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IL-12-Mediated NKRP1A Up-Regulation and Consequent Enhancement of Endothelial Transmigration of V2⁺ TCR⁺ T Lymphocytes from Healthy Donors and Multiple Sclerosis Patients¹

Alessandro Poggi^2,*, Maria Raffaella Zocchi, Paola Costa^*, Elisabetta Ferrero, Giovanna Borsellino, Roberta Placido, Simona Galgani^§, Marco Salvetti^¶, Claudio Gasperini^§,¶, Giovanni Ristori^¶, Celia F. Brosnan^|| and Luca Battistini

^* Laboratorio Immunopatologia, Istituto Nazionale per la Ricerca sul Cancro e Centro Biotecnologie Avanzate (IST-CBA), Genoa, Italy; Laboratorio Immunologia dei Tumori, Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele, Milan, Italy; Laboratorio Neuroimmunologia, Istituto di Ricovero e Cura a Carattere Scientifico Santa Lucia, Rome, Italy; ^§ Dipartimento di Neuroscienze "Lancisi," Ospedale S. Camillo, Rome, Italy; ^¶ Dipartimento di Scienze Neurologiche, Universita’ "La Sapienza," Rome, Italy; and ^|| Department of Pathology, Albert Einstein College of Medicine, Bronx, NY

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
T lymphocytes are thought to play a role in the pathogenesis of multiple sclerosis (MS) contributing to demyelinization and fibrosis in the central nervous system. In this study, we show that, in MS patients with active disease, the percentage of circulating V2⁺ T cells coexpressing NKRP1A is significantly increased compared with healthy donors. V2⁺ and V1⁺ T cells were sorted from MS patients and healthy volunteers and cloned. At variance with V1⁺ clones, all V2⁺ clones expressed NKRP1A, which was strongly up-regulated upon culture with IL-12; this effect was neutralized by specific anti-IL-12 Abs. No up-regulation of NKRP1A by IL-12 was noted on V1⁺ clones. RNase protection assay showed that IL-12R ß2 subunit transcript was significantly less represented in V1⁺ than V2⁺ clones. This finding may explain the different effect exerted by IL-12 on these clones. In transendothelial migration assays, V2⁺ NKRP1A⁺ clones migrated more effectively than V1⁺ clones, and this migratory potential was enhanced following culture with IL-12. Migration was strongly inhibited by the F(ab')₂ of an anti-NKRP1A Ab, suggesting that this lectin is involved in the migration process. We also show that, in freshly isolated PBMC from MS patients, the migrated population was enriched for V2⁺ NKRP1A⁺ cells. We conclude that the expression of NKRP1A on V2⁺ cells is associated with increased ability to migrate across the vascular endothelium and that this phenomenon may be regulated by IL-12 present in the microenvironment.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
It is now well accepted that multiple sclerosis (MS)³ is an immunologically mediated disease of the central nervous system (CNS). Active lesions are characterized by perivascular cuffs of lymphocytes and macrophages centered on postcapillary venules (1). Within the inflammatory infiltrate, all major subsets of lymphocytes have been identified. Studies using animal models of the primary demyelinating disease, have clearly shown that each of these different cell types, as well as resident activated glial cells, are capable of contributing to immune-mediated injury (2). Although the main effector cells are thought to belong to the ß TCR subset that express CD4, minor subsets of T cells may also contribute to the immune-mediated inflammatory process by functioning as sources of either proinflammatory or regulatory factors (3, 4). Included in these are T cells that express the TCR.

T lymphocytes represent a subset of peripheral T cells with peculiar phenotypic and functional characteristics (5). In fact, a portion of T lymphocytes share some surface markers with NK cells such as CD16, CD56, and different inhibitory NK cell receptors (NKR) for HLA class I Ags, suggesting that - and NK-mediated cell functions are regulated by similar mechanisms. In MS, T cells have been found in the lesions and in the cerebrospinal fluid (CSF), and PCR analysis and sequencing studies have shown that the major T cell subsets present in the MS lesion (6) differ from those in the CSF, suggesting specific functions for these cells in lesion development (7, 8). In more chronic MS lesions, T cells may become the most prevalent type of T lymphocytes detected (7). Although the exact function of these cells remains unknown, they have been shown to possess potent cytotoxic activity, including toxicity toward oligodendrocytes (9), and to produce cytokines, such as IFN-, and chemokines involved in the recruitment of cells of the monocyte/macrophage series (10). As such, they could significantly contribute toward inflammatory processes mediated by Th1-type cytokines (11). However, T cells must egress from the bloodstream and migrate into the CNS to exert their pathogenetic role. Several cell surface molecules contribute to lymphocyte endothelial transmigration, including NKRP1A (CD161), which is expressed by a fraction of CD4⁺ ß T lymphocytes and, among T cells, almost exclusively by the V2 subset (10, 12).

NKRP1A is a type II membrane glycoprotein with a C-type lectin domain, its coding gene mapping to chromosome 12 in the "NK gene complex" (13). Engagement of NKRP1A may modulate several cell functions, including transendothelial migration (12), in different lymphocyte subsets (14, 15, 16). Recently, we have demonstrated that IL-12 induces the up-regulation of NKRP1A expression in human NK cells and that, as a consequence, NKRP1A regulates NK activation (17). In the present study we provide evidence that NKRP1A^{+ +} T cells belonging to the V2 cell subset are strongly increased among peripheral blood lymphocytes in MS patients compared with healthy donors. More interestingly, NKRP1A expression is crucial for the endothelial transmigration of these cells, and this process is IL-12 regulated.

	Materials and Methods

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Donors

All biological samples were obtained from the Dipartimento di Neuroscienze "Lancisi," Ospedale S. Camillo, and the Dipartimento di Scienze Neurologiche, Universita’ "La Sapienza," according to protocols approved by the human experimentation committees of these two institutes. Blood samples were drawn from 15 patients with clinically active MS (patients in the relapsing phase or first episode of disease, primarily in the form of optic neuritis or internuclear ophthalmoplegia with abnormal magnetic resonance imaging brain scan and abnormal CSF) before treatment. Details of the MS patients are shown in Table I; none had received immunosuppressive therapy for at least 3 mo before entering the study.

The anti-NKRP1A mAb 191B8 (IgG2a), anti-CD16 mAb KD1 (IgG2a), anti-CD94 XA185 (IgG1), anti-CD94/NKG2A mAb Z199 (IgG1), anti-CD158a mAb EB6 (IgG1), anti-CD158b mAb GL183 (IgG1), anti-V1 mAb A13 (IgG1), and anti-V2 mAb BB3 (IgG1) were prepared as described (18, 19, 20). The anti-HLA class-I mAb W6/32 (IgG2a)-producing hybridoma was from the American Type Culture Collection (ATCC, Manassas, VA). 191B8 and W6/32 mAbs were purified from ascites fluids by affinity chromatography, and pepsin-digested F(ab')₂ fragments were prepared as described (12). PHA was from Life Technologies (Grand Island, NY). FITC- and phycoerythrin (PE)-conjugated IgG1-, IgG2a-, and IgM-specific goat anti-mouse (GAM) antisera were from Southern Biotechnology (Birmingham, AL). Cells were cultured in RPMI 1640 (Biochrom, Berlin, Germany) supplemented with FCS (HyClone, Logan, UT) and human AB serum (BioWhittaker, Walkersville, MA), L-glutamine, and penicillin-streptomycin (Biochrom). Recombinant human IL-2 was provided by Eurocetus (Milan, Italy). Recombinant human IL-12 and the anti-hIL-12 mAb were from R&D Systems Europe (Oxon, U.K.).

Isolation and cloning of T cells

PBMC from healthy donors or MS patients were isolated by Ficoll-Hypaque density gradient centrifugation. To obtain clones from healthy donors and MS patients, highly purified CD3⁺ TCR⁺ cells were obtained from PBMC (10) following staining with anti-V1 and anti-V2 mAbs and cell sorting using a MoFlo cell sorter (Cytomation, Fort Collins, CO). Cells were seeded at 1 cell/well in 96-microwell plates (Greiner, Nurtingen, Germany) in RPMI 1640, supplemented with 5% human AB serum, 5% FCS, 200 mM L-glutamine, 100 mM MEM nonessential amino acids, 2-ME, MEM sodium pyruvate, pen/strep (all from Life Technologies), PHA (1 µg/ml) and rIL-2 (25 U/ml). Cells were then expanded with IL-2 and restimulated every 3 wk with PHA and irradiated feeder cells according to standard procedures.

Immunofluorescence and cytofluorometric analysis

Single and double fluorescence stainings were performed as described elsewhere (19). Briefly, aliquots of 10⁵ cells were stained with the corresponding mAb followed by FITC- or PE-conjugated anti-isotype GAM antiserum. Control aliquots were stained with isotype-matched irrelevant mAbs followed by FITC- or PE-GAM or with the fluorescent reagent alone. In some experiments, cells cultured in IL-2 (25 U/ml) for 3 wk were recovered, washed twice in complete medium, and cultured for an additional 6 days in the presence of IL-2 (25 U/ml) or IL-12 (1 ng/ml), respectively. Samples were analyzed on a flow cytometer (FACSort, Becton Dickinson, Mountain View, CA) equipped with an argon ion laser exciting PE at 488 nm, and results are expressed as Log red fluorescence intensity (arbitrary units, a.u.) vs number of cells or vs Log green fluorescence intensity (a.u.) or as mean fluorescence intensity (MFI). Statistical analysis was performed using the Student t test and variance analysis.

Transmigration assay

Primary cultures of HUVEC were derived from umbilical cords, cultured in TC199 medium (Biochrom) supplemented with 10% FCS and 1% Nutridoma (Boehringer Mannheim, Milan, Italy) (21) and used at passage 3, after extensive washing. Endothelial confluent monolayers were tested for their integrity before the migration assay as described (21). The transmigration assay was performed as described (12, 22) using the Transwell cell culture chambers (polycarbonate membrane, 3-µm pore size, Costar, Cambridge, MA). In some experiments, V1⁺ or V2⁺ CD3/TCR⁺ T cell clones were preincubated for 30 min at 4°C with saturating amounts (5 µg/ml) of the F(ab')₂ fragment of either the anti-NKRP1A (191B8) or the anti-HLA class-I (W6/32) mAb and washed before the transmigration assay. After 2 h, migrated cells were recovered from the lower compartment, and their phenotype was analyzed. To quantitatively express the results of transmigration assays, V1⁺ or V2⁺ CD3/TCR⁺ T cell clones were labeled with ⁵¹Cr (NEN, Boston, MA) and added to the upper compartment of the Transwell chamber (10⁵/well in 24-well plates). At different time points (15 min, 30 min, 60 min), nonadherent cells were washed out, and migrated cells were recovered from the lower compartment and lysed with 100 mM Tris-HCl (pH 7.4) containing 0.1% Triton X-100. The radioactivity of the samples was measured in a gamma counter. Results are expressed as the percentage of migrating cells, calculated as described (12, 22). Statistical analysis was performed using the Student t test and variance analysis.

Ribonuclease protection assay

Total RNA was extracted from the T cell clones using TriReagent according to the manufacturer’s instructions (Molecular Research Center, Cincinnati, OH). Expression of mRNA for the cytokine receptors for IL-10, IL-11, and the ß1 and ß2 subunits of the IL-12 receptor were determined using a multiprobe protection assay (Riboquantä, PharMingen, San Diego, CA). Twenty micrograms of total RNA was hybridized to the hCR3 probe set containing [-³²P]UTP-labeled antisense RNA transcripts overnight at 43°C, and ssRNA was digested with an RNase A/T1 mixture using the RPA II kit (Ambion, Austin, TX) according to the manufacturer’s instructions. The samples were then analyzed on denaturing urea/polyacrylamide gels, and the protected bands were detected by autoradiography.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
A subset of V2 T cells coexpressing NKRP1A is expanded in peripheral blood of MS patients

T cells belonging to the V2 cell subset have been reported to localize in the chronic active lesions of MS (7). Moreover, circulating T cells are known to coexpress NKRs, including NKRP1A (10, 18). In the first set of experiments, we determined whether NKR expression on T cells was different in MS patients with active disease from that found in normal individuals. We used a panel of Abs that identify members of the C-type lectin family of NKRs (CD94 and NKRP1A) and the Ig supergene family of NKRs (p58.1 and p58.2). Thus, we analyzed the distribution of NKRP1A and other NK cell markers among T lymphocytes of 15 MS patients (Table I) in comparison with healthy donors. PBMC were isolated from peripheral blood of normal subjects or patients, and double immunofluorescence was performed using mAbs directed against NKRP1A and mAbs recognizing either V1 or V2 T cell subsets. In agreement with previous studies, we found that T cells express NKRP1A; interestingly, while in healthy donors NKRP1A is present on a fraction (about 20%) of both V1 and V2 subsets, in MS patients the proportion of V2 NKRP1A⁺ cells is significantly higher (>70% of the whole T cell population, p < 0.01) (Fig. 1). When the expression of other NK-related cell surface markers by V1 or V2 T cells was evaluated, we found that the fraction of V2 cells stained by the mAb Z199, which recognizes the inhibitory form of NKG2A/CD94 complex (20, 23), was decreased in MS patients (Fig. 1, p < 0.01). No significant difference between healthy donors and MS patients was observed in the expression of p58.1 (CD158a) and p58.2 (CD158b) molecules (Fig. 1).

View larger version (24K): [in this window] [in a new window]   FIGURE 1. Expression of NKRP1A and other NK receptors on T cells from MS patients and healthy donors. PBMC were isolated from peripheral blood, and double immunofluorescence was performed using mAbs directed against the indicated NK cell markers and mAbs recognizing either V1 or V2 T cell subsets, as described in Materials and Methods. Samples were run on a Facsort (Becton Dickinson) gated to exclude nonviable cells. Results are expressed as the percentage of V1 or V2 T cells coexpressing NK receptors and are the mean values ± SD from 15 MS patients and 10 healthy donors. *, p < 0.01.

NKRP1A expression is up-regulated on V2 T cells upon culture with IL-12

Since NKRP1A molecule can be up-regulated by IL-12 at the surface of NK cells (17), we asked whether this cytokine could exert the same effect on T cells as well. To this aim, 10 V1 and 20 V2 clones, obtained from MS patients or healthy donors and maintained in IL-2 (25 U/ml) for 3 wk, were washed and cultured for a further 6 days in either IL-2 (25 U/ml)- or IL-12 (1 ng/ml)-containing medium. Cells were then harvested, and indirect immunofluorescence was performed using the anti-NKRP1A 191B8 mAb. While NKRP1A expression did not change on day 1 or on day 6 of culture with IL-2 (data reported in Table II refer to day 6), exposure to IL-12 led to NKRP1A up-regulation in all of the V2 clones tested (20/20) both in normal donors and in MS patients (Table II, p < 0.01). Conversely, NKRP1A was not induced on the V1 clones examined (five from MS patients and five from healthy donors), since MFI of NKRP1A expression evaluated after 6 days of culture with IL-12 was not significantly different from that observed before treatment with this cytokine (Table II). Fig. 2 shows that the IL-12-mediated up-regulation of NKRP1A could be neutralized using an anti-IL-12 Ab. To explain the different effect exerted by IL-12, we addressed the question of whether IL-12R is differently expressed on V2 and V1 clones. Thus, we performed an RNase protection assay to evaluate transcription of the IL-12 receptor subunits in four V2 and in four V1 clones (Fig. 3 shows one representative clone for each subset). Interestingly, the IL-12 receptor ß2 subunit transcript was significantly less represented in V1 clones. Transcription of IL-10 and IL-11 receptors, analyzed for comparison, in V2 and V1 clones was superimposable.

View this table: [in this window] [in a new window]   Table II. IL-12 enhances NKRP1A expression on V2 clones from MS patients and healthy donors1

View larger version (28K): [in this window] [in a new window]   FIGURE 2. Up-regulation of NKRP1A expression on T cells upon culture with IL-12. T lymphocytes were isolated from peripheral blood of healthy donors as described in Materials and Methods, cultured either in IL-2 (25 U/ml, white column)- or in IL-12 (1 ng/ml)-containing medium, either in the absence (black column) or in the presence (gray columns) of anti-IL-12 antiserum at the indicated concentrations. Cells were then harvested, and indirect immunofluorescence was performed using the anti-NKRP1A 191B8 mAb, as described in Materials and Methods. Samples were run on a Facsort (Becton Dickinson) gated to exclude nonviable cells. Results are expressed as mean fluorescence intensity (MFI a.u.) and are the mean values ± SD from six independent experiments.

View larger version (69K): [in this window] [in a new window]   FIGURE 3. Ribonuclease protection assay for cytokine receptor mRNA expression. Total RNA was extracted from four V2 clones (lane 1 shows one representative clone) and four V1 clones (lane 2 shows one representative clone), and cytokine receptor mRNA expression was determined using a multiprobe RNA protection assay as described in Materials and Methods. The location of the protected fragments corresponding to specific cytokine receptors is shown on the right. The two housekeeping genes L32 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are included as internal probes to control for variabilities in loading.

T lymphocytes use NKRP1A to transmigrate across endothelial cells

The V2 subset, which we found to preferentially express NKRP1A, represents the major fraction among circulating T lymphocytes, while V1 T cells are mainly detected in peripheral tissues (5). Moreover, we have recently reported that NKRP1A expressed by a fraction of CD4⁺ T lymphocytes is involved in the migration of these cells across endothelial cell monolayers, independent of chemotactic stimuli (12). Therefore, we addressed the question of whether NKRP1A⁺ T lymphocytes display migratory properties comparable to those of CD4⁺ NKRP1A⁺ ß T cells. To this purpose, T cell clones derived from healthy donors and from MS patients were cultured in IL-2 alone (25 U/ml) or in IL-12 (1 ng/ml). On day 6, cells were assayed for transmigration through HUVEC monolayers using a double chamber Transwell system. At different time points, cells were recovered from the lower chamber, and the fraction of migrated cells was calculated (12).

Transendothelial migration of V2 lymphocytes was higher and faster than that of V1 cells, both in MS patients and healthy donors (Fig. 4, A and B); furthermore, migration was enhanced by treatment of V2, but not V1 cell clones with IL-12 (Fig. 4 and Table III), suggesting a relationship between up-regulation of NKRP1A and enhancement of transmigration. Since NKRP1A is involved in the transmigration process of CD4⁺ lymphocytes, we assessed the possible contribution of NKRP1A to transendothelial migration of T cells. When migration of the IL-12-treated clones was tested at 60 min after preincubation with the F(ab')₂ fragment of the anti-NKRP1A 191B8 mAb, a significant reduction of transendothelial migration was observed (Fig. 4, C and D). In contrast, no inhibitory effect was observed using the F(ab')₂ fragment of the anti-HLA class-I W6/32 mAb (Fig. 4, C and D).

View larger version (42K): [in this window] [in a new window]   FIGURE 4. Transendothelial migration of V1 or V2 T cell clones from one MS patient and a healthy donor (see also Table III). T cell clones were cultured in IL-2 (25 U/ml) alone or were exposed to IL-12 (1 ng/ml) for 6 days. Radiolabeled cells were assayed for transmigration through HUVEC monolayers using a double chamber Transwell system. After 15, 30, or 60 min, cells were recovered from the lower chamber and lysed, and radioactivity was counted in a gamma counter. Results are expressed as the percentage of migrated cells calculated as described in Materials and Methods. A and B, Time course of transendothelial migration of V1 or V2 T cell clones from a MS patients (A) and healthy donors (open symbols, clones cultured in IL-2; filled symbols, clones cultured in IL-12). C and D, Transendothelial migration at 60 min of the IL-12-treated clones depicted in A and B, in the absence (open columns) or presence of F(ab')2 fragments of the anti-NKRP1A 191B8 mAb (black columns) or of the anti-HLA class-I W6/32 mAb (hatched columns), both at 5 µg/ml/106 cells.

View this table: [in this window] [in a new window]   Table III. IL-2 enhances transendothelial migration by V2 clones from MS patients and healthy donors1

View larger version (51K): [in this window] [in a new window]   FIGURE 5. Transendothelial migration of resting T cells from one healthy donor (A and B) and an MS patient (C and D). PBMC were assayed for transmigration through HUVEC monolayers using a double chamber Transwell system. After 60 min, double immunofluorescence was performed on the whole PBMC population (A and C) or on migrated cells (B and D) using mAbs directed against V2 T cell subsets and the anti-NKRP1A mAb 191B8, as described in Materials and Methods. Samples were run on a Facsort (Becton Dickinson) gated to exclude nonviable cells. Results are expressed as log green fluorescence intensity (a.u.) vs log red fluorescence intensity (a.u.). The percentage of V2 T cells coexpressing NKRP1A is indicated in the upper right quadrant of each panel. Similar results were obtained using PBMC from three other MS patients and healthy donors.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Recent studies have shown an increased production of IL-12, which induces type 1 T helper cell responses (25, 26, 27), in progressive MS. This suggests a possible role of IL-12 in the pathogenesis of the disease (28, 29, 30). Our data clearly demonstrate that IL-12 is a potent inducer of NKRP1A expression on V2⁺ T cells and that this enhances transendothelial migration. Indeed, a strong up-regulation of NKRP1A surface expression was induced by culturing V2⁺ T cell clones in IL-12-containing medium, and this effect was abolished by adding anti-IL-12 mAb to the cultures. The increase in NKRP1A expression on NKRP1A⁺V2⁺ T cell clones parallels an increased ability to migrate across endothelium in vitro. We may speculate that NKRP1A⁺V2⁺ T cells from MS patients are recirculating lymphocytes that tend to localize to peripheral inflamed or injured tissues. Once in the site of the lesion, they up-regulate the expression of NKRP1A upon exposure to IL-12 produced by bystander inflammatory cells and use NKRP1A to migrate to regional lymph nodes, recirculate, and extravasate into the brain, leading to recrudescence of MS symptoms. This hypothesis is supported by the finding that infiltrating lymphocytes recovered at MS lesions belong to peripheral lymphocyte subsets that usually express NKRP1A, such as CD4⁺ ß T cells, V2⁺ T cells, and monocyte/macrophages (1, 2, 3, 4, 6, 7, 8).

An unexpected finding was that IL-12 did not induce NKRP1A up-regulation in the V1 T cell subset. We found different levels of the ß2 subunit of the IL-12 receptor transcript in the V1 and V2 T cell subsets, which may explain the different effect of IL-12 on V1 and V2 clones. It is interesting to note that expression of the ß2 subunit is regulated by IL-10 and TGF-ß, suggesting its central role in controlling IL-12 responsiveness (31). The decreased expression of the inhibitory form of CD94 in V2 T cells of MS patients suggests that CD94/NKG2A complex is less effective in delivering inhibitory signals to T cells, thus contributing to the increased sensitivity to inflammatory or differentiating stimuli, such as IL-12, at the site of lesion.

Recently it has been shown that NKRP1A is a costimulatory molecule for CD1d-restricted TCRß⁺NKRP1A⁺ T cells specific for galactosylceramides (32, 33). Both and NKRP1A⁺ T cells have been shown to recognize lipid Ags presented by CD1 molecules (34, 35). Since lipids, particularly glycolipids, are the major components of the myelin sheath, it is reasonable to consider that brain myelin lipid(s) may be involved in the immunopathology of MS. In this regard, it is interesting to note that a member of the CD1 family, CD1b, is expressed in active MS lesions (36). Interestingly, V2⁺ T cells are known to recognize nonprotein Ags (34). Thus, it is intriguing to hypothesize that, in the appropriate cytokine milieu, subsets of T cells specific for non-protein Ags up-regulate NKRP1A and transmigrate through the blood brain barrier to contribute to the immunological attack against myelin.

	Footnotes

 
¹ This work was partially supported by grants from the Italian Association for Cancer Research (AIRC), the Ministero della Sanità-Istituto Superiore di Sanità (ISS) "Progetto Sclerosi Multipla," and Progetto Ricerca Finalizzata 1998–2000; by Grant 1033 from Telethon; and by U.S. Public Health Service Grant NS 11920.

² Address correspondence and reprint requests to Dr. Alessandro Poggi, Laboratorio Immunopatologia, IST-CBA, Torre A1, Largo R. Benzi, 10, 16132-Genoa, Italy.

³ Abbreviations used in this paper: MS, multiple sclerosis; CNS, central nervous system; CSF, cerebrospinal fluid; GAM, goat anti-mouse (Ig); MFI, mean fluorescence intensity; PE, phycoerythrin; a.u., arbitrary units; NKR, NK cell receptor.

Received for publication October 22, 1998. Accepted for publication January 8, 1999.

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