Earlier, it has been convincingly established that exposure to ionizing radiation (IR) alters the T cell-mediated immunity in the long term. A number of studies have identified that irradiated individuals, including Techa Riverside residents, have more mutations in the genes of the T cell receptor (TCR mutations). An in-depth analysis of the immune status of such people allowed establishing the special role cytotoxic CD3⁺CD16⁺CD56⁺ lymphocytes play in the elimination of TCR mutant cells [1].

Regarding the impact of IR on T cell populations, there are data showing changes in the peripheral blood T helper indices. For example, 20 years after exposure, survivors of the Hiroshima and Nagasaki atomic bombing that received the dose greater than 1 Gy had the number of naive T helpers (CD4⁺CD45RA⁺ phenotype) decreasing [2]. The Chernobyl accident victims exhibited similar reactions: those who received high doses of radiation had less T-helpers in the peripheral blood [3]. A study that involved employees of Mayak Production Association (a nuclear industry production facility) has revealed a linear dose-dependent decrease in the number of peripheral blood T helpers [4]. A study of low dose occupational exposure that involved employees of Kozloduy, a Bulgarian nuclear power plant, yielded an assumption about a possible immune response shift from Th₁ to Th₂ [5]. However, a search for quantitative studies describing the effect chronic exposure to radiation has on various subpopulations of T helpers, including Th₁₇, Th₂₂, Th₉ etc., yielded no results.

This study aimed to investigate the quantitative characteristics of various subpopulations of T helpers in the peripheral blood of individuals chronically exposed to low-level radiation for a long period of time.

METHODS

The study of T helper subpopulations involved 102 people whose irradiation conditions and nature were described in detail earlier [6]. Techa Riverside residents were exposed to gamma radiation both externally and internally, mainly from ⁹⁰Sr and ¹³⁷Cs. The contamination came from the liquid radioactive waste discharged into the river by Mayak. The largest doses were absorbed by the red bone marrow (RBM) in 1950s–1960s; the associated key source of radiation was ⁹⁰Sr. We divided the chronically exposed people into two groups: exposed individuals (n = 54) and the control group (n = 48).  It is important to note that currently, among the Techa Riverside residents, people with the accumulated radiation dose exceeding 100 mGy (localization — stomach and RBM) suffer and die from malignant neoplasms, including leukemia, more often [6].

The study inclusion criteria were: permanent residence in one of the 41 Techa River villages from 01.01.1950 to 12.31.1960; availability of data on the established doses accumulated in the RBM, thymus and peripheral lymphoid organs (as measured with the TRDST 2016 dosimetric system) [7]; absence of autoimmune, oncological, acute or chronic (exacerbating) inflammatory diseases, hemoblastoses, renal or hepatic insufficiency, acute cerebrovascular accidents in the previous three months; no intake of antibiotics, glucocorticoids and cytostatics in the previous six months; no X-ray examinations in the previous six months.

The control group included people living in the similar socioeconomic conditions whose RBM radiation dose accumulated over their entire lives did not exceed 70 mGy [8] (tab. 1).

For the study, we sampled 9 ml of fasting blood of the participants into vacuum tubes filled with K3-EDTA (Greiner Bio-One; Austria). Flow cytometry enabled assessment of the relative number of T helper subpopulations by the level of expression of CD45RA (naive T helpers marker), CD62L (marker of directed cell migration to the peripheral lymphoid organs), CCR4, CCR6, CXCR3, and CXCR5. Into the flow cytometer (Beckman Coulter; USA) tube, we added 100 µl of the test sample, 5 µl of CD3 and CD4 monoclonal antibodies (Beckman Coulter, USA; conjugated with Krome Orange and Pacific Blue, respectively), 20 µl of CD45RA and CD62L monoclonal antibodies (Beckman Coulter, USA; conjugated with PE and FITC, respectively), 5 µl of monoclonal antibodies CCR4, CCR6, CXCR3 and CXCR5 (Beckman Coulter, USA; conjugated with APC, Per-CP-eFluorTM710, PE-Cyanine7 and PE-eFluor® 610 respectively). The samples were incubated for 20 minutes in a dark place at room temperature. Then, we added 1 ml of VersaLyse Lysing Solution (Beckman Coulter; USA) to the tube to remove erythrocytes, and then left the samples to incubate for 10 more minutes under the same conditions. After incubation, the samples were analyzed in a Navios flow cytometer (Beckman Coulter; USA).

The gating tactic we employed in the context of T helper subpopulations analysis relied on the identification of T helpers by the presence of CD3 and CD4 markers on the cell surface. Next, we divided the population of CD3⁺CD4⁺ cells into subpopulations of T helpers at different stages of differentiation by the presence of surface markers CD45RA and CD62L. Namely, we differentiated between phenotype CD3⁺CD4⁺CD45RA⁺CD62L⁺ naive T helpers (T_Naive), central memory phenotype CD3⁺CD4⁺CD45RA^-CD62L⁺ T helpers (T_CM), effector memory phenotype CD3⁺CD4⁺CD45RA^-CD62L^- T helpers (T_EM) and terminally differentiated phenotype CD3⁺CD4⁺CD45RA⁺CD62L^- T helpers (TEMRA). In the T_CM and T_EM populations, subpopulations of various T helpers were identified by the presence of CCR4, CCR6, CXCR3, and CXCR5 markers [9–11] (tab. 2).

For statistical data processing we employed SigmaPlot software (SYSTAT Software; USA). Kolmogorov-Smirnov test enabled verification of normalcy of distribution of the indicators. To compare the arrays of nonparametric data, we applied the Mann–Whitney U-test. First of all, the above parameters were evaluated for T helpers at different levels of differentiation, then we processed the data describing subpopulations of central and effector memory T helpers.

To identify dependencies, we used the Spearman's rank correlation coefficient and the Pearson correlation coefficient, as well as linear regression. The results were considered reliable at 5% significance level.

RESULTS

The currently adopted approach is to rely on CD markers expression to identify T helpers at different stages of differentiation. The literature offers detailed descriptions of the ability of TNaive not differentiated in the secondary lymphoid organs (antigen-dependent differentiation) to give rise to memory T cells and effector cells. T_CMs carry the CD62L adhesion molecule, which determines their ability to largely localize in the secondary lymphoid organs. T_EMs are not able to penetrate into the peripheral lymphoid organs, however, they carry a wide range of different adhesion and chemokine molecules on their surface, which aid their migration into tissues and organs. The ability of T_EMs to proliferate and differentiate is reduced, and the cells part of this population are the main producers of effector cytokines, such as IFNγ and IL4. TEMRA effector cells are considered to be the final stage of T lymphocyte differentiation process in the peripheral blood. The effector properties of TEMRA require no costimulation; they manifest under the action of cytokines produced by the inflamed tissue [12].

At the first stage of the study, we relied on the expression of CD45RA (surface marker) and CD62L (marker of directed cell migration to the peripheral lymphoid organs) to investigate the relative number of T helpers at different stages of differentiation: T_Naive, T_CM, T_EM, and TEMRA (tab. 3).

Comparison of the quantitative indicators of lymphocytes and T helpers at different stages of differentiation in the exposed and control groups revealed no significant differences (tab. 3).

The expression of CCR4, CCR6, CXCR3, and CXCR5 chemokine receptors allowed estimating the number of T helper populations in the T_CM and T_EM subpopulations. The Th₁, Th₂, Th₁₇ and Th₂₂, Th₉ subpopulations, as well as follicular T helpers, are predominantly found in the T_CM and T_EM populations. All these cells have unique developmental and regulatory pathways and play different roles in the immunity and immunity-mediated pathologies [13].

Tables tab. 4 and tab. 5 present the results of estimation of representation of various subpopulations of T helpers in the T_CM and T_EM populations, as well as the ratio of Th₁/Th₂ and Th₁/Th₁₇ in the exposed individuals group.

We discovered no significant differences in the T_CM population T helper subpopulation values between the exposed individuals and control groups.  

Comparison of the T_EM populations' T helper subpopulation indicators revealed no significant differences between the study groups.

To establish the long-term dependence of the number of different subpopulations of T helpers at different stages of differentiation in the peripheral blood of the exposed individuals we applied the Spearman's rank correlation coefficient and the Pearson correlation coefficient. "Long-term" here means that RBM, thymus, and peripheral lymphoid organs have accumulated the dose a long time ago. The analysis procedure covered both groups.

The correlation analysis did not reveal significant associations of the number of T helpers at different stages of differentiation in the peripheral blood of the exposed individuals with the dose accumulated by RBM, thymus and peripheral lymphoid organs.

Investigating the dependence of content of various T_CM population's T helper subpopulations on the radiation dose accumulated by the RBM, thymus and peripheral lymphoid organs, we discovered that the content of double positive Tfh₁₇ subpopulation depended significantly on the degree of irradiation of the thymus and peripheral lymphoid organs (p = 0.02; S = 0.23). However, linear regression analysis did not reveal significant dose-based dependences. As for the remaining studied subpopulations of T helpers, we also discovered no significant dependences on the dose values.

A similar analysis was carried out to investigate the dependences of the content of various T_EM population's T helper subpopulations on the degree of irradiation of RBM, thymus and peripheral lymphoid organs. No statistically significant dependences of the studied parameters of the T_EM populations on the dose values were found.

It is known that with age, immune system of the human beings undergoes involutional changes: the number of some subpopulations of T helpers goes down [14], the direction of differentiation changes [15], and their functioning is disrupted [16]. With this in mind, we have also investigated dependence of the content of various peripheral blood T helper subpopulations on age (tab. 6; the age was that reached by the participants at the time of the study).

Correlation analysis revealed a significant association between the decrease in the number of of T_Naive and age in the exposed group. The values were p = 0.009, S = –0.35 and p = 0.01, R = –0.34 (Spearman's rank correlation coefficient and Pearson correlation coefficient, respectively). No such association was registered in the control group. In the exposed group, we have also found that the number of T_EM grows up significantly with age (p = 0.04, S = 0.28 and p = 0.02, R = 0.33, Spearman's rank correlation coefficient and Pearson correlation coefficient, respectively). Nothing similar was discovered in the control group. These dependencies were investigated with a linear regression analysis (fig. 1; moving average graphs).   

We discovered a significant dependence of the content of T_EM subpopulation's double positive Tfh₁₇ (peripheral blood) on age in both groups, but these associations were multidirectional: in the exposed individuals, the number of cells of this population decreased significantly with age (p = 0.04; S = –0.28), and in the control group, on the contrary, the said number increased (p = 0.003, S = 0.32 and p = 0.05, R = 0.29, Spearman's rank correlation coefficient and Pearson correlation coefficient, respectively). Moreover, in the control group we revealed an age-dependent increase in the T_CM subpopulation's double positive Tfh₁₇ counts (p = 0.009, R = 0.38), while in the exposed group no such dependence was registered.

Linear regression analysis of the dependence of number of T_CM and T_EM subpopulations' double positive Tfh₁₇ on the age of the exposed group participant revealed no significant correlations; for the control group, the results are shown on fig. 2 (moving average graph).

The analysis of dependence of the content of various peripheral blood T helper subpopulations on the age of exposed individuals at the beginning of exposure showed the following correlations: the amount of T_Naive decreased as the age increased (p = 0.03, S = –0.34 and p = 0.04; R = –0.32, Spearman's rank correlation coefficient and Pearson correlation coefficient, respectively), while the number of T_EMs increased (p = 0.03, S = 0.35 and p = 0.04, R = 0.32, Spearman's rank correlation coefficient and Pearson correlation coefficient, respectively) (fig. 3).   

As for the remaining T helper populations, we discovered no significant dependences on the age reached at the time of the study and at the beginning of exposure.

DISCUSSION

T helper cells are critically important to the regulation of immune system: the range of their actions stretches from activating B-lymphocytes, cytotoxic T-lymphocytes and other cell populations to suppressing immune response.

However, in addition to supporting the functions of adaptive immunity, T helpers can also be involved in the development of autoimmune [17, 18] and oncological diseases [19, 20]. The development of chronic lymphocytic leukemia was reported to be associated with the spread of abnormal follicular T helper cells that elevate levels of cytokines and produce costimulatory factors that promote tumor cell proliferation [21]. Other studies [22, 23] have shown protumor activity of Th₁₇, which manifests in the production of immunosuppressive cytokines and chemokines in the tumor microenvironment, thus stimulating its growth and metastasis.

The balance of various subpopulations of T helpers plays an important role in the immune response. For example, cytokines produced by Th₂ cells block production of Th₁ cytokines and are their natural killer cells. In addition, Th1 cells can inhibit the differentiation and proliferation of basophils and eosinophils, the activity of which is controlled by the synthesis of Th₂ cytokines [24]. Ionizing radiation compromises the Th₁/Th₂ immune balance and tilts it towards Th₂ dominance, the unbalanced state potentially contributing to immune system dysfunction after exposure [25]. A number of studies report a shift in the Th₁/Th₂ balance towards Th₂ in the cases of hematological malignant neoplasms [26–28]. More recent papers describe another association of immunocompetent cells, Th₁/Th₁₇, which, when unbalanced, contributes to the development of autoimmune diseases, primarily rheumatoid arthritis [29, 30].

Investigating the long-term effects of exposure to radiation, we discovered no significant differences in the relative content of various peripheral blood T helper subpopulations, as well as the Th₁/Th₂ and Th₁/Th₁₇ associations, between the exposed individuals and control groups. However, in the exposed group, we did reveal some peculiarities (differences from the control group indicator values) in the dependence of individual populations of peripheral blood T helpers on the degree of irradiation of thymus, peripheral lymphoid organs and age.

In the exposed group, we established that the increase in the amount of peripheral blood T_CM population's double positive Tfh₁₇ depends on the dose accumulated by the thymus and peripheral lymphoid organs. Other studies that involved people exposed to radiation have also registered dose-dependent changes in the number of T helpers and their functional properties. Thus, the Hiroshima and Nagasaki atomic bombing survivors exhibited a dose-dependent decrease in the number of CD4⁺ T cells in peripheral blood [3], a higher occurrence of T cell receptor mutations (mainly in CD4⁺ T cells population) [31], and a dose-dependent shortening of the T helper telomere length at doses above 0.5 Gy [32].  In Mayak employees that were chronically exposed to radiation the T helper part of the immune system was also changed: a greater dose (2–4 Gy, external irradiation) translated into a smaller number of CD4⁺ cells [33]; the concentrations of some cytokines and chemokines changed, too. The results obtained allowed a conclusion that the identified changes in the parameters of immune systems of the examined individuals supported chronic inflammatory status and could contribute to the development of late radiation-induced pathologies, such as cardiovascular and malignant diseases [5, 34].

In addition to the dependences on radiation dose, agerelated changes were found in the groups. We discovered that the number of T_Naive in the peripheral blood of the exposed individuals decreased with age, and the amount of T_EMs increased, the latter capable of migrating through the vascular and tissue endothelium to inflammation foci and triggering a rapid immune response with the synthesis of predominantly effector cytokines [12]. These results are consistent with the literature data describing the decrease of naive T helper cell numbers with age in people older than 70–75 years [14]. In addition, naive T cells in older people grow prone to effector differentiation [35]. It should be noted that no such correlations were found in the control group, where the number of double positive Tfh₁₇ in the T_CM and T_EM subpopulations increased with age, while in the exposed group, on the contrary, the T_EM subpopulation's double positive Tfh₁₇ content was growing down very slightly with age. Follicular T helper cells express CXCR5 chemokine receptor, which allows them to migrate to its CXCL13 ligand in the B-cell follicle. Normal follicular T helper cells produce a unique range of cytokines and chemokines needed for supporting survival and proliferation of B-cells in the germinal centers [9, 21, 36, 37]. Thus, Tfh₁₇ produce IL21 and IL17, which are involved in enhancing the interaction between T and B cells and are necessary for the formation of germinal centers [38].

Thus, in the exposed individuals, the late effects after chronic exposure to radiation include some changes in the T helper part of the immune system, which depend both on the radiation dose and on the age reached at the time of the study. However, the limited sample recruited for this study disallows unequivocal conclusions at this stage and necessitates further research.

CONCLUSIONS

1. The long term effects of chronic exposure to radiation with the doses predominantly accumulated by the RBM (average dose — 567 ± 73 mGy), as registered in the Techa Riverside residents aged 67 through 84, do not include changes in the relative content of various T helper subpopulations in peripheral blood. 2. The relative amount of double positive Tfh₁₇ contained in the peripheral blood T_CM population of the exposed individuals positively correlated with the degree of irradiation of thymus and peripheral lymphoid organs (p = 0.02); however, there was no linear regression dependence registered. 3. The data obtained indirectly indicate intensification of involutional processes in the exposed individuals due to the number of T_Naive decreasing with age and the number of T_EM increasing therewith. In the exposed individuals group, the following late age-related dependences were revealed: decreasing relative amount of TNaive (p = 0.009) and double positive Tfh17 in the T_EM subpopulation (p = 0.04), and increasing relative content of T_EM (p = 0.04 ) in the peripheral blood. However, no similar dependencies were found in the control group.