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Human CD4-reactive antibodies from sle patients induce reversible inhibition of polyclonal t lymphocyte proliferation
ELSEVIER
Human CD4-Reactive Antibodies from SLE Patients Induce Reversible Inhibition of Polyclonal T Lymphocyte Proliferation Gordana Lenert and Petar Lenert ABSTRACT: We report on isolation of human polyclonal CD4-reactive antibodies of IgM and/or IgG isotypes from several SLE patients. These antibodies bound specifically to CD4-expressing cell lines and to rCD4 in ELISA and immunoblots. Saturation of CD4-binding sites occurred at antibody concentrations between 5 and 15 pgiml. Anti-CD4 antibodies, in a dose-dependent manner, suppressed the proliferative responses of human peripheral blood mononuclear cells (PBMC) to superantigens (Staphylococcal enterotoxins A and B), anti-CD3 antibodies, and mitogens (PWM and Con A, but not PHA). They could also inhibit the proliferation of highly purified human T cells induced by immobilized anti-CD3 antibodies. To promote their effects on T cells, human antiCD4 antibodies had to be present at lymphocyte cultures
ABBREVIATIONS systemic lupus erythematosus SLE peripheral blood mononuclear PBMC Stapbylococcal enterotoxin B SEB SEA Stapbylococcal enterotoxin A r recombinant
before or at the time of priming. There was no significant inhibition when antibodies were added more than 24 h following T cell activation. Substantial evidence that the immunosuppression induced by anti-CD4 antibodies was due to their direct effect on T cells was obtained. Downregulatory effect of anti-CD4 antibodies could be significantly reversed by addition of exogenous IL-2 and by preincubation with soluble recombinant (r)CD4. Interestingly, at least one affinity-purified anti-CD4 antibody could costimulate the T cell proliferation induced by superantigens or anti-CD3 antibodies, especially when used at subsaturating concentrations (l-4 pg/ml) and when added subsequently to the initiation of cultures. Hzlman Immunology49. 113-l 21 (1996)
TCR HRP PWM PHA Con A
cells
T cell receptor for antigen horseradish peroxidase pokeweed mitogen phytohemagglutinin concanavalin A
INTRODUCTION CD4 that
is an integral bears
the immunoglobulin on T cells (TCR) with
is well
recognition MHC
membrane
a significant
glycoprotein
homology supergene
correlated of antigenic
Class II molecules
of -55
to other
family
kDa
members
of
Cl]. Its expression the
T cell receptor
peptides
in association
with
on antigen-presenting
cells
{3]. Cell-tocell adhesion directly dependent on CD4/MHC Class II interaction was first shown by Doyle and Strominger [4] 121 and with
the ability
and later confirmed
to activate
by others
[5-71.
B cells
CD4
binds
to non-
From the Louis-Charles Simard ResearchCenter, Notre-Dame Hospital. University ofMontve’al. Mont&al, Quibec H2L 4Ml. Canah. Address reprint requeststo P. Lenert, Laboratoire de Rechercbeen Immunoregulation, M4211 -L, Centre de Rechexbe, H@ital Notre-Dame, 1560, rue SherbrookeEst, Mon.&al, Que’becH2L 4M 1, Canada. Received December 13, 1995; acceptedJanuary 25, 1996. Human Immunology 49, 113-12 1 (1996) 0 American Society for Histocompatibility and Immunogenetics,
1996
polymorphic determinants on j32 domain of MHC Class II molecules, thereby increasing the overall avidity of TCR for its ligand {S, 97. From the functional perspective, several studies in the past have shown that the transfection of human CD4 molecule into effector T cell hybridomas significantly enhances the proliferation and cytokine production, in response to human MHC Class II antigens expressed on target cells f2, 7). However, apart from the interaction with MHC Class II molecules, CD4 can also interact with the TCR/CD3 complex and with some other members of the Ig-supergene family [lo-12). Since simultaneous cross-linking of CD4 with the TCRi CD3 upregulates the T cell proliferative response, it has been proposed that CD4 participates in a formation of the multireceptor complex involved in optimal T cell activation [13-171. Intracellular domains of both CD4 0198~8859/96/$15.00 PII SOl9g-8859(96)00054-7
114
G. Lenert and P. Lenert
and CD8 interact with the p56& tyrosine kinase, and this association appears to be particularly important for the coreceptor function 131. CD4 is also the high-affinity receptor for the human immunodeficiency virus, a function that is carried out by residues 40-55 within a CD4Dl domain 15-71. Since high-affinity ligands for CD4, like HIV-gp120 and murine anti-CD4 mAbs, strongly inhibit T cell activation, even in the absence of any obvious interaction between MHC Class II determinants and CD4 13, 18241, we raised the question of whether human CD4reactive antibodies that occur increasingly in a subset of patients with SLE may similarly interfere with T cell activation events. We now report on successful isolation of several human anti-CD4 antibodies from SLE sera. These antibodies retained their ability to specifically recognize both cell-expressed and recombinant CD4 and gave saturable binding at concentrations between 5 and 15 pg/ml. Human anti-CD4 antibodies strongly suppressed the polyclonal T cell proliferation induced by different stimuli. This effect occurred only when antiCD4 antibodies were present before or at the time of priming and when used at saturating concentrations. Exogenous IL-2 could partially overcome this inhibitory effect, suggesting a possibility of the induction of classical functional energy.
MATERIALS
AND
METHODS
Affinity Purification Anti-CD4 Antibodies
of Human
Several affinity columns were made by coupling between 1 and 10 mg of rCD4 (Biogen, Cambridge, MA) to 1 ml of NHS-activated Sepharose (Hi-Trap, PharmaciaBiotech, Montreal, QC) according to the manufacturer’s recommendations. Samples of SLE sera showing high CD4 reactivity in ELISA and by FACS were diluted 1: 10 with 20 mM sodium phosphate, pH 7.0, precleared, and applied to a CD4 column at 0.8 ml/min flow rate (single passage). In some experiments, an intermediate step consisting of gamma-globulin purification by an E-Z-SEP (Pharmacia-Biotech) procedure was performed. In either case, desorption of anti-CD4 antibodies was achieved by using 0.1 M triethanolamine, pH 11.0. Eluates were immediately neutralized with sodium acetate, pH 4.15, concentrated using Spin-X UF concentrators (Costar, Cambridge, MA), and extensively dialyzed against PBS, pH 7.2. The purity of anti-CD4 antibodies was checked in both native PAGE and in SDS-12% PAGE, run under reducing conditions. Human polyclonal IgM and IgG (Calbiochem-Novabiochem, La Jolla, CA), together with myeloma proteins IgM, K (VIN), and IgGl, x (FITZ) (kindly provided by Dr. H. Spiegelberg, Scripps Clinic,
La Jolla, CA), were run in parallel. No bands other than those corresponding to human IgM and/or IgG or their reduced H and L chains were observed. The identity of these bands was further confirmed in Western blots. Concentration of IgG and IgM anti-CD4 antibodies was determined by ELISA. ELISA
for Anti-CD4
Antibodies
ELISA for detection of affinity-purified CD4-reactive antibodies was performed on Falcon 3911-MicroTest III assay plates (Becton-Dickinson Labware, Oxnard, CA). Plates were coated with 50 pliwell of rCD4 or control proteins (BSA, SEB, SEA, insulin), all diluted in 0.9% NaCl, pH 7.0, and used at 2.5-3 pgiml. After blocking the wells with PBS-0.25% BSA-0.05% Tween 20 (blocking buffer), anti-CD4 antibodies and control human polyclonal IgG and IgM, as well as myeloma proteins of IgG and IgM isotypes, were added at increasing concentrations and incubated for 1 h at 22°C. After washings with PBS-0.3% Tween 20, goat anti-human IgG (1:3.000) or anti-human IgM (1:3.000) HRPconjugated second-step Ab (Caltag, Cedarlane Labs., Hornby, ON) were added and incubated for an additional hour. An OPD substrate in citric/phosphate buffer, pH 5.0, containing 0.4 ml/liter of H,O, was used for detection of bound HRP. The color development was stopped with 3M H,S04, and absorbance at 492 nm was determined by using the Dynatech microplate reader. In competitive experiments, either IgG or IgM affinity-purified anti-CD4 Ab at 3 pg/ml (final concentration) werepreincubated in tubes for 30 min with increasing amounts of rCD4 or control proteins (BSA, SEB, SEA), transferred in duplicates to wells of microtiter plates previously coated with rCD4, and incubated for 1 h at 22°C. Bound antibodies were revealed as described above. Immunoblotting The reactivity of affinity-purified anti-CD4 antibodies with rCD4 and control proteins (e.g., SEB) was further tested in immunoblottings. One ug/band of total protein was applied to SDS-12% PAGE. Electrophoresis was performed for 1 h at 200 V (constant voltage). Gels were blotted onto nitrocellulose membrane (0.45 pm, Bio-Rad Labs., Mississauga, ON) for an additional hour at 4°C (100 V), in 12.5 mM Tris, pH 8.25, 96 mM glycine, 20% methanol. Nitrocellulose membranes were cut in individual strips, blocked for 1 h at 22°C with 1% BSAPBS, and incubated for 1 h at 22’C with affinity-purified anti-CD4 antibodies (5 pgiml) or control Ig. Following washings (three times for 5 min) with PBS-0.3% Tween 20, strips were further incubated with HRP-labeled goat anti-human IgG (1:3.000) or IgM (1:3.000) (Caltag), for
Inhibition
115
of T Cell Proliferation
1 h at 22°C.
4-Chloro-naphtol was used as a substrate. Development of colored reaction was stopped after lo15 min by rinsing with dH,O. Murine anti-Leu 3a mAb served as a positive control. FACS Analysis Human CD4-transfected and nontransfected HeLa cells at 5 x lo5 were incubated in 100 pl volumes, in PBS2% BSA-0.05% Na-azide, with human anti-CD4 antibodies or control Ig’s (used at 5 pg/ml), for 1 h on ice. Bound Ig’s were detected after an additional incubation for 30 min on ice with either FITC-labeled anti-human IgG or anti-human IgM antibodies (both used at 1:250, Cappel, Organon Teknika Inc., Scarborough, ON). Stained cells were analyzed using a Lysis II software on a FACSort (Beckton-Dickinson, Mont&al, QC) cell sorter. Cell Proliferation
Assays
Human PBMC were isolated from healthy laboratory personnel by Ficoll-Paque ET (Pharmacia-Biotech) gradient centrifugation. Purified T lymphocytes were obtained by E-rosetting with AET-treated sheep red blood cells. These cells were >97% CD2’ as demonstrated by flow cytometry. PBMC, 105, or purified T cells, at 100 $/well, were incubated in 96-well flat-bottom tissue culture plates (Falcon 3072, Microtest III, BecktonDickinson Labware, Lincoln Park, NJ) for 72 h. Culture medium consisted of RPM1 (Gibco, Grand Island, NY) supplemented with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 pggiml), HEPES buffer (15 mM), and FCS (10%). The following substances were used for PBMC stimulation: anti-CD3 (UCHT-1 antibody kindly provided by Dr. Delespesse, 10 pg/ml, precoated on plastic), PHA-P (SIGMA Chem. Co., St. Louis, MO, 5 pgiml), Con-A (SIGMA, lpg/ml), PWM (SIGMA, 1 pgiml), and microbial superantigensStaphylococcal enterotoxins A and B (SEA and SEB, at 50 and 125 ngiml, respectively, SIGMA). In most instances, human anti-CD4 antibodies (and control isotype matched human myeloma proteins) were used at I-20 pgiml and preincubated with cells for 24 h at 37”C, before polyclonal stimulation. In some experiments, between 10 and 100 U/ml of human recombinant IL-2, 200 U/ml of human recombinant IL-4 (BoehringerMannheim, Montreal, QC) or 10 pg/ml of soluble rCD4 was added. Cultures were incubated in triplicates at 37°C in 5% CO, in air and pulsed with 0.5 @/well of 13H] thymidine (6.7 CiimM, ICN, Irvine, CA) for the last 18 h. Incorporation of the L3H)thymidine was measured by liquid scintillation counting. Direct cytotoxic effect of anti-CD4 Ab on unstimulated human PBMC was excluded by trypan blue staining.
RESULTS Human Affinity-Purified Anti-CD4 Retain Their Specific CD4-Binding
Antibodies Properties
We recently observed that relatively high serum levels of antibodies recognizing the human CD4 specifically occur in a subset of SLE patients (-20%). To study their biological properties, we first made efforts to obtain them in highly purified form, by making several affinity columns with covalently linked rCD4 to Sepharose beads. Optimal isolation of anti-CD4 antibodies was achieved by single passing a diluted SLE sera at 0.8 ml/min flow rate over the rCD4 column (6 mg rCD4) and by using 0.1 M triethanolamine for elution (Fig. 1). These conditions were chosen in accordance with our recent observations that IgM anti-CD4 antibodies are highly sensitive to harsh acidic treatment. In cases of five SLE sera, we obtained relatively high yields of anti-CD4 antibodies, which ranged from 40 to 120 pg (from -0.5 ml of starting sera). Purified antibodies were of both IgM and IgG isotypes in two cases (Ab-79 and Ab-85) and solely IgM in two cases (Ab-21 and Ab-76), while in a single case antibodies of the IgG class were isolated (Ab-29). Preparation of Ab-79 also contained small amounts of IgA antibodies. The purity of antibody preparations was checked by both native PAGE and SDS-12% PAGE run under reducing conditions. We could observe only bands corresponding to either nonreduced IgG and/or IgM or their single heavy and light chains (data not shown). This was also confirmed in Western blottings.
10 Fraction
number
FIGURE 1 Affinity purification of human anti-CD4 antibodies. CD&&nity column was made by coupling 6 mg of rCD4 to NHS-activated Sepharose beads. Precleared SLE serum 85 was diluted 1:lO with 20 mM sodium phosphate and single-passed over a rCD4 column. After washings, specifically bound Ig were eluted by using 0.1 M triethanolamine and immediately neutralized with sodium acetate, pH 4.15. Ordinate refers to absorbance at 280 nM.
116
G. Lenert and P. Lenert
The anti-CD4 antibodies of both IgG and IgM isotypes gave specific and saturable binding to rCD4 in ELISA. Figure 2 shows the representative binding curves of Ab-85 on rCD4 and on control protein (BSA), in comparison to those given by human myeloma protein Vin (IgM, K) and human polyclonal IgM (CalbiochemNovabiochem) on rCD4, when tested over a range of concentrations. Other human anti-CD4 antibodies and control anti-Leu 3a mAb behaved similarly. We next tested human anti-CD4 antibodies for the binding to native CD4 expressed on cell membranes and to rCD4 in immunoblots (Figs. 3 and 4, respectively). In FACS analysis, anti-CD4 antibodies bound specifically to CD4expressing HeLa cells, while in immunoblots, we could observe reactivity toward a -45-kDa protein that corresponds to Mr of the truncated form of CD4. As specificity controls, we show the lack of binding of human anti-CD4 antibodies to SEB (-28 kDa) used at equivalent concentrations, as well as specific binding of murine anti-Leu 3a antibody to the 45-kDa band. Anti-CD4 Antibodies Inhibit, in a Dose-Dependent Fashion, the Polyclonal Proliferation of Human PBMC and Purified T Cells PBMC from seven different donors were preincubated with human anti-CD4 antibodies (7.5 pg/ml) and isotype-matched human myeloma proteins (e.g., IgM, K-Vin) for 24 h, which was followed by either mitogen (PHA, PWM, and Con A), superantigen (SEA and SEB), or anti-CD3 (UCHT-1)-mediated stimulation (Fig. 5). Data obtained with Ab-79 are shown. Human anti-CD4 antibodies, but not control Ig’s when used at equimolar concentrations, strongly inhibired the PBMC prolifera-
t
1/
I
--o-
Ab-850” BSA
--)_
IgM. k (Vin)
Fluorescence
intensity
(tog)
FIGURE 3 FACS analysis of human anti-CD4 antibody binding to CD4-expressing cells. CD4transfected and nontransfected HeLa cells were incubated for 1 h on ice with Ab-85 or control Ig’s (used at 5 pg/ml). FITC-conjugated goat anti-human IgM antibodies (1:250, Cappel) were used for detection of specifically bound IgM. The binding of control Ig’s at 5 pgiml to CD4-transfected cells was not different from that occurring on nontransfected HeLa cells and is not shown.
tion induced by various polyclonal activators. The inhibitory effect on superantigen-induced proliferation (-45%) was similar to that observed following anti-CD3 and PWM stimulation (49--53%) and slightly higher than that on Con A-induced proliferation (37%). Surprisingly, at the concentration indicated, Ab-79 failed to Lea
%a
21
29
76
79
85
hIlJc
rbrbrbrbrbrbrb 0
2 Ab
4
6
concentration
6
10
12
(pglml)
FIGURE 2 Saturable binding of Ab-85 to rCD4 in ELISA. Ab-85, control myeloma protein Vin (IgM, K), and human polyclonal IgM were tested in ELISA for binding to wells coated with either rCD4 or BSA. Under the assay conditions employed, only Ab-85, but not control Ig’s, gave specific binding to rCD4. Saturation of rCD4 binding sites in ELISA was achieved at Ab-85 concentrations higher than 3-5 pggiml. Similar data were obtained with other anti-CD4 antibodies.
FIGURE 4 Immunoblotting of affinity-purified anti-CD4 antibodies. The specific binding of human anti-CD4 antibodies (Ab-21, Ab-29, Ab-76, Ab-79, and Ab-85) and control anti-Leu 3a antibody (all used at 5 pg/ml) to the 45-kDa band (a) and lack of detectable binding to the 28 kD band (SEB) (b) blotted under identical conditions are shown. For comparison, myeloma protein Vin (IgM, K) failed to give significant binding to either band. Bound antibodies were revealed by 1 h incubation with HRP-labeled goat anti-human IgM antibodies, except in cases of Ab-29, which was revealed by incubation with HRP-labeled goat anti-human IgG and of anti-Leu 3a, with HRP-labeled goat anti-mouse IgG.
Inhibition
of T Cell Proliferation
0
117
40
120
80
Medi urn
+
Ab-79
PHA
+
Ab-65
PHA + Yin
+
Ab-21
PHA + Ab-79
*
Ab-76
--)-
Ab-29
---A--
IgM. k p/in)
PWM PWM + Vin PWM + Ab-79 01
Con-A Con-A
+ Vin
Con-A
+ Ab-79
1.2
2.5
5.0
Ab concentration
10.0
20.0
( v g/ m 1)
FIGURE 6 Dose dependency of anti-CD4 inhibition. Human PBMC were preincubated for 24 h at 37°C with five different anti-CD4 antibodies and control isotype-matched myeloma proteins added at increasing concentrations (ranging from 1 to 20 pg/ml), which was followed by stimulation with SEB (125 ng/ml) for 3 days. Lymphocyte proliferation was measured by determining C3H)thymidine incorporation, which was added 18 h prior to cell harvesting. Expressed are mean values of cpm (x 10-3) of three separate experiments.
SE4 SEA + Vin SEA + Ab- 79 SEB SEB + Vin SEB + Ab-79 anti-CD3 anti-CD3
+ Yin
anti-CD3
+ Ab- 79
Requirement for Anti-CD4 Phase of the Culture TdR Incorporation (mean x lC.f3+ SD)
FIGURE liferation. anti-CD4
5 Human anti-CD4 antibodies inhibit PBMC proHuman PBMC were preincubated for 24 h with antibodies (7.5 &ml) or isotype-matched myeloma
proteins and then stimulated for 3 days with immobilized anti-CD3 antibodies (10 &ml), PWM (1 pg/ml), PHA (5 pgiml), Con A (1 pgiml), SEB (125 ng/ml), or SEA (50 ngiml). Lymphocyte proliferation was measured by determining [- H}thymidine incorporation, which was added 18 h prior to cell harvesting. inhibit
PHA
by either induced pulsed
stimulation.
Ab-79
proliferation APC;
A similar
or Ab-85
of CD4’
data not shown,
degree
occurred
T cells
manuscript
of inhibition
on tetanus (with
toxoidantigen-
in preparation).
of antiCD4 antibodies and occurred in a dose-dependent fashion (Fig. 6). For example, in SEB-stimulated proliferation, an ED5,, for five different anti-CD4 antibodies was achieved at 7.5-15 pgiml. Interestingly, some anti-CD4 conantibodies (e.g., Ab-79), w h en used at subsaturating centrations (-1-4 pg/ml), costimulated the PBMC proliferation induced by superantigens or anti-CD3 antibodies. We next show that anti-CD4 antibodies, but not control Ig’s, similarly to their effects on nonseparated PBMC, could specifically inhibit the proliferation of highly purified T cells when stimulated with immobilized antiCD3 mAbs (Table 1). The inhibitory
effect
was due to specific binding
Antibodies
in the Early
Human anti-CD4 antibodies and control myeloma proteins Vin (IgM, K) and Huf (IgM, A) (all at 10 pg/ml) were added to SEB-stimulated cultures at different time intervals up to 48 h (Fig. 7). Proliferative response was measured after 72 h of SEB stimulation. To exhibit their inhibitory effects, human anti-CD4 antibodies had to be present at saturating concentrations 24 h before or at the time of polyclonal PBMC stimulation. Addition of antibodies to the cultures more than 24 h after SEB stimulation was not effective. The ability to add anti-CD4 antibodies during the last 48 h of culture without effect eliminates any potential for nonspecific cytotoxicity as an explanation for reduced thymidine incorporation. Inhibitory Effect of Anti-CD4 Antibodies on SEB-Induced Proliferation Is Due to Their Direct Binding to Human PBMC Several experiments were designed to ensure that the effect of anti-CD4 antibodies on SEB and anti-CD3induced proliferation of PBMC is a consequence of their TABLE
1
Human anti-CD4 anti-CD3-induced T cells
Condition
TdR incorporation (cpm + SD)
Medium Anti-CD3 Anti-CD3 + Ab-79 Anti-CD3
+ IgM,
Ab-79 inhibits proliferation of purified
355 f 103 55,747 + 18,470 K
(Vin)
14,446 58,994
Tk7,707 + 13,291
118
G. Lenert and P. Lenert
-24h
Oh
+24h
Addition
+
M-79
*
At-85
-f--
IgM. k (Vin)
--U--
IgM. I (HufJ
were present in cell cultures only before cell stimulation and washed out, significant inhibition of subsequent SEB-induced cell proliferarion still occurred (Table 2). However, when subsaturating amounts of some antiCD4 antibodies were used for preincubation and subsequently washed out, almost twofold enhancement of anti-CD3-mediated T cell proliferation occurred. Soluble rCD4 and IL-2 Overcome the Inhibitory Effect of Anti-CD4 Antibodies on SEB-Induced PBMC Proliferation
+48h
of Ab
FIGURE 7 Kinetics of anti-CD4 inhibition. Human PBMC (1 x 105/well) were stimulated with SEB (125 ngiml) for 3 days. Two affinity-purified anti-CD4 antibodies (Ab-79 and
Ab-85) and control myeloma proteins (Vin and Huf), all at 10 pgiml, were added to the cultures at different time points. Lymphocyte proliferation was assayed by determining the I- H)thymidine incorporation, which was added 18 h before cell harvesting. Ordinate shows the mean values of cpm (x 1O-i) from one out of three representative experiments.
direct binding to CD4-expressing T cells, and not to simple neutralization of the ligand. We first show that affinity-purified anti-CD4 antibodies at concentrations (5-10 pg/ml) that were used to inhibit T cell proliferation completely failed to bind to SEB, SEA, or BSA in ELISA (Fig. 8). Furthermore, soluble SEB or SEA could not compete the binding of anti-CD4 antibodies to rCD4 in a competitive ELISA, even at lOO-fold higher concentrations (-5% inhibition at SEB concentration of 300 pgiml). Finally, we show that when anti-CD4 antibodies
The addition of soluble r-CD4 to the SEB-stimulated cultures was able to block significantly the inhibitory effect of anti-CD4 antibodies on PBMC proliferation. This effect was achieved at rCD4 concentrations >10 uggiml (Table 3). As SEB stimulation depends on ability of superantigens to cross-link TCR with MHC Class II determinants, a finding that soluble rCD4 by itself failed to interfere with SEB stimulation of PBMC even at 100 pg/ml concentration could be of some interest. Since one of the possible mechanisms of anti-CD4 antibodies-mediated inhibition of cell proliferation could be due to reduced growth factor secretion (or alternatively increased consumption of IL-2), we further examined the effects of exogenous recombinant IL-2 (lo-100 U/ml) or IL-4 (200 U/ml) on anti-CD4-treated cultures. As shown in Table 4, the addition of IL-2 but not of IL-4 could partially reverse the inhibitory effect of anti-CD4 antibodies on anti-CD3-induced stimulation.
DISCUSSION We succeeded in affinity purification of human CD4reactive antibodies from several SLE patients sera. These antibodies, in a concentration-dependent manner, bound to both cell-expressed CD4 and rCD4. Saturation of CD4 binding sites occurred at anti-CD4 antibody concentrations between 5 and 15 pgiml. CD4-reactive antibodies were of both IgM and IgG isotypes and were not lightTABLE
I-r
2
Inhibitory effect of human anti-CD4 antibodies on SEB-stimulated proliferation is due to their direct binding to PBMC
Antibody”
rCD4
BSA
SE6
SEBlwashed PBMC
SEBinonwashed PBMC
Medium
SEA
FIGURE 8 Human anti-CD4 antibodies fail to bind to SEB or SEA in ELISA. The binding of an anti-CD4 antibody (Ab85) (used at 5 ug/ml) to rCD4, SEB, SEA, or BSA in ELISA is shown. Ab-85 was incubated for 1 h at 22°C on plates coated with either rCD4 or control proteins, which was followed by an additional 1 h incubation with isotype-specific HRPconjugated antibodies. Absorbance at 492 nM was determined in a Dynatech microplate reader.
NOW Ab-79 Ab-85 IgM, K (Vin)
459 650 334 400
i + f +
98,805 18,781 33,446 106,991
180 221 105 187
a Expressed are mean values P valueswere calculated with
+ SD of cpm the help
i f i k
7,660 4,360 18,682 6,805
from
of Student
‘ Ab-79
versus
IgM,
K; t = 8.52,
P < 0.0001.
'Ab-85
versus
IgM,
K; t = 5.55,
P = 0.0005.
three r test
94,443 51,208 60,649 93,800 different
f f + i
10,107
6,639h 9,797’ 9,071
expernnencs.
(two-talled).
Inhibition
119
of T Cell Proliferation
TABLE
3
Soluble rCD4 reverses anti-CD4-mediated inhibition of SEB-induced PBMC proliferation TdR incorporation (cpm f SD)
Condition Medium SEB SEB + Ab-79 SEB + Ab-79 SEB + rCD4
1,840 + 156 128,839 i- 11,106 74,710 + 4,593 104,381 + 6,680 124,827 + 12,064
+ rCD4
chain restricted. In several SLE patients, these antibodies occurred at serum concentrations higher than 50 pg/ml. When used at saturating concentrations, human antiCD4 antibodies strongly suppressed a number of T cell and T cell-dependent B cell-proliferative responses, induced by various polyclonal activators. The same was true when CD4’ T cell response to a tetanus toxoid was analyzed in sensitized persons (data not shown, manuscript in preparation). However, at least one anti-CD4 antibody (Ab-79), when added to cell cultures at subsaturating concentrations, costimulated the T cell proliferation promoted by superantigens/anti_CD3 antibodies. This effect was even more pronounced, when antibody (e.g., Ab-79) was added at the time of priming, or shortly afterward. From the theoretical standpoint, one or several diverse mechanisms could be operative in anti-CD4-mediated inhibition of T cell proliferation: blocking of the CD4I MHC Class II dependent cell adhesion, modulation of the CD4-lateral associations with TCR/CD3, and CD45 on T cells, sequestration or inappropriate signaling through p56Ick and priming for Fas-mediated apoptosis. CD4 molecule synergizes with the TCR in T helper cell activation, by laterally associating with the TCRKD3 complex and probably with CD45 and some other molecules {lo, 131. As a result of this, multireceptor complex is formed, which becomes highly competent to
TABLE
4
IL-2 but not IL-4 partially overcomes the inhibitory effect of anti-CD4 antibody on T cell proliferation induced by anti-CD3 antibodies
Condirion” Medium Anti-CD3 Anti-CD3 Anti-CD3
transduce activation signals. We now show that human anti-CD4 antibodies strongly suppressed the competence phase, but not the progression to DNA synthesis mediated by cytokineicytokine receptor interactions. Our data further suggest that the inhibition was most probably due to interference with CD4-lateral interactions, although additional effects on CD4/MHC Class II interaction and possible sequestration of the pS6kck could be operative too. This could prevent the PSblck, which is cytoplasmically associated with CD4, from phosphorylating the tyrosine residues in the 5 chain of the TCR complex, or by inducing an altered pattern of < chain phosphorylation. Consequently, this could block the activation of ZAP-70 and downstream signaling events, ultimately inhibiting the transcription of the IL-2 gene, and subsequent proliferation of stimulated cells (reviewed in [25]). Since the addition of exogenous IL-2 (but not of IL-4) to competent T cells partially restored the T cell proliferation, induction of reversible functional energy by human anti-CD4 antibodies should be considered as a primary mechanism of their action. We also show that following superantigen/anti-CD3 stimulation, IL-2iIL-4-mediated T cell DNA synthesis and cell proliferation was not sensitive to human anti-CD4 antibodies. There was a window of less than 24 h duration, during which simultaneous exposure to saturating amounts of anti-CD4 antibodies and polyclonal activators resulted in inhibition of PBMC proliferation. The inhibitory effect of anti-CD4 antibodies occurred in a monocyte-independent fashion, indicating a direct suppressive action on T cells. However, some contribution of monocyte-derived factors could not be ruled out completely. Furthermore, in the case of inhibition of PWM-stimulated B cell proliferation, one additional mechanism of immunosuppression induced by anti-CD4 antibodies could be due to direct interference with cellto-cell adhesion that occurs between activated CD4’ T cells and B cells.
Without
+ IL-2 + IL-4
317 68,120 80,792 73,244
f i I +
Abs 129 21,340 14,883 18,818
With 471 22,744 41,482 25,991
Ab-79 f + + +
104 13,888” 17,154 16,679J
With
IgM,
351 68,443 76,661 70,053
f + 2 f
K
(Vin)
49 23,320 18,687 22,905
a Expressed are mean values * SD of cpm from three different experiments. P values were calculated means of Wilcoxon slgned rank test (two-tailed). ’ Without
Abs versus with Ab-79,
P = 0.0039.
’ With
Ab-79
versus with Ab-79
+ IL-2, P = 0.0092.
“With
Ab-79
vetsus with Ab-79
+ IL-4, P > 0.05.
by
120
Several recent studies provided experimental evidence that CD4 cross-linking, in the absence of simultaneous TCR engagement, could prime T cells for activationdependent apoptosis [26-281. This required both functional p56kck and Fas-receptor expression [29, 301. Although we have not addressed this possibility directly, our preliminary experiments on CD4-expressing Jurkat cells (but not on CD4-negative variants) demonstrated that the sole presence of human anti-CD4 antibodies was sufficient to promote significant cell death by apoptosis, after 72 h of culture (data not shown). However, there was no evidence of enhanced cell death after 24-48 h of the culture. Moreover, an additional level of complexity in understanding the principal mechanisms involved in CD4-mediated T cell downregulation was provided by studies showing that anti-CD4 treatment could be beneficial in MRL-mice bearing Irp mutation and not expressing functional Fas receptor. So, anti-CD4 therapy in MRL-/‘Y/I@ mice dramatically reduced the frequency and severity of autoimmune disease, including glomerulonephritis 1311, development of CNS lesions, and arthritis [32). Furthermore, engagement of mutant forms of CD4 that have lost their association with p561ck still resulted in T cell downregulation in vitro 1331. While we have not addressed directly whether any particular Th-cell subset or specific cytokine profile could be preferentially affected by human anti-CD4 antibodies, others have observed that the Thl-type responses and activating memory T cell subsets demonstrate particular susceptibility to the action of murine anti-CD4 mAbs (34-37). Our findings may provide an explanation for some previously observed effects of antilymphocyte antibodies that occur in SLE on T and B cell activation (e.g., inhibitory effect on MLC and on antigen-lmitogen-induced IL-2R expression and lymphocyte proliferation, reviewed in [38}). SLE sera could also promote antigen modulation, inhibit both NK cell function and antibodydependent cell cytotoxicity, and reduce T suppressor function. The presence of these antibodies often correlates clinically with the active disease, neuropsychiatric manifestations, and decreased lymphocyte counts [38, 391. In conclusion, we demonstrate for the first time that human affinity-purified anti-CD4 antibodies, being of both IgG and IgM isotypes, can inhibit T cell proliferative responses by induction of a classical T cell energy. However, it remains to be determined in the future whether human anti-CD4 antibodies induce these effects by differentially affecting the cytokine secretion or, for example, by increasing the consumption of a particular cytokine. Another question, which needs further clarification, is whether human anti-CD4 antibodies may directly prime T cells for activation-induced apoptosis.
G. Lenert and P. Lenert
This will significantly advance our knowledge on pathogenesis of some T and B cell abnormalities that occur commonly in human SLE and in lupus-prone strains of mice. ACKNOWLEDGMENTS
We are grateful
to Drs. M. Zanetti, G. Delespesse, and E. Rasio for support; Dr. J-L. Seneca1 for the sera of SLE patients; Dr. R-P. Sekaly for providing the cell lines; and Dr. C. Demeure for help in FACS analysis. This research was supported in part by a Medical Research Council Invited Scientist Grant (P.L.).
REFERENCES 1. Maddon PJ, Littman DR, Godfrey M, Maddon DE, Chess L, Axe1 R: The isolation and nucleotide sequence of a cDNA encoding the T cell surface protein T4: a new member of the immunoglobulin gene family. Cell 42:93, 1985. 2. Gay D, Maddon P, SPkaly R, Talle MA, Godfrey M, Long E, Goldstein G, Chess L, Axe1 R, Kappler J, Marrack P: Functional interaction between human T-cell protein CD4 and the major histocompatibility complex HLA-DR antigen. Nature 328626, 1987. 3. Janeway CA Jr, Haque S, Smith LA, Saizawa K: The role of the murine L3T4 molecule in T cell activation: differential effects of anti-L3T4 on activation by monoclonal anti-receptor antibodies. J Mol Cell Immunol 3:12 1, 1987. between CD4 and 4. Doyle C, Strominger JL: Interaction class II MHC molecules mediates cell adhesion. Nature 330:256, 1987. 5. Lamarre D, Ashkenazi A, Fleury S, Smith DH, Sekaly R-P, Capon DJ: The MHC-binding and gpl20-binding functions of CD4 are separable. Science 245:743, 1989. 6. Clayton LK, Sieh M, Pious DA, Reinhetz EL: Identification of human CD4 residues affecting class II MHC versus HIV-l gp120 binding. Nature 339:549, 1989. 7. Lamarre D, Capon DJ, Karp DR, Gregory T, Long EO, SCkaly R-P: Class II MHC molecules and the HIV gp120 envelope protein interact with functionally distinct regions of the CD4 molecule. EMBO J 8:3271, 1989. 8. Cammarota G, Scheirie A, Takacs B, Doran DM, Knorr R, Bannwarth W, Guardiola J, Sinigaglia F: Identification of a CD4 binding site on the B2 domain of HLA-DR molecules. Nature 356:799, 1992. 9. Weber S, Karjalainen K: Mouse CD4 binds MHC class II with extremely low affinity. Int Immunol 5695, 1993. 10. Lenert P, Kroon D, Spiegelberg H, Golub ES, Zanetti M: Human CD4 binds immunoglobulins. Science 248:1639, 1990. interaction: 11. Lenert P, Zanetti M: CD4iimmunoglobulin implications for immune physiology and autoimmunity. Int Rev Immunol 7:237, 1991. D, Redoglia V, 12. Dianzani U, Bragardo M, Buonfiglio Funaro A, Portoles P, Rojo J, Malavasi F, Pileri A: Modu-
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lation of CD4 lateral interaction with lymphocyte surface molecules induced by HIV-l gp120. Eur J Immunol 25: 1306, 1995. 13. Janeway CA Jr: The T cell receptor as a multicomponent signalling machine: CD4/CD8 coreceptors and CD45 in T cell activation. Annu Rev Immunol 10:645, 1992. 14. Anderson P, Blue ML, Morimoto C, Schlossman SF: Cross-linking of T3 (CD3) with T4 (CD4) enhances the proliferation of resting T lymphocytes. J Immunol 139: 678, 1987. 15. Emmrich F, Kanz L, Eichmann K: Cross-linking of the T cell receptor complex with the subset-specific differentiation antigen stimulates interleukin 2 receptor expression in human CD4 and CD8 T cells. Eur J Immunol 17:529, 1987. 16. Anderson P, Blue ML, Schlossman SF: Comodulation of CD3 and CD4. Evidence for a specific association between CD4 and approximately 5% of the CD3:T cell receptor complexes on helper T lymphocytes. J Immunol 140: 1732, 1988. 17. Dianzani U, Shaw A, al-Ramadi BK, Kubo RT, Janeway CA Jr: Physical association of CD4 with the T cell receptor. J Immunol 148:678, 1992. 18. Bank I, Chess L: Perturbation of the T4 molecule transmits negative signal to T cells. J Exp Med 162:1294, 1985. 19. Wassmer PP, Chan C, Logdberg L, Shevach EM: Role of the L3T4-antigen in T cell activation. II. Inhibition of T cell activation by monoclonal anti-L3T4 antibodies in the absence of accessory cells. J Immunol 135:2237, 1985. 20. Owens T, Fazekas de St. Groth B: Participation of L3T4 in T cell activation in the absence of class II major histocompatibility complex antigens: inhibition by antiL3T4 antibodies is a function both of epitope density and mode of presentation of anti-receptor antibody. J Immuno1 138:2402, 1987. 2 1. Mann DL, Lasane F, Popovic M, Arthur LO, Robey WG, Blattner WA, Newman MJ: HTLV-III large envelope protein (gpl20) suppresses PHA-induced lymphocyte blastogenesis. J Immunol 138:2640, 1987. 22.
Diamond DC, Sleckman BP, Gregory T, Lasky LA, Greenstein JL, Burakoff SJ: Inhibition of CD4+ T cell function by the HIV envelope protein gp120. J Immunol 141:3715, 1988.
23. Mittler RS, Hoffmann MK: Synergism between HIV gp120 and gpl20-specific antibody in blocking human T cell activation. Science 245: 1380, 1989.
26. Newell MK, Haughn LJ, Maroun CR, Julius MH: Death of mature T cells by separate ligation of CD4 and the T-cell receptor for antigen. Nature 347:286, 1990. 27. Banda NK, Bernier J, Kurahara DK, Kurrle R, N, Sekaly R-P, Finkel TH: Crosslinking CD4 immunodeficiency virus gp120 primes T cells tion-induced apoptosis. J Exp Med 176:lO99,
Haigwood by human for activa1992.
28. Oyaizu N, McCloskey TW, Than S, Hu R, Kalyanaraman VS, Pahwa S: Cross-linking of CD4 molecules upregulates Fas antigen expression in lymphocytes by inducing interferon-y and tumor necrosis factor-a secretion. Blood 84: 2622, 1994. 29. Di Somma MM, Nuti S, Telford JL, Baldari CT: p561ck plays a key role in transducing apoptotic signals in T cells. FEBS Lett 363:101, 1995. 30. Wang Z-q, Dudhane A, Orlikowsky T, Clarke K, Li X, Darzynkiewicz Z, Hoffmann MK: CD4 engagement induces Fas antigen-dependent apoptosis of T cells in vivo. Eur J Immunol 24:1549, 1994. 31. Jabs DA, Kuppers RC, Saboori AM, Burek CL, Enger C, Lee B, Prendergast RA: Effects of early and late treatment with anti-CD4 monoclonal antibody on autoimmune disease in MRLIMP-lprilpr mice. Cell Immunol 154:66, 1994. 32. O’Sullivan FX, Vogelweid CM, Besch-Williford CL, Walker SE: Differential effects of CD4+ T cell depletion on inflammatory central nervous system disease, arthritis and sialadenitis in MRLilpr mice. J Autoimmun 8:163, 1995. 33. Zerbib AC, Reske-Kunz AB, Lock P, Sekaly R-P: CD4mediated enhancement or inhibition of T cell activation does not require the CD4:p561ck association. J Exp Med 179:1973, 1994. 34. Wang J, Yan T, Simmer B, Emmrich F: The effect of anti-CD4 on helper function of CD4,45RA+ versus CD4, 45RO+ T cells. Clin Exp Immunol 95:128, 1994. 35. Howie SE, Sommerfield AJ, Gray E, Harrison DJ: Peripheral T lymphocyte depletion by apoptosis after CD4 ligation in vivo: selective loss of CD44- and ‘activating’ memory T cells. Clin Exp Immunol 95:195, 1994. 36. Stumbles P, Mason D: Activation of CD4+ T cells in the presence of nondepleting monoclonal antibody to CD4 induces a Th2-type response in vitro. J Exp Med 182:5, 1995. 37. Mottram PL, Han WR, Purcell LJ, McKenzie IF, Hancock WW: Increased expression of IL-4 and IL-10 and decreased expression of IL-2 and interferon-gamma in long-surviving mouse heart allografts after brief CD4monoclonal antibody therapy. Transplantation 59:559, 1995.
24. Carrel S, Moretta A, Pantaleo G, Tambussi G, Isler P, Perussia B, Cerottini JC: Stimulation and proliferation of CD4+ peripheral blood T lymphocytes induced by an anti-CD4 monoclonal antibody. Eur J Immunol 18:333, 1988.
38. Winfield JB, Mimura T: Pathogenetic significance of anti-lymphocyte autoantibodies in systemic lupus erythematosus. Clin Immunol Immunopathol 63: 13, 1992.
25. Robey E, Allison JP: T cell activation: integration nals from the antigen receptor and costimulatory ecules: Immunol Today 16:306, 1995.
39. Denburg SD, Behmann SA, Carbotte RM, Denburg JA: Lymphocyte antigens in neuropsychiatric systemic lupus erythematosus. Arthritis Rheum 37:369, 1994.
of sigmol-
Human CD4-Reactive Antibodies from SLE Patients Induce Reversible Inhibition of Polyclonal T Lymphocyte Proliferation Gordana Lenert and Petar Lenert ABSTRACT: We report on isolation of human polyclonal CD4-reactive antibodies of IgM and/or IgG isotypes from several SLE patients. These antibodies bound specifically to CD4-expressing cell lines and to rCD4 in ELISA and immunoblots. Saturation of CD4-binding sites occurred at antibody concentrations between 5 and 15 pgiml. Anti-CD4 antibodies, in a dose-dependent manner, suppressed the proliferative responses of human peripheral blood mononuclear cells (PBMC) to superantigens (Staphylococcal enterotoxins A and B), anti-CD3 antibodies, and mitogens (PWM and Con A, but not PHA). They could also inhibit the proliferation of highly purified human T cells induced by immobilized anti-CD3 antibodies. To promote their effects on T cells, human antiCD4 antibodies had to be present at lymphocyte cultures
ABBREVIATIONS systemic lupus erythematosus SLE peripheral blood mononuclear PBMC Stapbylococcal enterotoxin B SEB SEA Stapbylococcal enterotoxin A r recombinant
before or at the time of priming. There was no significant inhibition when antibodies were added more than 24 h following T cell activation. Substantial evidence that the immunosuppression induced by anti-CD4 antibodies was due to their direct effect on T cells was obtained. Downregulatory effect of anti-CD4 antibodies could be significantly reversed by addition of exogenous IL-2 and by preincubation with soluble recombinant (r)CD4. Interestingly, at least one affinity-purified anti-CD4 antibody could costimulate the T cell proliferation induced by superantigens or anti-CD3 antibodies, especially when used at subsaturating concentrations (l-4 pg/ml) and when added subsequently to the initiation of cultures. Hzlman Immunology49. 113-l 21 (1996)
TCR HRP PWM PHA Con A
cells
T cell receptor for antigen horseradish peroxidase pokeweed mitogen phytohemagglutinin concanavalin A
INTRODUCTION CD4 that
is an integral bears
the immunoglobulin on T cells (TCR) with
is well
recognition MHC
membrane
a significant
glycoprotein
homology supergene
correlated of antigenic
Class II molecules
of -55
to other
family
kDa
members
of
Cl]. Its expression the
T cell receptor
peptides
in association
with
on antigen-presenting
cells
{3]. Cell-tocell adhesion directly dependent on CD4/MHC Class II interaction was first shown by Doyle and Strominger [4] 121 and with
the ability
and later confirmed
to activate
by others
[5-71.
B cells
CD4
binds
to non-
From the Louis-Charles Simard ResearchCenter, Notre-Dame Hospital. University ofMontve’al. Mont&al, Quibec H2L 4Ml. Canah. Address reprint requeststo P. Lenert, Laboratoire de Rechercbeen Immunoregulation, M4211 -L, Centre de Rechexbe, H@ital Notre-Dame, 1560, rue SherbrookeEst, Mon.&al, Que’becH2L 4M 1, Canada. Received December 13, 1995; acceptedJanuary 25, 1996. Human Immunology 49, 113-12 1 (1996) 0 American Society for Histocompatibility and Immunogenetics,
1996
polymorphic determinants on j32 domain of MHC Class II molecules, thereby increasing the overall avidity of TCR for its ligand {S, 97. From the functional perspective, several studies in the past have shown that the transfection of human CD4 molecule into effector T cell hybridomas significantly enhances the proliferation and cytokine production, in response to human MHC Class II antigens expressed on target cells f2, 7). However, apart from the interaction with MHC Class II molecules, CD4 can also interact with the TCR/CD3 complex and with some other members of the Ig-supergene family [lo-12). Since simultaneous cross-linking of CD4 with the TCRi CD3 upregulates the T cell proliferative response, it has been proposed that CD4 participates in a formation of the multireceptor complex involved in optimal T cell activation [13-171. Intracellular domains of both CD4 0198~8859/96/$15.00 PII SOl9g-8859(96)00054-7
114
G. Lenert and P. Lenert
and CD8 interact with the p56& tyrosine kinase, and this association appears to be particularly important for the coreceptor function 131. CD4 is also the high-affinity receptor for the human immunodeficiency virus, a function that is carried out by residues 40-55 within a CD4Dl domain 15-71. Since high-affinity ligands for CD4, like HIV-gp120 and murine anti-CD4 mAbs, strongly inhibit T cell activation, even in the absence of any obvious interaction between MHC Class II determinants and CD4 13, 18241, we raised the question of whether human CD4reactive antibodies that occur increasingly in a subset of patients with SLE may similarly interfere with T cell activation events. We now report on successful isolation of several human anti-CD4 antibodies from SLE sera. These antibodies retained their ability to specifically recognize both cell-expressed and recombinant CD4 and gave saturable binding at concentrations between 5 and 15 pg/ml. Human anti-CD4 antibodies strongly suppressed the polyclonal T cell proliferation induced by different stimuli. This effect occurred only when antiCD4 antibodies were present before or at the time of priming and when used at saturating concentrations. Exogenous IL-2 could partially overcome this inhibitory effect, suggesting a possibility of the induction of classical functional energy.
MATERIALS
AND
METHODS
Affinity Purification Anti-CD4 Antibodies
of Human
Several affinity columns were made by coupling between 1 and 10 mg of rCD4 (Biogen, Cambridge, MA) to 1 ml of NHS-activated Sepharose (Hi-Trap, PharmaciaBiotech, Montreal, QC) according to the manufacturer’s recommendations. Samples of SLE sera showing high CD4 reactivity in ELISA and by FACS were diluted 1: 10 with 20 mM sodium phosphate, pH 7.0, precleared, and applied to a CD4 column at 0.8 ml/min flow rate (single passage). In some experiments, an intermediate step consisting of gamma-globulin purification by an E-Z-SEP (Pharmacia-Biotech) procedure was performed. In either case, desorption of anti-CD4 antibodies was achieved by using 0.1 M triethanolamine, pH 11.0. Eluates were immediately neutralized with sodium acetate, pH 4.15, concentrated using Spin-X UF concentrators (Costar, Cambridge, MA), and extensively dialyzed against PBS, pH 7.2. The purity of anti-CD4 antibodies was checked in both native PAGE and in SDS-12% PAGE, run under reducing conditions. Human polyclonal IgM and IgG (Calbiochem-Novabiochem, La Jolla, CA), together with myeloma proteins IgM, K (VIN), and IgGl, x (FITZ) (kindly provided by Dr. H. Spiegelberg, Scripps Clinic,
La Jolla, CA), were run in parallel. No bands other than those corresponding to human IgM and/or IgG or their reduced H and L chains were observed. The identity of these bands was further confirmed in Western blots. Concentration of IgG and IgM anti-CD4 antibodies was determined by ELISA. ELISA
for Anti-CD4
Antibodies
ELISA for detection of affinity-purified CD4-reactive antibodies was performed on Falcon 3911-MicroTest III assay plates (Becton-Dickinson Labware, Oxnard, CA). Plates were coated with 50 pliwell of rCD4 or control proteins (BSA, SEB, SEA, insulin), all diluted in 0.9% NaCl, pH 7.0, and used at 2.5-3 pgiml. After blocking the wells with PBS-0.25% BSA-0.05% Tween 20 (blocking buffer), anti-CD4 antibodies and control human polyclonal IgG and IgM, as well as myeloma proteins of IgG and IgM isotypes, were added at increasing concentrations and incubated for 1 h at 22°C. After washings with PBS-0.3% Tween 20, goat anti-human IgG (1:3.000) or anti-human IgM (1:3.000) HRPconjugated second-step Ab (Caltag, Cedarlane Labs., Hornby, ON) were added and incubated for an additional hour. An OPD substrate in citric/phosphate buffer, pH 5.0, containing 0.4 ml/liter of H,O, was used for detection of bound HRP. The color development was stopped with 3M H,S04, and absorbance at 492 nm was determined by using the Dynatech microplate reader. In competitive experiments, either IgG or IgM affinity-purified anti-CD4 Ab at 3 pg/ml (final concentration) werepreincubated in tubes for 30 min with increasing amounts of rCD4 or control proteins (BSA, SEB, SEA), transferred in duplicates to wells of microtiter plates previously coated with rCD4, and incubated for 1 h at 22°C. Bound antibodies were revealed as described above. Immunoblotting The reactivity of affinity-purified anti-CD4 antibodies with rCD4 and control proteins (e.g., SEB) was further tested in immunoblottings. One ug/band of total protein was applied to SDS-12% PAGE. Electrophoresis was performed for 1 h at 200 V (constant voltage). Gels were blotted onto nitrocellulose membrane (0.45 pm, Bio-Rad Labs., Mississauga, ON) for an additional hour at 4°C (100 V), in 12.5 mM Tris, pH 8.25, 96 mM glycine, 20% methanol. Nitrocellulose membranes were cut in individual strips, blocked for 1 h at 22°C with 1% BSAPBS, and incubated for 1 h at 22’C with affinity-purified anti-CD4 antibodies (5 pgiml) or control Ig. Following washings (three times for 5 min) with PBS-0.3% Tween 20, strips were further incubated with HRP-labeled goat anti-human IgG (1:3.000) or IgM (1:3.000) (Caltag), for
Inhibition
115
of T Cell Proliferation
1 h at 22°C.
4-Chloro-naphtol was used as a substrate. Development of colored reaction was stopped after lo15 min by rinsing with dH,O. Murine anti-Leu 3a mAb served as a positive control. FACS Analysis Human CD4-transfected and nontransfected HeLa cells at 5 x lo5 were incubated in 100 pl volumes, in PBS2% BSA-0.05% Na-azide, with human anti-CD4 antibodies or control Ig’s (used at 5 pg/ml), for 1 h on ice. Bound Ig’s were detected after an additional incubation for 30 min on ice with either FITC-labeled anti-human IgG or anti-human IgM antibodies (both used at 1:250, Cappel, Organon Teknika Inc., Scarborough, ON). Stained cells were analyzed using a Lysis II software on a FACSort (Beckton-Dickinson, Mont&al, QC) cell sorter. Cell Proliferation
Assays
Human PBMC were isolated from healthy laboratory personnel by Ficoll-Paque ET (Pharmacia-Biotech) gradient centrifugation. Purified T lymphocytes were obtained by E-rosetting with AET-treated sheep red blood cells. These cells were >97% CD2’ as demonstrated by flow cytometry. PBMC, 105, or purified T cells, at 100 $/well, were incubated in 96-well flat-bottom tissue culture plates (Falcon 3072, Microtest III, BecktonDickinson Labware, Lincoln Park, NJ) for 72 h. Culture medium consisted of RPM1 (Gibco, Grand Island, NY) supplemented with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 pggiml), HEPES buffer (15 mM), and FCS (10%). The following substances were used for PBMC stimulation: anti-CD3 (UCHT-1 antibody kindly provided by Dr. Delespesse, 10 pg/ml, precoated on plastic), PHA-P (SIGMA Chem. Co., St. Louis, MO, 5 pgiml), Con-A (SIGMA, lpg/ml), PWM (SIGMA, 1 pgiml), and microbial superantigensStaphylococcal enterotoxins A and B (SEA and SEB, at 50 and 125 ngiml, respectively, SIGMA). In most instances, human anti-CD4 antibodies (and control isotype matched human myeloma proteins) were used at I-20 pgiml and preincubated with cells for 24 h at 37”C, before polyclonal stimulation. In some experiments, between 10 and 100 U/ml of human recombinant IL-2, 200 U/ml of human recombinant IL-4 (BoehringerMannheim, Montreal, QC) or 10 pg/ml of soluble rCD4 was added. Cultures were incubated in triplicates at 37°C in 5% CO, in air and pulsed with 0.5 @/well of 13H] thymidine (6.7 CiimM, ICN, Irvine, CA) for the last 18 h. Incorporation of the L3H)thymidine was measured by liquid scintillation counting. Direct cytotoxic effect of anti-CD4 Ab on unstimulated human PBMC was excluded by trypan blue staining.
RESULTS Human Affinity-Purified Anti-CD4 Retain Their Specific CD4-Binding
Antibodies Properties
We recently observed that relatively high serum levels of antibodies recognizing the human CD4 specifically occur in a subset of SLE patients (-20%). To study their biological properties, we first made efforts to obtain them in highly purified form, by making several affinity columns with covalently linked rCD4 to Sepharose beads. Optimal isolation of anti-CD4 antibodies was achieved by single passing a diluted SLE sera at 0.8 ml/min flow rate over the rCD4 column (6 mg rCD4) and by using 0.1 M triethanolamine for elution (Fig. 1). These conditions were chosen in accordance with our recent observations that IgM anti-CD4 antibodies are highly sensitive to harsh acidic treatment. In cases of five SLE sera, we obtained relatively high yields of anti-CD4 antibodies, which ranged from 40 to 120 pg (from -0.5 ml of starting sera). Purified antibodies were of both IgM and IgG isotypes in two cases (Ab-79 and Ab-85) and solely IgM in two cases (Ab-21 and Ab-76), while in a single case antibodies of the IgG class were isolated (Ab-29). Preparation of Ab-79 also contained small amounts of IgA antibodies. The purity of antibody preparations was checked by both native PAGE and SDS-12% PAGE run under reducing conditions. We could observe only bands corresponding to either nonreduced IgG and/or IgM or their single heavy and light chains (data not shown). This was also confirmed in Western blottings.
10 Fraction
number
FIGURE 1 Affinity purification of human anti-CD4 antibodies. CD&&nity column was made by coupling 6 mg of rCD4 to NHS-activated Sepharose beads. Precleared SLE serum 85 was diluted 1:lO with 20 mM sodium phosphate and single-passed over a rCD4 column. After washings, specifically bound Ig were eluted by using 0.1 M triethanolamine and immediately neutralized with sodium acetate, pH 4.15. Ordinate refers to absorbance at 280 nM.
116
G. Lenert and P. Lenert
The anti-CD4 antibodies of both IgG and IgM isotypes gave specific and saturable binding to rCD4 in ELISA. Figure 2 shows the representative binding curves of Ab-85 on rCD4 and on control protein (BSA), in comparison to those given by human myeloma protein Vin (IgM, K) and human polyclonal IgM (CalbiochemNovabiochem) on rCD4, when tested over a range of concentrations. Other human anti-CD4 antibodies and control anti-Leu 3a mAb behaved similarly. We next tested human anti-CD4 antibodies for the binding to native CD4 expressed on cell membranes and to rCD4 in immunoblots (Figs. 3 and 4, respectively). In FACS analysis, anti-CD4 antibodies bound specifically to CD4expressing HeLa cells, while in immunoblots, we could observe reactivity toward a -45-kDa protein that corresponds to Mr of the truncated form of CD4. As specificity controls, we show the lack of binding of human anti-CD4 antibodies to SEB (-28 kDa) used at equivalent concentrations, as well as specific binding of murine anti-Leu 3a antibody to the 45-kDa band. Anti-CD4 Antibodies Inhibit, in a Dose-Dependent Fashion, the Polyclonal Proliferation of Human PBMC and Purified T Cells PBMC from seven different donors were preincubated with human anti-CD4 antibodies (7.5 pg/ml) and isotype-matched human myeloma proteins (e.g., IgM, K-Vin) for 24 h, which was followed by either mitogen (PHA, PWM, and Con A), superantigen (SEA and SEB), or anti-CD3 (UCHT-1)-mediated stimulation (Fig. 5). Data obtained with Ab-79 are shown. Human anti-CD4 antibodies, but not control Ig’s when used at equimolar concentrations, strongly inhibired the PBMC prolifera-
t
1/
I
--o-
Ab-850” BSA
--)_
IgM. k (Vin)
Fluorescence
intensity
(tog)
FIGURE 3 FACS analysis of human anti-CD4 antibody binding to CD4-expressing cells. CD4transfected and nontransfected HeLa cells were incubated for 1 h on ice with Ab-85 or control Ig’s (used at 5 pg/ml). FITC-conjugated goat anti-human IgM antibodies (1:250, Cappel) were used for detection of specifically bound IgM. The binding of control Ig’s at 5 pgiml to CD4-transfected cells was not different from that occurring on nontransfected HeLa cells and is not shown.
tion induced by various polyclonal activators. The inhibitory effect on superantigen-induced proliferation (-45%) was similar to that observed following anti-CD3 and PWM stimulation (49--53%) and slightly higher than that on Con A-induced proliferation (37%). Surprisingly, at the concentration indicated, Ab-79 failed to Lea
%a
21
29
76
79
85
hIlJc
rbrbrbrbrbrbrb 0
2 Ab
4
6
concentration
6
10
12
(pglml)
FIGURE 2 Saturable binding of Ab-85 to rCD4 in ELISA. Ab-85, control myeloma protein Vin (IgM, K), and human polyclonal IgM were tested in ELISA for binding to wells coated with either rCD4 or BSA. Under the assay conditions employed, only Ab-85, but not control Ig’s, gave specific binding to rCD4. Saturation of rCD4 binding sites in ELISA was achieved at Ab-85 concentrations higher than 3-5 pggiml. Similar data were obtained with other anti-CD4 antibodies.
FIGURE 4 Immunoblotting of affinity-purified anti-CD4 antibodies. The specific binding of human anti-CD4 antibodies (Ab-21, Ab-29, Ab-76, Ab-79, and Ab-85) and control anti-Leu 3a antibody (all used at 5 pg/ml) to the 45-kDa band (a) and lack of detectable binding to the 28 kD band (SEB) (b) blotted under identical conditions are shown. For comparison, myeloma protein Vin (IgM, K) failed to give significant binding to either band. Bound antibodies were revealed by 1 h incubation with HRP-labeled goat anti-human IgM antibodies, except in cases of Ab-29, which was revealed by incubation with HRP-labeled goat anti-human IgG and of anti-Leu 3a, with HRP-labeled goat anti-mouse IgG.
Inhibition
of T Cell Proliferation
0
117
40
120
80
Medi urn
+
Ab-79
PHA
+
Ab-65
PHA + Yin
+
Ab-21
PHA + Ab-79
*
Ab-76
--)-
Ab-29
---A--
IgM. k p/in)
PWM PWM + Vin PWM + Ab-79 01
Con-A Con-A
+ Vin
Con-A
+ Ab-79
1.2
2.5
5.0
Ab concentration
10.0
20.0
( v g/ m 1)
FIGURE 6 Dose dependency of anti-CD4 inhibition. Human PBMC were preincubated for 24 h at 37°C with five different anti-CD4 antibodies and control isotype-matched myeloma proteins added at increasing concentrations (ranging from 1 to 20 pg/ml), which was followed by stimulation with SEB (125 ng/ml) for 3 days. Lymphocyte proliferation was measured by determining C3H)thymidine incorporation, which was added 18 h prior to cell harvesting. Expressed are mean values of cpm (x 10-3) of three separate experiments.
SE4 SEA + Vin SEA + Ab- 79 SEB SEB + Vin SEB + Ab-79 anti-CD3 anti-CD3
+ Yin
anti-CD3
+ Ab- 79
Requirement for Anti-CD4 Phase of the Culture TdR Incorporation (mean x lC.f3+ SD)
FIGURE liferation. anti-CD4
5 Human anti-CD4 antibodies inhibit PBMC proHuman PBMC were preincubated for 24 h with antibodies (7.5 &ml) or isotype-matched myeloma
proteins and then stimulated for 3 days with immobilized anti-CD3 antibodies (10 &ml), PWM (1 pg/ml), PHA (5 pgiml), Con A (1 pgiml), SEB (125 ng/ml), or SEA (50 ngiml). Lymphocyte proliferation was measured by determining [- H}thymidine incorporation, which was added 18 h prior to cell harvesting. inhibit
PHA
by either induced pulsed
stimulation.
Ab-79
proliferation APC;
A similar
or Ab-85
of CD4’
data not shown,
degree
occurred
T cells
manuscript
of inhibition
on tetanus (with
toxoidantigen-
in preparation).
of antiCD4 antibodies and occurred in a dose-dependent fashion (Fig. 6). For example, in SEB-stimulated proliferation, an ED5,, for five different anti-CD4 antibodies was achieved at 7.5-15 pgiml. Interestingly, some anti-CD4 conantibodies (e.g., Ab-79), w h en used at subsaturating centrations (-1-4 pg/ml), costimulated the PBMC proliferation induced by superantigens or anti-CD3 antibodies. We next show that anti-CD4 antibodies, but not control Ig’s, similarly to their effects on nonseparated PBMC, could specifically inhibit the proliferation of highly purified T cells when stimulated with immobilized antiCD3 mAbs (Table 1). The inhibitory
effect
was due to specific binding
Antibodies
in the Early
Human anti-CD4 antibodies and control myeloma proteins Vin (IgM, K) and Huf (IgM, A) (all at 10 pg/ml) were added to SEB-stimulated cultures at different time intervals up to 48 h (Fig. 7). Proliferative response was measured after 72 h of SEB stimulation. To exhibit their inhibitory effects, human anti-CD4 antibodies had to be present at saturating concentrations 24 h before or at the time of polyclonal PBMC stimulation. Addition of antibodies to the cultures more than 24 h after SEB stimulation was not effective. The ability to add anti-CD4 antibodies during the last 48 h of culture without effect eliminates any potential for nonspecific cytotoxicity as an explanation for reduced thymidine incorporation. Inhibitory Effect of Anti-CD4 Antibodies on SEB-Induced Proliferation Is Due to Their Direct Binding to Human PBMC Several experiments were designed to ensure that the effect of anti-CD4 antibodies on SEB and anti-CD3induced proliferation of PBMC is a consequence of their TABLE
1
Human anti-CD4 anti-CD3-induced T cells
Condition
TdR incorporation (cpm + SD)
Medium Anti-CD3 Anti-CD3 + Ab-79 Anti-CD3
+ IgM,
Ab-79 inhibits proliferation of purified
355 f 103 55,747 + 18,470 K
(Vin)
14,446 58,994
Tk7,707 + 13,291
118
G. Lenert and P. Lenert
-24h
Oh
+24h
Addition
+
M-79
*
At-85
-f--
IgM. k (Vin)
--U--
IgM. I (HufJ
were present in cell cultures only before cell stimulation and washed out, significant inhibition of subsequent SEB-induced cell proliferarion still occurred (Table 2). However, when subsaturating amounts of some antiCD4 antibodies were used for preincubation and subsequently washed out, almost twofold enhancement of anti-CD3-mediated T cell proliferation occurred. Soluble rCD4 and IL-2 Overcome the Inhibitory Effect of Anti-CD4 Antibodies on SEB-Induced PBMC Proliferation
+48h
of Ab
FIGURE 7 Kinetics of anti-CD4 inhibition. Human PBMC (1 x 105/well) were stimulated with SEB (125 ngiml) for 3 days. Two affinity-purified anti-CD4 antibodies (Ab-79 and
Ab-85) and control myeloma proteins (Vin and Huf), all at 10 pgiml, were added to the cultures at different time points. Lymphocyte proliferation was assayed by determining the I- H)thymidine incorporation, which was added 18 h before cell harvesting. Ordinate shows the mean values of cpm (x 1O-i) from one out of three representative experiments.
direct binding to CD4-expressing T cells, and not to simple neutralization of the ligand. We first show that affinity-purified anti-CD4 antibodies at concentrations (5-10 pg/ml) that were used to inhibit T cell proliferation completely failed to bind to SEB, SEA, or BSA in ELISA (Fig. 8). Furthermore, soluble SEB or SEA could not compete the binding of anti-CD4 antibodies to rCD4 in a competitive ELISA, even at lOO-fold higher concentrations (-5% inhibition at SEB concentration of 300 pgiml). Finally, we show that when anti-CD4 antibodies
The addition of soluble r-CD4 to the SEB-stimulated cultures was able to block significantly the inhibitory effect of anti-CD4 antibodies on PBMC proliferation. This effect was achieved at rCD4 concentrations >10 uggiml (Table 3). As SEB stimulation depends on ability of superantigens to cross-link TCR with MHC Class II determinants, a finding that soluble rCD4 by itself failed to interfere with SEB stimulation of PBMC even at 100 pg/ml concentration could be of some interest. Since one of the possible mechanisms of anti-CD4 antibodies-mediated inhibition of cell proliferation could be due to reduced growth factor secretion (or alternatively increased consumption of IL-2), we further examined the effects of exogenous recombinant IL-2 (lo-100 U/ml) or IL-4 (200 U/ml) on anti-CD4-treated cultures. As shown in Table 4, the addition of IL-2 but not of IL-4 could partially reverse the inhibitory effect of anti-CD4 antibodies on anti-CD3-induced stimulation.
DISCUSSION We succeeded in affinity purification of human CD4reactive antibodies from several SLE patients sera. These antibodies, in a concentration-dependent manner, bound to both cell-expressed CD4 and rCD4. Saturation of CD4 binding sites occurred at anti-CD4 antibody concentrations between 5 and 15 pgiml. CD4-reactive antibodies were of both IgM and IgG isotypes and were not lightTABLE
I-r
2
Inhibitory effect of human anti-CD4 antibodies on SEB-stimulated proliferation is due to their direct binding to PBMC
Antibody”
rCD4
BSA
SE6
SEBlwashed PBMC
SEBinonwashed PBMC
Medium
SEA
FIGURE 8 Human anti-CD4 antibodies fail to bind to SEB or SEA in ELISA. The binding of an anti-CD4 antibody (Ab85) (used at 5 ug/ml) to rCD4, SEB, SEA, or BSA in ELISA is shown. Ab-85 was incubated for 1 h at 22°C on plates coated with either rCD4 or control proteins, which was followed by an additional 1 h incubation with isotype-specific HRPconjugated antibodies. Absorbance at 492 nM was determined in a Dynatech microplate reader.
NOW Ab-79 Ab-85 IgM, K (Vin)
459 650 334 400
i + f +
98,805 18,781 33,446 106,991
180 221 105 187
a Expressed are mean values P valueswere calculated with
+ SD of cpm the help
i f i k
7,660 4,360 18,682 6,805
from
of Student
‘ Ab-79
versus
IgM,
K; t = 8.52,
P < 0.0001.
'Ab-85
versus
IgM,
K; t = 5.55,
P = 0.0005.
three r test
94,443 51,208 60,649 93,800 different
f f + i
10,107
6,639h 9,797’ 9,071
expernnencs.
(two-talled).
Inhibition
119
of T Cell Proliferation
TABLE
3
Soluble rCD4 reverses anti-CD4-mediated inhibition of SEB-induced PBMC proliferation TdR incorporation (cpm f SD)
Condition Medium SEB SEB + Ab-79 SEB + Ab-79 SEB + rCD4
1,840 + 156 128,839 i- 11,106 74,710 + 4,593 104,381 + 6,680 124,827 + 12,064
+ rCD4
chain restricted. In several SLE patients, these antibodies occurred at serum concentrations higher than 50 pg/ml. When used at saturating concentrations, human antiCD4 antibodies strongly suppressed a number of T cell and T cell-dependent B cell-proliferative responses, induced by various polyclonal activators. The same was true when CD4’ T cell response to a tetanus toxoid was analyzed in sensitized persons (data not shown, manuscript in preparation). However, at least one anti-CD4 antibody (Ab-79), when added to cell cultures at subsaturating concentrations, costimulated the T cell proliferation promoted by superantigens/anti_CD3 antibodies. This effect was even more pronounced, when antibody (e.g., Ab-79) was added at the time of priming, or shortly afterward. From the theoretical standpoint, one or several diverse mechanisms could be operative in anti-CD4-mediated inhibition of T cell proliferation: blocking of the CD4I MHC Class II dependent cell adhesion, modulation of the CD4-lateral associations with TCR/CD3, and CD45 on T cells, sequestration or inappropriate signaling through p56Ick and priming for Fas-mediated apoptosis. CD4 molecule synergizes with the TCR in T helper cell activation, by laterally associating with the TCRKD3 complex and probably with CD45 and some other molecules {lo, 131. As a result of this, multireceptor complex is formed, which becomes highly competent to
TABLE
4
IL-2 but not IL-4 partially overcomes the inhibitory effect of anti-CD4 antibody on T cell proliferation induced by anti-CD3 antibodies
Condirion” Medium Anti-CD3 Anti-CD3 Anti-CD3
transduce activation signals. We now show that human anti-CD4 antibodies strongly suppressed the competence phase, but not the progression to DNA synthesis mediated by cytokineicytokine receptor interactions. Our data further suggest that the inhibition was most probably due to interference with CD4-lateral interactions, although additional effects on CD4/MHC Class II interaction and possible sequestration of the pS6kck could be operative too. This could prevent the PSblck, which is cytoplasmically associated with CD4, from phosphorylating the tyrosine residues in the 5 chain of the TCR complex, or by inducing an altered pattern of < chain phosphorylation. Consequently, this could block the activation of ZAP-70 and downstream signaling events, ultimately inhibiting the transcription of the IL-2 gene, and subsequent proliferation of stimulated cells (reviewed in [25]). Since the addition of exogenous IL-2 (but not of IL-4) to competent T cells partially restored the T cell proliferation, induction of reversible functional energy by human anti-CD4 antibodies should be considered as a primary mechanism of their action. We also show that following superantigen/anti-CD3 stimulation, IL-2iIL-4-mediated T cell DNA synthesis and cell proliferation was not sensitive to human anti-CD4 antibodies. There was a window of less than 24 h duration, during which simultaneous exposure to saturating amounts of anti-CD4 antibodies and polyclonal activators resulted in inhibition of PBMC proliferation. The inhibitory effect of anti-CD4 antibodies occurred in a monocyte-independent fashion, indicating a direct suppressive action on T cells. However, some contribution of monocyte-derived factors could not be ruled out completely. Furthermore, in the case of inhibition of PWM-stimulated B cell proliferation, one additional mechanism of immunosuppression induced by anti-CD4 antibodies could be due to direct interference with cellto-cell adhesion that occurs between activated CD4’ T cells and B cells.
Without
+ IL-2 + IL-4
317 68,120 80,792 73,244
f i I +
Abs 129 21,340 14,883 18,818
With 471 22,744 41,482 25,991
Ab-79 f + + +
104 13,888” 17,154 16,679J
With
IgM,
351 68,443 76,661 70,053
f + 2 f
K
(Vin)
49 23,320 18,687 22,905
a Expressed are mean values * SD of cpm from three different experiments. P values were calculated means of Wilcoxon slgned rank test (two-tailed). ’ Without
Abs versus with Ab-79,
P = 0.0039.
’ With
Ab-79
versus with Ab-79
+ IL-2, P = 0.0092.
“With
Ab-79
vetsus with Ab-79
+ IL-4, P > 0.05.
by
120
Several recent studies provided experimental evidence that CD4 cross-linking, in the absence of simultaneous TCR engagement, could prime T cells for activationdependent apoptosis [26-281. This required both functional p56kck and Fas-receptor expression [29, 301. Although we have not addressed this possibility directly, our preliminary experiments on CD4-expressing Jurkat cells (but not on CD4-negative variants) demonstrated that the sole presence of human anti-CD4 antibodies was sufficient to promote significant cell death by apoptosis, after 72 h of culture (data not shown). However, there was no evidence of enhanced cell death after 24-48 h of the culture. Moreover, an additional level of complexity in understanding the principal mechanisms involved in CD4-mediated T cell downregulation was provided by studies showing that anti-CD4 treatment could be beneficial in MRL-mice bearing Irp mutation and not expressing functional Fas receptor. So, anti-CD4 therapy in MRL-/‘Y/I@ mice dramatically reduced the frequency and severity of autoimmune disease, including glomerulonephritis 1311, development of CNS lesions, and arthritis [32). Furthermore, engagement of mutant forms of CD4 that have lost their association with p561ck still resulted in T cell downregulation in vitro 1331. While we have not addressed directly whether any particular Th-cell subset or specific cytokine profile could be preferentially affected by human anti-CD4 antibodies, others have observed that the Thl-type responses and activating memory T cell subsets demonstrate particular susceptibility to the action of murine anti-CD4 mAbs (34-37). Our findings may provide an explanation for some previously observed effects of antilymphocyte antibodies that occur in SLE on T and B cell activation (e.g., inhibitory effect on MLC and on antigen-lmitogen-induced IL-2R expression and lymphocyte proliferation, reviewed in [38}). SLE sera could also promote antigen modulation, inhibit both NK cell function and antibodydependent cell cytotoxicity, and reduce T suppressor function. The presence of these antibodies often correlates clinically with the active disease, neuropsychiatric manifestations, and decreased lymphocyte counts [38, 391. In conclusion, we demonstrate for the first time that human affinity-purified anti-CD4 antibodies, being of both IgG and IgM isotypes, can inhibit T cell proliferative responses by induction of a classical T cell energy. However, it remains to be determined in the future whether human anti-CD4 antibodies induce these effects by differentially affecting the cytokine secretion or, for example, by increasing the consumption of a particular cytokine. Another question, which needs further clarification, is whether human anti-CD4 antibodies may directly prime T cells for activation-induced apoptosis.
G. Lenert and P. Lenert
This will significantly advance our knowledge on pathogenesis of some T and B cell abnormalities that occur commonly in human SLE and in lupus-prone strains of mice. ACKNOWLEDGMENTS
We are grateful
to Drs. M. Zanetti, G. Delespesse, and E. Rasio for support; Dr. J-L. Seneca1 for the sera of SLE patients; Dr. R-P. Sekaly for providing the cell lines; and Dr. C. Demeure for help in FACS analysis. This research was supported in part by a Medical Research Council Invited Scientist Grant (P.L.).
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of sigmol-