The binding of soluble HLA class I (sHLA-I) molecules to CD8 on EBV-specific CTL induced up-regulation of Fas ligand (FasL) mRNA and consequent sFasL protein secretion. This, in turn, triggered CTL apoptosis by FasL/Fas interaction. Molecular analysis of the biochemical pathways responsible for FasL up-regulation showed that sHLA-I/CD8 interaction firstly induced the recruitment of src-like p56lck and syk-like Zap-70 protein tyrosine kinases (PTK). Interestingly, p59fyn was activated upon the engagement of CD3/TCR complex but not upon the interaction of sHLA-I with CD8. In addition, sHLA-I/CD8 interaction, which is different from signaling through the CD3/TCR complex, did not induce nuclear translocation of AP-1 protein complex. These findings suggest that CD8 and CD3/TCR-mediated activating stimuli can recruit different PTK and transcription factors. Indeed, the engagement of CD8 by sHLA-I led to the activation of Ca2+ calmodulin kinase II pathway, which eventually was responsible for the NF-AT nuclear translocation. In addition, we found that the ligation of sHLA-I to CD8 recruited protein kinase C, leading to NF-κB activation. Both NF-AT and NF-κB were responsible for the induction of FasL mRNA and consequent CTL apoptosis. Moreover, FasL up-regulation and CTL apoptotic death were down-regulated by pharmacological specific inhibitors of Ca2+/calmodulin/calcineurin and Ca2+-independent protein kinase C signaling pathways. These findings clarify the intracellular signaling pathways triggering FasL up-regulation and apoptosis in CTL upon sHLA-I/CD8 ligation and suggest that sHLA-I molecules can be proposed as therapeutic tools to modulate immune responses.

Soluble HLA class I (sHLA-I)4 molecules circulate in serum in an immunologically functional form (1, 2, 3). The serum level of sHLA-I molecules significantly increases during activation of the immune system as in the course of acute rejection episodes following organ allograft, acute graft-vs-host-disease following bone marrow transplantation, autoimmune diseases, and viral infections (4, 5, 6, 7). It has been reported previously that sHLA-I molecules inhibit the cytolytic activity of alloreactive and virus-specific CTL (8, 9, 10, 11) and induce apoptosis in primary alloreactive CD8+ T cells (12, 13, 14). It has been proposed that these effects may be attributed to the binding of the α1 and/or α2 domain(s) of HLA-I H chain to the TCR. However, we and others (15, 16) have reported that the binding of the α3 domain of HLA-I H chain to CD8 by both classical (class Ia) and nonclassical (class Ib) sHLA-I molecules triggers apoptosis. Thus, at present, it is not clear whether sHLA-I can deliver an apoptotic signal in CTL through the direct engagement of CD8 without involving the CD3/TCR complex. Indeed, we have demonstrated that also NK cells, which do not express CD3/TCR complex, can undergo apoptosis upon binding of sHLA-I with CD8 (17, 18). Furthermore, sHLA-I triggered the up-regulation of FasL mRNA expression and secretion of FasL protein, which in turn led to cell apoptosis interacting with Fas at the cell surface in both T and NK cells (16, 17, 18, 19, 20). However, the cascade of signaling events that follow sHLA-I/CD8 ligation has not been clarified. Herein, we have demonstrated that sHLA-I induces CTL apoptosis without interacting with TCR. In addition, we characterized in detail the signal transduction pathways leading to FasL mRNA up-regulation and sFasL secretion after CD8 engagement by sHLA-I molecules in Ag-specific CTL. We found that the ligation of sHLA-I to CD8 led to activation of src-like p56lck and syk-like Zap-70 but not of src-like p59fyn, protein tyrosine kinases (PTK). Moreover, both the Ca2+/calmodulin/calcineurin and protein kinase C (PKC) Ca2+-independent signaling pathways were recruited upon the ligation of sHLA-I to CD8. This was responsible for the NF-AT and NF-κB activation, which eventually led to FasL up-regulation and CTL apoptosis.

The mAb W6/32 to a nonpolymorphic epitope of HLA-I H chains α3 domain and the mAb NAMB-1 to β2-microglobulin were a gift from S. Ferrone (Roswell Park Cancer Institute, Buffalo, NY). The anti-CD3 mAb OKT3, the anti-CD4 mAb OKT4, and the anti-CD8 α-chain mAb OKT8 were purchased from Ortho Diagnostics; the anti-CD16 mAb was purchased from Caltag Laboratories. The anti-human Fas mAb UB2 was purchased from Immunotech; the mAb NOK-1 and NOK-2 to different epitopes of human Fas ligand (FasL) were purchased from BD Pharmingen. mAb were conjugated to biotin (Pierce) according to the manufacturer’s procedure. Goat-anti-mouse Ig Abs (GAM-Ig) were purchased from Coulter. Annexin VFITC was purchased from Bender Systems, and propidium iodide was purchased from Sigma-Aldrich. The anti-lck and anti-fyn mAbs were purchased from Upstate Biotechnology; the anti-Zap-70 mAb and the phosphorylated and nonphosphorylated anti-I-κBα mAb were purchased from Alexis. PMA, sanguinarine, ionomicin, and fura 2-AM were purchased from Sigma-Aldrich. Soluble human rFasL was purchased from Alexis. The PTK inhibitor genistein, the calcineurin inhibitor cyclosporin A, the calcium chelator EGTA, and the PKC inhibitors bisindolylmaleimide I, Gö6976, and rottlerin were purchased from Calbiochem. The lysate of HeLa cells and of HeLa cells incubated with TNF-α was purchased from Santa Cruz Biotechnology. Recombinant TNF-α was purchased from Genzyme.

sHLA-I molecules were obtained from serum of healthy subjects by sequential precipitation with ammonium sulfate, ion exchange, and gel filtration chromatography as previously described (19) and purified by affinity chromatography using anti-HLA-I mAb W6/32 (10 mg/ml) coupled to cyanogen-bromide-activated Sepharose 4B (Pharmacia). sHLA-I A11 monomers and tetramers presenting either the IVT, CLG, or YVN peptide and sHLA-I A2 monomers presenting the CLG peptide were purchased from ProImmune; sHLA-I A11 monomers and tetramers mutated in the α3 domain presenting the IVT peptide were purchased from Beckman Coulter. The affinity scores of IVT, CLG, and YVN peptides for sHLA-I A11 allele are 124, 82, and 130, respectively (see the web site 〈http://hlaligand.ouhsc.edu〉).

The double determinant immunoassay was performed as previously described (21), with minor modifications, using anti-HLA-I mAb W6/32 and biotinylated anti-β2-microglobulin mAb NAMB-1 to measure sHLA-I Ags and anti-FasL mAb NOK-2 and biotinylated anti-FasL mAb NOK-1 to measure sFasL. The OD was read with a spectrophotometer at 490 nm against reagent blank (PBS/BSA 5%). Results were expressed as mean ± SD of triplicate wells.

Indirect immunofluorescence was performed by incubating 5 × 105 cells sequentially with mAb and with GAM-FITC. Each incubation was for 30 min at 4°C. Following three washings, cells were analyzed on an Epics Elite flow cytometer (Coulter).

EBV-specific CD8+ CTL were generated against autologous EBV transformed B cells according to Gavioli et al. (22). Briefly, 2 × 106 PBL were stimulated with 5 × 105 autologous irradiated (6000 rad) EBV-immortalized B cells. Responder cells were restimulated weekly by plating 1 × 106 cells with 3 × 105 irradiated EBV-immortalized B cells and adding IL-2 (50 U/ml) on day 2 and 4 after restimulation. After the third stimulation, cell lines were enriched for CD8+ T lymphocytes by negative selection with immunomagnetic beads (Unipath SpA) and cultured in the presence of IL-2. Experiments have been also performed generating EBV-specific CD8+ T cells with different IL-2 concentrations (0.1, 1, 10, 20, 30, 40, and 50 U/ml). The phenotype of the clones was assessed by flow cytometry. Activated CD8+ T lymphocytes and EBV-specific CTL expressed Fas as determined by flow cytometry after anti-Fas mAb UB2 staining.

Cytotoxic activity was tested in a standard 4-h 51Cr release assay. Appropriate EBV-transformed cells were labeled with 0.1 mCi/106 cells of Na251CrO4 for 1 h at 37°C. The cells were then pulsed with 5 μM IVTDFSVIK (IVT), CLGGLLTMV (CLG), or YVNVNMGLK (YVN) peptides for 1 h at 37°C and washed at least three times before addition of the effector cells. The cytotoxicity tests were run at 30:1, 10:1, and 3:1 E:T ratios in triplicate. Percentage of specific lysis was calculated as 100 × ((cpm sample − cpm medium)/(cpm Triton X-100 − cpm medium)).

Cells (106/ml) were cultured with RPMI 1640 culture medium supplemented with FBS (10%) or with culture medium containing sHLA-I molecules (4 μg/ml) for up to 72 h at 37°C in a 5% CO2 atmosphere. Additional experiments were performed incubating cells with OKT8 mAb (5 μg/106 cells) at 37°C for 15 min followed by cross-linking with GAM-Ig (5 μg/106 cells) at 37°C for 15 min. After 48 and 72 h of incubation, 5 × 105 cells were washed, and early apoptotic events were evaluated by the annexin V-labeling method according to the manufacturer’s protocol. Viable apoptotic cells were differentiated from necrotic cells by flow cytometry after propidium iodide staining of nonpermeabilized cells.

Cells were incubated for up to 10 min at 37°C with sHLA-I molecules (4 μg/ml) or with OKT8 mAb (10 μg/ml) or with OKT3 mAb cross-linked with GAM-Ig (5 μg/ml). Then, 40 μg of the cell extract obtained using 5× lysis buffer in presence of specific protease inhibitors (Na3VO4, aprotinin, leupeptin, NaF, PMSF, and Nonidet P-40) according to the basic protocol (23) was used to evaluate src and syk PTK activity by ELISA (PTK Assay kit; Sigma-Aldrich) using the synthetic random polymer substrate poly-Glu-Tyr. The activation of specific PTK was detected in the immunoprecipitate of 40 μg of the cell extract using the specific p56lck, p59fyn, and Zap-70 mAbs. The Src-PTK activity was evaluated by ELISA (Tyrosine Kinase Assay Kit 1; Pierce) using the biotinylated TK peptide 1 (KVEKIGEGTYGVVYK-amide), while the total PTK and Zap-70 activity was detected using the general PTK assay (Pierce). Some experiments were performed preincubating cells with PTK-phosphorylation inhibitor genistein (50 μg/ml) for 15 min at 37°C. PKC phosphotransferase activity was determined by the PKC Assay kit (Upstate Biotechnology) using [γ-32P]ATP-specific substrate (QKRPSQRSKYL) in 200 μg of crude cell extract after 1 h of incubation with sHLA-I molecules (4 μg/ml), OKT8 mAb (10 μg/ml), or OKT3 mAb cross-linked with GAM-Ig (5 μg/ml). The activation of specific PKC was detected in the immunoprecipitate of 40 μg of the cell extract using specific PKC-α, -δ, -ε (Upstate Biotechnology), and -θ (BD Transduction Laboratories) mAbs. Results are expressed as counts per minute. Some experiments were performed preincubating the cells with PKC inhibitors bisindolylmaleimide I (10 μM), Gö6976 (0.5 μM), and rottlerin (3 μM) for 15 min at 37°C.

Three short-interfering RNA containing 21 nt (ID no. 214477, 5′-GGAUCAGCUAUCAAUAGCCTT-3′; ID no. 214481, 5′-GGACCUUCUGGUGAAGCUCTT-3′; and ID no. 214479, 5′-CCAAGGAAAACCUCUUUUUTT-3′) were purchased from Ambion and were used to silencing PKC-θ human protein. Cells were transfected with a mixture of the PKC-θ-specific short-interfering RNA or GAPDH control by OligofectAMINE (Invitrogen Life Technologies). Forty-eight hours after transfection, cells were incubated with sHLA-I molecules (4 μg/ml) or with OKT8 mAb (10 μg/ml) or with OKT3 mAb cross-linked with GAM-Ig (5 μg/ml) for 1 h and processed for PKC phosphotransferase activity.

Calcium mobilization assay was performed loading the cells with the fura 2-AM (1 mM). Fluorescence was monitored with the LS-50B spectrofluorometer (PerkinElmer). The intracellular-free calcium concentration ([Ca2+]i) was calculated, as previously described (17), after addition of sHLA-I molecules (4 μg/ml) or OKT8 mAb (10 μg/ml) cross-linked with GAM-Ig (5 μg/ml). Some experiments were performed in the presence of the calcium chelator EGTA (2 mM) followed by CaCl2 (4 mM). Calmodulin kinase II phosphotransferase activity was evaluated by the Calmodulin Kinase II Assay kit (Upstate Biotechnology) using [γ-32P]ATP-specific substrate (KKALRRQETVDAL) in 200 μg of crude cell extract after 1 h of incubation with sHLA-I or OKT8 mAb cross-liked with GAM-Ig. Results are expressed as counts per minute. Some experiments were performed preincubating cells with the calcineurin inhibitor cyclosporin A (50 ng/ml) for 15 min at 37°C.

Total RNA was isolated from cell pellets using the RNAzol B method (Biotecx Laboratories) according to the manufacturer’s protocol. cDNA (corresponding to 2 μg of RNA) was synthesized from oligo(dT)-primed RNA in 20 μl of RT buffer and 200 U Moloney murine leukemia virus RT (PerkinElmer/Cetus) incubated at 42°C for 45 min and at 52°C for 45 min. cDNA (2 μl) was amplified by PCR in a total volume of 50 μl containing 2.5 mM MgCl2, 2 mM dNTP, 50 μM 5′ and 3′ oligonucleotide primers, and 2.5 U of Amply Taq Gold Polymerase (PerkinElmer/Cetus). The sequence (5′ to 3′) of the β-actin and FasL-specific primers used are as follows: β-actin, 5′-CATACTCCTgCTTgCTgATCC-3′; β-actin, 5′-ACTCCATCATgAAgTgTgACg-3′ (228-bp fragment); FasL, 5′-CAAgTCCAACTCAAggTCCATgCC-3′; and FasL, 5′-CAgTTgCATAgACTCgAgAgAgAC-3′ (350-bp fragment). Amplification was performed in a DNA thermal cycler (Eppendorf) using the following conditions: 94°C for 1 min, 60°C for 30 s, 72°C for 30 s (35 cycles), and 72°C for 10 min. PCR products were size fractionated by agarose electrophoresis and normalized according to the amount of β-actin detected in the same mRNA sample.

I-κBα phosphorylation was evaluated using cytoplasmic extracts prepared from 107 cells treated for 30 min with sHLA-I molecules (4 μg/ml) or OKT8 mAb (10 μg/ml) cross-linked with GAM-Ig (5 μg/106 cells). The extracts were then resolved on 10% SDS-PAGE and analyzed by Western blot using phosphorylated and nonphosphorylated anti-I-κBα mAb. The presence of proteins was detected by chemiluminescence method (ECL plus; Amersham Biosciences).

Preparations of nuclear extracts were obtained after lysis of 107 cells according to the method of Muller et al. (24). Protein concentration was determined by Bradford’s assay (Bio-Rad), and 5 μg of nuclear proteins was used to perform EMSA. The binding reaction was performed at room temperature for 30 min with 20,000 cpm of NF-AT and AP-1 double-stranded oligonucleotides (Santa Cruz Biotechnology) end-labeled with [γ-32P]ATP using T4 polynucleotide kinase (Amersham Biosciences). The nuclear translocation was analyzed after separation on a 5% polyacrylamide gel. NF-κB, NF-AT, and AP- 1/c-jun activation was also determined by highly sensitive ELISA-based assays (TransAM kits; Active Motif).

Gels were analyzed with the Alpha Imager IS55nc-5 apparatus (Alpha Innotech) using the Chemi5000 software to acquire the images and the GelPro 3.1 software to perform the densitometric analysis of the bands. Results of FasL mRNA PCR products are reported as percent variation of FasL band intensity as compared with β-actin band intensity in the same sample.

Data are expressed as mean ± SD. The statistical analysis of the data has been performed by one-way ANOVA followed by Bonferroni’s comparison test; a value of p < 0.05 was considered as significant.

We have shown previously that polyclonally activated peripheral blood CD8+ T cells undergo apoptotic death after incubation with sHLA-I molecules and that apoptosis is mediated by FasL mRNA up-regulation, sFasL secretion, and FasL/Fas interaction (16, 19, 20). However, it is not clear whether sHLA-I interacts with either CD8 or CD3/TCR complex. To clarify this point, CD8+ T cells were incubated with anti-CD8 α-chain mAb OKT8 before challenging with sHLA-I molecules. As shown in Table I, apoptosis and sFasL secretion of EBV-specific CTL exposed to sHLA-I were strongly inhibited by the preincubation with OKT8 mAb, suggesting that the CD8 molecule is directly involved in this process. Indeed, the ligation of CD8 with anti-CD8-specific mAb followed by its cross-linking using GAM-Ig led to a strong sFasL secretion and CTL apoptosis, while ex vivo-isolated resting CD8+ lymphocytes did not up-regulate FasL and did not undergo apoptosis after sHLA-I incubation (Table I). Apoptosis induction by sHLA-I molecules was not correlated with the IL-2 dose used to generate EBV-specific CD8+ CTL (Table II).

Table I.

Effect of PTK and PKC inhibitors and of calcium chelator EGTA on sFasL secretion and apoptosis triggered by sHLA-I/CD8 ligation

CellsPreincubationIncubationCoincubationsFasL (ng/ml)Apoptotic Cells (%)
AaBCDEF24 h48 h72 hAnnexin V+/PIAnnexin V+/PI+
1224 h48 h72 h24 h48 h72 h
Resting CD8+ T cells  sHLA-Ib − − − − − − − 0.0 0.0 0.0 10 
EBV-specific CTL  Medium − − − − − − − 0.0 0.5 0.3 13 
  sHLA-I − − − − − − − 580.0 173.0 16.9 26 69 0.1 21 
 OKT8 mAb Medium − − − − − − − 3.4 3.2 2.5 10 
 OKT8 mAb sHLA-I − − − − − − − 14.6 2.9 0.8 
  sHLA-I − − − − − − 95.0 3.4 1.0 16 
  sHLA-I − − − − − − 496.0 175.0 13.9 28 57 23 
  sHLA-I − − − − − − 4.3 0.6 0.0 16 
  sHLA-I − − − − − − 5.0 0.0 0.0 11 
  sHLA-I − − − − − − 96.0 4.9 0.3 16 23 
  sHLA-I − − − − − − 537.0 137.0 9.7 18 54 16 
  sHLA-I − − − − − − 14.0 0.5 0.0 14 25 
  OKT8-XLc − − − − − − − 637.0 209.0 8.2 40 72 27 
  OKY8-XL − − − − − − 107.0 8.5 1.3 18 11 
  OKT8-XL − − − − − − 597.0 194.0 10.7 38 68 0.1 16 
  OKT8-XL − − − − − − 1.8 1.0 0.0 23 13 
  OKT8-XL − − − − − − 0.0 0.0 0.0 19 12 
  OKT8-XL − − − − − − 19.2 5.7 0.1 11 31 15 
  OKT8-XL − − − − − − 497.0 103.0 3.2 16 59 13 
  OKT8-XL − − − − − − 21.0 7.5 0.0 13 33 13 
CellsPreincubationIncubationCoincubationsFasL (ng/ml)Apoptotic Cells (%)
AaBCDEF24 h48 h72 hAnnexin V+/PIAnnexin V+/PI+
1224 h48 h72 h24 h48 h72 h
Resting CD8+ T cells  sHLA-Ib − − − − − − − 0.0 0.0 0.0 10 
EBV-specific CTL  Medium − − − − − − − 0.0 0.5 0.3 13 
  sHLA-I − − − − − − − 580.0 173.0 16.9 26 69 0.1 21 
 OKT8 mAb Medium − − − − − − − 3.4 3.2 2.5 10 
 OKT8 mAb sHLA-I − − − − − − − 14.6 2.9 0.8 
  sHLA-I − − − − − − 95.0 3.4 1.0 16 
  sHLA-I − − − − − − 496.0 175.0 13.9 28 57 23 
  sHLA-I − − − − − − 4.3 0.6 0.0 16 
  sHLA-I − − − − − − 5.0 0.0 0.0 11 
  sHLA-I − − − − − − 96.0 4.9 0.3 16 23 
  sHLA-I − − − − − − 537.0 137.0 9.7 18 54 16 
  sHLA-I − − − − − − 14.0 0.5 0.0 14 25 
  OKT8-XLc − − − − − − − 637.0 209.0 8.2 40 72 27 
  OKY8-XL − − − − − − 107.0 8.5 1.3 18 11 
  OKT8-XL − − − − − − 597.0 194.0 10.7 38 68 0.1 16 
  OKT8-XL − − − − − − 1.8 1.0 0.0 23 13 
  OKT8-XL − − − − − − 0.0 0.0 0.0 19 12 
  OKT8-XL − − − − − − 19.2 5.7 0.1 11 31 15 
  OKT8-XL − − − − − − 497.0 103.0 3.2 16 59 13 
  OKT8-XL − − − − − − 21.0 7.5 0.0 13 33 13 
a

(A) Genistein (50 μg/106 cells) added at the beginning of incubation 1 or 2 h later (2), (B) EGTA (2, mM/106 cells), (C) cyclosporin A (50 ng/106 cells), (D) bisindolylmaleimide I (10 μM/106 cells), (E) Gö6976 (0.5 μM/106 cells), and (F) rottlerin (3 μM/106 cells) added at the beginning of incubation.

b

HLA-I molecules (4 μg/ml).

c

OKT8 mAb (5 μg/106 cells) cross-linked with GAM-Ig (5 μg/106 cells).

Table II.

Effect of IL-2 dose on apoptosis induction by sHLA-I in CD8+ T cellsa

IL-2 (μg/ml)Apoptotic Cells (%)b
Culture mediumsHLA-I (4 μg/ml)TNF-α (100 ng/ml)
0.1 2 ± 0.5 45 ± 4 46 ± 5 
1.0 1 ± 0.3 50 ± 6 43 ± 4 
10 1 ± 0.3 48 ± 5 43 ± 6 
20 0.5 ± 0.4 45 ± 3 46 ± 5 
30 1 ± 0.4 45 ± 2 44 ± 3 
40 1 ± 0.3 46 ± 2 44 ± 2 
50 0.5 ± 0.2 49 ± 4 39 ± 2 
IL-2 (μg/ml)Apoptotic Cells (%)b
Culture mediumsHLA-I (4 μg/ml)TNF-α (100 ng/ml)
0.1 2 ± 0.5 45 ± 4 46 ± 5 
1.0 1 ± 0.3 50 ± 6 43 ± 4 
10 1 ± 0.3 48 ± 5 43 ± 6 
20 0.5 ± 0.4 45 ± 3 46 ± 5 
30 1 ± 0.4 45 ± 2 44 ± 3 
40 1 ± 0.3 46 ± 2 44 ± 2 
50 0.5 ± 0.2 49 ± 4 39 ± 2 
a

Values are mean ± SD of four independent experiments.

b

sHLA-I and TNF-α were added at the beginning of the incubation, and apoptosis was evaluated 48 h later.

To better define whether sHLA-I molecules induced apoptosis of EBV-specific CTL through the engagement of either CD8 or CD3/TCR complex, we performed a series of additional experiments using haplotype-specific sHLA-I monomers or tetramers presenting peptides, which were either recognized or not by CD8+ EBV-specific CTL (Fig. 1,a). To this end, we generated HLA-A11-restricted CTL specific for the IVT peptide derived from EBV-encoded nuclear Ag EBNA-4 (HLA-A11 IVT) (22, 25). Indeed, this effector population lysed autologous target cells expressing HLA-A11 Ags pulsed with the specific IVT peptide but not those pulsed with the unrelated CLG and YVN peptides (Fig. 1,b). Of note, incubation for 24 h of EBV-specific CTL with sHLA-A11 molecules presenting either the specific IVT or the unrelated CLG or YVN peptides led to comparable levels of T cell apoptosis (Fig. 1,c). This finding suggests that recognition of the specific peptide via CD3/TCR complex is not required to deliver the apoptotic signal. It is known that the α3 domain of HLA-I molecules binds the CD8 α-chain (26). To demonstrate that HLA-I α3 domain is essential to trigger apoptosis, we incubated EBV-specific CTL with α3 domain-mutated sHLA A11 molecules presenting the specific IVT peptide. In these experimental conditions, EBV-specific CTL did not undergo apoptosis, suggesting that the interaction of sHLA-I α3 domain with CD8 was an essential requirement for apoptosis induction (Fig. 1,c). To confirm that apoptosis can be triggered in the absence of sHLA-I/TCR ligation, we performed experiments incubating HLA-A11-restricted CTL specific for the IVT peptide with sHLA-A2 molecules presenting the unrelated CLG peptide (25). As shown in Fig. 1,c, EBV-specific CTL underwent apoptosis when incubated with the sHLA-A2-CLG complex. Of note, the apoptotic effect induced by the sHLA-A2-CLG complex was similar to that of the haplotype-specific sHLAA11-IVT complex (Fig. 1,c). The covering of CD8 with anti-CD8-specific mAb strongly reduced (by 75%) the apoptosis of EBV-specific CTL induced by either sHLA-A11 IVT or sHLA-A2-CLG complexes (Fig. 1 c). Altogether, these findings indicate that sHLA-I molecules can trigger apoptosis of CD8+ T cells without interacting with the TCR and that their binding with CD8 is an essential requirement to deliver the apoptotic signal.

FIGURE 1.

Apoptosis induction by sHLA-I molecules in CD8+ T cells is not HLA restricted. a, Binding of sHLA-A11 monomers and tetramers presenting the EBV-specific IVT peptide, the unrelated EBV-specific CLG peptide, or the HBV-specific YVN peptide to HLA-A11-restricted, EBV-specific CTL. The HLA-A11 allele affinity scores for IVT, CLG, and YVN peptides are 124, 82, and 130, respectively, as indicated online (〈http://hlaligand.ouhsc.edu〉); b, lysis of HLA-A11 and HLA-A2 and EBV cell lines pulsed with the EBV-specific IVT and CLG peptides or with the HBV-specific YVN peptide by HLA-A11- and HLA-A2-restricted, EBV-specific CTLs. c, Apoptosis of HLA-A11-restricted, EBV-specific CTL after incubation with sHLA-A11 monomers presenting the EBV-specific IVT peptide (column 1), the unrelated EBV-specific CLG peptide (column 2) or the HBV-specific YVN peptide (column 3), sHLA-A11 monomers presenting EBV-specific IVT peptides after preincubation with OKT8 mAb (column 4), sHLA-A11 monomers mutated in the α3 domain presenting EBV-specific IVT peptides (column 5), sHLA-A2 monomers presenting EBV-specific CLG peptides (column 6), and sHLA-A2 monomers presenting EBV-specific CLG peptides after preincubation with OKT8 mAb (column 7). The percentage of spontaneous apoptotic cells is reported in the first column (column C).

FIGURE 1.

Apoptosis induction by sHLA-I molecules in CD8+ T cells is not HLA restricted. a, Binding of sHLA-A11 monomers and tetramers presenting the EBV-specific IVT peptide, the unrelated EBV-specific CLG peptide, or the HBV-specific YVN peptide to HLA-A11-restricted, EBV-specific CTL. The HLA-A11 allele affinity scores for IVT, CLG, and YVN peptides are 124, 82, and 130, respectively, as indicated online (〈http://hlaligand.ouhsc.edu〉); b, lysis of HLA-A11 and HLA-A2 and EBV cell lines pulsed with the EBV-specific IVT and CLG peptides or with the HBV-specific YVN peptide by HLA-A11- and HLA-A2-restricted, EBV-specific CTLs. c, Apoptosis of HLA-A11-restricted, EBV-specific CTL after incubation with sHLA-A11 monomers presenting the EBV-specific IVT peptide (column 1), the unrelated EBV-specific CLG peptide (column 2) or the HBV-specific YVN peptide (column 3), sHLA-A11 monomers presenting EBV-specific IVT peptides after preincubation with OKT8 mAb (column 4), sHLA-A11 monomers mutated in the α3 domain presenting EBV-specific IVT peptides (column 5), sHLA-A2 monomers presenting EBV-specific CLG peptides (column 6), and sHLA-A2 monomers presenting EBV-specific CLG peptides after preincubation with OKT8 mAb (column 7). The percentage of spontaneous apoptotic cells is reported in the first column (column C).

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To analyze the early intracellular events that lead to FasL mRNA up-regulation following CD8 ligation, we first investigated the involvement of PTK, which may be recruited upon lymphocyte activation by either the intracytoplasmic tail of CD8 α-chain, such as p56lck (27, 28, 29), or of the CD3/TCR complex, such as p59fyn (30, 31) and Zap-70 (32, 33, 34). We found that a peak of total PTK activity is detectable 5 min after the addition of sHLA-I molecules to EBV-specific CTL (Fig. 2,a). Moreover, CD8 cross-linking with anti-CD8 mAb followed by GAM-Ig led to an analogous peak of PTK activity, suggesting that sHLA-I molecules may trigger PTK activation through CD8 engagement (Fig. 2,a). Finally, CD3 engagements lead to early peak of PTK activation detectable 1 min after its cross-linking. Then, we investigated which PTK was specifically recruited upon sHLA-I/CD8 ligation. To this end, p56lck, Zap-70, or p59fyn PTK were immunoprecipitated by using specific mAbs, and PTK activity was evaluated. Interestingly, a peak of p56lck activity occurred from 3 to 5 min after sHLA-I incubation while Zap-70 activity peaked, as expected (35), ∼3 min later (Fig. 2,a). By contrast, no detectable activation of p59fyn was observed up to a 10-min incubation with sHLA-I, whereas p59fyn activation occurred 3–5 min after CD3 ligation (Fig. 2,a). The peak of PTK activity was always significantly inhibited by the addition of the PTK inhibitor genistein (36) at the beginning of the incubation (Fig. 2,a). The up-regulation of FasL mRNA expression and CTL apoptosis detectable after CD8/sHLA-I interaction were strongly inhibited when genistein was added to CD8+ CTL at the same time of sHLA-I molecules but not when genistein was added 2 h later, further confirming the critical role of PTK activation in CD8+ T cell apoptosis (Fig. 2,b and Table I). Taken together, these data confirm previous reports showing that CD8 cross-linking induces p56lck activation (36) and demonstrated that sHLA-I molecules are able, as with immobilized HLA-I molecules (36), to up-regulate p56lck activity through CD8 engagement. Moreover, they suggest that the PTKs recruited upon interaction of sHLA-I with CD8 are different from those involved in CD3-mediated signaling.

FIGURE 2.

sHLA-I/CD8 ligation triggers PTK activation and up-regulates FasL mRNA. a, Kinetics of total PTK, p56lck, p59fyn, and Zap-70 activity analyzed by ELISA method in activated CD8+ T cells after incubation with sHLA-I molecules, OKT8 mAb cross-linked with GAM-Ig (OKT8-XL), or OKT3-mAb cross-linked with GAM-Ig (OKT3-XL) in the absence (▧) and in the presence (▨) of genistein. The kinetics of total PTK, p56lck, p59fyn, and Zap-70 activity in activated CD8+ T cells grown in culture medium alone in absence (▦) and in presence (▩) of genistein is reported for comparison. The positive (▪) and negative (□) ELISA controls are also shown. The significance of the increase of PTK activity at the different times as compared with time 1 is indicated (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001). The significance of the inhibition of PTK activity by genistein at each time point is also shown (§, p < 0.001). Values are mean ± SD of five independent experiments. b, Analysis of FasL mRNA expression in resting CD8+ T cells (lane A); activated CD8+ T cells (lane B); activated CD8+ T cells incubated with sHLA-I molecules in the absence (lane C) and in the presence of genistein added at the same time (lane D) or 2 h later (lane E); and activated CD8+ T cells incubated with OKT8-XL in the absence (lane F) and in the presence of genistein added at the same time (lane G) or 2 h later (lane H). Numbers above the gels refer to the densitometric analysis of the bands (see Materials and Methods).  

FIGURE 2.

sHLA-I/CD8 ligation triggers PTK activation and up-regulates FasL mRNA. a, Kinetics of total PTK, p56lck, p59fyn, and Zap-70 activity analyzed by ELISA method in activated CD8+ T cells after incubation with sHLA-I molecules, OKT8 mAb cross-linked with GAM-Ig (OKT8-XL), or OKT3-mAb cross-linked with GAM-Ig (OKT3-XL) in the absence (▧) and in the presence (▨) of genistein. The kinetics of total PTK, p56lck, p59fyn, and Zap-70 activity in activated CD8+ T cells grown in culture medium alone in absence (▦) and in presence (▩) of genistein is reported for comparison. The positive (▪) and negative (□) ELISA controls are also shown. The significance of the increase of PTK activity at the different times as compared with time 1 is indicated (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001). The significance of the inhibition of PTK activity by genistein at each time point is also shown (§, p < 0.001). Values are mean ± SD of five independent experiments. b, Analysis of FasL mRNA expression in resting CD8+ T cells (lane A); activated CD8+ T cells (lane B); activated CD8+ T cells incubated with sHLA-I molecules in the absence (lane C) and in the presence of genistein added at the same time (lane D) or 2 h later (lane E); and activated CD8+ T cells incubated with OKT8-XL in the absence (lane F) and in the presence of genistein added at the same time (lane G) or 2 h later (lane H). Numbers above the gels refer to the densitometric analysis of the bands (see Materials and Methods).  

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To define in detail the biochemical pathways involved in the signal transduction following sHLA-I/CD8 interaction, we analyzed whether the engagement of CD8 induced an increase in intracellular-free calcium concentration ([Ca2+]i) and the activation of calcium calmodulin kinase II. As shown in Fig. 3,a, panel 2, [Ca2+]i increase was detectable in activated CD8+ T cells upon incubation with sHLA-I, and a similar calcium rise was observed upon CD8 cross-linking with GAM-Ig (Fig. 3,a, panel 3). The increase of [Ca2+]i following CD3/TCR complex ligation with anti-CD3-specific mAb reached its maximum level after ∼150–200 s (Fig. 3,a, panel 6), while [Ca2+]i increase after CD8 engagement was of lower degree and slower kinetics (Fig. 3 a, panel 3). These findings further reinforce the hypothesis that sHLA-I molecules are able to trigger signal transduction in CD8+ T cells independently of the engagement of CD3/TCR complex.

FIGURE 3.

sHLA-I/CD8 ligation activates Ca2+/calmodulin/calcineurin pathway. a, Intracellular-free calcium increases detected with spectrofluorometric analysis in activated CD8+ cells incubated with GAM-Ig as negative control (panel 1) and with sHLA-I molecules (panel 2); OKT8 cross-linked with GAM-Ig (OKT8-XL) (panel 3); sHLA-I or OKT8-XL in the presence of extracellular calcium chelator EGTA followed by CaCl2 (panels 4 and 5, respectively); and OKT3-XL as positive control (panel 6). The addition of stimulus is indicated in each panel by the arrow. b, Calmodulin kinase II activity assessed by enzyme assay in activated CD8+ T cells (▦) after incubation with sHLA-I molecules (▨), OKT8-XL (▩), or OKT3-XL (▧) (column A). Positive (▪) and negative (□) controls of the assay are also shown. The significance of the increase of calmodulin kinase II activity as compared with activated CD8+ T cells is indicated (∗, p < 0.001). The significance of the inhibition of calmodulin kinase II activity by EGTA or cyclosporin A (columns B or C, respectively, vs column A) is also shown (§, p < 0.001). Values are mean ± SD of three independent experiments. c, Analysis of FasL mRNA expression in activated CD8+ T cells grown in culture medium (lane A) or incubated with sHLA-I molecules (lane B); sHLA-I and cyclosporin A (lane C) or EGTA (lane D); OKT8-XL (lane E); and OKT8-XL and cyclosporin A (lane F) or EGTA (lane G). Numbers above the gels refer to the densitometric analysis of the bands (see Materials and Methods).

FIGURE 3.

sHLA-I/CD8 ligation activates Ca2+/calmodulin/calcineurin pathway. a, Intracellular-free calcium increases detected with spectrofluorometric analysis in activated CD8+ cells incubated with GAM-Ig as negative control (panel 1) and with sHLA-I molecules (panel 2); OKT8 cross-linked with GAM-Ig (OKT8-XL) (panel 3); sHLA-I or OKT8-XL in the presence of extracellular calcium chelator EGTA followed by CaCl2 (panels 4 and 5, respectively); and OKT3-XL as positive control (panel 6). The addition of stimulus is indicated in each panel by the arrow. b, Calmodulin kinase II activity assessed by enzyme assay in activated CD8+ T cells (▦) after incubation with sHLA-I molecules (▨), OKT8-XL (▩), or OKT3-XL (▧) (column A). Positive (▪) and negative (□) controls of the assay are also shown. The significance of the increase of calmodulin kinase II activity as compared with activated CD8+ T cells is indicated (∗, p < 0.001). The significance of the inhibition of calmodulin kinase II activity by EGTA or cyclosporin A (columns B or C, respectively, vs column A) is also shown (§, p < 0.001). Values are mean ± SD of three independent experiments. c, Analysis of FasL mRNA expression in activated CD8+ T cells grown in culture medium (lane A) or incubated with sHLA-I molecules (lane B); sHLA-I and cyclosporin A (lane C) or EGTA (lane D); OKT8-XL (lane E); and OKT8-XL and cyclosporin A (lane F) or EGTA (lane G). Numbers above the gels refer to the densitometric analysis of the bands (see Materials and Methods).

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To determine whether this effect depends on Ca2+ release from intracellular stores or on calcium influx from the extracellular medium through opening of calcium channels at the cell membrane, the calcium chelator EGTA (2 mM) was added to cells before either sHLA-I molecules or CD8 cross-linking. As shown in Fig. 3,a (panels 4 and 5), EGTA abolished, by chelating 1 mM Ca2+ present in the extracellular milieu, calcium influx induced by sHLA-I or CD8 cross-linking. Of note, the subsequent addition of a large excess of CaCl2 (4 mM) into the extracellular milieu evoked a prompt and strong calcium rise (Fig. 3,a, panels 4 and 5), indicating that CD8 engagement determined the opening of Ca2+ channels at the cell surface. Furthermore, CD8-mediated [Ca2+]i increase was followed by calmodulin kinase II activation (Fig. 3,b), which is known to be required for NF-AT nuclear translocation (38, 39). The involvement of [Ca2+]/calmodulin/calcineurin pathway in CD8+ T cell apoptosis was eventually supported by the significant inhibition (p < 0.001) of calmodulin kinase II activity and of FasL mRNA up-regulation, sFasL secretion, and apoptosis by either EGTA and cyclosporin A (Fig. 3,c and Table I).

It has been reported that the activation of [Ca2+]/calmodulin/calcineurin pathway leads to the dephosphorylation of NF-AT that translocates into the nucleus and associates with a nuclear component to form an active DNA binding complex (37, 38, 39). Thus, we analyzed the NF-AT nuclear translocation in EBV-specific CTL after incubation with sHLA-I molecules or CD8 cross-linking. Immunoenzymatic analysis demonstrated that a NF-AT/DNA complex was formed 1 h after CD8 engagement (Fig. 4, a and b). The specificity of this reaction was confirmed by the competitive inhibition of protein/DNA complex formation by the addition of an excess of NF-AT unlabeled probe (Fig. 4,a). Moreover, the formation of NF-AT/DNA complex was significantly inhibited (p < 0.001) by the calcineurin inhibitor cyclosporin A as well as by blocking [Ca2+]i increase with EGTA (Fig. 4 a). Altogether, these findings indicate that the engagement of CD8 with sHLA-I induces calmodulin kinase II activation, NF-AT nuclear translocation, and FasL up-regulation.

FIGURE 4.

sHLA-I/CD8 ligation induces NF-AT activation. a, NF-AT phosphorylation assessed by ELISA in activated CD8+ T cells grown in culture medium (negative control) and in cells incubated with OKT3 cross-linked with GAM-Ig (OKT3-XL) (positive control), sHLA-I or OKT8-XL, and sHLA-I or OKT8-XL in the presence of cyclosporin A, EGTA, bisindolylmaleimide I, and Gö6976. The significance of the increase of NF-AT activity as compared with negative control is indicated (∗, p < 0.001). The significance of the inhibition of NF-AT activity by cyclosporin A, EGTA, bisindolylmaleimide I, and Gö6976 vs sHLA-I or OKT8-XL is also shown (§, p < 0.01; §§, p < 0.001; n.s., not significant). Values are mean ± SD of four independent experiments. b, EMSA of NF-AT nuclear translocation in activated CD8+ T cells grown in culture medium alone (lane 1) and in cells incubated with sHLA-I molecules in the absence (lane 2) and in the presence of cyclosporin A (lane 3), EGTA (lane 4), bisindolylmaleimide I (lane 5), or Gö6976 (lane 6).

FIGURE 4.

sHLA-I/CD8 ligation induces NF-AT activation. a, NF-AT phosphorylation assessed by ELISA in activated CD8+ T cells grown in culture medium (negative control) and in cells incubated with OKT3 cross-linked with GAM-Ig (OKT3-XL) (positive control), sHLA-I or OKT8-XL, and sHLA-I or OKT8-XL in the presence of cyclosporin A, EGTA, bisindolylmaleimide I, and Gö6976. The significance of the increase of NF-AT activity as compared with negative control is indicated (∗, p < 0.001). The significance of the inhibition of NF-AT activity by cyclosporin A, EGTA, bisindolylmaleimide I, and Gö6976 vs sHLA-I or OKT8-XL is also shown (§, p < 0.01; §§, p < 0.001; n.s., not significant). Values are mean ± SD of four independent experiments. b, EMSA of NF-AT nuclear translocation in activated CD8+ T cells grown in culture medium alone (lane 1) and in cells incubated with sHLA-I molecules in the absence (lane 2) and in the presence of cyclosporin A (lane 3), EGTA (lane 4), bisindolylmaleimide I (lane 5), or Gö6976 (lane 6).

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Within the promoter region of the FasL gene are present binding sites not only for NF-AT but also for NF-κB nuclear protein (40). It has been demonstrated that PKC is involved in the phosphorylation of I-κBα (41, 42), then, phosphorylated I-κBα dissociates from the NF-κB/I-κBα complex leading to NF-κB nuclear translocation. Thus, we analyzed whether sHLA-I/CD8 engagement led to PKC activation and FasL mRNA up-regulation after binding of NF-κB to FasL promoter region. PKC activity significantly increased in EBV-specific CTL 1 h after the addition of sHLA-I molecules or CD8 cross-linking (Fig. 5,a). FasL mRNA up-regulation and sFasL secretion were prevented by the PKC inhibitor bisindolylmaleimide I, confirming that PKC activation is involved in CD8+ T cell apoptosis (Fig. 5,c and Table I). As PKC may mediate their effects via classical [Ca2+]-dependent isoforms (α, β, and γ) or via [Ca2+]-independent isoforms (δ, ε, η, θ, and ι) (43), we determined which PKC isoform was activated after CD8 engagement using Gö6976 that selectively inhibits α, β, and γ isoforms (44, 45) and rottlerin that selectively inhibits [Ca2+]-independent isoforms expressed in T cells (43). FasL mRNA up-regulation, sFasL secretion, and CTL apoptosis were inhibited by rottlerin while they were unaffected by Gö6976, indicating that [Ca2+]-independent PKC isoforms are mainly involved in sHLA-I/CD8-mediated signaling (Fig. 5,c and Table I). Additional experiments performed using RNAi assay and assessing PKC activity following immunoprecipitation of PKC isoforms suggested that PKC-θ is the isoform preferentially involved in sHLA-I-mediated signaling (Fig. 5, a and b). In addition, we found that phosphorylated I-κBα was detectable in the cytoplasm of activated CD8+ T cells 1 h after incubation with sHLA-I molecules or CD8 cross-linking (Fig. 6,a). Furthermore, NF-κB nuclear translocation occurred 1 h after CD8 ligation and was inhibited significantly (p < 0.05) by sanguinarine, which blocks I-κBα phosphorylation and degradation (46) (Fig. 6, a and b). Finally, NF-κB/DNA complex formation was inhibited significantly (p < 0.05) by the PKC-θ-specific inhibitor rottlerin, confirming the role of this PKC isoform in NF-κB nuclear translocation and binding to FasL promoter (Fig. 6,b). The involvement of PKC-θ, which leads to NF-AT nuclear translocation through the JNK pathway, was confirmed by the significant inhibition (p < 0.001) of NF-AT activation by bisindolylmaleimide I but not by Gö6976 (Fig. 4 a).

FIGURE 5.

sHLA-I/CD8 ligation triggers PKC activation. a, PKC activation assessed by enzyme assay in activated CD8+ T cells (▪) after incubation with sHLA-I molecules (▨), OKT8 mAb cross-linked with GAM-Ig (OKT8-XL) (▩), or OKT3-XL (▧) in the absence (column A) and in the presence of bisindolylmaleimide I (column B), Gö6976 (column C), rottlerin (column D), PKC-θ RNAi (column E), and GAPDH RNAi (column F). The negative control (□) of the assay is also shown. The significance of the increase of PKC activity as compared with activated CD8+ T cells is indicated (∗, p < 0.001). The significance of the inhibition of PKC activity by bisindolylmaleimide I, Gö6976, rottlerin, PKC-θ RNAi, and GAPDH RNAi (columns B, C, D, E, and F, respectively, vs column A) is also shown (§, p < 0.001; n.s., not significant). Values are mean ± SD of four independent experiments. b, PKC activation assessed by enzyme assay in immunoprecipitated PKC isoforms from activated CD8+ T cells after incubation with sHLA-I molecules (column A, total PKC; column B, PCK-δ; column C, PCK-ε; column D, PCK-θ; and column E, PKC-α). The negative (□) and positive (▪) controls of the assay are also shown. c, Analysis of FasL mRNA expression in activated CD8+ T cells (lane A) and in activated CD8+ T cells incubated with sHLA-I molecules in the absence (lane B) and in the presence of bisindolylmaleimide I (lane C), Gö6976 (lane D), and rottlerin (lane E); OKT8-XL in the absence (lane F) and in the presence of bisindolylmaleimide I (lane G), Gö6976 (lane H), and rottlerin (lane I). Numbers above the gels refer to the densitometric analysis of the bands (see Materials and Methods).

FIGURE 5.

sHLA-I/CD8 ligation triggers PKC activation. a, PKC activation assessed by enzyme assay in activated CD8+ T cells (▪) after incubation with sHLA-I molecules (▨), OKT8 mAb cross-linked with GAM-Ig (OKT8-XL) (▩), or OKT3-XL (▧) in the absence (column A) and in the presence of bisindolylmaleimide I (column B), Gö6976 (column C), rottlerin (column D), PKC-θ RNAi (column E), and GAPDH RNAi (column F). The negative control (□) of the assay is also shown. The significance of the increase of PKC activity as compared with activated CD8+ T cells is indicated (∗, p < 0.001). The significance of the inhibition of PKC activity by bisindolylmaleimide I, Gö6976, rottlerin, PKC-θ RNAi, and GAPDH RNAi (columns B, C, D, E, and F, respectively, vs column A) is also shown (§, p < 0.001; n.s., not significant). Values are mean ± SD of four independent experiments. b, PKC activation assessed by enzyme assay in immunoprecipitated PKC isoforms from activated CD8+ T cells after incubation with sHLA-I molecules (column A, total PKC; column B, PCK-δ; column C, PCK-ε; column D, PCK-θ; and column E, PKC-α). The negative (□) and positive (▪) controls of the assay are also shown. c, Analysis of FasL mRNA expression in activated CD8+ T cells (lane A) and in activated CD8+ T cells incubated with sHLA-I molecules in the absence (lane B) and in the presence of bisindolylmaleimide I (lane C), Gö6976 (lane D), and rottlerin (lane E); OKT8-XL in the absence (lane F) and in the presence of bisindolylmaleimide I (lane G), Gö6976 (lane H), and rottlerin (lane I). Numbers above the gels refer to the densitometric analysis of the bands (see Materials and Methods).

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FIGURE 6.

sHLA-I/CD8 ligation induces I-κBα phosphorylation and NF-κB nuclear translocation. a, Western blot analysis of nonphosphorylated (upper lane) and phosphorylated (lower lane) I-κBα molecule in the lysate of HeLa cells (lane 1, negative control), HeLa cells + TNF-α (lane 2, positive control), CD8+ blasts grown in culture medium (lane 3) and after incubation with sHLA-I (lane 4), OKT8 mAb cross-linked with GAM-Ig (OKT8-XL) (lane 5), sHLA-I in the presence of sanguinarine (lane 6), and OKT8-XL in the presence of sanguinarine (lane 7); b, NF-κB phosphorylation assessed by ELISA in activated CD8+ T cells incubated with culture medium (negative control), OKT3-XL (positive control), sHLA-I or OKT8-XL, and sHLA-I or OKT8-XL in the presence of bisindolylmaleimide I, Gö6976, rottlerin, and sanguinarine. The significance of the increase of NF-κB activity as compared with activated CD8+ T cells is indicated (∗, p < 0.01; ∗∗, p < 0.001). The significance of the inhibition of NF-κB activity by bisindolylmaleimide I, Gö6976, rottlerin, and sanguinarine vs sHLA-I or OKT8-XL is also shown (§, p < 0.05; §§, p < 0.01; §§§, p < 0.001; n.s., not significant). Values are mean ± SD of five independent experiments.

FIGURE 6.

sHLA-I/CD8 ligation induces I-κBα phosphorylation and NF-κB nuclear translocation. a, Western blot analysis of nonphosphorylated (upper lane) and phosphorylated (lower lane) I-κBα molecule in the lysate of HeLa cells (lane 1, negative control), HeLa cells + TNF-α (lane 2, positive control), CD8+ blasts grown in culture medium (lane 3) and after incubation with sHLA-I (lane 4), OKT8 mAb cross-linked with GAM-Ig (OKT8-XL) (lane 5), sHLA-I in the presence of sanguinarine (lane 6), and OKT8-XL in the presence of sanguinarine (lane 7); b, NF-κB phosphorylation assessed by ELISA in activated CD8+ T cells incubated with culture medium (negative control), OKT3-XL (positive control), sHLA-I or OKT8-XL, and sHLA-I or OKT8-XL in the presence of bisindolylmaleimide I, Gö6976, rottlerin, and sanguinarine. The significance of the increase of NF-κB activity as compared with activated CD8+ T cells is indicated (∗, p < 0.01; ∗∗, p < 0.001). The significance of the inhibition of NF-κB activity by bisindolylmaleimide I, Gö6976, rottlerin, and sanguinarine vs sHLA-I or OKT8-XL is also shown (§, p < 0.05; §§, p < 0.01; §§§, p < 0.001; n.s., not significant). Values are mean ± SD of five independent experiments.

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In the promoter region of the FasL gene, it has also been identified the AP-1 binding domain, which is recognized by Jun-Fos heterodimers and cooperates with NF-AT to regulate gene expression (47, 48). Thus, we analyzed whether CD8 ligation with sHLA-I-induced AP-1 nuclear translocation and activation. Of note, sHLA-I/CD8 ligation did not induce the binding of AP-1 to a promoter region on DNA. Similar results were obtained when CD8 was cross-linked with GAM-Ig (Fig. 7, a and b). On the contrary, CD3/TCR cross-linking or cell treatment with PMA and ionomicin triggered AP-1 nuclear translocation (Fig. 7, a and b). These findings suggest that AP-1 is not responsible for FasL mRNA up-regulation after sHLA-I/CD8 engagement and further support the idea that signaling elicited through CD8 ligation can activate transcription factors different from those recruited by CD3/TCR stimulation.

FIGURE 7.

sHLA-I/CD8 ligation does not activate AP-1 transcription factor. a, EMSA of AP-1 nuclear translocation in CD8+ blasts grown in culture medium (lane 1) and in blasts incubated with sHLA-I molecules (lane 2), OKT8 mAb cross-linked with GAM-Ig (OKT8-XL) (lane 3), PMA plus ionomycin (lane 4), and OKT3-XL (lane 5). b, AP-1/c-jun phosphorylation assessed by ELISA in CD8+ T cells (control) and in cells incubated with sHLA-I, OKT8-XL, PMA plus ionomycin, and OKT3-XL. The significance of the increase of AP-1 activity as compared with control cells is indicated (∗, p < 0.001). Values are mean ± SD of three independent experiments.

FIGURE 7.

sHLA-I/CD8 ligation does not activate AP-1 transcription factor. a, EMSA of AP-1 nuclear translocation in CD8+ blasts grown in culture medium (lane 1) and in blasts incubated with sHLA-I molecules (lane 2), OKT8 mAb cross-linked with GAM-Ig (OKT8-XL) (lane 3), PMA plus ionomycin (lane 4), and OKT3-XL (lane 5). b, AP-1/c-jun phosphorylation assessed by ELISA in CD8+ T cells (control) and in cells incubated with sHLA-I, OKT8-XL, PMA plus ionomycin, and OKT3-XL. The significance of the increase of AP-1 activity as compared with control cells is indicated (∗, p < 0.001). Values are mean ± SD of three independent experiments.

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Herein, we have demonstrated that sHLA-I molecules trigger apoptosis in activated CD8+ EBV-specific CTL by FasL/Fas interaction as a consequence of CD8 engagement. The apoptotic effect of sHLA-I is detectable in CD8+ EBV-specific CTL, which express Fas after IL-2 activation and not in “resting” CD8+ CTL, which are Fas negative before IL-2 addition. Furthermore, we have characterized the biochemical pathways involved in the up-regulation of FasL mRNA, which follows sHLA-I/CD8 ligation. Indeed, the engagement of CD8 by sHLA-I evoked [Ca2+]i influx and activation of calmodulin kinase II, which in turn induces translocation of NF-AT to the nucleus. Then, NF-AT eventually triggers up-regulation of FasL mRNA, which is responsible for the induction of CD8+ T cell apoptosis. In addition, CD8 engagement by sHLA-I molecules induces the activation of [Ca2+]-independent PKC and the consequent nuclear translocation of NF-κB, which is responsible for FasL mRNA up-regulation.

Of interest, we have provided several evidences that sHLA-I molecules can induce FasL up-regulation and apoptosis in CD8+ EBV-specific CTL upon direct interaction with CD8 Ag. First, sHLA-I-induced apoptosis was strongly reduced by the covering of CD8 with specific mAb, suggesting that sHLA-I should interact with CD8 to deliver the apoptotic signal. Second, CD8+ EBV-specific CTL did not undergo apoptosis when sHLA-I monomers presenting a haplotype-specific peptide were mutated in the α3 domain, which is known to interact with CD8 (26). Finally, EBV-specific CTL underwent apoptosis when incubated with sHLA-I monomers, which were both haplotype restricted and unrestricted, each presenting either specific or nonspecific peptides. These findings strongly suggest that the interaction with the CD3/TCR complex is not required to trigger apoptosis and indicate that the engagement of CD8 alone by sHLA-I molecules is sufficient to deliver the apoptotic signal in EBV-specific CTL. They are also in agreement with literature data indicating that CD4 engagement with specific mAbs induced FasL up-regulation and apoptotic cell death without TCR involvement (49, 50, 51, 52). Other authors have claimed that sHLA-I molecules induce apoptotic death in allele-specific and Ag-specific T cells upon interaction with the TCR (12, 13, 14). However, the percentage of apoptotic cells (70–80%) induced by sHLA-I in bulk CD8+ T cell lines is higher than the percentage of sHLA-I/Ag-specific cell complexes within the bulk population (14, 19, 20). Moreover, it has been reported that various sHLA-I isoforms may induce different immunoregulatory effects in animal models (53, 54). Therefore, differences in sHLA-I preparations used in the experiments might explain the inconsistent results reported in the literature.

We have also found that the interaction of sHLA-I with CD8, likewise that of CD4 with anti-CD4 mAbs (55), induced the activation of p56lck and Zap-70 but not the recruitment of p59fyn. By contrast, in agreement with literature data (30, 31), CD3/TCR-mediated stimulation induced the activation of p59fyn, which has been reported to be an early event during T cell anergization (56, 57). Altogether, these findings would suggest that the ligation of CD8, as with CD4 engagement (58, 59), does not play just a costimulatory role enhancing TCR/HLA-Ag complex interaction but can also mediate signal transduction.

As has been reported for several activating membrane receptors, the engagement of CD8 by sHLA-I molecules induced calcium channels opening at the T cell surface and [Ca2+]i influx (60). Then, [Ca2+]i increase activated calmodulin kinase II promoting NF-AT nuclear translocation and FasL mRNA up-regulation (37, 39, 60). It is known that calmodulin kinase II activation is essential to induce mRNA up-regulation of several proinflammatory cytokines, which play a key role in inducing and maintaining immune response (38, 60, 61, 62, 63). Our present data indicate that calmodulin kinase II is involved in the regulation of expression of proapoptotic proteins and thus may play a role in the down-regulation of immune responses.

In addition, sHLA-I/CD8 ligation led to the activation of [Ca2+]-independent PKC, which phosphorylate I-κBα leading to NF-κB nuclear translocation (40, 41, 42, 43) and activate NF-AT through the JNK pathway (61, 64). Among PKC, the θ isoform seems to be preferentially involved in sHLA-I signal transduction. Cyclosporin A, which inhibits calmodulin kinase II/calcineurin pathway, abrogated the sHLA-I-induced mRNA FasL up-regulation and CD8+ T cell apoptosis, while the specific [Ca2+]-independent PKC inhibitor rottlerin (64, 65) did exert a lesser inhibitory effect. These findings indicate that different but converging biochemical pathways are recruited upon sHLA-I/CD8 ligation. In our experimental conditions, AP-1 transcription factor did not appear to be activated following CD8 engagement by sHLA-I. Of note, AP-1 translocation was observed upon CD3/TCR cross-linking or PMA-ionomycin incubation, suggesting that AP-1 is not necessary for FasL mRNA induction after sHLA-I/CD8 ligation, although the AP-1 binding site is able to link to this transcription factor. Although AP-1 is required for FasL up-regulation in several experimental models (47, 48), the removal of the promoter distal region of FasL, which contains the AP-1 binding site or mutations in the AP-1 binding site, does not affect the inducibility of FasL gene transcription (39, 66). Taken together, these data support the hypothesis of the existence, in the promoter region of FasL gene, of NF-AT and NF-κB binding sites, which can function independently from the AP-1 cofactors.

The above reported findings, which have been obtained using Ag-specific CD8+ T cells, were confirmed in parallel experiments performed using polyclonally activated CD8+ T blasts. Fas/FasL interactions play a crucial role in apoptosis induction (67, 68, 69) and in the establishment of Ag-specific T cell tolerance both during the intrathymic negative selection and adult life (69, 70, 71). The role of sHLA-I molecules in immune regulation is still not completely defined. The available data indicate that they inhibit the activity of alloreactive CTL through receptor blockade (9, 10, 11) and induce apoptosis in activated CD8+Fas+ T lymphocytes and NK cells by Fas/FasL interaction (12, 13, 14, 15, 16, 17, 18, 19, 20). Therefore, the secretion of sHLA-I molecules seems to constitute the afferent arm of a network involved in the down-regulation of immune responses (4, 5, 6). In this regard, >30 years ago, Balner and Van Rood (72) demonstrated that the injection of donor serum that contains sHLA-I molecules into the recipient before a skin transplant resulted in prolongation of graft survival. More recent studies in animals undergoing liver and heart allografts have provided convincing evidence that the injection into the portal vein of hepatocytes transfected with the soluble form of MHC molecules induces tolerance and transplant acceptance (53, 54). To the best of our knowledge, no data are available for the human system. Nevertheless, the finding that the sHLA-I serum level increases in certain immunopathological conditions in vivo (4, 5, 6, 7) suggests that this factor might be involved in the control of immune responses to avoid the dangerous overexpansion of CTLs by inducing apoptosis of activated CD8 cells.

In conclusion, the data of the present work better define the molecular mechanisms that induce the up-regulation of the soluble form of Fas and trigger CD8+ T cell apoptosis after the binding of sHLA-I molecules to CD8. They also indicate that sHLA-I/CD8 interaction may down-modulate through a non-allo-restricted mechanism CD8+ T cells activated in the course of an immune response. Finally, as FasL/Fas-induced apoptosis plays a central role in the regulation of immune responses, it may be suggested that sHLA-I molecules, which have been demonstrated to up-regulate FasL in activated CD8+ T cells, might represent a new biologic tool for immunotherapeutic intervention.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by grants from Ministero dell’Università e della Ricerca Scientifica e Technologica National Program 2002 “Apoptosis in autoimmune and oncohaematological diseases: potential diagnostic and therapeutic perspectives” (Grant 20020653936), from University of Genoa (to F.I. and F.P.), from Comitato Interministeriale per la Programmazione Economica-Immunoterapia 2 Project, and from Ministero della Sanità 2002-2004 (to A.P.).

4

Abbreviations used in this paper: sHLA-I, soluble HLA class I; PTK, protein tyrosine kinase; PKC, protein kinase C; FasL, Fas ligand; GAM-Ig, goat-anti-mouse Ig Ab; RNAi, RNA interference method.

1
Van Rood, J. J., A. van Leeuwen, M. C. T. van Santen.
1970
. Anti HL-A2 inhibitor in normal human serum.
Nature
226
:
366
-367.
2
Charlton, R. K., C. M. Zmijewski.
1970
. Soluble HL-A7 antigen: localization in the β-lipoprotein fraction of human serum.
Science
170
:
636
-637.
3
Allison, J. P., M. A. Pellegrino, S. Ferrone, G. N. Callahan, R. A. Reisfeld.
1977
. Biologic and chemical characterization of HLA antigens in human serum.
J. Immunol.
118
:
1004
-1009.
4
Puppo, F., M. Scudeletti, F. Indiveri, S. Ferrone.
1995
. Serum HLA class I antigens: markers and modulators of an immune response?.
Immunol. Today
16
:
124
-127.
5
Puppo, F., S. Brenci, L. Lanza, O. Bosco, M. A. Imro, M. Scudeletti, F. Indiveri, S. Ferrone.
1994
. Increased level of serum HLA class I antigens in HIV infection: correlation with disease progression.
Hum. Immunol.
40
:
259
-266.
6
Puppo, F., F. Indiveri, M. Scudeletti, S. Ferrone.
1997
. Soluble HLA antigens: new roles and uses.
Immunol. Today
18
:
154
-155.
7
Tsuchiya, N., M. Shiota, A. Yamamuchi, K. Ito.
1996
. Elevated serum level of soluble HLA class I antigens in patients with systemic lupus erythematosus.
Arthritis Rheum.
39
:
792
-796.
8
Parham, P., C. Clayberger, S. L. Zorn, D. S. Ludwig, G. K. Schoolnik, A. M. Krensky.
1987
. Inhibition of alloreactive cytotoxic T lymphocytes by peptides from the α2 domain of HLA-A2.
Nature
325
:
625
-628.
9
Puppo, F., S. Brenci, E. Montinaro, L. Lanza, P. Contini, M. Scudeletti, F. Indiveri.
1994
. Inhibition of alloreactive cytotoxic T cell activity by HIV-positive sera: potential role of circulating soluble HLA class I molecules.
AIDS Res. Hum. Retroviruses
10
:
1061
-1064.
10
Dal Porto, J., T. E. Johansen, B. Catipovic, D. J. Parfiit, D. Tuveson, U. Gether, S. Kozlowski, D. T. Fearon, J. P. Schneck.
1993
. A soluble divalent class I major histocompatibility complex molecule inhibits alloreactive T cells at nanomolar concentrations.
Proc. Natl. Acad. Sci. USA
90
:
6671
-6675.
11
Gansuvd, B., M. Haghiara, B. Munkhbat, N. Kanai, N. Morita, N. Munkhtuvshin, J. Chargui, S. Kato, T. Hotta, K. Tsuji.
2000
. Inhibition of Epstein-Barr virus (EBV)-specific CD8+ cytotoxic T lymphocyte (CTL) activity by soluble HLA class I in vitro.
Clin. Exp. Immunol.
119
:
107
-114.
12
Zavazava, N., M. Krönke.
1996
. Soluble HLA class I molecules induce apoptosis in alloreactive cytotoxic T lymphocytes.
Nat. Med.
2
:
1005
-1010.
13
Hansen, B., E. Janssen, T. Machleidt, M. Krönke, N. Zavazava.
1998
. Purified truncated recombinant HLA-B7 molecules abrogate cell function in alloreactive cytotoxic T lymphocytes by apoptosis induction.
Transplantation
66
:
1818
-1822.
14
Smith, M. A., B. Naziruddin, N. J. Poindexter, A. E. Haynes, T. Howard, T. Mohanakumar.
2000
. Liver transplant recipient sera derived soluble HLA mediates allele specific CTL apoptosis.
Transplantation
69
:
157
-162.
15
Fournel, S., M. Aguerre-Girr, X. Huc, F. Lenfant, A. Alam, A. Toubert, A. Bensussan, P. Le Bouteiller.
2000
. Soluble HLA-G1 triggers CD95/CD95 ligand-mediated apoptosis in activated CD8+ cells by interaction with CD8.
J. Immunol.
164
:
6100
-6104.
16
Contini, P., M. Ghio, A. Poggi, G. Filaci, F. Indiveri, S. Ferrone, F. Puppo.
2003
. Soluble HLA-A, -B, -C and -G molecules induce apoptosis in T and NK CD8+ cells and inhibit cytotoxic T cell activity through CD8 ligation.
Eur. J. Immunol.
33
:
125
-134.
17
Spaggiari, G. M., P. Contini, R. Carosio, M. Arvigo, M. Ghio, D. Oddone, A. Dondero, M. R. Zocchi, F. Puppo, F. Indiveri, A. Poggi.
2002
. Soluble HLA class I molecules induce natural killer cell apoptosis through the engagement of CD8: evidence for a negative regulation exerted by CD94/NKG2A complex and KIR2D.
Blood
99
:
1706
-1714.
18
Spaggiari, G. M., P. Contini, A. Dondero, R. Carosio, F. Puppo, F. Indiveri, M. R. Zocchi, A. Poggi.
2002
. Soluble HLA class I induces NK cell apoptosis upon the engagement of killer activating HLA class I receptors through FasL/Fas interaction.
Blood
100
:
4098
-4107.
19
Puppo, F., P. Contini, M. Ghio, S. Brenci, M. Scudeletti, G. Filaci, S. Ferrone, F. Indiveri.
2000
. Soluble human MHC class I molecules induce soluble Fas ligand secretion and trigger apoptosis in activated CD8 Fas(CD95)+ T lymphocytes.
Int. Immunol.
12
:
195
-203.
20
Contini, P., M. Ghio, A. Merlo, S. Brenci, G. Filaci, F. Indiveri, F. Puppo.
2000
. Soluble HLA class I/CD8 ligation triggers apoptosis in EBV-specific CD8+ cytotoxic T lymphocytes by Fas/Fas-ligand interaction.
Hum. Immunol.
61
:
1347
-1351.
21
Russo, C., M. Fotino, A. Carbonara, S. Ferrone.
1987
. A double determinant immunoassay for HLA class I typing using serum as an antigen source.
Hum. Immunol.
19
:
69
-77.
22
Gavioli, R., P. O. de Campos-Lima, M. G. Kurilla, E. Kieff, G. Klein, M. G. Masucci.
1992
. Recognition of the Epstein-Barr virus-encoded nuclear antigens EBNA-4 and EBNA-6 by HLA-A11-restricted cytotoxic T lymphocytes: implication for down-regulation of HLA-A11 in Burkitt lymphoma.
Proc. Natl. Acad. Sci. USA
89
:
5862
-5866.
23
Coligan, J. E..
2000
. Immune-complex assays for tyrosine protein kinases. J. E. Coligan, and A. M. Kruisbeek, and D. H. Margulies, and E. M. Shevach, and W. Strober, eds.
Current Protocols in Immunology
11.4.1
-11.4.9. John Wiley & Sons, New York.
24
Muller, M. M., E. Schreiber, W. Schaffner, P. Matthias.
1989
. Rapid test for in vivo stability and DNA binding of mutated octamer binding proteins with “mini-extracts” prepared from transfected cells.
Nucleic Acids Res.
17
:
6420
25
Mastaroni, M., M. Bazzaro, F. Micheletti, R. Gavioli, R. Tomatis.
2001
. Peptide analogues of a subdominant epitope expressed in EBV-associated tumor: synthesis and immunological activity.
J. Med. Chem.
44
:
2370
-2373.
26
Salter, R. D., R. J. Benjamin, P. K. Wesley, S. E. Buxton, T. P. J. Garret, C. Clayberger, A. M. Krensky, A. M. Norment, D. R. Littman, P. Parham.
1990
. A binding site on the T cell co-receptor CD8 on the α3 domain of HLA-A2.
Nature
345
:
41
-46.
27
Veillette, A., M. A. Bookman, E. M. Horak, J. B. Bolen.
1988
. The CD4 and CD8 T cell surface antigens associated with the internal membrane tyrosine-protein kinase p56lck.
Cell
55
:
301
-308.
28
Barber, E. K., J. D. Dasgupta, S. F. Schlossmann, J. M. Trevillyan, C. E. Rudd.
1989
. The CD4 and CD8 antigens are coupled to a protein-tyrosine kinase (p56lck) that phosphorylates the CD3 complex.
Proc. Natl. Acad. Sci. USA
86
:
3277
-3281.
29
Kim, P. W., Z. Y. J. Sun, S. C. Blacklow, G. Wagner, M. J. Eck.
2003
. A zinc clasp structure tethers Lck to T cell coreceptors CD4 and CD8.
Science
301
:
1725
-1728.
30
Samelson, L. E., A. F. Phillips, E. T. Louong, R. D. Klausner.
1990
. Association of the protein-tyrosine kinase with the T cell antigen receptor.
Proc. Natl. Acad. Sci. USA
87
:
4358
-4362.
31
Sarosi, G. P., P. M. Thomas, M. Egerton, A. F. Phillips, K. W. Kim, E. Bonvini, L. E. Samelson.
1992
. Characterization of the T cell antigen receptor-p60fyn protein tyrosine kinase association by chemical cross-liking.
Int. Immunol.
4
:
1211
-1217.
32
Chan, A. C., M. Iwashima, C. W. Turck, A. Weiss.
1992
. Zap-70: a 70 kd protein-tyrosine kinase that associates with TCR ζ-chain.
Cell
71
:
649
-662.
33
Chan, A. C., B. A. Irving, J. D. Fraser, A. Weiss.
1991
. The z chain is associated with a tyrosine kinase and upon T cell antigen receptor stimulation associates with ZAP-70, a 70-kDa tyrosine phosphoprotein.
Proc. Natl. Acad. Sci. USA
88
:
9166
-9170.
34
Wange, R. L., A. N. T. Kong, L. E. Samelson.
1992
. A tyrosine phosphorylated 70-kDa protein binds a photoaffinity analogue of ATP and associates with both the ζ chain and CD3 components of the activated T cell antigen receptor.
J. Biol. Chem.
267
:
11685
-11688.
35
Burkhardt, A. L., B. Stealey, R. B. Rowley, S. Mahajan, M. Prendergast, J. Fargnoli, J. B. Bolen.
1994
. Temporal regulation of non-transmembrane protein tyrosine kinase enzyme activity following T cell antigen receptor engagement.
J. Biol. Chem.
269
:
23642
-23647.
36
Anel, A., A. M. O’Rourke, A. M. Kleinfeld, M. F. Mescher.
1996
. T cell receptor and CD8-dependent tyrosine phosphorylation events in cytotoxic T lymphocytes: activation of p56lck by CD8 binding to class I protein.
Eur. J. Immunol.
26
:
2310
-2319.
37
Norian, L. A., K. M. Latinis, G. A. Koretzky.
1998
. A newly identified response element in the CD95 ligand promoter contributes to optimal inducibility in activated T lymphocytes.
J. Immunol.
161
:
1078
-1082.
38
Dolmetsch, R. E., K. Xu, R. S. Lewis.
1998
. Calcium oscillation increase the efficiency and specificity of gene expression.
Nature
392
:
933
-936.
39
Latinis, K. M., L. A. Norian, S. L. Eliason, G. A. Koretzky.
1997
. Two NF-AT transcription factor binding sites participate in the regulation of CD95 (Fas) ligand expression in activated human T cells.
J. Biol. Chem.
272
:
31427
-31434.
40
Kasibhatla, S., L. Genestier, D. R. Green.
1999
. Regulation of Fas-ligand expression during activation-induced cell death in T lymphocytes via nuclear factor κB.
J. Biol. Chem.
274
:
987
-992.
41
Arendt, C. W., B. Albrecht, T. J. Soos, D. R. Littman.
2002
. Protein kinase C-θ: signalling from the center of the T cell synapse.
Curr. Opin. Immunol.
14
:
323
-330.
42
Lin, X., A. O’Mahony, Y. Mu, R. Geleziunas, W. Green.
2000
. Protein kinase C-θ participates in NF-κB activation induced by CD3-CD28 costimulation through selective activation of Iκ-B kinase β.
Mol. Cell. Biol.
20
:
2933
-2940.
43
Altman, A., N. Isakov, G. Baier.
2000
. Protein kinase Cθ: a new essential superstar on the T cell stage.
Immunol. Today
21
:
567
-573.
44
Villalba, M., S. Kasibhatla, L. Genestier, A. Mahboubi, D. R. Green, A. Altman.
1999
. Protein kinase Cθ cooperates with calcineurin to induce Fas ligand expression during activation-induced T cell death.
J. Immunol.
163
:
5813
-5819.
45
Martin-Baron, G., M. G. Kazanietz, H. Mischak, P. M. Blumberg, G. Kochs, H. Hug, D. Marmé, C. Schächtele.
1993
. Selective inhibition of protein kinase C isozymes by the indolocarbazole Gö6976.
J. Biol. Chem.
268
:
9194
-9197.
46
Chaturvedi, M. M., A. Kumar, B. G. Darnay, G. B. N. Chainy, S. Agarwal, B. B. Aggarwal.
1997
. Sanguinarine (pseudochelerytrine) is a potent inhibitor of NF-κB activation, Iκ-Bα phosphorylation, and degradation.
J. Biol. Chem.
272
:
30129
-30134.
47
Kasibhatla, S., S. Brunner, G. Genestier, E. Echeverri, A. Mahboubi, D. R. Green.
1998
. DNA damaging agents induce expression of Fas ligand and subsequent apoptosis in T lymphocytes via the activation of NF-κB and AP-1.
Mol. Cell
1
:
543
-551.
48
Eichhorst, S. T., M. Muller, M. Li-Weber, H. Schulze-Bergkamen, P. Angel, P. H. Krammer.
2000
. A novel AP-1 Element in the CD95 ligand promoter is required for induction of apoptosis in hepatocellular carcinoma cells upon treatment with anticancer drugs.
Mol. Cell. Biol.
20
:
7826
-7837.
49
Veillette, A., M.A. Bookman, E. M. Horak, L. E. Samelson, J. B. Bolen.
1989
. Signal transduction through the CD4 receptor involves the activation of the internal membrane tyrosine-protein kinase p56lck.
Nature
338
:
257
-259.
50
Gozalez-Garcia, A., L. R. Borlado, E. Leonardo, I. Mérida, C. Martinez, A. C. Carrera.
1997
. Lck is necessary and sufficient for Fas-ligand expression and apoptotic cell death in mature cycling T cells.
J. Immunol.
158
:
4104
-4112.
51
Newell, M. K., L. J. Haughn, C. R. Maroun, M. H. Julius.
1990
. Death of mature T cells by separate ligation of CD4 and the T cell receptor for antigen.
Nature
347
:
286
-289.
52
Eischen, C. M., B. L. Williams, W. Zhang, L. E. Samelson, D. H. Lynch, R. T. Abraham, P. J. Leibson.
1997
. Zap-70 tyrosine kinase is required for the up-regulation of Fas ligand in activation-induced T cell apoptosis.
J. Immunol.
159
:
1135
-1139.
53
Scherer, M. N., C. Graeb, S. Tange, C. Dyson, K.W. Jauch, E. K. Geissler.
2000
. Immunologic considerations for therapeutic strategies utilizing allogeneic hepatocytes: hepatocyte-expressed membrane-bound major histocompatibility complex class I sensitizes while soluble antigen suppresses the immune response in rats.
Hepatology
32
:
999
-1007.
54
Geissler, E. K., W. J. Korzun, C. Graeb.
1997
. Secreted donor-MHC class I antigen prolongs liver allograft survival and inhibits recipient anti-donor cytotoxic T lymphocyte responses.
Transplantation
64
:
782
-786.
55
Luo, K. X., B. M. Sefton.
1990
. Cross-linking of T cell surface molecules CD4 and CD8 stimulates phosphorylation of the lck tyrosine protein kinase at the autophosphorylation sites.
Mol. Cell. Biol.
10
:
5305
-5313.
56
Welke, J., N. Zavazava.
2002
. p59fyn is up-regulated in anergic CD8+ T cells.
Hum. Immunol.
63
:
834
-843.
57
Quill, H., M. P. Riley, E. A. Cho, J. E. Casnellie, J. C. Reed, T. Torigoe.
1992
. Anergic Th1 cells express altered level of the protein tyrosine kinases p56lck and p59fyn.
J. Immunol.
149
:
2887
-2893.
58
Miceli, M. C., P. von Hoegen, J. R. Parnes.
1991
. Adhesion versus coreceptor function of CD4 and CD8: role of the cytoplasmic tail in coreceptor activity.
Proc. Natl. Acad. Sci. USA
88
:
2623
-2627.
59
Miceli, M. C., J. R. Parnes.
1993
. Role of CD4 and CD8 in T cell activation and differentiation.
Adv. Immunol.
53
:
59
-122.
60
Bootman, M. D., P. Lipp, M. J. Berridge. The organisation and functions of local Ca2+ signals.
J. Cell Sci.
114
:
2213
-2222.
61
Wang, Y., L. Liang, W. J. Esselman.
2000
. Regulation of the calcium/NF-AT T cell activation pathway by the D2 domain of CD45.
J. Immunol.
164
:
2557
-2564.
62
Fladmark, K. E., O. T. Brustugun, G. Mellgren, C. Krakstad, R. Bøe, O. K. Vintermyr, H. Schulman, S. O. Døskeland.
2002
. Ca2+/calmodulin-dependent protein kinase II is required for microcystin-induced apoptosis.
J. Biol. Chem.
277
:
2804
-2811.
63
Harris, D. T., W. J. Kozumbo, J. E. Testa, P. A. Cerutti, J. C. Cerottini.
1998
. Molecular mechanisms involved in T cell activation. III. The role of extracellular calcium in antigen-induced lymphokine production and interleukin 2-induced proliferation of cloned cytotoxic T lymphocytes.
J. Immunol.
140
:
921
-927.
64
Villunger, A., N. Ghaffari-Tabrizi, I. Tinhofer, N. Krunböck, B. Bauer, T. Schneider, S. Kasibhatla, R. Greil, G. Baier-Bitterlich, F. Überall, et al
1999
. Synergistic action of protein kinase Cθ and calcineurin is sufficient for Fas ligand expression and induction of a crmA-sensitive apoptosis pathway in Jurkat T cells.
Eur. J. Immunol.
29
:
3549
-3561.
65
Monk, C. R., H. Kupfer, I. Tamir, A. Barlow, A. Kupfer.
1997
. Selective modulation of protein kinase Cθ during T cell activation.
Nature
385
:
83
-86.
66
Kasibhatla, S., L. Genestier, D. R. Green.
1999
. Regulation of Fas-ligand expression during activation-induced cell death in T lymphocytes via nuclear factor κB.
J. Biol. Chem.
274
:
987
-992.
67
Nagata, S..
1994
. Fas and Fas ligand: a death factor and its receptor.
Adv. Immunol.
57
:
129
-144.
68
Van Parijs, L., A. K. Abbas.
1996
. Role of Fas-mediated cell death in the regulation of immune responses.
Curr. Opin. Immunol.
8
:
355
-361.
69
Mountz, J. D., T. Zhou, J. Wu, W. Wang, X. Su, J. Cheng.
1995
. Regulation of apoptosis in immune cells.
J. Clin. Immunol.
15
:
1
-16.
70
Yonehara, S., Y. Nishimura, S. Kishil, M. Yonehara, K. Takazawa, T. Tamatani, A. Ishii.
1994
. Involvement of apoptosis antigen Fas in clonal deletion of human thymocytes.
Int. Immunol.
6
:
1849
-1856.
71
Lynch, D. H., F. Ramsdell, M. R. Alderson.
1995
. Fas and FasL in the homeostatic regulation of immune responses.
Immunol. Today
16
:
569
-574.
72
Balner, H., J. J. Van Rood.
1971
. Transplantation antigens in rhesus monkeys.
Nature
232
:
121