Cardiovascular diseases (CVD) are the leading cause of death worldwide. In Russia, CVD mortality accounts for more than 50%. At the same time, coronary heart disease (CHD) accounts for 54.6% in the structure of mortality from diseases of the circulatory system [1]. Atherosclerosis remains the main risk factor for CHD [2]. Atherosclerosis is a multifactorial disease characterized by polyetiology, where the formation of an atherosclerotic plaque with the development of local sites of intimal calcification is the final stage of atherosclerosis development. Considering that the most common theory of atherosclerosis occurrence is a reaction to damage with the activation of the primary immune response (innate immune response), and then with activation of the acquired or adaptive immune response [3], it seems interesting to study the indicators of the immune response in patients with proven coronary calcification for a more detailed understanding of the participation of immunocompetent cells in the calcification of coronary arteries. It is known that CHD and its complications lead to the development of disability among the population. In this regard, various tools for predicting fatal CHD complications have been developed for many decades, but currently, there is no unitary risk stratification tool. Perhaps it is related to the multifactorial nature of atherosclerosis. There are not only comorbidity risk factors, but also the involvement of the immune response in atherosclerosis progression. The rapid development of radiation diagnostics methods and other imaging techniques makes it possible to find new markers for the prediction of adverse CHD outcomes. Multispiral computed tomography of coronary arteries, predicting the risk of coronary events with calculation of the coronary index according to Agatston, is the most widespread method [4]. This method was used in various randomized, population-based, observational studies, such as ROBINSCA, MESA, RECALL, and Rotterdam studies, and showed a high prognostic role of the coronary index method, which made it possible to calculate more accurately the risk of coronary atherosclerosis complications [5-7].

The purpose of the research was to study and evaluate adaptive immune response in patients with coronary artery calcification depending on the coronary index calculated during spiral computed tomography of coronary arteries.

This study was conducted at the FSBEI of HE “Rostov State Medical University” of the Ministry of Health of Russia within the framework of the state assignment of the Ministry of Health of Russia. Patients with coronary atherosclerosis who underwent multispiral computed tomography synchronized with ECG were included in the study. The coronary index (CI) was calculated using the Agatston method, where the low risk was 0–10 U, the moderate risk was 11–100 U, the high risk was 101–400 U, and the very high risk was ≥401 U [8]. The protocol of the study was approved by the local ethics committee.

Sixty-two male patients participated in the study: 20 patients corresponding to the moderate risk group (group I), 20 people corresponding to a high risk group (group 2), and 22 volunteers (control) who did not have clinical and instrumental signs of coronary atherosclerosis according to spiral computed tomography (SCT) of coronary arteries comparable in age (55.2 ± 1.7; 56.9 ± 1.3 and 52.7 ± 1.1, respectively, pM = 0.34). Patients of both groups had the following atherosclerosis risk factors: smoking and arterial hypertension. Based on the pharmacological history, neither group of patients took lipid-lowering drugs. Diabetes mellitus, oncopathology, connective tissue diseases, and chronic viral infections were exclusion criteria. All participants in the study signed a voluntary informed consent to participate and to conduct the necessary studies. SCT of the coronary arteries and aorta (Revolution EVO spiral computed tomograph, GE Healthcare) was performed in order to randomize the groups. Coronary calcium assessment and Agatston index calculation were carried out using the Smart Score software application for calculating coronary calcium. Phenotyping of CD3+, CD3+CD4+, and CD3+CD8+ populations with calculation of the immuno-regulatory index was carried out to determine the quantitative composition of lymphocytes using the flow laser cytofluorimetry method. To investigate Granzyme B+, mononuclear cells were isolated from peripheral blood using centrifugation in a density gradient of Ficoll-Verografin. Isolated mononuclear cells were permeabilized by the Intra Prep Permeabilization Reagent (Immunotech) followed by staining with monoclonal antibodies to Granzyme B (Serotec). Samples were analyzed on a Cytomics FC 500 flow cytofluorimeter (Beckman Coulter). B cells (CD19+) were analyzed by flow cytofluorimetry (Cytomics FC 500, Beckman Coulter) using appropriate monoclonal antibodies. Quantitative determination of Ig A, M, and G levels was conducted by the method of radial immunodiffusion in the gel using monospecific antisera kits containing antibodies to the heavy chains of the corresponding immunoglobulins (FSUE SPA “Microgen”, Ministry of Health of the Russian Federation, Russia). The results were expressed in g/L. The method of serum precipitation in polyethylene glycol was used to determine circulating immune complexes. The results were recorded using a spectrophotometer at a wavelength of 450 nm and expressed in conventional units. Statistical analysis was performed using Statistica 12.0 software (StatSoft, USA). Indicators in groups were presented by median with interquartile range and sample mean with standard error. The Normality of value distribution in the samples was checked by the Shapiro-Wilk test. Analysis of variance and the Kruskal-Wallis test were used to compare the indicators in the three groups. A correction for the number of compared groups by Tukey was made in pairwise comparison.

As mentioned above, to clarify changes in the immune status, patients with atherosclerosis were divided into two groups: group I with a CI of 11–100 units and group II with a CI of 101–400 units. The median (Me) CI in group I was 33 U, in group II – 262 U, p-value=0.0001, which indicates a statistically significant difference of CI in both groups. At the same time, CI in the control group was 0 U. The comparative analysis in groups with atherosclerosis, where there was a significantly higher content of the relative number of CD3+ lymphocytes, but at the same time, groups I and II did not differ from each other in this indicator, is presented in Table 1. When evaluating T-lymphocyte subpopulations, there were no statistically significant changes in the content of CD3+CD4+ and CD3+CD8+ lymphocytes, which naturally did not lead to a change in the immuno-regulatory index either in comparison of groups with each other or in comparison with the control group. However, an increase in the cytotoxic potential of CD3+ CD8+ lymphocytes in group II compared to the control group and group I was noteworthy.

When conducting a comparative analysis of the indicators of the humoral component of the adaptive immune system, an increase in the relative content of B-lymphocyte subpopulation was noted in group II compared to the control group and group I, while in group I, there was only a tendency to increase without reaching statistical significance. The functional activity of CD19+ lymphocytes was manifested in class A hyperglobulinemia in both groups of patients with atherosclerosis, while the content of this immunoglobulin between groups I and II did not differ significantly. There were no differences in the content of immunoglobulins of classes M, G, and circulating immune complexes in the study groups (Table 2).

It should be noted that the development of calcification of the coronary arteries is associated with the progression of atherosclerosis, and therefore, there is predominantly intimate calcium deposition with the involvement of inflammatory cells of the immune response. Under the influence of cytokines, further phenotypic modulation of smooth muscle cells into chondrocyte-like cells occurs, which leads to the formation of calcification sites in atherosclerotic altered arteries [9]. The data obtained by us during the evaluation of the cell composition of adaptive immunity are comparable to the results of Farias-Itao et al., who showed a higher content of CD3+ lymphocytes in patients with atherosclerosis. Moreover, the more pronounced atherosclerosis was, the higher cell content was noted in the coronary calcification zone [10]. The authors also recorded a higher content of CD8+ lymphocytes and no fluctuations in the content of CD4+ lymphocytes. In our study, we did not note an increase in CD8+ lymphocytes, which may be due to their determination in the circulating blood, and not at the sites of atherosclerotic plaque formation. At the same time, the polarization of CD8+ lymphocytes by cytotoxic type was clearly noted, as evidenced by a higher content of intracellular granzyme B. This fact is also substantiated by the work of other authors and can have prognostic significance in coronary calcification [11]. An increased level of CD3+ lymphocytes without a significant change in CD4+ and CD8+ subpopulations is ambiguous and can indicate a redistribution of the cells in tissues and in systemic circulation.

When analyzing the humoral immunity, the detected increase in B-lymphocytes and hyperproduction of immunoglobulin A can be associated with more severe coronary calcification. This is also reflected in the Rotterdam study, which involved more than 8 thousand patients, where an increased level of immunoglobulin A was directly associated with coronary calcification, with high cardiovascular risk; the highest values of immunoglobulin A were observed with a CI of more than 400 U [12].

Thus, the identified changes in the adaptive immune response in patients with coronary calcinosis indicate its activation, in particular, the cytotoxic potential and functional activity of B cells, which is the foundation for the investigation of the immune response with the development of a universal screening tool.