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Departments of
*
Pathology and
Surgery, Leiden University Medical Center, Leiden, The Netherlands; and
Department of Surgery, University Hospital Rotterdam-Daniel den Hoed Cancer Center, Rotterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In NK cells, activation signals are initiated after binding to target cells. Adhesion molecules and activation structures such as CD161A (NKR-P1A) are thought to be responsible for the generation of these signals. However, inhibitory signals are induced after the binding of specific killer cell inhibitory receptors (KIRs) on NK cells to "self" MHC class I molecules on target cells (5, 6, 7). In the literature, CD45 has been described as being involved in NK cell-mediated lysis. mAbs against CD45 inhibited target cell lysis by NK cells (8, 9, 10, 11). Furthermore, CD45-negative NK cells failed to lyse tumor targets (12). Thus, CD45 also seems to play a regulatory role in the signal transduction pathway in NK cell-mediated lysis.
The aim of this study was to investigate the role of CD45 in rat NK function. We developed two new Abs directed against rat CD45 and used these to modulate CD45 activity in NK cells. We tested the effect of the mAbs in reversed Ab-dependent cell-mediated cytotoxicity assays, in tyrosine phosphorylation assays, and in cytotoxicity assays measuring the NK cell-mediated lysis of syngeneic target cells expressing different levels of MHC class I molecules.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mAbs OX18 (anti-MHC class I) (13, 14), OX1 (anti-CD45) (15), 3.2.3 (anti-CD161A) (16), WT.3 (anti-CD18) (17), and 3G7 (anti-gp42) (18) were used in this study. All Abs were of the IgG1 isotype. mAbs OX18 and OX1 were purchased from European Cell Culture Collection (Porton Down, Salisbury, U.K.). mAb 3.2.3. was kindly provided by Dr. W. H. Chambers (University of Pittsburgh Cancer Institute, Pittsburgh, PA), mAb 3G7 was a gift from Dr. W. Seaman (University of California, San Francisco, CA), and mAb WT.3 was provided by Dr. M. Miyasaka (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan). mAbs ANK74 and ANK12 were generated by immunizing female BALB/c mice i.p. with 107 cultured NK cells emulsified in CFA (Life Technologies, Paisley, U.K.), followed by one i.p. injection per week for 3 wk with 107 viable NK cells; spleen cells were fused with SP2/0 myeloma cells according to standard methods (19). mAbs ANK74 and ANK12 were selected for their ability to bind to viable NK cells and their inability to bind to viable syngeneic colon tumor cells (CC531) as measured by flow cytometry. The Abs were purified from hybridoma culture supernatant by protein A-Sepharose (Pharmacia, Uppsala, Sweden) chromatography according to the manufacturer’s instructions.
Production of F(ab')2 fragments
Purified F(ab')2 fragments of mAbs OX1, ANK12, and ANK74 were produced as described previously (17). The purity of the F(ab')2 fragments was determined by SDS-PAGE under nonreducing conditions and by Coomassie brilliant blue staining.
Generation of NK cells and T cells
NK cells and T cells were isolated and cultured as described
previously (19, 20). The NK cells were CD161A (rat NK marker)-positive
(
98%) and TCR-negative (98%). The T cells were TCR-positive
(95%).
Tumor cell lines
RNK16 (a rat LGL leukemia cell line) and its CD45-negative mutant RNK16-M45.1 (18) were maintained in a suspension culture in complete medium (CM), which consisted of RPMI 1640 medium (Life Technologies) supplemented with 10% heat-inactivated FCS (Life Technologies), 2 mM L-glutamine, 50 µg/ml streptomycin, and 50 U/ml penicillin in the presence of 50 µM 2-ME. Both cell lines were kindly provided by Dr. W. Seaman (University of California, San Francisco, CA). P815, a mouse mastocytoma, was maintained as a suspension culture in CM. CC531, a WAG rat colon carcinoma (21), and MCR86 and WRM, both Wag rat breast adenocarcinomas, were maintained as adherent cultures in CM.
Indirect immunofluorescence and flow cytometric analysis
Briefly, cells were incubated for 30 min at 4°C with an excess of one of the described mAbs and were washed twice with PBS with 0.5% v/w BSA (PBS/BSA). The cells were resuspended and incubated with an excess of goat anti-mouse IgG (GAM)-FITC (Southern Biotechnology Associates, Birmingham, AL) for 30 min at 4°C. Finally, the cells were washed twice, resuspended in PBS/BSA, and analyzed in a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Propidium iodide (1 µg/ml) was added to the cell suspension to distinguish vital cells from dead cells. A total of 5000 vital cells were analyzed. Data analysis was performed with Winlist software (Verity Software House, Topsham, ME). The results of the experiments are shown as mean fluorescence intensity (MFI).
51Cr release assay
The NK cell-mediated lysis of target cells was measured using a 51Cr release assay as described previously (17, 20). To test the effect of the Abs on lysis, the Abs were added to the assay in 50 µl in a final concentration of 4 µg/ml. Tests were conducted in triplicate at 4 E:T ratios (20, 10, 5, and 2.5), and the final result was presented as the mean of the triplicate.
Immunoprecipitation
NK cells (10 x 106 in 100 µl) were labeled with 125I by incubating the cells with 50 µl of lactoperoxidase (0.2 mg/ml) (Sigma, Bornem, Belgium), 500 µCi Na125I (5 ml) (Amersham, Little Chalfont, U.K.), and 15 µl 0.05% H2O2 at room temperature. Every 3 min, 15 µl 0.05% H2O2 was added. After 12 min, the labeling reaction was stopped by adding 1 ml of ice-cold PBS to the mixture. The cells were washed three times with PBS and subsequently lysed in 1 ml of lysis buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.2% NaDOC, and 1% Nonidet P-40 for 30 min on ice. Leupeptin (2 µg/ml) (Boehringer Mannheim, Mannheim, Germany) and 0.1% aprotinin (Sigma) were added to these buffers before use to prevent proteolysis. The cell lysates were precleared with protein G-Sepharose CL4B beads (protein G beads) (Pharmacia) by incubating cell lysates twice with 40-µl protein G beads for 1 h at 4°C. Simultaneously, 20-µl protein G beads were incubated with 10 mg of mAb for 1 h at 4°C. Next, 50 µCi of precleared [125I]-labeled cell lysate was incubated with the mAb-coated protein G beads for 2 h at 4°C. The beads were washed three times with lysis buffer and then once with PBS. The bound protein was eluted from the beads by boiling in SDS-PAGE buffer, which contained 0.5 M Tris-HCl (pH 6.8), 10% glycerol, and 1% SDS, with or without 5% 2-ME. The samples were then subjected to electrophoresis on 8.5% SDS-PAGE gels. The gels were stained with 2.5% Coomassie blue solution in 45% methanol and 5% acetic acid and destained by the same solution without Coomassie blue. The labeled proteins were analyzed by autoradiography. The gel was dried and exposed to x-ray films (Kodak, Rochester, NY) at -70°C.
Cross-immunoprecipitation was performed by using Sepharose-4CLB beads to which mAb OX1 was chemically cross-linked. After incubation of 125I-labeled lysates with these beads, the bound proteins were eluted from the beads with 0.1 M glycine buffer (pH 2.9) containing 1% Nonidet P-40 and 150 mM NaCl. The pH of the eluents was neutralized with a predetermined amount of 1 M Tris-HCl (pH 8.0). Each eluent was used for a second round of immunoprecipitation with mAb bound to protein G beads as described above.
Antiphosphotyrosine immunoblotting
NK cells (10 x 106) were incubated with 10 µg of the indicated Abs for 5 min in 1 ml of serum-free CM. Next, 10 µg of F(ab')2 fragments of GAM were added to the cells and incubated for an additional 5 min at 37°C. After incubation, the cells were lysed in a buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA, 1 mM Na3VO4, 1 mM NaF, 1 µg/ml leupeptin, and 0.1% aprotinin for 30 min on ice. A total of 100 µl of postnuclear supernatants from each sample were analyzed by SDS-PAGE on 5–15% gradient gels. The gels were transferred to Immobilon-P membranes (Millipore, Bedford, MA), blocked with 3% skim milk solution in PBS (PBS/milk) for 20 min at room temperature, and blotted with antiphosphotyrosine mAb 4G10 (1 mg/ml in PBS/milk) (Upstate Biologicals, Lake Placid, NY) overnight at 4°C, followed by enhanced chemiluminescence (Amersham) according to the manufacturer’s instructions and autoradiography (Kodak).
Statistical analysis
Statistical analysis was performed using Student’s t test. The level of significance was set at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the role of CD45 in NK cell function, we generated
two new mAbs against rat CD45. To test whether the selected Abs ANK74
and ANK12 were directed against CD45, we characterized the Ag
recognized by the two mAbs by immunoprecipitation and by flow
cytometry. For the flow cytometric analysis, we used the CD45-positive
rat NK cell line RNK16 and a CD45-negative mutant of this cell line.
mAb 3.2.3 (anti-CD161A) and 3G7 (anti-gp42) bound to both cell
lines as shown in Fig. 1
. The new mAbs
ANK74 and ANK12 and the anti-CD45 mAb OX1 bound to the RNK16 cells.
These mAbs did not bind to the CD45-negative cell line RNK16-M45.1,
indicating that mAbs ANK74 and ANK12 recognize CD45. To investigate
whether the molecular mass of the protein recognized by mAbs ANK74 and
ANK12 corresponded to CD45, we performed immunoprecipitation studies
using 125I-labeled NK cell lysates. mAb ANK74 was
found to recognize proteins of 180–220 kDa, whereas mAb ANK12
immunoprecipitated a protein of 220 kDa (Fig. 2, lanes 2 and 3).
To confirm biochemically that the mAbs ANK74 and ANK12 recognized CD45,
we performed a cross-immunoprecipitation study with the Ab OX1. The Ags
precipitated with mAb OX1 were eluted and again immunoprecipitated
using the mAbs ANK74 and ANK12. The Ags precipitated with mAb OX1 were
also precipitated by mAbs ANK74 and ANK12 (Fig. 2, lanes 6
and 7) and were not precipitated by a control Ab WT.3
(lane 8), which precipitated CD18 and the various
isoforms of CD11 (Fig. 2, lane 4). mAb ANK74 precipitated
the same proteins as mAb OX1; mAb ANK12 precipitated a protein of 220
kDa from the OX1 Ags. To demonstrate that ANK74 and OX1 recognize the
same Ags and that ANK12 recognizes a subpopulation of the OX1 Ags, NK
cell lysates were depleted from CD45 by incubating with OX1, ANK74, or
ANK12. After depletion, the lysates were used for immunoprecipitation
with the various Abs. We found that OX1 and ANK74 did not precipitate
Ags from OX1- or ANK74-depleted lysates, indicating that they recognize
the same Ags (data not shown). Both OX1 and ANK74 precipitated Ags from
ANK12-depleted lysates, except the ANK12 Ag (data not shown). These
data demonstrate that the CD45 isoform recognized by ANK12 is a
subpopulation from the CD45 isoforms recognized by OX1 and ANK74. In
addition, we performed competition assays using biotinylated OX1 Ab. NK
cells were incubated with OX1, ANK74, ANK12, or control Ab WT.3 and
subsequently incubated with biotinylated OX1 and PE-conjugated
streptavidin. We found that OX1 and ANK74 significantly inhibited the
binding of biotinylated OX1 (p = 0.03 and
0.04); ANK12 and control mAb WT.3 did not inhibit the binding of
biotinylated OX1 (p = 0.87 and 0.91). (Table I). The MFI of the biotinylated OX1 was
low, which was probably due to the masking of epitopes of OX1 by
biotin. These data demonstrate that the newly developed ANK74
recognized a constant part of CD45, as described for OX1, and that
ANK12 mAb recognized a specific isoform of CD45.
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The ability of the mAbs ANK12, ANK74, and OX1 to modulate
NK-mediated lysis in reversed Ab-dependent cell-mediated cytotoxicity
assays was investigated using the Fc receptor (FcR)-positive cell line
P815 as target cell line in 51Cr cytotoxicity
assays. The presence of mAb OX1 had no effect on the lysis of P815 by
NK cells (Fig. 3). mAb ANK74 slightly
increased the lysis of P815; however, this increase was not significant
(p > 0.05). In the presence of mAb ANK12, the
lysis of P815 was significantly increased (p <
0.01). Flow cytometry experiments demonstrated that the mAbs ANK74,
ANK12, and OX1 do not bind to P815 (data not shown).
F(ab')2 fragments of ANK12, unable to cross-link
CD45 via the FcR, did not increase the lysis of P815 (data not shown).
Also, lysis of the FcR-negative cell lines CC531, WRM, and MCR86 was
not affected by mAb ANK12 (see Fig. 5A). These data indicate
that NK cells can be triggered to lyse target cells by the
cross-linking of CD45 via mAb ANK12 and the FcR on target cells.
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). In contrast, only
minor changes in tyrosine phosphorylation were seen when CD45 was
cross-linked by mAbs ANK74 and OX1. These latter changes were also seen
when CD18 was cross-linked by mAb WT.3, suggesting that this is
background phosphorylation activity. These results demonstrate that
cross-linking of the ANK12 isoform of CD45 enhanced the cytolytic
activity of NK cells accompanied by the phosphorylation of specific
proteins in NK cells.
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To investigate whether the level of target cell MHC class I
expression is important for the regulatory role of CD45 in NK cell
function, we measured the effect of the anti-CD45 mAbs on the lysis
of the syngeneic tumor cell lines CC531, MCR86, and WRM. Cell lines
CC531 and MCR86 express low levels of MHC class I, whereas cell line
WRM expresses high levels of MHC class I (17). The anti-CD45 mAbs
ANK74 and OX1, but not ANK12, significantly inhibited the lysis of the
target cells CC531 and MCR86 (p < 0.01),
whereas the lysis of WRM was not inhibited (Fig. 5
A). Subsequently, we
investigated whether the anti-CD45 mAbs also inhibited lysis of the
tumor cell lines with masked MHC class I molecules. Tumor cells
preincubated with the anti-MHC class I mAb OX18 were lysed at
significantly higher levels by NK cells compared with untreated cells
(p < 0.001), as described previously (17)
(Fig. 5B). Addition of the mAbs ANK74 and OX1 resulted in a
significant inhibition of lysis of OX18-preincubated target cells,
including the tumor cell line WRM (p < 0.005)
(Fig. 5B). The inhibition of lysis by mAbs ANK74 and OX1 was
not FcR-mediated, because F(ab')2 fragments of
either mAb ANK74 or mAb OX1 also significantly inhibited lysis of
the tumor cell line CC531 (p < 0.01) (Fig. 6). These data demonstrate that the
inhibition of lysis by blocking of CD45 is only effective when target
cells express low or masked levels of MHC class I, indicating that CD45
plays a crucial regulatory role in the process of NK cell-mediated
target cell killing. Furthermore, our results may indicate that this
function of CD45 is isoform-dependent.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion, caused by reduction of
the phosphatase activity of CD45 and activation of tyrosine kinases.
Thus, inhibition of the phosphatase activity of specific isoforms of
CD45 may stimulate protein tyrosine kinase activity, which results in
activation of NK cells. In contrast, binding of mAb ANK74 or OX1 to the
common region of CD45 resulted in an inactivation of NK cell-mediated
lysis of syngeneic tumor cells. This may indicate that the isoform
recognized by ANK12 has a different function in NK regulation than the
other isoforms. Our experiments show that the binding of mAbs ANK74 and OX1 to the constant part of CD45 resulted in an inhibition of NK cell-mediated lysis; inhibition was most prominent when MHC class I expression on the syngeneic tumor cell lines was low or masked. These mAbs did not interfere with conjugation formation between NK cells and syngeneic target cells, demonstrating that the inhibition of NK cell lysis was not caused by blocking conjugation formation. The inhibition of NK cell lysis by these mAbs can also be explained by a possible role of CD45 in signal transduction after MHC class I recognition (i.e., that ANK74 and OX1 are able to trigger CD45 in a similar manner as might be seen for KIRs after binding to MHC class I). After binding to MHC class I, the KIR cytoplasmic domains have been described as phosphorylated, followed by an association of KIRs with PTPs (such as Src homology domain 2-containing PTP-1, PTP-1C, and HCP) and tyrosine phosphatase-mediated inhibition of NK cell activation (24, 25, 26, 27, 28). CD45 has been described as the major tyrosine phosphatase of lymphocytes, accounting for >90% of the total phosphatase activity in lymphocytes (29). Our results suggest that CD45 might be one of the tyrosine phosphatases that is recruited and activated after the binding of KIRs to MHC class I. We hypothesize that the binding of mAb ANK74 or OX1 to CD45 results in an inhibition of NK cell-mediated lysis by activation of the tyrosine phosphatase activity of CD45. Because we used a syngeneic model in which the KIR expressed by the NK cells binds to the specific MHC class I molecules on the target cells, the effect of the mAbs was only detected when syngeneic tumor cell lines with low or absent MHC class I expression were used as target cells. In this case, binding of NK cells and target cells did not result in cross-linking and activation of the KIRs on NK cells and stimulation of CD45 activity. Others also found that mAbs directed against CD45 inhibited NK cell-mediated lysis (8, 9, 10), indicating that binding of an Ab to CD45 may have stimulated the tyrosine phosphatase activity of CD45 in these studies, resulting in inhibition of lysis. Studies using CD45 exon 6-deficient mice demonstrated that CD45-negative NK cells had intact cytotoxic function; it was concluded that CD45 was not essential for cytotoxic activities (30). However, this study was done in an allogeneic model in which CD45 was not regulating NK cell-mediated lysis; in this case, the specific KIRs on NK cells will not trigger CD45, because they do not bind to the MHC class I molecules on the target cells.
In summary, we have demonstrated in this study that CD45 plays a key role in the regulation of NK cell-mediated lysis of target cells. By using Abs against this structure, we were able to stimulate as well as inhibit rat NK cells. We hypothesize that the regulatory role of CD45 is mediated by its tyrosine phosphatase activity and is directly linked to the function of KIRs. Further studies are necessary to investigate the specific role of the different isoforms of CD45 in regulation of NK function.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Peter J.K. Kuppen, Leiden University Medical Center, Department of Surgery, K6-R, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
3 Abbreviations used in this paper: PTP, protein tyrosine phosphatase; KIR, killer cell inhibitory receptor; CM, complete medium; GAM, goat anti-mouse IgG; MFI, mean fluorescence intensity; FcR, Fc receptor.
Received for publication August 31, 1998. Accepted for publication April 8, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production in NK cells. J. Immunol. 154:644.[Abstract]
production. J. Immunol. 155:5241.[Abstract]
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  D. G. T. Hesslein, R. Takaki, M. L. Hermiston, A. Weiss, and L. L. Lanier Dysregulation of signaling pathways in CD45-deficient NK cells leads to differentially regulated cytotoxicity and cytokine production PNAS, May 2, 2006; 103(18): 7012 - 7017. [Abstract] [Full Text] [PDF]
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