CELLULAR IMMUNOLOGY 173, 192–197 (1996)
ARTICLE NO. 0267
Secretion of IFN-g, IL-6, Granulocyte-Macrophage Colony-Stimulating
Factor and IL-10 Cytokines after Activation of Human Purified T
Lymphocytes upon CD38 Ligation
CLARA M. AUSIELLO,*,† ANDREA LA SALA,* CARLO RAMONI,‡ FRANCESCA URBANI,*
ADA FUNARO,§ AND FABIO MALAVASIØ
Departments of *Bacteriology and Medical Mycology and ‡Immunology, Istituto Superiore di Sanita’, 00161 Rome, Italy; †Institute CNR
of Tissue Typing and Dialysis, 67100 L’Aquila, Italy; §Laboratory of Cell Biology, Department of Genetics, Biology, and Medical
Chemistry, University of Torino, Torino Italy; and ØInstitute of Biology and Genetics, University of Ancona, Ancona, Italy
Received May 29, 1996; accepted July 16, 1996
Human CD38, a surface glycoprotein expressed by
different immunocompetent cells, is associated with
distinct transmembrane signaling molecules and plays
a key role in the synthesis of cyclic ADP-ribose, a cal-
cium-mobilizing compound. This study reports that
CD38 ligation by specific monoclonal antibodies (mAb)
in purified peripheral blood T cells is followed by se-
cretion of discrete cytokines. IL-6, granulocyte-macro-
phage colony-stimulating factor (GM-CSF), IFN-g, and
IL-10 mRNA expression were constant findings. Low
levels of IL-2 mRNA were also detected in CD38-acti-
vated T lymphocyte cultures of all subjects studied.
Low levels of IL-4 and IL-5 mRNA were detected in the
majority of CD38-activated T cultures. Moreover, CD38
mediated cytokine induction does not require T cell
proliferation or the addition of antigen presenting
cells. In conclusion, human CD38 runs an activation
pathway in purified T cells which operates through
the induction of a cytokine profile shared by Th1 or
Th2 cells. q 1996 Academic Press, Inc.
INTRODUCTION
Human CD38 is a type II transmembrane glycopro-
tein prevalently expressed by immature and activated
T and B lymphocytes, plasma cells, monocytes, periph-
eral blood NK cells, and, at lower epitope density, by
other cells and tissues (1–6). One of its unique charac-
teristics is the ability to perform as coreceptor, featur-
ing activation and proliferation signals in lymphocytes
(1, 3). It is physically associated (and involved in signal
transduction) with the surface receptors TcR/CD3, sur-
face Ig/CD19, and CD16, which are crucial molecules
for the function of T, B, and NK cells, respectively (6–
10). Second, it has been reported that CD38 mediates
a selectin-like binding to endothelial cells, thus acting
as an adhesion molecule (11).
0008-8749/96 $18.00
Copyright q 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.
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Like other surface receptors, human CD38 can be
shed: indeed, a soluble CD38 molecule was found both
in culture supernatants of activated T lymphocytes and
tumor cell lines and in vivo in biological fluids from
healthy and diseased individuals (12, 13).
Last, CD38 is also a complex bifunctional ectoen-
zyme, catalyzing the formation of cyclic ADP-ribose
(cADPR), a second messenger involved in the regula-
tion of calcium-induced calcium release flux in several
cell systems (14–16). A recently defined ligand for hu-
man CD38 molecule adds further complexity in placing
all these observations within the context of a surface
receptor (17).
Some of the results attributed to CD38 ligation could
be due to factors following immune cells activation via
CD38. In this context, cytokines released after CD38
activation could be responsible for some of the features
attributed to this pleiotropic molecule (1, 3, 6). A previ-
ous study has shown that CD38 triggering induces the
expression of a discrete pattern of cytokine in cultured
peripheral blood mononuclear cells (PBMC) (18). Here
we have evaluated the effects mediated by human
CD38 in purified resting T cells analyzed in terms of
messages and released cytokines. The interplay be-
tween CD38 and its ligand (17) has been simulated in
vitro by means of the specific IB4 mAb, an agonistic
anti-CD38 reagent (19).
MATERIAL AND METHODS
Monoclonal antibodies. IB4 (anti-CD38) and CBT3G
(anti-CD3) mAb were prepared (7, 19) and purified by
affinity chromatography on protein A–Sepharose
(Pharmacia, Uppsala, Sweden) and HPLC or hydroxyl-
apatite (19). The batches of mAb used were endotoxin-
free, as tested by reaction with the endotoxin kit
(Sigma, St. Louis, MO).
Preparation of PBMC and purified T lymphocytes.
PBMC were obtained from heparinized venous periph-
192
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 CD38 LIGATION AND CYTOKINE PRODUCTION IN T CELLS
eral blood samples from healthy donors by centrifuga-
tion on density gradient (Lymphoprep, Nyegaard, Oslo,
Norway). PBMC were washed twice in RPMI 1640 me-
dium (GIBCO BRL, Life Technologies, Inc., Gaithers-
burg, MD) and resuspended at 2 1 106/ml in RPMI
1640 medium supplemented with 5% pooled AB serum
and antibiotics [Penicillin (100 IU/ml), Streptomycin
(0.1 mg/ml); GIBCO], hereafter referred to as complete
medium (18). T cells were purified as previously de-
scribed (7, 20). Briefly, PBMC were deprived of adher-
ent cells by incubation on nylon wool columns, T cells
were then purified by positive selection using AET-
SRBC (experiments 1 and 2) (7) or by immunomagnetic
negative selection with Dynabeads magnetic particles
(Dynal, Oslo, Norway) conjugated with the relevant
mAb to eliminate B lymphocytes (Dynabeads M-450
Pan-B (CD19), monocytes (Dynabeads M-450 CD14),
and NK cells (B73.1 and 3G8 mAb and Dynabeads M-
450 sheep anti-mouse IgG) (experiments 3–6) (20).
Culture conditions and proliferation assays. Cyto-
kine mRNA in cultured cells from PBMC and T lym-
phocytes were measured in the presence of agonistic
mAb, i.e., anti-CD38 (IB4) and anti-CD3 (CBT3G) used
at predetermined optimal doses (20 mg/ml and 50 ng/
ml, respectively) (18). To isolate total RNA, cells were
cultured in 13-ml tubes (Falcon, Becton Dickinson, Lin-
coln Park, NJ) at 2 1 106 cells/ml in 3 ml complete
medium for 18 hr at 377C in a 5% CO2 . Culture super-
natants were collected and used to measure IFN-g, IL-
5, IL-6, and IL-10 cytokine secretion by ELISA tests
(Quantikine, R&D Systems, Minneapolis, MN). ELISA
sensitivities were 1 pg/ml for IL-5, 1.5 pg/ml for IL-
10, and 3 pg/ml for IL-6 and IFN-g. PBMC and T cell
proliferations were measured in triplicate in 2 1 105
cells in 0.2 ml complete medium in 96-well flat-bottom
trays (Falcon). Trays were incubated in 5% CO2 at 377C
and harvested on Day 4, after 18 hr culture in the
presence of 0.5 mCi/well of [3H]thymidine (NEN Du
Pont, Dreieich, Germany). Data are expressed as mean
cpm 1 1003 { SD (18).
Polymerase chain reaction (PCR)-assisted mRNA
amplification. Total cellular RNA was extracted fol-
lowing the guanidium–isothiocynate method (21).
RNA (1 mg in a 20-ml reaction volume) was transcribed
by using Moloney murine leukemia virus reverse tran-
scriptase, as previously reported (22) and following the
manufacturer’s instructions (GIBCO BRL). PCR was
carried out in a 20-ml volume, using 2 ml of cDNA, di-
luted 1:10 (GeneAmpkit, Perkin Elmer Cetus, Nor-
walk, CT) (23). The PCR amplification was carried out
starting from as low as 1 ng of original RNA. Cytokine
specific primer pairs were synthesized according to
published sequences (DNA Synthesizer, Applied Bio-
systems, Inc., Foster City, CA) (24, 25). PCR was per-
formed in a 9600 Perkin Elmer thermal cycler, each
cycle with 40 s denaturation at 947C, 40 s annealing
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193
at 627C, and 1 min extension at 727C. The PCR cycle
numbers for each primer pair, optimized to avoid the
PCR plateau effects (23), were 28 for IL-6, GM-CSF,
IL-2, and IFN-g and 30 for IL-4, IL-5, and IL-10. The
reaction product was visualized by electrophoresis us-
ing 10 ml of the reaction mixture. The specificity of
amplified cDNA sequences was validated by correspon-
dence to predicted size of genes and by restriction map-
ping (25). After electrophoresis, the relative density of
the ethidium bromide stained PCR reaction products
was determined by using a Pharmacia-LKB Ultrascan
XL densitometer. The actual densitometric values for
each cytokine mRNA were normalized, using b-actin
densitometry as the 100% reference and all other val-
ues expressed as the percentage of this figure (23).
Immunofluorescence analysis. Single or double im-
munofluorescence was performed using fluorescein
isothiocyanate (FITC)- or phycoerythrin (PE)-labeled
anti-CD3, anti-CD14, and anti-CD19 mAb (Becton
Dickinson, Mountain View, CA). Cells (5000) were ana-
lysed on a FACScan flow cytometer (Becton Dickinson)
gated to exclude non viable cells.
RESULTS
Flow cytometric analysis and proliferation of puri-
fied T cell populations. The working hypothesis was
that CD38 triggering is followed by a direct induction
of cytokine production in purified T cells, even in the
absence of antigen presenting cells. Table 1 shows
the results of the proliferation of purified T cells and
PBMC, from the six healthy donors studied, in re-
sponse to CD38 or CD3 stimulations. The same table
includes the phenotypic characterization of purified
T cells obtained either by positive selection (experi-
ments 1 and 2) or by negative selection (experiments
3 – 6). Both methods yielded an enrichment in T cells
of approximately 90% (range 88 – 96%). The ability of
CD38 or CD3 triggering to induce proliferation in
purified T cells was correlated to the presence of con-
taminating monocytes in the T cell preparations. For
example, a contamination of 2% of CD14 cells in T
cell preparation gives a 30% residual proliferation
compared to that induced in PBMC. As already re-
ported (7, 18), CD38 stimulation was able to induce
PBMC to proliferate in all the experiments per-
formed. The CD38-stimulated PBMC proliferation
was at least 38 times the proliferation of unstimu-
lated cultures. However, the degree of PBMC prolif-
eration was equal to or lower than that obtained via
CD3 stimulation in the same donor (Table 1).
Comparative analysis of cytokine mRNA expression
in cultured T cells and PBMC. Figure 1 reports a rep-
resentative profile of semiquantitative RT-PCR assays
of the cytokine mRNA expression in cultured purified
T cells and control PBMC challenged with anti-CD38
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194 AUSIELLO ET AL.

TABLE 1
Phenotypic Characterization and Proliferation of Purified Resting T Cells upon Ligation of CD38

T cell proliferationb PBMC proliferationb

Phenotypea [3H]thymidine incorporation [3H]thymidine incorporation
(% positive cells) (cpm { SD 1 103) (cpm { SD 1 103)

Experiment CD3 CD19 CD14 CD38 CD3 Control CD38 CD3 Control

1 95 õ1 1.2 13.3 { 2.1 (32)c 9.6 { 1.1 (23) 0.1 { 0.0 41.0 { 2.8 41.5 { 1.3 0.6 { 0.1
2 95 2 2 12.6 { 0.9 (30) 13.5 { 2.1 (32) 0.1 { 0.0 42.5 { 3.1 42.4 { 2.1 0.6 { 0.1
3 93 õ1 2 9.5 { 3.8 (18) 43.1 { 3.3 (34) 0.1 { 0.0 53.8 { 4.6 125 { 3.3 0.3 { 0.1
4 88 1.5 õ1 0.2 { 0.1 (5) 0.1 { 0.0 (0.0) 0.1 { 0.0 3.8 { 0.3 16.0 { 1.4 0.1 { 0.0
5 93 õ1 0 0.6 { 0.2 (1) 0.8 { 0.0 (0.1) 0.2 { 0.0 41.6 { 2.7 96.2 { 8.5 0.3 { 0.2
6 96 0 0 1.0 { 0.2 (3) 2.5 { 1.1 (7) 0.1 { 0.0 38.1 { 2.5 37.7 { 2.9 0.2 { 0.0
a
Determined by FACS analysis as specified under Materials and Methods.
b
Proliferation was measured after 4-day culture as specified under Materials and Methods.
c
Percentage residual proliferation of T cells as compared to PBMC.

mAb; the control was the analysis of effect induced by
anti-CD3 mAb. These experiments were performed in
cells obtained from the same donor. Figure 2 shows the
cumulative results of the comparative analysis in T
cells and PBMC, performed on cells derived from the
5 individuals included in the RT-PCR analysis. The
data are referred to the intensity of specific transcripts,
expressed as percentage of density relative to that of
b-actin, as determined by densitometry (23). The
mRNA was extracted after 18 hr of mAb challenge,
FIG. 1. Profile of cytokine mRNA expression in CD38-activated
purified T cells and PBMC. Purified T cells and PBMC from a repre-
sentative donor were cultured in the presence of anti-CD38 (20 mg/
ml) or anti-CD3 (50 ng/ml) mAb or unstimulated. After 18 hr, the
cells were harvested and RNA was extracted and reversetranscribed.
The cDNA obtained was assayed for the presence of specific cytokine
mRNA by PCR, as described under Materials and Methods. Reaction
products were run on 1% agarose gel in the presence of appropriate
molecular weight markers (arrow). b-actin was used as the positive
control.
based on previous experience which indicated that the
peak production of the cytokines under analysis is ap-
parent at 6 and 24 hr in culture (18).
CD38-mediated signaling constantly induced all T
cell cultures to express mRNAs for IL-6, GM-CSF, IFN-
g, IL-10, and IL-2, the latter at very low level. IL-4 and
IL-5 were expressed at low levels in the majority of
CD38-activated T cell cultures (four of five and three
of five, respectively). As already reported, CD38 trig-
gering induced all PBMC tested to express IL-6, GM-
CSF, and IL-10, while IFN-g was expressed by PBMC
in the majority of individuals (4/5) activated via CD38.
CD38-activated PBMC of the sample studied displayed
very low levels of mRNA transcripts of IL-2 (4/5), IL-4
(4/5), and IL-5 (3/5).
Comparing relative intensities of the transcripts in
purified T cells and PBMC, it was apparent that CD38-
activated T cells displayed an accumulation of mes-
sages for IFN-g mRNA, while those for IL-6 and IL-10
mRNA were decreased.
After activation via CD3, the messages for GM-CSF,
IFN-g, and IL-10 mRNA were apparent in cultured T
cells of all the donors tested, while the messages for
IL-6, IL-2, IL-4, and IL-5 mRNA were detected in the
majority of the donors (two of three). IL-6, GM-CSF,
IFN-g, IL-2, IL-4, IL-5, and IL-10 were expressed in
CD3-activated PBMC from all the donors tested, con-
firming previous results (18). The analysis of the rela-
tive intensities of the transcripts showed a decrease of
IL-6, IL-2, IL-4, IL-5, and IL-10 mRNA in CD3-acti-
vated T cells compared to that seen in PBMC samples.
No relevant differences were seen in the ability of
CD38 ligation to induce cytokines as compared to CD3
signaling, except for the expression of IL-6 mRNA,
which was more consistent in CD38-activated T cells.
This finding confirms previous experience with PBMC
cultures (18).
Untreated T cell cultures showed a constitutive

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 CD38 LIGATION AND CYTOKINE PRODUCTION IN T CELLS
FIG. 2. Cumulative results of cytokine mRNA expression in puri-
fied T cells upon CD38 ligation. Purified T cells and PBMC from five
donors were cultured in the presence of anti-CD38 (20 mg/ml) or anti-
CD3 (50 ng/ml) mAb or unstimulated. After 18 hr, the cells were
harvested and RNA was extracted and reverse transcribed. The
cDNA obtained was assayed for the presence of specific cytokine
mRNA by PCR, as described under Materials and Methods. Reaction
products were run on 1% agarose gel in the presence of appropriate
molecular weight markers (arrow). b-Actin was used as the positive
control. Data are expressed as percentage absorbance (mean { SEM)
using b-actin as comparative reference.
mRNA expression of GM-CSF and IFN-g even if only
in very low amounts. PBMC, in similar conditions, dis-
played a constitutive expression of IL-6, IFN-g, IL-4,
and IL-10 mRNA in the majority of cultures tested, but
the expression of these cytokines was clearly lower as
compared to the messages implemented by CD38 and
CD3 ligation.
Comparative analysis of cytokine secretion by puri-
fied T cells and PBMC in culture. The secretion of IL-
6, IL-5, IL-10, and IFN-g was measured by ELISA in
supernatants of CD38- and CD3-stimulated purified T
cells from the subjects studied in PCR-assisted mRNA
amplification using PBMC as reference (Table 2). The
supernatants were collected after 18 hr of culture, and
the data are expressed either as picograms per millili-
ter or as the number of positive cultures over the total
cultures examined. One conclusion is that there is a
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195
general concordance in the analyses of the cytokines
evaluated in terms of mRNA expression or release in
the supernatants. IL-6, IL-10, and IFN-g were consis-
tently found in culture supernatants of CD38-activated
purified T cells and the control PBMC. Similar results
were found in CD3-activated T cells and PBMC. IL-5
levels were almost not detectable in supernatants of
CD38-activated T cells or PBMC, but approached the
lower detection limits of the assay in CD3-activated
PBMC.
More striking differences were noted after compara-
tive analysis of released IL-6 and IL-10, cytokines
which are produced by monocytes and T cells. A
marked reduction in IL-6 and IL-10 secretion was in-
deed observed in CD38- or CD3-activated T cells as
compared to PBMC challenged in similar ways. Other
differences included an increased release of IFN-g in
CD38-activated T cells, as compared to PBMC acti-
vated in similar fashions. The comparison with the
same cell preparations in the absence of any ligation
showed that purified T cells display detectable levels
of IL-6 and IFN-g, while PBMC were characterized by
the presence of IL-6 and IL-10 in the supernatants.
DISCUSSION
CD38 stimulation specifically induces purified rest-
ing T lymphocytes to express the messages for cyto-
kines. The profile of cytokines induced in T cells via
CD38 was similar to that observed after CD3 signaling:
one relevant difference was the higher secretion of IL-
6 in T cells, confirming the results obtained in PBMC
(18). Cytokine secretion was characterized by higher
levels of IFN-g in T cell cultures upon CD38-ligation
than in PBMC similarly treated.
This study reveals two important facets of human T
lymphocytes activated via CD38. First, cytokine pro-
duction is not associated with T cell proliferation, indi-
cating a dissociation of the two events. Second, CD38
activation, like CD3 activation, induces a cytokine se-
cretion profile, which includes Th1- and Th2-type cyto-
kines (26).
Approximately 5% of freshly isolated T cells are
CD38/ (6, 13) and these are usually CD45RA/ (11).
Thus CD38 cells are actually mature resting T cells,
which respond poorly in vitro to stimuli acting via TcR/
CD3 (27, 28). The expression of CD38 in T cells is ap-
proximately 10-fold lower than in activated T lympho-
cytes and plasma cells (11).
A possible working hypothesis for explaining the in-
duction of cytokine secretion via CD38 activation of
resting mature T cells stems from similar experiences
derived from CD27–CD70 interaction (29, 30). CD27 is
a transmembrane glycoprotein expressed by peripheral
T cells, medullary T lymphocytes, B cells, and NK cells.
Similar to CD38, CD27 is expressed preferentially in
CD45RA/ subsets of naive CD4/ T cells (29). The ligand
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196 AUSIELLO ET AL.

TABLE 2
Cytokine Release in Purified T Cell upon CD38 Ligationa

CD38 CD3 None

T cells PBMC T cells PBMC T cells PBMC

IL-6 430 { 226 2/2 654 { 151 2/2 172 { 14** 2/2 514 { 30** 2/2 81 { 68 2/2 225 { 174 2/2
IFN-g 1048 { 370 4/4 894 { 402 4/4 766 { 361 3/4 1363 { 124 4/4 83 { 66 2/3 7{6 2/4
IL-5 0 0/2 0.6 1/2 0.3 1/2 4 2/2 0 0/2 0 0/2
IL-10 53 { 33* 2/2 519 { 102* 2/2 99 { 73 2/2 306 { 97 2/2 14 { 5 2/2 79 { 63 2/2
a
Immunoenzymatic assay of cytokine production in supernatants (18 hr) of CD38-activated T cells. PBMC and CD3 activation were
included as the control reference, while background was evaluated by addition of culture medium alone. Data are expressed as pg/ml (mean
{ SEM) and as the number of positive supernatants over total cultures examined.
* Student t test P Å 0.02.
** Student t test P Å 0.004.

for CD27 has been recently identified in the CD70 mole-
cule. CD70 is induced after activation on the CD45RO/
T cell. Morimoto et al. (30) provided evidence for di-
rect cellular communication between CD45RO/ and
CD45RA/ T cell subsets through CD27/CD70 ligation.
The contact is direct and leads to activation of
CD45RA/ T cells, as indicated by the upmodulation of
CD25 (30). In the culture conditions adopted in the
present study, CD38 ligation might mimic the single or
multiple ligands and directly activate CD45RA/ cells,
promoting the activation of CD45RO/ cells.
The secretion of significant amounts of cytokines
upon CD38 ligation either by Th1 (i.e., IL-2, IFN-g) or
by Th2 subsets (i.e., IL-6, IL-10) suggests the broad
activation of the molecule in T cells, a characteristic
shared with CD3. Actually, the ability of a T cell to
secrete Th1 or Th2 cytokines upon a same signal is not
surprising. In fact, it was recently reported that IL-12
is able to directly activate CD45RO//CD30/ cells and
to induce the secretion of IFN-g (31). Nonetheless,
CD30 T cells produce IL-5, which is generally consid-
ered to be a Th2 cytokine, thus indicating that CD30/
cells exhibit properties of both Th1 and Th2 cells (32).
These data coincide with recent views that consider the
clear-cut division of cytokine producing T cells into Th1
and Th2 subsets as an oversimplification (33). There is
strong evidence that a single naive peripheral T cell
can yield progeny of either Th1 or Th2 type, depending
upon the signals received during and after activation
(34, 35).
A constant finding in our studies on cytokine induc-
tion in immunocompetent cells activated via CD38 is
the overinduction of IL-6 gene expression. IL-6 is classi-
cally secreted by Th2 cells and among its many pleio-
tropic effects, IL-6 is also a myeloma cell growth factor
in vitro and in vivo (12). This cytokine is overproduced
in association with a poor prognosis in myeloma pa-
tients. In such patients the differentiation of B cells
into high rate Ig secreting mature plasma cells is im-
paired, likely due to abnormality in IL-6-mediated sig-
nal transduction (12, 36). High levels of soluble CD38
molecules are found in biological fluids of myeloma pa-
tients (12). IL-6 is also reported to play a role in HIV
pathophysiology: increased levels of the cytokine are
detected in the serum of patients (37–39) while in-
creased intratumoral expression of this cytokine is
found in AIDS lymphomas (40). Another symptom in
AIDS patients with poor prognosis is the presence of
high levels of soluble CD38 in the serum (14). Shedding
of CD38 is related to cell activation or interaction with
cytokines, and it can be an indication that this soluble
form serves as a receptor. The finding that elevated
amounts of IL-6 and soluble CD38 molecules are simul-
taneously presents in different pathological situations
can be an indication of a connection between the two
events. However, further studies are needed to confirm
and explain this work hypothesis.
ACKNOWLEDGMENTS
This work was supported by grants from the Special Project ACRO
(Consiglio Nazionale delle Ricerche, Rome, Italy), AIDS and ‘‘Pro-
getto Nazionale Tubercolosi’’ (Istituto Superiore di Sanita’, Rome,
Italy), Telethon (Rome, Italy), and AIRC (Milano, Italy).
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