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*
Amgen Institute, and Ontario Cancer Institute, Department of Medical Biophysics and Immunology, University of Toronto, Toronto, Ontario, Canada; and
Department of Oral Biochemistry, Kanagawa Dental College, Kanagawa, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IFN-
production, and expression of the
cytolytic effector molecules perforin and Fas-L were comparable among
IL-2-activated NK cells from LFA-1-/-,
CD44-/-, CD44-/-LFA-1-/-, and
control mice. However, CD44-/-, LFA-1-/-,
and CD44-/-LFA-1-/- IL-2-activated NK cells
showed impaired binding and conjugate formation with target cells. We
also show that hyaluronic acid is the principal ligand on tumor cells
for CD44-mediated cytotoxicity of IL-2-activated NK cells. These
results provide the first genetic evidence of the role of adhesion
receptors in IL-2-activated NK killing. These data also indicate that
distinct adhesion receptors cooperate to mediate binding between
effector and target cells required for the initiation of "natural"
cytotoxicity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many molecules that are crucial for the attachment and cytotoxic function of peptide/MHC-restricted CTLs have been identified (7, 8, 9, 10). CTL-mediated cytotoxicity can be divided into several stages: 1) recognition of and attachment to target cells by the effector cell; 2) interaction of the Ag-specific TCR with peptide/MHC complexes expressed on the target cell; and 3) induction of target cell apoptosis via perforin release or Fas-L/Fas interactions (7, 8, 9, 10). By contrast, adhesion molecules and specific target cell recognition structures that contribute to non-MHC-restricted cytolysis by NK cells remain largely unknown (1, 11).
In this study, we investigated the role of the adhesion receptors CD44 and LFA-1 in cytotoxic activity of IL-2-activated NK cells, using LFA-1-/- (12), CD44-/- (13), and CD44-/-LFA-1-/- gene-deficient mice. We report that both CD44 and LFA-1 have a crucial role in the cytotoxic activity of IL-2-activated NK cells. The cytotoxic machinery of mutant IL-2-activated NK cells appeared functional but the adhesion between activated killer cells and tumor targets was significantly impaired in the absence of CD44 and LFA-1 expression. Moreover, we provide evidence that hyaluronic acid (HA)2 expressed on tumor cells is the main ligand for CD44. These results provide the first genetic evidence that adhesion receptors have a crucial role in the binding and cytotoxic activity of non-MHC-restricted NK cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LFA-1 gene-deficient (12) and CD44 (13) gene-deficient mice have been described. CD44-/-LFA-1-/- double mutant mice were generated by the intercrossing of CD44-/- and LFA-1-/- mice. Mutant mice were back-crossed into a C57BL/6 background for at least six generations. Mice were screened for CD44 and LFA-1 deficiency using flow cytometry and PCR typing (12, 13). In all experiments, CD44+/+LFA-1+/+ and CD44+/-LFA-1+/- littermates were used as controls. All mice used were 6 to 8 wk old and maintained at the Ontario Cancer Institute in accordance with institutional guidelines.
Generation of IL-2-activated NK-effector cells
Spleens were isolated from CD44-/-, LFA-1-/-, and CD44-/-LFA-1-/- and littermate control mice. Splenocytes (2 x 106 cells/ml) were cultured in 1000 U/ml recombinant human IL-2 (Shionogi Chemical Institute, Osaka, Japan) in flat-bottom six-well plates (Costar, Cambridge, MA) in Iscove’s modified Dulbecco’s medium (IMDM; 10% FCS, 10-5 M ß-mercaptoethanol). After 4 days of culture, IL-2-activated NK cells were harvested from the cultures as described (14).
Cytotoxicity assay
Murine lymphoma YAC1 (H2a), EL4 (H2b), RMA (H2b), MHC-class I-deficient RMA-S (H2b), and fibrosarcoma MC57L (H2b) cell lines were used as target cells and maintained in culture with 10% heat-inactivated FCS in RPMI 1640 medium. All cell lines were obtained from American Type Culture Collection (Rockville, MD). Target cell lysis was assessed by 51Cr release. Briefly, 1 x 106 target cells were loaded with 51Cr for 1 h, washed, and plated in triplicate into round-bottom 96-well tissue culture plates (1 x 104 target cells/well). Target cells were incubated with lymphokine-activated NK cell-effector cells at the indicated effector/target (E/T) ratios in a total volume of 200 µl of IMDM (10% FCS) for 4 h at 37°C. IL-2-activated NK cytotoxicity was determined by the release of 51Cr into the supernatant using a gamma counter. Spontaneous lysis was defined as 51Cr release by target cells incubated in the absence of IL-2-activated NK cells. Total 51Cr release by target cells was determined after cell lysis with HCl. The percentage of specific lysis was based on the following equation: % specific lysis = [(experimental release - spontaneous release)/(total release - spontaneous release)] x 100.
Immunocytometry
Freshly isolated splenocytes and IL-2-activated NK cells from
CD44-/-, LFA-1-/-,
CD44-/-LFA-1-/-, and control mice were
resuspended in immunofluorescence staining buffer (PBS, 4% FCS, 0.1%
NaN3) and incubated with appropriate Abs (30 min, 4°C).
To block unspecific binding via FcRs, samples were preincubated with
a nonconjugated CD16/32 mAb (15 min, 4°C). The following
anti-mouse mAbs were used: anti-NK1.1 (PE conjugated, clone
NKR-PC1), anti-TCRß (FITC conjugated, clone H57-597),
anti-CD122 (FITC conjugated, clone TM-b1), anti-CD8 (PE
conjugated, clone 53-6.7), anti-CD16/32 (biotin conjugated, clone
2.4G2), anti-B220 (biotin conjugated, clone RA3-6B2),
anti-Thy1.2 (biotin conjugated, clone 53-2.1), anti-CD44
(biotin conjugated, clone IM7), anti-LFA-1/CD11a (biotin
conjugated, clone M17/4), anti-CD69 (biotin conjugated, clone
H1.2F3), anti-ICAM1 (FITC conjugated, clone 3E2), anti-ICAM2
(FITC conjugated, clone 3C4), anti-Fas (PE conjugated, clone Jo2)
(all from PharMingen, San Diego, CA), and anti-HA (biotin
conjugated; Seikagaku Kogyo, Tokyo, Japan). Biotinylated Abs were
visualized using streptavidin-RED670 (Life Sciences, Arlington Heights,
IL). All Abs were used at optimal concentrations determined in pilot
studies. All staining combinations were as indicated in the figure
legends. Cells were analyzed using a FACScalibur and CELLQuest software
(Becton Dickinson, Mountain View, CA).
Conjugate formation
IL-2-activated NK-effector cells were labeled with fluorescein diacetate (green fluorescence) and target cells labeled with hydroethidine (red fluorescence) for 1 h at 37°C and washed twice in PBS to remove unbound fluorochromes (15). Labeled target cells (5 x 105/ml) and IL-2-activated NK cells (2 x 106/ml) were then mixed together in a total volume of 200 µl and centrifuged for 10 min at 500 rpm. Pellets were incubated for 10 min at 37°C and resuspended in ice-cold PBS to stop conjugate formation. Binding between target and effector cells was determined using a microscope equipped with a UV light source (Zeiss) to visualize and document fluorochrome-labeled conjugates. The percentage of binding was calculated based on the following equation: % of conjugates formed = [(number of IL-2-activated NK cells bound to target cells)/(total number of IL-2-activated NK cells)] x 100. A minimum of 200 IL-2-activated NK-target cell conjugates were counted for each sample. The variability of conjugate formation was ±5% among different experiments.
Purification of NK1.1+ cells
Freshly isolated splenocytes or IL-2-activated splenocytes (1000
U of rIL-2, 4 day cultures, see above) were treated with magnetic beads
(Advanced Magnetics, Cambridge, MA) coupled with goat
anti-mouse-IgG (Cappel Organon, West Chester, PA) to remove surface
Ig (sIg)-positive B cells. To purify NK cells, sIg-negative cells were
incubated with anti-CD4 mAb (rat IgG, clone GK1.5) and anti-CD8
(rat IgG, clone 53.6.7) Abs followed by incubation with magnetic beads
coupled to goat anti-rat IgG (Cappel Organon). Residual cells were
then incubated with PE anti-NK1.1 and FITC anti-TCRß
(PharMingen), and NK1.1+TCRß - cells
were sorted using a FACStarPlus (Becton Dickinson). The
purity of sorted NK1.1+ cells was >98%.
Western blotting
Freshly isolated splenic and IL-2-activated
NK1.1+TCRß- cells were lysed in 10
mM EDTA, 1% SDS, 60% (v/v) glycerol, 0.5% bromphenol blue, and
protease inhibitors. In all, 10 µg of protein were separated by 10%
PAGE, transferred to PVDF membranes (Millipore, Bedford, MA), and
filters incubated in immunoblotting diluent solution (10% skim milk;
0.1% Tween-20 in PBS) at room temperature for 1 h to block
nonspecific Ab binding. Filters were incubated with a polyclonal
anti-Fas-L IgG Ab (C-178; Santa Cruz Biotechnology, Santa Cruz, CA)
at a dilution of 1:1000 at room temperature for 1 h and washed
three times in PBS/0.1% Tween-20. As a control, membranes were probed
with a polyclonal anti-actin antiserum (Sigma). Membranes were then
incubated with a secondary Ab (1:3000) at room temperature for 1 h
and washed three times in PBS/0.1% Tween-20. Immune complexes were
detected by enhanced chemiluminescence according to the manufacturer’s
protocol (Amersham, Arlington Heights, IL).
TNF- and IFN- production
IL-2-activated NK cells were generated from splenocytes as
described above. After a 4-day culture, purified
NK1.1+TCR- (1 x 106/ml)
cells were reseeded into 24-well tissue culture plates (IMDM; 10% FCS,
10-5 M ß-ME) and restimulated with 1000 U/ml of rIL-2
for 24 h. The amounts of IFN- and TNF- in the culture
supernatants were determined by ELISA (Genzyme, Cambridge,
MA).
Northern blotting
Total RNA was isolated from freshly isolated splenocytes and 4-day activated IL-2-activated NK cells using Trizol (Life Technologies, Grand Island, NY). Twenty micrograms of total RNA were subjected to electrophoresis on 1% agarose-formaldehyde gels, transferred to nylon membranes, and hybridized with probes to perforin, Fas-ligand, and ß-actin. Hybridized membranes were exposed and imaged. Perforin and Fas-L mRNA levels were compared with ß-actin mRNA using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Hyaluronidase treatment
To enzymatically remove HA bound to the cell membrane, target cells were treated with 20 µg of hyaluronidase (Sigma) for 60 min at 37°C. Cells were then washed twice and used as targets in cytotoxicity assays. Hyaluronase treatment did not affect target cell viability, 51Cr loading, spontaneous 51Cr release, or expression of other surface markers (not shown).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the role of the adhesion receptors LFA-1 and
CD44 in IL-2-activated NK cell activity, IL-2-activated NK cells were
generated from CD44-/-, LFA-1-/-, and
littermate control mice. IL-2-activated NK cytotoxicity against all
tested tumor cell lines, YAC1, EL4, RMA-S (Fig. 1
A), RMA, and MC57L
(not shown) was significantly reduced in the absence of CD44 or LFA-1.
A genetic double mutation of both CD44 and LFA-1
(CD44-/-LFA-1-/-) further reduced
IL-2-activated NK cell cytotoxicity (Fig. 1B),
indicating that CD44 and LFA-1 cooperate in IL-2-activated NK function.
It should be noted that CD44-/-, LFA-1-/-,
and CD44-/-LFA-1-/- mice display normal
lymphocyte development and normal numbers of leukocyte subpopulations
(12, 13, 16). These results indicate that CD44 and LFA-1 adhesion
receptors are required for IL-2-activated NK cell activity.
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To determine whether the impairment in IL-2-activated NK cell
function from CD44-/-, LFA-1-/-, and
CD44-/-LFA-1-/- mice was due to defective
in vitro differentiation of naive killer cells into IL-2-activated NK
cells, freshly isolated splenocytes and 4-day-activated killer cell
cultures were stained for the expression of activation and
differentiation markers. Since
NK1.1+TCRß- cells are the principal
cytotoxic effector cells of IL-2-activated splenocyte cultures, the
relative numbers and expression levels of NK1.1+ cells were
analyzed in freshly isolated and IL-2-activated spleen cells (Table I, Fig. 2A). In freshly
isolated NK1.1+TCRß- cells, there was no
apparent difference in the expression of CD16/32, B220, Thy1.2, and
CD69 surface molecules (Fig. 2B). Moreover, after 4
days of activation, the absolute and relative numbers of IL-2-activated
NK cells (Table I, Fig. 2A) and the expression and
induction of all tested surface markers (Fig. 2B) on
purified IL-2-activated NK1.1+TCRß- cells
were comparable among CD44-/-, LFA-1-/-,
CD44-/-LFA-1-/-, and control littermate
mice. These results imply that IL-2-activated NK cell differentiation
and activation are normal in the absence of CD44 and LFA-1
expression.
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in IL-2-activated
NK cells
It has been reported that perforin and Fas-L play a critical role
in IL-2-activated NK cell-mediated cytotoxicity against multiple target
cells (17). Moreover, it has been shown that IL-2-activated NK
cell-derived TNF- contributes to the cytotoxic effect (17, 18). We
tested whether the defect in CD44-/-,
LFA-1-/-, and CD44-/-
LFA-1-/- IL-2-activated NK cell cytotoxicity was due to a
lack of perforin and Fas-L expression or impaired production of
TNF-. As shown in Figure 3A, freshly isolated
splenocytes expressed only small amounts of perforin and Fas-L mRNA.
After activation with IL-2 for 4 days, perforin and Fas-L mRNA
expression were induced in CD44-/-,
LFA-1-/-, CD44-/-LFA-1-/-,
and control cells. However, there was no apparent difference in the
expression levels of perforin and Fas-L among the experimental groups.
Whereas purified NK1.1+TCRß- cells from
freshly isolated splenocytes expressed low levels of Fas-L protein,
expression of Fas-L protein was increased in
NK1.1+TCRß- cells purified from
IL-2-activated CD44-/-, LFA-1-/-,
CD44-/-LFA-1-/-, and control splenocyte
cultures (Fig. 3B). Furthermore, production of
TNF- and IFN- was comparable among IL-2-activated
NK1.1+TCRß- cells from
CD44-/-, LFA-1-/-,
CD44-/- LFA-1-/-, and control
mice (Fig. 4). These results imply that
the lack of CD44 and LFA-1 expression does not affect the induction of
the cytotoxic effector molecules perforin and Fas-L or the production
of the pro-inflammatory cytokines TNF- and IFN-.
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Our results implied that the absence of CD44 and LFA-1 does not
impair the differentiation of naive NK cells into IL-2-activated NK
cells and that the effector machinery required for IL-2-activated NK
cytotoxicity was normal in CD44-/-,
LFA-1-/-, and CD44-/-
LFA-1-/- mice. To test whether the reduction of
CD44-/-, LFA-1-/-, and
CD44-/-LFA-1-/- IL-2-activated NK
cytotoxicity was due to impaired binding to target cells, we visualized
IL-2-activated NK-target cell interactions in a direct binding assay
(Fig. 5, A and
B). Conjugate formation between IL-2-activated NK and
target cells, i.e., number of cells adhering to the target cells (Fig. 5B) and the total number of conjugates (not shown),
was significantly impaired in the absence of CD44 and LFA-1. The
reduction in IL-2-activated NK-target cell binding was even more
pronounced in IL-2-activated NK cells that lack both CD44 and LFA-1
molecules (Fig. 5B). It should be noted that
conjugate formation was tested after 10 min of incubation at 37°C.
Thus, our experiments do not reveal whether the lack of CD44 and LFA-1
surface expression alters conjugate stability. These data show that
CD44 and LFA-1 regulate adhesion between "natural" cytotoxic
effector and tumor cells.
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The principal ligands for LFA-1 are ICAM-1 and ICAM-2 (19),
whereas HA is a main ligand for CD44 (20). To examine whether target
cells expressed the ligands for LFA-1 and CD44, the expression of
ICAM-1, ICAM-2, and HA on target cells was determined by flow
cytometry. As shown in Figure 6, YAC1,
EL4, and RMA-S expressed high levels of ICAM-1 and ICAM-2 on the cell
surface. In addition, all of these cell lines expressed detectable
levels of HA on the cell surface (Fig. 6).
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, hyaluronidase treatment of
YAC1 cells reduced cytotoxicity of wild-type and LFA-1-/-
IL-2-activated NK cells, whereas the same treatment did not impair the
activity of CD44-/- and
CD44-/-LFA-1-/- killer cells. Similar
results were obtained using RMA, RMA-S, EL4, or MC57L cells as targets
(not shown). These data indicate that HA expressed on the surface of
tumor target cells is the main ligand for CD44 and that CD44-HA
interactions play a crucial role in the cytotoxic activity of
IL-2-activated NK cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and TNF-. These results provide the first
genetic proof that the adhesion receptors CD44 and LFA-1 have a
critical role in "natural" cytotoxicity.
IL-2-activated NK cells are defined by MHC-independent
cytotoxicity against a variety of tumor cells, including target cells
that are resistant to killing by resting NK cells (3, 4, 5, 6, 23, 24, 25).
Recently, it has been shown that various effector molecules, such as
TNF-, perforin, and the Fas-L/Fas death receptor system, are
involved in IL-2-activated NK cell cytotoxicity (17, 26). In
particular, the production and release of perforin appears to be the
principal cytotoxic mechanism utilized by NK cells (7, 17). Our
experiments indicate that CD44 and LFA-1 do not have any apparent role
in the differentiation of IL-2-activated NK cells and the induction of
cytotoxic effector molecules such as perforin, Fas-L, and TNF-, but
CD44 and LFA-1 control adhesion of activated killer cells to target
cells. CD44- and LFA-1-mediated adhesion appears to be a crucial
prerequisite for the attachment of specific surface receptors, which
then can initiate cytolysis. Although our data indicate that CD44 and
LFA-1 initiate attachment of killer cells to their targets, these
results do not preclude the possibility that the expression of yet
unknown specific surface receptors is impaired in the absence of CD44
or LFA-1.
The leukocyte integrin LFA-1 (CD11a/CD18) was initially defined by mAbs that inhibit CTL-mediated cytotoxicity in the absence of complement (27). Blocking studies using mAbs have shown that LFA-1 is involved not only in CTL-mediated cytotoxicity (21, 22), but also in a wide variety of other leukocyte functions. Thus, anti-LFA-1 Abs can inhibit T cell proliferation (27), NK-mediated cytotoxicity (28, 29), or adhesion of CTLs (30), suggesting that LFA-1 has an important role in lymphocyte function. However, LFA-1 gene-deficient mice displayed normal CTL responses against systemic choriomeningitis virus and vesicular somatitis virus infections (12), suggesting that the consequences of anti-LFA-1 mAbs may reflect nonspecific effects of Ab treatment, such as capping of other surface molecules. Moreover, in two different genetic mouse models of LFA-1 deficiency, it has been reported that LFA-1 expression is either important (12) or irrelevant (16) for cytotoxicity by freshly isolated splenic NK cells. The differences in cytotoxic activity of freshly isolated splenic NK cells between these two LFA-1 gene-targeted mouse strains are not known. Both strains are LFA-1 null mutations as determined by LFA-1 protein expression (12, 16). Hypothetically, distinct background genes present in both mutant mouse models might account for the observed discrepancies, that is, adhesion molecules expressed at different levels in distinct mouse backgrounds could contribute to NK cytotoxicity. Importantly, all mouse strains used in the experiments reported in this paper and the experiments reported by Schmits et al. (12) were back-crossed into a C57Bl/6 background for at least six generations. Alternatively, differences in NK activities between these two strains could be due to distinct in vivo activation protocols, since LFA-1 gene-deficient mice have a defect in cell migration in vivo (12, 16). Our data clearly show that LFA-1 has a crucial role in lymphokine-activated killer cell cytotoxicity and adhesion of IL-2-activated NK cells to their target cells.
The cell surface glycoprotein CD44 is an adhesion molecule for extracellular matrix proteins. CD44 has been implicated in lymphocyte recirculation, cell migration, cell-cell interactions, and tumor metastasis (31, 32). Moreover, several anti-CD44 mAbs have been characterized that either enhance or inhibit T and NK cell functions (33, 34). Little is known about the role of CD44 in IL-2-activated NK cytotoxicity. To address the role of CD44 in IL-2-activated NK cells function, we determined the cytotoxic activity of IL-2-activated NK cells from CD44-/- mice against various tumor cells. Our results showing that IL-2-activated NK cell cytotoxicity is reduced in the absence of CD44 expression provide the first genetic evidence that CD44 has a role in NK function.
Despite the crucial role of CD44 in various experimental systems, CD44 gene-deficient mice are apparently normal in all aspects of morphogenesis and leukocyte development (13). Moreover, no functional defects in lymphocyte proliferation, delayed-type hypersensitivity responses, or cytotoxic T cell effector functions after primary and secondary viral challenge have been identified in CD44-/- mice. However, CD44 is important for granuloma formation and the mobilization of granulocyte/macrophage-CFU from the bone marrow (13). Using the same gene-deficient mice, our study shows that CD44 has a role in IL-2-activated NK cytotoxicity. Since CD44 is expressed on all hematopoietic and most nonhematopoietic cells, it appears that different cytotoxic effectors have different requirements for CD44 expression.
Although a number of extracellular ligands, such as fibronectin, collagen types I and IV, chondroitin sulfate, and serglycin, have been described for CD44, its interaction with HA is the most thoroughly documented (20, 35, 36, 37, 38, 39). For instance, the adhesion of B cells to stromal cells can be blocked by hyaluronidase treatment (39, 40) and HA-binding CTLs have higher cytotoxic activity compared with a non-HA-binding CTL population (41). Nevertheless, many normal or transformed hematopoietic cells that express CD44 do not bind HA (42, 43), and the molecular interactions that govern CD44-dependent binding between effector and target cells in cell-mediated lysis are almost unknown. To determine whether the defect in CD44-/- IL-2-activated NK cell activity was due to a reduction of CD44-HA binding, we measured IL-2-activated NK cytotoxicity against hyaluronidase-treated target cells. The cytotoxic activity of wild-type and LFA-1-/- IL-2-activated NK cells was significantly reduced after hyaluronidase treatment. By contrast, hyaluronidase treatment of target cells did not alter the cytotoxic activity of IL-2-activated NK cells isolated from CD44-/- and CD44-/-LFA-1-/- mice. These results suggest that CD44 expressed on IL-2-activated NK cells acts as an HA receptor and that CD44-HA interactions are important for the conjugate formation between activated killer cells and tumor cells. However, our data do not preclude the possibility that CD44 can bind to other extracellular matrix proteins associated with the membrane of tumor cells.
The receptors that control the cytotoxic activity and attachment of non-MHC-restricted lymphokine-activated NK cells to tumor cells are not known. We provide genetic evidence in CD44-/-, LFA-1-/-, and CD44-/-LFA-1-/- mice that the cell adhesion receptors LFA-1 and CD44 regulate the cytotoxic activity of IL-2-activated NK cells against a variety of different tumor cell lines. Differentiation and the expression of the cytolytic machinery were normal in the absence of CD44 and LFA-1. However, CD44 and LFA-1 regulated the binding of activated killer cells to their targets. The defect in killer cytotoxicity and adhesion was enhanced in mice that carried a double mutation of both CD44 and LFA-1. HA was identified as the principal ligand on tumor cells for CD44-mediated adhesion of IL-2-activated NK cells. These data provide the first genetic evidence of the role of CD44 and LFA-1 in adhesion and cytotoxicity of lymphokine-activated killer cells.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: HA, hyaluronic acid; sIg, surface Ig.
Received for publication July 29, 1997. Accepted for publication February 12, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and tumor necrosis factor-. J. Immunol. 147:4398.[Abstract]
and common ß subunits. J. Cell Biol. 105:1873.This article has been cited by other articles:
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 K. Shingu, C. Kruschinski, A. Luhrmann, K. Grote, T. Tschernig, S. von Horsten, and R. Pabst Intratracheal Macrophage-Activating Lipopeptide-2 Reduces Metastasis in the Rat Lung Am. J. Respir. Cell Mol. Biol., March 1, 2003; 28(3): 316 - 321. [Abstract] [Full Text] [PDF]
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-CD3-sIg-
(not shown). The genotypes of cells are indicated on the
x-axis and expression of different surface molecules is
shown on the y-axis. Control Ab staining using
isotype-matched biotin-labeled control Abs corresponded to CD44 and
CD11a staining patterns in CD44-/-LFA-1+/+
(first upper panel: rows 2 and
4) and CD44+/+LFA-1-/- mice
(second upper panel: rows 3 and
4), respectively (not shown). One result representative of three
independent experiments is shown.
