Published July 1, 1991
Human Immunodeficiency Virus Type 1 gp120
Mimics a Hidden Monomorphic Epitope Borne by
Class I Major Histocompatibility Complex
Heavy Chains
By Fabio Grassi,* Raffaella Meneveri,* Martin Gullberg,t
Lucia Lopalco,* Giovanni Battista Rossi,S Paola Lanza,*
Claudio De Santis,* Goran Brattsand,$ Stefano Butto,S
Enrico Ginelli,* Alberto Beretta, 11 and Antonio G. Siccardi*
From the *Dipartimento di Biologia e Genetica per le Scienze Mediche, University di Milano,
20133 Milano, Italy; the # Unit for Applied Cell and Molecular Biology, University of Umea,
90187 Umed, Sweden; the aboratorio di Virologia, Istituto Superiore di Sanity, 00161 Roma,
Italy, and the Il Dipartimento di Biologia e Tecnologia, Istituto H S. Raffaeue,
20132 Segrate, Italy
s
Murine monoclonal antibodies (mAbs) M38 and L31 define two epitopes of a surface protein
of activated lymphocytes and monocytes. It has been shown that M38 also defines a crossreactive
epitope of human immunodeficiency virus type 1 (HIV1) gp120 (Beretta et al ., 1987. Eur. J.
Immunol. 17:1793) . The mAb inhibits syncytia formation driven by HIV-1-infected cells. The
surface protein was demonstrated to be a class I MHC ot chain, by sequence analysis of the
corresponding cDNA and by immunological means . The epitopes defined by mAbs M38 and
L31 are monomorphic and hidden (i.e., inaccessible to antibodies) on native HLA molecules
expressed by resting cells, but can be evidenced on denatured proteins by Western blot analysis.
The two epitopes become accessible after activation processes havebeen implemented, likely reflecting
a conformational alteration of at chains (such as that described by Schnabl et al. 1990. J Exp.
Med. 171:1431) . Consistent with molecular data are the results offunctional analysis, which indicate
that the molecule recognized by M38 and L31 is a gate for pleiotropic negative signals, since
the two mAbs were shown to inhibit monocyte antigen presentation and lymphocyte mitogenic
proliferation, respectively.
H
IV infection of susceptible cells starts with the interaction ofviral gp120 with the CD4 molecule (1-3) . Since
CD4 belongs to the Ig superfamily, whose members are involved in regulatory interactions essential for the immune
system function (4-6), the specificity ofgp120 for this molecule could be central for the virus in evading the host immune response.
Beside the depletion of CD4+ cells due to HIV cytopathic
effects, other gp120-related mechanisms for amplification of
immune damage have been proposed : killing ofbystander cells
induced by soluble gp120 (7, 8), anti-CD4 autoantibodies
induced by CD4-like idiotopes expressed within the env-specific
repertoire (9), synergism between gp120 and anti-gp120 antibodies in blocking T cell activation (10), and blockade of
the differentiation of CD4+CD8+ thymocytes into CD4+
CD8 - T lymphocytes secondary to gp120 binding to CD4
(11, 12).
53
CD4 is a ligand of class II MHC and interacts with other
proteins in the context of a multimolecular complex (13, 14) .
HIV might have evolved its specificity for CD4 by mimicry
of a physiological ligand of CD4 . Experimental evidence of
serological crossreactivity between viral envelope and human
epitopes have already appeared in two independent reports.
One case is the identification of homologous regions in HIV-1
gp41 and human class II MHC (/3-1 domain) . A fraction (36%)
of AIDS patient sera was shown to contain antibodies reacting
with a class II-derived peptide and with native class II antigens (15). Further, a gp120-specific mAb, M38, was shown
to crossreact with a surface protein of activated human monocytes endowed with negative signaling potentials (16).
The present work describes by biochemical, immunological, and genetical means the crossreactive human surface protein defined by mAb M38. Such a molecule resulted in being
a class I MHC heavy chain, present on activated immuno-
J . Exp. Med . ® The Rockefeller University Press " 0022-1007/91/07/0053/10 $2 .00
Volume 174 July 1991 53-62
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Summary
Published July 1, 1991
cytes in a form that differs from that of HLA molecules on
resting cells, likely corresponding to the conformational alteration of ca chains (not associated with 02m) recently described by Schnabl et al . (17) . We present evidence that such
conformationally altered molecules might be involved in functional regulatory mechanisms .
Materials and Methods
54
Results
Characterization of mAb L31. mAb L31 was derived from
the same HIV-immunized mouse that yielded M38 (16) . It
gp120 Mimics a Hidden Monomorphic Epitope
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Monoclonal Antibodies. mAbs OKT3, -4, -8, and -9 were purchased from Ortho Diagnostics Systems, Inc., (Raritan, NJ), and
Leu-3a was from Becton Dickinson & Co. (Mountain View, CA).
The anti TAC DMS-1 (18) and the anti-CD3 UCTH-1 (19) were
kindly provided by Drs. Smith and Beverly (ICRF, London), respectively. mAb 01 .65 (20), specific for a common determinant of
HLA-A, -B, -C molecules, and mAb R1 .30, specific for 02m (20),
were kind gifts of Dr. Malavasi (Department of Genetics, University of Torino, Italy), mAb CR11 .351, specific for a polymorphism
determinant of HLAA2 (21), was a kind gift of Dr. Russo (Columbia University, New York). M38, L31, and D28 were derived
from the same fusion (16) ; D28 was used as a control of irrelevant
specificity.
Cells. Human PBMC, isolated by Ficoll-Hypaque discontinuous
gradient centrifugation were cultured after stimulation with OKT3
and rabbit anti-mouse Ig antibodies (22) or with 5 Aglml Con
A to obtain activated lymphocytes . Rex cells, a human T lymphoblastoid cell line, were a kind gift of Dr. Acuto (Department of
Immunology, Pasteur Institute, Paris) . 81351 (LAV) cells, which
harbor a single integrated copy of a pot mutant of HIV-1 (LAV)
and produce defective, uninfectious virions (23), were a kind gift
of Dr. Folks (Center for Disease Control, Atlanta, GA). MOLT3
cells were a kind gift of Dr. De Rossi (Oncology Institute, University of Padova, Italy.
Cytofuorimetry. Lymphocytes of 8E51(LAV) cells were washed
and resuspended in RPMI 1640 containing 20% normal human
serum. Cells (106 cells/200 Al) were incubated on ice for 30 min
with the appropriate dilution of mAb. After two washings in RPMI
1640, FITC-conjugated rabbit anti-mouse Igs (Dakopatts a/s Glostrup, Denmark) were added and cells were incubated 30 min on
ice. Washed cells were fixed in 5% paraformaldehyde and applied
to a Spectrum III flow cytometer (Ortho Diagnostics Systems, Inc.) .
For the blocking experiment to demonstrate that M38 could bind
both the viral and the cellular antigens, Con A-activated lymphocytes were used : 1 leg of M38 (or 01.65) was preincubated (30 min
at room temperature) with an eightfold molar excess (5 Fcg) of
recombinant soluble gp160 (Transgene, Strasbourg) . A FACScana
(Becton Dickinson & Co .) was used for this experiment .
Radiolabelled mAb Cell Binding Assay. mAbs were purified from
ascitic fluids by (NH4)2SO4 precipitation and protein A-Sepharose
(Pharmacia Fine Chemicals, Piscataway, NJ) affinity adsorption and
radioiodinated using Enzymobeads (Bio-Rad Laboratories, Richmond, CA) and Na '25 1 (Amersham Corp ., Arlington Heights,
IL). For the cell binding assay, cells were resuspended at 5 x
106 /ml in RPMI 1640 supplemented by 5% rabbit serum; 100 Al
of the cell suspension was loaded onto a two-step gradient in 0.3ml tubes (lower layer: 90% FCS and 10% sucrose; upper layer:
80% silicon oil and 20% mineral oil) with appropriate dilutions
of radiolabeled mAbs and competitors . After a 30-min incubation
at 37°C, cell-bound and free radioactivity was determined after
centrifugal cell separation .
Radioiodination, Immunoprecipitation, andSDS/PAGE. Cells were
radioiodinated by the lactoperoxidase method and lysed in digitonin
lysis buffer (10 mM Tris-HCI, pH 7.5 ; 5 mM EDTA ; 0.15 M NaCl ;
2% digitonin; 10 MM NaF; 1 mM PMSF ; 10 mM iodoacetamide;
5 hg/ml leupeptin, and pepstatin A) . The lysates were adsorbed
on mAb-Affigel beads (Bio-Rad Laboratories) for 1 h at 4°C. After
four washes in Digitonin lysis buffer, the material was eluted from
the beads with SDS/PAGE sample buffer (with or without 2%
02m) . SDS/PAGE was run using vertical slab gels and 10%
acrylamide.
Immunoabsorption Assay. Activated lymphocytes (3 x 10'/ml)
were suspended in Triton lysis buffer (20 mM Tris-HCI, pH 7.4 ;
0.15 M NaCl ; 10 mM NaF; 2.5 mM EDTA ; 1% Triton X-100;
10 mM iodoacetamide; 1 mM PMSF ; 5 hg/ml leupeptin) at 4°C
for 30 min. Postnuclear supernatants were loaded on affinity columns
of Affigel (BioRad Laboratories) coupled to the indicated mAbs
(3 mg/ml of packed beads) . The first 3 ml effluent and the SDSeluted material were acetone precipitated and resuspended in
SDS/PAGE sample buffer for Western Blot analysis with "5I-M38
and "'I-L31.
Syncytia Formation Assay. 81351 (LAV) cells and MOLT3 cells
were grown in RPMI 1640 supplemented by 10% FCS to concentrations of 106 cells/ml, washed in medium, mixed in 1:2 proportions, and distributed in V-shaped microtiter wells (1 .6 x 105
cells/well) with the addition of mAbs or of antisera in appropriate
dilutions. After a 30-min incubation at 37°C, the cells were
sedimented by centrifugation and incubated as a pellet for 3 h at
37 ° C. Syncytia of dimensions of 10 to >100 cells were scored with
an inverted microscope after transfer of the resuspended pellet to
flat-bottomed well microtiter plates. In uninhibited control samples,
100-150 large Syncytia/well were counted.
Immunoselection of Recombinant Phages
"5I-M38 and -L31.
Recombinant phage plaques (2 x 106) of Agtll cDNA expression library from human PHA-stimulated T lymphocytes (HL1031b ;
Clontech Laboratories Inc., Palo Alto, CA) were screened according
to Young and Davis (24) with minor modifications : dry milk was
used to overcoat the filters, and directly labeled antibody (106
cpm/ml of either 1251-L31 or '2'I-M38, both at a specific activity
of 41 mCi/mg) was used instead of cold antibody and '251Staphylococcus Protein A.
Subdoning and Sequencing of cDNA . A phage clone, positive
with 1251-L31 and 125 1-M38 and negative with an irrelevant antibody, was chosen for further analysis . The insert was subdoned
into the EcoRI site of the plasmid vector pUC8, and the recombinant plasmid (PL208) was sequenced according to the method
of Sanger et al . (25) . A Data Bank search was done with the FASTN
algorithm to compare the DNA sequence with the EMBL data
bank .
Lymphocyte Proliferation Assays . Tetanus toxoid presentation experiments were performed as described (16); adherent monocytes
(2 x 104 /assay) were pulsed with tetanus toxoid (20 ng/assay), antibodies were used as 1:100 ascites and purified T cell (105/assay)
proliferation was evaluated at day 5 by ['H]thymidine incorporation .
Mitogenic proliferation of PBMC (2 x 10 5/assay) stimulated
by crosslinked OKT3 or by other mitogens was evaluated by
['H]thymidine incorporation at day 4. Antibodies were used as
1:200 ascites and added to the culture twice, at day 0 and at day
2. 11,2 production by human PBMC cultured for 18 h after stimulation by crosslinked OKT3 was evaluated as described by Gillis
et al . (26) .
Published July 1, 1991
O
d
J1
N
01
C
10
m
0
1
2
3
4
9
Days of culture
was selected for its strong reactivity in commercial HIVELISA
and was shown to bind the surface of activated human PBMC
by cell binding assays and by flow cytometry. Since these properties were similar to those described for M38, we compared
the specificities of the two antibodies in Western blot and
radioimmunoprecipitation assays.
The reactivity pattern of 125 1-L31 and -M38 on HIV-1,
HIV-2, and HTLV1 Western blot strips (Fig. 1 A) proved
to be similar, even though not identical: both antibodies reacted
with a 45-kD protein band, while only M38 recognized the
env product of HIV-1 (but not that of the other two
retroviruses) . Radioimmunoprecipitations of lysates from
surface-labeled activated lymphocytes demonstrated that both
L31 and M38 precipitate similar (likely identical) 45-kD bands
and fainter 90-kD bands (Fig . 1 B) .
The number of antibody binding sites on the lymphocyte
surface was found to increase 10-40-fold after polyclonal lymphocyte stimulation by OKT3 (Fig. 2 A) . The affinity of
the interaction (Kd = 5 nM) was deducted by Scatchard plot
analysis of equilibrium binding assays. The L31-defined antigen, undetectable at day 0 by flow cytometry analysis, was
detected on the majority of cells at day 3 (Fig . 2 B) . Conversely, Western blot analysis of whole cell lysates with L31
(Fig. 2 B) demonstrated quantitatively similar reactivities in
resting and activated lymphocytes. Analogous results were
obtained with M38 (data not shown) . These results indicate
that the protein is present in a fashion inaccessible to antibodies in resting cells . The epitopes recognized by the two
mAbs become accessible on the cell surface only as a consequence of mitogenic activation.
M38 and L31 React with the Same Cell Protein. To confirm
55
Grassi et al .
200
channel
Figure 2 . Differential reactivity of mAb L31 on live cells and on denatured proteins. (A) Binding of L31 to a cell surface antigen of activated
lymphocytes. PBMC were cultured after stimulation with OKT3 and rabbit
anti-mouse Ig antibodies. The number of 1251-L31 binding sites was determined by Scatchard plot analysis after subtraction of nonsaturable binding,
as detected in the presence of 100-fold molar excess of unlabeled L31 . (B)
L31-cytofluorimetry and Western blots on whole cell lymphocyte lysates
at day 0 and at day 3 after OKT3 stimulation.
the hypothesis that M38 and L31 recognize two epitopes of
the same cell protein, lysates from activated lymphocytes were
adsorbed onto four immunoaffinity columns bearing L31,
M38, and two control irrelevant isotype-matched mAbs,
respectively. Either effluents or SDS-released eluates were analyzed by immunoblotting with 125 1-M38 (Fig. 3 A) or 1251L31 (Fig. 3 B). The L31 column (but not those bearing control antibodies) caused a major immunodepletion in the
effluent (Fig. 3, lanes a) and an almost quantitative recovery
of the 45-kD band in the eluate (lanes b). The protein band
was decorated equally well by either antibody. Similar results
were obtained with the M38 column, although this antibody
was less efficient than L31 in retaining the 45-kD protein .
These results indicate that the cell protein defined by M38
is identical to that recognized by L31 .
Fine Specificity ofM38 and L31 . The interaction of M38
with the cellular antigen on the surface of activated lymphocytes (measured by flow cytometry) could be significantly reduced by soluble recombinant gp160; in the same conditions,
the binding of mAb 01 .65 (20), specific for a common determinant of HLA-A, -B, -C molecules was unaffected (Fig. 4) .
Also unaffected was the binding of L31 (data not shown) .
To further analyze the specificities of M38 and L31 with
respect to HIV 1, the two antibodies were used in gp120related assays on living cells . 8E51(LAV) cells and MOLTS
cells, sedimented together in Vshaped wells and incubated
as a pellet, form large syncytia . The assay can be inhibited
by anti-CD4 mAbs and by patients sera containing neutralizing
antibodies . The addition of mAb M38 resulted in a marked
inhibitory effect (Fig. 5 A), while L31 and a number of mAbs
(specific for HIV antigens or of irrelevant specificity) had no
effect at all . These data, together with the cytofluorimetry
findings on 8E51(LAV) cells, labeled by M38 and by other
anti-gp120 mAbs, while not by L31 (Fig. 5 B), confirm the
differential anti-gp120 specificity of M38 .
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Figure 1 . Reactivity pattern of mAbs M38 and L31 . (A) Western blot
of radioiodinated M38 (M and L31 (L) on HIV-1, HIV-2, and HTLV-1
commercial strips (Sorin Biomedica and BioRad Laboratories) . The reference AIDS serum (S) was revealed by 125 1-Protein A . (B) SDS-PAGE of
the materials immunoprecipitated by M38 (M, L31 (L), and UCTH-1
(L0 from lysates of OKT3-stimulated lymphocytes, surface labeled by 1251
(lactoperoxidase method) .
50 100
fluorescence
Published July 1, 1991
Figure 3 . Demonstration that mAbs
M38 and L31 recognize the same cellular protein . Activated lymphocyte lysates were loaded onto affinity columns
of Afgel coupled with the indicated
mAbs . The effluent and the SDS-eluted
material were acetone precipitated for
Western Blot analysis with 125 1-M38
(A) and 1251-L31 (B). Lanes a and b
were loaded with effluent and eluate,
respectively.
125
101
10 2
fluorescence channel
Figure 4 . Demonstration that the binding of mAb M38 (but not that
of mAb 01.65) to cells can be competed by gp160 . Indirect cytofluorimetry of activated lymphocytes with mAbs M38 or 01 .65 in the presence
(dotted lines) and in the absence (solid lines) of soluble recombinant gp160 .
56
recombinant a-galactosidase was confirmed by Western blot
analysis of IPTG-induced phage lysates (data not shown) .
The insert (1,549 bp) was subcloned into the vector pUC8,
and the recombinant plasmid (PL208) was then sequenced .
The insert sequence contained an open reading frame (between nucleotide residues 10 and 1108) (Fig. 6) coding for
a protein of 366 aminoacids. A data bank comparison of the
nucleotide sequence revealed a high degree of homology with
the class I MHC gene family. The sequence is not identical
to any of the already sequenced alleles, while maximal homology was found with the alleles of the C locus.
A direct comparison to all the available C alleles amino
acid sequences (27, 28) is seen in Fig. 7 . A 96% homology
was found with allele Cb-1. The protein contains the characteristic six domains of class I MHC heavy chains (leader peptide, ci 1, a 2, (x 3, transmembrane, and cytoplasmic domains) and the three C-specific residues (val-52, glu-183, and
glu-268) . The five cysteine residues and the glycosylation site
(gin-87) are conserved . Moreover, all but two variations from
the consensus are in residues that show the same amino acid
substitution in at least one other sequenced C allele ; the two
variations unique to this sequence are trp-14 and glu-49 . These
data strongly suggest that the sequenced polypeptide is a
product of an allele of the HLA-C locus.
M38 and L31 Recognize "Hidden" Epitopes of Class I MHC
a Chains. An immunological approach to confirm that the
protein defined by M38 and L31 is a class I MHC heavy chain
was based upon immunoprecipitations from detergent lysates
of Rex cells (reactive with both M38 and L31) using either
mAb 01 .65 (20), specific for a common determinant of HLAA,
-B, -C molecules, or mAb R1 .30, specific for 02m (20).
QmAb OKT4 was included as a control. Both precipitates and
supernatants were analyzed by immunoblotting with t251L31. As can be seen in Fig . 8, both antibodies precipitated
gp120 Mimics a Hidden Monomorphic Epitope
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Immunoselection of Recombinant Phages and Sequence Analysis of the cDNA of the Cellular Protein Defined by M38 and
L31 . To identify the cell protein defined by the two antibodies,
I-L31 was used to screen a commercial XGT11
cDNA expression library from PHA-activated human PBL .
Positive plaques were counter-screened with "'l-M38 and
with an irrelevant radioiodinated mAb. A clone, reactive with
both L31 and M38 and showing no binding with the isotypematched control antibody, was selected. The reactivity of phage
Published July 1, 1991
B
0
aC
79
0
100
M38
0
50
0
L31
8
1
1
2
Hg/assay
4
0.5
fluorescence channel
20 unrelated HLAtyped individuals . Further, SDS-denatured
molecules immunoprecipitated from resting lymphocyte lysates by mAb CR11.351(21), specific for HLAA2 molecules,
displayed similar reactivity when challenged with the two
antibodies. As a consequence of these findings, the two epi-
0001
I Signal peptide -->
I Alpha-1 -->
GAATTCGGGGGGGAGAMCGGGTCATGGCGCCCCGAACCCTCATCCTGCTGCTCTCGGGAGCCCTGGCCCTGACCGAGACCTGGGCCGGCTCCCACTCCA
0
TGAGGTATTTCTCCACATCCGTGTCCTGGCCCGGCCGCGGGGAGCCCCGCTTCATCGCAGTGGGCTACGTGGACGACACGCAGTTCGTGCGGTTCGACAG
0201
CGACGCCGCGAGTCCAAGAGGGGAGCCGCGGGAGCCGTGGGTGGAGCAGGAGGGGCCGGAGTATTGGGACCGGGAGACACAGAAGTACAAGCGCCAGGCA
0301
I Alpha-2 -->
CAGGCTGACCGAGTGAACCTGCGGAAACTGCGCGGCTACTACAACCAGAGCGAGGACGGGTCTCACACCCTCCAGAGGATGTTTGGCTGCGACCTGGGGC
0401
CGGACGGGCGCCTCCTCCGCGGGTATAACCAGTTCGCCTACGACGGCAAGGATTACATCGCCCTGAACGAGGATCTGCGCTCCTGGACCGCCGCGGACAC
0501
GGCGGCTCAGATCACCCAGCGCAAGTGGGAGGCGGCCCGTGAGGCGGAGCAGCGGAGAGCCTACCTGGAGGGCACGTGCGTGGAGTGGCTCCGCAGATAC
0301
I Alpha-3 -->
CTGGAGAACGGGAAGGAGACGCTGCAGCGCGCGGAACACCCAAAGACACACGTGACCCACCATCCCGTCTCTGACCATGAGGCCACCCTGAGGTGCTGGG
0701
CCCTGGGCTTCTACCCTGCGGAGATCACACTGACCTGGCAGTGGGATGGGGAGGACCAAACTCAGGACACCGAGCTTGTGGAGACCAGGCCAGCAGGAGA
0801
TGGAACCTTCCAGAAGTGGGCAGCTGTGGTGGTGCCTTCTGGAGAAGAGCAGAGATACACGTGCCATGTTCAGCACGAGGGGCTGCCGGAGCCCCTCACC
0901
I Transmembrane region -->
CTGAGATGGAAGCCGTCTTCCCAGCCCACCATCCCCATCGTGGGCATCGTTGCTGGCCTGGCTGTCCTGGCTGTCCTAGCTGTCCTAGGAGCTATGGTGG
1001
I Cytoplasmatic tail -->
CTGTTGTGATGTGTAGGAGGAAGAGCTCAGGTGGAAAAGGAGGGAGCTGCTCTCAGGCTGCGTCCAGCAACAGTGCCCAGGGCTCTGATGAGTCTCTCAT
1101
13' Untranslated sequence -->
CGCTTGTAAAGCC,U$GACAGCTGCCTGTGTGGGACTGAGATGCAGGATTTCTTCACACCTCTCCTTTGTGACTTCAAGAGCCTCTGGCATCTCTTTCTG
1201
CAAAGGCATCTGAATGTGTCTGCGTTCCTGTTAGCATAATGTGAGGAGGTGGAGAGACAGCCCACCCCCGTGTCCACCGTGACCCCTGTCCCCACACTGA
1301
CCTGTGTTCCCTCCCCGATCATCTTTCCTGTTCCAGAGAAGTGGGCTGGATGTCTCCATCTCTGTCTCAACTTCATGGTGCGCTGAGCTGCAACTTCTTA
1401
CTTCCCTAATGAAGTTAAGAACCTGAATATAAATTTGTTTTCTCAAATATTTGCTATGAAGGGTTGATGGATTAATTAAATAAGTCAATTCCTGGAAGTT
1501
GAGAGAGCAAATAAAGACCTGAGAACCTTCCAAAAACCCGCCCGAATTC
Figure 6. Nucleotide sequence of the insert of a recombinant phage immunoselected by the mAbs. The insert (1,549 bp) was subcloned into the
vector pUC8, and the recombinant plasmid (PL208) was then sequenced . The insert sequence contains an open reading frame (between nucleotide
residues 10 and 1108) coding for a protein of 366 amino acids bearing high homology to HLA-C alleles. the first and last codons of the open reading
frame are underlined. The boundaries of the six domains characteristic of class I MHC heavy chains are indicated' These sequence data are available
from EMBL/Gen-Bank/DDBJ under accession number X58536 .
57
Grassi et al .
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L31-reactive material. However, mAb 01.65 precipitated most
of the material, while only a fraction was bound by mAb
R1.30. The control experiments with mAb OKT4 displayed
all the L31-reactive material confined to the supernatant .
M38 and L31 did react with the activated lymphocytes of
Figure 5 . Differential activity of
mAbs M38 and L31 in gp120-related
assays . (A) Inhibition of syncytia formation between 8E51(LAV) and
MOLTS cells by M38 and lack of effect
by L31 . (B) Indirect cytofluorimetry of
8E51(LAV) cells with the two mAbs.
Published July 1, 1991
Signal peptide
CONS-C
Cb-1
Cb-2
Cw-1
Cw-2 .1
Cw-2 .2
Cw-3
PL208
MRVMAPRTLILLLSGALALTETWA
------------------------------------------------------------------------------L-------------------------------------
Alpha-1 domain
CONS-C
Cb-1
Cb-2
Cw-1
Cw-2 .1
Cw-2 .2
Cw-3
Cw-4
Cw-5
CwBL18
Cx52
PL208
CSHSMRYFYTAVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQTDRVSLRNLRGYYNQSEA
--------S-S------------------------------------------------------------------------------------------------------------------------------------------------------A---------------------K--F-S------------S--------------------------------------------------------------------------------S----H---------------------------GR--------------------------N--K------------------------S----H-------------------------------------------------------N--K---------G-------C-----------H------------------DE------------RK--------------P-------------------*-------D-------A------S------------------------------------------------A----------------D
--------------------H-------------Q-----------------------------------------N--K-----------------------------H----------------------------------------__---------A-----------------------------------ET------------R---------------------A----------------G-------S-S--W----------------------------------E-----------------------A---N--K---------D
Alpha-2 domain
GSHTLQRMYGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQWRAYLEGTCVEWLRRYLENGKETLQRA
------W-F--------------------------------------------------------R---------------------S----------------------------------------------------------------------------------------------------W-C----------------Y---------------------------------------R---------------------S---------------------------------------------------------------------------E--------------K---------------------------------------------------------------------------E---------------------II-------V------------H--------------------N------------------L------L---------K-------G--------S----------------------------------------------------A---L------L-----------------------------------------N-F---------------------K-----------------R--------------------K------------------------------------------------------------------------------------------------------------------------V--------------------------------------------------CV---------S-----------F--------------N-F---------------------------------------R--------------------------
Alpha-3 domain
CONS-C
Cb-1
Cb-2
Cw-1
Cw-2 .1
Cw-2 .2
Cw-3
Cw-4
CwBL18
Cx52
PL208
EHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQRWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRW
------------------------------------y,7----------------------------M--------------------------------------------------------------------------------------------------------------------------------------------------------W----------------------------M-----------------------------------------------------T------------------------------------------------------------------------------------------T--------------------------------------------------------------------------------------------------W-------------------------------------------------------P-----N---L------------------------------------------------K---------Q-------M-----Q-----S-------------------------_------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------W-----------------------K-------------------------------
Transmembrane region
Cytoplasmatic tail
CONS-C
Cb-1
Cb-2
EPSSQPTIPIVGIVAGLAVLAVLAVLGAVVAVVMCRRKSS
GGKGGSCSQAA
SSNSAQGSDESLIACK
----------------------------- M----------
-----------
----------------
Cw-2 .1
Cw-2 .2
Cw-3
Cw-4
Cw-5
CwBL18
Cx52
PL208
-------------------------------------------------------------------------------
---------------------------------------------------------------------------------
---------------------------------------------C------------ T--------------------------------------------------------------
Cw-1
---------- M---------V--------- TAM------G---------------------------------------------------------------------------------------------------------- M---------K---------------------------M-----------
-----------
AU
----------------
Figure 7 . Direct comparison of the deduced amino acid sequences of PL208 and of all the available HLA-C alleles . CONS-C is a consensus sequence
derived from the individual sequences.
topes defined by L31 and M38 may be truly classified as monomorphic and common determinants of HLA-A, -B, and -C
molecules . Such determinants, inaccessible to the antibodies
on the surface of resting lymphocytes, become available to
antibody reaction only as a consequence of mitogenic activation. These findings suggest that mitogenic activation induces the expression of class I MHC cx chains in two different
58
conformations. The conformation coprecipitable with 02m
and inaccessible to M38 and L31 is identical to that of resting
cells, while the second one is not coprecipitable with 02m
and bears the "hidden" epitopes now accessible to antibodies .
The above reported findings (and their interpretation) are
very similar to those of Schnabl et al. (17), who demonstrated
that activated T lymphocytes express class I MHC heavy chains
gp120 Mimics a Hidden Monomorphic Epitope
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CONS-C
Cb-1
Cb-2
Cw-1
Cw-2 .1
Cw-2 .2
Cw-3
Cw-4
Cw-5
CwBL18
Cx52
PL208
Published July 1, 1991
3 H-TdR (cpm .10-3)
A
0
w
1-
0)
oc
5
C
1:5
E 10
CNTR
N5-1
M38
L31
N
c
0
3 H-TdR
B
Ö
CNTR
NS-1
M38
L31
not associated with 02M . This was inferred on the basis of
the reactivity of mAb LA45 with SDS-denatured, but not
associated with 02m, heavy chains and with heavy chains expressed on the surface of activated (but not resting) lymphocytes .
The epitopes recognized by M38 and L31 seem to be similar
to that recognized by LA45 ; indeed, neither our antibodies
(Fig . 1 B), nor LA45, coprecipitated ß2m. Conversely, the
anti-ß2m antibody R1 .30 immunoprecipitated only a fraction of the L31-reactive material (Fig. 8), in close analogy
with the observation by Schnabl et al. (17) with mAb W6/32,
which immunoprecipitates only ß2m-associated heavy chains.
Functional Consequences of the Interaction of M38 and L31
with Immunocytes. Schnabl et al . (17) have suggested that
the conformational alteration of class I proteins upon T cell
activation might have some important functional implications . In light of our previous finding of antigen presentation inhibited by M38 (16), we investigated the functional
effects elicited by interaction of the two antibodies with immunocytes. Indeed, several mAbs reactive with class I monomorphic determinants were reported to influence lymphocyte proliferation in different experimental models (20, 29-31) .
Antigen presentation was not inhibited by the addition of
L31, at variance with the influence of M38 (Fig. 9 A) . Further, L31 inhibited lymphocyte proliferation induced by polyclonal mitogenic stimuli . The addition of L31 (but not of
M38) significantly inhibited the mitogenic response of human
lymphocytes challenged with either Con A or anti-CD3 (Fig.
9 B) . Such inhibition was concomitant to a reduction in IL-2
production (Fig. 9 C) . This result is concordant with the
finding that exogenously added IL-2 could reverse the inhibition of lymphocyte proliferation by L31 (data not shown) .
59
Grassi et al .
O
O
Ó
~
m
c
a
V
O
V
N
OKT9
5
L31
0
1 .25 2.5
5
Conc . of mAb (Ng/ml)
Figure 9 . Functional consequences of the interaction of mAbs M38 and
L31 with immunocytes . (A) Inhibition of antigen presentation by M38
and lack of inhibition by L31 and controls. Tetanus toxoid presentation
experiments were performed as described in Materials and Methods . (B)
Inhibition of mitogenic proliferation by L31 and lack of inhibition by M38
and controls . PBMC were stimulated by crosslinked OKT3 . [ 3 H]Thymidine incorporation was evaluated at day 4. Antibodies were used as 1 :200
ascites. (C) IL-2 production by human PBMC, cultured for 18 h after stimulation by crosslinked OKT3 in the presence of L31 or OKT9 (as a control) .
Discussion
The interaction of CD4 to class II MHC and to HIV-1
gp120 has been recently reported as involving different domains of the molecule (12, 32) . However, the finding that
soluble gp120 can inhibit CD4-class II MHC binding raises
the possibly that the CD4 binding sites for the two ligands
may be overlapping .
To verify the hypothesis that HIV could mimic a physiological ligand of CD4, we analyzed a panel of murine mAbs
raised against HIV-1 looking for crossreactivities between env
products and activated human PBMC. The expectation to
find a crossreactivity with class II MHC molecules was not
fulfilled : instead, we characterized mAb M38, which defines
an epitope shared by HIV-1 gp120 and by class I MHC heavy
chains . Such a determinant proved to be monomorphic,
common to HLAA, -B, -C molecules and accessible to the
antibody on activated, but not resting, immunocytes .
We had earlier reported that the anti-gp120 specificity of
mAb M38 was shared with an "activation protein" present
on human monocytes (16) . We then provided a tentative definition of the human target molecule as an "8041) protein",
which was the most prominent band in radioimmunoprecipitation from metabolically labeled cells. The 45-kD minor band
was also present, but was at the time interpreted as a nonspecific signal and overlooked . The findings obtained by several analytical approaches confidently demonstrate that the
reactive protein has a molecular mass of 45 kD. The previous
conclusions are likely to be referable to incomplete sample
reduction : dimerization of HLA molecules in tissue extracts
(33) and molecular masses of 90/45 kD under nonreducing
conditions have been reported for HLA molecules (17).
Here, we report the results obtained from the analysis of
L31, a mAb derived from the same fusion yielding M38. L31
proved to be specific for the same cell protein recognized
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Figure 8. Demonstration that the L31-defined epitope is present on denatured HLA chains. Postnuclear supernatants of Rex cell Triton lysates were
loaded on affinity columns of Affigel coupled with the indicated mAbs.
mAb 01 .65 is specific for a common determinant of HLA-A, -B, -C molecules ; mAb R1 .30 is specific for 02m and mAb OKT4 was included as
a control . The precipitate (p) and the supernatant (s) were subjected to
Western Blot analysis with 1251-1,31 .
(Cpm x 10-3 )
k
Published July 1, 1991
lymphocytes express class I MHC heavy chains not associated
with 02m, together with "conventional" 02m-associated
HLA molecules. Analogous results and conclusions were
reached by Smith and Barber in the mouse system (35) .
The "hidden"epitopes recognized by our mAbs M38 and
L31 seem to be, in all their properties, similar to that recognized by LA45 and, indeed, none of the three antibodies
coprecipitated 02m. Immunohistochemistry analysis of M38
and L31 reactivity indicates that the tissue distribution is completely different from that of mAbs specific for monomorphic
HLA-A, HLA-B, or for determinants common to HLA-A,
-B, -C products (P G. Natali, personal communication) .
Schnabl et al . (17) hypothesize that the loss of 02m and
the conformational alteration ofclass I MHC proteins upon
T cell activation might have some important functional implications. Indeed, this issue has been already addressed in
our previous work, where we showed that M38 inhibited
antigen presentation (16). We have now expanded that initial observation by studying the functional consequence of
the interaction of the two antibodies in lymphocyte proliferation assays. The results indicate that L31 treatment is associated with significant inhibitory effects, which are paralleled by a marked reduction of IL-2 production (Fig. 8). It
is noteworthy that the interactions ofthe two antibodies with
the target epitopes had distinct and independent effects .
The eveptual relevance of the crossreactivity between HIV-1
gp120 and 02M-less class I proteins is still obscure, but two
of the findings reported herein make it, in our opinion, a
worthwhile field of further research . These are : (a) the involvement ofthe M38-defined gp120 epitope in syncytia formation; and (b) the involvement of 02M-less class I proteins
in regulatory mechanisms related to fundamental immunocyte functions. Furthermore, it is known that lymphocyte
activation is required to make the cell susceptible to HIV-1
infection ; this could depend upon the requirement for activation products such as /32m-less class I proteins.
We thank M. Pelagi for handling the mAbs and F. Ciccomascolo for helping in the lymphocyte functional
assays . We thank A. De Rossi for substantial help in setting up the syncytia formation assay, Sorin Biomedica for the generous gift of reagents, and Transgene and Pasteur M6rieux for kindly supplying recombinant gp160 . We are grateful to F. Malavasi for critical reading ofthe manuscript and constructive suggestions. The secretarial assistance of S. Zanotta is gratefully acknowledged.
This work was supported by grants ofMinistero della Sanita "Progetto AIDS 1988" and "Progetto AIDS
1989", and by grants of Prodotti Genetici Srl ., Milano.
F. Grassi, L. Lopalco and P. Lanza have fellowships from Fondazione Anna Villa Rusconi, Byk Gulden
Italia, and Prodotti Genetici Srl ., respectively.
Address correspondence to Antonio G. Siccardi, Dipartimento di Biologia e Genetica per le Scienze Mediche,
University di Milano, Via G. B. Viotti 3/5, I-20133 Milano, Italy.
Received for publication 26 October 1990 and in revised form 1 April 1991.
60
gp120 Mimics a Hidden Monomorphic Epitope
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by M38 (Fig. 3), even though lacking the crossreactivity with
HIV-1 gp120 . The anti-gp120 specificity of M38 was documented by western blot analysis on SDS-denatured proteins
(Fig. 1) and by assays on live HIVinfected cells (i.e., inhibition of syncytia formation and cytofluorimetry) (Fig. 5). M38
inhibited syncytia formation between 8E51(LAV) and MOLT3
cells, a CD4-dependent reaction, although it did not inhibit
the infectivity of free virus, as already reported (16) . This
finding confirms that virus penetration and syncytia formation are distinct, discrete reactions, although with common
steps.
The cellular protein defined by M38 and L31 could be included among the products of class I MHC. This was inferred by sequence analysis ofthe cDNA (Figs. 6 and 7) from
a recombinant phage expressing the epitopes recognized by
the two antibodies and by immunoblotting with L31, the
precipitates of mAb 01.65 (anti-HLA-A, -B, -C), of mAb
R1.30 (anti-02m) (Fig. 8), and of mAb CR11.351 (antiHLAA2) (20, 21) . The two epitopes defined by L31 and M38
could be classified as monomorphic and common determinants
of HLA-A, -B, and -C molecules. These findings explain the
presence of the M38- and 1,31-defined protein in the retrovirus
preparations used for Western blots (Fig. 1); as demonstrated
by Gelderblom et al. (34), HLA proteins are present in
retroviral virions.
The class I molecule recognized by the two mAbs on the
surface of activated immunocytes could not be assimilated
to conventional HLA molecules, since the corresponding epitopes appear to be hidden in HLA molecules present on the
surface of resting immunocytes . A strikingly similar situation has recently been reported by Schnabl et al. (17). They
demonstrated the reactivity of mAb LA45 with SDS-denatured
(but not with 02m-associated) a chains and with a chains
expressed on the surface of activated (but not resting) lymphocytes. By differential immunoprecipitations and "independent capping" experiments, they concluded that activated T
Published July 1, 1991
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