Immunobiol., vol. 188, pp.

145-158 (1993)

lDepartment of Medical Biochemistry, University of Antwerp, Wilrijk, 2Laboratory of

Pathology and Immunology, Institute of Tropical Medicine, Antwerp, 3Eurogenetics, Tessen-
derlo, 4Dr. Willems Institute, Diepenbeek, Belgium, and 5 Istituto di Tumori, Genoa Univer-
sity, Genoa, Italy

Antibody Binding Profile of Purified and Cell-Bound CD26.

Designation of BTS/9 and TAS.9 to the CD26 Cluster

INGRID DE MEESTER 1, SIMON SCHARPE 1, GUIDO V ANHAM 2 , EUGENE

BOSMANS 3 , HARlE HEYLIGEN\ GREET VANHOOF 1 , and GIORGIO CORTES

Received October 6, 1992 . Accepted in Revised Form January II, 1993

Abstract

The CD26 activation antigen (A g) which is expressed on a subpopulation of human T cells

has been characterized as dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5). In this paper, we
describe the antibody binding profile of CD26/DPP IV, purified from human peripheral blood
lymphocytes. The purified molecule binds to the anti-Tal, anti-IF7 and anti-134-2C2 mono-
clonal antibodies (mAb), reported to react with cell-bound CD26 Ag. Among unclustered
mAb recognizing T cell antigens, two, anti-BTS/9 and anti-TAS.9 were found to react with
purified and cell-bound CD26 Ag. The classification of the BTS/9 Ag, the functional
properties of the BT5/9+ T cell subset, as well as the in vivo effect of anti-BTS/9 mAb
administration, are re-interpreted in the light of its specificity.
Applying the anti-TAS.9 mAb in three color FACS analyses, we demonstrated that
CD26+bright cells co-express CD45RO but not HLA-DR and CD38.

Introduction

A multiplicity of plasmamembrane proteins is involved in T cell activa-

tion. In addition to the classical T cell receptor-CD3 (TcR-CD3) signal
triggering, various surface molecules have been demonstrated to be
involved in co-stimulation.
One of these, the CD26 molecule, has been identified as the ectoenzyme
dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5), a highly specific serine-type
protease (1-3). It cleaves N-terminal dipeptides from peptides when a
proline or alanine resides at the penultimate position. By far the highest
efficiency is observed with the proline residue (4). The search for the
physiological role of lymphocytic DPP IV was encouraged by the observa-

Abbreviations: Ag = antigen, Can A = concanavalin A; DPP IV = dipeptidyl peptidase IV;

LCA = Lens culinaris lectin; mAb = monoclonal antibody; 4-Me-2-NA = 4-methoxy-2-
naphthylamine; PBL = peripheral blood lymphocyte; PBMC = peripheral blood mononuc-
lear cell; RCA/2o = Ricinus communis agglutinin 120; TcR = T cell receptor; WGA = Wheat
Germ ;lgglutinin
146 . 1. DE MEESTER et al.

tion that a remarkable number of cytokines and growth factors contains a

proline or alanine at its penultimate N-terminal position (5). These peptides
include the interleukins 1B, 2, 3, 5 and 6, granulocyte-macrophage-CSF,
tumor necrosis factor B, erythropoetin, insulin-like growth factor I and
growth hormone releasing hormone. Some of them have been demonstrated
to be cleaved by DPP IV (6,7). The CD26/DPP IV structure is involved in
cellular adhesion to the extracellular matrix and the molecule mediates
collagen-dependent T cell function (8-11).
The involvement of DPP IV /CD26 in T cell activation and proliferation
was proved by different experimental approaches (12) often using mAb
(13-18) and DPP IV specific inhibitors (19-22). Recently, direct evidence
for an integral role of the CD26 Ag in T cell activation was provided by
TANAKA et aI., who cloned the CD26 Ag and transfected a CD26 negative
human leukemia T cell line with a CD26 expression plasmid (23).
The availability of purified antigen and of a panel of well characterized
mAb, directed against distinct epitopes on this large membrane protein
remain important tools in studying the function of CD26. We have purified
and characterized the natural CD26/DPP IV from human peripheral blood
lymphocytes. In this study, we investigated the antibody binding profile of
the purified and cell-bound CD26 Ag, showing that BT5/9 (24) and TA5.9
(25) belong to the CD26 cluster of activation antigens. In addition, it was
demonstrated that the CD26+bright T cells co-express CD45RO, but not
HLA-DR and CD38.

Materials and Methods

Monoclonal antibodies
The production of anti-TA 5.9 (LYI2.CCl, IgG1) was described before (25). Shortly, the
clone LYI2.CCI was derived from the hybridization of mouse SP2/0 myeloma cells with
spleen cells of BALB/c mice immunized with phytohaemagglutinin stimulated peripheral
blood mononuclear cells (PBMC). The anti-BT5/9 (IgG3) mAb was obtained by immunizing
mice with human T cells activated in a secondary mixed lymphocyte culture and fusing the
spleen cells with the mouse myeloma P3X63Ag8Ul.
MAb reacting with the lymphocyte surface Ag CD3, CD4, CD8, CD25 (anti-Tac), CD38,
CD45RO, HLA-DR, Ta1(4EL-1C7, IgGl), IF7 (IgG1), Tp103 (CB1, IgG1), CD26 (134-
2C2, IgM) were used in these studies. They were extensively characterized before (17, 18,
26-28). MAb against CD3, CD4, CD8, CD38, CD45RO, and HLA-DR were purchased from
Becton Dickinson, Erembodegem, Belgium, and anti-Tal mAb was from Coulter Immunol-
ogy, Hialeach, FL, USA. The mAb 134-2C2 was a gift from Dr. O. VINAS (Hospital Clinic,
Barcelona, Spain). The anti-lF7 and anti-TpI03 mAb were provided by Dr. C. MORIMOTO
(Dana-Farber Cancer Institute, Boston, MA, USA) and Dr. M. HEGEN Q.-Gutenberg-
Universitat, Mainz, Germany), respectively. The anti-Tac mAb was kindly provided by Dr.
TH. WALDMAN (NIH, Bethesda, MD, USA).

Enzyme .1ssay
DPP IV activity was determined using the fluorogenic substrate Gly-Pro-4-methoxy-2-
naphthylamide (Gly-Pro-4-Me-2-NA) (29). One unit of enzyme activity was defined as the
amount of enzyme catalyzing the formation of 1 [.tmol of assay-product per min under assay
conditions.
 CD26 Antibody Binding . 147

Dipeptidyi peptidase IV purification

DPP IV was purified from peripheral blood mononuclear cells essentially as described
before (30). The following modifications were introduced: The active fractions of the initial
anion exchange step were pooled and applied at a flow rate of 0.2 mllmin, onto a series of two
immobilized metal-ion-affinity columns (Chelating Sepharose 6B), the first loaded with Zn2+
(6 mg ZnCb/ml chelating gel) and the second loaded with Cu 2+ (4 mg CUS04' 5H 2 0/mi
chelating gel). After sample loading, the Zn2+ -column, which did not bind DPP IV, was
disconnected. Recovery of DPP IV from the Cu 2+ column was as described. Ricinus communis
agglutinin affinity chromatography replaced the previously used concanavalin A-based separa-
tion. Elution of bound proteins was performed with 0.20 molillactose.
The final ion exchange step was carried out on a HR SIS Mono Q column equilibrated with
20 mmolll Tris-HCI, pH 7.4, at a flow rate of 1 mllmin. Elution was performed with a linear
gradient of NaCI.

Binding of DPP IV to immobilized mAb

Before starting the binding experiments, the influence of the mAb on the enzymatic activity
of DPP IV was evaluated by preincubation of DPP IV with different concentrations of each of
the mAb.
The optimal dilution factor for subsequent tests was estimated by dilution of the ascites or
supernatant preparations in phosphate-buffered saline (111; 1110; 11100; 1/1000) before
spotting onto the nitrocellulose membrane. Immobilization of 10 [!I of a 1110 dilution resulted
in optimal signals, while control antibodies remained negative. An exception was made for
anti-1F7, where we used a 11100 dilution as proposed by Dr. C. MORIMOTO.
The mAb were spotted directly onto a strip of nitrocellulose (Bio-Rad, Richmond, CA,
USA) and allowed to air dry at room temperature for 15 min. The membranes were then
blocked for 1 h at 37°C in 3 % (w/v) BSA in Tris-HCI 20 mmolll, pH 7.4, and washed with
1 molll NaCI in the same buffer. Incubation with 300 [!I of a purified DPP IV solution was
carried out for 1 h at 37°C and overnight at 8°C. After washing as described above, the strip
was brought into 475 [!I Tris-HCI SO mmolll, pH 8.3, supplemented with 25 [!I of the substrate
Gly-Pro-4-Me-2-NA (final concentration 2 mmolll) to measure bound DPP IV activity. At
different time intervals, a 25 [!I sample was taken from the incubation vial, and diluted in 500 [!I
citrate (100 mmol/l citrate pH 4.0) to stop the enzymatic activity. Fluorescence was measured
at 340 and 425 nm for excitation and emission wavelength, respectively. The fluorescence
intensities resulting from overnight incubation at 8°C proved to be the most reliable values for
comparison of DPP IV binding to the different mAb.
The influence of the mAb anti-BT5/9 and anti-TA5.9 on enzyme binding by the anti-Tal,
134-2C2, and anti-1F7 mAb was investigated by preincubating the DPP IV preparation with
anti-BT5/9 ascites or anti-TA5.9 (10 % v/v) during 1 h at room temperature. As controls, the
blanks 1 and 2, we used a mouse ascites and purified mouse IgG 1 of irrelevant specificity. The
further procedure was as described above. To test the influence of different concentrations
anti-BT5/9 mAb on the binding of lymphocytic DPP IV onto immobilized anti-TA5.9 mAb,
purified lymphocytic DPP IV was preincubated during 1 h at room temperature with 10 %
(v/v) of different anti-BT5/9 or control ascites dilutions (from 112 to 1/100) prior to incubation
with immobilized anti-TA5.9 mAb.
The ability of different lectins [Wheat Germ Agglutinin (WGA), Lens cuiinaris lectin
(LCA), and Ricinus communis agglutinin 120 (RCA 120 )] to compete with the mAb for binding
of DPP IV was investigated. Partially purified DPP IV was preincubated with different
concentrations (0.1-100 [!g/ml) of the respective lectins during 1 h at room temperature.
Afterwards the binding to the immobilized anti-TA5.9 mAb and anti-BT5/9 mAb was carried
out as described.

Binding of mAb to immobilized DPP IV

Native and SDS denatured preparations of lymphocytic DPP IV (5-10 [!I) were spotted
directly onto nitrocellulose and allowed to air dry for 15 min. The following monoclonal
antibodies were tested for binding (incubation overnight, 8°C) in a 11100 dilution: anti-Tal,
148 . I. DE MEESTER et al.

anti-TA5.9, anti-BT5/9, 134-2C2, CBl, anti-lF7. Blocking was performed by immersing the
membrane in a solution of dried skim milk powder (5 % w/v in tris-buffered saline) for at least
1 h at room temperature, with rocking. In the control experiment, we omitted the first
antibody during the procedure, while all other steps were carried out as described. Biotiny-
lated F(ab)2 anti-mouse Ig (Dakopatts, Copenhagen, Denmark) was used as second antibody.
The incubation with avidin and biotinylated horseradish peroxidase (Dakopatts) was carried
out during 50 min at room temperature.
After each step, the membranes were washed three times in tris-buffered saline. Bound
horseradish peroxidase was detected by a chemiluminescence technique, using «ECL Western
blotting reagents» (Amersham International, Amersham, UK). All steps of the procedure were
carried out in the dark room. The incubation was performed for precisely 1 min at room
temperature. The membrane was exposed to Hyperfilm-ECL autoradiography film (Amers-
ham International).

Immunofluorescence
Two and three color experiments were performed on whole blood with a FACScan (Becton
Dickinson) using Lysis I software. In two color experiments, anti-TA5.9 FITC was combined
with anti-CD4 phyco-erythrin (PE) or anti-CD8 PE. In standard three color experiments,
anti-TA5.9 FITC was combined with anti-CD4 peridin-chlorophyll-protein or anti-CD8
peridin-chlorophyll-protein and anti-CD45RO-PE, anti-CD38-PE or anti-HLA-DR-PE.
Three color experiments with anti-Tac were performed as follows. First 0.1 ftg unconjugated
anti-Tac was incubated with 50 ftl whole blood. The blood was washed after 30 min and 1 ftl
F(ab)2 goat anti-mouse PE was added for another 20 min. After a second wash-cycle and
blocking of free binding sites on cell-bound goat anti-mouse with 5 ftl mouse serum, anti-
TA5.9 FITC and anti-CD4 peridin-chlorophyll-protein or anti-CD8 peridin-chlorophyll-
protein were added. After appropriate incubations, whole blood was washed, erythrocytes
were lysed and white cells were fixed.

Results

Purification of dipeptidyl peptidase IV

The CD26/DPP IV preparation used in this study was enriched 8000-
fold from PBMC and exhibited a specific activity of 55 U/g protein.
Lymphocytic DPP IV showed a high affinity for Ricinus communis12o
(RCA 120 ) and Lens culinaris agglutinin. Therefore, we included the former
in our purification scheme. After electrophoresis and subsequent silver-
staining, three bands could be visualized, one small band at 179 000, and
two bands corresponding to relative molecular masses of 120000, and
100000 respectively. In this electrophoresis system, DPP IV migrates at a
Mr of 100000. Some biochemical properties of lymphocytic DPP IV/CD26
were reported before (30-32).

Binding between purified DPP IV/CD26 antIgen and monoclonal anti-

bodies
The study of the influence of the mAb on DPP IV activity revealed that
none of them blocked the enzymatic activity (data not shown). The binding
of purified lymphocytic DPP IV to several immobilized mAb showed in
Figure 1 represents the hydrolysis of Gly-Pro-4-Me-2-NA by the lym-
 CD26 Antibody Binding . 149

100

!
~
80
"to
l!
:e
,!
60

."
u
u
u
u 40
~
CI
c:"
20

o
CBI IF7 Tal 134·2C2 BT5/9 TAS.9 blank I blank2

immobilized monoclonal antibody

Figure 1. The binding of DPP IV, purified from human lymphocytes, to different immobilized
mAb (mentioned under each column) is depicted. As blanks, a monoclonal antibody and an
ascites both of irrelevant specificity, were used. The DPP IV binding is analyzed by measuring
the hydrolysis of Gly-Pro-4-Me-2-NA and is expressed in arbitrary fluorescence units,
obtained after overnight incubation of the membranes at 8°C.

phocytic DPP IV bound onto the immobilized mAb. The actlVlty is

expressed in arbitrary fluorescence units. The unclassified T cell mAb anti-
BT5/9 and anti-TA5.9 bind a high amount of DPP IV, a first indication that
they might belong to the CD26 cluster. We could also confirm here that
DPP IV belongs to the group of molecules recognized by the anti-lF7
mAb. However, the mAb CBl, also known as anti-Tpl03 reacted only
weakly with purified DPP IV. The anti-DPP IV/CD26 mAb included in
our investigations, were able to detect nitrocellulose bound DPP IV. For
each of the mAb tested, the reaction with native Ag was identical or more
intense compared to SDS denatured samples. The mAb anti-Tal, anti-
TA5.9, 134-2C2, and anti-lF7, detected spots of purified lymphocytic DPP
IV, containing about 160 femtomol of DPP IV.

Influence of the an ti-BT519 and anti-TA5.9 mAb on the binding of DPP IV

to immobilized anti-CD26 mAb
The influence of the anti-TA5.9 and anti-BTS/9 mAb on enzyme binding
by the anti-Tal, 134-2C2, and anti-lF7 mAb was investigated. As can be
noticed from Figure 2A, the anti-TA5.9 mAb decreases the binding of DPP
IV onto immobilized 134-2C2 mAb to a significant extent. The effect of
anti-BTS/9 on the binding to the different immobilized mAb is, under our
experimental conditions, even more extensive (Fig. 2B): for immobilized
anti-TAS.9 and 134-2C2, the fluorescence produced by bound DPP IV
diminished with more than 60 %. A decrease in binding to anti-Tal was
also observed.
150 . 1. D E MEESTER et al.

....
120

100
~
~
:!
..
80
....
!.
..::.
60
co

..:!
~
40

c
20

0
IF7 134·2C2 Tal BT519

~ 100
u
u
c
~
~ 80

...~
0
60
~
u
u

.."
c:
u 40
~
0
::l
C 20

0
IF7 I 34·2C2 Tal TAS .9

Figure 2. The influence of soluble anti-TA5.9 (Fig. 2A, top) and anti-BT5/ 9 (Fig. 2B, bottom)
mAb on the binding of DPP IV to several immobilized mAb is represented. The influence of
the soluble anti-TA5.9 and anti-BT5/ 9 on the binding of DPP IV to immobilized mAb
(mentioned under each column) is tested. The DPP IV binding is analyzed by measuring the
hydrolysis of Gly-Pro-4-Me-2-NA obtained after overnight incubation of the membranes at
8 OC and is expressed as a percentage of the binding in the absence ofanti-BT5/9 or anti-TA5 .9
mAb respectively. The mean ± SD for three different experiments is given.

In a next step we investigated the influence of different dilutions of anti-

BT5/9 ascites on the binding of lymphocytic DPP IV onto immobilized
anti-TA5.9 (Fig. 3). Anti-BT5/9 inhibits binding of DPP IV to immobilized
anti-TAS.9, while anti-TA5.9 does not influence DPP IV binding to
immobilized anti-BT5/9 to a similar extent.

Influence of lectins on the binding of DPP IV onto immobilized anti- TA5. 9

and anti-BT519 mAb
In order to detect whether the antibodies recognize a peptide or a
carbohydrate epitope on the DPP IV molecule, we investigated the ability
of three DPP IV binding lectins (WGA, LCA, and RCA 120 ) to compete
 CD26 Antibody Binding . 151

120

-..."
~
100

..
~ 80

...
~

" 60
!.
..
...."
0
40

20
"
C

0
0 0, 1 0 ,2 10

relative concentration BT5/9 (% v/v)

Figure 3. The influence of different relative concentrations of anti-BT5/9 mAb on binding of

lymphocytic DPP IV to immobilized anti-TA5.9 mAb. The DPP IV binding is analyzed by
measuring the hydrolysis of Gly-Pro-4-Me-2-NA obtained after overnight incubation of the
membranes at 8°C and is expressed as the percentage fluorescence generated by bound DPP IV
as compared to a sample without anti-BT5/9 as the 100 percent. The relative concentrations
tested, ranged from 0.1 to 10 percent (v/v) of anti-BTS/9 ascites present in the sample.

with the mAb for binding. Purified CD26/DPP IV was preincubated with
different concentrations of the lectins, ranging from 0.1 to 100 [tg/ml. None
of the lectins, even at the highest concentration under study, could prevent

140

....
~
120
~
~ 100
~
.
...-!!
0
80

!. 60
.::"... 40
~
""
l:: 20

0
reference WGA LCA RCA
Leclins

Figure 4. The influence of different lectins (WGA, wheat germ agglutinin; LCA, Lens culinaris
agglutinin; RCA, Ricinus communis agglutinin) on the binding of DPP IV onto immobilized
anti-BTS/9 (first column) and anti-TAS.9 mAb (second column) is represented. DPP IV was
preincubated with the lectins (concentrations ranging from 0.1 to 100 [!g/ml) before measuring
the binding to the different mAb. Results of experiments with lectin concentrations of 100 [!gl
ml are depicted. The DPP IV binding is analyzed by measuring the hydrolysis of Gly-Pro-4-
Me-2-NA obtained after overnight incubation of the membranes at 8°C and is expressed as a
percentage (+SD, n~3) of the binding in the absence of lectins during preincubation.
152 . I. DE MEESTER et al.

o dim+
40 S bright+
,..---

30
~
U
u

...+
.,;
20

...-<
"I-
10

0
CD4+ CD8+

D - 2J

Figure 5. TA5.9 expression in CD4+ (n=23) and CD8+(n=42) T cells. The % dim and
bright TA5.9 + cells within each T cell population is given in the first and second column,
respectively. The median values and the 25th and 75th percentiles are represented. n represents
the number of samples analyzed.

CD26/DPP IV from binding to these mAb. Therefore we assume that the

anti-TA5.9 and anti-BT5/9 mAb do not recognize carbohydrate structures
on the lymphocytic CD26 Ag. The results of the lectin competition studies
are presented in Figure 4.

Expression of the TA5.9 Ag on peripheral blood lymphocytes

Using the anti-TA5.9 mAb, lymphocytes can be divided in three popula-
tions: a negative group, a dim + group with weak expression of the TA5.9
Ag and a bright + group with strong expression of this Ag. The TA5.9 Ag is
present on ± 50 % of the CD4+ and on ± 25 % of the CD8+ cells as is
illustrated in Figure 5. The difference between CD4+ and CD8+ was mainly
in the dim TA5.9 population, whereas the proportion of TA5.9+bright
was ± 10 % in both CD4+ and CD8+ cells. The associations of strong
TA5.9 (CD26) expression with other activation and/or memory markers on
CD4+ and CD8+ T cells was investigated by performing three color flow
cytometry experiments. The results of these studies are summarized in
Figure 6. The TA5.9+bright populations co-express CD45RO but not HLA-
DR and CD38. Tac was only relatively over-represented in the subsets with
strong TA5.9 expression.

Discussion

By means of the DPP IV specific immunosorbent assay, two unclassified

T cell mAb, anti-TA5.9 and anti-BT5/9, were found to bind CD26/DPP
IV, purified from human peripheral blood lymphocytes. Both mouse
 CD26 Antibody Binding · 153

100
o negldim TAB

~~::= ~ Q bright TAB

80 ~~=:;~
;~;~:\
;:;:=
.!! 60 :::::
uu
.
:.
.; 40 ~l~l~
':;:;
.,.'"
0

';~:
','
20

0
CD45RO CD25 HlA·DR CD)8

100
,
,, ..;.. o neg/dim TAS.9

80 G bright TAS.9

. ;',"
,>
','

.
U 60
u

.~
.;
0 40 ::'::::
.,.'" .. ' ..' .. ',

20 m~m
':~:~:~
' .. ;:;,
0
CD45RO CD25 HlA·DR CD)8

Figure 6. Expression (as percentages of positive cells) of activation markers within the TA5.9
negative/dim or bright + subsets o f CD4 + T cells (Fig. 6A, top) and CD8 + T cells (Fig. 6B,
bottom). The median values and the ranges are represented (n, number of samples
analyzed = 5).

antibodies, respectively of the IgG 1 and IgG3 isotype, also recognize cell-
bound CD26 and proved to be suitable for flow cytometry experiments.
Competitive binding assays revealed that the mAb anti-TAS. 9 and anti-
BTS/ 9 recognize close or partially overlapping epitopes on the g lycoprotein
CD26/DPP IV, which are partially identical or very close to the one bound
by the 134-2C2 mAb. The anti-1F7 mAb is likely to bind a different epitope
of the CD26/DPP IV molecule, while the influence on anti-TAl binding is
complex.
During the last decade, the BTS/9 Ag was already studied in detail (24,
33-42). The 4th workshop on leukocyte differentiation antigens reported
for the anti-BTS/9 mAb, the recognition of a T cell Ag of 120 kDa on SDS
154 . I. DE MEESTER et al.

PAGE after immunoblotting (43). For the CD26 Ag, molecular masses
between 100 and 120 kDa have been reported (3). The studies on BTS/9
expression in normal and pathological conditions, and on the functional
behavior of positive cells, add supporting evidence for the involvement of
this molecule in cellular immunity. The Ag is expressed on approximately
lS-20 % of resting T lymphocytes and its expression increases following
T cell activation both in vivo and in vitro. The BTS/9+ subset includes all
the cells with helper activity for pokeweed mitogen driven B cell differenti-
ation. In addition, the peripheral blood BTS/9+ population contains the
cells capable of proliferative responses to soluble antigens and to allogeneic
cells (24). The BTS/9+ subset is remarkably increased in several pathological
conditions such as Hashimoto thyroiditis, Graves' disease, and sarcoidosis
(34, 3S, 39). An increase in T cells bearing the Tal (CD26) Ag was detected
in untreated Graves' disease patients, in rheumatoid arthritis and in multiple
sclerosis (44-46). On the other hand, a decrease in BTS/9 positive lym-
phocytes was observed in patients with common variable hypo gamma-
globulinaemia (36). Independently, the monitoring of BTS/9+ and of
CD26+ T cells had been proposed as objective measures of abnormal
immunologic activity. The finding that the anti-BTS/9 mAb reacts with
CD26, constitutes an important increase in knowledge on the clinical
significance of the analysis of CD26 expression. The further study of this
activation marker may prove useful for investigating the pathogenesis of T
cell mediated autoimmune diseases. Injection with the mAb BTS/9 has been
used for the prevention and treatment of acute graft-versus-host disease
after allogeneic bone marrow transplantation (40). The use of BTS/9+ cell
depleted bone marrow for allogeneic bone marrow transplantation in
leukemia patients, resulted in a lower incidence of acute and chronic graft-
versus-host disease, but relapse rates were higher (42).
DANG et al. (16) showed that the Tal (CD26) molecule is an alternate
mediator of human lymphocyte activation. Triggering via Tal (CD26) is
shown to be functionally interconnected to CD3/TcR activation mecha-
nisms, because modulation of the CD3/TcR complex inhibits anti-Tal
mediated cytolysis without affecting Tal surface expression. Likewise, the
soluble anti-BTS/9 mAb can induce monocyte-depleted T lymphocytes to
proliferate in the presence of suboptimal doses of pokeweed mitogen, but
this effect was strongly inhibited by antibody-induced modulation of the
CD3/TcR complex. These findings add supporting evidence for the state-
ment that the molecules recognized by anti-BTS/9, anti-Tal and anti-1F7
belong to the CD26 family of lymphocyte surface structures, which can
provide costimulatory signals for CD3/TcR mediated T cell activation and
proliferation. By using CD26 transfected Jurkat cells, it was recently shown
that crosslinking of the CD26 and CD3 Ag resulted in enhanced intracellu-
lar Ca-mobilization and interleukin-2 production (23). TORIMOTO et al.
(47) demonstrated that lF7 (CD26) may be closely associated with the
CD4S protein tyrosine phosphatase on the T cell surface. Their experimen-
tal results support the option that the interaction with CD4S leads to
 CD26 Antibody Binding· 155

enhanced tyrosine kinase actlVlty, ~ chain phosphorylation and T cell

activation. MUNoz et al. observed tyrosine phosphorylation on a number of
proteins after triggering of CD26 with the 134-2C2 mAb (48). This protein
tyrosine phosphorylation seems to play a major role in CD26 mediated
T cell proliferation.
Although the CD45RO expression is commonly used to characterize
«memory» cells (49), it is not the only antigen associated with memory
function. Several studies have shown that the response to recall antigen is
restricted to DPP IV/CD26 positive cells and therefore it has been sug-
gested that CD26+ T lymphocytes represent the memory pool of the
immune system (48, 50, 51). We demonstrated in this study that CD26 +bright
cells are only a subset of the CD45RO+ T cells and that they lack HLA-
DR and CD38. In a recent investigation on CD26 expression on memory
and naive T cells, ULMER et al. argue against the hypothesis that CD26+
T cells are memory cells (52). The discussion on the value of CD26 as a
«memory marker» does not exclude the functional involvement of CD26+
T cells in the response to soluble antigen. This is supported by recent
findings of a diminished CD26 expression on T cells in HIV -infected
persons (51, 53), and the impairment of their T cells to proliferate against
soluble antigen (53).
In summary, we have studied the antibody binding to purified and cell-
surface bound lymphocytic CD26 and we have designated the BTS/9 and
TA5.9 Ag to the CD26 cluster. Our observation that the CD26+bright T cells
co-express the CD45RO antigen but not the CD38 or HLA-DR, might
indicate that these cells constitute a functionally distinct T cell subset. We
suggest that the analysis of CD26 expression on lymphocytes may contrib-
ute to a better understanding of the functional immune status of patients
with either an augmented or decreased cellular immune reactivity.
Moreover, CD26 specific monoclonal antibodies might also have
therapeutic applications, since at least one of them (anti-BT5/9) has already
shown immunomodulatory effects in vivo.

Acknowledgements
We would like to thank Dr. M. HEGEN, Dr. C. MORIMOTO, Dr. O. VINAS, and Dr. TH.
WALDMAN for providing antibodies. We are grateful to Dr. M. VAN SANDE, Dr. D. HENDRIKS
and Dr. F. GOOSSENS for the valuable discussions and comments and to N. LAMOEN for her
excellent technical assistance.
This work was supported by grants VLAB/074b of the Flemish Biotechnology Program and
IUAP-II of the Belgian Science Policy Programming. I. DE MEESTER is a Senior Research
Assistant of the Belgian National Fund for Scientific Research.

References

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Dr. INGRID DE MEESTER, Department of Medical Biochemistry (Bldg S-6), University of

Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium