Functional modulation of expanded CD8+ synovial fluid T cells by NK cell receptor expression in HLA-B27-associated reactive arthritis

Nicolas Dulphy1, Claire Rabian1, Corinne Douay1, Odile Flinois1, Saddek Laoussadi2, Jens Kuipers3, Ryad Tamouza1, Dominique Charron1 and Antoine Toubert1

1 Laboratoire d‘Immunologie et d‘Histocompatibilité, INSERM U396, Centre G. Hayem, Université Paris VII, Institut Universitaire d’Hématologie, Hôpital Saint-Louis, Avenue C. Vellefaux, 75475 Paris Cedex 10, France 2 Service de Rhumatologie A, Hôpital Cochin, 75679 Paris Cedex 14, France 3 Division of Rheumatology, Medical School, 30625 Hannover, Germany

Correspondence to: A. Toubert; E-mail: toubert{at}histo.chu-stlouis.fr
Transmitting editor: T. Sasazuki


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The aim of this study was to determine whether NK cell receptor (NKR) expression could modulate cytotoxicity of oligoclonal CD8+ T cells present in the synovial fluid (SF) of HLA-B27-reactive arthritis (ReA) patients, especially in a TCRBV1 population shared among different patients and cytotoxic toward HLA-B27. A CD8+ T cell line, two TCRBV1 lines and clones were isolated from the SF of an HLA-B27+ ReA patient, and tested with mAb specific for Ig-like (KIR2DL1, KIR2DL2, KIR3DL1 and ILT2) and CD94 C-type lectin NKR. Transcripts for NKG2 subunits (NKG2A–2E) associated with CD94 were also evaluated. Function was tested in a 51Cr-release cytotoxic assay. We found stable but distinct levels of CD94/NKG2 complexes at the surface of T cell lines and clones. Different NKG2 members could be associated with CD94, either inhibitory (NKG2A/B) or activating (NKG2C). The inhibitory ILT2 receptor could also be differently expressed, but other Ig-like NKR were negative. Functionally, one TCRBV1 line and clones with a high CD94/NKG2A expression did not lyse B27+ targets. Another TCRBV1 line with the same TCRBV1 rearrangement had a low expression of CD94/NKG2A, but expressed NKG2C transcripts and was cytotoxic toward HLA-B27. HLA-B27 is a ligand for ILT2 and we observed an inhibitory effect of ILT2 engagement on B*2705 targets in blockade experiments. Altogether, these data indicate a high degree of heterogeneity in the expression of NKR by intrasynovial CD8+ T cells which could modulate their cytotoxicity and play a role in the control of this HLA class I-associated autoimmune disease.

Keywords: autoimmunity, MHC, oligoclonal T cell expansions, spondyloarthropathies


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Spondyloarthropathies (SA) constitute a group of inflammatory rheumatic diseases of special interest because of a strong association with the HLA class I molecule HLA-B27 (1,2). Predisposing genetic factors, of which the molecule HLA-B27 (and its worldwide prevalent subtype B*2705) is the major one, interplay with environmental factors in the pathogenesis. This is best evidenced by the occurrence of reactive arthritis (ReA) following outbreaks of either Shigella, Yersinia or Salmonella infections. The three-dimensional structure of HLA-B27 is known as well as peptide-binding rules and the definition of HLA-B27 subtypes (3,4). More recently, a peculiar misfolding of the HLA-B27 heavy chain has been suggested as a potential mechanism in the disease pathogenesis (5).

The disease association with an HLA class I molecule is a strong rationale to study T cell functions at the inflammatory site. In SA, the arthritogenic peptide hypothesis proposes that an antigenic peptide derived from an autoantigen or from a triggering bacteria would be presented in an HLA-B27-restricted manner to CD8+ T cells. Indeed, synovial fluid (SF) T cell lines and clones with cytotoxic activity toward HLA-B27 or HLA-B27-presented bacterial peptides have been described in this disease (68). Analysis of the TCR ß chain complementarity-determining region 3 (CDR3) size diversity (‘spectratyping’ or ‘Immunoscope’ methods) is a powerful tool to define T cell expansions suggestive of an antigen-driven response. We previously described {alpha}ß oligoclonal expansions in SF CD8+ T lymphocytes defined by BV–BJ gene transcripts common in different patients (6). Identical CDR3 sequences were observed in TCRBV1 expansions in two different patients and with a previously reported sequence of a cytolytic clone derived in vitro from a Salmonella triggered ReA patient (8). Notably, shared TCRBV1 usage and junctional sequences in intrasynovial T cell expansions have now been found in six ReA-affected patients investigated in three different laboratories. A canonical CDR3 sequence could be defined which characterizes these shared expanded T cells as TCRBV1/23–CASSVG(V/I/L)(Y/F)STDTQYF–J2S3 (May et al., submitted). This strong argument in favor of the arthritogenic peptide hypothesis prompted us to focus on functional studies of this T cell population.

HLA class I molecules have the dual function of presenting intracellular peptides to CD8+ T cells, and of modulating the activity of NK cells and of subsets of {alpha}ß and {gamma}{delta} T cells upon recognition of receptors called NK receptors (NKR) (911). NKR are divided in two structural groups, killer cell Ig-like receptors (KIR) and C-type lectin-like receptors. NKR are able to activate or inhibit lysis from the effector cell depending on their intracytoplasmic sequence and several NKR may be expressed on a given NK or T cell (9,11). In humans, KIR belong to a multigenic and multiallelic family among which KIR2DL1 and 2DL2 recognize products of HLA-Cw alleles, and KIR3DL1 interacts with multiple HLA-B molecules of the Bw4 allotype (1114). Lectin-like CD94/NKG2 heterodimers are specific for the non-classical class I molecule HLA-E which presents peptides from signal sequences of classical HLA class I molecules (15). The nature of the NKG2 subunit can modulate the function of the CD94/NKG2 complex, CD94/NKG2A and CD94/NKG2C being respectively inhibitory or activating receptors. Leukocytes possess a third kind of inhibitory or activating receptor, the Ig-like transcripts (ILT) family (16). Among those, ILT2 is a four Ig-domain protein expressed by NK and T cells (17). Its inhibitory activity was shown with a wide spectrum of ligands including HLA-B*2705 (1820).

NKR are not NK-specific markers. T cell NKR expression, especially CD94/NKG2A, has been reported, mainly in CD8+ oligoclonal populations of the CD28CD45RACD45RO+ memory phenotype (21). The expression of several NKR on a single T cell and the broad specificity of NKR for multiple MHC class I molecules explains the complexity of the biological significance of NKR on T cells (22). Diverse NKR phenotypes have been reported on T cells presenting the same {alpha}ß TCR (2325), increasing the heterogeneity of T cells that constitute a clone as defined by the TCR. In rheumatoid arthritis (RA), expanded CD4+CD28 T cells notably express activating KIR molecules which could participate in a clonal outgrowth of autoreactive T cells and tissue injury (26,27). Therefore we asked whether NKR could modulate the activity of CD8+ intrasynovial T cell expansions in this typical example of HLA class I-associated autoimmune disease. To answer that question, we studied SF T cell lines and clones isolated from an HLA-B27 ReA patient (PG) with defined in situ CD8+ oligoclonal expansions cytotoxic toward HLA-B27 (6). We observed a heterogeneous expression of some inhibitory and activatory receptors correlating with the HLA-B27-restricted cytotoxic activity. These results indicate that the expression of different NKR could modulate T cell function at inflammatory sites in HLA-B27-associated arthritis.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cell culture
Patient PG (HLA-B27+) was affected by a recent-onset form of ReA after a bacterial enteric infection (6). SF lymphocytes were obtained under informed consent from knee effusions during SF analysis before steroid administration. A CD8+-purified SF cell line (PG-CD8) and subsequent cell lines were cultured in medium supplemented with rIL-2 (Promocell, Heidelberg, Germany) at 150 IU/ml, purified phytohemagglutinin (PHA)-L at 0.5 µg/ml (Leukoagglutinin; Sigma, St Louis, MO) and irradiated (50 Gy) allogeneic feeder cells in which HLA-B27+ cells had been excluded. Cells were maintained by adding rIL-2 at 150 IU/ml twice a week, and were re-stimulated with irradiated feeder cells and PHA-L every 2 weeks. TCRBV1+ cells were purified from PG-CD8 in two successive runs by means of a primary TCRBV1 mAb (BL37.2; Immunotech, Marseille, France) and secondary magnetic beads (Dynal, Oslo, Norway). This cell-sorting protocol was performed 2 times to obtain the BV1-1 and BV1-2 cell lines. T cell clones were separated from PG-CD8 and during the purification of BV1-1 after limiting dilution into 96 V-bottom wells plate at 0.3 cells/well. Clones were expanded and stimulated as described above.

Antibodies and flow cytometry analysis
mAb specific for TCRBV1 (BL37.2), KIR2DL1 (EB6), KIR2DL2 (GL183), CD94 (HP-3B1), CD45RO (UCHL1), CD3 (UCHT1) and CD94/NKG2A (Z199) were purchased from Immunotech (Marseille, France). mAb specific for CD28 (L293), CD56 (NCAM16.2), CD8 (SK1) and KIR3DL1 (DX9) were supplied by Becton Dickinson (Mountain View, CA). ILT2-specific mAb (HP-F1) was kindly provided by Dr F. Navarro and Dr M. Lopez-Botet. W6/32 (monomorphic HLA class I-specific antibody) and OKT3 (anti-CD3) ascites were produced in the laboratory. Flow cytometry analysis was performed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) as previously described (6). For peripheral blood lymphocytes (PBL) or SF analysis (CellQuest software; Becton Dickinson), 104 events were gated on CD3 or CD8 expression.

RNA extraction, cDNA synthesis and T cell repertoire analysis
After at least 24 h of culture, T cell lines were washed twice in PBS and RNA was extracted from 10 x 106 cells by lysis in guanidium thiocyanate buffer. cDNA synthesis and Immunoscope TCRBV analysis were performed as previously described (6) for the T cell lines.

CD94- and NKG2-specific PCR
The CD94 and NKG2A/B, D and E primers were used as described (28), and NKG2C primers were as in (29). Aliquots of 0.5 µl of cDNA synthesis reaction were amplified in 25 µl reactions with each pair of primers. The final concentration was 0.5 mM for each primer, 0.2 mM dNTP and 1 mM MgCl2 in Taq polymerase buffer in the presence of 1 U of Taq polymerase (Promega, Madison, WI). The PCR cycle profile was denaturation at 94°C for 30 s, annealing for 45 s and primer extension at 72°C for 45 s for 40 cycles, and a final polymerization step of 5 min at 72°C, on a DNA thermal cycler (model 2400; Perkin-Elmer, Norwalk, CT). Annealing temperatures were 55°C for CD94 amplification, 63°C for NKG2A/B amplification, 50°C for NKG2C amplification, 58.5°C for NKG2D amplification and 59.5°C for NKG2E amplification. PCR amplification products were analyzed in a 2% agarose gel stained with ethidium bromide.

51Cr-release assay
Cytotoxicity was measured by a standard 5 h 51Cr-release assay and the Epstein–Barr virus-transformed B lymphoblastoid cell lines (B-LCL) used as targets have been previously described (6). In blocking experiments, targets were preincubated for 1 h with a 1/100 dilution of HP-F1 ascites specific for ILT2 or the isotypic IgG1 control (Sigma) as in (6).


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Synovial fluid T cell lines with the same TCRB rearrangement have different cytotoxic activities
We described previously shared SF CD8+ oligoclonal expansions characterized by a TCRBV1–J2S3 rearrangement of the TCRB chain in three different HLA-B27 ReA patients (6). This observation has recently been extended in two other independent laboratories (May et al., submitted) arguing for a pathogenic role of this T cell population in agreement with the ‘arthritogenic peptide’ hypothesis. To study the function of the expanded TCRBV1 cells, we established three T cell lines from the SF of patient PG. CD8+ T cells were sorted from SF and expanded in short-term in vitro cultures (PG-CD8) to preserve the in vivo observed TCR repertoire and function (6). In addition, two TCRBV1 cell lines were independently sorted and expanded (BV1-1 and BV1-2) from PG-CD8. Immunoscope profiles of the dominant TCRBV1 expansion were similar in both lines (Fig. 1) and with the previously identified ex vivo shared oligoclonal expansion (6). We tested the cytotoxic activity of the three polyclonal T cell lines by 51Cr-release assay. Targets were B*2705 B-LCL, including the autologous PG-EBV, ADA, HOM-2 and A16, these last two lines sharing B*2705 only as a HLA class I-restriction element with the effector cells. PG-CD8 was able to kill ADA, HOM-2 and A16, but neither the autologous nor the irrelevant B-LCL COX (Fig. 2) (6). A different pattern of lysis was observed for BV1-1 and BV1-2. BV1-2 recognized HOM-2 with a lytic efficiency comparable to that of PG-CD8, but lysis of ADA was strongly reduced and A16 was not lysed at all. None of these targets was lysed by BV1-1 (Fig. 2). Thus, there was an important difference in the cytotoxic function between these TCRBV1+ lines selected from the same CD8+ SF T cell expansion. The pattern of reactivity was stable repeatedly in three different experiments. The HLA class I-restricted recognition of PG-CD8 and BV1-2 was confirmed by the blockade of HOM-2 lysis with the HLA class I-specific mAb W6/32 [(6) and data not shown]. Targets expressed B*2705 at the same level and the lytic potential of BV1-1 was checked in redirected lysis experiments with a CD3-specific mAb (data not shown). These data suggested that TCRBV1–J2S3 cells could play a pathogenic role due to their HLA-B27-restricted cytotoxicity. However, these T cells may present a different pattern of cytotoxicity depending on the target cell. We therefore hypothesized that NKR expression could modulate the TCR-mediated B27-directed lysis.



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Fig. 1. TCR ß chain CDR3 size analysis in clinical samples (PBL and SF) and lines (PG-CD8, and TCRBV1+ lines BV1-1 and BV1-2) from patient PG. BV1-BC PCR products were copied in run-off reactions primed with BC and BJ fluorescent internal primers. The 10-amino-acid CDR3 size of the oligoclonal TCRBV1-BJ2S3 expansion is indicated on the x-axis. Percentages indicate the frequency of the whole TCRBV1 family (left) or of the oligoclonal BV1-BJ2S3 expansion (right) within PBL, right knee SF lymphocytes (SF), the CD8 (PG-CD8), and the two BV1 sorted lines BV1-1 and BV1-2. They were calculated as ratios of the fluorescence intensity of the peaks to the total signal from the 22 BV-BC or 13 BV1-BJ semiquantitative PCR amplifications as in (6).

 


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Fig. 2. Cytotoxic activity of PG-CD8, BV1-1 and BV1-2 cell lines tested on the B*2705+ B lymphoblastoid cell lines (B-LCL) A16, ADA, HOM-2, PG-EBV and on the irrelevant COX B-LCL. B*2705 is the only HLA class I molecule shared between the effector T cell lines and B-LCL targets A16 and HOM-2. Results are expressed as the percentage of specific lysis in a 5 h 51Cr-release assay at an effector:target (E:T) cell ratio of 25:1. One representative experiment out of three is shown.

 
NKR cell surface expression on SF CD8+ T cells
To ascertain that differences in NKR expression would not be due to in vitro culture, we investigated NKR expression at the surface of fresh SF T cells in comparison to PBL in patient PG (Fig. 3). The lectin-like NKR CD94 and the inhibitory complex CD94/NKG2A were over-expressed 10-fold in joints in comparison to PBL. Expression of CD94 and CD94/NKG2A was 3.9 and 2% respectively in peripheral T cells, and 38.6 and 26.2% in the right knee SF T cells. A more in-depth phenotypic analysis was performed on the various CD8+ SF cell lines. NKR KIR2DL1 and KIR2DL2, specific for HLA-C allotypes (30), and p70 or KIR3DL1, specific for HLA-Bw*04 molecules such as B*2705 (12,14,31), were not detected on PG-CD8 (Fig. 4A and B). The CD94 member of the lectin-like heterodimer was present on ~30% of PG-CD8 cells with a CD45RO+CD28 memory phenotype. This heterogeneous profile was also observed with mAb Z199 specific for the CD94/NKG2A inhibitory complex which labeled 20% of cells. Therefore, CD94 and CD94/NKG2A expression levels were similar in CD8+ T cells from SF without in vitro cell culture and in the CD8+ sorted T cell line. About 15% of cells expressed a low level of ILT2, an inhibitory ligand for many HLA class I molecules including HLA-B27. CD94 and the inhibitory heterodimer CD94/NKG2A were respectively expressed on 70 and 60% of Vß1+ cells, whereas only 30% of Vß1 cells were CD94+ and 17% CD94/NKG2A+ (Fig. 4A). Thus the inhibitory receptor CD94/NKG2A was selectively expressed on the major TCRBV1+ intrasynovial T cell expansions of this patient. However, although sharing a similar TCRB rearrangement, expression levels of the lectin-like NKR and of ILT2 were clearly different on both BV1 cell lines with a balance between CD94/NKG2A and ILT2 (Fig. 4B). NKR expression was stable at the surface of these lines. BV1-1 cells expressed CD94 and CD94/NKG2A at a high level, and with a very homogeneous profile, while the majority of the BV1-2 cell line expressed a low to intermediate level of CD94 and CD94/NKG2A. Conversely, ILT2 was expressed on 16% of BV1-1 cells at a low level, while 88% of BV1-2 cells were highly positive for ILT2.



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Fig. 3. Three-color flow cytometry analysis of PBL, right knee (SF-R) and left knee (SF-L) SF lymphocytes of the ReA-affected patient PG. In total, 104 events were analyzed and results expressed as percentages of cells staining above the background level. Percentages of CD8bright cells were obtained on the gated CD3+ T cells and the other phenotypic markers (CD45RO, CD28, CD56 and CD94) on the gated CD8bright population within CD3+ T cells. NKG2A refers to the CD94/NKG2A complex stained by mAb Z199.

 


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Fig. 4. (A) Percentages of CD94- and CD94/NKG2A-expressing cells within TCRBV1+ and TCRBV1populations from the PG-CD8 SF T cell line (PG-CD8) measured by two-color flow cytometry analysis. PG-CD8 cells were incubated with the BV1 region-specific mAb BL37.2 and with mAb specific for NKR of the Ig superfamily (KIR2DL1 and KIR2DL2) or of the C-lectin type (CD94 and CD94/NKG2A). (B) Flow cytometry analysis on PG-CD8, BV1-1 and BV1-2 cell lines of Ig-like (KIR2DL1, KIR2DL2 and KIR3DL1), lectin-like NKR (CD94 and CD94/NKG2A) and ILT2. KIR2DL1, KIR2DL2 and KIR3DL1 expression was negative in both TCRBV1 cell lines according to results obtained with PG-CD8 (data not shown). Background staining of Ig isotypic control is indicated by dotted lines. Fluorescence staining (logarithmic scale) is indicated on the x-axis and the relative cell number on the y-axis.

 
Different expression of NKG2 members in CD94/NKG2 complexes
Different NKG2 molecules can be associated with CD94: NKG2A but also NKG2C or E (32). The CD94/NKG2C complex recognizes HLA-E as does CD94/NKG2A (33), but NKG2C provides an activating signal in contrast to NKG2A (32,34,35). In the absence of specific mAb, RT-PCR was performed to assess NKG2 transcripts in the different lines (Fig. 5). PG-CD8 expressed each NKG2 transcript of the lectin-like heterodimer. NKG2C was not detected in BV1-1, which expressed the inhibitory NKG2A transcript. BV1-2 expressed both NKG2A and C. NKG2E transcripts were present in all three cell lines. NKG2D, presumably expressed at the cell surface as a homodimer (36), was also detected at the mRNA level in all lines. Therefore, expression of NKG2A and NKG2C transcripts was heterogeneous on T cell lines with the same TCRBV1–BJ2S3 rearrangement.



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Fig. 5. PCR analysis of C-lectin-like NKR expression (CD94 and NKG2-A/B to NKG2E transcripts) in the three T cell lines (PG-CD8, BV1-1 and BV1-2) and two clones (PG862 and PG865) derived from line PG-CD8. Products of the NKG2A amplification also contained the NKG2B spliced transcript.

 
NKR heterogeneity at the T cell clonal level
One hundred T cell clones were isolated from lines PG-CD8 and BV1-1. Immunofluorescence analysis confirmed the results obtained with the polyclonal T cell lines (Figs 4B and 6). Thirty-five clones isolated from BV1-1 and clone PG865 had the same phenotype, with a strong expression of the inhibitory complex CD94/NKG2A and a low level of ILT2 (Fig. 6). In addition, RT-PCR performed on PG865 showed the same expression of NKG2 transcripts as in BV1-1 (Fig. 5). A larger heterogeneity was observed in the phenotypes of other clones purified from PG-CD8 (Fig. 6). For instance, PG846 and PG864 expressed an intermediate level of CD94/NKG2A, and ~80% of cells were positive for ILT2. In PG848, 44% of cells were positive for ILT2 at a low level, while CD94 and CD94/NKG2A were expressed at an intermediate level in ~75% of cells. Clone PG862 had an intermediate expression of CD94, 60% of cells were positive for ILT2 and the CD94/NKG2A complex was absent at the cell surface as well as by RT-PCR. Finally, PG83, PG86 and PG861 were negative for CD94 in RT-PCR. CD94 and ILT2 were not expressed at the surface of the clone PG83 (Fig. 6). This reflected at the clonal level the heterogeneity of CD94/NKG2 and ILT2 expression observed in the SF T cell lines.



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Fig. 6. NKR expression and functional analysis of T cell clones. CD94, CD94/NKG2A or ILT2 cell surface expression was checked by flow cytometry analysis as described in Fig. 4(B). Clones PG86 and PG861 tested in PCR as in Fig. 5 were negative for CD94 transcripts (Neg.). Cytotoxic activities were measured by 51Cr-release assays and expressed as the percentage of HOM-2-specific lysis (B*2705) at an E:T cell ratio of 25:1. BV1-1 clones PG865, PG846 and PG848 did not lyse HOM-2 (Neg.). nd, not done.

 
Different cytotoxic activities according to NKR phenotypes
Functional differences could correlate with differences in NKR phenotypes. At the level of T cell lines, PG-CD8 and BV1-2 were the only T cell lines able to kill HOM-2. Both had a low surface level of the inhibitory complex CD94/NKG2A, but expressed transcripts for the activating molecule NKG2C. Conversely, BV1-1 was negative for NKG2C transcripts, expressed high levels of CD94/NKG2A and was unable to kill any HLA-B27 target cell. Also, T cell clones confirmed and extended the results obtained with polyclonal cell lines. BV1-1 clones behaved similarly to the line. The comparison of clones PG83 and PG865 is especially informative as both share the TRCBV1 canonical CDR3 sequence (May et al., submitted): BV1–SVGIFSTDTQ–J2S3 (PG83) and BV1–SVGLYSTDTQ–J2S3 (PG865). Clones able to recognize HOM-2 did not express the CD94/NKG2A inhibitory complex due to the absence of either CD94 (PG83, PG86, PG861) or NKG2A (PG862) transcripts (Figs 5 and 6). Clones with a high (BV1-1 clones and PG865) or intermediate (PG846, PG864 and PG848) CD94/NKG2A expression were not cytotoxic toward HOM-2 (Fig. 6). Repeated attempts to recover a cytotoxic activity by blocking the CD94/NKG2A complex were unsuccessful, suggesting the occurrence of additional inhibitory signals. Among those, the contribution of ILT2, a known inhibitory ligand for B*2705 (19,20), was evaluated in the presence of the specific mAb HP-F1 in the BV1-2 line expressing high ILT2 levels. Addition of HP-F1 during the assay increased the lytic efficiency of the BV1-2 cell line against HOM-2 (Fig. 7). These results suggested that ILT2 could directly inhibit the function of TCRBV1+ cells recruited at the inflammatory site through binding to HLA-B*2705.



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Fig. 7. Cytotoxic activity of the BV1-2 cell line against HOM-2 target cells (B*2705) expressed as percentage of specific lysis. Before addition of the effector cells, targets were incubated for 1 h with the ILT2-specific mAb HP-F1 or an IgG1 isotypic control mAb (1/100 diluted ascites). One representative experiment of three is shown. Cytotoxicity on the irrelevant cell line COX was unchanged in the presence of HP-F1.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We show in this paper that, in addition to TCR diversity, NKR could participate to the heterogeneity of SF T cells due to the stable surface expression of different NKR on clonally expanded CD8+ {alpha}ß populations. This is in line with recent data reported in T cell clones isolated from normal donors (23,24) and from RA patients in the CD4+CD28null population (27), but is to our knowledge the first example in the case of HLA-B27-associated arthritis. This observation was based on data from a representative ReA patient studied in depth with regard to the in situ T cells TCR diversity. It could be of more general relevance as we observed a significantly increased CD94/NKG2A expression in memory CD45RO+CD8bright SF cells compared to PBL in six additional HLA-B27 SA-affected patients (data not shown). Functional implications of NKR expression on T cells is still incompletely defined, especially because of the possible expression of several NKR with activating or inhibitory functions on the same T cell population. Regulatory functions of NKR on oligoclonal {alpha}ß T cell expansions have been reported in the case of HIV infection (37,38), in tumor immunity (39,40), and more recently in the context of human autoimmune MHC class II-associated diseases, celiac disease (41) and RA (26,27). This is clearly different from NK T cells which express in CD4+ or double-negative CD4CD8 populations a restricted pattern of TCR usage (Vß11/V{alpha}24–J{alpha}Q) and recognize CD1d molecules (42).

We observed a heterogeneous expression of the CD94/NKG2 dimer and ILT2 on T cell lines, supporting the following conclusions. First, CD94/NKG2A is probably the main regulatory factor because a high or intermediate expression level correlated with a lack of cytotoxicity as seen on line BV1-1 and clones isolated from it, as well as with clones PG865, PG846, PG864 and PG848 isolated from PG-CD8 (Fig. 6). Second, both T cell lines expressing activating NKG2C transcripts (PG-CD8 and BV1-2) were able to lyse a HLA-B*2705+ target. Therefore, we propose that a low expression of the CD94/NKG2A inhibitory dimers and the reciprocal expression of the activating CD94/NKG2C dimers could be permissive for the TCR-mediated lysis of HLA-B27 targets. Indeed, CD94/NKG2A expression was totally absent both at the cell surface and at the transcript level in clones PG862, PG83, PG86 and PG861, all cytotoxic toward HLA-B27. Furthermore, we directly evidenced the role of the inhibitory receptor ILT2, a ligand for HLA-B*2705 able to down-regulate the cytotoxicity of an ex vivo derived SF T cell line as shown in blockade experiments (Fig. 7). In conclusion, these results illustrate different ways to modulate the cytotoxicity of oligoclonal CD8+ synovial T cells depending on the expression of inhibitory (CD94/NKG2A, ILT2) or activating (CD94/NKG2C) receptors.

Which ligand could be recognized by NKR expressed on these T cells and how could this be relevant to the pathogenesis of ReA? Fully matured HLA-B27 complexes are ligands for ILT2 (43). We may argue that the inhibitory ILT2 signal could be switched-off if HLA-B27 molecules are modified by a bacterial or self-peptide (44,45) or because of an excess of B27 homodimeric free heavy chains unable to bind ILT2 (43). The other NKR likely to play a role is CD94/NKG2, a major ligand of the non-classical HLA class I molecule HLA-E (33). Expression of HLA-E at the cell surface requires the availability of a nonamer peptide derived from certain HLA class I signal sequences (46), preferably with a methionine residue at position 2 (47). The signal sequence derived from HLA-B*2705 expresses a threonine at P2 and is expected to bind weakly to HLA-E (48). However, increased HLA-E expression could occur in the case of HLA class I heavy chain overexpression, as during an inflammatory process. Notably, a high HLA-B27 expression is a distinctive feature of the B27 transgenic rat model (49), whereas the B27 heavy chain has been directly involved in the murine B27 transgenic model (50). A peculiar B*2705 property possibly related to disease pathogenesis is also the heavy chain misfolding (5), which could increase the turnover rate of the molecule and HLA-E expression. Finally, given that the murine counterpart of HLA-E, Qa-1, is able to bind Salmonella peptides (51), it is also possible that bacterial peptides could be presented by HLA-E and increase its cell surface expression. In that view, the immune response would result from the balance between a TCR-mediated CD8+ recognition of a B27-presented antigenic peptide or of misfolded B27 heavy chains and the lectin-like CD94/NKG2A inhibitory signal delivered by HLA-E molecules. Expression of activating CD94/NKG2C dimers adds a supplementary degree of complexity to the functional modulation of these intrasynovial cytolytic T cells. Co-expression of NKG2C and a CD94dim phenotype correlated with B27-restricted cytotoxicity (lines PG-CD8 and BV1-2). This may suggest that the CD94dim population in CD8+ expanded cells would be associated preferentially with NKG2 activating molecules, e.g. NKG2C. As lectin-like NKR expression on T cells is usually restricted to memory CTL, CD94/NKG2 expression on T cell could be considered rather as a mechanism regulating the inflammatory response. We show here, as was also the case for celiac disease, another MHC-associated autoimmune disease (41), that lectin-like NKR are over-expressed on T cells in situ. Depending on the balance between NKG2 subunits expressed, these complexes will have either an inhibitory or an activating effect on T cell function. Therefore we may hypothesize that a preferential expression of NKG2C on some T cell clones as shown here could increase their lytic potential and keep the autoimmune process uncontrolled.

Finally, we may comment on the regulation of CD94/NKG2 complex and ILT2 expression on these oligoclonal T cell populations. Regulation of ILT2 expression is still poorly understood, but that of CD94/NKG2A is better known (52). Cytokines produced locally in the inflamed joint could play a direct role in CD94/NKG2 expression, especially IL-15 which can induce CD94/NKG2A expression in alloantigen-activated CD8+ T cells (53). In line with this hypothesis, abnormalities of IL-15 expression have been described in RA patients and inflammatory bowel diseases which may be associated with arthritis (41,54).

In conclusion, these results highlight the functional heterogeneity of clonally expanded intrasynovial T cells in HLA-B27 arthritis. This may also suggest future therapeutic perspectives directed toward the expression of CD94/NKG2A, CD94/NKG2C or ILT2 molecules in order to modulate the function of in situ antigen-driven CD8+-activated T cells.


    Acknowledgements
 
This work was supported in part by the Association de Recherche sur la Polyarthrite (ARP), EUROAS (BMH4-CT98-3605) and Assistance Publique–Hôpitaux de Paris (DRC AOB94028). We thank Drs V. Braud, F. Navarro, M. Lopez-Botet and E. Clave for helpful discussions, F. Berenbaum for providing clinical samples, and M. Busson for help in statistical analysis. The technical assistance of I. Fournier is greatly acknowledged.


    Abbreviations
 
B-LCL—Epstein–Barr virus-transformed B lymphoblastoid cell line

CDR3—complementarity-determining region 3

ILT—Ig-like transcript

KIR—killer cell Ig-like receptor

NKR—NK receptor

PBL—peripheral blood lymphocyte

PHA—phytohemagglutinin

RA—rheumatoid arthritis

ReA—reactive arthritis

SA—spondyloarthropathies

SF—synovial fluid


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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