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International Immunology, Vol. 13, No. 1, 85-93, January 2001
© 2001 Japanese Society for Immunology

Inhibition of thymidine synthesis by folate analogues induces a Fas–Fas ligand-independent deletion of superantigen-reactive peripheral T cells

Kamel Izeradjene, Jean-Pierre Revillard and Laurent Genestier

Laboratory of Immunopharmacology, Institut National de la Santé et de la Recherche Médicale U503, Claude Bernard University, Hopital E. Herriot, 5 Place d'Arsonval, 69437 Lyon Cedex 03, France

Correspondence to: L. Genestier


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Methotrexate (MTX), a folate antagonist with multiple enzymatic targets, is used in the treatment of malignancies as well as in autoimmune and chronic inflammatory diseases, and ZD1694 (tomudex), a water-soluble quinazoline specific inhibitor of thymidylate synthase (TS), is used in the treatment of adenocarcinomas. In this study, we investigated the effects of these folate analogues on superantigen (SAg)-reactive peripheral T cells in vivo. In BALB/c mice, staphylococcal enterotoxin B (SEB)-induced cytokine secretion, IL-2R (CD25) expression and early deletion of a fraction of SEB-reactive Vß8+ T cells were not impaired by either MTX (7 mg/kg/day) or tomudex (5 mg/kg/day). However, both MTX and tomudex prevented Vß8-selective T cell expansion and accelerated their peripheral elimination. Administration of thymidine (500 mg/kg/12 h) completely abrogated this effect, indicating that inhibition of TS but not that of other folate-dependent enzymes was the main mechanism involved. Furthermore, a marked increase of apoptotic cells restricted to the Vß8+ T cell subset indicated that proliferation inhibition was associated with apoptosis. In contrast with peripheral Vß8+ T cell deletion, MTX and tomudex did not prevent the increase of Vß8+ thymocytes triggered by SEB. Experiments in C57BL/6-lpr/lpr mice further demonstrated that deletion of Vß8+ T cells induced by folate analogues was independent of Fas–Fas ligand interaction. Our results provide evidence that folate analogues may selectively delete dividing peripheral T cells through TS inhibition, but do not interfere with other events triggered by SAg.

Keywords: apoptosis, deletion, methotrexate, superantigen, tomudex


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Methotrexate (MTX) is an anticancer drug included as a component of combination chemotherapy regimens used in the treatment of several malignancies, such as acute lymphoblastic leukemia and non-Hodgkin's lymphoma. MTX is also widely prescribed as an immunosuppressive agent in the treatment of autoimmune diseases, including rheumatoid arthritis (1). The combination of MTX with cyclosporin A and/or prednisone (2,3) or FK506 (4) is effective for prevention of acute graft-versus-host disease. MTX has also been used as an adjunct therapy for persistent mild cardiac allograft rejection (5).

MTX has multiple enzymatic targets which may account for these biological effects. Indeed, MTX and MTX polyglutamates are potent inhibitors of dihydrofolate reductase (DHFR) which allows the reduction of dihydrofolates in tetrahydrofolates. The decrease of the reduced folate pool results in an indirect inhibition of thymidylate synthase (TS) which catalyzes the production of the pyrimidine nucleotide dTMP from dUMP (6). In addition, polyglutamated MTX may directly inhibit TS catalytic activity (7). In TS-deficient human carcinoma cells, a thymine-less state may result in growth arrest and/or Fas–Fas ligand (FasL)-dependent apoptosis (8,9). Polyglutamated MTX also inhibits 5-aminoimidazole-4-carboxamide ribonucleotide transformylase (10) and ecto-5'- nucleotidase (11), resulting in increased release of adenosine by fibroblasts, endothelial cells or other cell types (10,11). Adenosine is a potent endogenous anti-inflammatory purine nucleotide which modulates several polymorphonuclear neutrophil activities such as superoxide anion generation, adhesion and phagocytosis by interacting with A2 receptors (12,13). More recently, MTX has been shown to inhibit the first step of de novo purine biosynthesis by blocking the amidophosphoribosyltransferase (14). This effect would lead to inhibition of de novo purine biosynthesis and increase of de novo pyrimidine synthesis in T lymphocytes stimulated by phytohemagglutinin (14).

We previously demonstrated that MTX induces apoptosis of in vitro activated but not resting T cells from human peripheral blood (15). MTX-induced apoptosis required progression to the S phase of the cell cycle, was Fas–FasL independent and was not significantly inhibited by addition of adenosine deaminase, indicating that adenosine was not involved (15). Thus, beyond the anti-inflammatory properties of adenosine, MTX may inhibit T cell proliferation and induce clonal deletion in vitro.

Since MTX has several enzymatic targets, we also conducted in parallel experiments with the quinazoline-based antifolate derivative tomudex which is a specific TS inhibitor (16). Like MTX, tomudex is converted to polyglutamated derivatives, and Glu4 and Glu5 compounds remain at least 24 h after removal of extracellular drug (17).

Although numerous in vitro studies have been devoted to the biochemical and immunological properties of MTX and tomudex, their effect on normal T cells during in vivo activation has not been investigated. For instance, their capacity to modulate cytokine secretion, and to induce proliferation arrest and/or apoptosis in vivo is not known. To address these questions, we took advantage of the bacterial superantigen staphylococcal enterotoxin B (SEB) which selectively binds to T cells without processing requirement (18), therefore allowing a precise identification of the reactive T cells. Upon SEB injection in BALB/c mice, Vß8+ T cells are first activated and produce large amounts of cytokines that may lead to lethal T cell shock (19). Subsequently, a fraction of Vß8+ T cells is deleted by apoptosis and the remaining Vß8+ T cells expand, reaching a plateau value at day 2–3 (20–23). Thereafter, Vß8+ T cells are deleted to fall back to normal values (22,23). The remaining Vß8+ T lymphocytes become anergic and fail to proliferate in vitro upon ligand-specific re-stimulation (24). Thus SAg as outlined here can be used to analyze complex T cell responses in vivo, and we took advantage of this model to study the in vivo effect of MTX and tomudex.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
C57BL/6 homozygous for the lpr mutation (lpr) and BALB/c mice were from IFFA CREDO (L'Arbresle, France). BALB/c mice were used at 8–12 weeks of age. The lpr mice were used at the age of 8 weeks, at a time when they do not show any clinical signs of disease.

Reagents and antibodies
Phycoerythrin (PE)- and FITC-conjugated anti-CD4 (CT-CD4), anti-CD8 (CT-CD8a), biotin- or PE-conjugated anti-IL-2R mAb (PC61 5.3), streptavidin–TriColor and PE-coupled anti-B220 (RA3-6B2) were purchased from Caltag (South San Francisco, CA). FITC-conjugated anti-Vß8 (F23.1) and FITC-coupled anti-Vß6 (RR4-7) were from PharMingen (San Diego, CA). MTX and SEB were purchased from Sigma (St Louis, MO). Tomudex was obtained from Zeneca (Reims, France).

Injection protocol
BALB/c mice were given 50 µg SEB i.p, or 100 µg SEB i.v. for C57BL/6 lpr mice, in a volume of 0.2 ml of PBS. The day of SEB injection was considered as day 0 of our experiments. MTX and tomudex were given i.p. diluted in PBS at a dose of 7 and 5 mg/kg/day respectively. Tomudex was formulated in 0.05 M NaHCO3 and the pH was adjusted to 9 with NaOH. The drugs were administered daily starting from 2 days before SEB injection and continued until the mice were sacrificed, unless otherwise indicated. In some groups, mice were treated with thymidine (500 mg/kg/12 h) administered twice daily by i.p. injection. In control mice, thymidine, MTX, tomudex and/or PBS were administered in the same dose and/or volume as in the experimental groups.

Flow cytometry analysis
Mice were sacrificed, mesenteric lymph nodes and thymuses were removed, and single-cell suspensions were prepared. Two- and three-color staining with FITC-, PE- or biotin-labeled mAb was done simultaneously for 30 min on ice. Cells were subsequently washed twice with PBS containing 1% BSA and 0.2% NaN3 (PBS/BSA/azide) and biotinylated mAb restained with streptavidin–TriColor from Caltag for 30 min on ice. After washing twice with PBS, cells were fixed with 1% formaldehyde in PBS/BSA/azide. In order to assess the peripheral deletion in lpr mice, 50 µl of fresh blood obtained from the intraorbital sinus was incubated with the appropriate antibodies for 30 min at 4°C. Following red blood cells lysis in a specific buffer (1.53 M NH4Cl, 48.4 M EDTA and 10 mM KHCO3), cells were washed twice and fixed. Analysis was performed on a FACScan flow cytometer (Becton Dickinson, Pont de Claix, France) using the CellQuest program.

Cytokine assays
Blood samples were drawn at various time points after i.p. injection of SEB. After clotting, sera were immediately recovered by centrifugation and samples were stored at –70°C until tested for cytokine content. Tumor necrosis factor (TNF)-{alpha} and IFN-{gamma} ELISAs were performed on serum samples as specified by the manufacturer (PharMingen). The sensitivity limit of the assay was 1 U/ml for IFN-{gamma} and 0.31 ng/ml for TNF-{alpha}.

Measurement of apoptosis
Annexin V alexa (Boehringer Mannheim, Mannheim, Germany) was used as specific apoptotic marker (25) under the conditions described by the manufacturer. Briefly, cells were washed in HEPES buffer (10 mM HEPES–NaOH, pH 7.4, 140 mM NaCl and 5 mM CaCl2) and then incubated in a 1:500 dilution of annexin V alexa solution for 5 min, followed by flow cytometry. For analysis of apoptosis by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay (26), cells were stained for Vß8 and Vß6 expression, fixed and permeabilized, and DNA degradation was assessed using TUNEL reaction mix containing 20 U terminal deoxyribosyl transferase, 10 mM biot-dUTP and 1mM cobalt chloride (Boehringer Mannheim). The cells were washed twice with cold PBS/BSA and incubated with streptavidin–PE for 20 min on ice. Apoptotic cells were identified as PE+ by flow cytometry. The percentage of CD4+ and CD8+ T cells that express B220 was also used as a marker associated with apoptosis as previously described in this model (27,28).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
MTX and tomudex do not interfere with the early steps of T cell activation.
In BALB/c mice, SEB injection induces the activation of Vß8+ T cells, resulting in the rapid release of several cytokines including IL-2, TNF-{alpha} and IFN-{gamma} (19,29). TNF-{alpha} levels in the serum of mice peaked at 2 h and those of IFN-{gamma} at 4 h following SEB injection. Neither peak levels nor kinetics of TNF-{alpha} and IFN-{gamma} were modified in MTX- and tomudex-treated mice (Fig. 1A and BGo).



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  		Fig. 1. MTX and tomudex do not inhibit SAg-induced T cell activation in vivo. Mice were injected i.p. with SEB on day 0 and MTX (7 mg/kg/day) or tomudex (5 mg/kg/day) treatments were started 2 days before SEB injection and continued until day 2. (A and B) Blood was collected and the serum content of TNF-{alpha} and IFN-{gamma} was determined by ELISA. (C) Mesenteric lymph node cells were removed, stained with anti-Vß8–FITC and anti-CD25–PE, and the percentage of CD25+ among Vß8+ T cells was determined. Data are the mean ± SEM of two experiments.

 
SEB activation also induces blast transformation and expression of the IL-2R {alpha} chain (CD25) on Vß8+ T cells (30). To determine whether folate analogues interfere with early T cell activation marker expression, MTX- or tomudex-treated mice were injected with SEB and lymph node T cells were analyzed for cell size and the expression of CD25 by flow cytometry. Most of the Vß8+ T cells in all groups of mice were blasts 24 h after SEB injection (data not shown) and the majority (~90%) of Vß8+ T cells were CD25+ 9 h after SEB injection, irrespective of whether animals were pretreated with MTX, tomudex or PBS (Fig. 1CGo). CD25 expression rapidly declined and after 48 h almost all Vß8+ T cells were CD25– in all groups of mice. Vß8– T cells showed no significant changes in either cell size or expression of CD25 during the observed period (data not shown). We conclude that MTX and tomudex do not interfere with SAg-induced blast transformation, cytokine secretion and CD25 expression on activated Vß8+ T cells.

MTX and tomudex do not alter SEB-driven early deletion of Vß8+ peripheral T cells
Within the first 12–24 h after injection of SEB, there is a complete disappearance of Vß8+ T cells from peripheral blood and a 50% decrease of Vß8+ percentage in lymph nodes both in CD4+ and CD8+ subsets (20,21). The mechanisms of this initial deletion which involves glucocorticoid receptors (21) are still unknown and the effect of folate analogues was investigated. Mice treated with MTX or tomudex were injected with SEB and 16 h later mesenteric lymph node cells were analyzed. The data detailed in Table 1Go depict representative results of a set of experiments in which MTX and tomudex did not modify the selective loss of SEB-reactive Vß8+ T cells in vivo.


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  		Table 1. MTX and tomudex do not inhibit SEB-induced early T cell deletion
 
Enhanced peripheral elimination of SEB-reactive T cells in MTX- and tomudex-treated mice
After SEB injection, Vß8+ T cells expand until day 2–3, then this subpopulation is deleted between days 4 and 7 to reach a level below pretreatment values (22,23). To test the effect of folate analogues on the SEB-driven sequential expansion and deletion of Vß8+ T cells, MTX and tomudex were administered to mice starting 2 days before the SEB challenge, followed by daily injections of these drugs throughout the experiment. As demonstrated in Fig. 2Go, administration of MTX or tomudex fully inhibited the expansion of the CD4+Vß8+ and CD8+Vß8+ T cells induced by a single injection of SEB. At day 7 the deletion of CD8+Vß8+ T cells was still more pronounced in MTX and tomudex-treated than in control mice. However, the elimination of CD4+Vß8+ was not further increased by MTX or tomudex treatments 7 days post SEB injection. The effect of MTX- and tomudex was restricted to SEB-activated T cells, as shown by the unaltered frequency of Vß6+ T cells (Fig. 2Go) and Vß8– T cells (not shown). This effect was therefore quite unlikely to be due to a non-specific decrease in T cell dividing precursors in bone marrow or thymus. To further exclude this possibility, we checked thymus cellularity and percentage of CD4+CD8+ and CD4+CD8– or CD4–CD8+ in mice treated with MTX (7 mg/kg/day) or tomudex (5 mg/kg/day) from day 0 to day 4. In the absence of SEB, the percentages of CD4+CD8+, CD4+CD8– or CD4–CD8+ thymocytes in the presence of MTX or tomudex were equivalent to that observed in untreated mice (Fig. 3AGo). In addition, the number of thymocytes was not affected by either MTX or tomudex treatments (data not shown), suggesting that these drugs do not induce direct thymic depletion. On the other hand, SEB profoundly modified the number of Vß8+ cells comprised in each thymocyte subset. In Fig. 3Go(B), there was a marked increase in Vß8+ thymocytes among CD4+CD8+, CD4+CD8– and CD4–CD8+ subsets 48 h after injection of SEB alone. This effect was not affected by MTX or tomudex treatments.



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  		Fig. 2. MTX and tomudex prevent SEB-induced expansion of Vß8+ T cells. Mice were treated as in Fig. 1Go. Treatment with MTX or tomudex was continued until sacrifice. Mice were sacrificed at indicated times, and mesenteric lymph node cells were removed and stained for Vß8, Vß6, CD4 and CD8 expression. The percentage of CD4+Vß8+, CD8+Vß8+ and total Vß6+ lymphocytes is plotted. Note that only viable cells are gated. Data are the mean ± SEM of four mice in each condition.

 


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  		Fig. 3. Neither thymocyte differentiation nor SEB-induced changes are modified by MTX and tomudex treatment. (A) Mice were injected i.p. with PBS (left panel), with MTX (7 mg/kg/day) (middle panel) or tomudex (5 mg/kg/day) (right panel) for 4 days, and thymic CD4/CD8 profiles were analyzed by staining with an anti-CD4–FITC and an anti-CD8–PE mAb. Percentages of cells in each area are shown. (B) Mice were injected i.p. with SEB on day 0 and MTX (7 mg/kg/day) or tomudex (5 mg/kg/day) treatments started 2 days before SEB injection were continued until individual mice were sacrificed at 2 days after SEB injection. Thymocytes were triple-labeled with TriColor-conjugated anti-CD4, PE-conjugated anti-CD8 and FITC-conjugated anti-Vß8 mAb, and the percentage of Vß8+ cells in each thymocyte subset (left panel) was analyzed by FACScan. Right panel: absolute counts of Vß8+ thymocytes among double-negative, double-positive and single-positive subsets.

 
Given the observation that Vß8+ T cell deletion following SEB treatment was enhanced in MTX- and tomudex-treated mice, we determined whether MTX and tomudex merely inhibited T cell proliferation or actually triggered apoptosis of peripheral Vß8+ T cells. To this end, lymph node cells from mice pretreated with MTX, tomudex or PBS were isolated 36 h following SEB injection, and subsequently apoptosis was determined by staining with annexin V and by TUNEL assay. At this time, deletion induced by SEB did not occur yet as demonstrated by the low percentage of annexin V+ Vß8+ cells in PBS-treated mice injected with SEB (Fig. 4AGo). However, the Vß8+ subset from MTX or tomudex plus SEB-treated mice contained an increased percentage of apoptotic cells (~50%) as revealed by annexin V staining (Fig. 4AGo) or by TUNEL assay (~45%) (Fig. 4BGo), whereas the percentage of annexin V+ or TUNEL+ cells among Vß6+ T cells did not increase after MTX or tomudex treatments. Furthermore, the percentage of CD4+ and CD8+ T cells that express B220, a marker associated with apoptosis in this model (27,28), was raised from 15.7 to 31.1% in mice treated with folate analogues (Fig. 4CGo). Most of the B220+ CD4+ and CD8+ T cells were stained by annexin V, and therefore represented T cells undergoing apoptosis (Fig. 4CGo, right panel).



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  		Fig. 4. Enhancement of T cell apoptosis by combination of SEB challenge with MTX or tomudex treatment. Mice were treated as in Fig. 1Go, and lymph node cells were collected 36 h after SEB challenge and immediately analyzed for annexin V binding and for expression of Vß8 and Vß6 (A). Lymph node cells were cultured for 12 h at 37°C, and then stained for expression of Vß8, Vß6 and for nicked DNA by the TUNEL assay (B). Lymph node cells were simultaneously stained with anti-CD4, anti-CD8, anti-B220 and annexin V (C). Numbers indicate the proportion of annexin V+ within Vß8–, Vß8+ and Vß6+, and apoptotic cells detected by TUNEL within Vß8+ and Vß6+ T cells, and CD4/8 versus B220 staining. Histograms in (C) show the distribution of annexin V+ cells among CD4/CD8+B220+ and B220– cells. Representative data from four experiments with three or four mice for each condition.

 
Only expanding Vß8+CD8+ are deleted upon iterative stimulation by SEB in the presence of folate analogues
As shown previously, repeated injections of SEB cause in vivo activation, i.e. IL-2R expression, in both CD4+Vß8+ and CD8+Vß8+ T cells, but only CD8+ T lymphocytes initiate a vigorous expansion reaching peak value after 48 h (our data not shown and 31). Therefore, we investigated the effect of TS inhibitors on Vß8+ T cell response to multiple additional injections of SEB. As shown in Fig. 5Go(A), repeated injections of SEB in MTX- and tomudex-treated mice greatly enhanced the elimination of Vß8+CD8+ T cells, so that their frequency decreased to <5% of total CD8+ T cells. Unlike that of CD8+Vß8+ T cells, the deletion of CD4+Vß8+ T cells was not further increased (Fig. 5BGo).



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  		Fig. 5. Marked enhancement of CD8+Vß8+ T cell deletion with multiple injections of SEB in MTX- and tomudex-treated mice. SEB (50 µg) was injected at 4-day intervals twice (SEBx2) or up to 5 times (SEBx5) and the mice were sacrificed 4 days after the last injection. MTX (7 mg/kg/day) and tomudex (5 mg/kg/day) treatments were begun 2 days before the first SEB injection and continued until sacrifice. (A) Dot-plots show two-color flow cytometry analysis of BALB/c lymph node cells of SEBx5 control mice, MTX–(SEBx5)- and tomudex–(SEBx5)-injected mice. (B) Upper and lower panels represent the relative numbers of gated CD4+Vß8+ and CD8+Vß8+ respectively. In untreated mice, the percentages of CD4+Vß8+ and CD8+Vß8+ were 18.7 ± 0.9 and 26.6 ± 1.5 respectively. In MTX- or tomudex-treated mice not injected with SEB the percentages of CD4+Vß8+ and CD8+Vß8+ were 18.9 ± 2.6 and 26.6 ± 1.7, and 20.8 ± 3.0 and 26.1 ± 3.0 respectively. Data are the mean ± SEM of three mice in each condition.

 
MTX and tomudex induce T cell deletion by inhibition of TS
In contrast to tomudex which is a selective inhibitor of TS (17), MTX inhibits multiple folate-dependent enzymes that may account for several biological activities (6,7,10,11). Therefore we investigated the contribution of TS inhibition to tomudex- and MTX-induced deletion of Vß8+ T cells. To this end, thymidine was co-administrated with MTX and tomudex twice a day, and percentages of CD4+Vß8+ and CD8+Vß8+ were determined 48 h after SEB injection. Surprisingly thymidine completely reversed both MTX- and tomudex-induced deletion of both CD4+Vß8+ and CD8+Vß8+ T cells (Fig. 6Go), demonstrating that, at least in vivo, TS is the principal target of MTX.



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  		Fig. 6. Thymidine completely abrogates MTX- and tomudex-induced deletion of Vß8+ T cells after SEB injection. Mice were injected with MTX or tomudex started 2 days before SEB immunization. Thymidine (500 mg/kg/12h) was injected twice a day, until the mice were sacrificed. Lymph node cells were removed 48 h after the injection of SEB, and staining was performed with anti-Vß8–FITC mAb and either anti-CD4–PE or anti-CD8–PE mAb. Data are the mean ± SEM of three mice.

 
MTX- and tomudex-induced peripheral T cell deletion is independent of Fas–FasL interactions
Knowing that MTX-mediated apoptosis of cell lines was reported to depend on Fas–FasL interactions (32,33) and that defective TS induces a Fas-dependent apoptosis in human carcinoma cell lines (9), we examined whether MTX and tomudex could induce peripheral deletion of Vß8+ T cells in lpr mice defective in Fas expression (34). In the experiment shown in Fig. 7Go, administration of SEB in wild-type animals (C57BL/6) induced the expansion of Vß8+ T cells which was maximal after 2 days followed by the deletion of these cells between day 3 and 10. This Vß8+ T cell deletion was not observed in the Fas-defective lpr mice where Vß8+ T cells continued to expand throughout the course of the experiment. These observations confirm that Fas–FasL interactions participate in peripheral deletion of Vß8+ T cells induced by SEB (35). We then examined the response to SEB in lpr mice that received daily injections of MTX or tomudex. Unlike SEB control mice, MTX or tomudex-treated mice displayed a marked deletion of Vß8+ T cells observed within 2 days post SEB injection (Fig. 7Go). The percentage of Vß6+ T cells was also determined during the course of the experiment and did not show any variations, confirming the specificity of the deletion in the C57BL/6 wild-type and lpr mice (Fig. 7Go). Thus, MTX and tomudex induce in vivo peripheral deletion of Vß8+ T cells by a Fas-independent pathway.



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  		Fig. 7. MTX- and tomudex-induced deletion of Vß8+ T cells in SEB-treated mice is independent of Fas–FasL interaction. C57BL/6 wild-type or lpr mice were treated in vivo with MTX (7 mg/kg/day) or tomudex (5 mg/kg/day) 2 days before i.v. injection of SEB. At the times indicated, FACS analysis was performed on peripheral blood samples to determine the number of Vß8+ and Vß6+ T cells expressed as a percent of the total CD4+ and CD8+ T cells. The percentage of Vß6+ T cells was determined as a control.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
MTX was shown to exert a wide range of anti-inflammatory activities that are primarily mediated by the release of adenosine (10,13). Such anti-inflammatory properties do not exclude a genuine immunosuppressive activity, in as much as adenosine deaminase deficiency is associated with severe combined immunodeficiency disease (36). Our study demonstrates that treatment with two folate analogues, MTX and tomudex, does not modify the early events of SEB-induced activation but markedly accelerates and enhances the deletion of SEB-reactive Vß8+ T cells. Without folate analogues treatment, the partial deletion observed in SEB-treated mice is limited and preceded by an expansion phase of the Vß8+ T cells (22,23) (Fig. 2Go). Furthermore, apoptosis of SEB-activated T cells occurs after a discrete number of cell divisions in vivo (22). However, in MTX- and tomudex-treated mice, SEB-induced deletion occurs earlier, before the expansion phase and is more extensive than with SEB treatment alone. In addition, repeated injections of SEB in MTX- and tomudex-treated mice greatly enhanced the elimination of Vß8+CD8+ T cells, so that their frequency decreased to <5% of total CD8+ T cells. In contrast, repeated injections of SEB in MTX- and tomudex-treated mice induced no further deletion of Vß8+CD4+ T cells, in agreement with their lack of proliferation despite the expression of CD25. Altogether, these results suggest that T cell deletion induced by folate analogues in SEB-injected mice only occurs in cycling cells and probably at the beginning of the S phase of the cell cycle according to in vitro studies (15). Knowing that cell division is required for acquisition of specific B and T cell functions such as Ig isotype switch in activated B cells (37), cytokine gene expression by Th cells (38) and differentiation from naive to effector and memory cytotoxic T lymphocytes (39), folate analogues may be considered as major immunosuppressive agents.

Our results clearly demonstrate that deletion induced by folate analogues in SEB-treated mice is strictly restricted to SEB-reactive peripheral T cells and involves apoptosis resulting from TS inhibition. Indeed, thymidine administration completely abrogated deletion induced by MTX and tomudex. We may conclude that among all the possible targets of MTX, inhibition of TS both by a direct effect (7) and/or as a consequence of DHFR blockade (6) represents the major metabolic pathway at least in vivo. This is in contrast with the cytostatic effect observed when MTX is co-incubated with phytohemagglutinin on peripheral blood lymphocytes in vitro (14). In this study, the blockade in the G0/G1 phase of the cell cycle induced by MTX was correlated with the inhibition of the first step of the de novo purine biosynthesis. However, complete reversibility of proliferation arrest by addition of guanosine was not documented (14). Whether proliferation inhibition could represent an additional mechanism accounting for the lack of expansion of Vß8+ T cells in our model could not be formally excluded, but it is unlikely in view of the major deletion of CD8+ T cells. Cyclosporin A was also reported to inhibit SEB-induced T cell expansion in vivo (40). Whereas the authors speculated on the mechanism by which cyclosporin A enhances peripheral deletion, they did not formally demonstrate that this effect was attributed to apoptosis. Similarly, leflunomide did not affect initial activation events but prevented the expansion of SEB- reactive T cells (41). Knowing that leflunomide also blocks pyrimidine nucleotide synthesis by inhibiting dihydro-orotate dehydrogenase (42), it is likely that it shares similar mechanisms of action with the folate analogues used in our study.

In agreement with a previous report (43), a single injection of SEB in adult mice triggered proliferation of Vß8+ thymocytes. The lack of deletion of those cells in MTX- or tomudex-treated mice may reflect poor diffusion of the drugs to the thymic tissue or, alternatively, intrinsic resistance of thymocytes due to the elevated nucleotide pools.

In the experiment shown in Fig. 7Go, the Vß8+ T cell deletion was not observed in the Fas-defective lpr mice where Vß8+ T cells continued to expand throughout the course of the experiment. Based on our results, we postulate that Fas-mediated T cell apoptosis plays a major role in peripheral elimination of activated T cells. Several studies using lpr mice have shown a requirement for Fas in peripheral T cell apoptosis induced by superantigens or soluble peptides (35,44). However, Fas-mediated apoptosis is certainly not the only pathway for the deletion of mature T cells after antigenic stimulation. Other studies have revealed that peripheral T cell apoptosis can proceed independently of Fas (45,46). Thus, mechanisms in addition to Fas signaling contribute to peripheral T cell deletion. TNFR1 signaling may be implicated, as it shares apoptotic signaling pathways with Fas (47). Elimination of TNFR1 in lpr mice accelerates the onset of the lpr phenotype (48) and further evidence has suggested that TNF is involved in peripheral T cell deletion (49,50). Whether these Fas-independent pathways may be involved in TS inhibitor-induced apoptosis require further investigations.

In contrast to actively dividing cells such as human colon carcinoma cell lines in which a thymine-less state induced a Fas–FasL-dependent apoptosis (9), deletion of Vß8+ T cells induced by MTX and tomudex still occurred in Fas-defective lpr mice, demonstrating that in vivo as well as in vitro (15) peripheral T cell apoptosis induced by TS inhibitors is independent of Fas–FasL interaction.

Recent findings provided novel insights into the mechanisms by which CD4+ and CD8+ T cells from HIV-1-infected patients are deleted after stimulation through the TCR. Indeed, in these conditions cell loss observed between 48 and 72 h could be attributed to an impaired ability of T cells from HIV-1-infected patients to synthesize nucleotides through the de novo pathway (51). In contrast with that we observed after treatment by TS inhibitors, where apoptotic cell death has been clearly characterized by annexin V staining, TUNEL assay and B220 expression, the cell loss observed in HIV patients seems to be associated with necrotic death (51) which may be explained by the resultant ATP depletion (52).

Finally, our study directly demonstrates by staining of Vß8+ and Vß6+ T cells that folate analogue-induced apoptosis is strictly restricted to cycling T cells. In these conditions, it is remarkable that thymic populations and other peripheral T cell subsets were not affected. Other studies will be necessary to know whether these results could be applied to T and B cell responses to specific antigens. In particular, it will be interesting to study whether prolonged treatment in the presence of sustained antigenic stimulation (e.g. allograft response or autoimmune disorders) results in a restriction of a specific antigenic repertoire or a selection of clones resistant to TS inhibitors. Since no hematological toxicity appeared in our experiments, the data obtained in mouse are likely to be applicable to patients treated with TS inhibitors as part of cancer chemotherapy. Whether similar mechanisms will operate at low doses of MTX is suggested by our previous observations that peripheral blood lymphocytes from rheumatoid arthritis patients treated with MTX underwent apoptosis upon in vitro stimulation by phytohemagglutinin (15).


   Acknowledgments
 
This work was supported by grants from INSERM, by Région Rhône Alpes no. H098730000 (J.-P. R.) and by Fondation pour la Recherche Médicale (J.-P. R.).


   Abbreviations
 
DHFR dihydrofolate reductase
FasL Fas ligand
MTX methotrexate
PE phycoerythrin
SEB staphylococcal enterotoxin B
TNF tumor necrosis factor
TS thymidylate synthase
TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling

   Notes
 
Transmitting editor: A. Fischer

Received 10 May 2000, accepted 5 October 2000.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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