Soluble human MHC class I molecules induce soluble Fas ligand secretion and trigger apoptosis in activated CD8+ Fas (CD95)+ T lymphocytes
Francesco Puppo,
Paola Contini,
Massimo Ghio,
Sabrina Brenci,
Marco Scudeletti,
Gilberto Filaci,
Soldano Ferrone1 and
Francesco Indiveri
Department of Internal Medicine, University of Genoa, Viale Benedetto XV no. 6, 16132 Genoa, Italy
1 Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
Correspondence to:
F. Puppo
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Abstract
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In the present study, we have evaluated the apoptotic effect of soluble human MHC class I (sHLA-I) antigens on CD8+ T lymphocytes. sHLA-I antigens and ß2-microglobulin-free HLA class I heavy chains, isolated from serum, induced apoptosis on phytohemagglutinin-activated CD8+ T lymphocytes in autologous and allogeneic combinations. The extent of CD8+ T cell apoptosis depends on the degree of activation, time of incubation with sHLA-I antigens and amount of sHLA-I antigens added to the cultures. Apoptosis is induced by the interaction of Fas (CD95)+ cells with soluble Fas ligand which is released following binding of sHLA-I antigens to CD8 molecules. These results suggest that sHLA-I antigens may regulate immune responses by inducing apoptosis in activated CD8+ T cells.
Keywords: HLA, immunomodulation
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Introduction
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It has been known for some time that besides being expressed on nucleated cells (1) immunologically functional ß2-microglobulin (ß2m)-associated HLA class I heavy chains (sHLA-I) are present in serum (24). More recently the presence of ß2m-free HLA class I heavy chains (free heavy chains) in serum has also been described (5,6). The level of ß2m-associated as well as -free heavy chains is significantly increased in serum of patients with an activation of their immune system such as those suffering from an acute rejection episode following organ allografts, acute graft versus host disease following bone marrow transplantation, autoimmune diseases or viral infections (7 and personal data). Because of the statistically significant association with clinical parameters, the level of sHLA-I antigens has been suggested to represent a useful marker to predict the evolution of HIV infections and to monitor the clinical course of allografts (8,9). The potential involvement of sHLA-I antigens in the outcome of allografts is suggested by at least three lines of evidence. The early findings by van Rood and his collaborators that skin allograft survival is prolonged following plasma administration implies that sHLA-I antigens may have a tolerogenic effect (10). More recently, sHLA-I antigens have been found to be immunogenic in allogeneic combinations (11). Lastly, sHLA-I antigens have been shown to inhibit the cytotoxicity of alloreactive cytotoxic T lymphocytes (1215) and to induce apoptosis of primary alloreactive CD8+ T cells through selective stimulation of the TCR (16).
Whether sHLA-I antigens and the increase in their serum level in patients with activation of their immune system play a role in the regulation of the immune response in autologous combinations is not known. In the present study, we have addressed these questions by investigating the interaction of sHLA-I antigens and free heavy chains with T cells, and the functional consequences of these interactions.
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Methods
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mAb and conventional antisera
The anti-ß2m-associated HLA class I heavy chains
3 domain mAb W6/32, the anti-ß2m-associated and anti-ß2m-free HLA class I heavy chains
3 domain mAb TP25.99, the anti-HLA-A3
1 domain mAb LGIII-220.6, the anti-HLA-A3
2 domain mAb TP11, the anti-ß2m-free HLA class I heavy chains mAb HC-10, the anti-ß2m mAb NAMB-1, the anti-HLA class II mAb LGII-612.14, the anti-ICAM-1 mAb VF27-516.1, and the anti-human Fas ligand (FasL) neutralizing mAb 4H9 were developed and purified as described (1724 and unpublished results). The anti-CD3 mAb OKT3, the anti-CD4 mAb OKT4 and the anti-CD8
chain mAb OKT8 were purchased from Ortho (Milan, Italy). The anti-human TCR mAb TCR-1
/ß was purchased from Becton Dickinson (San Jose, CA). The anti-CD8 ß chain mAb CD8ß and the anti-human Fas mAb UB2 were purchased from Immunotech (Marseilles, France). The anti-human Fas mAb CH11 and ZB4 were purchased from Kamiya (Thousand Oaks, CA). The mAb NOK-1 and NOK-2 to different epitopes of human soluble FasL were purchased from PharMingen (San Diego, CA). mAb were labeled with 125I utilizing the chloramine T method (25) or conjugated to biotin (Pierce, Rockford, IL) according to the manufacturer's procedure. FITC-conjugated goat anti-mouse Ig antibodies (GAMFITC) were purchased from Coulter (Hialeah, FL). Annexin-Vbiotin and streptavidinR-phycoerythrin were purchased from Boehringer Mannheim (Monza, Italy).
Cells
Human peripheral blood mononuclear cells were obtained from healthy donors as described (26). CD8+ T and CD4+ T cells were purified by negative selection on anti-CD4 and anti-CD8 mAb-coated magnetic beads (Dynal, Oslo, Norway) respectively. Purity of CD4+ and CD8+ T cell preparations as assessed by flow cytometric analysis was at least 90%. CD8+ and CD4+ T cells were activated with phytohemagglutinin (PHA) (10 µg/ml) in RPMI 1640 medium (Gibco/BRL, Gaithersburg, MD) supplemented with 10% FBS (Gibco/BRL) for up to 72 h at 37°C in a 5% CO2 atmosphere. Cultured human B lymphoid cells HUT 78 and human T lymphoid cells Jurkat (CD2+, CD3+, CD8, CD95+, HLA class I ) were grown in RPMI 1640/FBS 10% medium at 37°C in a 5% CO2 atmosphere.
Antigen preparations
sHLA-I molecules were purified from serum of healthy subjects by sequential precipitation with ammonium sulfate (60, 40 and 20%). Supernatant of the 20% fraction was sequentially applied in a low/medium-pressure chromatography system (BioLogic; BioRad, Milan, Italy) to strong anionic ion exchange Sourse 30 Q, strong cationic ion exchange Sourse 30 S and gel filtration HiPrep Sephacryl S-200 columns (Pharmacia, Uppsala, Sweden), and then purified by affinity chromatography on anti-HLA class I mAb W6/32 (10 mg/ml) coupled to cyanogen bromide-activated Sepharose 4B (Pharmacia). Free heavy chains were eluted from sHLA-I molecules with acetic acid (0.1 N, pH 2) for 5 min at room temperature (27). The solution was then neutralized with 1 M Tris buffer, pH 11.0 (Sigma, Milan, Italy). Following four sequential 4 h incubations at room temperature with anti-ß2m mAb NAMB-1 coupled to cyanogen bromide-activated Sepharose 4B to remove free ß2m, the antigen preparation was dialyzed against PBS (pH 7.4) overnight at 4°C. The purity of sHLA-I molecules and free heavy chains preparations was analyzed by one-dimensional PAGE under non-reducing/non-denaturing or reducing/denaturing conditions followed by silver staining or immunoblotting with 125I-labeled anti-HLA class I mAb TP25.99, as described (25).
Soluble HLA class II antigens were purified from HUT 78 cells by affinity chromatography on anti-HLA class II mAb LGII-612.14, as described (28). Soluble CD8 antigen was purchased from T Cell Diagnostics (Cambridge, MA). Soluble human recombinant FasL was purchased from Alexis (Läufelfingen, Switzerland).
Immunoassays
The double-determinant immune assay (DDIA) was performed as described (29) utilizing anti-HLA class I mAb W6/32 and biotinylated anti-ß2m mAb NAMB-1 to measure sHLA-I antigens, anti-HLA class I heavy chains mAb TP25.99 and biotinylated anti-free heavy chains mAb HC-10 to measure free heavy chains, and anti-sFasL mAb NOK-2 and biotinylated anti-sFasL mAb NOK-1 to measure sFasL. The binding of sHLA-I molecules to CD8 was assessed as follows. Polyvinylchloride 96-well plates (Becton Dickinson, Oxnard, CA) were coated overnight at 4°C with anti-CD8 ß chain mAb CD8ß (15 µg/ml) in bicarbonate buffer (0.01 M, pH 9.5), washed with PBS/0.2% Tween 20 and blocked with PBS/5% BSA. Soluble CD8 molecules (500 U/ml), biotinylated or non-biotinylated anti-CD8
chain mAb OKT8 (5 µg/ml), sHLA-I molecules (4 µg/ml) and biotinylated anti-ß2m mAb NAMB-1 (5 µg/ml) were sequentially added in 100 µl volume to microtiter plates for 1 h at 37°C. After washings with PBS/0.2% Tween 20, streptavidinHPC (Pierce, Rockford, IL) was added for 1 h at 37°C and the reaction was developed for 15 min at room temperature in the dark with o-phenylenediamine (40 µg/ml) in phosphate citrate buffer (pH 5.0) supplemented with 0.04% (v/v) H2O2 30%. The optical density (OD) was read with a spectrophotometer at 490 nm against reagent blank (PBS/5% BSA). Results were expressed as mean ± SD of triplicate wells.
Indirect immunofluorescence (IIF) was performed by incubating 5x105 cells sequentially with mAb and with GAMFITC. Each incubation was for 30 min at 4°C. Following three washings, cells were analyzed on an Epics Elite flow cytometer (Coulter). Results are expressed as fluorescence intensity.
Induction and detection of apoptosis
PHA-activated T cells (106/ml) or Jurkat cells that are susceptible to FasL-mediated apoptosis were washed and then cultured in 96-well U-bottomed plates (Becton Dickinson) with RPMI 1640/ 10% FBS culture medium alone or with culture medium containing apoptosis inducing stimuli for up to 72 h at 37°C in a 5% CO2 atmosphere. After 48 h of incubation, 5x105 cells were washed and early apoptotic events were evaluated by Annexin-V labeling method according to the manufacturer's protocol. Viable apoptotic cells were differentiated from dead cells by flow cytometry after propidium iodide (PI) staining of non-permeabilized cells. At the end of incubation, 1x106 cells were washed and analyzed on a Epics Elite flow cytometer (Coulter) after permeabilization and PI staining, by DNA gel electrophoresis and by electron microscopy utilizing a Siemens Elmiskop 101 transmission electron microscope (Siemens, Iselin, NJ), as described (3032).
Isolation of RNA and reverse transcription
Total RNA was isolated from cell pellets by using the RNAzol B (Biotecx, Houston, TX) method according to the manufacturer's protocol. cDNA (corresponding to 2 µg of RNA) was synthesized from oligo(dT)-primed RNA in 20 µl reverse transcriptase buffer and 200 U MMLV reverse transcriptase (Perkin-Elmer Cetus, Emeryville, CA) incubated at 42°C for 45 min and at 52°C for 45 min.
PCR amplification
The PCR mixture contained 2 µl of cDNA, 2.5 mM MgCl2, 2 mM dNTP, 50 µM 5' and 3' oligonucleotide primers, and 2.5 U Amply Taq Gold Polymerase (Perkin-Elmer Cetus). The PCR mixture was amplified by 35 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 30 s and extension at 72°C for 30 s in 0.2 ml thin-walled tubes in a total volume of 50 µl. Primer sequences used were: ß-actin 5'-GTGGGG- CGCCCCAGGCACCA, ß-actin 3'-CTCCTTAATGTCACGCACGATTTC (548 bp fragment); Fas 5'-ATGCTGGGCATCT- GGACCCT, Fas 3'-GCCATGTCCTTCATCACACAA (335 bp fragment); and FasL 5'-CAAGTCCAACTCAAGGTCCATGCC, FasL 3'-CAGAGAGAGCTCAGATACGTTGAC (350 bp fragment) (33,34). PCR products were size-fractionated by agarose electrophoresis and normalized according to the amount of ß-actin detected in the same mRNA sample.
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Results
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Immunochemical profile of sHLA-I molecules
Silver staining detected a 56 kDa band when sHLA-I molecules isolated from serum were electrophoresed under nonreducing/non-denaturing conditions (Fig. 1
, lane 1). This band contains HLA class I heavy chains and ß2m, since it reacts with anti-HLA class I mAb TP25.99 in immunoblotting (Fig. 1
, lane 2) and with anti-ß2m mAb NAMB-1 in the DDIA (data not shown). Immunoblotting with mAb TP25.99 of sHLA-I molecules electrophoresed under reducing/denaturing conditions detected the known isoforms of serum HLA class I antigens with a mol. wt of 44, 39 and 35 kDa (35) (Fig. 1
, lane 3). Similar results were obtained when the free heavy chains isolated from the HLA class I molecular complex by dissociation of ß2m were analyzed (Fig. 1
, lane 4). The free heavy chains reacted in the DDIA performed with mAb TP25.99 and mAb HC-10 which recognize determinants expressed on free heavy chains but did not react with anti-ß2m mAb NAMB-1 (data not shown).

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Fig. 1. Immunochemical profile of sHLA-I molecules. sHLA-I molecules purified from serum and separated by PAGE under non-reducing/non-denaturing (lanes 1 and 2) and reducing/denaturing (lane 3) conditions followed by silver staining (lane 1) or immunoblotting with anti-HLA class I mAb TP25.99 (lanes 2 and 3) are shown. Free heavy chains obtained by dissociation of ß2m from sHLA-I molecules and separated by PAGE under non-reducing/non-denaturing conditions followed by silver staining are also shown (lane 4).
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Induction by sHLA-I molecules of apoptosis in PHA-activated CD8+ T lymphocytes
Incubation for up to 72 h of sHLA-I molecules with autologous PHA-activated CD8+ T cells induced apoptosis in >90% of cells as indicated by Annexin-V labeling (Fig. 2
A) and by staining with the DNA intercalating dye PI (data not shown). Apoptosis of target cells was confirmed by the laddered pattern of DNA gel electrophoresis (Fig. 2C
, lane 2) and by cell morphology (data not shown). Similar results were obtained when PHA-activated CD8+ T cells were incubated for 72 h with allogeneic sHLA-I molecules or with free heavy chains (Fig. 2B
). The apoptotic effect of sHLA-I molecules is specific for the CD8+ T cell subset since PHA-activated CD4+ T lymphocytes did not undergo apoptosis when cultured with sHLA-I molecules as indicated by DNA gel electrophoresis (Fig. 2C
, lane 4) and by PI staining (data not shown). The number of CD8+ T cells undergoing apoptosis is influenced by the extent of PHA activation of T cells, by the incubation time with sHLA-I molecules and by the concentration of sHLA-I molecules (Fig. 3A and B
). The apoptosis-inducing activity of sHLA-I molecules is higher than that of well-known apoptotic stimuli such as methylprednisolone, anti-CD3 mAb and anti-TCR mAb (Fig. 3C
). Soluble HLA class II antigens, anti-HLA class I mAb, anti-CD8 mAb and anti-ICAM-1 mAb did not induce apoptosis in PHA-activated CD8+ T cells (Fig. 3C
). The apoptotic effect of sHLA-I antigens was inhibited by their preincubation with an anti-HLA class I
3 domain mAb and with soluble CD8 molecules as well as by preincubation of target cells with an anti-CD8
chain mAb (Table 1
). Apoptosis was unaffected by anti-CD8 mAb if added to cells preincubated for 1 h with sHLA-I molecules (data not shown). These results suggest that apoptosis is triggered by the interaction between the sHLA-I
3 domain and CD8
chain. This possibility is also supported by the binding of sHLA-I antigens to CD8 molecules which have been immobilized to a solid support by an anti-CD8 ß chain mAb and by the blocking of this binding by an anti-CD8
chain mAb (Table 2
).

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Fig. 2. Detection of apoptosis induced by sHLA-I molecules in PHA-activated CD8+ T cells. (A) Flow cytometric analysis after Annexin-V labeling of PHA-activated CD8+ T cells following incubation with culture medium or with sHLA-I molecules (4µg/ml) for 48h (a and b respectively). (B) Percentage of PHA-activated CD8+ T cells that undergo apoptosis following incubation with autologous sHLA-I molecules, allogeneic sHLA-I molecules and free heavy chains for 72 h. (C) DNA gel electrophoresis of PHA-activated CD8+ and CD4+ T cells following incubation with culture medium (lanes 1 and 3 respectively) or with sHLA-I molecules (lanes 2 and 4 respectively) for 72 h.
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We also investigated whether a sHLA-ITCR interaction might contribute to the apoptotic effect of sHLA-I molecules. To this end PHA-activated CD8+ T cells were preincubated with an anti-CD8
chain mAb before adding sHLA-I molecules. In these experimental conditions, in which the
1 and
2 domains of sHLA-I molecules can bind to the TCR, whereas the
3 domain cannot bind to the CD8
chain, sHLA-I molecules do not trigger apoptosis in target cells (Table 1
). In additional experiments the binding of the
1 and
2 domains of sHLA-I molecules to the TCR was selectively blocked. The apoptotic effect of sHLA-A3 molecules on HLA-A3 PHA-activated CD8+ T cells was unaffected by preincubation with an anti-HLA-A3
1 domain mAb and was partially inhibited by preincubation with an anti-HLA-A3
2 domain mAb (Table 1
). These findings suggest that, under our experimental conditions, sHLA-ITCR interaction in the absence of sHLA-ICD8 interaction is unable to induce apoptosis in PHA-activated CD8+ T cells.
Role of FasFasL interactions in the induction by sHLA-I molecules of apoptosis in CD8+ T cells
The role of FasFasL interactions in sHLA-I-induced apoptosis of PHA-activated CD8+ T cells was then investigated. The apoptosis-inducing activity of sHLA-I molecules was inhibited by preincubating with an anti-human Fas neutralizing mAb CD8+ T cells which had acquired Fas (CD95) following a 72 h PHA activation (Fig. 4
). Additional experiments determined whether the production of a soluble form of FasL (sFasL) (22,36) was involved in the apoptosis-inducing activity of sHLA-I molecules. Supernatants harvested from cultures of PHA-activated CD8+ T cells incubated with sHLA-I molecules for 48 h induced apoptosis in Fas+ Jurkat cells (Fig. 5
). The apoptosis-inducing activity was increased in supernatants incubated with an anti-human Fas neutralizing mAb before incubation with sHLA-I molecules. This finding reflects the inhibition of the binding of sFasL to PHA-activated Fas+ CD8+ T cells by anti-human Fas neutralizing mAb (Fig. 5
). The apoptosis-inducing activity of supernatants was abolished by preincubation with the an anti-human FasL mAb and by preincubation of Jurkat cells with an anti-human Fas neutralizing mAb (Fig. 5
). The presence of sFasL molecules in supernatants is further supported by its detection by DDIA (0.86 ± 0.24 µg/ml). Supernatants harvested from cultures of PHA-activated CD8+ T cells in which the binding of the sHLA-I
3 domain to CD8
chain was blocked by preincubation with the appropriate mAb did not contain apoptosis-inducing activity (data not shown). These findings indicate that, in our experimental conditions, sHLA-ITCR interaction in the absence of sHLA-ICD8 interaction does not induce sFasL secretion by PHA-activated CD8+ T cells. Moreover, Fas mRNA was detected in CD8+ T cells following PHA activation and FasL mRNA was detected in PHA-activated CD8+ T cells following incubation with sHLA-I molecules (Fig. 6
). Taken together, these findings strongly suggest that sFasL is produced after the binding of sHLA-I antigens to PHA-activated CD8+ T cells and mediates apoptosis in Fas+ CD8+ T cells.

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Fig. 4. Inhibition of sHLA-I-induced apoptosis by anti-Fas mAb. Detection by IIF with anti-human Fas mAb UB2 (2.5 µg/ml) of Fas expression on freshly isolated (solid line) and PHA-activated (dashed line) CD8+ T cells (a). Flow cytometric analysis after PI staining of PHA-activated CD8+ T cells following a 72 h incubation with culture medium (b) or sHLA-I molecules (4 µg/ml) (c). The apoptotic effect of sHLA-I molecules is blocked by preincubation of PHA-activated CD8+ T cells for 1 h at 37°C with anti-human Fas neutralizing mAb ZB4 (100 ng/ml) (d). Apoptotic cells with hypodiploid DNA are shown as a black area and the percentage of such cells is indicated.
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Fig. 5. Supernatants from cultures of PHA-activated CD8+ T cells incubated with sHLA-I molecules induce apoptosis in Fas+ Jurkat cells. Flow cytometric analysis after PI staining of Jurkat cells (1x105 cells) following a 72 h incubation with supernatants (100 µl/well) from cultures of: (a) PHA-activated CD8+ T cells, (c) PHA-activated CD8+ T cells incubated with sHLA-I molecules (4 µg/ml) for 48 h and (d) PHA-activated CD8+ T cells preincubated for 1 h at 37°C with anti-human Fas neutralizing mAb ZB4 (100 ng/ml) and then incubated with sHLA-I molecules (4 µg/ml) for 48 h. The apoptotic activity of the latter supernatant is blocked either by its preincubation for 1h at 4°C with anti-human FasL neutralizing mAb 4H9 (5µg/ml) (e) or by the preincubation for 1h at 37°C of Jurkat cells with anti-human Fas neutralizing mAb ZB4 (100 ng/ml) (f). The percentage of Jurkat cells that undergoes apoptosis following a 72 h incubation with anti-human Fas apoptosis inducing mAb CH11 (100 ng/ml) is shown as positive control (b). Apoptotic cells with hypodiploid DNA are shown as a black area and the percentage of such cells is indicated.
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Fig. 6. RT-PCR detection of Fas and FasL mRNA. Total cellular RNA was extracted, reverse transcribed and amplified with Fas and ß-actin primers (upper panel) or with FasL and ß-actin primers (lower panel) in the same tubes. RNA was isolated from resting CD8+ T cells (lane 3), PHA-activated CD8+ T cells (lane 5) and PHA-activated CD8+ T cells incubated with sHLA-I molecules (4 µg/ml) for 2 h (lane 4). RNA was also isolated from Jurkat cells and Jurkat cells activated with PMA (10 ng/ml) and ionomycin (500 ng/ml) for 4 h as positive control (lanes 1 and 2 respectively).
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Discussion
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Antigenic peptides are presented to the TCR in the context of the
1 and
2 domains of HLA class I antigens (37). During antigen presentation the
3 domain binds to the
chain of CD8 molecules (38). However, the
3 domain may also bind to CD8 without the simultaneous binding of the
1 and
2 domains to the TCR (39). To the best of our present knowledge, the possibility that sHLA-I antigens might bind to CD8 has never been investigated. The results of the present study indicate that sHLA-I antigens purified from serum interact through their
3 domain with the
chain of CD8 molecules and that this interaction triggers apoptosis in PHA-activated CD8+CD95+ T cells. The observation that apoptosis is induced by sHLA-I antigens in autologous and allogeneic combinations as well as by ß2m-free HLA class I heavy chains further supports the hypothesis that soluble HLA class I heavy chains interact with CD8 molecules through the
3 domain also after dissociation of ß2m. Moreover, apoptosis induction is inhibited by the preincubation of sHLA-I antigens with W6/32 mAb which blocks the binding between HLA class I
3 domain and CD8
chain (40) as well as by preincubation of PHA-activated CD8+ T cells with anti-CD8
chain mAb. By contrast, apoptosis is not inhibited by the preincubation of sHLA-I antigens with an anti-
1 domain mAb, whereas it is partially inhibited by the preincubation with an anti-
2 domain mAb. This latter finding suggests that the interaction between the
2 domain of HLA class I antigens and the
chain of CD8 molecules might be also involved in apoptosis induction. This hypothesis is in agreement with previously published data showing that the residues of the
2 domain which interact with the CD8
chain are located outside from the region of interaction of the TCR and point down towards a cavity composed of the
1
2 platform, the loops of the
3 domain and ß2m (41,42). The interaction between HLA class I antigens
3 domain and CD8
chain is required for the positive selection of CD8+ T cells within the thymus and for T cell activation which follows the trimolecular ligation of TCR, antigen and MHC (38,40,4347). Our data suggest that an apoptotic signal may be delivered to CD8+ T cells after CD8 ligation by sHLA-I antigens. The cytoplasmic tail of CD8
chain which associates with tyrosine kinase p56lck and ZAP-70 tyrosine kinase might be involved in the intracellular transduction of the apoptotic signal (44,4850).
Zavazava and Krönke recently reported that sHLA-I molecules purified from spleen lymphocytes induce apoptosis in alloreactive cytotoxic T lymphocytes and suggest that TCR ligation might play a pivotal role in sHLA-I-induced cell death (16). The results of the present work suggest that sHLA-ICD8 interaction plays a major role in apoptosis induction of autologous and allogeneic PHA-activated CD8+ T cells. In these experimental conditions sHLA-ITCR interaction seems unable to deliver an apoptotic signal in the absence of sHLA-ICD8 ligation.
FasFasL interactions play a crucial role in inducing apoptosis (36,51). It has been reported that CD4 co-receptor cross-linking prior to TCR engagement triggers apoptosis in CD4+ T cells (52). Our data indicate that CD8 ligation by sHLA-I antigens induces the release of soluble FasL by PHA-activated CD8+ T lymphocytes. This conclusion is supported by several lines of evidence. First, FasL mRNA expression is up-regulated in PHA-activated CD8+ T cells after incubation with sHLA-I molecules. Second, the supernatant of PHA-activated CD8+ T cells incubated with sHLA-I molecules contains functional sFasL molecules which induces apoptosis in Fas+ Jurkat cells. Third, the apoptosis-inducing capacity of the supernatant is inhibited by the preincubation of target cells with anti-Fas neutralizing mAb and by the preincubation of supernatant with anti-FasL mAb. The secretion of sFasL after CD8 molecule ligation by sHLA-I antigens may therefore add to other known mechanisms of CD8 mediated cytotoxicity (51,5356).
Apoptosis induced by FasFasL interactions plays a crucial role for the establishment of antigen-specific T cell tolerance both during the intrathymic negative selection and during adult life (5762). The amount of sHLA-I molecules that induces apoptosis in PHA-activated CD8+ T cells is analogous to the level found in plasma of patients with an activation of their immune system such as those suffering from viral infections, acute rejection episodes following organ allografts or acute graft versus host disease following bone marrow transplantation (7). Increased serum levels of sFasL have been found in several clinical conditions like HIV-1 infection (63), large granular lymphocytic leukemia and NK cell lymphoma (22), and graft versus host disease (64). Elevated levels of functional sHLA-I and sFasL molecules have been detected in blood components and might play a role in the immunomodulatory effect of allogeneic transfusions (65). Moreover, CD8+ T lymphocytes undergoing apoptosis are present in lymph nodes of HIV-1+ patients (66) and in graft infiltrating mononuclear cells during acute rejection episodes (67,68).
Therefore, sHLA-I antigens secreted during immune system activation may bind to CD8 molecules on activated CD8+ T cells and induce sFasL secretion. Then, sFasL may act in an autocrine and/or paracrine way triggering apoptosis in activated CD8+CD95+ T cells. If so, serum sHLA-I molecules may represent an important efferent arm of the network to control the expansion of CD8+ T lymphocytes and to down-regulate immune responses.
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Acknowledgments
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This work was supported by grants from Ministero della SanitàIstituto Superiore di Sanità, Progetto di Ricerche sull'AIDS 19981999, from MURST National Program `Meccanismi umorali e cellulari di modulazione dell'immunoflogosi' (no. 9706117821-001) and by PHS grant CA67108 awarded by the National Cancer Institute, DHHS. We wish to thank Dr S. Nagata (Osaka Bioscience Institute, Osaka, Japan) for the kind gift of mAb 4H9.
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Abbreviations
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ß2 mß2-microglobulin |
DDIA double-determinant immune assay |
FasL Fas ligand |
free heavy chains ß2m-free HLA class I heavy chains |
IIF indirect immunofluorescence |
PHA phytohemagglutinin |
PI propidium iodide |
sFasL soluble FasL |
sHLA-I soluble HLA class I |
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Notes
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Transmitting editor: L. Moretta
Received 12 July 1999,
accepted 19 October 1999.
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