ryabova

South Ural State Medical University

Email: lianarabowa@rambler.ru

PhD, MD (Medicine), Professor, Department of Life Safety, Disaster Medicine, Emergency and Emergency Medical Care

Russian Federation, 64, st. Vorovskogo, Chelyabinsk, 454048

References

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