Survival of activated human T lymphocytes is promoted by retinoic acid via induction of IL-2
Nikolai Engedal1,
Aase Ertesvag1 and
Heidi Kiil Blomhoff1
1 Department of Medical Biochemistry, University of Oslo, PO Box 1112, Blindern, 0317 Oslo, Norway
The first two authors contributed equally to this work.
Correspondence to: H. K. Blomhoff; E-mail: h.k.blomhoff{at}basalmed.uio.no
Transmitting editor: J. Borst
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Abstract
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At the end of an immune response, most activated T cells spontaneously undergo programmed cell death (apoptosis). In the present study we show that all-trans retinoic acid (atRA), a major vitamin A metabolite, can inhibit the spontaneous apoptosis of activated human T lymphocytes in vitro. Isolated peripheral blood T lymphocytes were activated by 12-O-tetradecanoyl phorbol 13-acetate and cultured for up to 11 days without any further stimuli. With time, a gradual increase in cell death was observed. This spontaneous death of activated T cells was apoptotic, as demonstrated by cell shrinkage, DNA fragmentation and depolarization of the mitochondrial membrane. In the presence of physiological concentrations of atRA, the percentage of T cells exhibiting these apoptotic features was significantly reduced. After 5 days of stimulation, the percentage of TUNEL+ T cells decreased from 28 to 12% in the presence of atRA. The anti-apoptotic effect of atRA was mimicked by the retinoic acid receptor (RAR)-selective agonists 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid and AM-580, and totally abrogated by the RAR-selective antagonist Ro 41-5253. Cytokines of the IL-2 family have been shown to improve the survival of activated T cells. Strikingly, we found that the ability of atRA to inhibit apoptosis was significantly correlated with its ability to increase the production of IL-2. Furthermore, a blocking anti-IL-2 receptor antibody completely abrogated the anti-apoptotic effect of atRA. Together, these results suggest that retinoic acid inhibits spontaneous apoptosis of activated T lymphocytes through a RAR-dependent increase in IL-2 production.
Keywords: activated human T lymphocyte, apoptosis, IL-2, retinoic acid, retinoic acid receptor
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Introduction
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A typical immune response involves the activation and clonal expansion of antigen-specific T lymphocytes, resulting in rapid accumulation of a large amount of effector T cells that are instrumental in the elimination of the infectious agents. Following a peak of expansion, the number of effector cells declines rapidly, a period commonly referred to as the contraction phase (1,2). During this phase, the majority of activated T cells die through apoptosis (3). Two distinct pathways have been shown to be responsible for the death of activated T cells in vivo: activation-induced cell death (AICD) and activated T cell autonomous death (ACAD) (4). In contrast to AICD, which is initiated by the activation of death receptors as a result of repeated antigenic stimulation (5,6), ACAD proceeds after a single exposure to antigen and is driven by intrinsic signals in the activated T cell that are independent of death receptors to induce apoptosis (7). The elimination of effector cells during the contraction phase is primarily caused by ACAD, whereas AICD appears to play only a minor, if any, role in this process (7,8).
The regulation of ACAD is incompletely understood. Experiments performed in mice have demonstrated that inflammatory cytokines such as tumor necrosis factor-
, IFN-
and cytokines of the IL-2 family may inhibit ACAD in vivo (9,10). Moreover, IL-2 family cytokines as well as type I IFN (
and ß) were shown to directly prevent the death of activated T cells in vitro (10,11).
Numerous studies have shown that vitamin A strengthens immune responses in both animals and humans (1214). The biological effects of vitamin A in the immune system are mainly mediated by its acidic derivatives, and in particular by the two isomers all-trans- and 9-cis retinoic acid (atRA and 9cRA) (1517). AtRA is a ligand for retinoic acid receptor (RAR), a nuclear receptor that regulates transcription of RA-responsive genes through heterodimerization with the retinoid X receptor (RXR) (1821). Whereas atRA only binds to RAR, 9cRA has high affinity for both RAR and RXR (2224). Previously, we have shown that both atRA and 9cRA activate the cell-cycle machinery in normal T cells (25), indicating that vitamin A may stimulate immune responses through a direct potentiation of T cell activation. Vitamin A has also been shown to have the potential of regulating T cell death. First, RA and other retinoids can directly induce apoptosis in adult T cell leukemia cells (26,27). Second, a number of reports have shown that RA can inhibit AICD in thymocytes, T cell hybridomas and mature human T lymphocytes (2830).
In the current study we assessed whether atRA could directly influence apoptosis of activated T cells in an in vitro model system resembling ACAD. We show that physiological concentrations of atRA significantly reduced spontaneous apoptosis of isolated peripheral T lymphocytes activated by 12-O-tetradecanoyl phorbol 13-acetate (TPA). Moreover, we demonstrate that the protective effect of atRA was mediated by a RAR-dependent increase in IL-2 production.
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Methods
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Reagents
TPA, atRA, 9cRA, 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid (TTNPB), AM-580 and propidium iodide (PI) were purchased from Sigma (St Louis, MO). SR11217 was provided by Dr Marcia I. Dawson (Burnham Institute, La Jolla, CA) and Ro 41-5253 was provided by Dr M Klaus (Hoffman-La Roche, Basel, Switzerland). The retinoid compounds were dissolved in ethanol or DMSO, flushed with argon and stored in lightproof containers at 20°C. Experiments with retinoids were performed in subdued light. JC-1 and 5- (and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR) were dissolved in DMSO to concentrations of 2 mg/ml and 5 µM respectively, and stored at 20°C. Recombinant human IL-2 was purchased from R & D Systems (Minneapolis, MN). Anti-IL-2 receptor antibodies were purified from supernatant of the mouse hybridoma cell line 2A3A1H (ATCC, Manassas, VA) as recommended by the manufacturer.
T lymphocyte isolation and culture
T lymphocytes were negatively selected from peripheral blood of healthy human blood donors (Blood Bank, Ullevaal Hospital, Oslo, Norway) as previously described (25). The purity of the T cell populations varied between 85 and 95%, as analyzed by flow cytometry using FITC-conjugated antibodies against CD3. T cells were cultured at an initial density of 1.5 x 105 cells/0.2 ml in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10 % heat-inactivated FBS (Life Technologies), 2 mM glutamine, 125 U/ml penicillin and 125 µg/ml streptomycin in 96-well flat-bottomed microtiter plates at 37°C in a humidified incubator with 5% CO2.
Determination of cell viability and apoptosis
Cell viability was determined by a PI-exclusion test. Because the intact membrane of live cells excludes charged dyes like PI, short-term incubation of these dyes results in selective labeling of dead cells, whereas live cells have no or minimal uptake (31). Cells were incubated with 5 µg/ml PI for 10 min prior to flow cytometric analysis using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Data were analyzed using CellQuest software (BD Biosciences). DNA strand breaks were detected by the TUNEL assay, and the mitochondrial membrane potential was assayed by JC-1 staining and flow cytometry as previously described (32).
Flow cytometric analysis of CD69 expression
Isolated T lymphocytes (5 x 105 cells) were pelleted, washed with PBS and resuspended in 90 µl PBS into which 10 µl FITC anti-human CD69 (BD PharMingen; #555530) was mixed. After 15 min at room temperature in the dark, cells were washed twice with PBS and 10,000 cells were analyzed by flow cytometry. As a control for non-specific binding, TPA-treated T cells were also stained with an isotype-matched (IgG1) anti-keyhole limpet hemocyanin (which is not expressed in human cells) antibody (BD PharMingen; #349041) using the same protocol.
CFSE-staining experiments
Freshly isolated T lymphocytes were stained with CFSE as described by the manufacturer with some minor modifications. Briefly, cells were washed once with PBS, resuspended in PBS containing 0.5 µM CFSE and incubated at 37°C for 10 min. Subsequently, cells were washed twice with PBS containing 20% FBS, resuspended in RPMI 1640 containing 20% FBS and incubated for 2 h at 37°C. Finally, cells were washed once with RPMI 1640 containing 10% FBS, before the experimental treatments were initiated. After 5 days, cells were stained with PI and 40,000 cells were analyzed by flow cytometry.
IL-2 and IL-4 secretion assays
Isolated T lymphocytes (3 x 105) were treated with TPA in the absence or presence of atRA and cultured as described above. At various time points, cells were pelleted by centrifugation and the amount of IL-2 or IL-4 secreted into the supernatants was determined by an enzyme-linked immunosorbent assay (R & D Systems; #D2050 or #D4050 respectively) according to the manufacturers procedure.
Statistical analysis
SPSS11.0 for windows was used to perform the Wilcoxon signed-rank test, independent and paired samples t-tests, and bi-variant correlation analyzes as indicated in the legends to the figures.
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Results
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AtRA inhibits spontaneous cell death of activated T lymphocytes
In an effort to understand how vitamin A affects the immune function of normal T lymphocytes, we recently found that atRA has a potent co-stimulatory action on T cell proliferation when combined with the protein kinase C-activating phorbol ester TPA (25). In the present study, we used the same system to assess the effect of atRA on another process that critically influences T cell function, namely the spontaneous apoptosis of activated T cells. Purified T cells from five blood donors were left untreated or stimulated with TPA in the absence or presence of 100 nM atRA and cell death was monitored by a PI-exclusion test (31) over a time period of 11 days. As shown in Fig. 1, under each experimental condition the amount of cell death gradually increased with time. However, when cells were treated with atRA, spontaneous cell death was slowed down. Thus, at day 5 and 8, cell death was significantly (P < 0.05) reduced in cells treated with TPA and atRA, compared to cells treated with TPA only (Fig. 1). Virtually the same results were obtained with a lower dose of atRA (10 nM) (data not shown). At day 11, the protective effect of atRA was diminished (Fig. 1). Treatment with atRA alone did not affect the spontaneous death of non-stimulated T lymphocytes (data not shown). Since atRA-mediated survival was most pronounced after 5 days of stimulation, we used this time point in the following experiments.

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Fig. 1. AtRA inhibits spontaneous cell death of activated T cells. Isolated T cells were left untreated (white bars), or stimulated with 4 x 109 M TPA in the absence (grey bars) or presence of 100 nM atRA (black bars). At the indicated time points, cell viability was determined by PI staining and flow cytometric analysis as described in Methods. The data are presented as average percentages of cell death ± SEM (*P < 0.05, Wilcoxon signed-rank test, n = 5).
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Activated T cells die through apoptosis
In general, cells die through one of two alternative pathways: apoptosis, a programmed and highly ordered form of cellular destruction, or necrosis, a passive and unordered form of cell death (33). In the experiments described above, cell death was determined by the inability of dying cells to exclude the fluorescent dye PI. This is a feature shared by both necrotic and late apoptotic cells (31). Therefore, in order to determine the nature of the spontaneous cell death described in Fig. 1, we specifically analyzed apoptotic cell death by the TUNEL assay. In this assay the fragmentation of genomic DNA, a hallmark of apoptosis (33), is detected. As shown in Fig. 2, spontaneous T cell death at day 5 (Fig. 2A) was to a large extent accompanied by DNA strand breaks (Fig. 2B and C) and, importantly, the percentage of TUNEL+ cells was clearly reduced (from 28 to 12% on average, see Fig. 2B) in cells treated with atRA.

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Fig. 2. AtRA reduces DNA fragmentation and mitochondrial membrane depolarization in activated T cells. Isolated T cells were treated as indicated in the legend to Fig. 1 and cell death (AC) or mitochondrial membrane potential (D) was measured after 5 days. (A and B) Cells were stained with PI or subjected to the TUNEL assay as described in Methods, followed by flow cytometric analysis. Cell death is given as average percentage of PI+ (A) or TUNEL+ (B) cells ± SEM (n = 3). (C) The dot-blots presents the flow cytometric analysis of cells from one of the donors presented in (A) and (B). (D) The cells were stained with JC-1 as described in Methods, followed by flow cytometric analysis. The percentages of cells with depolarized mitochondrial membrane potential are indicated. One representative experiment out of four is shown. RA = atRA.
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Another characteristic apoptotic event is the disruption of the mitochondrial membrane potential, 
m (34,35). We monitored 
m by flow cytometric analysis of T cells stained with the cell-permeable, fluorescent dye JC-1 (36). This compound forms red-fluorescent aggregates in mitochondria with a normal 
m. An apoptosis-associated decrease in 
m, however, leads to the disintegration of these aggregates, resulting in decreased red fluorescence concomitant with increased green fluorescence, due to the emergence of green-fluorescent JC-1 monomers. As shown in Fig. 2(D), after 5 days, a high proportion of T cells contained mitochondria with depolarized membranes. Moreover, atRA treatment led to a marked reduction in the percentage of cells with depolarized mitochondrial membranes.
From the experiments described above we concluded that the spontaneous cell death we observed in activated T cells was due to apoptosis and not necrosis. This was further confirmed by examining the forward- and side-scatter profiles of the cells. Throughout the time period in the experiment described in Fig. 1, a population of T cells with both decreased forward scatter and increased side scatter (characteristic for apoptotic cells, since apoptotic cells typically shrink and become more granular) accumulated, and at the same time there were no signs of a population of cells with increased forward scatter (characteristic for necrotic cells, since necrotic cells typically swell) (data not shown).
AtRA inhibits spontaneous apoptosis of activated, not resting, T cells
Although TPA-stimulated T cells display an increased incorporation of thymidine, indicating increased DNA synthesis and S phase entry (25), one cannot exclude the possibility of the presence of a subpopulation of resting T cells in the TPA-treated cell cultures. Therefore, one could argue that the atRA-mediated inhibition of cell death we observed was due to enhanced survival of a subpopulation of resting T cells rather than protection of activated T cells from apoptosis. To explore this possibility, we examined the expression of CD69, a well-known activation marker that is rapidly up-regulated after T cell activation (37). T cells were stimulated with TPA for 24 h, stained with a FITC-conjugated antibody against CD69 and subjected to flow cytometric analysis. As shown in Fig. 3, virtually all TPA-stimulated T cells exhibited an increased expression of CD69 compared to untreated cells. The expression of CD69 in TPA-stimulated cells was maintained for at least 11 days (data not shown). Thus, we concluded that the effect of atRA on the spontaneous apoptosis of activated T cells was not due to the inhibition of apoptosis in a subpopulation of resting T cells.

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Fig. 3. Activation of T cells with TPA induces CD69 expression in virtually all cells. Isolated T cells were left untreated (A) or stimulated with 4 x 109 M TPA (B and C) for 24 h. Subsequently cells were harvested and stained with a FITC-conjugated anti-CD69 antibody as described in Methods (A and B). (C) As a control, TPA-treated T cells were stained with a FITC-conjugated, isotype-matched antibody against an irrelevant protein. One representative experiment of three is shown.
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The effect of atRA on T cell survival is independent of its effect on T cell proliferation
We have previously shown that atRA stimulates the cell cycle machinery in T lymphocytes (25). If one supposes that proliferating T cells are more viable than non-proliferating T cells, atRA would, by increasing the number of proliferating and viable cells, dilute out the apoptotic cells and thereby decrease the percentage of apoptotic T cells in the culture. In that case, the putative anti-apoptotic effect of atRA that we observed could simply be the result of its action as a growth promoter and not as a true apoptosis inhibitor. In order to resolve this problem, we used CFSE staining of the cells to discriminate between proliferating and non-proliferating cells, and at the same time be able to determine the viability of the different cell populations. CFSE readily diffuses into cells where it, after cleavage by endogenous esterases, reacts with intracellular amines, forming fluorescent conjugates that are retained by cells for several weeks without affecting their viability (38). Since the fluorescent conjugates are equally inherited by daughter cells, cell divisions can be tracked by the sequential halving of cellular fluorescence.
Apoptotic cells also retain CFSE (39), although they in our experiments displayed some loss of CFSE fluorescence intensity (Fig. 4A and data not shown). We harvested CFSE-labeled T cells at various time points and monitored cell viability by the PI-exclusion test. Dot-blots from one representative experiment are shown in Fig. 4(A). We gated the cells into two groups: undivided cells (the population of cells with high CFSE fluorescence) and divided cells (cells with less than half of the fluorescence of the undivided cells). The gatings were set in order to accommodate for the loss of CFSE fluorescence by dying cells. As expected, untreated T cells did not undergo division at all (Fig. 4A, upper panels). In contrast, in T cell cultures that were either stimulated with TPA or co-stimulated with TPA and atRA, a gradual increase in the number of divided cells was evident (Fig. 4A, middle and bottom panels respectively). Moreover, the number of cells that had divided was noticeably higher in the presence of atRA. This result was confirmed when the average percentage of divided cells over time was calculated from four independent experiments (Fig. 4B). Whereas treatment of T cells with TPA alone generated <15% divided cells after 7 days, the presence of atRA led to the production of
30% divided cells within 5 days (Fig. 4B). These results are consistent with our previously suggested role of atRA as a T cell growth promoter (25).

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Fig. 4. AtRA inhibits apoptosis of both divided and undivided T cells. Isolated T cells were labeled with CFSE as described in Methods and left untreated or stimulated with TPA (T) (4 x 109 M), atRA (RA) (100 nM) or rIL-2 (I) (0.5 ng/ml) as indicated. At the indicated time points, cells were stained with PI as described in Methods and analyzed by flow cytometry. (A) Dot-blots from one representative kinetic experiment out of four. The percentages of PI+ cells among the divided and undivided cell populations, respectively, are indicated. (B) The percentage of T cells that had divided was determined for each experimental condition (white bars = untreated, grey bars = TPA, black bars = TPA + atRA) at each time point. Average percentages of divided cells ± SEM of four independent experiments is shown. (C) The percentage of apoptotic cells was determined among both the cells that had divided and those that had not divided at day 5. The data are presented as the average percentage of apoptotic cells ± SEM (**P < 0.01, *P < 0.05, paired samples t-test, n = 7).
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Interestingly, however, and in line with findings in T cell lines (40), we found that the divided cells underwent apoptosis to at least the same extent as the undivided cells (Fig. 4A). Moreover, atRA inhibited cell death in both groups (Fig. 4A). Consistent with the results that we described in Fig. 1, the effect of atRA was most pronounced at day 5. In order to assess the statistical significance of the differences seen at day 5 we determined the percentage of apoptotic cells in both the divided and the undivided cell populations in T cells obtained from seven different blood donors. As shown in Fig. 4(C), atRA significantly (P < 0.01) reduced apoptosis both in the divided and the undivided cell populations. Again, there were no significant differences between the viability of divided and undivided T cells. Together, these data strongly indicate that atRA exhibits a true anti-apoptotic effect as well as a growth promoting effect on human T cells.
T cells from different donors can be divided into low and high responders to atRA
It is generally accepted that the cellular responses of primary T lymphocytes can vary substantially between different blood donors. Of the five donors examined in Fig. 1, we noticed that T cells from two of the donors responded strongly to atRA, regarding inhibition of apoptosis, whereas the three others responded more moderately. In order to determine whether this might represent a pattern of responses to atRA, we examined cell death at day 5 in T lymphocytes from a large number of blood donors. From a total of 38 donors, a small, but still statistical significant (P < 0.01), decrease in apoptosis was noticed in cells co-stimulated with TPA and atRA as compared to cells treated with TPA alone (Fig. 5A). On average, a 16% inhibition of apoptosis was observed. However, the responses to atRA varied considerably between T cells from different donors. To explore what could be the common feature(s) in the T cells that showed a strong anti-apoptotic response to atRA, we divided the 38 donors into two groups: (i) donors whose T cells exhibited a lower than average response to atRA (low responders) and (ii) donors whose T cells showed a higher than average response to atRA (high responders). Whereas, on average, there was no significant inhibition of apoptosis in the low responders (18 donors), the average inhibition of apoptosis mediated by atRA in the high responders (20 donors) was 29% (Fig. 5B). We next searched for candidate molecules that could explain the differences between these two groups.

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Fig. 5. AtRA promotes the survival of activated T cells through induction of IL-2. T cells were left untreated or stimulated with TPA (4 x 109 M), atRA (100 nM) or recombinant IL-2 (505000 pg/ml) as indicated. Cell death was measured by PI staining, followed by flow cytometric analysis of the cells. In (AC) cell death is given as the average percentage of apoptotic cells ± SEM (* P < 0.01, paired samples t-test). (A) The result from 38 donors is shown. (B) The cells from the 38 donors were divided into two groupsthose that responded less than average to atRA (low responders, n = 18) and those that responded stronger than average to atRA (high responders, n = 20). The average cell deaths in the two groups are shown. (C) The effect of recombinant IL-2 on apoptosis was assessed. The results from eight donors are presented. (D) IL-2 production after 24 h of treatment was measured in culture supernatants from cells of the two groups presented in (B). The results are given as the average concentration of IL-2 ± SEM (*P < 0.01, independent samples t-test). T = TPA, RA = atRA.
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A high response to atRA is associated with a high production of IL-2
As already mentioned, the mechanisms responsible for the elimination of activated T cells in vivo are poorly understood. However, the general view is that T cell death proceeds due to the absence of life-sustaining cytokines (1,7,41). Screening of a panel of various agents for their effect on the spontaneous cell death in vitro of in vivo activated mouse T lymphocytes, revealed that only members of two cytokine families, the type I IFN family (IFN-
/ß) and the IL-2 family (IL-2, -4, -7 and -15), had an apoptosis-inhibiting effect (10,11). Since, to our knowledge, T cell activation does not lead to any significant secretion of either IFN-
, -ß nor IL-7 or IL-15, we considered IL-2 and IL-4 to be likely candidates for mediating the anti-apoptotic effect of atRA. Of these two, IL-4 was shown to be the most potent survival factor in activated mouse T cells (10). Whereas we have previously shown that atRA can rapidly increase the production of IL-2 in T cells (25), we, in the present study, found only negligible levels of IL-4 expression within the first 7 days of culture (data not shown). We therefore hypothesized that atRA mediated its anti-apoptotic effects on activated T lymphocytes by increasing IL-2 levels. First, we assessed if rIL-2 could inhibit spontaneous apoptosis of activated T cells in our model system. Isolated human T cells were stimulated with TPA in the absence or presence of increasing concentrations of rIL-2 and cell death was measured after 5 days. As shown in Fig. 5(C), a gradual decrease in apoptosis was observed after addition of increasing amounts of rIL-2, reaching a plateau level at
2501000 pg/ml of rIL-2. At these concentrations of rIL-2, apoptosis was inhibited by
25%. A similar reduction in cell death in the presence of rIL-2 was noted when examining apoptosis with the TUNEL assay (data not shown). Moreover, similar to atRA, rIL-2 inhibited apoptosis both in divided and undivided T cells (Fig. 4C), indicating that the anti-apoptotic effect of rIL-2 was not a result of its action as a T cell growth promoter.
Next, we determined the effect of atRA on IL-2 secretion in the two groups defined above. As shown in Fig. 5(D), atRA enhanced the level of IL-2 secretion in both groups. However, the average net increase in IL-2 production was significantly higher (P < 0.01) in the group of high responders (210 pg/ml) than in the low responding group (60 pg/ml). In Fig. 5(C) we noted that, whereas 250 pg/ml rIL-2 inhibited apoptosis by 24%, 50 pg/ml rIL-2 was not sufficient to significantly inhibit cell death. This suggests that the amount of IL-2 produced by atRA determines whether or not the T cells are protected from apoptosis. Thus only in the group of high responders were inhibiting levels of IL-2 reached (Fig. 5D).
The induction of IL-2 by atRA correlates with its ability to inhibit apoptosis
In both of the groups described above we noticed that the extent of IL-2 secretion in response to atRA varied considerably. Therefore, we wished to test whether there was an overall correlation between the amount of IL-2 produced in the presence of atRA and its ability to inhibit apoptosis. To that end, we prepared a scatter plot with the percentage apoptotic cells on the y-axis and total IL-2 production on the x-axis, and performed a correlation analysis. As shown in Fig. 6, there was a significant correlation between the extent of apoptosis and IL-2 production in the presence of atRA (Pearson correlation coefficient rp = 0.43, P < 0.01, n = 38).

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Fig. 6. Correlation between production of IL-2 and apoptosis in atRA-treated T cells. The scatter plot presents the percentage of apoptotic cells versus IL-2 production (ln[IL-2 secretion (pg/ml)]) of cells treated with TPA and atRA. The results from the 38 donors presented in Fig. 5(A) are shown. Pearson correlation coefficient, rp = 0.43, P < 0.01.
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IL-2 is essential for atRA-mediated inhibition of apoptosis
To test whether IL-2 was obligatory for atRA-mediated survival, we made use of an anti-IL-2 receptor antibody that binds to the IL-2 receptor and blocks receptor signaling. As evident from Fig. 7, the anti-IL-2 receptor antibody completely (P < 0.01) reversed the apoptosis-inhibiting effect of atRA measured at day 5 after stimulation. Collectively, these data strongly point to a crucial role for IL-2 in the atRA-mediated inhibition of spontaneous apoptosis of activated T cells.

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Fig. 7. Inhibition of IL-2 signaling abrogates the anti-apoptotic effect of atRA. T cells were left untreated or stimulated with TPA (4 x 109 M), atRA (100 nM) or anti-IL-2 receptor antibody (10 µg/ml) as indicated. After 5 days of culture, cells were stained with PI and subjected to flow cytometric analysis as described in Methods. The data are presented as the average percentage of apoptotic cells ± SEM (*P < 0.01, paired samples t-test, n = 8). aIL-2R = anti-IL-2 receptor antibody.
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The anti-apoptotic effect of atRA involves RAR
atRA is generally believed to mediate its cellular effects through binding to RAR and thereby regulate the transcriptional activity of target genes (20,21). Whereas atRA only binds to RAR, 9cRA has high affinity for both RAR and RXR (2224). Previously we have shown that 9cRA and a specific RAR agonist, TTNPB, both increased IL-2 production to the same extent as atRA in primary T lymphocytes stimulated with TPA (25). Thus, if our hypothesis that atRA mediates its survival signals through IL-2 is valid, we would expect 9cRA and RAR agonists to inhibit spontaneous apoptosis to the same extent as atRA. As shown in Fig. 8(A), 9cRA as well as the RAR
agonist, AM-580, and the pan-RAR agonist, TTNPB, indeed displayed the same potency as atRA in preventing apoptosis. Interestingly, a specific RXR agonist, SR11217, also exhibited some protective effect (Fig. 8A) probably due to the small increase in IL-2 production we have observed with this compound (25). These data indicated that atRA inhibits spontaneous apoptosis of activated T cells through a RAR-dependent pathway. To test the dependency of RAR activity for the survival effect of atRA, we stimulated T cells with TPA and atRA in the absence or presence of a RAR-selective antagonist, Ro 41-5253, and determined the percentage of apoptotic cells after 5 days. In T cells from eight blood donors, Ro 41-5253 completely (P < 0.01) abrogated the anti-apoptotic effect of atRA (Fig. 8B). Collectively, these results suggest that the inhibitory effect of atRA on spontaneous apoptosis of activated T cells is mediated by a RAR-dependent increase in the production of IL-2.

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Fig. 8. The anti-apoptotic effect of atRA is mediated by RAR. (A) T cells were left untreated (M) or stimulated with 4 x 109 M TPA (T) in the absence or presence of atRA (RA), 9cRA (9c), AM-580 (AM), TTNPB (TT) or SR11217 (SR) (all at a final concentration of 100 nM) as indicated. After 5 days, cells were stained with PI and analyzed by flow cytometry. The percentage of apoptotic cells in one representative experiment out of four is shown. (B) T cells were stimulated with TPA (4 x 109 M) in the absence or presence of atRA (10 nM) and Ro 41-5253 (10 µM). After 5 days, cells were stained with PI and analyzed by flow cytometry. The data are presented as the average percentage of apoptotic cells ± SEM (*P < 0.01, paired samples t-test, n = 8).
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Discussion
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Although vitamin A has for a long time been known to positively modulate the immune system, the mechanisms involved still remain poorly understood. Relatively few studies have focused on the possible direct effects of RA, the most important vitamin A metabolite in the immune system, on mature lymphocytes. We have previously shown that RA inhibits both cell activation and spontaneous apoptosis of B lymphocytes (4244). On the other hand, we, and others, have reported that atRA potentiates the activation of T lymphocytes (25,4547) and it has also been demonstrated that RA can inhibit AICD of mature T lymphocytes (30). At the end of an immune response, most activated T cells die through a process that has recently been termed ACAD (7). ACAD is believed to occur when activation-induced survival signals fade some time after T cell activation (1,7,41). In the current study, we for the first time address the issue of whether atRA may influence this process. As a model system to study the effect of atRA on ACAD we activated freshly isolated T lymphocytes from healthy human blood donors in the absence or presence of physiological concentrations of atRA and followed their fate without any further stimuli for up to 11 days. We demonstrated that atRA significantly inhibited the spontaneous death of these cells.
Although ACAD is considered to proceed as a result of the absence of survival signals rather than being initiated by an extrinsic apoptosis-inducing molecule, ACAD is nevertheless an active, apoptotic process (3,7). The spontaneous cell death observed in our system was not a passive, necrotic-like event caused by a general lack of nutritional constituents in the cell medium. Rather, we showed that culturing of activated T lymphocytes without any further stimuli resulted in a cellular destruction process that exhibited several salient features of apoptosis such as cell shrinkage, DNA fragmentation and depolarization of the mitochondrial membrane potential. Importantly, atRA had an inhibitory effect on all the apoptotic features examined. The apoptosis-inhibiting effect of atRA was not due to protection of resting cells, since virtually all TPA-treated T cells displayed a sustained increased expression of the activation marker CD69. Thus, in a model system that resembles ACAD, atRA promotes the survival of activated T cells. Interestingly, however, and in contrast to what we have previously shown in primary human B lymphocytes (44), the spontaneous apoptosis of non-stimulated T cells was not inhibited by atRA.
We have previously shown that atRA stimulates the cell-cycle machinery and cell-cycle entry in T lymphocytes (25). Therefore, instead of acting as a true anti-apoptotic factor, an alternative explanation of the effect of atRA on T cell survival in our system could be that atRA merely dilutes out dead cells by proliferating, viable cells. Two important findings argue against this notion. First, since we, by the use of a combination of CFSE and PI staining, found that the divided cells died to the same extent as the undivided cells, atRA could not reduce the percentage of apoptotic cells by simply increasing the number of T cells that divide. Second, we showed that atRA inhibited apoptosis in the undivided cell population where there is no possibility of a dilution of dead cells by proliferating cells. Thus, we conclude that atRA exhibits a true anti-apoptotic activity as well as a growth promoting activity in human T cells.
In the present study, we showed that atRA increased IL-2 production in T cells and that specific inhibition of IL-2 signaling by the use of blocking anti-IL2R antibodies completely abrogated the anti-apoptotic effect of atRA. This suggests that atRA inhibited apoptosis by enhancing IL-2 secretion. Alternatively, increased IL-2 production may be dispensable for the anti-apoptotic effect of atRA and instead atRA might influence IL-2 signaling per se. Two lines of evidence argue against the latter hypothesis. First, there was a significant overall correlation between the amount of IL-2 produced in the presence of atRA and its ability to inhibit apoptosis. Second, a concentration of rIL-2, comparable to that induced by atRA, could fully substitute for the effect of atRA on apoptosis. Thus, enhanced secretion of IL-2 may be both necessary and sufficient for the anti-apoptotic effect of atRA on activated T cells. This also suggests that the protective effect of atRA is not dependent on other cytokines, e.g. IL-4, which was previously reported to potently inhibit apoptosis of activated murine T cells (10). In line with this, we did not observe any increase in IL-4 production in the presence of atRA.
Why do T cells from some blood donors respond stronger to atRA than T cells of others? When we divided the 38 donors examined into two groups according to the degree of atRA responsiveness towards apoptosis of activated T cells, we found that the average net increase in IL-2 production was significantly higher in the group of high responders than in the group of low responders and only in the high responders were apoptosis-inhibiting levels of IL-2 reached. We do not know why atRA is more effective in promoting IL-2 production in T cells from some donors than in T cells from others. We have previously shown that atRA increases IL-2 secretion through a RAR-dependent mechanism (25) and recently we have found that atRA enhances the amount of IL-2 mRNA (data not shown), indicating that atRA regulates IL-2 production at the transcriptional level. Since, to our knowledge, there does not exist any RAR-responsive elements in the IL-2 promoter, it seems likely that atRA regulates IL-2 mRNA levels through a mechanism that is dependent on other intracellular factors. We believe that variability in the functional activities of such factors may explain why T cells from some donors express more IL-2 in response to atRA than others. Furthermore, we argue that this variability in IL-2 production is the reason why atRA has a stronger anti-apoptotic effect in T cells from some blood donors than in T cells from others.
Once IL-2 binds to the IL-2 receptor, it is rapidly internalized and degraded (48,49). When examining the amount of IL-2 protein in the cell culture medium, we found that co-treatment of T cells with TPA and atRA initially led to a gradual increase in IL-2 with time. Maximum levels were reached after 12 days. Thereafter, we observed a steady decline in IL-2 levels, approaching the detection limit of the IL-2 assay (
10 pg/ml) after 67 days (data not shown). Since atRA seems to be totally dependent on IL-2 for its anti-apoptotic effect on activated T cells, this can explain why the protective effect of atRA decreases after day 8. However, a delay in the cell death of activated T cells may nevertheless significantly strengthen immune function, since it could prolong the duration of T cell responses. Moreover, increased survival of activated T cells could also contribute to the generation of memory cells. Since memory T cells are believed to originate from the pool of effector cells (50,51), the number of memory cells generated will depend on the number of effector cells that survive the elimination of T cells at the end of an immune response. The latter is determined by the initial burst size and the extent of cell death (1,52). Since atRA can both potentiate the proliferation of T cells (25) and delay the spontaneous apoptosis of activated T cells, as shown in the current study, it is conceivable that vitamin A may promote the generation of memory cells by directly affecting life and death of peripheral T cells.
In conclusion, the data presented in this paper show that atRA inhibits spontaneous apoptosis of activated T cells, and that this effect is strictly correlated with the ability of atRA to produce IL-2. The results suggest that atRA could extend the lifespan of activated T cells, and consequently vitamin A may prolong T cell responses as well as promote the generation of memory T cells. The latter aspect may in fact explain the recently reported observations that vitamin A increases the efficiency of certain vaccines such as diphtheria, measles and polio type 1 vaccines (5356).
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Acknowledgements
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We are grateful to Dr Maria I. Dawson for providing the RXR-selective agonist SR11217, Dr M. Klaus for providing the RAR-selective antagonist Ro 41-5253 and Hilde R. Haug for excellent technical assistance. This work was supported by the Norwegian Cancer Society, Freia Research Foundation, Jahre Research Foundation and the Blix Family Legacy.
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Abbreviations
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9cRA9-cis retinoic acid
ACADactivated T cell autonomous death
AICDactivation-induced cell death
atRAall-trans retinoic acid
CFSE5- (and -6)-carboxyfluorescein diacetate succinimidylester
PIpropidium iodide
PKCprotein kinase C
RAretinoic acid
RARretinoic acid receptor
RXRretinoid X receptor
TPA12-O-tetradecanoyl phorbol 13-acetate
TTNPB4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid.
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