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Anti-CD26 monoclonal antibodies can reversibly arrest human T lymphocytes in the late G1 phase of the cell cycle
Immunobiol., vol. 188, pp. 36-50 (1993) 1 Department 2
of Immunology and Cell Biology, Forschungsinstitut Borstel, Borstel, and Department of Internal Medicine, Medical Academy of Magdeburg, Magdeburg, Germany
Anti-CD26 Monoclonal Antibodies Can Reversibly Arrest Human T Lymphocytes in the Late G1 Phase of the Cell Cycle TAILAMATTERN1, SIEGFRIEDANSORGE2 , HANS-DIETERFLAD1, and ARTUR J. ULMER1 Received AprilS, 1992 . Accepted in Revised Form October 12, 1992
Abstract Three different anti-CD26 monoclonal antibodies (mAbs) are described, which specifically inhibited proliferation of human T lymphocytes after stimulation with PHA, tetanus toxoid or soluble anti-CD3 mAb. Anti-CD26 mAbs induced in T cells a dose-dependent shift of the maximum of DNA synthesis, which was due to a transitory arrest of cells in the cell cycle. This cell cycle arrest was found to occur in the late phase of G 1 since the expression of the T cell activation markers CD25-, CD71-, and HLA-DR-positive cells was the same in anti-CD26 mAb-containing and control cultures. Propidium iodide staining further confirmed the assumption that the arrest occurs in the first round of the cell cycle before S phase cells were detectable. Because the cells were arrested before consuming IL-2 in the S phase, we observed in accumulation of IL-2 in anti-CD26 mAb-containing cultures, whereas IFN-gamma production by PHA-activated T lymphocytes was reduced. These data indicate that CD26 is involved in the processes of T cell activation and proliferation.
Introduction Recently, a new surface marker of human T cells was described, the T cell activation marker CD26, which was thought to be likewise involved in processes of T cell activation (1). CD26 was found to be identical to the ectoenzyme dipeptidyl peptidase IV (DPP IV, E.C. 3.4.14.5) (2). This enzyme can be demonstrated in several mammalian tissues (3), but within the hematopoetic system only on T lymphocytes. DPP IV was characterized by a highly restricted substrate specificity, i.e. cleavage of dipeptides from the free amino terminus of peptides after L-proline or L-alanine (4). Abbreviations: DPP IV = dipeptidyl peptidase IV; FCS = fetal calf serum; GCSF = granulocyte colony-stimulating factor; Gly-Pro pNA = glycylproline-p-nitroanilide; 3HTdR = eH)thymidine; IFN-gamma = interferon-gamma; IL-l{3 = interleukin 1~; IL-2 = interleukin 2; IL-6 = interleukin 6; L T = lymphotoxin; MNC = mononuclear cells; mAbs = monoclonal antibodies; PHA = phytohemagglutinin
Inhibition of T Lymphocytes by Anti-CD26 . 37
Although the natural substrate of DPP IV is still unknown, there exist, with regard to their amino acid sequences, many hypothetical substrates for this enzyme within the immune system, e.g. interleukin 1B(IL-1 B), interleukin 6 (IL-6), granulocyte colony-stimulating factor (GCSF), lymphotoxin (LT), or substance P. During the last years we have phenotypically and functionally characterized CD26-positive and -negative T cells. While the expression of CD26 is associated with the ability to produce high amounts of IL-2 and IFNgamma and to proliferate considerably after PHA stimulation (5), CD26negative T cells showed better helper functions for the differentiation and Ig synthesis of B cells after pokeweed mitogen stimulation (6). Our studies further revealed that CD26 is expressed de novo on CD26negative as well as on CD26-positive T cells after stimulation with various mitogens or antigens (7). From comparative studies regarding the expression of other T cell activation markers like the IL-2 receptor and HLA-DR, we assumed that CD26 arises during G 1 of the cell cycle after the expression of IL-2 receptor and HLA-DR. These studies and further investigations with specific inhibitors of DPP IV by SCHON et al. (1, 8-11) led to the assumption that CD26 may be directly involved in the activation of T cells. Other authors have examined the effects of various anti-CD26 mAbs on T cell stimulation and have found different effects: Neither mitogenic nor comito genic effects have been described for the anti-Tal mAb (12, 13). Costimulatory effects of anti-CD26 mAbs in an IL-2/IL-2 receptor-dependent pathway have been found by others (14-16). FLEISCHER et al. (17, 18) and DANG et al. (19) reported on the mitogenic effects of anti-CD26 mAbs and on the activation of effector functions of cytotoxic T cells. We have now investigated the influence of two other anti-CD26 mAbs on the proliferation of human T cells in vitro.
Material and Methods Isolation of cells Human peripheral blood mononuclear cells (MNC) were isolated from heparinized blood of healthy donors by density gradient centrifugation, as described elsewhere (20). For some experiments, T lymphocytes were isolated by adherence of MNC to nylon wool (Leuko Pak, Fenwal Laboratories, Morton Grove, IL, USA) in a 1 x 20 cm column (Bio-Rad Laboratories, Miinchen, Germany) according to the methods described by JULIUS et al. (21). In brief, 1 x 10 8 MNC in 3 ml Hanks' balanced salt solution (BSS) supplemented with 10% heat-inactivated (56 DC, 30 min) fetal calf serum (FCS) were loaded on the column at room temperature. Subsequently, the column was rinsed with 25 ml Hanks' BSS + 10 % FCS at a flow rate of about 0.5 mllmin to elute non-adherent cells. These non-adherent cells consisted of more than 97 % CDr T lymphocytes. Monocytes were isolated from MNC by adherence to disposable falcon tissue culture flasks. The non-adherent cells were discarded, and the adherent cells were isolated by being scraped off with a rubber policeman in ice-cold RPM!. More than 95 % of the adherent cells were monocytes as determined by cytochemical evaluation of the unaphthyl acetate esterase pattern.
38 . T. MATTERN, S. ANSORGE, H.-D. FLAD, and A.
J. ULMER
Cell culture MNC, T cells or T cells + 10 % autologous adherent cells were suspended at a concentration of 2 x lOs Iml for stimulation with PHA and 1 x 106 Iml for stimulation with soluble anti-CD3 or tetanus toxoid, respectively, in RPMI 1640 medium supplemented with antibiotics and 10 % FCS. Because FCS (as well as human serum) contains soluble CD26, which itself binds the anti-CD26 antibodies and thus requires relative high concentrations of antibodies, a batch of FCS was used which contains only low amounts of soluble CD26. Cells were cultured at a final volume of 50 [!l/well in half area microtiter plates (Costar, Cambridge, MA, USA). The cells were stimulated by phytohemagglutinin (PHA 1 flg/ml, Wellcome, Beckenham, Kent, UK), soluble anti-CD3 (BMA 030, 12.5 ng/ml, Behringwerke AG, Marburg, Germany), or tetanus toxoid (1 U/ml, Behringwerke AG). The anti-CD26 mAb Til 19-4-7 (ascites, kindly provided by Prof. FELLER, Inst. of Pathology, University of Wiirzburg, Germany), anti-Tal (4ELlC7, purified antibody, Coulter Clone, Hialeah, FL, USA), and M5 (ascites) were added to the cultures at different times and concentrations. After incubation for 1 to 4 d the cultures were pulsed with 37 kBq (1 [!Ci) eHJthymidine eHTdR) per well for the last 8 h of culture. Cells were harvested onto glass filters, and 3HTdR incorporation was determined by scintillation counting. The results represent the mean of triplicate wells, and SD were less than 15 %. Induction and detection of lymphokines Cells were stimulated and cultured as described above. After the indicated culture period, supernatants were harvested for lymphokine determination. IL-2 activity was determined by measuring the proliferation of the IL-2-dependent CTL-6 line, as described elsewhere (22). IFN-gamma production was measured in an ELISA system, as described in more detail by ENNEN et al. (23). Anti-human IFN-gamma mAb, Clone 69, was a generous gift from Dr. GALLATI (Hoffmann-LaRoche, Basel, Switzerland). Immunofluorescence staining of cells The following antibodies were used: Anti-CD25: Tue 69-FITC, Biotest, Offenbach, Germany; anti-CD71: anti-human transferrin receptor FITC, Becton Dickinson, San Jose, CA, USA; Ki-67: kindly provided by Dr. J. GERDES, Forschungsinstitut Borstel, Germany; anti-HLA-DR: anti-human HLA-DR FITC, Becton Dickinson; FITC labelled F(ab'h fragments of goat anti-mouse IgG Oackson Immunoresearch Laboratories, West Grove, PA, USA). For immunofluorescence staining, cells were harvested and labelled with monoclonal antibodies and analyzed in a cell sorter. Direct or indirect immunofluorescence staining was performed in ice-cold PBS (containing 0.1 % sodium azide) using the mAbs at optimal concentrations. After the first incubation with the specific mAb for 20 min the cells were washed by centrifugation on a FCS gradient (200 x g, 10 min), resuspended in 100 [!l PBS, and, in case on indirect staining, incubated for further 20 min in presence of the second FITC labelled antibody. The cells were washed again in a FCS gradient and resuspended in PBS. Labelled cells were analyzed in a Cytofluorograf (System SOH, Ortho Diagnostic System, Inc., Westwood, MA, USA). Propidium iodide staining of cells 1 x 106 MNC were suspended in 200 [!l of citrate buffer, pH 7.6 (250 mM sucrose, 40 mM trisodiumcitrate x 2 H 2 0, 5 % v/v DMSO in distilled water), and stored at -80°C until analysis. 1800 [!l of trypsin solution, pH 7.6 (0.03 mg/ml trypsin [Sigma T-0134J in 3.4 mM trisodiumcitratex2 H 2 0, 0.1 % v/v Nonidet P-40 [Sigma N-3516J, 1.5 mM spermine tetrahydrochloride [Sigma S-2876J, 0.05 mM Tris in distilled water), were mixed with the thawed cells, and the trypsin was allowed to react for 10 min at room temperature. Then 1500 [!l of trypsin inhibitor (0.5 mg/ml trypsin inhibitor [Sigma T-9253J and 0.1 mg/ml ribonuclease A [Sigma R-4875] in 3.4 mM trisodiumcitrate x 2 H 2 0, 0.1 % v/v Nonidet P-40 [Sigma N3516J, 1.5 mM spermine tetrahydrochloride [Sigma S-2876J, 0.05 mM Tris in distilled water,
Inhibition of T Lymphocytes by Anti-CD26 . 39 pH 7.6) were mixed with the cells and incubated for further 10 min at room temperature. Cells were then washed by centrifugation (10 min, 4°C, 1800 x g) and resuspended in 350 ftl citrate buffer. They were mixed with 150 ftl of a propidium iodide solution (0.4 mg/ml propidium iodide [Sigma P-4170J, in 3.4 mM trisodiumcitrate x 2 H 2 0, 0.1 % v/v Nonidet P-40 [Sigma N-3516J, 5 mM spermine tetrahydrochloride [Sigma S-2876J, 0.05 mM Tris in distilled water, pH 7.6), wrapped with tinfoil for light protection and kept for 15 min on ice bath. Cells were washed again and measured with 3 h after addition of propidium iodide. Cells were measured in a Cytofluorograf (System SOH, Ortho Diagnostic System, Inc.). For internal G OIl standard, frozen MNC of the same donor were used.
Results
Inhibition of proliferation by anti-CD26 antibodies Human MNC were stimulated polyclonally with PHA or soluble antiCD3 or monoclonally with the recall antigen tetanus toxoid, respectively. The anti-CD26 mAbs TIl 19-4-7, Tal or M5 were given at the beginning of the cultures. As shown in Table 1, upper rows, the addition of anti-CD26 mAb resulted in a clear and significant (p < 0.0027) inhibition of DNA synthesis. Crosslinking of CD26 by means of anti-CD26 mAb plus goat anti-mouse F(ab')2 fragments did not influence the inhibition affected by anti-CD26 mAb (Table 1, middle part). Goat anti-mouse fragments themselves were not mitogenic and did not influence PHA-induced DNA synthesis (data not shown). Similar to these results, we found that depletion of adherent cells or addition of a defined number of adherent cells to T cells did not influence the inhibitory capacity of the anti-CD26 mAbs (Table 1, lower rows), indicating that crosslinking of the CD26 molecules by Fc Table 1. Anti-CD26-mediated inhibition of DNA synthesis of MNC, T cells, or T cells plus adherent cells after polyclonal or monoclonal stimulation
+ anti CD26
Stimulus
cells
No. of Exp.
- anti CD26
PHA anti CD3 tet. toxoid
MNC MNC MNC
10 10 5
16.7 ± 5.3 10.9 ± 6.0 8.6 ± 8.4
5.3 ± 2.5'" 2.5 ± 2.1" 1.9 ± 1.4'"
PHA
MNC
3
34.2 ± 5.5
11.0 ± 2.5'"
PHA PHA PHA
MNC T + 10% Adh. T cells
3 3 3
15.9 ± 6.3 12.6 ± 4.7 9.4 ± 5.2
9.4 ± 2.6'" 7.4 ± 3.2'" 3.6 ± 1.Y
+ anti CD26 + GaM n.t. n.t. n.t. 12.2 ± 3.1 n.t. n.t. n.t.
Human peripheral MNC, T cells plus 10 % autologous, adherent cells were stimulated with PHA (1 ftg/ml, 2 x lOS cells/ml), soluble CD3 (BMA 030, 12.5 ng/ml, 1 x 106 cells/ml), or tetanus toxoid (1 U/ml, 1 x 106 cells/ml). The cells were cultured with or without anti-CD26 mAbs (TIl 19-4-7, 1:50 ascites dilution, Tal, 10 ftg/ml, or M5, 1:200 ascites dilution). For crosslinking experiments, PHA -stimulated cells were cultured in the presence or absence of anti-CD26 (TIl 19-4-7, 1:50 ascites dilution) and goat anti-mouse F(ab' 2) fragments (GaM, 1:100). DNA synthesis was measured after 96 h of culture by 3HTdR incorporation for the last 8 h of culture. ". p < 0.0027
.40 . T. MATTERN, S. ANSORGE, H.-D. FLAD, and A.
fo
J. ULMER
. - . PHA + TII19-4-7 + DPP fII A- -A PHA + DPP fII 20 0 - 0 TII19-4-7 + DPP fII ll. - -ll. DPP fII alone
A-
10
_-A...
.___..
... "'A7-X- - -A- - -A
.--.................
o o
20
50
100
200
500
DPP fII concentration (ng/ml) Figure 1. Effect of DPP IV on the inhibitory activity of anti-CD26 mAbs on PHA-induced proliferation. Peripheral MNC (2 x 10s/ml) were stimulated with PHA (1 flg/ml) in the presence or absence of anti-CD26 mAb TIl 19-4-7 (1 :50, ascites dilution). In parallel cultures purified DPP IV from human placenta was added in concentrations as indicated. DNA synthesis was measured on day 4 of culture by 3HTdR incorporation for the last 8 h of culture.
receptor-positive adherent cells was not required for inhibition. All three anti-CD26 mAbs tested were found to reduce proliferation in a dosedependent manner (data not shown).
Specificity of inhibition To prove the specificity of the inhibition of T cell proliferation mediated by the anti-CD26 mAbs, purified DPP IV was used as an antagonist to antiCD26 mAbs. As shown in Figure 1, the inhibition of DNA synthesis after PHA stimulation by anti-CD26 mAbs could be neutralized by addition of increased concentrations of the purified antigen DPP IV. Amounts of 100 ng/ml purified DPP IV totally neutralized the inhibition affected by TIl 194-7 (1 :50 ascites dilution, corresponding to 5 ~g/ml total protein). In control experiments we could show that anti-CD26 mAbs alone or antiCD26/DPP IV immune complexes were not mitogenic for T lymphocytes. Furthermore, soluble DPP IV itself neither influenced PHA-induced DNA synthesis nor was able to stimulate proliferation by itself at all concentrations tested (Fig. 1). Unspecific cytotoxicity of the anti-CD26 mAbs could be excluded, since the viability of the cells after 96 h of culture with or without anti-CD26 mAbs was not affected and furthermore, anti-CD26 mAbs did not inhibit the proliferation of the CD26-negative cell lines Wehi and K562 (data not given). Kinetics of DNA synthesis after PHA stimulation and addition of anti-
CD26 mAbs Experiments were carried out to examine the effects of anti-CD26 mAbs on the kinetics of DNA synthesis after PHA-induced T cell activation (Fig.
Inhibition of T Lymphocytes by Anti-CD26 . 41
0-0 PHA alone
o
20
. - . PHA + 111 19-4-
'/'0('"·"
0~.. . . . . . /0
10
/
0 '. '-0--0__ : : .
/.\
.'.
O+-__~~--~-+--+_~~~--+__T~~ 0-0 PHA alone
10 ,:) I
o
. - . PHA + Ta1
5
10
5
days of culture Figure 2. Kinetics of DNA synthesis after stimulation of MNC with PHA in absence or presence of anti-CD26 mAbs. Human peripheral MNC (2 x lOs/ml) were stimulated with PHA (1 ltg/ml). Anti-CD26 mAbs (TIl 19-4-7, 1:50, ascites dilution, Tal, 10 ltg/ml, or M5, 1:200, ascites dilution) were added at the beginning of the culture. For control Ig isotypespecific antibodies (both in a 1:20 dilution) were added. DNA synthesis was measured after a culture time as indicated by 3HTdR incorporation for the last 8 h.
2). DNA synthesis after PHA stimulation in the absence of anti-CD26 mAbs showed a maximum on day 4 of culture and then diminished. The addition of anti-CD26 mAbs resulted in a shift of DNA synthesis. The proliferation was now maximal on day 5 or 6 of culture. It should be emphasized that DNA synthesis is significantly inhibited by anti-CD26 mAbs when investigated on day 4 of culture (see also Table 1). On the other hand, analysis of DNA synthesis on day 5 or 6 of culture shows an enhancement caused by anti-CD26. Similar results were found in experi-
42 . T. MATTERN, S. ANSORGE, H.-D. FLAD, and A.
ULMER
~
0
WOIhed after 8811
0
20
i
10
0
J.
o"0"-e__e
~ 0
2
3
4-
'-0---
S
6
7
8
9
10
days of culture Figure 3. Kinetics of PHA-induced DNA synthesis of human MNC with and without antiCD26 mAbs and after washing off the antibody effects. Human peripheral MNC (2 x 10s/ml) were stimulated with PHA (1 f!g/ml). The anti-CD26 mAb TIl 19-4-7 (1:50, ascites dilution) was added at the beginning of culture. After 24 h, 48 h, or 96 h, the cells (of the antibody-free as well as of the antibody-containing cultures) were harvested, washed 2 times in fresh medium, and then recultured in fresh, PHA containing medium. DNA synthesis was measured after various culture times by 3HTdR incorporation for the last 8 h. A: Kinetics of DNA synthesis with and without anti-CD26 mAbs in undisturbed cultures. B: Kinetics of DNA synthesis with or without anti-CD26 mAbs in cultures, washed after 24 h of culture. C: Kinetics of DNA synthesis with or without anti-CD26 mAbs in cultures, washed after 48 h of culture. For details see above. D: Kinetics of DNA synthesis with or without anti-CD26 mAbs in cultures, washed after 96 h of culture.
ments with the recall antigen tetanus toxoid or soluble anti-CD3 antibody. In Figure 2 we further demonstrate that isotype-specific control antibodies could not mimick the effects of anti-CD26 mAbs. These experiments strongly indicate that the effects of anti-CD26 mAbs were specific for the CD26 antigen. To investigate the reversibility of the effect of anti-CD26 mAbs, a twostep culture system was employed. Cells were incubated with PHA in the
presence or absence of anti-CD26 mAbs, washed after different culture times, and then recultured with fresh PHA -containing medium. The kine-
Inhibition ofT Lymphocytes by Anti-CD26 . 43
tics of DNA synthesis after this procedure were measured. Figure 3 shows the typical results of such an experiment. Cells incubated in undisturbed cultures show the kinetics of DNA synthesis, as described above, with a shift of maximal proliferation from day 4 to day 6 of culture in the presence of the anti-CD26 mAb TIl 19-4-7 (Fig. 3A). Cells, prestimulated with PHA for 24 or 48 h, then washed and cultured further in presence of PHA, show a maximum of DNA synthesis on day 5 of culture independently of whether they were precultured in presence of anti-CD26 mAbs or not (Fig. 3B and C). On the other hand, when cells were preincubated for four days in the presence of anti-CD26 mAb-containing medium and then washed, kinetics of DNA synthesis showed the same shift as undisturbed cultures (Fig. 3D). In contrast, when new anti-CD26 mAbs were added daily to PHAstimulated cultures, we found a clear and nearly complete inhibition of DNA synthesis without affecting the viability of the cells (data not given).
L ymphokine production after addition of anti-CD26 mAbs These experiments were carried out to investigate whether the observed shift in the DNA synthesis after anti-CD26 mAb addition is due to a reduced lymphokine production. MNC were stimulated with PHA and cultured with or without anti-CD26 mAbs for various times. Production of IL-2 was measured in a CTL-6 proliferation assay, while IFN-gamma production was measured in an ELISA system. Table 2 shows the results of five independent experiments. Cultures with anti-CD26 mAb Tal reveal again the shift in the kinetics of DNA synthesis (data not given). To our surprise IL-2 release was enhanced in cultures with anti-CD26 mAbs at all culture times tested. In contrast, IFN-gamma release was found to be reduced by addition of anti-CD26 mAbs up to day 5 of culture. However,
Table 2. Effects of anti-CD26 mAbs on the kinetics of PHA-induced IL-2 and IFN-gamma production days of culture Lymphokine
anti CD26
IL-2 (Ulml)
+ IFN-y (ng/ml)
-
+
2
3
4
6
5±1 6±1
11 ± 1 11 ± 1
9±3 12 ± 6
6±2 17± 8
2±1 7±2
0 5±2
0 3± 1
3±2 1± 1
6±3 2±1
12 ± 8 4±3
18 ± 9 6±3
10 ± 5 8±4
8±3 10 ± 2
4±1 10 ± 3
5
8
Human peripheral MNC (2 x 10 s/ml) were stimulated with PHA (1 flg/ml) in the presence or absence of anti-CD26 mAb (TIl 19-4-7, 1:50 ascites dilution, Tal, 10 flg/ml, or M5 1:200 ascites dilution). The supernatants were harvested after different culture periods. IL-2 production was measured in the CTL-6 assay and IFN-gamma production in an ELISA system. Data are expressed as mean of five independent experiments ± SEM.
44 . T. MATTERN, S. ANSORGE, H.-D. FLAD, and A.
0-0 PHA alone . - . PHA + Tal 50
g .!!
"ii (.)
0
GI
.. '0 .. .! .2:
~
0
50
Q.
E ;:I z
0
/
A
~.
--
.~
."".,,-._e-==e CD25
Q' -<;2 PHA alone . - . PHA + Tal
B
~~7e=::::::8
e--==i~ CD71
O-Q PHA alone . - . PHA+Tal
50
o
e..,.:::::::::::e
J. ULMER
~-
o
e HLA-DR
i-i--' e-e-=i:::::::::::-2
3
4
5
6
days of culture
Figure 4. Kinetics of the expression of different T cell activation markers on PHA-stimulated MNC after addition of anti-CD26 mAbs. A: Kinetics of CD25 expression (IL-2 receptor). B: Kinetics of CD71 expression (transferrin receptor). C: Kinetics of HLA-DR expression. For cell staining see Materials and Methods.
it remained detectable at that low level during later culture times, whereas it dropped in control cultures with PHA alone. Influence of anti-CD26 mAbs on the cell cycle The observation that IL-2 production after anti-CD26 addition was enhanced and not inhibited might indicate that the cells could be preactivated by PHA in a normal way, but were stopped somewhere in the cell cycle before DNA synthesis occurred. We have, therefore, investigated the effects of anti-CD26 mAbs on the expression of typical T cell activation markers like CD25, CD71, or HLA-DR, and the DNA content on the single cell level (measured by propidium iodide staining). Figures 4A, B, and C show the kinetics of CD25, CD71 or HLA-DR expression, respectively. Although DNA synthesis was shifted, as normally found by addition of anti-CD26 mAbs (data not given), the cells from cultures with or without anti-CD26 mAbs and PHA were not distinguishable with respect to their expression of CD25, CD71, or HLA-DR. Also no differences between anti-CD26 mAb-treated and untreated cells could be observed with respect to the density of the activation markers on the cell surface (data not shown). Anti-CD26 mAbs alone were not able to induce expression of activation markers (data not shown). These data indicate that the cells seemed to be stopped by anti-CD26 mAbs within the cell cycle after expression of the IL-2 receptor CD25 (early G 1 phase), HLA-DR and the
Inhibition ofT Lymphocytes by Anti-CD26 . 45 Table 3. Cell cylce analysis of PHA-stimulated cells, cultured with or without anti-CD26 mAbs, measured by propidium iodide staining days of culture anti CD26
0
G OIl phase positive cells
+
99.3% 99.3%
S phase positive cells
+
G 2 phase positive cells
+
2
4
94.5% 99.3%
87.7% 97.4%
78.9% 87.9%
0.5% 0.5%
5.1 % 0.5%
10.9% 2.2%
16.6% 9.7%
0.2% 0.2%
0.4% 0.2%
1.4 % 0.4%
4.5% 2.4%
Human peripheral MNC (2 x lOs/ml) were stimulated with PHA (1 ltg/ml) in the presence of anti-CD26 mAb (Tal, 10 ltg/ml). Cells were harvested and stained with propidium iodide as described in Materials and Methods.
transferrin receptor CD71 (late G 1 phase), but prior to DNA synthesis in the S phase of the cell cycle. To verify this assumption, we stained the cells with propidium iodide (Table 3). Cells were frozen after culture at -80°C and stained simultaneously after thawing with propidium iodide. For internal Go standard, fresh MNC of the same donor were frozen and stained with propidium iodide. At day zerQ all cells were found to be in the G OIl phase of the cell cycle. After 24 h of culture with PHA alone the number of cells within the S phase slowly increased, and after further 24 h the first cells within the G2 phase were detectable. To the contrary, cells stimulated with PHA and anti-CD26 mAbs showed a clearly prolonged G OIl phase of the cell cycle. Cells within the S phase were for the first time detectable after 48 h of culture, while G 2 cells were first detectable after 96 h of culture. Discussion CD26 (or DPP IV) is an activation marker on T lymphocytes and thought to be involved in the processes of T cell activation. In this report we demonstrate the inhibitory effects of three different anti-CD26 mAbs (TIl 19-4-7, Tal, and MS) on the processes of PHA-induced T cell activation, as measured by DNA synthesis, cytokine production, expression of activation markers, and cell cycle analysis. We could show that the addition of anti-CD26 mAbs together with PHA resulted in a significant and donor-independent inhibition, as proven by several individual experiments shown in Table 1. The inhibition was not mediated by monocytes or by crosslinking via Fe receptor binding of mAb, since purified T lymphocytes show the same or even enhanced reduction of DNA synthesis as MNC or T lymphocytes plus adherent cells.
46 . T.
MATTERN,
S.
ANSORGE,
H.-D.
FLAD,
and
A.
J. ULMER
The specificity of the anti-proliferative effects of anti-CD26 mAbs is supported by the following points: i) The inhibition was not due to unspecific toxic effects of the antibody solutions themselves, because neither was the viability of cells influenced by anti-CD26 mAbs nor was the DNA synthesis of CD26-negative cell lines affected (data not given). ii) The inhibition was a specific event of the binding between surface-bound CD26 and anti-CD26 mAbs, because normal levels of DNA synthesis could be reconstituted by the addition of purified soluble DPP IV from human placenta in a dose-dependent way (Fig. 1). iii) We could further show that anti-CD26 mAbs did not influence thymidine incorporation, because addition of anti-CD26 mAb together with 3HTdR at day 4 of PHA-stimulated cultures did not result in a decreased 3HTdR incorporation (data not given). iv) Washing-off the added antibodies after 24 or 48 h of culture resulted in the same response as in control cultures without anti-CD26 mAbs (Fig. 3). v) IL-2 production (Table 2) and the expression of CD25, CD71, or HLADR (Fig. 4) were not inhibited by anti-CD26 mAbs. The manifestation of the inhibitory effect of anti-CD26 mAbs could be clarified in more detail by kinetic studies. All three anti-CD26 mAbs tested caused a shift of DNA synthesis after stimulation with PHA (Fig. 2). On the basis of these experimental data we raised the following hypothesis: - The binding of anti-CD26 mAbs to surface CD26 on resting or preactivated T cells causes the cells to rest in their cell cycle. Anti-CD26 mAbs were exhausted by internalization or shedding of the anti-CD26 mAb/ CD26 complexes. After reexpressing new CD26 on their surface, arrested cells in the absence of sufficient amounts of anti-CD26 mAbs became able to pass through the cell cycle in a normal manner. This hypothesis was verified by the following results. The daily addition of anti-CD26 mAbs resulted in a total reduction of DNA synthesis without affecting the viability of the cells (data not shown). Furthermore, we were not able to detect specific antibodies in the supernatants of T cells, stimulated for 48 h in the presence of PHA and anti-CD26 mAbs, as measured in an ELISA system with solid-phase bound CD26 (data not shown). To determine the time point at which anti-CD26 mAbs were effective in preventing stimulation of DNA synthesis, kinetic experiments were performed. After different times of culture the anti-CD26 mAbs were washed out, and the cells were further cultured only in the presence of PHA (Fig. 3). This experiment indicates that anti-CD26 mAbs were effective during a period preceding DNA synthesis after PHA stimulation, but that they did not affect an early event (e.g. signal transduction for activation). The assumption that no early signal transduction but a later event was involved, was confirmed by the observation that the release of IL-2 (Table 2) and the expression of CD25, CD71, and HLA-DR (Fig. 4) were not reduced. All these events have been reported to occur during distinct points of the G 1 phase of the cell cycle (25-27). Therefore, anti-CD26 mAbs have an effect in the late G 1 phase before DNA synthesis (S phase).
Inhibition of T Lymphocytes by Anti-CD26 . 47
This assumption was directly confirmed by cell cycle analysis, using propidium iodide staining of the DNA. In the absence of anti-CD26 mAbs we can already show after 24 h of PHA stimulation detectable amounts of cells in the S phase (Table 3). To the contrary, nearly no S phase cells were detectable in anti-CD26 mAb-treated cells within the first 48 h after PHA stimulation. This lack of Grpositive cells in anti-CD26 mAbs-containing cultures during the first 48 h of culture indicates that the cells were arrested in the first round of the cell cycle. It is notable that the expression of the transferrin receptor, CD71, which is thought to be the «point of no return» (26), was not affected. The uncoupling of transferrin receptor expression from DNA synthesis by anti-CD26 mAbs refers to an undefined restriction point in the cell cycle, which may occur after transferrin receptor expression in the late G b but prior to the initiation of DNA replication in S phase. IL-2 and IFN-gamma release by PHA-stimulated T lymphocytes were affected by anti-CD26 mAbs in an opposite way. The content of IL-2 in anti-CD26 mAbs-containing cultures was enhanced during all days investigated, and no shift of the kinetics of IL-2 release was observed (Table 2). We assume that cells cultured with anti-CD26 mAbs consume less IL-2, because IL-2 is known to be produced in the late G 1 phase of the cell cycle and is consumed in the early S phase (27). As the T cells were arrested by anti-CD26 mAbs prior to the S phase, we have reason to assume that they do not consume but accumulate IL-2. In contrast to IL-2, IFN-gamma release induced by PHA was inhibited by the addition of anti-CD26 mAbs (Table 2). However, contrary to cultures without anti-CD26 mAbs more IFN-gamma was detectable on day 6 to 7 of culture. The production of IFN-gamma mRNA and the release of IFN-gamma are known to be IL-2dependent (28). While we could show that IL-2 was enriched in the supernatants of cells cultured in the presence of anti-CD26 mAbs, the reduced IFN -gamma content could not be simply explained by a lack of IL2 production. Specific synthetic DPP IV inhibitors, which inhibit the hydrolytic activity of this enzyme, have previously been reported to inhibit PHA-induced DNA synthesis (8, 9). This observation indicates that the enzymatic activity of CD26 is involved in the process of cell cycling (11). To look into the mechanism of the inhibitory action of anti-CD26 mAbs, we have tested whether these antibodies could inhibit the enzymatic activity of DPP IV. For testing the hydrolytic activity of DPP IV the artificial substrate GlyPro-pNA was used. No inhibitory effect of our anti-CD26 antibodies on the enzymatic activity of DPP IV could be observed, neither on purified soluble DPP IV nor on surface-bound DPP IV (data not shown). It should be noted, however, that a substrate with a very low molecular weight was used. The cleavage of a natural, higher molecular substrate might be inhibited by anti-CD26 mAbs by steric hindrances. Because of this restriction, the mechanism by which anti-CD26 mAbs inhibit T cell proliferation still remains an unresolved question.
48 . T. MATTERN, S. ANSORGE, H.-D. FLAD, and A.
J. ULMER
It has been reported by others that anti-CD26 mAbs did not influence the PHA-induced DNA synthesis (13-18). For these findings we have the following explanations: i) All these experiments were normally done in the presence of FCS. FCS contains soluble DPP IV, sometimes at high concentrations. Soluble DPP IV inhibits the binding of anti-CD26 mAbs to the cell surface-bound DPP IV in a competitive manner (Fig. 1). Therefore, higher quantities of anti-CD26 mAbs than used by these authors might be necessary. ii) As discussed above, anti-CD26 mAbs can arrest T cells in the cell cycle, which results in a shift of the maximum of DNA synthesis. Different culture conditions, as cell number, PHA concentration etc, may result in different kinetics of DNA synthesis. iii) The antibodies used by other authors might detect different epitopes on the enzyme. These divergent epitopes on DPP IV might have different effects on the enzymatic activity and/or the signal transduction by CD26. Also controversly discussed by different authors is the mitogenic activity of anti-CD26 mAbs. The three tested anti-CD26 mAbs described in this paper were found not to be mitogenic for human resting MNC (Fig. 1). These observations were corroborated by PLANA et al. (16) with the antiCD26 mAb 134-2C2, by Fox et al. with Tal (12), and DANG et al. with lF7 (14). The anti-CD26 mAb CB.1 could induce DNA synthesis on T cell clones in the presence of FcR+ cells (but not in the presence of monocytes), while Tal failed to do so (17,18). On the other hand, DANG et al. (15) could show that Tal is able to induce DNA synthesis in clones of T cells in the presence of FcR+ cells. The reason for all these contradictory results remains unclear. However, these data and our results indicate the participation of CD26 in T cell activation and proliferation. Acknowledgements The authors gratefully acknowledge the excellent technical assistance of Mrs. B. RIEKENS as well as the kind help in cell sorter analysis by Dr. M. ERNST and Mrs. R. BERGMANN. This work was supported by Deutsche Forschungsgemeinschaft, grant No. FlI04/6-1.
References 1. SCHON, E., H.-W. MANSFIELD, H.-U. DEMUTH, A. BARTH, and S. ANSORGE. 1985. The dipeptidyl peptidase IV, a membrane enzyme involved in the proliferation of T lymphocytes. Biomed. Biochem. Acta 44: K9. 2. ULMER, A. J., T. MATTERN, A. C. FELLER, E. HEYMANN, and H.-D. FLAD. 1990. CD26 antigen is a surface dipeptidyl peptidase IV (DPP IV) as characterized by monoclonal antibodies clone TIl 19-4-7 and 4ELlC7. Scand. J. Immunol. 31: 429. 3. HEYMANN, E. and R. MENTLEIN. 1984. BeeinfluBt Dipeptidylpeptidase IV Blutdruck und Gerinnung? Klin. Wschr. 62: 2. 4. BARTH, A. 1980. Untersuchungen zum Funktionsmechanismus der Dipeptidylpeptidase. IV. Ein Beitrag zur Chemie der enzymatischen Katalyse. Z. Chemie 20: 39. 5. SCHOLZ, W., R. MENTLEIN, E. HEYMANN, A. C. FELLER, A. J. ULMER, and H.-D. FLAD. 1985. Interleukin 2 production by human T lymphocytes identified by antibodies to dipeptidyl peptidase IV. Cell. Immunol. 93: 199.
Inhibition of T Lymphocytes by Anti-CD26 . 49 6. GRUBER, M., W. SCHOLZ, and H.-D. FLAD. 1988. Influence of human T lymphocytes identified by antibodies to dipeptidyl peptidase IV on differentiation of human B lymphocytes stimulated with Staphylococcus aureus Cowan I and pokeweed mitogen. Cell. Immunol. 113: 423. 7. MATTERN, T., W. SCHOLZ, A. C. FELLER, H.-D. FLAD, A. J. ULMER. 1991. Expression of CD26 (dipeptidyl peptidase IV) on resting and activated human T-lymphocytes. Scand. J. Immunol. 33: 737. 8. SCHON, E., E. EICHMANN, R. GRUNOW et al. 1986. Dipeptidyl peptidase IV in human T lymphocytes. An approach to the role of a membrane peptidase in immune system. Biomed. Biochem. Acta 45: 1523. 9. SCHON, E., S. JAHN, S. T. KIESSIG, H.-D. DEMUTH, K. NEUBERT, A. BARTH, R. VON BAEHR, and S. ANSORGE. 1987. The role of dipeptidyl peptidase IV in human T lymphocyte activation. Inhibitors and antibodies against dipeptidyl peptidase IV suppress lymphocyte proliferation and immunoglobulin synthesis in vitro. Eur. J. Immunol. 17: 1821. 10. SCHON, E., H.-D. DEMUTH, E. EICHMANN et al. 1989. Dipeptidyl peptidase IV in human T lymphocytes. Impaired induction of interleukin 2 and gamma interferon due to specific inhibition of dipeptidyl peptidase IV. Scand. J. Immunol. 29: 127. 11. SCHON, E., E. EICHMANN, S. JAHN, J. Kopp, H.-D. VOLK, and S. ANSORGE. 1988. On the role of dipeptidyl peptidase IV (DP IV) in the immune system. Relations to the cell cycle and lymphokine production of activated lymphocytes. Biol. Zbl. 107: 141. 12. Fox, D. A., R. E. HUSSEY, and K. A. FITZGERALD et al. 1984. Tal, a novel 105 kD human T cell activation antigen defined by a monoclonal antibody. J. Immunol. 133: 1250. 13. HAFLER, D. A., D. A. Fox, D. BENJAMIN, and H. L. WEINER. 1986. Antigen reactive memory T cells are defined by Tal. J. Immunol. 137: 414. 14. DANG, N. H., Y. TORIMOTO, and K. SUGITA et al. 1990. Cell surface modulation of CD26 by anti-1F7 monoclonal antibody. J. Immunol. 145: 3963. 15. DANG, N. H., Y. TORIMOTO, K. DEUSCH, S. F. SCHLOSSMANN, and C. MORIMOTO. 1990. Comito genie effect of solid-phase immobilized anti-1F7 on human CD4 T cell activation via CD3 and CD2 pathway. J. Immunol. 144: 4092. 16. PLANA, M., O. VINAS, O. DE LA CALLE-MARTIN et al. 1991. Induction of interleukin 2 (IL 2) and interferon-gamma and enhancement of IL 2 receptor expression by a CD26 monoclonal antibody. Eur. J. Immunol. 21: 1085. 17. FLEISCHER, B., D. J. SCHENDEL, and D. VON STELDERN. 1986. Triggering of the lethal hit in human cytotoxic T lymphocytes: a functional role for a 103-kDa T cell-specific activation antigen. Eur. J. Immunol. 16: 741. 18. SCHREZENMEIER, H. and B. FLEISCHER. 1988. A regulatory role for the CD4 and CD8 molecules in T cell activation. J. Immunol. 141: 398. 19. DANG, N. H., D. A. HARER, S. F. SCHLOSSMANN, and J. B. BREITMEYER. 1990. FcRmediated crosslinking of Tal (CDw26) induces human T lymphocyte activation. Cell Immunol. 125: 42. 20. BOYUM, A. 1968. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Lab. Clin. Invest. 21: 77. 21. Julius, M. H., E. Simpson, and L. A. HERZENBERG. 1973. A rapid method for the isolation of functional thymus-derived murine lymphocytes. Eur. J. Immunol. 3: 645. 22. BRANDT, E., A. ALTMAN, M. GRONEFELD, A. J. ULMER, and H.-D. FLAD. 1986. Functional and molecular characterization of a monoclonal antibody against human interleukin 2. Immunobiol. 172: 33. 23. ENNEN, J., M. ERNST, and H.-D. FLAD. 1989. The effect of interleukin 2 on Fe-gamma receptor function of human monocytes requires specific intercellular interaction. Immunobiol. 179: 17. 24. POSCHEL, G., R. MENTLEIN, and E. HEYMANN. 1982. Isolation and characterization of dipeptidyl peptidase IV from human placenta. Eur. J. Biochem. 126: 359. 25. MATSUI, Y., H. M. SHAPIRO, M. J. SHEEHY et al. 1986. Differential expression of T cell differentiation antigens and major histocompatibility antigens on activated T cells during the cell cycle. Eur. J. Immunol. 16: 248. 26. NECKERS, L. M. and J. COSSMAN. 1983. Transferrin receptor induction in mitogen-
50 . T. MATTERN, S. ANSORGE, H.-D. FLAD, and A.
J. ULMER
stimulated human T lymphocytes is required for DNA synthesis and cell division and is regulated by interleukin 2. Proc. Nat!' Acad. Sci. USA 80: 3494. 27. STADLER, B. M., S. F. DOUGHERTY,].]. FARRAR, and].]. OPPENHEIM. 1981. Relationship of cell cycle to recovery of IL 2 activity from human mononuclear cells, human and mouse T cell lines. J. Immuno!. 127: 1936. 28. BURDACH, S., N. ZESSACK, D. DILLOO, M. SHATSKY, D. THOMPSON, and L. LEVITT. 1991. Differential regulation of lymphokine production by distinct subunits of the T cell interleukin 2 receptor. ]. Clin. Invest. 87: 2114. Dr. ARTHUR]. ULMER, Forschungsinstitut Borstel, Parkallee 22,2061 Borstel, Germany
of Immunology and Cell Biology, Forschungsinstitut Borstel, Borstel, and Department of Internal Medicine, Medical Academy of Magdeburg, Magdeburg, Germany
Anti-CD26 Monoclonal Antibodies Can Reversibly Arrest Human T Lymphocytes in the Late G1 Phase of the Cell Cycle TAILAMATTERN1, SIEGFRIEDANSORGE2 , HANS-DIETERFLAD1, and ARTUR J. ULMER1 Received AprilS, 1992 . Accepted in Revised Form October 12, 1992
Abstract Three different anti-CD26 monoclonal antibodies (mAbs) are described, which specifically inhibited proliferation of human T lymphocytes after stimulation with PHA, tetanus toxoid or soluble anti-CD3 mAb. Anti-CD26 mAbs induced in T cells a dose-dependent shift of the maximum of DNA synthesis, which was due to a transitory arrest of cells in the cell cycle. This cell cycle arrest was found to occur in the late phase of G 1 since the expression of the T cell activation markers CD25-, CD71-, and HLA-DR-positive cells was the same in anti-CD26 mAb-containing and control cultures. Propidium iodide staining further confirmed the assumption that the arrest occurs in the first round of the cell cycle before S phase cells were detectable. Because the cells were arrested before consuming IL-2 in the S phase, we observed in accumulation of IL-2 in anti-CD26 mAb-containing cultures, whereas IFN-gamma production by PHA-activated T lymphocytes was reduced. These data indicate that CD26 is involved in the processes of T cell activation and proliferation.
Introduction Recently, a new surface marker of human T cells was described, the T cell activation marker CD26, which was thought to be likewise involved in processes of T cell activation (1). CD26 was found to be identical to the ectoenzyme dipeptidyl peptidase IV (DPP IV, E.C. 3.4.14.5) (2). This enzyme can be demonstrated in several mammalian tissues (3), but within the hematopoetic system only on T lymphocytes. DPP IV was characterized by a highly restricted substrate specificity, i.e. cleavage of dipeptides from the free amino terminus of peptides after L-proline or L-alanine (4). Abbreviations: DPP IV = dipeptidyl peptidase IV; FCS = fetal calf serum; GCSF = granulocyte colony-stimulating factor; Gly-Pro pNA = glycylproline-p-nitroanilide; 3HTdR = eH)thymidine; IFN-gamma = interferon-gamma; IL-l{3 = interleukin 1~; IL-2 = interleukin 2; IL-6 = interleukin 6; L T = lymphotoxin; MNC = mononuclear cells; mAbs = monoclonal antibodies; PHA = phytohemagglutinin
Inhibition of T Lymphocytes by Anti-CD26 . 37
Although the natural substrate of DPP IV is still unknown, there exist, with regard to their amino acid sequences, many hypothetical substrates for this enzyme within the immune system, e.g. interleukin 1B(IL-1 B), interleukin 6 (IL-6), granulocyte colony-stimulating factor (GCSF), lymphotoxin (LT), or substance P. During the last years we have phenotypically and functionally characterized CD26-positive and -negative T cells. While the expression of CD26 is associated with the ability to produce high amounts of IL-2 and IFNgamma and to proliferate considerably after PHA stimulation (5), CD26negative T cells showed better helper functions for the differentiation and Ig synthesis of B cells after pokeweed mitogen stimulation (6). Our studies further revealed that CD26 is expressed de novo on CD26negative as well as on CD26-positive T cells after stimulation with various mitogens or antigens (7). From comparative studies regarding the expression of other T cell activation markers like the IL-2 receptor and HLA-DR, we assumed that CD26 arises during G 1 of the cell cycle after the expression of IL-2 receptor and HLA-DR. These studies and further investigations with specific inhibitors of DPP IV by SCHON et al. (1, 8-11) led to the assumption that CD26 may be directly involved in the activation of T cells. Other authors have examined the effects of various anti-CD26 mAbs on T cell stimulation and have found different effects: Neither mitogenic nor comito genic effects have been described for the anti-Tal mAb (12, 13). Costimulatory effects of anti-CD26 mAbs in an IL-2/IL-2 receptor-dependent pathway have been found by others (14-16). FLEISCHER et al. (17, 18) and DANG et al. (19) reported on the mitogenic effects of anti-CD26 mAbs and on the activation of effector functions of cytotoxic T cells. We have now investigated the influence of two other anti-CD26 mAbs on the proliferation of human T cells in vitro.
Material and Methods Isolation of cells Human peripheral blood mononuclear cells (MNC) were isolated from heparinized blood of healthy donors by density gradient centrifugation, as described elsewhere (20). For some experiments, T lymphocytes were isolated by adherence of MNC to nylon wool (Leuko Pak, Fenwal Laboratories, Morton Grove, IL, USA) in a 1 x 20 cm column (Bio-Rad Laboratories, Miinchen, Germany) according to the methods described by JULIUS et al. (21). In brief, 1 x 10 8 MNC in 3 ml Hanks' balanced salt solution (BSS) supplemented with 10% heat-inactivated (56 DC, 30 min) fetal calf serum (FCS) were loaded on the column at room temperature. Subsequently, the column was rinsed with 25 ml Hanks' BSS + 10 % FCS at a flow rate of about 0.5 mllmin to elute non-adherent cells. These non-adherent cells consisted of more than 97 % CDr T lymphocytes. Monocytes were isolated from MNC by adherence to disposable falcon tissue culture flasks. The non-adherent cells were discarded, and the adherent cells were isolated by being scraped off with a rubber policeman in ice-cold RPM!. More than 95 % of the adherent cells were monocytes as determined by cytochemical evaluation of the unaphthyl acetate esterase pattern.
38 . T. MATTERN, S. ANSORGE, H.-D. FLAD, and A.
J. ULMER
Cell culture MNC, T cells or T cells + 10 % autologous adherent cells were suspended at a concentration of 2 x lOs Iml for stimulation with PHA and 1 x 106 Iml for stimulation with soluble anti-CD3 or tetanus toxoid, respectively, in RPMI 1640 medium supplemented with antibiotics and 10 % FCS. Because FCS (as well as human serum) contains soluble CD26, which itself binds the anti-CD26 antibodies and thus requires relative high concentrations of antibodies, a batch of FCS was used which contains only low amounts of soluble CD26. Cells were cultured at a final volume of 50 [!l/well in half area microtiter plates (Costar, Cambridge, MA, USA). The cells were stimulated by phytohemagglutinin (PHA 1 flg/ml, Wellcome, Beckenham, Kent, UK), soluble anti-CD3 (BMA 030, 12.5 ng/ml, Behringwerke AG, Marburg, Germany), or tetanus toxoid (1 U/ml, Behringwerke AG). The anti-CD26 mAb Til 19-4-7 (ascites, kindly provided by Prof. FELLER, Inst. of Pathology, University of Wiirzburg, Germany), anti-Tal (4ELlC7, purified antibody, Coulter Clone, Hialeah, FL, USA), and M5 (ascites) were added to the cultures at different times and concentrations. After incubation for 1 to 4 d the cultures were pulsed with 37 kBq (1 [!Ci) eHJthymidine eHTdR) per well for the last 8 h of culture. Cells were harvested onto glass filters, and 3HTdR incorporation was determined by scintillation counting. The results represent the mean of triplicate wells, and SD were less than 15 %. Induction and detection of lymphokines Cells were stimulated and cultured as described above. After the indicated culture period, supernatants were harvested for lymphokine determination. IL-2 activity was determined by measuring the proliferation of the IL-2-dependent CTL-6 line, as described elsewhere (22). IFN-gamma production was measured in an ELISA system, as described in more detail by ENNEN et al. (23). Anti-human IFN-gamma mAb, Clone 69, was a generous gift from Dr. GALLATI (Hoffmann-LaRoche, Basel, Switzerland). Immunofluorescence staining of cells The following antibodies were used: Anti-CD25: Tue 69-FITC, Biotest, Offenbach, Germany; anti-CD71: anti-human transferrin receptor FITC, Becton Dickinson, San Jose, CA, USA; Ki-67: kindly provided by Dr. J. GERDES, Forschungsinstitut Borstel, Germany; anti-HLA-DR: anti-human HLA-DR FITC, Becton Dickinson; FITC labelled F(ab'h fragments of goat anti-mouse IgG Oackson Immunoresearch Laboratories, West Grove, PA, USA). For immunofluorescence staining, cells were harvested and labelled with monoclonal antibodies and analyzed in a cell sorter. Direct or indirect immunofluorescence staining was performed in ice-cold PBS (containing 0.1 % sodium azide) using the mAbs at optimal concentrations. After the first incubation with the specific mAb for 20 min the cells were washed by centrifugation on a FCS gradient (200 x g, 10 min), resuspended in 100 [!l PBS, and, in case on indirect staining, incubated for further 20 min in presence of the second FITC labelled antibody. The cells were washed again in a FCS gradient and resuspended in PBS. Labelled cells were analyzed in a Cytofluorograf (System SOH, Ortho Diagnostic System, Inc., Westwood, MA, USA). Propidium iodide staining of cells 1 x 106 MNC were suspended in 200 [!l of citrate buffer, pH 7.6 (250 mM sucrose, 40 mM trisodiumcitrate x 2 H 2 0, 5 % v/v DMSO in distilled water), and stored at -80°C until analysis. 1800 [!l of trypsin solution, pH 7.6 (0.03 mg/ml trypsin [Sigma T-0134J in 3.4 mM trisodiumcitratex2 H 2 0, 0.1 % v/v Nonidet P-40 [Sigma N-3516J, 1.5 mM spermine tetrahydrochloride [Sigma S-2876J, 0.05 mM Tris in distilled water), were mixed with the thawed cells, and the trypsin was allowed to react for 10 min at room temperature. Then 1500 [!l of trypsin inhibitor (0.5 mg/ml trypsin inhibitor [Sigma T-9253J and 0.1 mg/ml ribonuclease A [Sigma R-4875] in 3.4 mM trisodiumcitrate x 2 H 2 0, 0.1 % v/v Nonidet P-40 [Sigma N3516J, 1.5 mM spermine tetrahydrochloride [Sigma S-2876J, 0.05 mM Tris in distilled water,
Inhibition of T Lymphocytes by Anti-CD26 . 39 pH 7.6) were mixed with the cells and incubated for further 10 min at room temperature. Cells were then washed by centrifugation (10 min, 4°C, 1800 x g) and resuspended in 350 ftl citrate buffer. They were mixed with 150 ftl of a propidium iodide solution (0.4 mg/ml propidium iodide [Sigma P-4170J, in 3.4 mM trisodiumcitrate x 2 H 2 0, 0.1 % v/v Nonidet P-40 [Sigma N-3516J, 5 mM spermine tetrahydrochloride [Sigma S-2876J, 0.05 mM Tris in distilled water, pH 7.6), wrapped with tinfoil for light protection and kept for 15 min on ice bath. Cells were washed again and measured with 3 h after addition of propidium iodide. Cells were measured in a Cytofluorograf (System SOH, Ortho Diagnostic System, Inc.). For internal G OIl standard, frozen MNC of the same donor were used.
Results
Inhibition of proliferation by anti-CD26 antibodies Human MNC were stimulated polyclonally with PHA or soluble antiCD3 or monoclonally with the recall antigen tetanus toxoid, respectively. The anti-CD26 mAbs TIl 19-4-7, Tal or M5 were given at the beginning of the cultures. As shown in Table 1, upper rows, the addition of anti-CD26 mAb resulted in a clear and significant (p < 0.0027) inhibition of DNA synthesis. Crosslinking of CD26 by means of anti-CD26 mAb plus goat anti-mouse F(ab')2 fragments did not influence the inhibition affected by anti-CD26 mAb (Table 1, middle part). Goat anti-mouse fragments themselves were not mitogenic and did not influence PHA-induced DNA synthesis (data not shown). Similar to these results, we found that depletion of adherent cells or addition of a defined number of adherent cells to T cells did not influence the inhibitory capacity of the anti-CD26 mAbs (Table 1, lower rows), indicating that crosslinking of the CD26 molecules by Fc Table 1. Anti-CD26-mediated inhibition of DNA synthesis of MNC, T cells, or T cells plus adherent cells after polyclonal or monoclonal stimulation
+ anti CD26
Stimulus
cells
No. of Exp.
- anti CD26
PHA anti CD3 tet. toxoid
MNC MNC MNC
10 10 5
16.7 ± 5.3 10.9 ± 6.0 8.6 ± 8.4
5.3 ± 2.5'" 2.5 ± 2.1" 1.9 ± 1.4'"
PHA
MNC
3
34.2 ± 5.5
11.0 ± 2.5'"
PHA PHA PHA
MNC T + 10% Adh. T cells
3 3 3
15.9 ± 6.3 12.6 ± 4.7 9.4 ± 5.2
9.4 ± 2.6'" 7.4 ± 3.2'" 3.6 ± 1.Y
+ anti CD26 + GaM n.t. n.t. n.t. 12.2 ± 3.1 n.t. n.t. n.t.
Human peripheral MNC, T cells plus 10 % autologous, adherent cells were stimulated with PHA (1 ftg/ml, 2 x lOS cells/ml), soluble CD3 (BMA 030, 12.5 ng/ml, 1 x 106 cells/ml), or tetanus toxoid (1 U/ml, 1 x 106 cells/ml). The cells were cultured with or without anti-CD26 mAbs (TIl 19-4-7, 1:50 ascites dilution, Tal, 10 ftg/ml, or M5, 1:200 ascites dilution). For crosslinking experiments, PHA -stimulated cells were cultured in the presence or absence of anti-CD26 (TIl 19-4-7, 1:50 ascites dilution) and goat anti-mouse F(ab' 2) fragments (GaM, 1:100). DNA synthesis was measured after 96 h of culture by 3HTdR incorporation for the last 8 h of culture. ". p < 0.0027
.40 . T. MATTERN, S. ANSORGE, H.-D. FLAD, and A.
fo
J. ULMER
. - . PHA + TII19-4-7 + DPP fII A- -A PHA + DPP fII 20 0 - 0 TII19-4-7 + DPP fII ll. - -ll. DPP fII alone
A-
10
_-A...
.___..
... "'A7-X- - -A- - -A
.--.................
o o
20
50
100
200
500
DPP fII concentration (ng/ml) Figure 1. Effect of DPP IV on the inhibitory activity of anti-CD26 mAbs on PHA-induced proliferation. Peripheral MNC (2 x 10s/ml) were stimulated with PHA (1 flg/ml) in the presence or absence of anti-CD26 mAb TIl 19-4-7 (1 :50, ascites dilution). In parallel cultures purified DPP IV from human placenta was added in concentrations as indicated. DNA synthesis was measured on day 4 of culture by 3HTdR incorporation for the last 8 h of culture.
receptor-positive adherent cells was not required for inhibition. All three anti-CD26 mAbs tested were found to reduce proliferation in a dosedependent manner (data not shown).
Specificity of inhibition To prove the specificity of the inhibition of T cell proliferation mediated by the anti-CD26 mAbs, purified DPP IV was used as an antagonist to antiCD26 mAbs. As shown in Figure 1, the inhibition of DNA synthesis after PHA stimulation by anti-CD26 mAbs could be neutralized by addition of increased concentrations of the purified antigen DPP IV. Amounts of 100 ng/ml purified DPP IV totally neutralized the inhibition affected by TIl 194-7 (1 :50 ascites dilution, corresponding to 5 ~g/ml total protein). In control experiments we could show that anti-CD26 mAbs alone or antiCD26/DPP IV immune complexes were not mitogenic for T lymphocytes. Furthermore, soluble DPP IV itself neither influenced PHA-induced DNA synthesis nor was able to stimulate proliferation by itself at all concentrations tested (Fig. 1). Unspecific cytotoxicity of the anti-CD26 mAbs could be excluded, since the viability of the cells after 96 h of culture with or without anti-CD26 mAbs was not affected and furthermore, anti-CD26 mAbs did not inhibit the proliferation of the CD26-negative cell lines Wehi and K562 (data not given). Kinetics of DNA synthesis after PHA stimulation and addition of anti-
CD26 mAbs Experiments were carried out to examine the effects of anti-CD26 mAbs on the kinetics of DNA synthesis after PHA-induced T cell activation (Fig.
Inhibition of T Lymphocytes by Anti-CD26 . 41
0-0 PHA alone
o
20
. - . PHA + 111 19-4-
'/'0('"·"
0~.. . . . . . /0
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10
5
days of culture Figure 2. Kinetics of DNA synthesis after stimulation of MNC with PHA in absence or presence of anti-CD26 mAbs. Human peripheral MNC (2 x lOs/ml) were stimulated with PHA (1 ltg/ml). Anti-CD26 mAbs (TIl 19-4-7, 1:50, ascites dilution, Tal, 10 ltg/ml, or M5, 1:200, ascites dilution) were added at the beginning of the culture. For control Ig isotypespecific antibodies (both in a 1:20 dilution) were added. DNA synthesis was measured after a culture time as indicated by 3HTdR incorporation for the last 8 h.
2). DNA synthesis after PHA stimulation in the absence of anti-CD26 mAbs showed a maximum on day 4 of culture and then diminished. The addition of anti-CD26 mAbs resulted in a shift of DNA synthesis. The proliferation was now maximal on day 5 or 6 of culture. It should be emphasized that DNA synthesis is significantly inhibited by anti-CD26 mAbs when investigated on day 4 of culture (see also Table 1). On the other hand, analysis of DNA synthesis on day 5 or 6 of culture shows an enhancement caused by anti-CD26. Similar results were found in experi-
42 . T. MATTERN, S. ANSORGE, H.-D. FLAD, and A.
ULMER
~
0
WOIhed after 8811
0
20
i
10
0
J.
o"0"-e__e
~ 0
2
3
4-
'-0---
S
6
7
8
9
10
days of culture Figure 3. Kinetics of PHA-induced DNA synthesis of human MNC with and without antiCD26 mAbs and after washing off the antibody effects. Human peripheral MNC (2 x 10s/ml) were stimulated with PHA (1 f!g/ml). The anti-CD26 mAb TIl 19-4-7 (1:50, ascites dilution) was added at the beginning of culture. After 24 h, 48 h, or 96 h, the cells (of the antibody-free as well as of the antibody-containing cultures) were harvested, washed 2 times in fresh medium, and then recultured in fresh, PHA containing medium. DNA synthesis was measured after various culture times by 3HTdR incorporation for the last 8 h. A: Kinetics of DNA synthesis with and without anti-CD26 mAbs in undisturbed cultures. B: Kinetics of DNA synthesis with or without anti-CD26 mAbs in cultures, washed after 24 h of culture. C: Kinetics of DNA synthesis with or without anti-CD26 mAbs in cultures, washed after 48 h of culture. For details see above. D: Kinetics of DNA synthesis with or without anti-CD26 mAbs in cultures, washed after 96 h of culture.
ments with the recall antigen tetanus toxoid or soluble anti-CD3 antibody. In Figure 2 we further demonstrate that isotype-specific control antibodies could not mimick the effects of anti-CD26 mAbs. These experiments strongly indicate that the effects of anti-CD26 mAbs were specific for the CD26 antigen. To investigate the reversibility of the effect of anti-CD26 mAbs, a twostep culture system was employed. Cells were incubated with PHA in the
presence or absence of anti-CD26 mAbs, washed after different culture times, and then recultured with fresh PHA -containing medium. The kine-
Inhibition ofT Lymphocytes by Anti-CD26 . 43
tics of DNA synthesis after this procedure were measured. Figure 3 shows the typical results of such an experiment. Cells incubated in undisturbed cultures show the kinetics of DNA synthesis, as described above, with a shift of maximal proliferation from day 4 to day 6 of culture in the presence of the anti-CD26 mAb TIl 19-4-7 (Fig. 3A). Cells, prestimulated with PHA for 24 or 48 h, then washed and cultured further in presence of PHA, show a maximum of DNA synthesis on day 5 of culture independently of whether they were precultured in presence of anti-CD26 mAbs or not (Fig. 3B and C). On the other hand, when cells were preincubated for four days in the presence of anti-CD26 mAb-containing medium and then washed, kinetics of DNA synthesis showed the same shift as undisturbed cultures (Fig. 3D). In contrast, when new anti-CD26 mAbs were added daily to PHAstimulated cultures, we found a clear and nearly complete inhibition of DNA synthesis without affecting the viability of the cells (data not given).
L ymphokine production after addition of anti-CD26 mAbs These experiments were carried out to investigate whether the observed shift in the DNA synthesis after anti-CD26 mAb addition is due to a reduced lymphokine production. MNC were stimulated with PHA and cultured with or without anti-CD26 mAbs for various times. Production of IL-2 was measured in a CTL-6 proliferation assay, while IFN-gamma production was measured in an ELISA system. Table 2 shows the results of five independent experiments. Cultures with anti-CD26 mAb Tal reveal again the shift in the kinetics of DNA synthesis (data not given). To our surprise IL-2 release was enhanced in cultures with anti-CD26 mAbs at all culture times tested. In contrast, IFN-gamma release was found to be reduced by addition of anti-CD26 mAbs up to day 5 of culture. However,
Table 2. Effects of anti-CD26 mAbs on the kinetics of PHA-induced IL-2 and IFN-gamma production days of culture Lymphokine
anti CD26
IL-2 (Ulml)
+ IFN-y (ng/ml)
-
+
2
3
4
6
5±1 6±1
11 ± 1 11 ± 1
9±3 12 ± 6
6±2 17± 8
2±1 7±2
0 5±2
0 3± 1
3±2 1± 1
6±3 2±1
12 ± 8 4±3
18 ± 9 6±3
10 ± 5 8±4
8±3 10 ± 2
4±1 10 ± 3
5
8
Human peripheral MNC (2 x 10 s/ml) were stimulated with PHA (1 flg/ml) in the presence or absence of anti-CD26 mAb (TIl 19-4-7, 1:50 ascites dilution, Tal, 10 flg/ml, or M5 1:200 ascites dilution). The supernatants were harvested after different culture periods. IL-2 production was measured in the CTL-6 assay and IFN-gamma production in an ELISA system. Data are expressed as mean of five independent experiments ± SEM.
44 . T. MATTERN, S. ANSORGE, H.-D. FLAD, and A.
0-0 PHA alone . - . PHA + Tal 50
g .!!
"ii (.)
0
GI
.. '0 .. .! .2:
~
0
50
Q.
E ;:I z
0
/
A
~.
--
.~
."".,,-._e-==e CD25
Q' -<;2 PHA alone . - . PHA + Tal
B
~~7e=::::::8
e--==i~ CD71
O-Q PHA alone . - . PHA+Tal
50
o
e..,.:::::::::::e
J. ULMER
~-
o
e HLA-DR
i-i--' e-e-=i:::::::::::-2
3
4
5
6
days of culture
Figure 4. Kinetics of the expression of different T cell activation markers on PHA-stimulated MNC after addition of anti-CD26 mAbs. A: Kinetics of CD25 expression (IL-2 receptor). B: Kinetics of CD71 expression (transferrin receptor). C: Kinetics of HLA-DR expression. For cell staining see Materials and Methods.
it remained detectable at that low level during later culture times, whereas it dropped in control cultures with PHA alone. Influence of anti-CD26 mAbs on the cell cycle The observation that IL-2 production after anti-CD26 addition was enhanced and not inhibited might indicate that the cells could be preactivated by PHA in a normal way, but were stopped somewhere in the cell cycle before DNA synthesis occurred. We have, therefore, investigated the effects of anti-CD26 mAbs on the expression of typical T cell activation markers like CD25, CD71, or HLA-DR, and the DNA content on the single cell level (measured by propidium iodide staining). Figures 4A, B, and C show the kinetics of CD25, CD71 or HLA-DR expression, respectively. Although DNA synthesis was shifted, as normally found by addition of anti-CD26 mAbs (data not given), the cells from cultures with or without anti-CD26 mAbs and PHA were not distinguishable with respect to their expression of CD25, CD71, or HLA-DR. Also no differences between anti-CD26 mAb-treated and untreated cells could be observed with respect to the density of the activation markers on the cell surface (data not shown). Anti-CD26 mAbs alone were not able to induce expression of activation markers (data not shown). These data indicate that the cells seemed to be stopped by anti-CD26 mAbs within the cell cycle after expression of the IL-2 receptor CD25 (early G 1 phase), HLA-DR and the
Inhibition ofT Lymphocytes by Anti-CD26 . 45 Table 3. Cell cylce analysis of PHA-stimulated cells, cultured with or without anti-CD26 mAbs, measured by propidium iodide staining days of culture anti CD26
0
G OIl phase positive cells
+
99.3% 99.3%
S phase positive cells
+
G 2 phase positive cells
+
2
4
94.5% 99.3%
87.7% 97.4%
78.9% 87.9%
0.5% 0.5%
5.1 % 0.5%
10.9% 2.2%
16.6% 9.7%
0.2% 0.2%
0.4% 0.2%
1.4 % 0.4%
4.5% 2.4%
Human peripheral MNC (2 x lOs/ml) were stimulated with PHA (1 ltg/ml) in the presence of anti-CD26 mAb (Tal, 10 ltg/ml). Cells were harvested and stained with propidium iodide as described in Materials and Methods.
transferrin receptor CD71 (late G 1 phase), but prior to DNA synthesis in the S phase of the cell cycle. To verify this assumption, we stained the cells with propidium iodide (Table 3). Cells were frozen after culture at -80°C and stained simultaneously after thawing with propidium iodide. For internal Go standard, fresh MNC of the same donor were frozen and stained with propidium iodide. At day zerQ all cells were found to be in the G OIl phase of the cell cycle. After 24 h of culture with PHA alone the number of cells within the S phase slowly increased, and after further 24 h the first cells within the G2 phase were detectable. To the contrary, cells stimulated with PHA and anti-CD26 mAbs showed a clearly prolonged G OIl phase of the cell cycle. Cells within the S phase were for the first time detectable after 48 h of culture, while G 2 cells were first detectable after 96 h of culture. Discussion CD26 (or DPP IV) is an activation marker on T lymphocytes and thought to be involved in the processes of T cell activation. In this report we demonstrate the inhibitory effects of three different anti-CD26 mAbs (TIl 19-4-7, Tal, and MS) on the processes of PHA-induced T cell activation, as measured by DNA synthesis, cytokine production, expression of activation markers, and cell cycle analysis. We could show that the addition of anti-CD26 mAbs together with PHA resulted in a significant and donor-independent inhibition, as proven by several individual experiments shown in Table 1. The inhibition was not mediated by monocytes or by crosslinking via Fe receptor binding of mAb, since purified T lymphocytes show the same or even enhanced reduction of DNA synthesis as MNC or T lymphocytes plus adherent cells.
46 . T.
MATTERN,
S.
ANSORGE,
H.-D.
FLAD,
and
A.
J. ULMER
The specificity of the anti-proliferative effects of anti-CD26 mAbs is supported by the following points: i) The inhibition was not due to unspecific toxic effects of the antibody solutions themselves, because neither was the viability of cells influenced by anti-CD26 mAbs nor was the DNA synthesis of CD26-negative cell lines affected (data not given). ii) The inhibition was a specific event of the binding between surface-bound CD26 and anti-CD26 mAbs, because normal levels of DNA synthesis could be reconstituted by the addition of purified soluble DPP IV from human placenta in a dose-dependent way (Fig. 1). iii) We could further show that anti-CD26 mAbs did not influence thymidine incorporation, because addition of anti-CD26 mAb together with 3HTdR at day 4 of PHA-stimulated cultures did not result in a decreased 3HTdR incorporation (data not given). iv) Washing-off the added antibodies after 24 or 48 h of culture resulted in the same response as in control cultures without anti-CD26 mAbs (Fig. 3). v) IL-2 production (Table 2) and the expression of CD25, CD71, or HLADR (Fig. 4) were not inhibited by anti-CD26 mAbs. The manifestation of the inhibitory effect of anti-CD26 mAbs could be clarified in more detail by kinetic studies. All three anti-CD26 mAbs tested caused a shift of DNA synthesis after stimulation with PHA (Fig. 2). On the basis of these experimental data we raised the following hypothesis: - The binding of anti-CD26 mAbs to surface CD26 on resting or preactivated T cells causes the cells to rest in their cell cycle. Anti-CD26 mAbs were exhausted by internalization or shedding of the anti-CD26 mAb/ CD26 complexes. After reexpressing new CD26 on their surface, arrested cells in the absence of sufficient amounts of anti-CD26 mAbs became able to pass through the cell cycle in a normal manner. This hypothesis was verified by the following results. The daily addition of anti-CD26 mAbs resulted in a total reduction of DNA synthesis without affecting the viability of the cells (data not shown). Furthermore, we were not able to detect specific antibodies in the supernatants of T cells, stimulated for 48 h in the presence of PHA and anti-CD26 mAbs, as measured in an ELISA system with solid-phase bound CD26 (data not shown). To determine the time point at which anti-CD26 mAbs were effective in preventing stimulation of DNA synthesis, kinetic experiments were performed. After different times of culture the anti-CD26 mAbs were washed out, and the cells were further cultured only in the presence of PHA (Fig. 3). This experiment indicates that anti-CD26 mAbs were effective during a period preceding DNA synthesis after PHA stimulation, but that they did not affect an early event (e.g. signal transduction for activation). The assumption that no early signal transduction but a later event was involved, was confirmed by the observation that the release of IL-2 (Table 2) and the expression of CD25, CD71, and HLA-DR (Fig. 4) were not reduced. All these events have been reported to occur during distinct points of the G 1 phase of the cell cycle (25-27). Therefore, anti-CD26 mAbs have an effect in the late G 1 phase before DNA synthesis (S phase).
Inhibition of T Lymphocytes by Anti-CD26 . 47
This assumption was directly confirmed by cell cycle analysis, using propidium iodide staining of the DNA. In the absence of anti-CD26 mAbs we can already show after 24 h of PHA stimulation detectable amounts of cells in the S phase (Table 3). To the contrary, nearly no S phase cells were detectable in anti-CD26 mAb-treated cells within the first 48 h after PHA stimulation. This lack of Grpositive cells in anti-CD26 mAbs-containing cultures during the first 48 h of culture indicates that the cells were arrested in the first round of the cell cycle. It is notable that the expression of the transferrin receptor, CD71, which is thought to be the «point of no return» (26), was not affected. The uncoupling of transferrin receptor expression from DNA synthesis by anti-CD26 mAbs refers to an undefined restriction point in the cell cycle, which may occur after transferrin receptor expression in the late G b but prior to the initiation of DNA replication in S phase. IL-2 and IFN-gamma release by PHA-stimulated T lymphocytes were affected by anti-CD26 mAbs in an opposite way. The content of IL-2 in anti-CD26 mAbs-containing cultures was enhanced during all days investigated, and no shift of the kinetics of IL-2 release was observed (Table 2). We assume that cells cultured with anti-CD26 mAbs consume less IL-2, because IL-2 is known to be produced in the late G 1 phase of the cell cycle and is consumed in the early S phase (27). As the T cells were arrested by anti-CD26 mAbs prior to the S phase, we have reason to assume that they do not consume but accumulate IL-2. In contrast to IL-2, IFN-gamma release induced by PHA was inhibited by the addition of anti-CD26 mAbs (Table 2). However, contrary to cultures without anti-CD26 mAbs more IFN-gamma was detectable on day 6 to 7 of culture. The production of IFN-gamma mRNA and the release of IFN-gamma are known to be IL-2dependent (28). While we could show that IL-2 was enriched in the supernatants of cells cultured in the presence of anti-CD26 mAbs, the reduced IFN -gamma content could not be simply explained by a lack of IL2 production. Specific synthetic DPP IV inhibitors, which inhibit the hydrolytic activity of this enzyme, have previously been reported to inhibit PHA-induced DNA synthesis (8, 9). This observation indicates that the enzymatic activity of CD26 is involved in the process of cell cycling (11). To look into the mechanism of the inhibitory action of anti-CD26 mAbs, we have tested whether these antibodies could inhibit the enzymatic activity of DPP IV. For testing the hydrolytic activity of DPP IV the artificial substrate GlyPro-pNA was used. No inhibitory effect of our anti-CD26 antibodies on the enzymatic activity of DPP IV could be observed, neither on purified soluble DPP IV nor on surface-bound DPP IV (data not shown). It should be noted, however, that a substrate with a very low molecular weight was used. The cleavage of a natural, higher molecular substrate might be inhibited by anti-CD26 mAbs by steric hindrances. Because of this restriction, the mechanism by which anti-CD26 mAbs inhibit T cell proliferation still remains an unresolved question.
48 . T. MATTERN, S. ANSORGE, H.-D. FLAD, and A.
J. ULMER
It has been reported by others that anti-CD26 mAbs did not influence the PHA-induced DNA synthesis (13-18). For these findings we have the following explanations: i) All these experiments were normally done in the presence of FCS. FCS contains soluble DPP IV, sometimes at high concentrations. Soluble DPP IV inhibits the binding of anti-CD26 mAbs to the cell surface-bound DPP IV in a competitive manner (Fig. 1). Therefore, higher quantities of anti-CD26 mAbs than used by these authors might be necessary. ii) As discussed above, anti-CD26 mAbs can arrest T cells in the cell cycle, which results in a shift of the maximum of DNA synthesis. Different culture conditions, as cell number, PHA concentration etc, may result in different kinetics of DNA synthesis. iii) The antibodies used by other authors might detect different epitopes on the enzyme. These divergent epitopes on DPP IV might have different effects on the enzymatic activity and/or the signal transduction by CD26. Also controversly discussed by different authors is the mitogenic activity of anti-CD26 mAbs. The three tested anti-CD26 mAbs described in this paper were found not to be mitogenic for human resting MNC (Fig. 1). These observations were corroborated by PLANA et al. (16) with the antiCD26 mAb 134-2C2, by Fox et al. with Tal (12), and DANG et al. with lF7 (14). The anti-CD26 mAb CB.1 could induce DNA synthesis on T cell clones in the presence of FcR+ cells (but not in the presence of monocytes), while Tal failed to do so (17,18). On the other hand, DANG et al. (15) could show that Tal is able to induce DNA synthesis in clones of T cells in the presence of FcR+ cells. The reason for all these contradictory results remains unclear. However, these data and our results indicate the participation of CD26 in T cell activation and proliferation. Acknowledgements The authors gratefully acknowledge the excellent technical assistance of Mrs. B. RIEKENS as well as the kind help in cell sorter analysis by Dr. M. ERNST and Mrs. R. BERGMANN. This work was supported by Deutsche Forschungsgemeinschaft, grant No. FlI04/6-1.
References 1. SCHON, E., H.-W. MANSFIELD, H.-U. DEMUTH, A. BARTH, and S. ANSORGE. 1985. The dipeptidyl peptidase IV, a membrane enzyme involved in the proliferation of T lymphocytes. Biomed. Biochem. Acta 44: K9. 2. ULMER, A. J., T. MATTERN, A. C. FELLER, E. HEYMANN, and H.-D. FLAD. 1990. CD26 antigen is a surface dipeptidyl peptidase IV (DPP IV) as characterized by monoclonal antibodies clone TIl 19-4-7 and 4ELlC7. Scand. J. Immunol. 31: 429. 3. HEYMANN, E. and R. MENTLEIN. 1984. BeeinfluBt Dipeptidylpeptidase IV Blutdruck und Gerinnung? Klin. Wschr. 62: 2. 4. BARTH, A. 1980. Untersuchungen zum Funktionsmechanismus der Dipeptidylpeptidase. IV. Ein Beitrag zur Chemie der enzymatischen Katalyse. Z. Chemie 20: 39. 5. SCHOLZ, W., R. MENTLEIN, E. HEYMANN, A. C. FELLER, A. J. ULMER, and H.-D. FLAD. 1985. Interleukin 2 production by human T lymphocytes identified by antibodies to dipeptidyl peptidase IV. Cell. Immunol. 93: 199.
Inhibition of T Lymphocytes by Anti-CD26 . 49 6. GRUBER, M., W. SCHOLZ, and H.-D. FLAD. 1988. Influence of human T lymphocytes identified by antibodies to dipeptidyl peptidase IV on differentiation of human B lymphocytes stimulated with Staphylococcus aureus Cowan I and pokeweed mitogen. Cell. Immunol. 113: 423. 7. MATTERN, T., W. SCHOLZ, A. C. FELLER, H.-D. FLAD, A. J. ULMER. 1991. Expression of CD26 (dipeptidyl peptidase IV) on resting and activated human T-lymphocytes. Scand. J. Immunol. 33: 737. 8. SCHON, E., E. EICHMANN, R. GRUNOW et al. 1986. Dipeptidyl peptidase IV in human T lymphocytes. An approach to the role of a membrane peptidase in immune system. Biomed. Biochem. Acta 45: 1523. 9. SCHON, E., S. JAHN, S. T. KIESSIG, H.-D. DEMUTH, K. NEUBERT, A. BARTH, R. VON BAEHR, and S. ANSORGE. 1987. The role of dipeptidyl peptidase IV in human T lymphocyte activation. Inhibitors and antibodies against dipeptidyl peptidase IV suppress lymphocyte proliferation and immunoglobulin synthesis in vitro. Eur. J. Immunol. 17: 1821. 10. SCHON, E., H.-D. DEMUTH, E. EICHMANN et al. 1989. Dipeptidyl peptidase IV in human T lymphocytes. Impaired induction of interleukin 2 and gamma interferon due to specific inhibition of dipeptidyl peptidase IV. Scand. J. Immunol. 29: 127. 11. SCHON, E., E. EICHMANN, S. JAHN, J. Kopp, H.-D. VOLK, and S. ANSORGE. 1988. On the role of dipeptidyl peptidase IV (DP IV) in the immune system. Relations to the cell cycle and lymphokine production of activated lymphocytes. Biol. Zbl. 107: 141. 12. Fox, D. A., R. E. HUSSEY, and K. A. FITZGERALD et al. 1984. Tal, a novel 105 kD human T cell activation antigen defined by a monoclonal antibody. J. Immunol. 133: 1250. 13. HAFLER, D. A., D. A. Fox, D. BENJAMIN, and H. L. WEINER. 1986. Antigen reactive memory T cells are defined by Tal. J. Immunol. 137: 414. 14. DANG, N. H., Y. TORIMOTO, and K. SUGITA et al. 1990. Cell surface modulation of CD26 by anti-1F7 monoclonal antibody. J. Immunol. 145: 3963. 15. DANG, N. H., Y. TORIMOTO, K. DEUSCH, S. F. SCHLOSSMANN, and C. MORIMOTO. 1990. Comito genie effect of solid-phase immobilized anti-1F7 on human CD4 T cell activation via CD3 and CD2 pathway. J. Immunol. 144: 4092. 16. PLANA, M., O. VINAS, O. DE LA CALLE-MARTIN et al. 1991. Induction of interleukin 2 (IL 2) and interferon-gamma and enhancement of IL 2 receptor expression by a CD26 monoclonal antibody. Eur. J. Immunol. 21: 1085. 17. FLEISCHER, B., D. J. SCHENDEL, and D. VON STELDERN. 1986. Triggering of the lethal hit in human cytotoxic T lymphocytes: a functional role for a 103-kDa T cell-specific activation antigen. Eur. J. Immunol. 16: 741. 18. SCHREZENMEIER, H. and B. FLEISCHER. 1988. A regulatory role for the CD4 and CD8 molecules in T cell activation. J. Immunol. 141: 398. 19. DANG, N. H., D. A. HARER, S. F. SCHLOSSMANN, and J. B. BREITMEYER. 1990. FcRmediated crosslinking of Tal (CDw26) induces human T lymphocyte activation. Cell Immunol. 125: 42. 20. BOYUM, A. 1968. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Lab. Clin. Invest. 21: 77. 21. Julius, M. H., E. Simpson, and L. A. HERZENBERG. 1973. A rapid method for the isolation of functional thymus-derived murine lymphocytes. Eur. J. Immunol. 3: 645. 22. BRANDT, E., A. ALTMAN, M. GRONEFELD, A. J. ULMER, and H.-D. FLAD. 1986. Functional and molecular characterization of a monoclonal antibody against human interleukin 2. Immunobiol. 172: 33. 23. ENNEN, J., M. ERNST, and H.-D. FLAD. 1989. The effect of interleukin 2 on Fe-gamma receptor function of human monocytes requires specific intercellular interaction. Immunobiol. 179: 17. 24. POSCHEL, G., R. MENTLEIN, and E. HEYMANN. 1982. Isolation and characterization of dipeptidyl peptidase IV from human placenta. Eur. J. Biochem. 126: 359. 25. MATSUI, Y., H. M. SHAPIRO, M. J. SHEEHY et al. 1986. Differential expression of T cell differentiation antigens and major histocompatibility antigens on activated T cells during the cell cycle. Eur. J. Immunol. 16: 248. 26. NECKERS, L. M. and J. COSSMAN. 1983. Transferrin receptor induction in mitogen-
50 . T. MATTERN, S. ANSORGE, H.-D. FLAD, and A.
J. ULMER
stimulated human T lymphocytes is required for DNA synthesis and cell division and is regulated by interleukin 2. Proc. Nat!' Acad. Sci. USA 80: 3494. 27. STADLER, B. M., S. F. DOUGHERTY,].]. FARRAR, and].]. OPPENHEIM. 1981. Relationship of cell cycle to recovery of IL 2 activity from human mononuclear cells, human and mouse T cell lines. J. Immuno!. 127: 1936. 28. BURDACH, S., N. ZESSACK, D. DILLOO, M. SHATSKY, D. THOMPSON, and L. LEVITT. 1991. Differential regulation of lymphokine production by distinct subunits of the T cell interleukin 2 receptor. ]. Clin. Invest. 87: 2114. Dr. ARTHUR]. ULMER, Forschungsinstitut Borstel, Parkallee 22,2061 Borstel, Germany