Proc. Natl. Acad. Sci. USA
Vol. 84, pp. 4205-4209, June 1987
Functional characterization of an antigen involved in an early step
of T-cell activation
(T lymphocytes/monoclonal antibodies/interleukin 1)
M. ELISABETTA COSULICH*t, ANNA RUBARTELLI*, ANGELA RISSO*t, FEDERICO COZZOLINO*,
AND ANTONIO BARGELLESI*t
*Istituto Scientifico Tumori; and tIstituto di Chimica Biologica, Viale Benedetto XV, 10 16132 Genova, Italy
Communicated by Matthew D. Scharff, January 30, 1987
monoclonal antibody MLR3, is described that is present on
activated T lymphocytes and thymocytes but not on resting T
lymphocytes. Immunoprecipitation of radiolabeled mem-
branes from an activated T-cell line showed that the MLR3-
binding molecule has a molecular size of 28-34 kDa. Im-
munofluorescence analysis showed that the appearance of the
MLR3 antigen is an early event and precedes that of the
interleukin 2 receptor both in T lymphocytes and thymocytes.
The proliferative response of resting T cells to OKT3-Sepha-
rose and interleukin 1 or accessory cells, but not the interleukin
2-dependent proliferation, was inhibited by the addition of
MLR3 monoclonal antibodies. Similar results were also ob-
tained in an interleukin 1-dependent human thymocyte prolif-
eration assay. In addition when MLR3-positive cells were
cultured with purified interleukin 1, MLR3 surface antigen
expression was not observed. ThusMLR3 monoclonal antibody
appears to recognize an antigen involved in an early step of
T-cell activation related to interleukin 1-dependent functions
and on both T lymphocytes and thymocytes.
An activation antigen, identified by the
T-cell activation is a multistep process induced by the
interaction of the Ti-T3 complex with the antigen in the
presence of self-major histocompatibility complex-encoded
molecules (1-3). Triggering ofthis complex ultimately results
in cell proliferation mediated by the interaction ofinterleukin
2 (IL-2) with its receptor on the cell surface (4-7). Between
the interaction with the antigen and the IL-2-mediated pro-
liferation, a series of signals are delivered by factors such as
colony stimulating factor, interleukin 1 (IL-1), IL-2, and
interferon-y (8-11). At the same time, anumber ofmembrane
structures with receptor function are expressed on the
surface ofactivated T cells and mediate communication with
the environment (6, 7, 12-15). In antigen-mediated T-cell
activation, the collaboration with accessory cells (AC) (mac-
rophages and B cells) is crucial, at least for the HLA class
II-restricted T cells, since AC provide the system with both
membrane determinants and soluble factors necessary for a
successful completion of the entire T-cell activation process
(3, 16-18). Among soluble factors, IL-1 seems to play a key
role in T-cell activation (19, 20); however, the precise role of
AC membranes and AC-derived cytokines is still debated and
its definition awaits a more subtle dissection (18, 21-24).
Monoclonal antibodies (mAbs) that recognize molecular
structures expressed on T-cell membranes in early phases of
activation and that are able to interfere with theirfunction are
important tools in dissecting the multiple events occurring
after antigen triggering. In this paper, we have studied the
mouse mAb MLR3 that recognizes an antigen present on
activated but not on resting T cells (25). Our data show that
MLR3 mAb is able to inhibit the polyclonal T-cell prolifer-
ation induced by anti-T3 mAb (OKT3) coupled to Sepharose
and by IL-1 or AC but fails to affect IL-2-dependent prolif-
eration of T blasts or T-cell clones. This mAb also inhibits
thymocyte proliferation to suboptimal doses ofphytohemag-
glutinin (PHA) and IL-1. The relevancy of the molecular
structures recognized by the MLR3 mAb in T-cell activation,
and its possible correlation with the IL-1 receptor complex is
MATERIALS AND METHODS
Antibodies. MLR3 mAb (a mouse IgGi) (25) a kind gift
from G. Corte was purified from ascites by 40% ammonium
sulfate precipitation followed by gel filtration with AcA 34
Ultragel. Anti-IL-2 receptor and Leu-11 mAbs were obtained
from Becton Dickinson. OKT3 and OKM1 were purchased
from Ortho Diagnostics. OKT3 and OKM1 cell lines were
obtained from the American Type Culture Collection. Puri-
fied immunoglobulin was derived from ascites as described
above. Purified OKT3 and MLR3 mAbs were coupled to
activated Sepharose 4B (Pharmacia) under sterile conditions
(following the supplier's instructions). BT2/9 (26) (an anti-
HLA class II mAb) was purified from ascites fluid as
described above. Clone 21 (T40), a kind gift of L. Moretta
(Genova) is equivalent to 3A1 mAb (27). A fluorescein
isothiocyanate-conjugated goat anti-mouse antiserum was
prepared as described (28) and used as a second reagent in
indirect immunofluorescence assays.
Cell Preparation. Peripheral blood mononuclear cells
(PBL) were isolated from heparinized blood by standard
Ficoll/Hypaque centrifugation. T cells were separated by
double-rosettes formation with neuraminidase-treated sheep
erythrocytes (29). Nonrosetting cells were either irradiated
with 4000 rad (1 rad = 0.01 Gy) and used as the source ofAC
or cultured at a concentration of106 cells per ml inRPMI 1640
medium supplemented with 10% (vol/vol) fetal calfserum for
48 hr at 37°C. Supernatants were then collected, filtered, and
frozen until used. Rosetting cells were additionally depleted
of monocytes by two cycles of adherence to plastic Petri
dishes (Costar, Cambridge, MA) for 90 min at 37°C. Nonad-
herent cells were recovered and incubated for 1 hr at 37°C
with a mixture of various mAbs (BT2/9 at 10,gg/ml,OKM1
at 10 ,ug/ml, and anti-Leu-llb at 10 ,ug/ml) and rabbit
complement (Cedarlane Laboratories, Homby, ON, Canada)
at a final dilution of 1:3 (vol/vol). By this procedure it was
possible to eliminate activated T cells, monocytes, and large
granular lymphocytes from the cell suspensions. Recovered
cells were >99% OKT3 positive, as determined by immuno-
fluorescence. Thymuses were obtained from children 6
Abbreviations: AC, accessory cells; mAb, monoclonal antibody;
IL-1 and IL-2, interleukin 1 and 2, respectively; PHA, phytohemag-
glutinin; PBL, peripheral blood mononuclear cells; PMA, phorbol
myristate acetate; pIL-1, purified IL-1.
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Immunology: Cosulich et al.
months to 3 years of age undergoing cardiac surgery. A
single-cell suspension was obtained avoiding contamination
with peripheral blood cells. PHA-stimulated lymphoblasts
(PHA blasts) were obtained from PBL (106 cells per ml)
cultured for 6 days at 370C in 24-well plates (Costar) with
PHA-P (Difco) at a final dilution of 1:1000.
Cell Cultures. To evaluate the proliferative response, cells
were cultured in triplicate in 96-well flat-bottomed plates
(Costar) containing RPMI 1640 supplemented with 10%
(vol/vol) fetal calf serum, 1:100 dilution of nonessential
amino acids, 2 mM L-glutamine, and gentamycin at 50 ug/ml.
Cultures were incubated at 370C in a humidified atmosphere
of 95% air/5% CO2. The allogeneic mixed lymphocytes
reaction was performed as described (28). Briefly, 105 PBL
per well were cultured for 7 days with 4 x 104 4000-rad
irradiated, allogeneic non-T cells. PBL (105 cells per well)
were stimulated with PHA-P diluted 1:1000 (final dilution).
Six-day PHA blasts were cultured at 5 x 104 cells per well
with recombinant IL-2 at 25 units/ml (Genzyme, Norwalk,
CT) for 3 days. Four IL-2-dependent T-cell clones (a kind gift
ofB. Parodi, Genova) were cultured for 3 days at 104 cells per
well with 25 units ofrecombinant IL-2 (Genzyme). Resting T
cells, purified as above, were cultured at 105 cells per well for
3 days with or without OKT3 coupled to Sepharose (1:100,
final dilution) in the presence or absence of 5 units/ml of
purified IL-1 (pIL-1) (Genzyme) or 2 x 104 irradiated autol-
ogous non-T cells or 25% (vol/vol) AC supernatant.
Thymocytes were cultured for 3 days at 5 x 105 cells per well
and stimulated with PHA-P at a final dilution of 1:7500 in the
presence or absence of pIL-1 at 5 units/ml. The effect of
MLR3 mAb or control mAb on the proliferative response of
cells in the various assays described above was studied by
adding various doses ofMLR3 or T40 mAbs at the beginning
of culture (in triplicate), as detailed above. Proliferative
response was evaluated by adding 1 uCi (1 Ci = 37 GBq) of
[3H]thymidine (Amersham) to each well during the last 16 hr
of cultures. [3H]Thymidine incorporation was assessed by
harvesting cells onto glass fiber filters and measuring the
radioactivity in a liquid scintillation counter.
Flow Cytometric Analysis. PBL, thymocytes, or resting T
cells (106 cells) were cultured in 24-well plates (Costar) for
various periods oftime in the presence or absence ofPHA-P
at a 1:1000 dilution or OKT3-Sepharose at a 1:100 dilution.
Jurkat cell line (obtained from L. Moretta, Genova) was
stimulated under the same culture conditions with PHA-P
diluted 1:1000 alone or PHA-P and phorbol myristate acetate
(PMA) at 30 ng/ml (Sigma) or PHA-P and pIL-1 at 15
units/ml for 16 hr. Cells were then stained with MLR3 or
anti-IL-2 receptor mAbs in the presence of human immuno-
globulin to inhibit the binding to Fc receptors, followed by
fluoresceinated goat anti-mouse immunoglobulin antiserum
(28). Control aliquots were incubated with the fluorescent
antibodies alone. All samples were then analyzed on a flow
cytometer (Ortho Spectrum III) gated to exclude nonviable
Membrane Radioiodination and NaDodSO4/PAGE. Mem-
brane radioiodination was carried out according to a modi-
fication of the technique of Marchalonis et al. (30) and
Rubartelli et al. (31). Briefly, 107 viable cells were washed
four times in 10 ml ofPBS (0.02M sodium phosphate, pH 7.2)
and resuspended in 250Alof PBS; subsequently, 50 ,ul of
lactoperoxidase at 2 mg/ml (Sigma) and 0.3 mCi of carrier
free 1251 (Amersham) were added. At 10-min intervals, 20 ,ul
of 0.03% H202 was added three times. Labeled cells were
washed three times in cold PBS and lysed with 1% Triton
X-100 (final dilution). Labeled samples were first incubated
with normal mouse immunoglobulin coupled to Sepharose
and subsequently with protein A-Sepharose (Pharmacia) to
remove any nonspecific binding. Samples were then incu-
bated with MLR3 or BT2/9 mAb at 10 ,g/ml as a control
followed by goat anti-mouse immunoglobulin at 10,ug/ml and
protein A-Sepharose for 1 hr. Immunoprecipitates were
washed three times in PBS containing 1% Triton X-100 and
eluted by boiling in Laemmli sample buffer with 1%
NaDodSO4 and 5% (vol/vol) 2-mercaptoethanol for 2 min.
NaDodSO4/PAGE was performed by a modification of the
method ofLaemmli as described (31, 32). Gels were dried and
autoradiographed using Trimax XD film (Ferrania 3M,
Genova, Italy). 14C-labeled marker proteins, phosphorylase b
(92.5 kDa), bovine serum albumin (69 kDa), ovalbumin (46
kDa), and carbonic anhydrase (30 kDa), were obtained from
New England Nuclear.
Immunofluorescence Analysis ofMLR3 Surface Expression.
It has been reported (25) that MLR3 mAb recognizes struc-
ture(s) present on the membrane of activated but not resting
T cells. Therefore, resting and PHA-stimulated PBL or
thymocytes were used in atime-course experiment to analyze
the kinetics of appearance of MLR3 antigen in comparison
with the expression of IL-2 receptor. Confirming the data
(25), both resting PBL and thymocytes did not express MLR3
antigen on their surface (Fig. 1 A and B); however, MLR3-
positive cells steadily began to appear in the same cell
population during PHA stimulation. After 18 hr of culture,
the number of MLR3-positive cells was higher than that of
IL-2 receptor-positive cells; after48 hr, the numberofMLR3-
and IL-2 receptor-positive cells was similar; and after 64 hr,
IL-2 receptor-positive cells still increased while MLR3-
thymocytes cultured with PHA (1:7500, final dilution) (B), and T cells cultured with OKT3-Sepharose (1:100, final dilution) (C). A monoclonal
antibody IgG1 specific for human IgM has been used as negative control. e, MLR3; *, IL-2 receptor. The data are the mean + SEM of four
Time-course analysis of MLR3 antigen and IL-2 receptor appearance on PBL cultured with PHA (1:1000, final dilution) (A),
Proc. Natl. Acad. Sci. USA 84(1987)
Proc. Natl. Acad. Sci. USA 84 (1987)
receptor (D-F) antigens on Jurkat cells unstimulated (A and D) or
stimulated for 20 hr with PHA (B and E) or PHA and PMA (C and
F). The percentages of positive cells were as follows: A, 5%; B,
68.5%; C, 98.5%; D, 0%; E, 1.3%; F, 25.4%.
Immunofluorescence analysis of MLR3 (A-C) and IL-2
positive cells were stationary
decreased (Fig. 1 A and B). To further characterize the
stimulus needed for triggering the expression of MLR3
antigen, purified resting T cells were stimulated with OKT3-
Sepharose and analyzed at different intervals for MLR3
antigen and IL-2 receptor (Fig. 1C). A time-related increase
in the percentage of MLR3-positive cells was recorded; in
contrast IL-2 receptor-positive cells only reached 10%, a
finding in agreement with reports indicating (20, 21) that
T3-cross-linking by itself is not sufficient to induce the
appearance of IL-2 receptor on resting T cells. Further
evidence for the difference between MLR3 antigen and IL-2
receptor came from immunofluorescence analysis of the
Jurkat cell line. In the absence of stimuli, 5% of the cells
expressed the MLR3 antigen at low density but no IL-2
receptor (Fig. 2 A and D). After PHA stimulation, MLR3-
positive cells sharply increased, while few, if any, IL-2
receptor-positive cells appeared (Fig. 2 B and E). By con-
trast, stimulation by PHA and PMA resulted in the expres-
sion ofboth antigens on a large number of cells (Fig. 2 C and
F). To exclude a structural identity between MLR3 and HLA
or, in some experiments,
class II antigens, activated T cells were incubated with a
rabbit anti-human HLA class II antiserum followed by a
fluorescein isothiocyanate-conjugated sheep anti-rabbit im-
munoglobulin antiserum, in the presence or absence of an
excess ofunlabeledMLR3 orBT2/9 mAb. While BT2/9mAb
(that recognizes a common epitope on DQ, DR, and DP
molecules) reduced the immunofluorescence staining, no
inhibition was obtained with MLR3 mAb (data not shown).
The above data suggest that MLR3 antigen is distinct from
both IL-2 receptor and class II antigens and that, following
activation, its expression precedes that of IL-2 receptor.
Functional Analysis oftheMLR3 Antigen. Since the kinetics
of surface expression of the MLR3 antigen could suggest its
involvement in the process ofT-cell activation, the effects of
MLR3 mAb on T-cell proliferation were investigated in
different experimental systems. Highly purified resting T
cells were stimulated by allogeneic non-T cells or by OKT3-
Sepharose in the presence or absence of AC supernatant or
pIL-1 or with autologous non-T cells; the effect of MLR3
mAb on their proliferation was evaluated. The addition of
MLR3 mAb caused a marked reduction, or a complete
abrogation, of the response (Table 1). Control cultures with
the T-cell lineage-specific mAb T40 or with mouse IgGl
yielded negative results (data not shown). By contrast the
IL-2-dependent proliferation ofT-cell clones or ofshort-term
T-cell lines from PHA stimulation was totally unaffected by
the addition of MLR3 mAb. Since immunofluorescence
staining had shown that a proportion of thymocytes ex-
pressed the MLR3-binding molecule following PHA stimu-
lation, an IL-i-dependent thymocyte proliferation assay was
used to investigate the role ofthe MLR3-binding molecule in
another T-cell activation system. Table 2 shows that MLR3
mAb inhibited the thymocyte proliferation stimulated by
PHA and IL-1, while the T-lineage-specific control mAb T40
was completely uneffective. In these experiments the MLR3-
binding molecule appeared to be involved in IL-i-dependent
processes; therefore, to rule out the possibility that MLR3
mAb reacts with soluble IL-1, purified IL-1 was extensively
absorbed with MLR3 mAb coupled to Sepharose. Table 2
shows that, following absorption with MLR3-Sepharose, the
IL-1 activity was completely unaffected.
Modulation ofMLR3 Antigen on Jurkat Cells Cultured with
IL-1. Jurkat cells were activated by culturing cells with PHA
overnight in the presence or absence of pIL-1 and tested by
flow cytometric analysis for MLR3-antigen expression. Sixty
Effect of the MLR3 mAb on T-cell activation
[3H]Thymidine incorporation, cpma
42,580 ± 4,860
23,514 ± 2,928
61,684 ± 5,520
31,829 ± 3,025
38,225 ± 3,150
75,240 ± 6,230
5,320 ± 2,832
6,847 ± 1,484
36,480 ± 2,816
78,312 ± 7,410
Resting T lymphocytesb
Allogeneic non-T cellsc
aData are mean±SD of triplicate cultures from four experiments.
bPurified T cells (101 cells) were cultured for 3 days in the presence or absence of MLR3 mAb at 25
cAilogeneic non-T cells (4 x 104 cells) were irradiated and cultured with 105 resting T cells for 6 days.
dOKT3-Sepharose (T3-Seph) was added to each well at final dilution of 1:100.
epIL-1 was at 5 units/ml, final dilution.
fAutologous non-T cells (104 cells) were added.
gUnstimulated normal macrophage supernatant (MO) at 25% (vol/vol).
hT cells (104 cells) from a representative T-cell clone plus IL-2 at 25 units/ml were cultured for 3 days.
T blasts (104 cells) from 6-day PHA cultures and IL-2 at 15 units/ml were cultured for 3 days.
Immunology:Cosulich et al.
Proc. Natl. Acad. Sci. USA 84 (1987)
Effect of MLR3 mAb on thymocytes proliferation in response to PHA and IL-1
[3H]Thymidine incorporation, cpm
Thymocytes (5 x 105 cells) were cultured for 3 days. [3H]Thymidine (1 ,Ci per well) was added during the last 16 hr of
cultures. Purified MLR3 or T40 mAbs (25 ,ug/ml, final dilution) were added. PHA-P was added at a final dilution of 1:7500.
Purified IL-1 was at 5 units/ml, final dilution. Data are the mean of triplicate cultures. SD was always ±15%. pIL-1
MLR3-ads., pIL-1 was extensively adsorbed with MLR3 mAb coupled to Sepharose before the addition to the cultures.
ND, not determined.
percent of the Jurkat cells were MLR3-positive after activa-
tion with PHA alone (Fig. 3A); by contrast, pIL-1 (Fig. 3B)
but not recombinant IL-2 (Fig. 3C) caused a dramatic
reduction in the number of positive cells. These results
suggest that IL-1 interferes with the expression of MLR3
antigen in activated Jurkat cells.
Biochemical Characterization of the MLR3-Binding MQle-
cule. Characterization ofthe MLR3 molecule was carried out
by membrane radioiodination of Jurkat cells, cultured in the
presence or absence of PHA and PMA, followed by immu-
noprecipitation with MLR3 mAb coupled to Sepharose and
NaDodSO4/PAGE. The molecule immunoprecipitated by
MLR3 mAb from activated Jurkat cells was detected as a
broad band with the following two major components: a
majorband of28 kDa and a minorband of34 kDa (Fig. 4, lane
2). By contrast MLR3 mAb failed to precipitate the 28- or
34-kDa band or any other distinct, labeled material from
membranes ofuntreated Jurkat cells (Fig. 2, lane 1), afinding
in agreement with the low number of slightly positive,
untreated cells seen by immunofluorescence. No bands were
immunoprecipitated by a control mAb (anti-DR, data not
It is generally accepted that resting T cells activated by
triggering of Ti-T3 complex require signals from accessory
cells to undergo proliferation (3, 16-20). Whether AC mainly
provide soluble factors or other membrane determinants are
crucial for an optimal T-cell activation is still a matter of
debate. Furthermore, the precise role of IL-1 is also contro-
versial: While it is accepted that IL-1 induces IL-2 production
by lectin-stimulated T cells (33, 34), it is notcleqrt hether
cross-linking of Ti-T3 complex is sufficient for driving the
expression ofIL-2 receptor (35, 36) orwhether IL-1 is needed
(19, 20). The thorough dissection ofthe functional ^events that
lead to T-cell proliferation has been impeded so far by the
lack ofknowledge ofIL-1 receptors and related structures on
sion on Jurkat cells after stimulation with PHA in the absence (A) or
presence (B) of pIL-1 at 15 units/ml or recombinant IL-2 at 25
units/ml (C). The percentages of positive cells were as follows: A,
59.5%; B, 7.2%; C, 57.5%.
Immunofluorescence analysis of MLR3 antigen expres-
T lymphocytes. This report describes amAb that identifies an
antigen expressed by activated but not resting human T
lymphocytes and thymocytes that affects the proliferative
response ofT cells in various experimental systems known to
require IL-1. Immunofluorescence analysis ofMLR3 antigen
shows that this molecule is not related to the IL-2 receptor
because the appearance of MLR3 molecule on the T-cell
surface always precedes that of the IL-2 receptor; further-
more, functional characterization showed that MLR3 mAb is
able to inhibit T-cell proliferation both in allogeneic mixed
lymphocyte reaction and in PHA stimulation but not in
IL-2-dependent systems, suggesting its involvement in some
early step of T-cell activation. To clarify this point, we
examined the role ofMLR3 antigen in the OKT3-Sepharose
activation ofT cells. Our data clearly show that MLR3 mAb
is able to abrogate the proliferative response ofresting T cells
to OKT3-Sepharose and IL-1, indicating that the MLR3
molecule is an activation antigen that could be related to the
IL-1 receptor. It is worth stressing that although IL-1 can
bypass accessory cell requirement, the degree of prolifera-
tion supported by non-T cells was always higher than that
obtained with IL-1 alone, possibly due to the clustering ofthe
reacting cells (23, 24). The putative role of MLR3 molecule
in IL-1 function is indicated also by the evidence that this
structure is expressed very early after PLiA stimulation on
thymocytes and that MLR3 mAb is able to inhibit the
IL-1-dependent proliferation of PHA-treated thymocytes.
Further support to this hypothesis comes from the observa-
tion that incubation of MLR3-positive Jurkat cells with IL-1
leads to the disappearance of the antigen from the cell
membranes, as shown by immunofluorescence. Whether
IL-1 causes a modulation of MLR3-binding molecule, pos-
sibly by internalization or by shedding or else directly
or PHA- and PMA-stimulated (lane 2) Jurkat cells (107 cells) were
membrane radioiodinated, and cell lysates were immunoprecipitated
with MLR3 mAb and electrophoresed on a 10% NaDodSO4/poly-
acrylamide gel under reducing conditions. Molecular size markers
are indicated on the left.
Characterization ofMLR3 antigen. Unstimulated (lane 1)
Immunology:Cosulich et al.
Proc. NatL. Acad. Sci. USA 84 (1987) Download full-text
competes with MLR3 mAb binding to the cell membrane
remains to be elucidated. The partial characterization of the
molecule(s), carried out by membrane radioiodination,
showed that MLR3 mAb immunoprecipitates a broad band
with two major components of28 kDa and 34 kDa. This band
represents different degrees ofglycosylation ofa single sharp
22-kDa band (unpublished data). The possible correlation of
the MLR3 antigen with HLA class II product was excluded
by the following evidence: (i) Lack ofcross-reaction between
MLR3 and HLA class II antigens demonstrated by the
inhibition of immunofluorescence staining on activated T
cells. (ii) Lack of expression of class II antigen by activated
Jurkat cells (37) that are positive for MLR3 antigen and, by
contrast, lack of expression of MLR3 antigen on resting B
cells that are strongly stained by anti-HLA class II antibody
(data not shown). Our data show that MLR3 is an antigen that
is distinct from other activation molecules, to our knowledge,
so far characterized. Although a structural correlation be-
tween the MLR3 molecule and the IL-1 receptor has not been
established, the interference of MLR3 mAb with IL-1 func-
tions suggests that the MLR3 antigen is strictly involved in
IL-1-dependent processes of both peripheral blood T lym-
phocytes and thymocytes. The Mr ofthe MLR3 antigen is not
apparently in agreement with the Mr ofthe described putative
IL-1 receptors (38-40), but the various experimental systems
used may account for the discrepancies in that the cross-
linkers, which are generally used, could link different mole-
cules. However, the heterogeneity observed in the Mr ofIL-1
receptor by different investigators delineate the complexity
of this receptor molecule.
These data suggest that MLR3 antibody identifies an
activation molecule involved in IL-1-dependent processes on
both peripheral blood T lymphocytes and thymocytes and
that MLR3 mAbs will be a useful tool for analyzing the early
events of T-cell activation related to IL-1 function.
We thank the department of cardiosurgery of the Ospedale Gaslini
for providing us thymuses; Drs. R. Franchini and R. Bologna, from
the Centro Trasfusionale of the Ospedale Gaslini, for the use of the
fluorescence-activated cell sorter; Dr. B. Parodi for providing us with
T-cell clones; and Dr. L. Moretta for the kind gift of mAbs. We also
thank Drs. G. Corte, C. E. Grossi, and R. Sitia for helpful discussion.
We are grateful to Ms. G. Tirelli for secretarial assistance. This work
was supported in part by grants from the Consiglio Nazionale delle
Ricerche, Progetto Finalizzato Oncologia (59/85.02018.44) and from
the Progetto Finalizatto Ingegneria Genetica e Basi Molecolari delle
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