Ex vivo stimulation of cord blood mononuclear cells by dexamethasone and interleukin-7 results in the maturation of interferon-γ-secreting effector memory T cells

Abstract

The effects of dexamethasone phosphate and interleukin-7 upon the proliferation of T-cells and the production of interferon-γ in the newborn's cord blood mononuclear cell cultures were studied. The capability of dexamethasone to enhance T-cell proliferation induced by anti-CD3 with interleukin-7 in some newborn cord blood mononuclear cell cultures was identified. Dexamethasone suppressed production of interferon-γ in 68-h cell cultures stimulated with anti-CD3 both in the presence of interleukin-7 and without it. However, a 68-h cultivation of newborn blood cells with dexamethasone, anti-CD3 and interleukin-7 resulted in the accumulation of T-lymphocytes capable of producing interferon-γ after restimulation. As a result of it the amount of interferon-γ producing CD7⁺ T-cells and the concentration of interferon-γ in cultural supernatants were maximal in the cell cultures incubated with anti-CD3, interleukin-7 and dexamethasone during the first 68 h and subsequently restimulated with phorbol 12-myristate 13-acetate and ionomycin. The stimulation of neonatal or adult blood cells by dexamethasone, anti-CD3 and interleukine-7 also causes a decrease in the number of naïve T-cells and central memory cells and an increase in the number of effector memory CD7⁺CD45RA⁺CD62L⁻ cells in cultures. It is possible that these effects are caused by the influence of dexamethasone on IL-7 receptor expression: it is known that IL-7 receptor alpha-chain gene is a glucocorticoid-inducible gene.

Introduction

The activation, proliferation and death of immune system cells are controlled by a variety of factors: intermediate direct cell—to—cell interactions, cytokines, secreted by the immune system cells, as well as factors related to the neuroendocrinal system. Among the latter, glucocorticoids are the best studied neuroendocrinal regulators. Glucocorticoids and their synthetic analogue, dexamethasone, are now known to inhibit the proliferation T-lymphocytes and the production of a broad spectrum of cytokines, including IFN-γ[1–6]. Some cytokines, however, can modify the effect of dexamethasone upon T-cells. It was shown that dexamethasone in the presence of IL-2 and anti-IL-10 mAb stimulates the proliferation and maturation of naive mouse T-lymphocytes in vitro[7]. We have shown earlier that IL-7 can also change the effect of dexamethasone phosphate (DEX) on neonatal T-cells. DEX in the presence of IL-7 does not decrease, but rather increases T-cell proliferation in some cord blood mononuclear cell (CBMC) cultures [8]. IL-7 is not only actively produced in the organism of a newborn, but also performs a number of specific functions. IL-7 is known to be a key cytokine regulating the thymocyte development. Moreover, this cytokine plays a crucial role in the maintenance of polyclonal populations of naive T-lymphocytes and memory T-cells and therefore controls the overall size of the T-cell pool [9–13]. IL-7 is an optimal growth and survival factor for nonactivated naive T-cells and for activated T-lymphocytes of newborn's cord blood [14,15].

The production and functions of glucocorticoids in fetus and newborn organisms also possess a number of characteristic features [16]. During the latter two thirds of gestation in humans, the fetal adrenal glands are disproportionately enlarged and exhibit extraordinary growth and steroidogenic activity. The fetal adrenal cortex produces a large amount of the androgenic C₁₉ steroid, dehydroepiandrosterone sulphate and glucocorticoid cortisol, which promotes the maturation of fetal organ system, including the lungs, liver, thyroid and gut.

The present work studies the influence of DEX and IL-7 upon the proliferation of T-cells and the maturation of IFN-γ-producing T-lymphocytes and effector memory T-cells in neonatal CBMC cultures. In order to examine the maturation of T-cells in the culture the quantity of naïve T-lymphocytes and three subpopulations of memory T-cells, differing in expression of CD45RA and CD62L, was defined. Human naïve T-cells express CD45RA [17,18] and the lymph node homing receptors CCR7 and l-selectin CD62L [19,20]. Naïve T-lymphocytes travel to T-cell areas of secondary lymphoid organs in search of antigen presented by dendritic cells [17,19–21]. Once activated, they proliferate vigorously and generate memory/effector cells. A fraction of primed T-lymphocytes persists as circulating memory cells that can confer protection and give, upon secondary challenge, a qualitatively different and quantitatively enhanced response [17,21]. Memory T-cells are not a homogeneous population. Recently, memory CD8⁺ T-cells were classified into three distinct populations, based on phenotype [17,21–26]: a central memory population, which is CD45RA⁻CCR7⁺CD62L⁺CD28⁺IL-2⁺IFN-γ⁻; and two effector memory populations, namely the preterminally differentiated CD45RA⁻CD62L⁻CCR7⁻ and the terminally differentiated CD45RA⁺CD62L⁻CCR7⁻ populations. The two latter effector memory populations are targeted to inflammatory tissues. These cells contain perforin, secrete IFN-γ and tumour necrosis factor-α, are cytotoxic, and are capable of rapid effector function in response to antigenic stimulation [21,27–29]. Central memory cells are targeted to the lymph nodes, produce primarily IL-2 and produce effector cytokines only after a secondary challenge [21]. CD4⁺ memory cells subdivide into three populations too: nonpolarized central memory CD45RA⁻CCR7⁺CD62L⁺ cells, CD45RA⁻CD62L⁻CCR7⁻ and CD45RA⁺CD62L⁻CCR7⁻ effector memory cells [29,30].

Materials and methods

Proliferation assay

For blood sampling from newborns and adult control subjects, performed for the purpose of the study, all adult donors and newborn's mothers gave written informed consent. CBMC were obtained from 43 newborn children in different clinical condition. During the gestation period the mothers of the examined babies suffered from the following diseases: bacterial or viral infections, pyelonephritis, preeclampsia. The children had a 25–40 weeks of intra-uterine development, with the average gestation time of 35·8 weeks. Prematurely born babies comprised 55·8% of all newborns. The weight of babies varied from 650 g to 4200 g. The average body weight was 2710 g. The average loss of body weight during the 5 postnatal days was 8·73%. The adult group consisted of 16 healthy mothers of sound newborns with normal gestation time. They were 20–32 years of age.

CBMC and adult's PBMC were obtained from sterile blood by centrifugation on a layer of Hystopaque-1077 (Sigma Chemical Co., St. Louis, MO, USA), washed twice and resuspended in Dulbecco's modified Eagle's medium (DMEM) (Sigma Chemical Co.) with 10% of FCS (Biolot, St. Petersburg, Russia), 0·584 g/1 of l-glutamine and 10 mg/1 of gentamycine (Sigma Chemical Co). T-cells were activated with anti-CD3 mAb ICO-90 (Medbiospecter, Moscow, Russia). The final concentration of anti-CD3 mAb was 1 µg/ml. Human rIL-7 (Sigma Chemical Co.) was used at the concentration of 10 ng/ml. Dexamethasone phosphate (KRKA, Slovenia) was added to cultures to achieve the concentration of 0·4 µg/ml. To estimate the proliferation we placed the cell suspension with or without anti-CD3 mAb, DEX and IL-7 into 96-well flat-bottom culture plates (Evergreen Scientific, Los Angeles, CA, USA). For purposes of control microcultures without added mAb, IL-7 and DEX were used. The total amount of cell suspension was 200 µl per well, the final cell concentration was 5 × 10⁵/ml. Each culture variant was placed in three wells. The cells were cultivated at 37 °C in the atmosphere containing 5% of CO₂.

0·5 µCi of ³H-methylthymidine (Isotop, St. Petersburg, Russia) was added into the cultures 48 h after the beginning of cultivation. 24 h later the plates were frozen, thawed, the cells were transported on glass filter paper (ICN Biomedicals Inc, Irvine, CA, USA) by cell harvester and analysed using Beckman scintillation counter.

Detecting intracellular IFN-γin activated T-lymphocytes

The newborn group consisted of 12 healthy babies with normal gestation time. The adult group consisted of 12 healthy mothers of sound newborns. Two different protocols of T-cell activation were used.

Short-term activation

PBMC or CBMC were resuspended to 10⁶/ml in DMEM with 10% of FCS, and 0·1% (V/V) of GolgiStop (BD Pharmingen, San Jose, CA, USA). Cells were stimulated with 25 ng/ml of PMA (Sigma Chemical Co.) and 1 µg/ml of ionomycin (Sigma Chemical Co.) in the presence of DEX (0·4 µg/ml) or without DEX. The cells were incubated at 37 °C, 5% CO₂ for 4 h. Unstimulated control cell cultures were incubated with GolgiStop, without PMA, ionomycin or DEX for 4 h. After incubation the cells were fixed, permeabilized by saponin (Sigma Chemical Co.) and stained with FITC-conjugated anti-CD7 mAb (Medbiospecter, Moscow, Russia) and PE-conjugated anti-IFN-γ mAb (BD Immunocytometry Systems, San Jose, CA, USA) or appropriate isotype control reagents. The samples were washed and analysed using flow cytometry.

Long-term activation

PBMC or CBMC were resuspended in DMEM with l-glutamine, gentamycine and 10% of FCS. The cells were incubated with or without anti-CD3 mAb (1 µg/ml), IL-7 (10 ng/ml) and DEX (0·4 µg/ml). Micro-cultures without mAb, IL-7 and DEX were used for purposes of control. The final cell concentration was 10⁶/ml. The cell cultures were cultivated at 37 °C, 5% of CO₂ for 68 h. Then cells were harvested, washed and restimulated with 25 ng/ml of PMA and 1 µg/ml of ionomycin in the presence of GolgiStop for 4 h. Finally cells were harvested, fixed, permeabilized and stained with anti-CD7-FITC and anti-IFN-γ-PE or isotype control reagents. Samples were washed and analysed using flow cytometry.

IFN-γ ELISA

CBMC and PBMC were stimulated with anti-CD3 and IL-7 with or without DEX according to the long-term activation protocol. Cells were cultivated at 37 °C, 5% of CO₂ for 68 h. Then cell culture supernatants (CSN) were harvested and frozen, cells were washed and subsequently cultivated in medium containing PMA (25 ng/ml) and ionomycin (1 µg/ml) for 24 h. After the second cultivation CSN were harvested again. ELISA-IFN-gamma test-system (Citikine, St. Petersburg, Russia) was used for measuring IFN-γ in CSN.

Immunophenotyping

The newborn group consisted of 6 healthy babies with normal gestation time. The adult group consisted of 6 healthy mothers of sound newborns. Direct immunofluorescence staining was performed on cell from CBMC or PBMC cultures using a combination of three (CD7/CD45RA/CD62L) directly labelled mAb. mAb were purchased from Becton-Dickinson, USA (CD7-CyChrome, CD62L-PE) and Medbiospecter, Russia (CD45RA-FITC). In brief, CBMC and PBMC were cultivated without stimulation or with anti-CD3 (2 and 100 ng/ml), with or without IL-7 (10 ng/ml) and DEX (0·4 µg/ml) at 37 °C, 5% of CO₂ for 68 h. Then 5 × 10⁵ cells from each culture were added to a premixed solution of mAb or to solution appropriate isotype control reagents. After a 30-min incubation at room temperature in the dark, samples were washed twice and resuspended in PBS with 2% of paraformaldehyde. Analysis was performed in a FACScalibur (Becton-Dickinson) instrument. For each sample, cells were subsequently gated into the lymphocyte population and CD7⁺ lymphocytes, and 10 000 events were acquired in the gate. The results of experiments were registered as a percent of CD45RA⁺CD62L⁺ naïve T cells and CD45RA⁻CD62L⁺, CD45RA⁻CD62L⁻ and CD45RA⁺CD62L⁻ memory cells amongst CD7⁺ cells.

Apoptosis assay

The apoptosis was measured by the number of cells with partial DNA loss (hypodiploid cells). CBMC and PBMC were cultivated without stimulation, with anti-CD3 (100 ng/ml) with or without IL-7 (10 ng/ml) and DEX (0·4 µg/ml) at 37 °C, 5% of CO₂ for 48 or 68 h. Then 5 × 10⁵ cells from each culture was fixed by 70% ethanol, washed and stained by propidium iodide solution (propidium iodide, 5 µg; sodium citrate, 100 mg; glass-distilled water, 100 ml; Nonidet P-40, 30 µl). Analysis was performed in a FACScalibur (Becton-Dickinson) instrument.

Statistics

The normality of parameter distribution was checked and then common parametrical criteria were calculated. All data are presented as mean ± standard deviation.

Results

Dexamethasone in the presence of IL-7 can enhance the proliferation of activated newborn's T-lymphocytes

In the present work we have analysed the influence of DEX upon the proliferation of neonatal CBMC and adult's PBMC. As was expected, DEX suppressed the proliferation of activated T-cells in CBMC and PBMC cultures. However, the addition of IL-7 to the cultures changed the effect of DEX on newborn's T-cells. DEX did not decrease, but rather increased the proliferation induced by anti-CD3 together with IL-7 in CBMC cultures from the 23 newborns. These children comprise group 1 (Fig. 1). For group 1 the proliferation level in cultures with ant-CD3 mAb, IL-7 and DEX was 1·84 ± 0·24 times higher, than in cultures with ant-CD3 and IL-7 without DEX. The effect of DEX upon the proliferation of activated T-cells in the presence of IL-7 was maximal in the blood of a prematurely born baby with hypoxic lesion of central nerve system. In this case, DEX enhanced proliferation by a factor of 7·54. In cultures of CBMC from other 20 children DEX insignificantly decreased the proliferation, induced by anti-CD3 mAb together with IL-7 (group 2, Fig. 1). In all adult PBMC cultures DEX did not stimulate proliferation, induced by anti-CD3 together with IL-7.

Fig. 1.

Proliferation of newborn CBMC and adult PBMC without stimulators and in the presence of anti-CD3 mAb (1 µg/ml), DEX (0·4 µg/ml) and IL-7 (10 ng/ml). The composition of reagents, added to the cultures is given to the right of the figure. The newborns are divided into groups according to the DEX action upon CBMC proliferation with anti-CD3 and IL-7. The Y axis is for counts per min (Cpm). The significant difference between the proliferation with DEX and without DEX: *P < 0·05 and **P < 0·01 (pair t-test). †marks significant difference (P< 0·05) between the group 1 and 2 (t-test).

The anthropometrical and clinical data of newborns in groups 1 and 2 did not differ significantly (data not shown). The average gestation time in group 1 was 35·9 ± 0·93 weeks and in group 2 36·05 ± 0·75 weeks. Forty-eight percent of children from group 1 and 30% of children from group two were sent to specialized children's hospitals directly from maternity hospitals where they were born, as they needed constant medical supervision and care. Two babies from group 1 died on the 4th and 6th day after birth. The severe clinical condition of children from both group 1 and group 2 was due to premature birth, asphyxia, hypoxic lesion of central nerve system, peri-natal encephalopathia and pneumonia.

The effect of DEX upon the production of IFN-γ

The number of IFN-γ producing T-lymphocytes was measured by intracellular cytokine detection. CD7 membrane molecule was used as a T-cell marker because this molecule can be well kept on a membrane of T-lymphocytes activated by anti-CD3 mAb or PMA and ionomycin. The results of experiments were registered as a percent of IFN-γ producers amongst CD7⁺ cells.

The number of IFN-γ producers was extremely small in unstimulated 4-h cultures of neonatal CBMC or adult PBMC (Fig. 2). Activation with PMA and ionomycin increased the number of IFN-γ producing adult's T-lymphocytes by a factor of 36·5. Activation of neonatal CBMC led to an increase in the number of IFN-γ producing T-lymphocytes by a factor of 19·6. The percentage of IFN-γ producing T-lymphocytes was significantly smaller in stimulated neonatal cell cultures than in adult's cell cultures (P< 0·005). DEX did not influence the amount of IFN-γ producing T-cells in 4-h stimulated CBMC and PBMC cultures (Fig. 2).

Fig. 2.

(a) Two-colour flow cytometric analysis of neonatal CBMC (upper row of panels) and adult PBMC (lower row of panels) for CD7 and IFN-γ. All cell samples were cultivated with GolgiStop for 4 h. Left column of panels: unstimulated cells. Middle colums of panels: cells stimulated with PMA and ionomycin. Right column of panels: cells stimulated with PMA and ionomycin in the presence of DEX. After cultivation the cells were fixed, permeabilized, stained with anti-CD7-FITC and anti-IFN-γ-PE and analysed by flow cytometry. Lymphocytes were gated. Histogram A: the average percentage of IFN-γ⁺CD7⁺ T-cells (share of IFN-γ⁺CD7⁺ cells amongst CD7⁺ cells, %, y axis) in unstimulated (□) and stimulated by PMA and ionomycin with (▪) or without (□) DEX 4- h cultures of neonatal CBMC or adult PBMC.

The activation of T-cells by anti-CD3 antibodies increased the concentration of IFN-γ in CSN from 68-h adult's PBMC cultures by 4 times (Fig. 3). Anti-CD3 stimulation of neonatal T-cells increased IFN-γ concentration only by 2 times. Anti-CD3 together with IL-7 induced a strong enhancement of IFN-γ production in neonatal cell cultures as well as in adult's cell cultures. DEX suppressed IFN-γ production in 68-h cell cultures both in the presence of IL-7 and without it.

Fig. 3.

Concentration of IFN-γ (pg/ml, Y axis) in CSN from 68- h adult PBMC cultures (□) and neonatal CBMC cultures (□). The composition of stimulators and reagents, added into the culture is given in X axis. IFN-γ concentration in CSN was measured by ELISA. *significant difference (P< 0·05) between the cultures with DEX and without DEX. †significant difference (P< 0·05) between the cultures with IL-7 and without IL-7.

However, the cultivation of CBMC with DEX, anti-CD3 mAb and IL-7 for 68 h resulted in accumulation of T-lymphocytes capable of producing IFN-γ after restimulation with PMA and ionomycin. In this experiment, the cells were cultivated with or without anti-CD3, IL-7 and DEX for 68 h. Then the cells from primary cultures were washed and restimulated with PMA and ionomycin for 4 h. For 11 out of 12 newborns studied in this experiment, the number of IFN-γ producers was maximal in cell cultures incubated with anti-CD3, IL-7 and DEX during the first 68 h (Fig. 4). The number of IFN-γ producing T cells in these cultures was 4·39 ± 1·0 times higher than in CBMC cultures stimulated with anti-CD3 during the first 68 h of cultivation (P< 0·005) and in 4·27 ± 1·47 times higher than in cultures stimulated with anti-CD3 and IL-7 (P< 0·005, Fig. 5a). The number of IFN-γ producers in restimulated cultures of adult's PBMC was significantly higher than in the cultures of neonatal cells. The addition of DEX to primary anti-CD3 stimulated cultures of adult's PBMC reduced the number of IFN-γ producers. DEX did not influence the number of IFN-γ producing adult's T-cells stimulated with anti-CD3 and IL-7.

Fig. 4.

Two-colour flow cytometric analysis of restimulated neonatal CBMC for CD7 and IFN-γ. CBMC were cultivated with compositions of reagents (pointed above panels) during the first 68 h. Then cells from primary cultures were washed and subsequently cultivated with PMA and ionomycin in the presence of Golgi Stop for the 4 h. After cultivation the cells were fixed, permeabilized by saponin, stained with anti-CD7-FITC and anti-IFN-γ-PE and analysed by flow cytometry. Lymphocytes were gated.

Fig. 5.

The percentage of IFN-γ⁺CD7⁺ T-cells (A) and IFN-γ concentrations (C) in restimulated cell cultures. The percentage of effector memory CD45RA⁺CD62L⁻ cells among CD7⁺ lymphocytes in primary 68-h cultures (B). The composition of reagents, added into primary cultures is given to the bottom of the figures. The number of CD7⁺CD45RA⁺CD62L⁻ lymphocytes measured by three-colour flow cytometry. In order to measure the number IFN-γ⁺CD7⁺ lymphocytes the cells from primary cultures were washed and subsequently cultivated with PMA, ionomycin and GolgiStop for 4 h. After incubation the cells were fixed, permeabilized, stained with anti-CD7-FITC and anti-IFN-γ-PE and analysed by flow cytometry. In order to measure the concentration of IFN-γ the cells from primary cultures were washed and restimulated with PMA and ionomycin for 24 h. After the second cultivation we harvested CSN and analysed by ELISA. Statistic significance of the follow-up values in cultures with DEX in comparison with values in cultures without DEX: *P < 0·05 and **P < 0·005 (pair t-test).

In order to measure the concentration of IFN-γ in CSN from restimulated cultures, the cells were cultivated with or without anti-CD3, IL-7 and DEX for 68 h. The cells were then washed and cultivated with PMA and ionomycin without GolgiStop for 24 h. The concentrations of IFN-γ in CSN from restimulated cell cultures are shown in Fig. 5c. Among the neonatal CBMC cultures the concentration of IFN-γ was greatest in the cultures incubated with anti-CD3, IL-7 and DEX during the first 68 h. The concentration of IFN-γ in these cultures was 6·03 ± 2·91 times higher than in cultures stimulated with anti-CD3 during the first 68 h (P< 0·01) and 2·13 ± 0·53 times higher than in cultures stimulated with anti-CD3 and IL-7 (P< 0·05). In the restimulated adult PBMC cultures DEX strongly inhibited anti-CD3 induced IFN-γ production. The concentrations of IFN-γ in CSN from adult PBMC cultures incubated with anti-CD3, IL-7 and DEX during the first 68 h were slightly lower than in cultures incubated with anti-CD3 and IL-7 without DEX (the difference is not significant).

Quantification of T-lymphocyte subsets after stimulation with anti-CD3, IL-7 and DEX

The activation of T-cells did not influence substantially the concentration of naïve CD7⁺CD45RA⁺CD62L⁺ T-lymphocytes in 68-h newborn and adult cell cultures (Fig. 6a). Neither did DEX cause any significant changes in the concentration of these cells. The cultivation of newborn and adult cells with IL-7 and DEX led to a substantial decrease in the number of naïve T-lymphocytes in the cultures.

Fig. 6.

The percentage of naïve CD45RA⁺CD62L⁺ T cells, central memory CD45RA⁻CD62L⁺ cells, CD45RA⁻CD62L⁻ cells and CD45RA⁺CD62L⁻ cells among CD7⁺ lymphocytes in primary 68-h (a) neonatal CBMC cultures and (b) adult PBMC cultures. The composition of IL-7 and DEX, added into cultures is given to the bottom of the figures. The y axis is for anti-CD3 mAb concentration (ng/ml). Statistic significance of the follow-up values in comparison with baseline values of unstimulated cultures *P < 0·05.

The activation of T-lymphocytes with anti-CD3 mAb at a concentration of 100 ng/ml caused a significant decrease in the concentration of central memory T-cells with CD7⁺CD45RA⁻CD62L⁺ phenotype in the culture (Fig. 6b). The addition of DEX prevented a drop in the concentration of these cells. IL-7 with or without DEX induced a significant drop in the concentration of central memory cells in adult PBMC cultures and an almost total disappearance of these cells in newborn CBMC culture.

The stimulation of newborn and adult blood cells with IL-7 led to a significant decrease in the concentration of CD7⁺CD45RA⁻CD62L⁻ effector memory cells (Fig. 6c). A similar effect was observed in newborn cell cultures when IL-7 together with DEX was added to them. At the same time, in adult cell cultures in the presence of IL-7 and DEX the concentration of CD7⁺CD45RA⁻CD62L⁻ effector memory cells increased (the differences are statistically insignificant).

Finally, the concentration of adult CD7⁺CD45RA⁺CD62L⁻ effector memory cells increased under activation, IL-7, IL-7 with DEX and, especially notably, under the combination of anti-CD3 activation, IL-7 and DEX (Fig. 6d). For newborns a substantial increase in the concentration of these cells was observed only in the presence of IL-7 and DEX.

The decrease of naïve T-lymphocytes concentration and the disappearance of central memory cells from CBMC cultures when stimulated with IL-7 together with DEX were not accompanied by an increase in cell death. The level of apoptosis in the control, nonactivated CBMC cultures and in cultures stimulated with IL-7 and DEX with or without anti-CD3, did not differ (Table 1). At the same time, the highest number of CD7⁺CD45RA⁻CD62L⁺ cells was present in the cultures where DEX without IL-7 was added, i.e. in cultures with a significant increase in apoptosis. We did not find any significant differences of apoptosis rates between the cultures with different stimulator combinations in 68-h CBMC cultures and in 48- and 68-h PBMC cultures (data not shown). The percentage of CD7⁺ cells increased in all CBMC cultures stimulated by IL-7 with or without anti-CD3 or DEX (Table 1).

Table 1. The action of anti-CD3, IL-7 and DEX upon apoptosis rate in CBMC and PBMC cultures and number of CD7⁺ T-cells.

Stimulators	The number of hypodiploid cells in 48-h CBMC cultures (%)	The percentage of CD7+ cells among lymphocytes in 68-h CBMC cultures (%)	The percentage of CD7+ cells among lymphocytesin 68-h PBMC cultures (%)
Without stimulators	9·00 ± 1·21	66·20 ± 5·49	65·50 ± 8·89
DEX (0·4 µg/ml)	10·58 ± 1·23*	53·85 ± 8·99	56·02 ± 7·23
Anti-CD3 (100 ng/ml)	11·13 ± 1·51*	66·50 ± 3·11	70·65 ± 5·86
Anti-CD3+ DEX	12·41 ± 1·70*	72·45 ± 2·56	74·36 ± 3·97
IL-7 (10 ng/ml)	7·21 ± 1·62*	78·17 ± 3·15*	72·57 ± 3·69
IL-7 + DEX	8·80 ± 1·93	″84·60 ± 2·45**	71·93 ± 0·89
IL-7 + anti-CD3	7·82 ± 1·21	79·85 ± 4·06*	77·25 ± 5·57
IL-7 + anti-CD3+ DEX	9·45 ± 1·91	78·82 ± 5·53*	72·83 ± 5·11

Statistic significance of the follow-up values in comparison with baseline values of unstimulated cultures:

^*P < 0·05;

^**P < 0·005.

Discussion

It was shown that DEX is able to enhance T-cell proliferation induced by anti-CD3 and IL-7 in some neonatal CBMC cultures. DEX inhibited the production of IFN-γ in 68-h cell cultures stimulated with anti-CD3 both in the presence of IL-7 and without it. However, the cultivation of neonatal CBMC with DEX, anti-CD3 mAb and IL-7 for 68 h resulted in accumulation of T-lymphocytes capable of producing IFN-γ after restimulation with PMA and ionomycin. The stimulation of CBMC or PBMC by DEX, anti-CD3 mAb and IL-7 also causes a decrease in the number of naïve T-cells and central memory cells and an increase in the number of effector memory CD7⁺CD45RA⁺CD62L⁻ T cells in cultures. A decrease in the concentration of naïve T-cells and central memory cells is not accompanied by an increase in cell death. Apparently, these changes of T-cell subsets are caused by the differentiation of cells into more mature CD7⁺CD45RA⁺CD62L⁻ effector memory cells. It is known that part of terminally differentiated CD45RA⁺CD62L⁻ T-cells are strong producents of IFN-γ. The concentration of CD7⁺CD45RA⁺CD62L⁻ cells in primary cultures, the number of IFN-producers after restimulation and the concentration of the IFN they produce change concurrently dependent on the conditions of simluation (Fig. 6), which suggests that the increase in IFN-production, induced by anti-CD3, IL-7 and DEX, is related to the maturation of T-lymphocytes to the stage of terminally differenciated effector memory cells.

The literature on the combined effects of IL-7 and glucocorticoids on T-lymphocytes mostly deals with the ability of IL-7 to disable the glucocorticoid-induced effects. IL-7 inhibits DEX-induced apoptosis in early thymocyte progenitor cells, CD4⁺CD8⁺ thymocytes and in mature, peripheral T cells [31,32]. IL-7 blocked DEX-induced processing of caspase-3 and caspase-7, dephosphorylation of the serine-threonine kinase Akt/PKB and its target, the Ser(136) residue in Bad, the loss of anti-apoptotic proteins Bcl-x(L) and inhibitor of apoptosis protein-2 [32]. For discussing the mechanisms by which IL-7 disables glucocorticoid-induced effects, of particular interest is the information that IL-7 and IL-18 increased expression of the beta isoform of the human glucocorticoid receptor, as a putative dominant negative regulator of glucocorticoid action [33]. These facts, however, can not explain the positive effects of glucocorticoids and IL-7 on T-cell function identified by us. It is possible that these effects are caused by the influence of glucocorticoids on IL-7 receptor expression. Earlier Franchimont et al. [34] identified IL-7 receptor alpha-chain gene as a glucocorticoid-inducible gene. The up-regulation of IL-7Ralpha by glucocorticoids in human PBMC was associated with enhanced IL-7-mediated signalling and IL-2R expression.