| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Adult Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115;
Institut National de la Santé et de la Recherche Médicale, Unit 448, Faculte de Medicine de Creteil, Paris, France; and
Gastroenterology Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
/ß+CD4-CD8+CD11b+CD28-CD45RO+CD56-CD101+CD103+
(Eß7 integrin). In addition, BY55 was
expressed on most CD8+CD28- peripheral blood T
cells. These phenotypic relationships suggest that
CD8+CD28+ precursor CTL may terminally
differentiate into CD8+CD28-BY55+
effector CTL and that some of the peripheral blood
CD8+CD28- subset may represent recirculation
from mucosal epithelial immune sites. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The BY55 mAb was developed following immunization with YT2C2, a human
leukemia cell line with NK activity, to further delineate lymphocytes
with cytotoxic activity (16). The BY55 mAb was shown to be unique and
to immunoprecipitate an 80-kDa cell surface structure (16). Of the
greatest importance, the BY55 mAb stains all lymphocytes exhibiting
cytotoxic activity (16, 17). Depletion of BY55+ lymphocytes
removed both NK lytic activity and CTL activity (16, 17, 18). BY55 mAb
bound to only 10 to 25% of E-rosette+ PBLs and not to
normal B or myeloid cells. The BY55 mAb recognizes most lymphocytes
with an NK-like phenotype (CD16+, CD56+) and a
sizable proportion of CD3+CD8+ T cells with
TCR/ß+ or 
/
+, but not
CD4+ T cells (16, 17). BY55 expression was lost from the
cell surface within 1 h following in vitro activation of E+PBLs with PMA, and BY55 expression did not reappear even after 3
days of culture (16). In cord blood, all BY55+ lymphocytes
have an NK-like phenotype, and BY55+ CD8+ CTL
are not yet present (18). In HIV-infected individuals, the number of
CD8+ T lymphocytes increased by twofold compared with that
in normal individuals, but the number of
BY55+CD8+ T cells was increased even further
(fourfold), suggesting a role for BY55+ CTL in the response
to HIV (18). In contrast, the BY55+ NK cell population was
decreased following HIV infection.
To further study the BY55 molecular structure, we cloned the gene encoding BY55 by COS cell expression cloning. The cDNA sequence predicts a cysteine-rich, glycosylphosphatidylinositol (GPI)-anchored protein of 181 amino acids with a single Ig-like domain weakly homologous to KIR. Further, RNA blot analysis confirmed that BY55 expression is highly restricted to NK and T cells. BY55 was expressed on the CD56dim, CD16+ subset of NK cells, which have high cytolytic activity, but was not expressed on the CD56bright, CD16- subset of NK cells, a subset with high proliferative but low cytolytic capacity. BY55 protein was expressed on all intestinal intraepithelial lymphocytes (iIEL) which are CD8+CD28-CD11b+ cells, similar to the subset of BY55+CD8+CD28-CD11b+ peripheral blood T cells. Overall, our results further demonstrate that the BY55 cell surface molecule delineates functional cytotoxic lymphocytes. These phenotypic relationships suggest that CD8+CD28+ precursor CTL may terminally differentiate into CD8+CD28-BY55+ effector CTL and that some of the peripheral blood CD8+CD28- subset may represent recirculation from mucosal epithelial immune sites.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
FITC- and PE-conjugated class- and subclass-specific goat
anti-mouse Ig secondary Ab were purchased from Southern
Biotechnology (Birmingham, AL). BY55 mAb was used at a 1/500 dilution
of clarified ascitic fluid (16). Anti-CD8, 7PT 3F9, IgG2a;
anti-CD11b, Mo1, IgM; anti-CD14: Mo2, IgM; anti-CD16, 3G8,
IgG1; anti-CD19, B4, IgG1; anti-CD20: B1, IgG2a (19),
anti-CD28, 9.3, IgG2a (20) (Dr. C. June, Naval Research Institute,
Bethesda, MD); anti-MHC class II, 9-49, IgG2a; and anti-CD94,
NKH3 (21), have been described previously. CD8 /ß-specific mAb
2T8-5H7 was provided by Dr. Ellis Reinherz (Dana-Farber Cancer
Institute, Boston, MA). TCR/ß-specific Ab BMA031 and
TCR/-specific Ab IMMU510 were obtained from Immunotech
(Westbrook, ME). Anti-Eß7 integrin (CD103)
mAb (Eß7-2, IgG1) (22) was provided
by Dr. Gary Russell (Brigham and Women’s Hospital, Boston, MA).
Fluorochrome-conjugated NKH-1 (CD56, IgG1), CD16 (IgG1), and
unconjugated control IgM were obtained from Coulter (Hialeah, FL). All
other Abs were purchased from Coulter.
Phenotypic analysis
Expression of cell surface molecules was determined by direct or indirect labeling using standard methodology. Irrelevant isotype-matched Abs (IgG subclasses or IgM) were used as negative controls. FITC- or PE-conjugated goat anti-mouse IgG or IgM were used for unconjugated mAbs. Samples were analyzed in a Coulter Elite flow cytometer.
Chemicals
Protease inhibitors, DTT, iodoacetamide, and
n-octyl-ß-D-glucoside were obtained
from Sigma (St. Louis, MO). Protein A-Sepharose 4B was purchased from
Repligen (Cambridge, MA). ECL Western blot reagents were obtained from
Amersham (Arlington Heights, IL). [-32P]dATP and
-dCTP were obtained from New England Nuclear (Boston, MA).
COS cell expression cloning
A cDNA library was constructed in the pCDM8 vector using NK cell poly(A)+ RNA pooled from four different donors. For the first round of selection, COS cells were transfected via the DEAE-dextran procedure (23) with 0.2 µg of NK plasmid library DNA/100-mm dish. After 40 h, cells were harvested, incubated with BY55 mAb (1/500 dilution of ascites), washed, and panned on anti-IgM-coated plates as previously described (23, 24). Episomal DNA was prepared from adherent cells, reintroduced into Escherichia coli, and transfected into COS cells by polyethylene glycol-mediated fusion of spheroplasts (23), and the panning with BY55 was repeated. Individual plasmid DNAs were transfected into COS cells via the DEAE-dextran procedure (4 µg/100-mm dish) and analyzed after 72 h for cell surface expression of BY55 Ag by indirect immunofluorescence and flow cytometry. Nine of ten plasmids showed cDNA inserts of two different sizes, 1.3 or 1.4 kb. COS cells transfected with the miniprep plasmid DNA from eight of these clones bound BY55 but not control mouse IgM.
DNA sequence analysis
Both strands of one clone from the 1.3- and 1.4-kb human BY55 cDNA insert groups and of mBY55 (Integrated molecular analysis of genomes and their expression consortium clone accession no. AA276726) were sequenced with synthetic oligonucleotide primers and dye-labeled terminator/Taq polymerase chemistry and analyzed with an automated fluorescent DNA sequencer (Applied Biosystems, Foster City, CA). The accession numbers for the human and mouse BY55 sequences reported in this paper are AF060981 and AF060982, respectively. Homology searches of GenBank and EST databases were performed with the gapped BLAST program (25). Sequence comparisons and signal cleavage predictions were generated with the GAP and Sig_cleave programs of the Genetics Computer Group (Madison, WI) using the default parameters.
Northern and Southern blot analyses
Poly(A)+ RNAs were prepared as described from
various cell lines, denatured with formaldehyde, electrophoresed, and
blotted onto nitrocellulose (26). Human tissue Northern blots were
obtained from Clontech (Palo Alto, CA). The 1.3-kb BY55 cDNA was
labeled by random oligonucleotide priming using
-32P-labeled dCTP and dATP and a random labeling kit
following the protocol recommended by the supplier (Boehringer
Mannheim, Indianapolis, IN). Hybridization, washing, and
autoradiography were performed as previously described (27).
Stable transfection
CHO and 300.19 cells were stably transfected by electroporation with linearized BY55 plasmid DNA and the drug selection plasmids pGK-hygromycin or pSV2-neomycin, respectively. Following drug selection, transfected cells expressing BY55 Ag were selected by FACS sorting with BY55 mAb, and single cells expressing BY55 were cloned by limiting dilution.
Cell surface biotinylation and immunoprecipitation
Cells were biotinylated by a sulfosuccinimidobiotin (Sulfo-NHS-biotin, Pierce, Rockford, IL) procedure. Briefly, after three washes in PBS, cells were suspended at 10 x 106/ml in PBS with 0.1 M HEPES (pH 8.0) and 0.1 µg/ml of Sulfo-NHS-biotin. After a 40-min incubation at room temperature with occasional shaking, cells were washed three times with RPMI 1640 at 4°C. Cells were lysed in a lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 60 mM n-octyl-ß-D-glucoside, and 0.05% Triton X-100 in the presence of the protease inhibitors aprotinin (1 U/ml), benzamidine hydrochloride (10 µg/ml), leupeptin (1 µg/ml), pepstatin (10 µg/ml), soybean trypsin inhibitor (10 µg/ml), and PMSF (1 mM) by rocking at 4°C for 45 min. After centrifugation, the lysate supernatant was precleared first by incubation with protein A-Sepharose 4B beads and second by incubation with a control rabbit polyclonal antiserum plus protein A-Sepharose 4B. Immunoprecipitation was performed by an overnight incubation with BY55 or a control IgM Ab plus goat anti-mouse IgM coupled Sepharose-4B (Zymed Laboratories, South San Francisco, CA). After five washes with a wash buffer containing 50 mM Tris-HCl (pH 8.3), 0.5 M NaCl, and 0.5% Nonidet P-40, protein was either eluted directly by boiling for 10 min in SDS sample buffer containing 5 mM ß-ME or alternatively was reduced with 10 mM DTT in wash buffer with a 1-h incubation at 60°C and carboxyamidomethylated with 100 mM iodoacetamide at 90°C, and the protein was eluted as before. The immunoprecipitates were resolved by 10% SDS-PAGE and blotted electrophoretically onto Immobilon membrane (Millipore, Bedford, MA). The membrane was blocked overnight with 5% dried milk in PBS plus 0.05% Tween-20, and the protein bands were developed with horseradish peroxidase-conjugated streptavidin and ECL reagents.
Phosphatidylinositol-specific phospholipase C (PIPLC) treatment
Cells were washed with cold RPMI three times, incubated with phosphatidylinositol-specific phospholipase C (0.4 U/ml) plus 1 mM PMSF for 1 h at 37°C, and washed again three times with cold RPMI. Monophosphatidylinositol-specific phospholipase C (recombinant grade) was obtained from Oxford Glyco System (Rosedale, NY). Control cells were treated similarly but without PIPLC. Cells were then stained with either BY55 or control Abs and PE-conjugated secondary Ab and analyzed by FACS.
Isolation of iIEL
The iIEL were purified essentially as described previously (28). Briefly, mucosa of surgically removed small intestinal specimen was separated from the submucosa by dissection. After washing several times with RPMI 1640, the mucosa was cut into 1-cm2 pieces. Tissues were washed three times with HBSS (without calcium or magnesium) and 1 mM DTT by shaking in a water bath at 37°C for 30 min. The iIELs were then eluted from the tissue with 0.75 mM EDTA in HBSS (without calcium or magnesium) containing 10 mM HEPES (pH 7.4), penicillin, and streptomycin by incubating in a shaking water bath at 37°C for three separate 45-min periods. The eluted iIELs were pooled and washed in culture medium (RPMI 1640, 10% FCS, 2 mM glutamine, 1% nonessential amino acids, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 10 mM HEPES, pH 7.4) and kept at 4°C overnight. The iIELs were further purified by centrifugation through a 40/60% Percoll gradient (Pharmacia, Piscataway, NJ) and isolation of the iIELs at the 40%/60% interface. Purified iIELs were then washed in RPMI 1640.
Isolation of CD8+CD28- PBL
PBMC were isolated from fresh whole blood by centrifugation over Ficoll-Hypaque. Monocytes were depleted by adherence to plastic flasks for 2 h to overnight at 37°C. Cells were incubated with Abs specific for NK cells (anti-CD16 (3G8, IgG1) plus anti-CD56 (3B8, IgG1)), B cells (anti-CD19 (B4, IgG1) plus anti-CD20 (B1, IgG2a)), macrophages (anti-CD14 (Mo2, IgM)), CD4+ T cells (anti-CD4 (T4, IgG1)), and CD28+ T cells (anti-CD28 (9.3, IgG2a)) and NK cells, B cells, macrophages, CD4+ T cells, and CD28+ T cells were depleted using goat anti-mouse IgG and IgM Ab-coated magnetic beads (PerSeptive Diagnostics, Framingham, MA).
Isolation and culture of human NK cells
PBMC were isolated by Ficoll-diatrizoate density gradient centrifugation from cytophoresis buffy coats obtained from normal volunteer donors. Adherent mononuclear cells were depleted by incubation on sterile scrubbed nylon wool columns for 60 min at 37°C. Enriched NK cells were obtained by negative selection using T1/24T6G12 (CD5), T3/RW2 (CD3), and MY4 (CD14) mAb together with immunomagnetic beads as described previously (29). Highly purified CD56bright and CD56dim NK subsets were isolated from populations of enriched NK cells by cell sorting as previously described (30). Basal culture medium was RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 µg/ml streptomycin, 100 µg/ml gentamicin, and 15% heat-inactivated FCS. Activation and in vitro expansion of NK cells using leukocyte-conditioned medium (LCM) and ionomycin were performed as previously described (31). Polyclonal NK cell cultures were maintained at cell concentrations of 1 to 2 x 106/ml by addition of basal medium supplemented with 10 to 15% LCM.
Proliferation assays
Sorted NK cells were plated at 30,000 cells/well (1.5 x 105 cells/ml) in 96-well microtiter plates (Flow Laboratories, McLean, VA) with the indicated concentrations of IL-2 and/or mAb. The final dilution of mAb-containing ascites added to the cultures was 1/500. In some experiments, sorted NK cells were cultured together with 5,000 irradiated (10,000 cGy) K562 stimulator cells at an NK to stimulator cell ratio of 5:1. Cells were cultured for 96 h, followed by a 16-h pulse with 1 µCi [3H]thymidine.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
COS cell expression cloning using BY55 mAb and a cDNA library
constructed from NK cell mRNA resulted in the isolation of 1.3- and
1.4-kb cDNAs. On transient transfection into COS cells, both the 1.3-
and 1.4-kb cDNAs directed the expression of a cell surface protein that
was recognized by the BY55 mAb, but not by isotype-matched control IgM
(Fig. 1
).
|
A shows a comparison of the
predicted amino acid sequences of human and murine BY55. BY55 has two
hydrophobic domains, one at the amino terminus and the other at the
carboxyl terminus (Fig. 2B). The amino terminal hydrophobic
domain (human, amino acids 1–25) has the characteristics of a
secretory signal sequence with a predicted cleavage site after the
glycine at position 25. The carboxyl-terminal hydrophobic domain
comprises the last 15 (human) or 18 (mouse) amino acids of the open
reading frame. This is shorter than expected for a membrane anchor and
is not followed by charged amino acids as is usual for transmembrane
domains, but is characteristic of a GPI membrane anchor signal sequence
(32). BY55 and GPI-anchored human CD58 (LFA-3) and rat CD48 share the
sequence SSG at position -6 from the carboxyl-terminal hydrophobic
domain with the GPI linkage predicted to be added to the first serine
at position 159, following cleavage after these amino acids. Thus the
mature, unmodified human BY55 polypeptide would have 134 amino acids
and a predicted m.w. of 14,901. The BY55 polypeptide has two (human) or
three (mouse) potential sites for N-linked glycosylation.
Surprisingly, the hydrophobic signal and carboxyl domains are more
conserved (68 and 86%, respectively) than the mature extracellular
domain (62%). This may indicate that the hydrophobic signal and
carboxyl domains contain important information for intracellular
sorting or cell surface localization.
|
C). The 68-amino acid distance between the
Ig cysteines at positions 44 and 112 is more characteristic of an Ig-V
domain than an Ig-C domain. The BY55 structure is unusual for an Ig
superfamily member in having an additional cysteine immediately
following the second Ig cysteine. This dicysteine is found only in
three 
Ig-V domains and one other Ig superfamily member, IGSF1
(accession no. AF034198). BY55 also has two cysteines between the Ig
cysteines, potentially looping out a short region between amino acids
61 and 68. This unusual loop is found in only a few Ig superfamily
members, such as CTLA4 and polymeric Ig receptor. In summary, BY55 is
cysteine rich, with six cysteines in the mature polypeptide, including
the first amino acid of the predicted mature polypeptide, and is
composed of a single Ig-like domain. Thus, BY55 has ample capacity for
forming intra- and interchain disulfide bonds. Expression of BY55 mRNA
Two mRNA transcripts of 1.5 and 1.6 kb were identified by
hybridization with BY55 cDNA (Fig. 3),
suggesting that the 1.3- and 1.4-kb cDNAs, along with poly(A) tails,
represent full-length cDNAs. Northern blot analysis of T, B,
NK, and myeloid cell lines showed that BY55 mRNA was detected only in
human NK cells and NKL, an NK-like cell line (36). BY55 mRNA was not
expressed in Raji, a Burkitt’s lymphoma B cell line; LBL-DR7, a
lymphoblastoid B cell line; NALM6, a pre-B cell line; the T cell
leukemia cell lines Jurkat, Rex, CEM, HPB-ALL, and Peer4; the HTLV-I
transformed T cell lines SPP, MT2, and H9; or the myeloid cell line
U937 (Fig. 3). In human tissues, BY55 mRNA was expressed only in
spleen, PBL, and small intestine. As described below, BY55 expression
in small intestine is in gut-associated lymphocytes. BY55 mRNA was not
expressed in thymus, prostate, testis, ovary, heart, brain, placenta,
lung, liver, skeletal muscle, kidney, or pancreas (Fig. 4, A and B). BY55
mRNA was not detectable in colon; however, the amount of RNA in this
lane was less, as judged by the weak intensity of the control
hybridization to the housekeeping gene G6PD.
|
|
BY55 mAb has been shown to immunoprecipitate an 80-kDa protein
from YT2C2, an NK-like cell line (16). Initial immunoprecipitation
experiments using BY55 mAb and NKL cells, BY55-transfected CHO cells,
or BY55-transfected 300.19 (a murine B cell line) also identified an
80-kDa polypeptide (Fig. 5, lane
1, and data not shown). In addition, there was a faint amount of a
27-kDa polypeptide. The size predicted for the mature BY55 polypeptide
plus two N-linked glycosylations would be approximately 25
kDa. Thus, the observed 27-kDa band is close to the predicted size but
the 80-kDa band is far larger than expected. Since BY55 protein has six
cysteines in the mature polypeptide, there is ample possibility for the
formation of intra- and interchain disulfide bonds that might be
resistant to reduction or reform during electrophoresis (37) and
increase the apparent m.w. We tested this hypothesis by reducing the
BY55 immunoprecipitate with DTT, carboxyamidomethylating it with
iodoacetamide, and resolving it by SDS-PAGE (Fig. 5, lanes
2–6). Following reduction and carboxyamidomethylation, the
relative amount of the 27-kDa band was greatly increased relative to
that of the 80-kDa band in BY55 immunoprecipitates from NKL cells and
BY55-transfected 300.19 cells. Nevertheless, some 80-kDa band remained
in both. These data are most consistent with BY55 being a tightly
disulfide cross-linked multimer. BY55 is currently defined by a single
IgM mAb, and definitive protein analysis must await the development of
high affinity IgG mAbs.
|
To test whether BY55 is anchored to the membrane by a
glycosylphosphatidylinositol linkage, we incubated NKL cells and
BY55-transfected CHO cells with PIPLC and then analyzed cells for the
expression of BY55 on the cell surface. PIPLC cleaved BY55 protein from
the cell surface of NKL cells (Fig. 6,
top panel) and BY55-transfected CHO cells (Fig. 6, second panel). The level of cleavage was almost 100% as
opposed to some other GPI-anchored proteins that are incompletely
removed by PIPLC (38, 39). As controls for PIPLC activity and the
absence of protease activity, PIPLC was shown to cleave GPI-anchored
hamster CD87 but not membrane-anchored complement regulatory protein
(CRRY; Fig. 6, bottom panels).
|
As shown in Figure 4, BY55 mRNA was expressed at high levels in
the small intestine. As the small intestine has abundant gut-associated
lymphoid tissue, we stained freshly isolated human small iIEL with BY55
mAb. Strikingly, BY55 was expressed on almost all iIEL (Fig. 7). The iIEL are a unique subpopulation
of lymphocytes lining the gut, and almost all are
CD3+TCR/ß+CD56- (Fig. 7, top panel). For comparison, we stained PBL. Consistent with
previous reports (16, 18), only 21% of peripheral blood
CD3+ T cells expressed BY55. Most peripheral blood NK cells
expressed BY55, with 59% of CD56+ PBL expressing BY55.
|
/ß+CD4-CD8+CD28-CD56-,
iIEL were almost all positive for Eß7
integrin (CD103), CD11b (complement receptor type 3; data not shown),
CD45RO, CD101, and the /ß CD8 heterodimer (Fig. 8).
|
The phenotype of iIEL, specifically that they were
CD8+CD28-CD11b+, is similar to a
subset consisting of about half the peripheral blood CD8+ T
cells (40). To compare these populations,
CD8+CD28- peripheral blood T cells were
prepared by depletion of macrophages and NK, B, and CD4- and
CD28-positive cells. The CD8+CD28- peripheral
blood T cells uniformly expressed BY55 and CD11b but lacked
Eß7 and CD101 (Fig. 9 and data not shown). Expression of
Eß7 is induced by TGF-ß and so may be a
consequence of the intestinal microenvironment. Thus, iIEL share
some, but not all, markers with the CD8+CD28-
peripheral blood T cell subset. This confirms previous work showing
that in PBL the CD3+CD8+ subset, which does not
express CD28, is predominantly BY55+ (41). We have also
reported that most CD101+ T cells in peripheral blood are
CD28+ (41, 42).
|
Human CD56+ NK cells can be divided into
CD16+CD56dim and
CD16-CD56bright subsets (9, 30, 31, 38, 43)
(Fig. 10). The
CD16+CD56dim NK cell subset is highly cytolytic
but does not proliferate well in response to known signals that
costimulate NK cell proliferation (31, 43). In contrast, the
CD16-CD56bright NK cell subset proliferates
vigorously in response to mitogenic cytokines and costimulatory
signals; however, it shows little lytic activity (21, 31, 43). We
examined the expression of BY55 in these NK cell subsets and found that
BY55 was expressed by the CD16+CD56dim NK cell
subset and was absent from the CD16-CD56bright
NK cell subset (Fig. 10). This is consistent with our previous reports
showing the association of BY55 with cytolytic lymphocytes (16, 17).
Following in vitro activation and expansion with IL-2, sorted
CD56bright cells remained BY55-. Sorted
CD56dim cells were still 50% BY55+ after in
vitro activation, and expression of BY55 paralleled that of CD16 (data
not shown).
|
). In
contrast, sorted BY55-CD56bright NK cells
proliferated vigorously in response to increasing doses of IL-2, and
this was augmented by costimulatory signals provided by anti-CD94
mAb. BY55 mAb neither augmented nor inhibited the proliferative
response of either CD56bright or CD56dim NK
cell subsets. K562 cells can costimulate the proliferation of human NK
cells in response to IL-2 (31). In other experiments, inclusion of BY55
had no effect on the proliferation of CD56dim NK cells in
response to IL-2 and irradiated K562 cells (data not shown). Following
in vitro activation and expansion, reculture of sorted NK cell subsets
with IL-2 in the presence of BY55 had no significant effect on the
proliferative response of these activated NK cell subsets (data not
shown). Consistent with previous reports (16, 17), the presence of BY55
mAb did not augment or inhibit NK cell lysis (data not shown). Taken
together, these results strongly strengthen our previous observation
that BY55 is expressed only by NK cytotoxic cells, which proliferate
poorly in response to IL2 alone.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The BY55 cDNA encodes a polypeptide of 181 amino acids with two predicted N-linked glycosylation sites. Further, because the carboxyl-terminal hydrophobic domain was short and not bounded by charged residues, we investigated whether the BY55 molecule is anchored to the cell surface via GPI anchorage. Removal of BY55 protein from the cell surface by PIPLC treatment confirms that BY55 is indeed GPI linked. The BY55 carboxyl-terminal hydrophobic domain is novel, but the GPI addition site is similar to that of CD58. Gpi membrane anchorage of BY55 would allow rapid movement across the membrane, and cleavage of the GPI anchor would explain its rapid loss from the cell surface following PMA activation.
With the cleavage of both the secretory signal sequence of about 25 amino acids at the amino terminus and a GPI anchor signal sequence of about 22 amino acids at the COOH terminus, the resultant mature polypeptide would be 134 amino acids. We have previously reported that an 80-kDa protein was immunoprecipitated by BY55 mAb from cell surface iodinated cells (16). We show here that BY55 mAb also immunoprecipitates an 80-kDa protein from biotin-labeled NKL cells and BY55-transfected cell lines. This molecular mass is far larger than that predicted for mature BY55 polypeptide, with two N-linked glycosylations, approximately 25 kDa. Since BY55 protein has six cysteines in the mature polypeptide, there is ample possibility for forming intra- and interchain disulfide bonds, which might increase the apparent m.w. Reduction and alkylation of the immunoprecipitated BY55 gave rise to a band of 27 kDa, whereas reduction alone led to an 80-kDa species, suggesting that BY55 is a tightly disulfide-linked multimer. Moreover, we could eliminate the possibility that the 80-kDa predominant band immunoprecipitated by BY55 mAb is an associated chain similar to what has been described for CD94 associated protein (44), as this 80-kDa band was present in all immunoprecipitates, including those from BY55-transfected CHO cells and 300.19 murine B cells.
BY55 mRNA expression shows a remarkably restricted pattern. BY55 mRNA was expressed only in NK cells, NKL cells, spleen cells, and T cells, but not in any T, B, or myeloid cell line tested. In human tissues, BY55 mRNA expression also showed a very restricted distribution and was expressed only in spleen, small intestine, and peripheral blood, but not in thymus, prostate, testis, ovary, heart, brain, placenta, lung, liver, skeletal muscle, kidney, or pancreas. The failure to detect BY55 expression in thymus may be a consequence of the low number of differentiated effector CTL in the thymus and suggests that further differentiative signals are required for BY55 expression in the periphery.
BY55 mRNA expression in human small intestine prompted us to
investigate the cell surface phenotype of the lymphoid subsets isolated
from small intestine. Two anatomically distinct lymphocyte populations
are found in the intestinal mucosa, iIEL and lamina propria
lymphocytes. Approximately one iIEL is present for every eight
epithelial cells in the human small intestine, which, considering the
vast area of the intestinal epithelium, makes the intestinal immune
system by far the largest immune organ in the body. The iIELs in situ
in nonpathologic conditions express some granzyme A, but not
significant levels of Fas ligand, granzyme B, or perforin; however,
they rapidly acquire cytolytic potential after in vitro cultivation
with mitogens (45, 46, 47). Reports in mouse and man have indicated that
IEL may function as cytolytic effector cells in pathologic conditions
in vivo (48, 49). The iIEL have been shown to express immunoregulatory
cytokines, such as IL-1ß, IL-2, IL-8, IFN-, TNF-, and TGF-ß
(45). The biologic functions of iIEL may include roles in defense
against gut microorganisms, oral tolerance, and local
immunosurveillance against epithelial cell injury and neoplastic
transformation. The phenotypic properties of iIEL,
CD8+CD45RO+ with a limited array of TCR/ß,
suggests that they are memory T cells that recognize a limited number
of Ags in the context of MHC class I or class I-like molecules (28, 46).
Interestingly, almost all the iIELs, which are CD8+
TCR/ß T cells, expressed BY55. In addition, as previously
reported, fresh iIELs expressed phenotypic markers of memory T cells
such as CD45RO (50, 51, 52), Eß7 integrin
(CD103) (53), and CD101 (54), while they lacked the CD28 costimulatory
receptor (54). The lack of expression of CD28 and the weak response to
anti-CD3 cross-linking alone (55, 56, 57) suggest that they use
alternative costimulatory pathways, perhaps through CD2 (55) or CD101
(54). We previously showed that CD101+ cells represent a
subset of peripheral blood CD8+CD28+ cells (41, 42). In addition, CD101 expression, similar to CD103 and CD45RO, is
induced during activation. In contrast, we showed that BY55 is
down-modulated after activation in short or long term culture (16).
Besides the difference in phenotype between the cells expressing BY55
in PB and iIEL, both cell populations are able to exert cytotoxic
function (17, 46, 57). This is in agreement with our earlier studies
showing that in fresh PBL, only sorted
CD8+BY55+ cells exhibit CTL activity in an
anti-CD3-redirected lysis assay (17). Thus, it appears that BY55
delineates effector cytotoxic CTL in both circulating blood and
intestinal tissue.
These results show that a subset of peripheral blood T cells has a
striking phenotypic similarity to iIELs (BY55+,
CD28-, CD11b+, CD8+, TCR/ß).
Both iIELs and peripheral blood CD8+28- T
cells have been shown to have an oligoclonal distribution of TCR
ß-chain usage (28, 58). We are critically testing the relationship
between the blood and iIEL subsets by examining whether the oligoclonal
distribution of TCR ß-chain usage of iIEL is also found in peripheral
blood CD8+28- T cells. Similar TCR usage would
validate assaying this blood CD8+28- subset as
a reflection of gut pathology.
BY55 was expressed on the CD56dim, CD16+ subset of NK cells, which have high cytolytic activity. In contrast, BY55 was not expressed and was not induced on the CD56bright, CD16- subset of NK cells, a subset with high proliferative, but low cytolytic, capacity (31, 38, 43). Recently, several KIR/killer activating receptor structures defined as receptors for HLA class I molecules have been identified on NK cells and a subset of CD8+ TCR+ cells. These receptors have been shown to provide either inhibitory or activating signals following their ligation (reviewed in Refs. 12–15). Interestingly, the KIR/killer activating receptor can be coexpressed on the same NK cell, but they do not presently identify the whole circulating NK subset. BY55 is expressed on all the lytic NK cells, whereas each KIR is expressed on only a few percent of the total NK population. The homology of BY55 to the KIR suggests that it may have a role in NK target recognition. Nevertheless, the single BY55 mAb available does not inhibit or enhance allogeneic or NK cell lysis (16).
We show here that the CD8+28- and
CD8+BY55+ peripheral blood T cell populations
are essentially the same. In HIV-infected individuals, CD8+
T lymphocytes increased by twofold compared with those in normal
individuals, and the level of BY55+CD8+ T cells
was increased even further (fourfold), suggesting a role for
BY55+ CTL in the response to HIV (18). In contrast, the
BY55+ NK cell population was decreased following HIV
infection. The CD8+28- T cell subset contains
the active anti-HIV cytolytic cells and high level IFN-
producers (59, 60, 61, 62, 63). In contrast, the noncytotoxic antiviral activity
that inhibits HIV replication is in the CD8+28+
subset (64). Progression of HIV infection leads to an expansion of the
CD8+28- T cell subset (65). During SIV
infection in monkeys, the expanded population of blood
CD8+28- T cells has been shown to be cytolytic
and to arise not from peripheral lymph node but from mucosal sites such
as intestine and lung (66, 67). Thus, the expanded
BY55+CD8+ T cell population in the peripheral
blood of HIV-infected individuals probably represents recirculation
from mucosal epithelial immune sites.
Abundant evidence shows that precursor cytolytic T cells require B7-mediated costimulation to develop into effector cytolytic cells (68, 69). This implies that the precursor cytolytic T cells must express CD28 to receive the costimulatory signal. Considerable evidence indicates that in vivo effector cytolytic T cells have lost CD28 expression (59, 60, 61, 62, 63). This implies a differentiative pathway in which precursor cytolytic CD8+CD28+ T cells receive a B7 costimulatory signal and develop into CD8+CD28- effector cytolytic T cells. Azuma et al. (63) have also shown that some CD8+CD28- T cells can regain CD28 expression. Some intriguing possibilities are suggested by the phenotypic analysis of BY55. While iIEL and the CD8+CD28- peripheral blood subset clearly share some markers (CD3+CD8+CD11b+CD28-), others are different (CD101, CD103). The phenotypic similarities of iIEL and CD8+CD28- peripheral blood T cells suggest that some iIEL may migrate into peripheral blood and vice versa. This has recently been documented in SIV infection (66). Thus, the BY55+CD8+CD28- T cell population may represent a terminally differentiated population with active cytolytic but limited proliferative capacity.
In cord blood, BY55+CD8+ T cells are not yet present, and all BY55+ lymphocytes have an NK-like phenotype (18). The CD8+CD28- T cell subset expands in both percentage and absolute number as humans age (70). It has been suggested that the CD8+CD28- T cell subset may be in a state of replicative senescence based on shorter telomere lengths (71), and in HIV infection, telomeres in these cells are even shorter (72, 73). This leads to the idea that BY55+CD8+ T cells in HIV infection reflect the massive commitment of the immune system to antiviral cytolysis, but that these cells also represent a population with limited expansion possibilities, perhaps reflecting clonal exhaustion.
It is an interesting observation that BY55 is expressed only in the subpopulation of T and NK cells that are programmed for the final effector function, i.e., cytolysis. When considering all these observations, it seems that iIELs, cytolytic BY55+CD8+ peripheral blood T lymphocytes, and BY55+ NK cells all share some common properties, viz., poor or null proliferative response (31, 55, 56, 57), activated lymphocyte phenotype (16, 18, 50, 74), and capacity for cytolytic activity (16, 17, 45, 46, 57). Another important characteristic shared by iIELs and BY55+ T lymphocytes is their expansion in certain disease conditions. For example, BY55+CD8+ T lymphocytes showed a fourfold increase in HIV infection (18). The peripheral blood CD8+CD28- T cell subset is expanded following bone marrow transplantation (75), during chemotherapy (76), and in hereditary hemochromatosis (77), perhaps because it may be extrathymically derived (52). Similarly, increased numbers of iIELs have been reported in diseases of mucosal delayed-type hypersensitivity, allograft rejection, graft-vs-host disease, parasite infections, enteral challenge after immunization, and celiac disease (48, 49).
The restricted expression of BY55 on cytolytic lymphocytes and its homology to KIR suggest a role in target recognition. BY55 would not be expected to deliver an inhibitory signal as it lacks an immunoreceptor tyrosine-based inhibition motif; however, GPI-anchored proteins can signal by associating with kinases (78). The molecular cloning of BY55 will facilitate additional studies on its function in cytolytic lymphocytes.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Gordon J. Freeman, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115. E-mail address:
3 Abbreviations used in this paper: KIR, killer inhibitory receptors; GPI, glycosylphosphatidylinositol; iIEL, intestinal intraepithelial lymphocytes; ECL, enhanced chemiluminescence; PIPLC, phosphatidylinositol-specific phospholipase C; LCM, leukocyte-conditioned medium; CRRY, complement regulatory protein.
Received for publication February 19, 1998. Accepted for publication May 19, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RIII (CD16) on human natural killer cells and neutrophils: cell type-specific expression of two genes that differ in single nucleotide substitutions. J. Exp. Med. 170:481.Eß7 integrin. Eur. J. Immunol. 24:2832.[Medline]
/ß cells in the lamina propria but proliferation (Ki-67) of /ß and / cells in the epithelium. Eur. J. Immunol. 23:505.[Medline]
/ß or /) and CD3- lymphocytes isolated from normal human gut epithelium display phenotypical features different from their counterparts in peripheral blood. Eur. J. Immunol. 20:1097.[Medline]
/ß or TCR / are largely unresponsive to proliferative signals mediated via conventional stimulation of the CD3-TCR complex. Int. Immunol. 3:563. in asymptomatic HIV-infected patients. AIDS Res. Hum. Retroviruses 12:135.[Medline]
subunits in lymphocytes. Proc. Natl. Acad. Sci. USA 93:6053.This article has been cited by other articles:
|
|
 |
|
  M. Nikolova, J.-D. Lelievre, M. Carriere, A. Bensussan, and Y. Levy Regulatory T cells differentially modulate the maturation and apoptosis of human CD8+ T-cell subsets Blood, May 7, 2009; 113(19): 4556 - 4565. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. C. Cheung, M. W. Steinberg, L. M. Oborne, M. G. Macauley, S. Fukuyama, H. Sanjo, C. D'Souza, P. S. Norris, K. Pfeffer, K. M. Murphy, et al. Unconventional ligand activation of herpesvirus entry mediator signals cell survival PNAS, April 14, 2009; 106(15): 6244 - 6249. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. C. Vendel, J. Calemine-Fenaux, A. Izrael-Tomasevic, V. Chauhan, D. Arnott, and D. L. Eaton B and T Lymphocyte Attenuator Regulates B Cell Receptor Signaling by Targeting Syk and BLNK J. Immunol., February 1, 2009; 182(3): 1509 - 1517. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Giustiniani, A. Bensussan, and A. Marie-Cardine Identification and Characterization of a Transmembrane Isoform of CD160 (CD160-TM), a Unique Activating Receptor Selectively Expressed upon Human NK Cell Activation J. Immunol., January 1, 2009; 182(1): 63 - 71. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Rabot, H. El Costa, B. Polgar, A. Marie-Cardine, M. Aguerre-Girr, A. Barakonyi, S. Valitutti, A. Bensussan, and P. Le Bouteiller CD160-activating NK cell effector functions depend on the phosphatidylinositol 3-kinase recruitment Int. Immunol., April 1, 2007; 19(4): 401 - 409. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Giustiniani, A. Marie-Cardine, and A. Bensussan A Soluble Form of the MHC Class I-Specific CD160 Receptor Is Released from Human Activated NK Lymphocytes and Inhibits Cell-Mediated Cytotoxicity J. Immunol., February 1, 2007; 178(3): 1293 - 1300. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Buttner, S. Mosig, A. Lechtermann, H. Funke, and F. C. Mooren Exercise affects the gene expression profiles of human white blood cells J Appl Physiol, January 1, 2007; 102(1): 26 - 36. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Fons, S. Chabot, J. E. Cartwright, F. Lenfant, F. L'Faqihi, J. Giustiniani, J.-P. Herault, G. Gueguen, F. Bono, P. Savi, et al. Soluble HLA-G1 inhibits angiogenesis through an apoptotic pathway and by direct binding to CD160 receptor expressed by endothelial cells Blood, October 15, 2006; 108(8): 2608 - 2615. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Maeda, C. Carpenito, R. C. Russell, J. Dasanjh, L. L. Veinotte, H. Ohta, T. Yamamura, R. Tan, and F. Takei Murine CD160, Ig-Like Receptor on NK Cells and NKT Cells, Recognizes Classical and Nonclassical MHC Class I and Regulates NK Cell Activation J. Immunol., October 1, 2005; 175(7): 4426 - 4432. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Dardalhon, A. S. Schubart, J. Reddy, J. H. Meyers, L. Monney, C. A. Sabatos, R. Ahuja, K. Nguyen, G. J. Freeman, E. A. Greenfield, et al. CD226 Is Specifically Expressed on the Surface of Th1 Cells and Regulates Their Expansion and Effector Functions J. Immunol., August 1, 2005; 175(3): 1558 - 1565. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Barakonyi, M. Rabot, A. Marie-Cardine, M. Aguerre-Girr, B. Polgar, V. Schiavon, A. Bensussan, and P. Le Bouteiller Cutting Edge: Engagement of CD160 by its HLA-C Physiological Ligand Triggers a Unique Cytokine Profile Secretion in the Cytotoxic Peripheral Blood NK Cell Subset J. Immunol., November 1, 2004; 173(9): 5349 - 5354. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. McCarty, M. L. Shinohara, L. Lu, and H. Cantor Detailed analysis of gene expression during development of T cell lineages in the thymus PNAS, June 22, 2004; 101(25): 9339 - 9344. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Krenacs, M. J. Smyth, E. Bagdi, T. Krenacs, L. Kopper, T. Rudiger, A. Zettl, H. K. Muller-Hermelink, E. S. Jaffe, and M. Raffeld The serine protease granzyme M is preferentially expressed in NK-cell, gamma delta T-cell, and intestinal T-cell lymphomas: evidence of origin from lymphocytes involved in innate immunity Blood, May 1, 2003; 101(9): 3590 - 3593. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Le Bouteiller, A. Barakonyi, J. Giustiniani, F. Lenfant, A. Marie-Cardine, M. Aguerre-Girr, M. Rabot, I. Hilgert, F. Mami-Chouaib, J. Tabiasco, et al. Engagement of CD160 receptor by HLA-C is a triggering mechanism used by circulating natural killer (NK) cells to mediate cytotoxicity PNAS, December 24, 2002; 99(26): 16963 - 16968. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Nikolova, A. Marie-Cardine, L. Boumsell, and A. Bensussan BY55/CD160 acts as a co-receptor in TCR signal transduction of a human circulating cytotoxic effector T lymphocyte subset lacking CD28 expression Int. Immunol., May 1, 2002; 14(5): 445 - 451. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. M. Morales, A. Christ, S. M. Watt, H. S. Kim, K. W. Johnson, N. Utku, A. M. Texieira, A. Mizoguchi, E. Mizoguchi, G. J. Russell, et al. Regulation of Human Intestinal Intraepithelial Lymphocyte Cytolytic Function by Biliary Glycoprotein (CD66a) J. Immunol., August 1, 1999; 163(3): 1363 - 1370. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Agrawal, J. Marquet, G. J. Freeman, A. Tawab, P. L. Bouteiller, P. Roth, W. Bolton, G. Ogg, L. Boumsell, and A. Bensussan Cutting Edge: MHC Class I Triggering by a Novel Cell Surface Ligand Costimulates Proliferation of Activated Human T Cells J. Immunol., February 1, 1999; 162(3): 1223 - 1226. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
