Dr C. Morimoto, Division of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. E-mail: firstname.lastname@example.org
CD26 is T cell costimulatory molecule with dipeptidyl peptidase IV (DPPIV) enzyme activity located in its extracellular region. The expression of CD26 is enhanced after activation of T cells, while it is preferentially expressed on a subset of CD4+ memory T cells in the resting state. In this paper, we demonstrate that binding of the soluble anti-CD26 monoclonal antibody (mAb) 1F7 inhibits human T-cell growth and proliferation in both CD26-transfected Jurkat T-cell lines and human T-cell clones by inducing G1/S arrest, which is associated with enhancement of p21Cip1 expression. This effect depends on the DPPIV enzyme activity of the CD26 molecule. Moreover, we show that expression of p21Cip1 after treatment with the anti-CD26 mAb 1F7 appears to be induced through activation of extracellular signal-regulated kinase (ERK) pathway. These data thus suggest that anti-CD26 treatment may have potential use in the clinical setting involving activated T cell dysregulation, including autoimmune disorders and graft-vs.-host disease.
CD26 is a 110 000 MW cell-surface glycoprotein that possesses dipeptidyl peptidase IV (DPPIV; EC 184.108.40.206) activity in its extracellular domain, and plays an important role in T-cell activation.1,2 Recently identified as the adenosine deaminase (ADA) binding protein, CD26 regulates ADA surface expression, with the CD26/ADA complex perhaps playing a key role in the catalytic removal of local adenosine to regulate immune function.3 Although constitutively expressed in the liver, intestine and kidney, CD26 expression level is tightly regulated on T cells, and its density is markedly enhanced after T cell activation.1,4 In the resting state of T cells, CD26 is expressed on a subset of CD4+ memory T cells, and this CD4+ CD26high T-cell population has been shown to respond maximally to recall antigens.1,5 In fact, CD26 itself is involved in the signal transduction process of T cells.1 Cross-linking of CD26 and CD3 with immobilized monoclonal antibodies (mAbs) can induce T-cell activation and interleukin (IL)-2 production.1,2,6 Moreover, anti-CD26 antibody treatment of T cells leads to a decrease in the surface expression of CD26 via its internalization, and this modulation of CD26 on T cells results in an enhanced proliferative response to anti-CD3 or anti-CD2 stimulation.7 While ligation of the CD26 molecule by the anti-CD26 mAb 1F7 induces increased tyrosine phosphorylation of signalling molecules such as CD3-zeta, extracellular signal-regulated kinase (ERK), p56lck, and ZAP-708,9 we showed previously that the anti-CD26 mAb 1F7 inhibits tetanus-toxoid induced T-cell proliferation.10 In normal T cells, engagement of CD26 results in increased phosphorylation of proteins involved in T-cell signal transduction, mediated in part through the physical association of CD26 and CD45 in lipid rafts.11 Besides being a key immunoregulatory molecule, CD26 may have a potential role in the development of certain neoplasms, including aggressive T-cell haematological malignancies.12,13
In eukaryotic cells, cell cycle progression is controlled at the G1/S checkpoint by a group of related enzymes known as the cyclin-dependent kinases (CDKs), which are positively regulated by their physical association with regulatory subunits called cyclins.14,15 However, enzymatic activities of the CDK-cyclin complexes are negatively regulated by a set of proteins termed CDK inhibitors.14 The p21 (waf1, Cip1) CDK inhibitor (CDKI) blocks multiple cyclin–CDK complexes through its physical association with these structures.15,16 In addition, through its direct interaction with proliferating cell nuclear antigen (PCNA), p21Cip1 can inhibit DNA replication.17 Various stimuli such as cellular damage, serum factors, and phorbol esters, can induce p21Cip1 expression in both p53-dependent and p53-independent manners, depending on the stimuli.16,18,19
In this paper, we demonstrate that binding of soluble anti-CD26 mAb 1F7 inhibits proliferation of CD26 Jurkat transfectants and T-cell clones derived from human peripheral blood. Moreover, anti-CD26 binding results in cell cycle arrest at the G1/S checkpoint, associated with increased p21Cip1 protein and mRNA levels. Finally, we show that ERK pathways appear to play a role in the enhancement of p21Cip1 expression following anti-CD26 mAb treatment. These data thus suggest that anti-CD26 treatment may have potential use in the clinical setting involving activated T cell dysregulation, including graft-versus-host disease (GVHD) and autoimmune disorders.
Materials and methods
Preparation and culture of cells
Human T-cell clones were established by in vitro stimulation of human peripheral blood lymphocytes according to the methods described previously.20 Human Jurkat T-cell line was obtained from the American Type Culture Collection (Rockville, MD). The Jurkat cell lines include: (1) wild type CD26-transfected Jurkat cell lines (J. C26/DP+); (2) Jurkat cell lines transfected with mutant CD26 containing an alanine at the putative catalytic serine residue at position 630, resulting in a mutant CD26-positive/DPPIV-negative Jurkat transfectant (J.C26/DP–); and (3) non-transfected parental Jurkat cells (Jwt).21,22 Jurkat transfectants were incubated at 37° at a concentration of 1 × 106/ml in culture media, consisting of RPMI-1640 (Life Technologies Inc., Grand Island, NY) supplemented with 10% FCS, penicillin (100 units/ml), streptomycin (100 µg/ml) (Life Technologies Inc.), and G418 (500 µg/ml) (Sigma-Aldrich, St. Louis, MO). Non-transfected parental Jurkat cells were maintained in the same culture media without G418. Human peripheral blood mononuclear cells (PBMC), collected from healthy adult volunteers, were isolated by centrifugation on Ficoll/Paque (Amersham Pharmacia Biotech., Piscataway, NJ). To obtain a highly purified T-cell population, PBMC were separated into an E rosette-positive population and were used as resting T cells as determined by flow cytometric analysis (FACScalibur™, Nippon Becton Dickinson Co., Ltd, Tokyo, Japan) using an FITC-labeled anti-CD3 mAb (BD PharMingen, San Diego, CA), with purity being > 95%. T cell clones were maintained in culture media containing IL-2 (10 ng/ml; PeproTech EC Ltd, London, UK), and restimulated every 2–3 weeks with irradiated (30 Gy) allogeneic PBMC (1·0 × 105/ml).20 Viability of cells was examined using trypan blue (Sigma-Aldrich) dye exclusion method.
Antibodies and reagents
Anti-CD26 mAbs, 1F7 and 5F8, and isotype control mAb 4B4 (CD29 mAb) were previously described.10,23,24 Anti-CD3 mAb (OKT3) was described elswhere.25 The following antibodies and reagents were purchased from BD PharMingen: anti-p21Cip1 anti-p27Kip1 anti-p53, anti-p18INK4c, anti-p19INK4d, anti-cyclin D1, anti-CDK4, anti-CDK-6, and anti-ERK. Mouse anti-β-actin was purchased from Sigma-Aldrich, and anti-phosphorylated ERK was from Santa Cruz (Delaware Avenue, CA). The source and working concentration of reagents used for cell stimulation and inhibition of signal transduction are as follows: OKT3 (0·05 µg/ml), PMA (10 ng/ml; Sigma-Aldrich), Nocodazole (500 ng/ml from 1 mg/ml stock solution in DMSO; Sigma-Aldrich), PD98059 (10 µm from 10 mm stock solution in DMSO; BIOMOL, Plymouth Meeting, PA), and U0126 (10 µm from 10 mm stock solution in DMSO; Cell Signalling Technology Inc., Beverly, MA). Cells were treated with each inhibitor 30 min before initiation of culture with mAbs.
Flow cytometry analysis
All procedures were carried out at 4°, and flow cytometry (FCM) analyses were performed with FACSCalibur™ (Nippon Becton-Dickinson) using standard CELLQuest™ acquisition/analysis software (Becton-Dickinson). Cells were stained with the appropriate antibodies, and washed twice with ice-cold phosphate-buffered saline (PBS) prior to FCM analysis.
Cell cycle analysis
Cells (1 × 106/well) were incubated in media alone or in the presence of 1F7, 5F8 or isotype control mAb (4B4) at indicated concentrations with or without Nocodazole at 37°. In experiments using inhibitors, 1 × 106 cells were incubated with various inhibitors at the indicated concentrations for 30 min at 37° prior to incubation with anti-CD26 mAbs. At the appropriate time interval, after washed in ice-cold PBS twice, cells were fixed with ice-cold 70% ethanol for 30 min, then resuspended in 500 µl solution containing 50 µg/ml propidium iodide (Sigma-Aldrich) and 50 µg/ml RNaseA (Sigma-Aldrich) for 30 min at 37°. Samples were then analysed by FACSCaliburTM within 1 hr after preparation. After gating out cell debris and fixation artifacts, FCM analysis allowed for the discrimination of DNA contents. G0/G1, S and G2/M populations were quantified using the ModFiT™ program (Becton-Dickinson).
Preparation of cell lysates and Western blot analysis
After incubation at 37°, cells were harvested from wells, washed with PBS and lysed in RIPA lysis buffer, consisting of 1% NP-40, 0·5% sodium deoxycholate, 0·1% sodium dodecyl sulphate (SDS), 5 mm ethylenediaminetetraacetic acid (EDTA), 10 mm Tris–HCl (pH 7·4), 0·15 m NaCl, 1 mm phenylmethylsulphonyl fluoride (PMSF), 0·5 mm NaF, 10 µg/ml aprotinin and 0·02 mm Na3VO4. For detecting phosphotyrosine proteins, cells after incubation were washed with ice-cold phosphate-buffered saline (PBS) containing 5 mm EDTA, 10 mm, NaF, 10 mm Na pyrophosphate and 0·4 mm Na3VO4. Cells were centrifuged and then solubilized in lysis buffer (1% NP-40, 0·5% sodium deoxycholate, 5 mm EDTA, 50 mm Tris–HCl (pH 8·0), 0·15 m NaCl, 1 mm PMSF, 10 mm iodacetamide, 10 mm NaF, 10 µg/ml aprotinin and 0·4 mm Na3VO4). After removal of precipitation by ultracentrifugation, cell lysates were then submitted to SDS–polyacrylamide gel electrophoresis (PAGE) analysis on an appropriate concentration gel under reducing condition using a mini-Protean II system (Bio-Rad Laboratories, Hercules, CA). For immunoblotting, the proteins were transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA) in 25 mm Tris, 192 mm glycine, and 20% methanol, and the membrane was blocked for 1 hr at room temperature in PBS with 0·05% Tween-20 containing 5% non-fat milk. Specific antigens were probed by the corresponding mAbs, followed by horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin (Amersham Pharmacia). Western blots were visualized by the enhanced chemiluminescence technique (NEN, Boston, MA).
In vitro cell proliferation assay
Cell proliferation was determined using [3H]thymidine (ICN Radiochemicals, Irvine, CA) incorporation. All proliferation assays of each experiment were performed in triplicate. 0·2 × 106 of cells in each microplate well were incubated in the presence of media alone or in the presence of 1F7 (1 µg/ml) at 37° with or without stimulation of OKT3 and PMA. After being incubated for 72 hr, cells were pulsed with [3H]thymidine (1 µCi/well) for the final 8 hr of culture. Cells were then harvested onto a glass filter (Wallac, Turk, Finland), and radioactivity was counted using a liquid scintillation counter (Wallac). [3H]thymidine uptake was expressed as the mean c.p.m. of triplicate samples.
Student's t-test was used to determine whether the difference between control and sample was significant (P < 0·05 being significant).
Anti-CD26 mAb treatment inhibits cell cycle progression at the G1/S checkpoint
We first examined the effect of soluble anti-CD26 antibody binding on cell cycle of Jurkat cells transfected with cDNA of a CD26 with DPPIV active domain (J.C26/DP+), and without DPPIV active domain (J.C26/DP–) as established previously.21,22 To better visualize cell cycle effects, we treated cells with Nocodazole, which arrests cells in M phase unless they are arrested in G0/G1 phase. Cell viability validated by trypan blue dye exclusion method remained to be ≥ 95% in the presence or absence of Nocodazole. As shown in Fig. 1(a, b), the addition of anti-CD26 mAb 1F7 to J.C26/DP+ resulted in blockade of cell cycle progression at the G1/S checkpoint. Of note is the fact that cell cycle arrest at the G1/S checkpoint was not observed in J.C26/DP– or parental Jurkat (Jwt) (Fig. 1b). In Fig. 1(c), G2/M accumulation by Nocodazole was observed in 1F7-non-treated J.C26/DP+, but not in 1F7-treated J.C26/DP+. This effect of G2/M accumulation by Nocodazole was also observed in J.C26/DP– and Jwt in the presence or absence of 1F7 (Fig. 1c). On the other hand, S phase was not influenced by 1F7 treatment (Fig. 1d). These findings suggested that the effect of cell cycle progression at the G1/S checkpoint was dependent on the enzymatic activity of DPPIV intrinsic to the CD26 molecule.
Enhancement of p21 expression associated with cell cycle arrest at the G1/S check point following anti-CD26 mAb treatment
Close examination of the cellular response of Jurkat T cells to 1F7 by FCM analysis revealed that J.C26/DP+ exhibited an approximately 25% increase in G1 arrest 6 hr after initiation of culture with 1F7 (Fig. 2a). At 12 and 24 hr after 1F7 treatment, J.C26/DP+ gradually lost their initial G0/G1 arrest. To clarify the status regarding S phase, we also performed cell cycle analysis using BrdU (and anti-BrdU antibody) and 7-amino-actinomycin D staining (BD PharMingen's BrdU Flow KitTM). In fact, no other effects on cell cycle than arrest at G0/G1 phase were observed (data not shown), as examined by propidium iodide staining in Figs 1 and 2(a). Notably, in J.C26/DP–, cell cycle arrest was not observed. These findings again suggested that the effect on cell cycle progression at the G1/S checkpoint was dependent on the enzymatic activity of DPPIV. The effect of 1F7 was dose dependent at concentrations 0·1–10·0 µg/ml (data not shown). It should be noted that another anti-CD26 mAb 5F8 recognizing a distinct CD26 epitope from 1F7 had no such effect as observed with 1F723 (data not shown).
Because cell cycle arrest can be accompanied by increases in CDKI and/or decreases in cyclins or CDKs, we next examined the expression of various cell cycle regulatory proteins following 1F7 binding. Compared to incubation under control conditions (media alone or 4B4 as isotype-matched control mAb treatment), treatment of J.C26/DP+ with 1F7 resulted in increased expression of p21Cip1 shown by Western blotting analysis of the relative levels of the protein (Fig. 2b). However, treatment of J.C26/DP– with 1F7 did not result in increased expression of p21Cip1. Enhanced p21Cip1 expression was detected within 6 hr of treatment with 1F7, and then gradually decreased, being compatible with the cell cycle analysis shown in Fig. 2(a). In contrast to p21Cip1 the expression of cyclin D1, CDK4, CDK6, p27Kip1 p53 antip18INK4c, and p19INK4d, which are associated with G1-regulation cyclin complex, did not change at 6 hr after treatment with 1F7 (Fig. 2c). It should be noted that the expression of these proteins did not change between 0 and 24 hr after initiation of culture with 1F7 (data not shown). These results suggested that 1F7 stimulation led to the up-regulation of p21Cip1 and cell cycle arrest at the G1/S checkpoint through the DPPIV enzymatic activity of CD26. Because p21Cip1 was found first as an inhibitor of cyclin/CDK complex leading to changes in the phosphorylation state of retinoblastoma (Rb) protein15,16 we examined the phosphorylation status of Rb protein in our system by Western blotting, and did not observe differences among phosphorylation states (data not shown).
The mitogen activated protein (MAP) kinase/ERK (MEK)-ERK pathway plays an important role in 1F7-mediated cell cycle arrest at the G1/S checkpoint
Recently, we showed that CD26 molecules are present in membrane lipid rafts and that ligation of CD26 by 1F7 increases the recruitment of CD26 molecules to rafts.11 T-cell receptors (TCR) in lipid rafts also interacts with other signalling molecules26,27 thereby inducing increased tyrosine phosphorylation of signalling molecules. CD26 is involved in essential T-cell signalling events through its physical and functional association with key cellular structures.1,2,6 Other studies demonstrated that hyperactivation of the Raf-MEK-ERK pathway in T cells and other cell lineages led to alterations in the expression of key cell cycle regulators and cell cycle arrest at the G1/S check point.28–30 We therefore examined whether tyrosine phosphorylation of signalling molecules related to CD26 in T cells leads to increased expression of p21Cip1. Probing for tyrosine phosphorylated proteins through Western blot analysis (4G10), we found that 1F7 treatment of J.C26/DP+ induced tyrosine phosphorylation of proteins with molecular weights of approximately 40 000 MW at 5–10 min after initiation of culture (data not shown). However, no induction in tyrosine phosphorylation was observed following 1F7 treatment in J.C26/DP– and Jwt. These changes were not observed in experiments using isotype-matched control mAb 4B4. To characterize the 40 000 MW phosphorylated protein, we examined the phosphorylation status of ERK, as previous work showed that the Raf-MEK-ERK pathway mediates anti-CD3 mAb-induced G1 arrest.30 As shown in Fig. 3(a), ERK proteins are phosphorylated following treatment of J.C26/DP+ with 1F7. To confirm these results, we next examined the effect of inhibiting the MEK-ERK pathway on p21Cip1 expression. Cells were treated with 1F7 for 6 hr in the absence or presence of the MEK-specific inhibitor PD98059 (Fig. 3b). The enhanced expression of p21Cip1 associated with phosphorylation of ERK was clearly inhibited by the presence of the MEK inhibitor. It should be noted that equal loading of the gel lanes in Fig. 3(c) was confirmed by probing the Western blots with an antibody that recognizes ERK. These results suggested that induction of p21Cip1 following 1F7 treatment was mediated via the MEK-ERK pathway.
To further determine the role of the MEK-ERK pathway in cell cycle regulation of T cells after treatment with 1F7, we performed cell cycle analysis by FCM in the absence or presence of the MEK-specific inhibitors PD98059 and U0126. Consistent with results regarding p21Cip1 expression in Fig. 3(C), G0/G1 arrest of 1F7-treated J.C26/DP+ was disrupted by the presence of the MEK-specific inhibitors (Fig. 3c), which was not observed in J.C26/DP– and Jwt. These findings strongly suggested that anti-CD26 treatment induced cell cycle arrest at G1/S checkpoint in T cells by activating the MEK-ERK pathway, leading to enhanced expression of the CDKI p21Cip1.
Anti-CD26 mAb 1F7 treatment inhibits proliferation of T-cell clones
Up-regulation of p21Cip1 has been described during T-cell proliferation and in CD4+ memory T cells of autoimmune-prone BXSB mice.31,32 Moreover, p21Cip1-deficient mice accumulated abnormal amounts of CD4+ memory T cells and developed loss of tolerance towards nuclear antigens.32 In view of these findings, to define the biological effect of 1F7-mediated p21Cip enhancement on the proliferation of human peripheral T cells, we next examined the effect of soluble anti-CD26 antibody binding on proliferation of human T-cell clones derived from PBMC. As shown in Fig. 4(a), the addition of 1F7 to human T-cell clones resulted in a reduction of cellular proliferation, as assayed by [3H]thymidine uptake. Of note is the fact that there was no inhibitory effect following treatment with the anti-CD26 mAb 5F823,33 or an isotype control antibody 4B4. Similar to results described above in experiments using Jurkat transfectants, p21Cip1 expression in T-cell clones was also enhanced following treatment with 1F7 (Fig. 4b). 1F7 effect of enhanced p21Cip1 expression was also observed in phytohaemagglutinin (PHA) blast T cells, albeit to a lesser degree, but not in resting T cells (Fig. 4b). These results suggested that in activated T cells such as T-cell clones and PHA blast T cells, T-cell proliferation was inhibited by the treatment of 1F7 via the induction of p21Cip1.
In this paper, we demonstrated that anti-CD26 mAb 1F7 binding led to cell cycle arrest at the G1/S check point, and that engagement of CD26 induced G1 arrest on CD26 Jurkat transfectants through enhanced expression of the cell cycle regulatory protein p21Cip1. This effect was mediated by activation of the MEK-ERK pathway. In addition to CD26 Jurkat transfectants, we showed that inhibition of proliferation and enhancement of p21Cip1 expression was observed in T-cell clones and PHA blast T cells derived from human PBMC.
The antigen sensitivity of class II MHC-restricted human CD4+ T-cell clones is demonstrated to increase gradually with time after stimulation. This is manifested in a requirement of less antigen in culture, as well as decreased numbers of peptide–MHC complexes per antigen-presenting cells (APC) for T-cell activation, and increased resistance to inhibition by class II MHC blockade.34 Previously, we showed that the increase in antigen sensitivity was accompanied by increased cell-surface expression of CD26, leucocyte function-associated-1 (LFA-1) and very late antigen-1 (VLA-1), whereas the expression of TCR and a series of other T-cell surface molecules remains unchanged.5 We also showed that the late-memory T-cell phenotype occurred among T cells activated in vivo. Moreover, using appropriate mAbs, we indicated that treatment by CD26 mAb with MHC blockade contributed to inhibition of proliferation of activated memory T cells.5 Other researchers also demonstrated that anti-CD26 mAbs (TII 19-4-7, Ta-1, and M5) reversibly arrested PHA-stimulated PBMC in the late G1 phase.35 However, the molecular mechanism of this inhibitory effect of T-cell proliferation by anti-CD26 mAb has not been clear at the moment. The data presented in this paper demonstrated that anti-CD26 mAb 1F7 inhibits T-cell proliferation via cell cycle arrest at G1/S check point and induction of p21Cip1 by activation of the MEK-ERK pathway. Besides its diverse functions, as reported previously,1,2,6 our findings thus indicate that CD26 is functionally linked to cell cycle regulation and provide additional evidence of the integral role played by this intriguing molecule in key cellular processes and subsequent function.
Our recent studies have demonstrated that CD26 molecules in T cells exist in membrane lipid rafts, and that cross-linking of CD26 with anti-CD26 mAbs induces aggregation of CD26 molecules into lipid rafts. This process results eventually in the activation of T cells through tyrosine phosphorylation of signalling molecules, such as Cbl, ZAP-70, ERK, p56Lck and CD3-zeta.11 TCR also exerts its signalling effects through the recruitment of various surface and cytosolic adapter proteins into lipid rafts.26,27 As negative regulators of TCR signalling, Rap1, Raf and Cbl-b have been shown to aggregate in lipid rafts.28,29,36 Relating to this point, it has been demonstrated that increased intensity of Raf-MEK-ERK signalling can elicit cell cycle arrest at G1/S check point associated with an increase in the expression of p21Cip1. Meanwhile, high dose of anti-CD3 mAb induced cell cycle arrest by activating the Raf-MEK-ERK pathway, leading to the expression of p21Cip1 in T cells and a failure to down-regulate the expression of p27Kip.1,29,30 In spite of our failure to observe changes in the phosphorylation state of Rb protein after 1F7 treatment, p21Cip1 may have a complex role in T cells, and is associated with differentiation, proliferation and systemic immunity.37 Moreover, G1/S arrest in our system was observed transiently, which was the same result as shown by Mattern et al.35 In fact, after 36–48 hr without Nocodazole, cells re-entered into cell cycle (data not shown). In addition, apoptotic events were not observed in our system using annexin V and propidium iodide analysis by FCM (data not shown). It remains unclear how p21Cip1 performs its broader functions in T cells, since p21Cip1 is involved in complex interactions and networks.38 Although we found that ERK was phosphorylated after 1F7 treatment, leading to a subsequent increase in the expression of p21Cip1 further experiments will focus on the identification of the precise molecular mechanisms involved in signalling of the CD26–ERK–p21Cip1 pathway.
Accumulating evidence suggests that DPPIV enzyme activity plays an essential role in CD26-mediated T-cell costimulation as well as T cell immune responses.1,2,6 Our results showed that DPPIV enzyme activity plays a role in the induction of p21Cip1 following treatment of T cells with the anti-CD26 mAb 1F7. It is reported that CD26/DPPIV regulates various cellular functions by cleaving selected chemokines at the N-terminus to modify their biological functions.6,39,40 In view of its ability to cleave certain biological factors as a serine protease, it is conceivable that DPPIV enzyme activity of CD26 appears to regulate phosphorylation of ERK and induction of p21Cip1 through cleavage of relevant biological factors in T cells. Further studies are in progress to determine the CD26/DPPIV-associated factors responsible for regulating the expression of p21Cip1.
Our findings that 1F7 has a more potent effect than 5F8 demonstrated that engagement of selected epitopes of CD26 is an important factor in mediating cell cycle arrest, inhibiting cell proliferation and inducing p21Cip1 expression following mAb treatment. It should also be noted that 1F7 has a strong comitogenic capacity whereas 5F8 has no such activity.33 Thus, the epitopes recognized by 1F7 and 5F8 on the CD26 molecule have distinct functional effects. Further studies are required to define the relationship between the exact epitope of CD26 and cell cycle arrest in association with p21Cip1 induction.
Activated memory T cells express high levels of CD26, and this phenotype of late-memory T cells is associated with both in vivo and in vitro increased antigen sensitivity.5In vivo studies revealed that a large number of CD26+ T cells are found in inflamed tissues of patients with multiple sclerosis and rheumatoid arthritis41–43 suggesting that CD26+ T cells function as effector T cells. In view of these findings, CD26 can potentially be a target for immunotherapy. In fact, anti-CD26 treatment was reported to be effective in decreasing the incidence of steroid-resistant acute GVHD after allogeneic bone marrow transplantation44,45 although the precise mechanism involved in these clinical results is not yet elucidated. Our data therefore suggest that cell cycle regulation of activated T cells via CD26 might be useful for controlling acute GVHD by inhibiting cellular proliferation. Taken together with the observation that transfection of p21Cip1 gene enhanced cyclosporin A-mediated inhibition of lymphocyte proliferation46 anti-CD26 mAb therapy may serve as an alternative strategy to induce immunosuppression, one that is potentially less toxic than the side effects currently seen with conventional agents. Cell-cycle check point studies have led to the identification of a number of therapeutic anticancer targets.47 Based on studies demonstrating that expression of CD26 was restricted to aggressive T-cell lymphoblastic lymphoma/acute lymphoblastic leukaemia and CD30+ anaplastic large cell lymphoma (ALCL)13 CD26-targeted therapy may also be effective when used in combination with antineoplastic drugs for selected tumours. Supporting this hypothesis are the recent findings from our group demonstrating that 1F7 exhibits in vitro and in vivo antitumour effect in the treatment of CD26+ CD30+ ALCL cell line Karpas 299.48
The ability to inhibit T-cell proliferation and induce G0/G1 arrest through the use of anti-CD26 mAb may therefore lead to the eventual development of new reagents targeting CD26+ activated T cells, including immunotherapy of allogeneic organ transplant resistant to current treatment modalities. Furthermore, given its enhanced expression and its potential role in the pathophysiology of selected cancers and autoimmune diseases, CD26-targeted treatment may similarly prove to be effective in these clinical settings.
This study was supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture, and Ministry of Health, Labor and Welfare, Japan, and from the National Institutes of Health, grant AR33713. Dr Kei Ohnuma is a research fellow of the Japan Society for the Promotion of Science. Dr Nam H. Dang is the recipient of a grant from the MD Anderson Cancer Center Physician-Scientist Program and the V Foundation.