Changes in sensitivity of peripheral lymphocytes of autoimmune gld mice to FasL-mediated apoptosis reveal a mechanism for the preferential deletion of CD4CD8B220+ T cells

Sheng Xiao1,6, Xingmin Zhang4, Koren K. Mann1,5, Satoshi Jodo3, Li Li6, Wael N. Jarjour6, Ann Marshak-Rothstein2, David H. Sherr5 and Shyr-Te Ju1,6

1 Department of Pathology and Laboratory Medicine, 2 Department of Microbiology and 3 Department of Medicine, Boston University School of Medicine, Boston, MA 02118, USA 4 Center for Neurologic Diseases, Harvard Medical School, Boston, MA 02115, USA 5 Department of Environmental Health, School of Public Health, Boston University School of Medicine, Boston, MA 02118, USA 6 Department of Medicine, Division of Rheumatology and Immunology, University of Virginia, Charlottesville, VA 22908, USA

Correspondence to: S.-T. Ju; E-mail: sj8r{at}virginia.edu
Transmitting editor: R. Geha


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
During thymic selection ‘mis-selected’ CD8+ T cells exit to the periphery where they are deleted by a Fas/FasL-mediated mechanism, presumably as a result of activation by self-antigens. In the absence of functional FasL, as is the case in autoimmune gld mice, these ‘mis-selected’ T cells develop into unique Thy1+CD4CD8 TCR{alpha}ß+B220+ lymphocytes [abnormal double negative T (DN T) cells]. Using bioactive FasL-bearing vesicles [FasL vesicle preparation (FasL VP)], we were able to induce acute apoptosis in freshly isolated lymphocytes and to demonstrate that peripheral lymphocytes of gld mice become more sensitive to the FasL-mediated apoptosis as they age. Furthermore, flow cytometric analyses indicated that within this peripheral lymphocyte population, the abnormal DN T cells were preferentially eliminated. The exquisite sensitivity of these abnormal DN T cells is attributed to their increased membrane Fas expression with a concomitant reduction of cytosolic FLIPL. Our data support the hypothesis that specific components of the Fas-mediated apoptotic pathway are modulated in favor of the elimination of auto-reactive T cells as well as those CD8+ T cells that are ‘mis-selected’ in the thymus and escape to the periphery.

Keywords: apoptosis, DN T cells, Fas, FLIP, IL-2, T cell deletion


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD95 (Fas), a type I transmembrane protein and a member of the tumor necrosis factor receptor (TNFR) family, is widely expressed by a variety of tissues and cells (1,2). The physiological ligand for Fas is CD178 (FasL). It is a type II transmembrane protein of the TNF super-family and is expressed mainly by activated T cells (1,37). Engagement of Fas by FasL results in Fas aggregation, which enables the binding of the cytosolic adaptor protein Fas-associated death domain (FADD) and procaspase-8 to form a complex known as the death-inducing signaling complex (DISC) (810). Concentrated procaspase-8 in DISC initiates an auto- activation process that generates active caspase-8. Depend ing on the activation of caspase 8 or the participation of mitochondria, an acute (type I) or a slow (type II) apoptosis pathway can be activated (11,12). Both apoptosis processes, however, can be inhibited by FLICE/caspase-8 inhibitory protein (FLIP), which competitively inhibits the binding of procaspase-8 to FADD before the bifurcation of the type I and type II pathways (1316). FLIP is expressed as both long (FLIPL) and short (FLIPS) isoforms, and both inhibit Fas-mediated apoptosis (13,16).

Fas-mediated apoptosis is critically involved in the regulation of lymphocyte development, function, and homeostasis. In mice, a defect in the fas (lpr mice) or fasl (gld mice) gene causes lymphoproliferation, autoantibody formation with autoimmune manifestations, and the accumulation of abnormal double negative T (DN T) cells, which display the unique surface phenotype of CD4CD8Thy-1+TCR{alpha}ß+B220+ (1720). The equivalent human disorder is known as autoimmune lymphoproliferative syndrome (ALPS), Canale–Smith syndrome, or lymphoproliferative syndrome with autoimmunity. Patients with ALPS develop diffuse nonmalignant lymphadenopathy due to the accumulation of the abnormal DN T cells (2126).

Introduction of a B2m or a CD8 {alpha} gene defect into lpr mice inhibits the development of the abnormal DN T cells, suggesting that CD8+ T cells are the major precursors of abnormal DN T cells (2729). These abnormal DN T cells are derived from a population of ‘mis-selected’ CD8+ T cells that are normally generated during lymphocyte differentiation in the thymus and then exit to the periphery (30). Activation of these ‘mis-selected’ CD8+ T cells in the periphery without engaging CD8 molecules results in the methylation of the CD8 gene and inhibition of CD8 synthesis, rendering the CD4CD8 phenotype. Activation also induces FasL expression and up-regulation of Fas. Subsequent interaction between Fas and FasL deletes abnormal DN T cells. Due to the lack of Fas-mediated apoptosis in gld and lpr mice, abnormal DN T cells accumulate in these hosts (30). In this regard, the mechanism that deletes the abnormal DN T cells is likely to be similar if not identical to the mechanism that deletes auto-reactive T cells in the periphery.

While abnormal DN T cells from lpr mice express FasL (31), direct evidence that the unique DN T cells become more sensitive to FasL-mediated apoptosis and the reasons accounting for their increased sensitivity have not been demonstrated. Recently, we developed a novel reagent termed FasL vesicle preparation (FasL VP). FasL VP are bio-vesicles containing full-length FasL capable of delivering a powerful Fas-mediated apoptotic signal (3236). FasL VP can induce acute apoptosis in many targets that are resistant to anti-Fas mAb. Apoptosis induced by FasL VP is physiologically relevant because FasL VP utilize membrane FasL identical to that expressed on activated T cells (32,33). Here, we demonstrate that FasL VP can induce apoptosis in freshly isolated normal lymphocytes within 6 h of interaction. We show that abnormal DN T cells in the lymph nodes and spleens of gld mice are exquisitely sensitive to FasL VP. Further analysis suggests that the exquisite sensitivity of abnormal DN T cells is due to an increased Fas expression and a decreased FLIPL level. Our study provides strong evidence that ‘mis-selected’ CD8+ T cells that exit to the periphery following thymic selection are eliminated because they are sensitized to a deletion mechanism that involves not only increased FasL expression but also membrane Fas up-regulation and cytosolic FLIPL down-regulation.

Whether such a coordinated program regulating the Fas-mediated apoptotic pathway is involved in the deletion of the conventional MHC-restricted auto-reactive T cells in the periphery is discussed.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
B6, B6.lpr, and MRL mice were obtained from the Jackson Laboratories, Bar Harbor, ME. MRL.gld mice were obtained from our own breeding colony at Boston University Medical Campus. The Jo2 anti-Fas mAb was purchased from BD PharMingen, San Diego, CA.

FasL VP
FasL VP was prepared as described (3236). Briefly, a retroviral packaging cell line carrying the hfasl gene (hFasL-PA317) was prepared (32). Clones were selected by G418 (0.4 mg/ml, GibcoBRL, Grand Island, NY) and tested for FasL-mediated cytotoxicity. A clone expressing strong FasL- mediated cytotoxicity was expanded in 25 ml of culture medium and culture supernatant was collected 2 days after cells reached 75% confluence. Supernatant was centrifuged at 13 000 r.p.m. with a Sorvall RC-5B centrifuge for 30 min to remove cells and cell debris. Vesicle particles were prepared from the cell-free supernatant by ultracentrifugation at 25 000 r.p.m. for 4 h using a Beckman ultracentrifuge (model L5-m55, Beckman Coulter, Fullerton, CA) and SW25 rotor. The pellet (FasL VP) was suspended with 0.5 ml of culture medium and sterilized through a 0.45 µm filter for use in cytotoxicity assays. The concentration of FasL was determined to be about 1.5 µg/ml using a capture ELISA kit (Oncogen, Boston, MA). The same procedure was carried out using culture supernatant of PA317 cell line carrying the human ckrox gene (encoding a transcription factor) to prepare vesicles (designated Krox VP). Krox VP was used as a control (32).

Cytotoxicity assays
Single cell suspensions were prepared from mouse lymph nodes, spleens and thymi. Cells were mildly labeled with Na251CrO4 (New England Nuclear, Boston, MA) and used as targets in a 6 h cytotoxicity assay. Various doses of Jo2 mAb or FasL VP plus polybrene (Sigma Co., St Louis, MO; 4 µg/ml) were cultured with 2 x 105 target cells in a 96-well plate. Culture supernatants were collected after 6 h and released radioactivity was measured with a {gamma}-scintillation counter (LKB, Turku, Finland). Cytotoxicity was calculated according to the formula: (c.p.m. release of sample – background c.p.m. release)/(total c.p.m. release – background c.p.m. release) x 100%. Background c.p.m. release was determined by culturing target cells in medium only. Total c.p.m. release was determined by the addition of 0.2% NP-40 at the beginning of culture. Cytotoxicity was also conducted with unlabeled lymphocytes. Target cells (6 x 106) were cultured with various reagents for 6 h in a 6-well plate. Viable cells were purified by Ficoll-Hypaque (Pharmacia Biotech AB, Uppsala, Sweden) gradient centrifugation, washed, counted and used for surface phenotype determination by flow cytometric analysis.

Enrichment of the abnormal DN T cells and CD8+ T cells
Lymphocytes prepared from gld lymph nodes were depleted of single positive T cells and B cells by treatment with a mixture of GK.1.5 anti-CD4 mAb, 53-6.7 anti-CD8 mAb and J11D anti-CD24 mAb at 37°C for 30 min, followed by the addition of rabbit complement (37). This treatment yields a viable cell population consisting of >90% abnormal DN T cells as assessed by flow cytometry. To purify CD8+ T cells, lymphocytes prepared from gld lymph nodes were incubated with anti-CD8 MicroBeads (Miltenyi Biotec, Inc., Auburn, CA) at 4°C for 15 min and then washed twice with phosphate-buffered saline, pH 7.2. CD8+ cells were separated with a positive selection column according to the manufacturer’s protocol.

RNA extraction and RT–PCR
Total RNA of lymphocyte preparations was extracted with TRIzol Reagent (Gibco BRL). RNA preparations (4 µg) were used as templates for cDNA synthesis in a reaction mixture containing 150 ng of oligo(dT) primer and 50 units of M-MuLV reverse transcriptase (New England BioLabs, Inc., Beverly, MA) in 25 µl of reaction mixture. Aliquots of 3 µl were then diluted in 50 µl of PCR buffer containing 2 mM MgCl2, 2.5 units of Taq polymerase (Promega, Madison, WI), and 20 nmol of primers. All primers were synthesized by Integrated DNA Technology Inc. (Coraville, IA). The primers used in this study are as follows: Fas primers (forward, 5'-ATCCGAGCTAGGAGGCGGTTCATGAAAAC-3'; reverse, 5'-GGAGGTTCTAGATTCAGGGTCATCCTG-3'), FasL primers (forward, 5'-CAGCTCTTCCACCTGCAGAAGG-3'; reverse, 5'-AGATTCCTCAAAATTGATCAGAGAGAG-3'), ß-actin primers (forward, 5'-GTGGGCCGCTCTAGGCACCAA-3'; reverse, 5'-CTCTTTGATGTCACGCACGATTTC-3') (6), FLIP primers (forward, 5'-GTCACATGACATAACCCAGATTGT-3'; reverse, 5'-GTACAGACTGCTCTCCCAAGCACT-3') (38), and Bcl-xL primers (forward, 5'-GTGGAAGAGAACGGGGCTGAGG-3'; reverse, 5'-ATGTGGTGGAGCAGAGAAGG-3') (39).

The conditions for PCR were 1 min at 94°C, 1 min at 60°C and 1 min at 72°C for 30 cycles. The samples were then resolved by electrophoresis through a 2% agarose gel, stained with ethidium bromide and visualized under UV light illumination.

Flow cytometry
Lymphocytes (106) were stained with appropriate combinations of fluorescent antibodies (detailed in figure legends), and analyzed using a FACScan equipped with CellQuest analysis software (Becton Dickinson, Mountain View, CA). All fluorescent antibodies were purchased from BD PharMingen, San Diego, CA.

Western blots
Cell lysates were prepared from purified CD8+ T cells and abnormal DN T cells. Western blotting using antibodies against FLIPL (H-150, Santa Cruz Biotechnology Inc., Santa Cruz, CA) was conducted to determine FLIPL expression (40). Western blotting for ß-actin was included as an internal control using antibodies against ß-actin (AC-15, Sigma Co.).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Induction of acute apoptosis of lymphocytes of normal mice
We have recently shown that FasL VP is a powerful cytotoxic agent and its apoptosis-inducing ability is enhanced further by the presence of polybrene (3235). In the presence of polybrene, even FasL VP-resistant targets become sensitive (32,33). We first compared Jo2, a well characterized and commonly used anti-Fas mAb, with FasL VP plus polybrene for the ability to kill lymphocytes freshly obtained from the lymph nodes, spleens and thymi of 6-week old B6 and B6.lpr mice. Freshly obtained lymphocytes were 51Cr-labeled and used as targets in a 6 h cytotoxicity assay. A remarkable contrast of strength in the induction of apoptosis was observed (Fig. 1). While Jo2 was relatively ineffective even at a concentration as high as 10 µg/ml, a mixture of FasL VP (containing as little as 30 ng/ml of FasL protein) plus polybrene was able to kill a significant portion of the freshly isolated lymphocytes.



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Fig. 1. FasL VP but not Jo2 anti-Fas mAb kill lymphocytes freshly isolated from normal mice. Lymphocytes were isolated from thymi (Thy), lymph nodes (LN) and spleens (SP) of B6 or B6.lpr mice. These cells were mildly labeled with 51Cr, and 2 x 105 cells were mixed with various amounts of FasL VP (stock solution contains ~1.5 µg/ml of FasL as determined by FasL-specific ELISA) or Krox VP in the presence of 4 µg/ml of polybrene in a 96-well plate. The ability of various amounts of Jo2 anti-Fas mAb (stock solution contains 40 µg/ml in culture medium) to kill labeled targets was also determined. Cell death was determined after incubation for 6 h as described in Methods. Background release was <35% in all cases. Similar results were obtained in three independent experiments.

 
Lymphocytes from the thymi, lymph nodes and spleens from normal mice were all sensitive to FasL VP. It appears that thymocytes were the most sensitive, followed by lymph node lymphocytes, and then by splenic lymphocytes. The killing was specific because lymphocytes from the thymi, lymph nodes and spleens from B6.lpr mice were resistant to FasL VP under the same conditions. Moreover, Krox VP, a vesicle preparation that does not contain FasL (32), did not kill lymphocytes from B6 mice under identical conditions (Fig. 1).

Lymphocytes from old gld lymph nodes are highly sensitive to FasL VP
As the autoimmune disease in MRL.gld mice progresses, the proportion of the abnormal DN T cells increases. In addition, they accumulate primarily in the lymph nodes, with modest numbers accumulating in the spleens and none appearing in the thymi (41). To determine whether abnormal DN T cells are more sensitive to FasL-induced apoptosis, lymphocytes from MRL.gld mice of various ages were examined for their susceptibility to FasL VP (Fig. 2).



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Fig. 2. Change in sensitivity to FasL-induced apoptosis in lymphocytes from various tissues as gld mice age. Lymphocytes were isolated from thymi (Thy), lymph nodes (LN) and spleens (SP) of 6-, 16- or 24-week old MRL.gld mice. Cytotoxicity assays were carried out as described in Fig. 1. Background release was <35% in all cases. Similar results were obtained in five independent experiments.

 
Lymphocytes obtained from 6-, 16- and 24-week old MRL.gld mice were compared. Like lymphocytes from normal mice, lymphocytes from 6-week old MRL.gld mice were sensitive to FasL VP; with thymocytes being the most sensitive, followed by lymph node cells and then splenic lymphocytes. In contrast, lymph node lymphocytes from 16- and 24-week old MRL.gld mice displayed increased susceptibility to FasL VP as evidenced by a progressive increase in the percentage of specific Cr-release. By contrast, there was a small increase in sensitivity to FasL VP for splenic lymphocytes of both the 16- and 24-week old mice while the sensitivity of thymocytes to FasL VP was not increased. It is known that the abnormal DN T cells accumulate primarily in the lymph nodes and, to a lesser extent, in the spleen while there is little accumulation in the thymus (41). This correlation suggests abnormal DN T cells are highly sensitive to FasL VP and are more so than thymocytes.

Abnormal DN T cells are preferentially eliminated by FasL VP
We treated 16-week old MRL.gld lymphocytes obtained from lymph nodes, spleen and thymi with FasL VP, and determined by flow cytometric analysis which subset was preferentially eliminated. After 6 h of culture, viable cells of various treatment groups were purified and analyzed with a mixture of FITC anti-Thy-1 mAb and PE anti-B220 mAb. Untreated or cells treated with Krox VP were used as controls (Fig. 3). Following treatment with Krox VP, abnormal DN T cells, as determined by their co-expression of Thy-1 and B220, remained as the major lymphocyte population in the lymph nodes. The same result was observed with untreated control groups (data not shown). In contrast, nearly all of the abnormal DN T cells, which represented ~70% of the original untreated population, were eliminated by the treatment with FasL VP. It should be emphasized that the preferential killing of these abnormal DN T cells is remarkably effective in view of the fact that this population represented >70% of the lymphocytes in the lymph nodes and that >80% of them were killed. The data indicate that abnormal DN T cells are the most sensitive targets to FasL VP. This population, which represents ~16% of the splenic lymphocytes, was also preferentially eliminated by FasL VP, but remained unchanged following incubation with Krox VP. The preferential elimination of abnormal DN T cells in the lymphocytes of lymph nodes and spleen contrasts with results obtained using thymocytes. Consistent with their virtual lack of abnormal DN T cells, FasL VP did not cause a significant change in staining pattern.



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Fig. 3. FasL VP preferentially eliminates abnormal DN T cells. Lymphocytes were obtained from lymph nodes, spleens and thymi of individual 16-week old MRL.gld mice. Lymphocytes (6 x 106) were cultured with 1 ml of FasL VP or Krox VP in the presence of 4 µg/ml of polybrene for 6 h in 4 ml in wells of a 6-well plate. Viable cells were recovered on a Ficoll–Hypaque gradient, washed once and stained with FITC anti-Thy1 (FL1 height) plus PE anti-B220 (FL2 height) mAb. Killing of lymphocytes was similar to that described in Fig. 2. Despite significant killing within each population being noted with FasL VP, only the elimination of the abnormal DN T cells in a preferential and remarkable manner was observed. Similar results were obtained in two other experiments.

 
Abnormal DN T cells of old MRL.gld mice express an increased level of Fas and a decreased level of FLIPL
It is possible that the increased sensitivity of the abnormal DN T cells to FasL VP is the result of an increased Fas expression and/or a decreased expression of anti-apoptotic molecules during the differentiation process. Therefore, we compared the expression levels of several critical components of the Fas-mediated apoptosis pathway in MRL.gld CD8+ T cells, the precursors of abnormal DN T cells, and in the abnormal DN T cells themselves. The CD8+ T cells and the abnormal DN T cells were enriched from the lymph nodes of 16-week old MRL.gld mice as described in the Methods. Staining of the viable cells using PE anti-B220 mAb and FITC anti-Thy-1 mAb indicated that >90% of the enriched population expressed the abnormal Thy-1+B220+ phenotype. Staining of the purified T cells using PE anti-CD8 mAb and FITC anti-CD4 mAb indicated that >90% of the purified lymphocytes were CD8+CD4 T cells. First, we compared between the mRNA levels of Fas, FasL, Bcl-xL and FLIP by RT–PCR in abnormal DN T cells and CD8+ T cells. We observed that abnormal DN T cells expressed an increased level of Fas and FasL mRNA (gld mice express a mutated and nonfunctional FasL), a decreased expression of FLIP mRNA, and little change in the expression of Bcl-xL (Fig. 4a). The increased expression of Fas protein on the abnormal DN T cells was demonstrated by fluorescence staining using PE-Jo2 mAb. The increase in staining was observed specifically with Jo2 mAb and not with an isotype control (Fig. 4b). The decreased expression of FLIPL in the abnormal DN T cells was confirmed by western blotting of cell lysates. While there is a strong reduction of FLIPL in the abnormal DN T cells, their ß-actin level was comparable to that of CD8+ T cells (Fig. 4c). These data are consistent with the interpretation that the preferential killing of the abnormal DN T cells by FasL VP is the result of an increase in cell surface Fas and the concomitant decrease of the cytoplasmic anti-apoptotic protein FLIPL.



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Fig. 4. Comparing the expression levels of Fas and FLIPL between the abnormal DN T cells and CD8+ T cells from old MRL.gld mice. The CD8+ T cells and the abnormal DN T cells were purified from 16-week old MRL.gld mice and analyzed: (a) RT-PCR analysis of Fas, FasL, Bcl-xL, FLIP and ß-actin; (b) Fluorescence staining for surface expression of Fas using PE-Jo2 mAb (dark line and dark area) or PE-hamster isotype control mAb (dashed lines); (c) western blot analysis of FLIPL and ß-actin in cell lysate.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Tolerance to self-antigens is controlled by a central mechanism that deletes high affinity auto-reactive T cells during lymphocyte differentiation in the thymus and by peripheral mechanisms that contain the development of auto-reactive T cells in the peripheral environment. Thymocytes may escape negative selection but still become auto-reactive in the periphery due to changes either in their activation threshold or in the presentation of auto-antigens. Pestano et al. (30) have identified ‘mis-selected’ CD8+ T cells as one class of such cells. Studies using gld mice have demonstrated that in the absence of FasL-induced apoptosis, these ‘mis-selected’ CD8+ T cells lose CD8 expression, gain B220 expression, and accumulate in the periphery. These data imply that abnormal DN T cells that accumulate in gld mice are derived from ‘mis-selected’ CD8+ T cells and that these ‘mis-selected’ CD8+ T cells are deleted in the periphery by a Fas/FasL-mediated mechanism in normal mice.

The present study provides direct evidence that abnormal DN T cells are the most sensitive lymphocyte population to FasL-induced apoptosis. The induction of acute, Fas- mediated apoptosis in primary lymphocytes in vitro is difficult in part due to their relatively low Fas expression and a lack of cross-linking reagents that are capable of delivering a strong Fas-mediated apoptotic signal. Using the FasL VP that we have recently developed (3236), we were able to induce significant levels of apoptosis of freshly isolated mouse lymphocytes within 6 h. We found that, as autoimmune disease progresses with age, the peripheral lymphocytes become appreciably more sensitive to FasL VP. This increase in sensitivity to FasL VP correlates with the age-dependent accumulation and tissue distribution of the abnormal DN T cells. Indeed, flow cytometric analyses demonstrated that abnormal DN T cells are the most sensitive population in gld lymph nodes and spleens. It is important to note that gld lymphocytes lack functional FasL. Therefore, the increased sensitivity of lymphocytes from old gld mice is entirely attributed to the apoptosis induced by FasL VP and not by any FasL associated with target lymphocytes that might have been activated in the autoimmune condition.

To determine why abnormal DN T cells are exquisitely sensitive to FasL-induced apoptosis, we compared the expression of Fas, FLIPL and Bcl-xL in CD8+ lymphocytes and abnormal DN T cells. We found that the high sensitivity of the abnormal DN T cells to FasL VP correlated with both an increase of cell surface Fas expression and a decrease of cytosolic FLIPL protein. It is known that membrane Fas expression levels do not necessarily correlate with sensitivity to FasL-induced apoptosis and that the intracellular component, particularly FLIP, is a critical negative regulator of the Fas-mediated apoptotic pathway (42,43). The simultaneous up-regulation of Fas and down-regulation of FLIP strongly suggests that a concomitant regulation of Fas and FLIPL expression is the reason for the exquisite sensitivity of abnormal DN T cells to FasL-induced apoptosis.

Our results raise the interesting question as to how the change of Fas and FLIPL expression is regulated in the abnormal DN T cells that do not respond to either TCR cross-linking or exogenous IL-2. It has been shown that ‘mis-selected’ CD8+ T cells must undergo activation and several rounds of proliferation before differentiating into abnormal DN T cells (30). Indeed, ‘mis-selected’ CD8+ T cells that exit the thymus were found to express B220 and IL-2 receptor (44). Therefore, it is possible that fas gene expression is increased prior to the transition from the activated and IL-2-responsive CD8+B220+ T cell stages to the abnormal DN T cell stages. We have reported that there is a high level of Sp1 and a moderate increase in NF-{kappa}B levels in the nuclei of the abnormal DN T cells compared to that seen in lymphocytes from normal mice (37). It is possible that both Sp1 and NF-{kappa}B are responsible for the increase of cell surface Fas level by increasing fas gene activation. This is supported by a study demonstrating that Sp1 is responsible for basic fas gene transcriptional activity and that NF-{kappa}B is required for activation-dependent fas transcriptional up-regulation (45). Indeed, the level of fas mRNA in abnormal DN T cells is higher than that in CD8+ T lymphocytes (Fig. 4a). Also, we have reported that Sp1 and NF-{kappa}B were up-regulated in recently activated T cells treated with IL-2 and that the expression pattern of the two transcriptional factors is similar to that present in abnormal DN T cells (37,46).

IL-2 treatment has been shown to increase surface Fas expression in T cells and to facilitate Fas-mediated apoptosis in T cells (47,48). These observations are consistent with the interpretation that an IL-2-mediated expansion is involved in the development of the abnormal DN T cells following the activation of ‘mis-selected’ CD8+ T cells. In addition to the increase in Fas and FasL expression, we also observed that FLIPL protein levels were significantly decreased in abnormal DN T cells. It has been reported that IL-2 increases FasL transcription/expression and suppresses FLIPL transcription/expression, two of several critical components that regulate T cell apoptosis (38). The fact that the abnormal DN T cells have increased Fas/FasL and decreased FLIPL further supports the concept that IL-2 is involved in the transition from activated CD8+ T cells to abnormal DN T cells. In addition, CD8+B220+IL-2R+ cells have been identified. These cells represent ‘mis-selected’ T cells that are activated in the periphery before the transition into the pool of abnormal DN T cells (44). We have introduced an Il2 null gene into lpr mice and observed a dramatic reduction of abnormal DN T cells in these double mutant mice (49). Because these mice lack a Fas/FasL-mediated apoptosis pathway, this clearly demonstrates that IL-2 is involved in the development/expansion of the abnormal DN T cells. In this regard, it is interesting to note that while abnormal DN T cells are not responsive to IL-2, the expression pattern of Fas, FasL and FLIPL is similar to that in Con A-activated T cells that had been further treated with IL-2.

Because ‘mis-selected’ CD8+ T cells are activated by self-antigens without engagement of CD8 co-receptor, they are rapidly eliminated in normal mice (30). These abnormal DN T cells are not observed in other lupus-prone autoimmune mice that do not have a defect in the fas or fasl gene. The ‘mis-selected’ CD8+ T cells survive in gld and lpr mice but they rapidly differentiate into the abnormal DN T cells that do not respond to stimulus mediated through TCR or IL-2. Therefore, it is unlikely that the abnormal DN T cells are directly responsible for the autoimmune disease in these mice. Although the abnormal DN T cells strongly express FasL (31), it is not functional in gld mice and it does not have a target in lpr mice, consistent with the idea that it is not critical for autoimmune disease development. These considerations suggest that there is a distinct functional difference between conventional auto-reactive T cells and the ‘mis-selected’ abnormal DN T cells. Despite the fact that ‘mis-selected’ CD8+ T cells do not contribute to autoimmunity, they can be viewed as auto-reactive because they are activated by self-antigens, albeit without CD8 engagement, in the periphery in hosts that are not deliberately immunized (30). Based on the studies of Pestano et al. (30) and Trimble et al. (44), these ‘mis-selected’ T cells are deleted by Fas/FasL-mediated ‘activation-induced cell death’ (AICD). Our in vitro study provides a mechanistic explanation for the preferential deletion of these cells based on AICD.

Because AICD is a critical mechanism for the deletion of conventional auto-reactive T cells, the question of whether a similar or identical sensitization mechanism is induced in conventional auto-reactive T cells can be raised. It is important to emphasize that conventional auto-reactive T cells, unlike the ‘mis-selected’ CD8+ T cells, use CD4 or CD8 as co-receptor for activation. Furthermore, there is no specific surface marker for these auto-reactive T cells, prohibiting a parallel comparison with the ‘mis-selected CD8+ T cells’ in old gld mice. Our work with the abnormal DN T cells, however, suggests that FasL VP can be used to answer this important question once a specific surface marker for auto-reactive T cells in gld mice is identified.


    Acknowledgements
 
This work is supported by NIH grant AI-36938 (S.T.J.), AR-35230 (A.M.S.), ES-06086 (D.H.S.), and Lupus Foundation of America (S.X.). We thank Mr Derek V. Chan for scientific and editorial comments.


    Abbreviations
 
DN T—Double negative T

FADD—Fas-associated death domain

FasL—CD178 or Fas ligand

FasL VP—FasL vesicle preparation

FLIP—FLICE/caspase-8 inhibitory protein


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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