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RADIATION RESEARCH 159, 109–116 (2003)0033-7587/03 $5.00q 2003 by Radiation Research Society.All rights of reproduction in any form reserved.

Late Effects of the Chernobyl Radiation Accident on T Cell-MediatedImmunity in Cleanup Workers

Oleg Kuzmenok,a,c Michail Potapnev,b Svetlana Potapova,c Victoria Smolnikova,c Valeriy Rzheutsky,d

Alexandr A. Yarilin,e Wilson Savinoa and Igor M. Belyakovf,1

a Laboratory on Thymus Research, Department of Immunology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Av. Brasil,4365-Manguinhos 21045-900 Brazil; b Belarussian Center for Pediatric Oncology and Hematology, P.O. Lesnoye, Minsk District 223052 Republic

of Belarus; c Belarussian Research Institute of Hematology & Blood Transfusion, 160 Dolginovsky Tract, Minsk 223059 Republic of Belarus;d Belarussian Dispensary of Radiation Medicine, Philimonova Str, Minsk 220000 Belarus; e Institute of Immunology, Kashirskoye Shosse 24-2,

Moscow, Russia; f Molecular Immunogenetics and Vaccine Research Section Metabolism Branch, National Cancer Institute, National Institutes ofHealth, Bethesda, Maryland 20892

Kuzmenok, O., Potapnev, M., Potapova, S., Smolnikova, V.,Rzheutsky, V., Yarilin, A. A., Savino, W. and Belyakov, I. M.Late Effects of the Chernobyl Radiation Accident on T Cell-Mediated Immunity in Cleanup Workers. Radiat. Res. 159,109–116 (2003).

The main goal of this investigation was to evaluate the ab-normal T-cell immunity in cleanup workers who took part inthe cleanup after the Chernobyl accident in 1986. Peripheralblood mononuclear cells (MNCs) of apparently healthy clean-up workers (n 5 134) were used to analyze the phenotype andproliferative response to mitogens in vitro. Evaluation of theMNC phenotype of cleanup workers did not reveal a signifi-cant disturbance in the T-cell subpopulation content exceptfor an increase in CD31CD161561 (NKT) cells. Immunophe-notyping of phytohemagglutinin (PHA)-activated MNCs dem-onstrated suppression of CD41 T-cell propagation and aug-mentation of CD81 T-cell propagation in vitro compared tocontrol individuals. DNA synthesis in the MNCs of cleanupworkers was markedly inhibited after activation for 3 dayswith suboptimal concentrations of PHA, pokeweed mitogenand PMA. In contrast to control individuals, the monocytesof cleanup workers were able to stimulate the proliferation ofT cells from healthy individuals but inhibited the proliferationof T cells from cleanup workers. This study affords a betterunderstanding of the response of MNCs to stimulation withsuboptimal concentrations of PHA and provides an approachto a more accurate analysis of the immunological disordersfound after exposure to radiation from Chernobyl-relatedactivities. q 2003 by Radiation Research Society

INTRODUCTION

The accident at the Chernobyl nuclear power station innorthern Ukraine on April 26, 1986 resulted in both acute

1 Address for correspondence: Molecular Immunogenetics and VaccineResearch Section, Metabolism Branch, National Cancer Institute, Nation-al Institute of Health, Bldg. 10, Rm. 6B-12 (MSC1578), Bethesda, MD20892; e-mail: belyakov@box-b.nih.gov.

and long-lasting harmful effects on human health. About300,000 persons (cleanup workers or ‘‘liquidators’’) partic-ipated in the cleanup activities at the Chernobyl nuclearpower station. During the first year after the accident, acuteradiation sickness was observed in 41 persons due to ex-posure to a high (4–9 Gy) external dose of radiation (1).All other categories of liquidators received significantlylower doses of radiation. The mean doses received wereeither 10 cGy (2) or 25 cGy (3). The results of cytogeneticanalyses of more than 500 liquidators performed by Schev-chenko et al. showed that the mean dose received was be-tween 13.6 and 41.4 cGy (4). In the present study, we ex-amined a group of liquidators who received a mean doseof 15–50 cGy.

The human immune system is involved in the regulationof most pathological processes. Impaired immunity pro-motes disease progression and, in some cases, disease ini-tiation. Therefore, numerous studies have been done toevaluate both the acute and late effects of radiation on theimmune system of victims of the Chernobyl accident (5–10). It was reported that a marked reduction in T-cell con-tent was observed in affected persons during the first yearafter the accident (5, 8). One to 2 years later, a tendency toa return to a normal level of T cells was observed (10).Five to 10 years after the accident, different groups of in-vestigators reported decreased (8, 9) or normal (11) levelsof T cells and T-cell subpopulations in the peripheral bloodof cleanup workers and other groups of persons affected bythe Chernobyl accident. Functional assessments in vitrodemonstrated a consistent decrease in the rate of lympho-cyte proliferation (7, 11). Abnormalities in the blood mono-cyte and natural killer (NK) cell content and function werealso found in cleanup workers in the period after the Cher-nobyl accident (8, 12, 13). A recent study by Kusunoki etal. (14) indicated that the reduction in the PHA responsein atomic bomb survivors is dependent on a decrease inIL2-producing CD4 T cells. When exogenous IL2 was add-

110 KUZMENOK ET AL.

ed to the peripheral blood lymphocytes of the heavily ex-posed individuals, the proliferative response to the mitogenwas restored (14).

The purpose of this study was to evaluate late T-cellimmune abnormalities in a selected, apparently healthygroup of cleanup workers who now live in Belarus. Wefound that the main subpopulations of the peripheral lym-phocyte population (CD31, CD41, CD81 and CD191) werenot different from those in control individuals. Immuno-phenotyping and in vitro cell culturing revealed suppressionof cell proliferative capacity and an abnormal pattern ofsubpopulation distributions in the T cells of cleanup work-ers after activation with plant lectins (phytohemagglutinin,pokeweed mitogen, etc.). Suppression of PHA-induced T-cell proliferation depends predominantly on the low acces-sory function of monocytes. These results may be usefulfor a better understanding of the long-term immunologicaldisorders that occur after exposure to low doses of irradi-ation.

EXPERIMENTAL PROCEDURES

Study Subjects

One hundred thirty-four Belarussian cleanup workers were screened in1997–1999. Taking into account the data in the literature indicating thatthe results of dosimetry of the persons investigated were sometimes in-accurate or incorrect (15), we used the four most informative factors whenwe created our group for study—the date of arrival of workers at Cher-nobyl, the distance from the Chernobyl nuclear power station, the dura-tion of their stay, and the nature of their work. The median period oftheir stay at the nuclear power station was 2 weeks from May to October1986. According to Shantyr et al., the level of radiation exposure did notchange significantly within the periods of 0–15, 60–350, 351–700 andmore than 700 days after April 26, 1986 (15). Taking into account ex-ternal dosimetry data obtained from the Research and Clinical Instituteof Radiation Medicine and Endocrinology, Minsk, we estimated a meandose received of 15–50 cGy in our group of liquidators. All of the sub-jects were examined in the Republican Dispensary of Radiation Medicine,Ministry of Health, Belarus. This study was reviewed and approved bythe Ethics Committee of the Belarussian Dispensary of Radiation Medi-cine. The group included only cleanup workers who had no clinical man-ifestation of health disorders during and just after work at the Chernobylnuclear power station. At the time of observation, none of the cleanupworkers had complaints or signs of acute illness. The control group com-prised 89 persons matched for similarity of age and health status whodid not take part in work at the Chernobyl nuclear power station and whowere not inhabitants of the radioactively polluted regions of Belarus.

Flow Cytometry

Fresh heparinized blood from cleanup workers and control individualswas used. Immunophenotyping was performed with whole blood or cul-tured mononuclear cells as indicated. Surface antigens were detected bya double-color immunofluorescence assay combining fluorescein isothy-ocyanate (FITC) or phycoerythrin (PE)-conjugated monoclonal antibodies(16). These included CD3-FITC, CD4-FITC, CD8-PE, CD16CD56-PE,HLA-DR-PE, CD19-PE and CD14-PE (Becton Dickinson). All the mono-clonal antibodies were used at concentrations titrated for optimal staining.Treatment of leukocytes with monoclonal antibodies was done accordingto the manufacturer’s instructions. Irrelevant fluorochrome-conjugatedmurine isotype-matched monoclonal antibodies were always included inthe staining protocols as negative controls. Cells were fixed in parafor-

maldehyde solution (2%) within 48 h and were held in this solution untilthey were analyzed by flow cytometry. Detection of fluorescence ofstained cells was performed using a FACScan flow cytometer (BectonDickinson) equipped with an argon laser tuned to 488 nm. Calibrationbeads were used for monitoring and optimizing the instrument settings.Data were acquired with LYSIS II software (Becton Dickinson). Forwardlight scattering (FSC), orthogonal light scattering (SSC), and fluorescencesignals (FL-1-FITC, FL-2-PE) were sorted in listmode data files. For datastandardization, gated acquisition of living lymphocyte populations wasperformed routinely. A minimum of 50,000 cells were analyzed and atleast 5,000 gated events were measured for each sample. All data wereanalyzed using PAINT-A-GATE software (Becton Dickinson). While thephenotype of cultured lymphoid cells was being evaluated, the initialpopulations of mononuclear cells were separated on density gradients asdescribed below. All the cell samples were washed with RPMI 1640medium without serum before they were stained with monoclonal anti-bodies and analyzed by flow cytometry (17, 18).

Preparation of Mononuclear Cells and Assessment of Proliferation

Peripheral blood mononuclear cells (MNCs) were separated by gradientcentrifugation through Ficoll/Hypaque (Pharmacia Biotech) from freshlyobtained heparinized blood from cleanup workers or control individuals.MNCs were washed and suspended in RPMI 1640 medium (Sigma) con-taining 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin,and 10% heat-inactivated AB serum (serum of three to five healthy in-dividuals of the IY blood group), referred to as complete medium. MNCsat a starting concentration of 1 3 106 cells/ml were cultured in triplicatein 96-well flat-bottomed tissue culture plates (Costar) for 3 days at 378Cin a humidified atmosphere of 95% air/5% CO2. The following polyclonalactivators were added: phytohemagglutinin (PHA; Serva), concanavalinA (Con A; Serva), and pokeweed mitogen (PWM; Gibco). Both optimal(10 mg/ml) and suboptimal (0.5 mg/ml) doses were used for each mitogen.In some experiments, the monoclonal antibody anti-CD3 (mg/ml) and IL2(Cetus, 10 and 100 IU/ml) were added as described previously (9). Forthe last 16 h cells were incubated with 37 kBq/well of [3H]dThd (Izotop,Russia). After the culture was terminated, the cells were transferred tofilters with a cell harvester (Skatron, Norway). The radioactivity of thecell samples was counted in a b-particle counter (LS 1301, Becton Dick-inson).

T-Cell and Monocyte Purification and Culturing

T cells were purified by double-rosetting of MNCs with sheep redblood cells pretreated with 2-aminoethylisothiouronium bromide. Thephenotypically defined purity of T cells was 90–93% (estimated as CD31

cells). T cells were resuspended at a concentration of 1.0 3 106 cells/mland plated with an equal volume of medium containing PHA into wellsof flat-bottomed tissue culture plates. Monocytes were obtained as plastic-adherent cells after incubation of MNCs in complete medium in petridishes (Flow Laboratories) for 1 h at 378C (19). Monocytes were detachedusing Versene solution (Flow Laboratories), washed and counted. Flowcytometry analysis revealed 70–80% CD141 cells in the monocyte pop-ulation.

To determine the accessory function of monocytes in PHA-induced T-cell proliferation in cleanup workers, purified T lymphocytes (0.5 3 106 cells/ml) were mixed with monocytes at an optimal ratio of 5:1 and werecultured in wells of a flat-bottomed plate for 3 days at 378C in a humid-ified atmosphere of 95% air/5% CO2. PHA at suboptimal (0.5 mg/ml) oroptimal (10.0 mg/ ml) concentrations was added to activate cells. All thecell samples were cultured in triplicate. Cell radiolabeling, harvesting andcounting of radioactivity were performed as described above.

Statistical Analysis

All data were analyzed using the null hypothesis that the mean changesbetween the two groups were equal. The results are presented as means

111LATE EFFECTS OF THE CHERNOBYL ACCIDENT ON T CELLS

TABLE 1Phenotypic Characterization of Peripheral Blood

Lymphoid Cells of Cleanup Workers

Cell surfacemarkers

Cleanup workers(percentage ofpositive cells)

(n 5 66)

Control individuals(percentage ofpositive cells)

(n 5 19)

CD31

CD41

CD81

CD191

CD161561

CD4181

CD31CD161561

62.0 6 1.437.6 6 1.129.8 6 1.08.4 6 1.2

15.2 6 1.01.7 6 0.26.7 6 0.4*

59.0 6 0.637.4 6 0.529.5 6 0.59.5 6 0.3

17.8 6 0.51.8 6 0.13.9 6 1.2

* P , 0.05 compared to control individuals; the data are presented asmeans 6 SE.

TABLE 2Surface Molecule Expression on In Vitro Activated Lymphoid Cells of Cleanup Workers

and Control Individuals

Surface marker

Percentage of positive cells in population of lymphoid cells

Freshly isolatedCultured for 72 h

in mediumActivated for 72 h

with PHA (10 mg/ml)

CD31

(control, n 5 12)(cleanup workers, n 5 16)

62.0 6 1.857.7 6 2.8

63.1 6 3.464.8 6 2.8

51.4 6 4.836.4 6 3.1*

CD41

(control, n 5 12)(cleanup workers, n 5 16)

39.3 6 2.932.0 6 2.1

40.7 6 2.440.7 6 2.4

31.7 6 5.713.1 6 2.5*

CD81

(control, n 5 12)(cleanup workers, n 5 16)

25.4 6 1.632.0 6 2.1*

24.3 6 1.833.0 6 3.4

35.6 6 5.655.5 6 2.5*

CD161CD561

(control, n 5 12)(cleanup workers, n 5 16)

22.8 6 2.821.7 6 2.4

13.2 6 2.015.9 6 2.3

31.0 6 7.443.8 6 5.7

CD191

(control, n 5 12)(cleanup workers, n 5 16)

6.9 6 1.16.3 6 1.6

9.7 6 2.07.5 6 1.6

4.5 6 0.93.9 6 1.1

* P , 0.05 compared to control subjects; the data are presented as means 6 SE.

6 SE. The significance between the results for different groups was es-timated using a two-tailed Student’s t test and Fisher’s exact test.

RESULTS

Phenotype of Peripheral Blood Lymphoid Cells inCleanup Workers

A broad spectrum of immune abnormalities has been de-tected in cleanup workers and other groups of exposed per-sons early after the Chernobyl accident (5–10). However,no long-term consequences of the accident on the immunesystem have been reported. Therefore, we used several ap-proaches to determine immune dysfunction in cleanupworkers. We started with phenotypic analysis of peripheralblood lymphocytes. As shown in Table 1, the total amountsof T cells (CD31), B cells (CD191), and NK cells (CD161

CD561) were the same in cleanup workers and control in-dividuals. Only the number of CD31CD161CD561 (NK/T)cells was significantly increased (P , 0.05) in the periph-eral blood of cleanup workers. Thus no definite effects ofChernobyl radiation on the NK cell population could bedetected. The same results were obtained for A-bomb sur-vivors after a single radiation exposure ($1.0 Gy) (20, 21).

In some cases, calculation of the mean fluorescence in-tensity allowed us to detect differences in surface moleculeexpression. For example, mean fluorescence intensity forCD81 expression on CD41CD81 cells of cleanup workerswas 375 6 173 arbitrary units, whereas in control individ-uals it was 846 6 115 arbitrary units. There were no dif-ferences in the mean fluorescence intensity of CD3, CD4or CD8 surface molecular expression on the main T-cellsubpopulations between cleanup workers and control indi-viduals (data not shown).

Phenotype of Lymphoid Cells of Cleanup WorkersActivated In Vitro

Since surface molecule expression is under the controlof both internal and external factors, we designed our ex-periment to evaluate the level of lymphocyte marker ex-pression on lymphocytes that had been cultured for 3 days.As shown in Table 2, after in vitro incubation in completemedium without activator, the content of CD31 T cells inthe MNC population of cleanup workers increased to thelevel of that in control individuals. These data argue forsuppression of CD41 and CD31 molecule expression on Tlymphocytes in cleanup workers, probably because therewere suppressive soluble factors in their blood. The contentof B lymphocytes (CD191) and NK cells (CD161CD561)

112 KUZMENOK ET AL.

TABLE 3Proliferative Response of T Cells of Cleanup Workers to Mitogens In Vitro

Mitogen

[3H]dThd incorporation (cpm) by mononuclear cells, cultured for 3 days with:

MediumSuboptimal dose

of stimulatorStimulation

indexOptimal dose of

stimulatorStimulation

index

PHA

Controls

Cleanup workers

377 6 29(n 5 89)

396 6 27(n 5 134)

6889 6 600(n 5 89)

4179 6 484**(n 5 134)

18.2

10.6

36,167 6 1724(n 5 89)

24,726 6 1503**(n 5 134)

95.9

62.4

Con A

Controls453 6 76 2144 6 672 4.7 12,927 6 3768 28.5

(n 5 13) (n 5 10) (n 5 10)Cleanup workers 396 6 64

(n 5 13)1064 6 243

(n 5 10)2.6 13,168 6 4008

(n 5 10)33.3

PWM

Controls

Cleanup workers

319 6 25(n 5 40)

386 6 30(n 5 43)

9907 6 1080(n 5 24)

5941 6 756**(n 5 24)

31.1

15.4

10,056 6 926(n 5 33)

9226 6 1162(n 5 33)

31.5

23.9

PMA

Controls

Cleanup workers

286 6 31(n 5 19)

306 6 36(n 5 20)

1918 6 503(n 5 19)

773 6 258*(n 5 20)

6.7

2.5

6166 6 1108(n 5 19)

3396 6 638*(n 5 20)

21.6

11.1

IL2

Controls

Cleanup workers

300 6 88(n 5 7)

404 6 49(n 5 24)

1141 6 322(n 5 20)

1554 6 346(n 5 25)

3.8

3.8

2870 6 964(n 5 20)

5900 6 1300(n 5 24)

9.6

14.6

a-CD3

Controls

Cleanup workers

374 6 28(n 5 9)

271 6 28(n 5 42)

n.d.

n.d.

3943 6 1039(n 5 8)

2125 6 436(n 5 42)

10.5

7.8

Notes. Polyclonal activators PHA, Con A and PWM were used at suboptimal concentration (0.5 mg/ml) or optimal concentration (10 mg/ml), PMAat suboptimal (5 ng/ml) or optimal (50 ng/ml) concentration, IL2 at suboptimal (10 IU/ml) or optimal (100 IU/ml) concentration, IL2, anti-CD3 atoptimal concentration (1:300). * P , 0.05, ** P , 0.01; the data are presented as means 6 SE. n.d., not determined. The stimulation index is theratio between proliferative response after stimulation of T cells with mitogen to spontaneous proliferative response (no mitogen).

was the same in initial and cultured samples of mononu-clear cells of both cleanup workers and control individuals.It should be emphasized that there was abnormal up-regu-lation (P , 0.05) of the percentage of CD81 T cells in 3-day cultured MNCs of cleanup workers compared to con-trol individuals.

After culture for 3 days with the polyclonal activatorPHA at the optimal concentration (10 mg/ml), the mono-nuclear cells of both cleanup workers and control individ-uals displayed different patterns in the content of lympho-cyte subpopulations. We observed an evident decline (P ,0.05) in the number of CD31 and CD41 T cells in cleanupworkers compared to control individuals. In contrast, thepercentages of CD81 T cells and CD161CD561/NK cellswere increased more dramatically in cleanup workers com-pared to control individuals. This argues for a consistent

imbalance of T-cell differentiation in vitro under the influ-ence of PHA in cleanup workers.

Proliferative Response of Lymphoid Cells to Mitogens

Our next task was to evaluate the functional activity ofthe peripheral mononuclear cells of cleanup workers. MNCproliferation in vitro was used as the primary assay of ac-tivity; this assay also serves as a general indicator of thedegree of cooperation between T cells, B cells, and mono-cytes. We used both optimal and suboptimal concentrationsof polyclonal T-cell activators PHA, ConA and PMA, andof PWM, which mainly activates B cells. As shown in Ta-ble 3, the response of the mononuclear cells of cleanupworkers was significantly decreased with optimal concen-trations of PHA (P , 0.01) and PMA (P , 0.05) compared

113LATE EFFECTS OF THE CHERNOBYL ACCIDENT ON T CELLS

FIG. 1. Proliferative response of the T cells of cleanup workers andhealthy individuals to a suboptimal concentration of phytohemagglutinin(0.5 mg/ml) in the presence of allogeneic monocytes from healthy indi-viduals and cleanup workers. The data are presented as means 6 SE forcleanup workers (n 5 18) and healthy individuals (n 5 16). Differencesfrom the control group: **P , 0.01 FIG. 2. Distributions of the levels of PHA-induced proliferative re-

sponses of mononuclear cells from healthy individuals (panel A) andcleanup workers (panel B). The x axis is the amount of [3H]dThd incor-poration (cpm), and the y axis is the number of individuals with thatlevel.

to the response of control individuals. The proliferative re-sponses of MNCs to suboptimal concentrations of PHA (P, 0.01), PWM (P , 0.01) and PMA (P , 0.05) were alsoreduced in the group of cleanup workers. The level of ConA-induced MNC proliferation was not significantly de-creased for either concentration in cleanup workers.

It is of interest that the TCR-restricted proliferative re-sponse of T cells to anti-CD3 monoclonal antibodies wasnot decreased significantly (Table 3) compared to the great-er suppression of the response of mononuclear cells seenearlier (22). We were surprised to find increased MNC pro-liferation in response to an optimal concentration of thecytokine IL2, which may indicate the up-regulation ofCD25 on these cells. Autocrine secretion of IL2 along withthe up-regulation of CD25 on MNCs can lead to malignan-cy (7, 9).

Monocyte-Dependent Suppression of T-Cell Proliferationby PHA in Cleanup Workers

T-cell activation with activators/antigens (especially atsuboptimal concentrations) is dependent on cooperation be-tween the antigen-presenting cells and the T cells (23). Weevaluated the accessory function of monocytes/macrophag-es in the proliferative response of purified T cells to sub-optimal concentrations of PHA. As shown in Fig. 1, therewere no differences in the levels of spontaneous prolifera-tion of T cells from cleanup workers and the control group.The PHA-stimulated response of the T cells from cleanupworkers was decreased compared to the control group (P, 0.05).

Allogeneic monocytes purified from healthy individuals

marginally augmented the proliferative response of T cellsfrom both cleanup workers and control individuals, but theincrease in the response was not statistically significant.Other results were obtained when allogeneic monocytesfrom cleanup workers were used (see Fig. 1). The level ofproliferation of T cells from healthy individuals was in-creased 2.4-fold by allogeneic Mf from cleanup workerscompared to the 1.5-fold increase found using an allogeneicMf from healthy individuals (P , 0.01). Analogous butsmaller differences were observed when an optimal con-centration (10 mg/ml) of PHA was used (data not shown).

These results indicate that monocytes from cleanupworkers have a stronger co-stimulating effect on T-cell re-sponses in both groups. These data suggest that differencesin the cleanup worker Mf may have a strong impact on T-cell function.

Immune Abnormalities as Criteria for Further Monitoringof Cleanup Workers

Our findings of a reduced function of T cells (prolifera-tive response to polyclonal activators in vitro) were used toevaluate the types of immune abnormalities in the cleanupworkers. As shown in Fig. 2, the pattern of distribution ofthe level of MNC proliferative responses to PHA (0.5 mg/ml) among cleanup workers (panel B) was markedly dif-ferent from that of control individuals (panel A). The num-ber of middle-range (normal type) responders was reducedin cleanup workers compared to control individuals. The

114 KUZMENOK ET AL.

numbers of persons with low and high levels of T-cell pro-liferation were markedly increased in cleanup workers. Thenumber of ‘‘nonresponders’’ (with a level of [3H]dThd in-corporation lower than 500 cpm) was 3/89 in the controlgroup compared to 27/134 in the cleanup workers (P ,0.002, Fisher’s exact test). The number of ‘‘over-respond-ers’’ (with a level of [3H]dThd incorporation higher than20,000 cpm) was 0/89 in the control group compared to 4/134 in the cleanup workers (P 5 0.11, Fisher’s exact test).

DISCUSSION

Delayed biological effects of radiation as a result of theChernobyl accident in 1986 have been studied extensively.Numerous signs of immune system dysfunction in exposedpersons have been detected (5–13, 20, 21). The T-cell sys-tem has been the main point of interest because of its keyrole in immunoregulation. Our data show that 13 years afterthe Chernobyl accident, the numbers of cells in the phe-notypically defined T-cell subpopulations do not differ sig-nificantly in cleanup workers and nonexposed control in-dividuals. It is clear that the dynamics of the changes inCD41 cells are dependent on dose and time. Interestingly,other authors found conflicting results concerning the con-tent of CD41 and CD81 subpopulations. One group re-ported a decrease in the level of both CD31 and CD41 Tcells in the peripheral blood of cleanup workers 10 yearsafter the accident (12). Another reported that the percentageof CD41 cells was increased significantly in heavily irra-diated men while the percentage of CD81 cells tended todecrease with dose (6).

To determine factors influencing the expression of thesemolecules, we scored the percentage of T-cell subpopula-tions before and after culturing in vitro. Our data (Table 2)clearly demonstrate that cleanup workers initially had lowernumbers of CD31 and CD41 T cells and that the percent-ages of these cell populations returned to normal levels af-ter culturing for 3 days (without activation). In control in-dividuals, the content of CD31 and CD41 T cells was thesame before and after culturing in vitro. This suggests thatin cleanup workers the surface expression of T-cell markersis under the control of serum-inhibitory factors. These sol-uble factors are not detected functionally in the serum ofhealthy individuals, but they can be found in patients withoncological and other diseases (C. Healy, personal com-munication). Moreover, the serum of cleanup workers hasbeen reported to inhibit PHA-induced proliferation of hu-man lymphocytes (25). Also, after low doses of radiation,antiepithelial autoantibodies (which can be detected in theserum) can induce dysfunction of the thymus and thymus-dependent T-cell development (7). As was shown by Chenet al. (26), g irradiation alone does not stimulate T-cellproliferation. However, radiation enhances PHA-inducedCD69 expression (which does not lead to proliferation) at2 h, and T-cell proliferation was inhibited at day 3 (26).

We also demonstrated that the abnormal pattern of acti-

vation-induced expression of T-cell-restricted markers re-flects an intrinsic defect in the T-cell subpopulation matu-ration in cleanup workers. In a special series of experi-ments, we evaluated the content of CD31, CD41 and CD81

T cells in mononuclear cells cultured for 3 days with PHA.As shown in Table 2, marked suppression of CD41 T-cellmaturation and augmentation of CD81 T cells and CD161

CD561 NK cells was found in cultures of PHA-stimulatedmononuclear cells of cleanup workers. We speculate thatthis activation-induced deviation in the maturation of T-cellsubpopulation may be one of the consistent characteristicsof impaired T-cell immunity in cleanup workers.

Among another phenotypic findings in our study was theaugmented level of peripheral blood CD31CD161CD561

NK/T cells in cleanup workers compared to control indi-viduals. Other authors detected other small T-cell subpop-ulations (CD41CD81 T cells, CD41CD251 T cells,CD31CD41 T cells, etc.) as evidence of the disturbance ofthe T-cell system in persons exposed to radiation (11, 27,28). These small Th1 subpopulations can be sources of anabnormal pattern of cytokines (29, 30).

The functional assessment of T cells is of special valuebecause of its key role in immune regulation of humanpathology. To detect impaired T-cell function, we increasedthe sensitivity of the cell proliferation assay by using sub-optimal concentrations of polyclonal activators in vitro.This approach was used to demonstrate the decreased T-cell proliferation that was induced by plant lectins (PHA,Con A, PWM) and PMA (activator of protein kinase C) incleanup workers compared to control individuals. The sen-sitivity of proliferative assays using optimal concentrationsof polyclonal activators was lower. It is known that thesemitogens activate T cells through classical (plant lectins,anti-CD3) and alternative (PMA) pathways (22, 31). Ourdata confirmed previous results (7, 25) indicating a markedsuppression of the T-cell proliferative response in cleanupworkers through classical (TCR-dependent) and alternative(TCR-independent) pathways. In our study, several types ofmitogens (plant lectins, monoclonal antibodies to CD3,PMA) served as differential probes to evaluate T-cell func-tion in vitro. Our data show the impairment of both theclassical and alternative pathways of T-cell proliferation invitro in cleanup workers compared to control individualsmore than 10 years after the Chernobyl accident. Analogousresults were obtained for cleanup workers 5–7 years afterthe Chernobyl accident (22).

It is becoming clear that the response of MNCs to mi-togens in victims of the Chernobyl accident and in A-bombsurvivors depends on production of IL2. Using limiting di-lution assays, Kusunoki et al. (14) showed that the PBMCsfrom heavily exposed individuals contained a low numberof IL2-producing CD4 T cells and had a decreased responseof MNCs to mitogens. The response to the mitogen is great-ly dependent on both CD41 and CD81 cells (24). The effectof radiation on the functional activity of CD81 cells is lesswell known. However, the results of Kajioka et al. indicate

115LATE EFFECTS OF THE CHERNOBYL ACCIDENT ON T CELLS

that the CD81 T-cell subset in mice is more radioresistantthan the CD41 T-cell subset (32). Carloni et al. recentlyshowed that PHA stimulation of human MNCs could rescuelymphocytes from X-ray-induced apoptosis (33).

Plant lectin-induced T-cell proliferation is an accessorycell-dependent immune reaction. Therefore, we comparedthe function of monocytes from cleanup workers and con-trol individuals as antigen-presenting cells in a T-cell pro-liferative assay in an allogeneic system. We were surprisedto find that the response of purified T cells (.90% CD31

cells) from cleanup workers and control persons to PHAdid not differ significantly in the presence of antigen-pre-senting cells (monocytes) from healthy individuals. At thesame time we observed a difference in the effect of mono-cytes from cleanup workers on the proliferation of T cellsfrom cleanup workers and control individuals. Monocytesfrom cleanup workers significantly augmented the level ofproliferation of T cells from control individuals but inhib-ited proliferation of T cells from cleanup workers. It is dif-ficult to explain this result. However, it should be men-tioned that one of the well-described effects of the Cher-nobyl accident on the immune system of cleanup workersis consistently increased levels of monocytes in peripheralblood and disregulation of the production of IL1B and TNFin vitro by mononuclear cells (12). However, a recent studyby Lankford et al. (34) indicated that antigen-presentingcells are resistant to low doses of UV light, while the sur-face markers and cytokine production of T cells were se-lectively affected (34).

We speculate that there are three possible targets for thedetection of impaired T-cell function in cleanup workers:(1) serum inhibitory factors that down-regulated T-cell sur-face antigen expression, (2) an intrinsic defect of CD31

(predominantly CD41) T-cell activation-induced maturationin vitro and an augmented level of small T-cell subpopu-lations in vivo; (3) impaired monocyte function as antigen-presenting cells in the T-cell proliferation assay. The mainquestion that arises is whether this exists in all the cleanupworkers. To answer this question, we compared the distri-bution of the levels of PHA-induced lymphocyte prolifer-ative responses in vitro among cleanup workers and controlindividuals. As shown in Fig. 2, the histogram of T-cellproliferative responses in cleanup workers was markedlyskewed to the left compared to the histogram for controlindividuals. A comparison of the two histograms revealedthe disappearance of the middle-range (normal) respondersand a substantial increase in the number of nonresponders(sixfold) and over-responders (more than fourfold). It is ofinterest that histogram analyses of some other immunolog-ical parameters (number of CD41 T cells, etc.) demonstrat-ed a similar distribution for cleanup workers (data notshown). This type of distribution of the level of lymphocyteproliferation in cleanup workers in response to T-cell mi-togen PHA indicates that statistical analyses based on scor-ing average or mean parameters does not completely reflectthe effect of the Chernobyl radiation accident on the im-

mune system of cleanup workers; additional approachesshould be applied. In addition, the proposal of some authors(35) regarding the predominantly Th2 immune polarizationin people suffering from the effects of radiation accidentsneeds to be corrected because of the existence of differ-ential groups of persons with opposite immune parameters.In this regard, the group of nonresponders can be consid-ered as persons with Th2 dominance and the group of over-responders can be considered as individuals with a predom-inant Th1 type of immune reactivity. The number of non-responders is 6.75-fold higher than the number of over-responders. Fujiwara et al. found an elevated level ofautoantibodies in Japanese A-bomb survivors who were ex-posed to similar doses of radiation (36). These facts reflectthe predominance of the Th2 type of immune reactivity invictims of radiation accidents.

Thus we conclude that T-cell-related immune deviationsare seen in cleanup workers in the late period after theChernobyl accident. These deviations are predominantlyfunctional and can serve as more accurate immunologicalcriteria for the observation of cleanup workers.

ACKNOWLEDGMENTS

This work was performed according to an agreement with the Dispen-sary of Radiation Medicine, Ministry of Health, Belarus. It was supportedin part by Belarussian Soros Foundation Grant B 96-18-2421-2, Inter-national Open Society Grant RSS 1355/1997, and United Nations De-partment of Humanitarian Affairs Grant Health/BEL 04/98.

Received: May 2, 2002; accepted: August 28, 2002

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