The Th1-Specific Costimulatory Molecule,
M150, Is a Posttranslational Isoform of
Lysosome-Associated Membrane Protein-1
This information is current as
of June 18, 2017.
Durbaka V. R. Prasad, Vrajesh V. Parekh, Bimba N. Joshi,
Pinaki P. Banerjee, Pradeep B. Parab, Samit Chattopadhyay,
Anil Kumar and Gyan C. Mishra
J Immunol 2002; 169:1801-1809; ;
doi: 10.4049/jimmunol.169.4.1801
http://www.jimmunol.org/content/169/4/1801
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References
The Journal of Immunology
The Th1-Specific Costimulatory Molecule, M150, Is a
Posttranslational Isoform of Lysosome-Associated Membrane
Protein-11
Durbaka V. R. Prasad,* Vrajesh V. Parekh,* Bimba N. Joshi,* Pinaki P. Banerjee,*
Pradeep B. Parab,* Samit Chattopadhyay,* Anil Kumar,† and Gyan C. Mishra2*
T
he hallmark of the adaptive immune system is its ability
to induce as well as regulate its responses to a given infectious agent. Activation of CD4⫹ T cells minimally requires two distinct signals to be provided by an APC. Although the
first signal is provided by the MHC class II molecule presenting
the appropriate Ag-derived peptide, the second signal is delivered
by a class of accessory molecules that are also known as costimulatory molecules (1). A number of such costimulatory molecules
have now been discovered, along with their counterreceptors on T
cells. The most prominent ones include B7.1 and B7.2 that interact
with CD28 on T cells (2– 4), OX40 ligand-OX40 (5, 6), 4-1BBL4-1BB (7, 8), and B7RP-1 that bind to inducible costimulatory (9,
10). On the basis of their ability to secrete specific lymphokines,
CD4⫹ T cells have been divided into Th1 and Th2 subsets. Th1
lymphocytes contributing to cellular immunity are characterized
by the production of IL-2 and IFN-␥, whereas Th2 lymphocytes
produce IL-4, IL-5, IL-10, and IL-13, which are thought to be
mainly involved in humoral immunity (11, 12). Th1- and Th2associated cytokines tend to be reciprocally regulatory. IFN-␥ inhibits Th2-associated functions (13), while IL-4 and IL-10 have
negative effect on Th1-associated functions (14). It has been postulated that these two helper cell subsets are not only functionally
different, but also show qualitative and quantitative distinctions in
their requirements for costimulation (15).
*National Centre For Cell Science, Pune, India; and †School of Biotechnology, Devi
Ahilya Vishwavidyalaya, Indore, India
Received for publication October 23, 2001. Accepted for publication June 17, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Department of Biotechnology, Government of India.
D.V.R.P. and P.P.B. are the recipients of Senior Research Fellowship from National
Centre for Cell Science, Department of Biotechnology, Pune, India. V.V.P. is a recipient of Senior Research Fellowship, and B.N.J. is a recipient of Research Associateship from Council for Scientific and Industrial Research, New Delhi, India.
2
Address correspondence and reprint requests to Dr. Gyan C. Mishra, National Center for Cell Science, Ganeshkhind Road, Pune 411007, Maharashtra, India. E-mail
address: [email protected]
Copyright © 2002 by The American Association of Immunologists, Inc.
In addition to ensuring the activation of T cells, accumulating
evidence suggests that costimulatory molecules may also play a
role in regulating the qualitative aspects of T cell responses. For
example, at least when expressed on activated B lymphocytes,
B7.1/CD28 has been shown to predominantly activate the Th1
subset of CD4⫹ T cells (16 –18), whereas B7.2/CD28 appears to
bias toward Th2 responses (19 –21). Similarly, 4-1BB-4-BBL interactions preferentially contribute toward the development of Th2
responses. The recently identified costimulatory molecule B7RP-1,
which is known to induce IFN-␥ production, is widely expressed
on B cells and macrophages (10). In this context, a new costimulatory molecule, B7-DC, which is specifically present on dendritic
cells, has also been described. This molecule induces Th1-specific
polarization (22).
In an earlier study, we have reported the isolation of a 150-kDa
protein (M150) from the surface of activated macrophages. This
protein was shown to possess costimulatory activity, and was capable of stimulating T cell proliferation. In addition, it was also
able to induce secretion of lymphokines that are typical of Th1
responses (23). Furthermore, we showed that macrophages predominantly use M150, relative to B7.1 for costimulating proliferation and IFN-␥ production from Th cells (24). M150 was also
shown to restore normal Th1 function upon bystander costimulation in diseases like leishmaniasis and tuberculosis, where Th1-like
responses are supressed (25, 26). However, the biochemical identity of M150 has not been elucidated so far.
In the present study, we identify M150 as a specific posttranslational isoform of constitutively produced lysosome-associated
membrane protein-1 (LAMP-1).3 Interestingly, the costimulatory
activity of this molecule depends upon its unique pattern of glycosylation that is generated only in activated macrophages. Thus,
M150 represents a novel example of a housekeeping protein that
can also function as a costimulatory molecule, and adds to the
expanding list of APC-restricted costimulatory molecules with a
biased influence on T cell function.
3
Abbreviations used in this paper: LAMP-1, lysosome-associated membrane protein-1; CHO, Chinese hamster ovary.
0022-1767/02/$02.00
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In an earlier report, we had shown a 150-kDa protein termed as M150, isolated from the surface of activated macrophages, to
possess costimulatory activity for CD4ⴙ T cells. Significantly, this protein was found to specifically elicit Th1 responses. In this
study, we characterize M150, which belongs to a unique subset of the lysosome-associated membrane protein-1 glycoprotein.
Interestingly, the costimulatory activity of M150 depends on its posttranslational modification, which has a distinct glycosylation
pattern restricted to macrophages. Furthermore, it has been demonstrated that in addition to stimulating Th1-specific responses,
M150 is also capable of driving differentiation of naive CD4ⴙ T cells into the Th1 subset. This altered posttranslational modification of housekeeping protein appears to represent a novel pathway by which APCs can additionally regulate T cell
responses. The Journal of Immunology, 2002, 169: 1801–1809.
1802
GLYCOSYLATION MODULATES THE COSTIMULATORY PROPERTY OF LAMP-1
Materials and Methods
Macrophage membrane preparation and purification of M150
Sequencing of M150
The internal and amino-terminal sequencing were conducted independently
at two different facilities. Typically, the purified M150 was electrophoresed
on 10% SDS-PAGE and blotted on to polyvinylidene difluoride membrane
using 10 mM CAPS buffer (US Biochemicals, Cleveland, OH) containing
10% methanol at 200 mA for 2 h at 4°C. The blot then was subjected to
tryptic digestion and the peptides thus obtained were sequenced for internal
sequencing at the protein sequencing facility of Worcester Foundation for
Experimental Biology. The protein obtained by FPLC was subjected to
N-terminal sequencing at the sequencing facility of Purdue University
(West Lafayette, IN).
Heteroduplex analysis
Primers were designed from the internal stretches of the M150 protein that
was sequenced earlier. Sense primer 5⬘-GAGATCTACACAATGGACTC-3⬘
and antisense primer 5⬘-GAGTCCATXGTGTAGATCTC-3⬘ were custom
made (Life Technologies, Grand Island, NY). The cDNA of LAMP-1 cloned
from mouse embryo 3T3 cDNA library was a gift from Dr. J. T. August
(John Hopkins University, Baltimore, MD). PCR was performed using
these primers at an annealing temperature of 55°C. RT-PCR was done
using the same primers from RNA isolated from thioglycolate-elicited
macrophages using S.N.A.P. total RNA isolation kit (Invitrogen, San Diego, CA). A total of 500 ng of total RNA was used to perform RT-PCR
using Titan one tube RT-PCR system (Roche, Indianapolis, IN). The product was subcloned in PCR cloning vector. After transformation and plating
from several different colonies, PCR was performed from plasmid preparations using the same primers. A total of 0.5 ␮g of each PCR product was
mixed with 0.5 ␮g of PCR product obtained from the cDNA construct of
LAMP-1. Heteroduplex analysis was conducted in TNE buffer, final concentration Tris:NaCl:EDTA equals 1/10/0.1 mM (30). The PCR product
mixtures were made to a final volume of 10 ␮l and heated at 95°C for 5 min
followed by rapid cooling in an ice bath for 60 min. The sample was mixed
with loading dye and electrophoresed on an 8% native PAGE using TBE
buffer. DNA pattern was visualized by ethidium bromide staining.
mAb preparation
Lewis rats 8 –10 wk old, obtained from National Institute of Immunology
(New Delhi, India) were immunized by i.p. injections of thioglycolateelicited macrophages (107 cells/mice) from BALB/c mice. Three booster
doses were given after 21 days of primary immunization at an interval of
2 wk each. The spleen was removed on the third day after the final booster
dose and the cells were fused to SP2/01-AG14 (American Type Culture
Collection, Manassas, VA) using PEG 1500 (Roche). Hybrids were selected in hypoxanthine/aminopterin/thymidine medium (Life Technologies). The supernatants were screened for M150 reactivity both by ELISA
and Western blot analysis. Positive clones were subcloned by limiting dilution and the isotype of Abs secreted by individual clones was determined
FACS analysis
The affinity-purified mAb (G1) against M150 and control IgM (normal rat
IgM, affinity purified by sheep anti-rat IgM; Pierce) were FITC conjugated.
PE-conjugated anti-Mac-1, anti-B220, anti-CD4, anti-CD8, normal IgG2a,
normal IgG 2b, and FITC-labeled anti-LAMP-1 Ab, FITC-labeled normal
rat IgG2a (isotype control for anti-LAMP-1), and Fc block were purchased
from BD PharMingen. Splenocytes and macrophages (106 cells), either
resting or activated with IFN-␥ (5 ng/ml for 12 h) from C57 BL/6 mice,
were first incubated with Fc block for 20 min at 4°C and the splenocytes
were then dually stained either with FITC-labeled anti-M150 or antiLAMP-1 along with various PE-conjugated CD markers. Incubations were
done at 4°C in FACS buffer (2% FCS, 0.5% BSA in PBS, pH 7.2) and
washed using the same buffer three times before incubation with the secondary Ab. Finally, cells were washed and fixed in 1% paraformaldehyde
in PBS and staining was analyzed by flow cytometry (FACSVantage; BD
Biosciences, Mountain View, CA). Macrophages were used for the competitive binding experiment between the anti-LAMP-1 and anti-M150 Abs.
Western blotting
The samples were electrophoresed using 10% SDS-PAGE (SE 260; Amersham Pharmacia Biotech, Uppsala, Sweden) and transferred onto a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) using 20
mM Tris containing 125 mM glycine (pH 7.5) and 20% methanol at 200
mA for 2 h at 4°C. The membrane was blocked with 2% BSA in TBST (50
mM Tris-Cl, 150 mM NaCl, 0.05% Tween 20, pH 8). Incubations and
washings of primary and secondary Abs were done with TBST at room
temperature. Blots were developed using diaminobenzidine (Sigma-Aldrich, St. Louis, MO).
Generation of LAMP-1-Fc fusion protein
The pCEP4-hFc vector that encodes the Fc portion of human IgG1 was a gift
from Drs. T. W. Mak and Dr. S. K. Yoshinaga (Amgen, Thousand Oaks, CA).
For cloning LAMP-1 in pCEP4-hFc vector, 370 aa from the amino-terminal of
LAMP-1 were fused in frame to the sequence encoding the Fc portion in the
amino terminus region. The forward and reverse primers, 5⬘-CGCAAGCTT
ATGCGGCCCCCGCGCGCG-3⬘ and 5⬘-CGCGCGGCCGCGTTGTTACC
ATCCTGAACACACTC-3⬘ were used for cloning the LAMP-1 truncated
gene into the HindIII and NotI sites within the multiple cloning sites of
pCEP4. The coding sequence of LAMP-1 from N terminus up to the transmembrane domain, devoid of the region spanning the membrane, was incorporated into pCEP4-Fc. The highly purified pCEP4-LAMP-1-Fc plasmid was then transfected into Chinese hamster ovary (CHO) cells and the
mouse macrophage cell line P388D1 using FuGene 6 transfection reagent
(Roche). The transfected cells were grown in serum-free media containing
insulin-transferrin-selenium-A (Life Technologies). Soluble secreted fusion proteins were purified from culture supernatants using protein A agarose affinity column chromatography (Roche).
Deglycosylation of transfected fusion proteins
A total of 5 ␮g of fusion proteins, i.e., CHO-LAMP-1-Fc or macrophageLAMP-1-Fc, were treated with 20 mU endoglycosidase H (Roche) for 16 h
at 37°C using 0.5 M sodium citrate buffer (pH 5.5) containing 0.1 M 2-ME
and 0.1% SDS.
T cell proliferation, cytokine, and blocking assays
Spleens from 6- to 8-wk-old female BALB/c obtained from experimental
animal facility of our institute were used to make single-cell suspensions of
splenocytes. RBCs were lysed using hemolytic Gey’s solution. Nonadherent cells were collected from supernatants after allowing cells to adhere to
plastic petri dishes (Corning Glass, Corning, NY) at 37°C in the presence
of 5% CO2 for 2 h. The CD4⫹ T cells were enriched by passing through
a nylon wool column (Robbins Scientific, Sunnyvale, CA). Finally, CD4⫹
T cells were purified to at least 98% purity by negative selection using the
CD4⫹ T cell enrichment mixture (StemCell Technologies, Vancouver,
British Columbia, Canada). The T cells were cultured in RPMI 1640 (Life
Technologies) supplemented with penicillin (70 ␮g/ml), streptomycin (100
␮g/ml), glutamine (4 mM), 2-ME (50 mM), sodium pyruvate (1 mM),
HEPES (20 ␮M), and heat-inactivated 10% FCS (Life Technologies). The
purified T cells (105) were cultured along with anti-CD3, either in presence
or absence of varying concentrations of Fc fusion proteins for 72 h in
96-well, flat-bottom plate (Costar, Cambridge, MA). These cells were then
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The peritoneal exudate cells were harvested from BALB/c mice (6 – 8 wk
old) injected 4 days previously with 2 ml of 3% thioglycolate (Difco,
Detroit, MI). The peritoneal exudate cells were washed with cold HBSS
and macrophages were obtained by adhering for 1 h at 37°C on plastic petri
dishes and the purity of macrophage was ⬎98%, as analyzed by their
reactivity with anti-mac-1 Ab. The macrophages were washed thrice and
pellet was frozen overnight at ⫺70°C. The cells were thawed and homogenized in the presence of 20 mM Tris-Cl (pH 7.4) and 1 mM EDTA along
with protease inhibitor mixture (10 ␮g/ml leupeptin, 10 ␮g/ml aprotinin,
10 ␮g/ml pepstatin, 10 ␮g/ml antipain, 10 mM iodoacetamide, 10 ␮g/ml
chymostatin, and 1 mM PMSF). The nuclear fraction was removed by
centrifuging at 700 ⫻ g for 10 min at 4°C. The supernatant was subjected
to centrifugation at 110,000 ⫻ g for 2 h at 4°C. The pellet was solubilized
overnight in 20 mM Tris-Cl (pH 7.5) containing 1% Triton X-100, 20%
glycerol and protease inhibitor mixture, and recentrifuged at 100,000 ⫻ g
for 1 h at 4°C to remove the insoluble debris. The membrane proteins (27,
28) in the supernatant were separated on 10% SDS-PAGE (29). M150 was
eluted from the gel by crushing the gel pieces containing the protein band
followed by overnight incubation with 100 mM NH4 HCO3, 50 mM TrisCl, 0.1 mM EDTA, and 150 mM NaCl (pH 8.0). SDS from the protein
solution was removed by passing through Extracti-D gel column (Pierce,
Rockford, IL) and the protein content was estimated using the bicinchoninic acid kit (Pierce). The protein was further purified by fast performance
liquid chromatography on Mono-Q column and the purity was ascertained
by two-dimensional electrophoresis before subjecting it to protein
sequencing.
using mAb-based Rat Ig isotyping kit (BD PharMingen, San Diego, CA).
For the present study, anti-M150 Abs were produced as ascites, and purified by sequential chromatography over Sepahcryl S 300, followed by goat
anti-rat IgM affinity chromatography.
The Journal of Immunology
pulsed with [3H]thymidine (1 ␮Ci/well) during the final 12 h of the culture
period, following which the incorporated radioactivity was determined by
liquid scintillation counting. For blocking experiments, anti-LAMP-1
(ID4B) and IgG2a isotype (BD PharMingen), along with affinity-purified
anti-M150 and normal rat IgM were used. In parallel experiments, supernatants from such cultures were also collected for determination of IL-2,
IFN-␥, IL-4, and/or IL-13 by ELISA (R&D Systems, Minneapolis, MN).
RT-PCR for c-Maf and T-bet
Total RNA was prepared from 106 CD4⫹ T cells per well cultured in
24-well plate for 24 h either in the presence of CHO-LAMP-1-Fc or macrophage-LAMP-1-Fc and 1 ␮g/ml of soluble anti-CD3 (BD PharMingen).
The total RNA was isolated using S.N.A.P. total RNA isolation kit (Invitrogen). A total of 500 ng of total RNA was used to perform RT-PCR using Titan
one tube RT-PCR System (Roche). The T-bet primers used were 5⬘-ATGG
GCATCGTGGAGCCGGGCT-3⬘ and 5⬘-ACTTGGACCACACAGGTGGT
TG-3⬘, cMaf primers were 5⬘-ACTGAACCGCAGCTGCGCGGGGTCAG-3⬘
and 5⬘-CTTCTCGTATTTCTCCTTGTAGGCGTCC-3⬘ (31), which were
custom made (Gemini Biotech, Alachua, FL). The control primers used were
dihydrofolatereductase set for RT-PCR (Stratagene, La Jolla, CA).
Results
In our earlier studies, we have isolated a 150-kDa protein from the
membrane fraction of activated murine macrophages. This protein,
termed as M150, was demonstrated to provide costimulatory activity to CD4⫹ T cells. Interestingly, expression of M150 on the
surface of the activated macrophages was found to specifically
elicit Th1 responses from CD4⫹ T cells, at least when assessed at
the level of cytokine production in culture supernatants. Furthermore, surface expression of M150 could be detected on activated
macrophages but not on activated B cells. Thus, M150 constitutes
a macrophage restricted costimulatory molecule that specifically
drives Th1 responses. Therefore, it was of interest to establish the
biochemical identity of this cell surface protein.
Using a previously established procedure, a homogenous preparation of M150 was obtained from the plasma membrane fraction
of activated macrophages (Fig. 1, A and B) and the purified M150
was subjected to internal and amino-terminal sequencing by two
independent preparations at two different sequencing facilities.
The three partial sequences thus obtained were subsequently used
in a search for homologous sequences within the protein databases.
Surprisingly, all of the three partial sequences yielded 100% identity with segments of the murine LAMP-1 protein. As seen in Fig.
1C, the amino terminus of M150 was identical with that of
LAMP-1, and the two internal sequences obtained were found to
correspond to segments between aa 128 –139 and 288 –293 of the
LAMP-1 protein. These results strongly suggested that M150 is
either identical with, or highly homologous to LAMP-1 (32, 33).
To further confirm this, and to check whether there is any alternate form of LAMP-1 gene existing in macrophages with internal sequence differences as compared with 3T3 LAMP-1 cDNA, a
heteroduplex analysis was performed. For this, corresponding oligonucleotide segments from the internal amino acid sequences of
M150 were used as primers to amplify the corresponding segment
from cDNA of LAMP-1. In parallel, these primers were also used
FIGURE 1. Electrophoretic analysis of M150 protein and its characterization. A, SDS-PAGE of plasma membrane proteins isolated from thioglycolate
exudate macrophages. Macrophage membrane proteins were prepared as described in Materials and Methods. In brief, macrophages (98% mac-1 positive)
were homogenized and after removal of the debris by low-speed centrifugation, the supernatant was subjected to ultra centrifugation at 110,000 ⫻ g. The
pellet was solubilized and it was recentrifuged at 100,000 ⫻ g. The supernatant containing the membrane fraction was separated on a 10% SDS-PAGE and
was stained with Coomassie blue. Lane 1, Molecular mass markers; lane 2, Surface membrane proteins of macrophage; lane 3, Gel-purified M150 under
reducing condition. B, Two-dimensional electrophoresis of M150. The purified M150 (as shown in A, lane 3) was further purified by fast performance liquid
chromatography on a Mono-Q column and subjected to two-dimensional electrophoresis. The M150 was resolved by isoelectro focusing across a pH
gradient of 3.5–9.5 (first dimension) and then electrophoresed on a 10% SDS-PAGE (second dimension). C, Sequence identity of partial amino acid
sequences of M150 with LAMP-1. Sequence data of N-terminal and internal amino acid sequences of M150 in comparison with LAMP-1. Peptide sequence
1 was derived from the amino-terminal end of M150. Peptide sequences 2 and 3 are two internal sequences derived by tryptic digestion of M150 using
a different preparation as reported by us earlier (23). D, Heteroduplex analysis. Heteroduplex analysis was conducted as described in Materials and Methods.
The 500-bp PCR product generated from LAMP-1 cDNA was mixed with PCR product obtained from different clones (representing the RT-PCR product
of thioglycolate-elicited macrophages). Duplex DNA thus generated was electrophoresed on 8% native PAGE and visualized by ethidium bromide staining.
Lanes 1–3, Results for 3 of the 40 clones tested are shown as representatives.
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The protein M150 displays amino acid sequence homology with
LAMP-1
1803
1804
GLYCOSYLATION MODULATES THE COSTIMULATORY PROPERTY OF LAMP-1
to amplify the cDNA, isolated from thioglycolate-elicited macrophages. The amplified product was then cloned into an appropriate
vector and used for transformation. The plasmid DNA from these
colonies was then PCR amplified and subjected to a heteroduplex
analysis with the PCR product derived from cDNA of LAMP-1. It
may be mentioned in this study that mutations or sequence variations are easily detected by gel electrophoresis of heteroduplexes,
where minute sequence variations appear as blebs on the gel. In
contrast, identical sequences occur as homoduplexes. All of the
40 clones tested in hetroduplex analysis exhibited homoduplex
formation. Results of three of these clones are shown in Fig. 1D
as a representative example. Therefore, these results strongly
suggest that M150 protein is in fact LAMP-1 protein, which is
an activation-dependent cell surface glycoprotein also known as
CD107a (34).
Differential expression of anti-M150 and anti-LAMP-1 reactivity
in splenocyte subsets
The above results support that M150 and LAMP-1 are products of
the same gene. Therefore, it is surprising that while LAMP-1 is a
ubiquitous protein expressed in a variety of cell types, the expression of M150 is restricted to the surface of activated macrophages.
To probe further for a possible basis for this distinction, we next
examined the expression of M150 and anti-LAMP-1 reactivity in
splenocyte subsets using specific Ab conjugates by FACS analysis.
As shown in Fig. 2A, surface reactivity with anti-LAMP-1 Ab was
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FIGURE 2. Differential surface expression of LAMP-1 and M150 on APCs and T cells. A, Flow cytometric analysis showing surface expression of
LAMP-1 and M150 on splenocytes. Either resting (columns 1 and 2) or IFN-␥ activated (columns 3 and 4) splenocytes were analyzed for surface expression
of LAMP-1 and M150. FITC-labeled anti-LAMP-1 and anti-M150 Abs were used along with PE-labeled Abs to the markers of B cells (B220), macrophages
(mac-1), and T cells (CD4 and CD8) to double stain the splenocytes. PE-conjugated isotype controls of Abs were used for staining B cells (rat IgG2a),
macrophages (rat IgG2b), CD4⫹ (rat IgG2b), and CD8⫹ T cells (rat IgG2a). Isotype controls for anti-LAMP-1 and anti-M150 Abs used were FITC-labeled
normal rat IgG2a and normal rat IgM, respectively. a, The representative data of isotype controls used for staining splenocytes. b, Results for dual staining
with anti-B220 PE and either FITC-conjugated anti-LAMP-1 or anti-M150 Abs. c, Dual staining was also performed using anti-Mac-1 PE along with either
FITC-conjugated anti-LAMP-1 or FITC-labeled anti-M150 Abs. d, Staining of anti-CD4 PE- and FITC-labeled anti-LAMP-1 or anti-M150 Abs on
splenocytes. e, The binding of anti-LAMP-1 FITC and anti-M150 FITC to CD8⫹ splenocytes. The experiments were done using the identical compensation
throughout. In all of these experiments, ⬎97% of the cells were viable as revealed by the propidium iodide staining (data not shown). B, Anti-LAMP-1
and anti-M150 recognize distinct epitopes on macrophages. Upper panel, The cells (106 cells/sample) were incubated simultaneously with 1.5 ␮g of
anti-LAMP-1-FITC in the presence of purified anti-M150 at 5, 10, 25, and 50 times molar concentrations. Conversely, when cells were stained with
anti-M150 FITC (as depicted in lower panel) 5, 10, 25, and 50 times molar concentrations of unlabeled anti-LAMP-1 was used as the competitor for the
FITC-tagged anti-M150. Isotype controls for the anti-LAMP-1-FITC and anti-M150-FITC were FITC-conjugated normal rat IgG2a and normal rat IgM,
respectively.
The Journal of Immunology
Differential glycosylation gives antigenic distinctness to LAMP-1
The existence of antigenic variance between M150 and LAMP-1
was also confirmed by Western blot analysis of the membrane
fraction of activated macrophages using either anti-M150 or antiLAMP-1 Abs as probes. Probing with either polyclonal or monoclonal anti-M150 Ab (G1) detected a band that centered around a
molecular mass of 150 kDa. As opposed to this, anti-LAMP-1 Ab
identified a lower band ranging from 120 –145 kDa (Fig. 3A). Previous reports have demonstrated that the size heterogeneity of
LAMP-1 is due to heterogeneous glycosylation (32). These studies
also revealed that the size distribution of LAMP-1 differs significantly, depending upon the cell type examined. In other words, the
glycosylation pattern of LAMP-1 appears to vary depending upon
the cell type in which it is expressed (35).
To further confirm the possible cell type-specific differences in
glycosylation of LAMP-1, we expressed a truncated version of
LAMP-1 protein as LAMP-1-Fc chimera either in CHO cells, or in
the mouse macrophage cell line, P388D1. The transfected fusion
proteins were then purified and subjected to a Western blot analysis. As shown in Fig. 3B, nonidentical forms of the chimeric
protein were produced in CHO and P388D1 cells, at least as detected by anti-LAMP-1 and anti-M150 mAbs. The product of CHO
cells displayed a molecular mass between 105 and 120 kDa; in
contrast, the product from P388D1 cells was significantly of higher
molecular mass, ranging from 130 –150 kDa. Interestingly, antiM150 mAb did not show reactivity with LAMP-1 protein expressed in CHO cells while recognizing the LAMP-1 expressed in
macrophages (Fig. 3B, compare lanes 5 and 6). In contrast, antiLAMP-1 recognized a discrete number of lower molecular mass
proteins from LAMP-1-transfected CHO and P388D1 cells as
shown in Fig. 3B, lanes 2 and 3, respectively. A comparison of the
protein bands (Fig. 3B, lanes 3 and 6) recognized by anti-LAMP-1
and anti-M150 Abs suggest that the M150 is a specific form which
constitutes only a small fraction of the total LAMP-1 produced in
macrophages.
To compare the protein core of these two molecular species, the
expressed products from both CHO and P388D1 cells were first
deglycosylated using endoglycosidase H. Surprisingly, this procedure completely eliminated the reactivity of the anti-LAMP-1 and
anti-M150 Abs. Thus, the resultant deglycosylated proteins could
be detected only with anti-Fc Ab.
Therefore, these results collectively suggest that both Abs preparations are directed against the glycosylated portion of the molecules. Thus, from these results it can also be inferred that the
expression of LAMP-1-Fc chimera in CHO and P388D1 cells results from qualitatively distinct glycosylation patterns. Furthermore, it is this variation in glycosylation that accounts for the
observed difference in antigenicity, at least with respect to antiM150 Ab. In other words, the anti-M150 mAb appears to be directed against a LAMP-1 subset that is specifically produced only
in macrophages.
The LAMP-1-Fc chimera displays costimulatory activity when
expressed in macrophages
We next examined whether the altered glycosylation pattern of the
LAMP-1-Fc chimera also confers altered function for the protein
molecule when expressed in P388D1 vs CHO cells. Therefore,
purified CD4⫹ T cells were stimulated with varying concentration
of anti-CD3 Ab in the presence of a fixed concentration of either
CHO-LAMP-1-Fc or macrophage-LAMP-1-Fc proteins. A proliferative response was observed only in cultures that included macrophage-LAMP-1-Fc, but not in those where CHO-LAMP-1-Fc
was added (Fig. 4A).
It is possible that the difference in activity observed for macrophage-LAMP-1-Fc and CHO-LAMP-1-Fc in Fig. 4A simply reflects differences in dose requirements for the two chimeric products. To verify this, T cells were stimulated with a constant dose of
1 ␮g/ml of anti-CD3 Ab in the presence of increasing concentrations of either macrophage-LAMP-1-Fc or CHO-LAMP-1-Fc. For
these experiments, a dose-dependent proliferation of CD4⫹ T cells
was obtained with macrophage-LAMP-1-Fc (Fig. 4B). In contrast
to this, CHO-LAMP-1-Fc was unable to induce T cell proliferation
at any of its tested concentrations (Fig. 4B). Furthermore, the specificity of the macrophage-LAMP-1-Fc-dependent T cell response
is also evident from the fact that this effect could be inhibited by
addition of anti-M150 Ab, but not by anti-LAMP-1 Ab (Fig. 4B).
We have previously demonstrated that M150 serves as a costimulatory molecule that specifically elicits cytokines typical of
the Th1 subset of CD4⫹ T cells. As shown in Fig. 4C, the macrophage-LAMP-1-Fc product also reproduced this activity when
used in conjunction with anti-CD3 for stimulation of CD4⫹ T
cells. Significant levels of both IL-2 and IFN-␥ were detected in
the supernatants from these cultures (Fig. 4C). In contrast, stimulation in presence of CHO-LAMP-1-Fc failed to induce detectable
levels of Th1 representative cytokines (Fig. 4C). It is pertinent to
mention that no detectable levels of Th2 representative cytokines
were obtained from the culture supernatants of Th cells activated
either by CHO-LAMP-1-Fc or macrophage LAMP-1-Fc. Collectively, these results categorically identify that LAMP-1 can indeed
display Th1-specific costimulatory activity, but this activity is contingent upon its expression in macrophages.
Macrophage-LAMP-1-Fc induces expression of T-bet in naive
CD4⫹ T cells
It is now becoming evident that differentiation of CD4⫹ T cells
into either the Th1 or Th2 commitment pathways is regulated by
the activation of independent transcription factors (36). Subsetspecific transcription factors have been identified and their role in
distinct T cell differentiation is being actively explored. For example, STAT-6 has been shown to synergize with an antigenic
stimulus leading to the up-regulation of GATA3, a potent inducer
of Th2 differentiation (31). The transcription factor c-Maf, which
negatively regulates Th1 differentiation, has now been shown to be
responsible for the tissue-specific expression of IL-4 (37). Recently, T-bet was identified as a transcription factor specific to Th1
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displayed by activated splenic B cells, macrophages, and CD8⫹ T
cells. In contrast, anti-M150 reactivity was restricted only to the
Mac-1⫹ subset of activated splenocytes. This suggests that M150
is antigenically distinct from LAMP-1. However, as depicted in
Fig. 2A, the anti-M150 and anti-LAMP-1 Abs have been found to
bind only with the IFN-␥-activated macrophage populations (i.e.,
the Mac-1⫹ populations of the splenocytes). Therefore, we tested
the possibility of these two Abs for competing for the same epitope
of LAMP-1 present on macrophage membrane. To address this
issue, IFN-␥-activated peritoneal exudate macrophages were
stained with anti-M150 FITC-labeled Ab keeping the unlabeled
anti-LAMP-1 Ab as a competitor of its specific site and vice versa.
Fig. 2B demonstrates that there is no significant difference in the
binding of either of the labeled Abs in the presence of the other as
competitor, which conclusively proves that anti-LAMP-1 and antiM150 Abs do not compete for binding to IFN-␥-activated macrophages. Thus, this result also clearly substantiates that antiLAMP-1 and anti-M150 bind to different epitopes of LAMP-1.
However, NK T cells, dendritic cells, and monocytes were negative for staining with anti-LAMP-1 and anti-M150 Abs (data not
shown).
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GLYCOSYLATION MODULATES THE COSTIMULATORY PROPERTY OF LAMP-1
cells, and demonstrated to act by controlling the expression of
IFN-␥ (38). Given the Th1-specific costimulatory activity observed for M150 in our earlier study (23), as well as in the present
report (Fig. 4C), we further examined whether it could be implicated in selectively driving differentiation of CD4⫹ T cells.
CD4⫹ T cells were stimulated with anti-CD3 in the presence of
either macrophage-LAMP-1-Fc or CHO-LAMP-1-Fc from which
the total RNA was isolated. This was then analyzed for the presence of specific mRNA for c-Maf and T-bet by RT-PCR. Consistent with the absence of any activity of CHO-LAMP-1-Fc, no expression of either c-Maf or T-bet could be detected in cells
stimulated with this protein. In contrast, costimulation with macrophage-LAMP-1-Fc yielded a significant induction of T-bet
mRNA, with no concomitant effect on c-Maf expression (Fig. 5).
Therefore, this selective effect of macrophage-LAMP-1-Fc on Tbet expression may implicate M150, a modified glycosylated form
of LAMP-1, as a costimulatory molecule that specifically drives
the naive CD4⫹ T cells toward Th1 differentiation.
Discussion
The development of a productive T cell response initially depends
upon appropriate costimulatory signals that are provided by the
APC. Costimulatory molecules present on the surface of activated
APCs are now known to act by binding to specific receptors on T
cells which, in turn, initiate intracellular signaling pathways that
lower the threshold for T cell activation. Although it was earlier
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FIGURE 3. Western blot analysis showing differential glycosylation pattern of LAMP-1 in macrophages. A, Western blot analysis of macrophage
membrane fractions. Surface membrane proteins from peritoneal exudate macrophages were isolated as described in Materials and Methods. These were
then resolved by 10% SDS-PAGE and were transferred onto a nitrocellulose membrane and probed with different primary Abs. This was followed by
incubation with biotinylated anti-rat and streptavidin-HRP. Bound secondary Ab was revealed using diaminobenzidine as the substrate. Lanes 1, 4, and 7,
Protein molecular mass markers. Lanes 2 and 3, Position of M150 when the blots were probed with polyclonal anti-M150 and control rat sera, respectively.
Lanes 5 and 6, The blots probed with anti-LAMP-1 and an isotype control rat IgG2a, respectively. Lanes 8 and 9, Position of M150 on the blots probed
with monoclonal anti-M150 and isotype control rat IgM Abs, respectively. B, Western blot analysis of purified LAMP-1-Fc fusion proteins. Purified
LAMP-1-Fc proteins (LAMP-1 fused with Fc), obtained from transfected CHO and P388D1 cells, were run on SDS-PAGE and transferred to a nitrocellulose membrane. Lanes 1 and 4, Biotinylated protein molecular mass markers. Lanes 2 and 5 contain CHO-LAMP-1-Fc, and lanes 3 and 6 contain
macrophage-LAMP-1-Fc proteins. Left panel (lanes 2 and 3) was probed with anti-LAMP-1 Ab (ID4B) and right panel (lanes 5 and 6) was probed with
anti-M150 monoclonal (G1) Ab. C, Deglycosylation of purified LAMP-1-Fc fusion proteins. Lane 1, Molecular mass markers; lanes 2 and 3, Deglycosylated products of CHO-LAMP-1-Fc and macrophage-LAMP-1-Fc proteins, respectively, separated on 10% SDS-PAGE. Note the identical mobility of
both the proteins. D, lanes 1 and 2, Deglycosylated CHO-LAMP-1-Fc and macrophage-LAMP-1-Fc products (shown in C) probed with anti-Fc Ab.
The Journal of Immunology
1807
thought that CD80 and CD86 were the principal costimulatory
molecules involved in regulation of immune responses, more recent studies have revealed that they constitute members of a family
of related molecules. As discussed earlier, several novel costimulatory molecules have now been identified, some of which display
selectivity either at the level of APC-restricted expression or at the
level of Th subset preferences. Thus, immune regulation appears to
be far more complex than hitherto suspected, the outcome being
presumably dependent on a tightly controlled network of interactions between the various costimulatory molecules and their appropriate receptor on T cells. Therefore, T cell activity may well
represent a temporally regulated process, which at any given time
will be dependent on both the spectrum and surface densities of
individual costimulatory molecules that are expressed on the APC
surface.
In a recent study, we had isolated a novel protein M150 from the
plasma membrane of activated macrophages that possessed costimulatory activity (23). We also demonstrated further that the
activity of M150 dominates over that of B7.1 when both are expressed on the surface of activated macrophages. This was demonstrated both at the level of enhanced alloreactivity, as well as
increased IFN-␥ production in cocultures with T cells (24).
The present report is the outcome of our subsequent efforts to
further characterize the M150 protein. Surprisingly, our results revealed that M150 was in fact the ubiquitously expressed LAMP-1,
albeit in a uniquely glycosylated form. This was initially suggested
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FIGURE 4. CD4⫹ T cell proliferation assay and cytokine analysis in response to LAMP-1-Fc. A, Induction of CD4⫹ T cell proliferation induced by
macrophage-LAMP-1-Fc. T cell proliferation assay was performed in the presence of increasing concentrations of soluble anti-CD3 Ab, without (䡺) or
with (o) Fc control, or with CHO-LAMP-1-Fc (p) and macrophage-LAMP-1-Fc, i.e., M␾-LAMP-1-Fc (f), used at a concentration of 2 ␮g/ml. Proliferation was measured by [3H]thymidine incorporation during the last 16 h of the 72-h culture. The data represent the mean of three independent experiments
and SD was obtained by nonbiased or (n-1) method. B, Anti-M150 blocks the T cell proliferation induced by macrophage LAMP-1-Fc. The T cell
proliferation assay was performed (as described for A) in presence of soluble anti-CD3 Ab (1 ␮g/ml) and Fc protein (䡺), CHO-LAMP-1-Fc (o), and
M␾-LAMP-1-Fc (f) at the indicated concentrations. To describe the effect of various Abs in the blocking assay, the bars are labeled as a, b, c, and d, which
represent anti-M150, normal rat IgG2a, anti-LAMP-1, and normal rat IgM, respectively. The proliferation induced by macrophage LAMP1-Fc at 0.5 ␮g/ml
concentration in the presence of anti-CD3, was inhibited by only anti-M150 Ab (a), where as its isotype control, rat IgM (d) was found to have no effect.
No effect of anti-LAMP-1 Ab (c) or its isotype control rat IgG2a (b) on the T cell proliferation was noticed. The anti-M150 Ab blocked the proliferation
to a significant level induced by macrophage-LAMP-1-Fc. The data represent the mean of three independent experiments and SD was obtained by nonbiased
or (n-1) method. C, Induction of Th1 type cytokines by macrophage-LAMP-1-Fc. Culture supernatants from the T cell proliferation assay with increasing
concentrations of purified macrophage LAMP1-Fc in the presence of anti-CD3 (1 ␮g/ml) show the presence of IL-2 (f) and IFN-␥ (䡺). There were no
detectable levels of these cytokines when CHO-LAMP-1-Fc was used to costimulate the Th cells. IL-4 and IL-13 were not detectable in these supernatants.
The data represent the mean of three independent experiments and SD was obtained by nonbiased or (n-1) method.
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GLYCOSYLATION MODULATES THE COSTIMULATORY PROPERTY OF LAMP-1
Acknowledgments
FIGURE 5. Induction of T-bet, a Th1-specific transcription factor. Total
RNA was prepared from CD4⫹ T cells activated in presence of anti-CD3
(1 ␮g/ml) and with CHO-LAMP-1-Fc (2 ␮g/ml), or with macrophageLAMP-1-Fc for 24 h and analyzed for the messages for c-Maf and T-bet by
RT-PCR. Lanes 1 and 2, Equal loading controls which are the RT-PCR
product for dihydrofolatereductase (DHFR) from the RNA prepared from
T cells activated with CHO-LAMP-1-Fc protein and M␾-LAMP-1-Fc fusion proteins, respectively. Lanes 3 and 4, The RT-PCR products of c-Maf
and T-bet mRNA, respectively, from T cells activated with CHO-LAMP1-Fc. Lane 5 and 6, The RT-PCR product of c-Maf and T-bet mRNA
derived from M␾-LAMP-1-Fc-activated T cells, respectively.
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