The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ohnuma, K.
Right arrow Articles by Morimoto, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ohnuma, K.
Right arrow Articles by Morimoto, C.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Substance via MeSH
The Journal of Immunology, 2001, 167: 6745-6755.
Copyright © 2001 by The American Association of Immunologists

Soluble CD26/Dipeptidyl Peptidase IV Induces T Cell Proliferation Through CD86 Up-Regulation on APCs1

Kei Ohnuma*, Yasuhiko Munakata{dagger}, Tomonori Ishii{ddagger}, Satoshi Iwata*, Seiji Kobayashi*, Osamu Hosono*, Hiroshi Kawasaki*, Nam H. Dang§ and Chikao Morimoto2,*

* Division of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan; Departments of {dagger} Molecular Diagnostics and {ddagger} Rheumatology and Hematology, Graduate School of Medicine, Tohoku University, Japan; and § Department of Lymphoma/Myeloma, M. D. Anderson Cancer Center, Houston, TX 77030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD26 is a T cell costimulatory molecule with dipeptidyl peptidase IV enzyme activity in its extracellular region. We have previously reported that the addition of soluble CD26 (sCD26) resulted in enhanced proliferation of peripheral blood T lymphocytes induced by the recall Ag, tetanus toxoid (TT). However, the mechanism involved in this immune enhancement has not yet been elucidated. In this paper, we demonstrate that the enhancing effect of sCD26 on TT-induced T cell proliferation occurred in the early stages of immune response. The cells directly affected by exogenously added sCD26 are the CD14-positive monocytes in the peripheral blood. Mannose-6 phosphate interfered with the uptake of sCD26 into monocytes, suggesting that mannose-6 phosphate/insulin-like growth factor II receptor plays a role in the transportation of sCD26 into monocytes. When sCD26 was added after Ag presentation had taken place, enhancement in TT-induced T cell proliferation was not observed. In addition, enhancement of TT-mediated T cell proliferation by sCD26 does not result from trimming of the MHC-bound peptide on the surface of monocytes. Importantly, we also showed that exogenously added sCD26 up-regulated the expression of the costimulatory molecule CD86 on monocytes through its dipeptidyl peptidase IV activity, and that this increased expression of CD86 was observed at both protein and mRNA level. Therefore, our findings suggest that sCD26 enhances T cell immune response to recall Ag via its direct effect on APCs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The widely distributed 110-kDa cell surface glycoprotein CD26 has known dipeptidyl peptidase IV (DPPIV,3 EC 3.4.14.5; Ref. 3) activity in its extracellular domain (1, 2). This enzyme is capable of cleaving amino-terminal dipeptides with either L-proline or L-alanine at the penultimate position. CD26/DPPIV has two physiological forms in human (3). One is the membrane-bound form preferentially expressed on the CD4+ helper/memory T cell population, whereas the other is the soluble form found in serum. Accumulating evidence suggests that DPPIV enzyme activity plays an essential role in the immune response (4). Cross-linking of CD26 and CD3 with solid-phase immobilized mAbs can induce T cell costimulation and IL-2 production by either human CD4+ T cells or Jurkat T cell lines transfected with CD26 cDNA (5, 6). In addition, anti-CD26 Ab treatment of T cells leads to a decrease in the surface expression of CD26 via its internalization, and such modulation results in an enhanced proliferative response to anti-CD3 or anti-CD2 stimulation, as well as enhanced tyrosine phosphorylation of signaling molecules such as CD3{zeta} and p56lck (7). Moreover, we have shown that DPPIV enzyme activity is required for the CD26-mediated T cell costimulation (8). More recently, we have shown that internalization of CD26 after cross-linking is mediated in part by the mannose-6-phosphate (M6P)/insulin-like growth factor II receptor (IGF-IIR), and that the interaction of CD26 and M6P/IGFIIR plays a role in CD26-induced T cell costimulation (9).

Maximal T cell activation requires both an Ag-specific stimulus provided by an MHC peptide complex and a costimulatory signal (10). Engagement of CD28 on the surface of T cells by B7-1 (CD80) or B7-2 (CD86) expressed on APCs provides a potent costimulatory signal (10, 11, 12, 13, 14). CD28-B7 interactions lead to T cell proliferation, differentiation, and cytokine secretion (14, 15). In contrast, engagement of CTLA-4 on activated T cells by B7-1 or B7-2 results in an inhibition of T cell responses (16, 17, 18). However, only CD28 is constitutively expressed and, hence, it has an important role in the generation of an immune response (19, 20, 21, 22, 23, 24).

In our previous report, we have shown that recombinant soluble CD26 (sCD26) enhanced proliferative responses of PBLs to stimulation with the soluble Ag, tetanus toxoid (TT) (25). In addition, this enhancing effect required DPPIV enzyme activity (8, 25). However, the precise mechanism for enhancement of TT-induced T cell proliferation by sCD26 remains unresolved.

In the present study, we demonstrated that the target cells of sCD26 were the CD14-positive monocytes in the peripheral blood and that M6P/IGF-IIR played a role in transporting sCD26 into the monocytes. Importantly, we found that sCD26 could up-regulate the expression of the CD86 but not CD80 or HLA-DR Ag on monocytes. These results suggest that sCD26 can be a useful agent in potentiating immune response in selected clinical settings.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation and activation of human lymphocyte populations

Human PBMC, collected from healthy adult volunteers who were immunized with TT within two years before donation, were isolated by centrifugation on Ficoll/Paque (Amersham Pharmacia Biotech, Piscataway, NJ). PBMC were directly used for the time-course studies. For the reconstitution experiments, PBMC were further purified into T cell fractions and APC fractions. To obtain a highly purified T cell population, PBMC were separated into an E rosette-positive (E+) population and were used as resting T cells as determined by flow cytometric analysis (FACSCalibur; Nippon BD Biosciences, Tokyo, Japan) using an FITC-labeled anti-CD3 mAb (BD PharMingen, San Diego, CA) with purity being >95%. To obtain APC, an E rosette-negative (E-) population was adhered to plastic plates for 4 h at 37°C, and adherent cells were used as APC. Monocytes were either purified by a flow cytometer on the basis of PE-labeled anti-CD14 mAb (BD PharMingen)-oriented parameter, or by negative selection through the use of immunomagnetic beads coated with an anti-CD3, CD7, CD19, CD45RA, CD56, and IgE mAb (Miltenyi Biotec, Auburn, CA) with purity being >95%. PBMC were cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies) and TT (Calbiochem, La Jolla, CA) at different concentrations for 16 h, followed by sCD26 pulses at various time intervals. T cells were also cultured with the standard medium (10% FCS-RPMI 1640 with penicillin and streptomycin) in the presence or absence of sCD26. Monocytes were incubated with TT at different concentrations for 16 h in the standard medium, followed by the addition of sCD26 in the culture medium at different times. To avoid interference by nonspecific activation of monocytes due to contamination, polymyxin B sulfate (20 IU/ml; Sigma-Aldrich, St. Louis, MO) was added to all media and reagents used for APC/monocytes experiments.

All cells were preincubated in the standard medium for 24 h to minimize the risk of potential interference from sCD26 present in human serum (8). In these experiments, cells were incubated in 96-well plates (200 µl/well; Falcon, Franklin Lakes, NJ). For the time-course study, 1.0 x 105 PBMC were incubated with 0.5 µg/ml TT for 16 h. Then, 0.5 µg/ml sCD26 was added to the well and was incubated for 0, 6, 12, 24, 48, 72, and 96 h. The day when the first sCD26 was added to the culture well was defined as day 0 of culture. All assays in proliferation experiments were performed on day 7 of culture after cells were incubated at 37°C in a 5% CO2 humidified atmosphere. For the reconstitution study, 0.5 x 106 of monocytes were preincubated with TT at a concentration of 0.5 µg/ml with different concentrations of sCD26 for 24 h. Monocytes were washed with PBS, and then 1 x 104/well of preincubated monocytes were subjected to the assay with 1 x 105/well of purified T cells from the same donor as the prepared monocytes. For fixation study, as prefixed stimulation study, after being treated with 0.05% glutaraldehyde for 30 s at room temperature, followed by being washed three times with PBS, 0.5 x 106 of monocytes were incubated with TT at a concentration of 0.5 µg/ml and with 0.5 µg/ml sCD26. As postfixed stimulation study, 0.5 x 106 of monocytes were initially incubated with TT at a concentration of 0.5 µg/ml and with 0.5 µg/ml sCD26. Subsequently, the preincubated monocytes were treated with 0.05% glutaraldehyde for 30 s at room temperature, followed by being washed three times with PBS. Monocytes (1.0 x 104/well) were then subjected to the assay with 1.0 x 105/well of purified T cells, which originated from the same donor as the prepared monocytes.

Preparation of sCD26

sCD26 with DPPIV (sCD26/DPPIV+) was produced according to the method described previously (24). Briefly, the expression vector RcSR{alpha}-26 days 3–9, which contains a deletion of the coding sequence for amino acids 3–9 of CD26, was transfected into a dihydrofolate reductase-deficit Chinese hamster ovary (CHO) cell line, DXB-11 by electroporation, together with pMT-2 providing the dihydrofolate reductase gene. Mutant sCD26 without DPPIV (sCD26/DPPIV-) was produced in the same method except that RcSR{alpha}-26d3-9 was further modified to yield RcSR{alpha}-26d3-9 S630A, which contains a point mutation at the active site of the DPPIV enzyme (Ser630 was replaced by Ala) by site-directed mutagenesis using the oligonucleotide. The transfected CHO cells, which produce either sCD26 or mutant sCD26, were cultured in serum-free CHO-S-SFM II medium (Life Technologies) supplemented with 1 µM methotrexate (Sigma-Aldrich). The culture supernatant was collected and subjected to affinity chromatography on adenosine deaminase-Sepharose according to the methods described previously (9).

mAbs and reagents

The source and working concentration of the mAbs used as primary Abs for flow cytometry are as follows: PE-conjugated anti-CD3 (UCHT1, mouse IgG1; 10 µg/ml; BD PharMingen), anti-CD14 (Mo-2, mouse IgM; 10 µg/ml; Beckman Coulter, Miami, FL), anti-CD19 (HIB19, mouse IgG1; 10 µg/ml; BD PharMingen), and anti-CD56 (NKH1, mouse IgG1; 10 µg/ml; Beckman Coulter); FITC-labeled anti-CD80 (BB1, mouse IgM; 10 µg/ml; BD PharMingen), anti-CD86 (IT2.2, mouse IgG2; 10 µg/ml; BD PharMingen), and anti-HLA-DR (L243, mouse IgG2; 10 µg/ml; BD PharMingen). Oregon green-conjugated sCD26 (sCD26-OG) was made with FluoReporter Oregon green protein labeling kit (Molecular Probes, Eugene, OR) according to the manufacturer’s instruction. These Oregon green-conjugated proteins were used at a concentration of 1 µg/ml. Biotinylated anti-CD14 (Mo-2; IgM; 10 µg/ml) was purchased from Beckman Coulter. mAbs for blocking assays were obtained as follows: CD80 (BB-1), CD86 (IT2.2), and HLA-DR (L243) from were BD PharMingen; the chimeric protein of human CTLA-4 and murine Ig (CTLA-4 Ig) and relevant control murine Ig were purchased from Ancell (Bayport, MN); and mouse anti-human M6P/IGFIIR mAb was kindly provided by Dr. V. Horcjsi (Academy of Science of the Czech Republic, Praha, Czech Republic). Texas red-conjugated anti-human M6P/IGF-IIR mAb (M6P/IGF-IIR-red) was made with FluoReporter Texas red protein labeling kit (Molecular Probes) according to the manufacturer’s instructions. In all experiments, relevant control mAbs of the same Ig isotype were included (IgG1 (MOPC-21), IgG2 (G155–178), and IgM (G155–228) were purchased from BD PharMingen). Egg white lysozyme was purchased from Wako Pure Chemical (Osaka, Japan) and was conjugated with Oregon green.

T cell proliferation assay

T cell proliferation induced by APC was measured by [3H]TdR (ICN Radiochemicals, Irvine, CA) uptake. Seven-day-incubated cells were pulsed with 1 µCi/well of [3H]TdR 8 h before harvesting onto a glass-fiber filter (Wallac, Turk, Finland), and the incorporated radioactivity was quantified by a liquid scintillation counter (Wallac).

Flow cytometric analysis

Assessment of the PBMC population that takes up sCD26-OG was performed with PE-conjugated anti-CD3, anti-CD14, anti-CD19, and anti-CD56 mAbs (10 µg/ml). For these experiments, 1 x 106 PBMC/well were incubated in standard medium containing sCD26-OG for 24 h with or without preincubation with TT. Cells were then washed twice in ice-cold PBS, followed by incubation for 2 min in acidic PBS (pH 3.0) to strip any sCD26-OG attached to the cell surface. Cells were subsequently washed in ice-cold PBS and were incubated with mouse Ig isotypes (1 µg/ml) to block nonspecific binding; this was followed by reaction with PE-conjugated mAbs.

In experiments assessing the expression of CD80, CD86, and HLA-DR on purified monocytes (0.5 x 106 monocytes/well), monocytes were preincubated with or without TT (0.5 µg/ml) for 16 h. Following incubation with sCD26 (0.5 µg/ml) for 24 h, FITC-conjugated CD80, CD86, and HLA-DR mAbs (10 µg/ml) were used with PE-conjugated anti-CD14 (10 µg/ml) to gate exclusively on the monocyte population. In experiments assessing the effect of M6P on cellular uptake of sCD26, purified monocytes (0.5 x 106 monocytes/well) were incubated with the appropriate concentration of M6P in the presence or absence of sCD26-OG for 24 h in standard medium. Cells were then washed twice in ice-cold PBS, followed by incubation for 2 min in acidic PBS (pH 3.0) to strip any sCD26-OG attached to the cell surface. Cells were washed in ice-cold PBS and were incubated with mouse Ig isotypes (1 µg/ml) to block nonspecific binding; this was followed by reaction with PE-conjugated anti-CD14 (10 µg/ml) to gate exclusively on monocyte population.

Flow cytometric analysis of 10,000 viable cells was conducted on FACSCalibur (BD Biosciences, Mountain View, CA). Each experiment was repeated at least three times, and the results were provided in the form of a histogram of a representative experiment, or increased mean percent ± SE fluorescent intensity compared with control or untreated cells.

Confocal laser microscopy

For fluorescent microscopy experiments of uptake of sCD26 by monocytes, purified monocytes were incubated with sCD26-OG (1 µg/ml) with or without preincubation with TT (0.5 µg/ml). Cells were then washed in ice-cold PBS twice and were incubated in acidic PBS (pH 3.0) to strip any sCD26-OG attached to the cell surface. The cells were then attached to microslide glass (Matsunami Glass, Tokyo, Japan) and fixed with 3% paraformaldehyde in PBS for 15 min at room temperature. Cells were blocked with mouse Ig isotypes (1 µg/ml) for 30 min at 4°C, followed by incubation with biotinylated anti-CD14 (10 µg/ml) for 30 min at 4°C, then washed with ice-cold PBS twice and incubated with streptavidin-conjugated Texas red conjugate (1/200; Beckman Coulter) for 30 min at 4°C. In experiments of sCD26 uptake via M6P/IGF-IIR on monocytes, purified monocytes were incubated with or without TT for 16 h, and sCD26-OG and M6P/IGF-IIR-red were added to the culture well after being blocked with mouse Ig isotypes (1 µg/ml) for 30 min at 4°C. Cells were then incubated for 30 min at 4°C. For detection of cell surface colocalization, cells were washed in ice-cold PBS twice and were attached to microslide glass, followed by fixation with 3% paraformaldehyde in PBS for 15 min at room temperature. For detection of intracellular incorporation, cells were incubated for 24 and 36 h at 37°C in a 5% CO2 humidified atmosphere, and were then washed in ice-cold PBS twice and incubated in acidic PBS (pH 3.0) to strip any sCD26-OG attached to the cell surface. The cells were then attached to microslide glass and fixed with 3% paraformaldehyde in PBS for 15 min at room temperature. Confocal microscopy was performed with an Olympus IX70 confocal microscope with 60 objective lenses (Olympus, Tokyo, Japan), using laser excitation at 496 and 568 nm. The widths of Oregon green and Texas red emission channels were set to maximize specificity.

Relative quantitative RT-PCR assay

In experiments assessing expression of CD86 mRNA, purified TT-treated monocytes (0.5 x 106 monocytes/well) were incubated with sCD26 (0.5 µg/ml) at the appropriate time intervals. RNA was then extracted by the use of TRIzol reagent solution according to the manufacturer’s instruction (Life Technologies). cDNA was produced by using Thermo-Script II reverse transcriptase (Life Technologies) with oligo(dT)12–18 primers. The quantities of mRNA were adjusted equally by using PCR of {beta}-actin. Forward primer was 5'-CAAGAGATGGCCACGGCTGCT-3' (cDNA position 746–766), and reverse primer was 5'-TCCTTCTGCATCCTGTCGGCA-3' (cDNA-position 1000–1020)) as the internal control. CD86 mRNA was amplified with primers designed to amplify the entire coding sequence of CD86 (forward primer was 5'-ATGGGACTGAGTAACATTCTCTTTGTGATGGCCT-3' (cDNA position 149–181 and linker), and reverse primer was 5'-CTCGAGTTAAAAACATGTATCACTTTTGTCGCATGA-3' (cDNA position 1090–1120 and linker)). The PCR were performed as follows: 94°C for 4 min, then denaturing at 94°C for 30 s, annealing at 64°C for 1 min, and extending at 72°C for 30 s at the different cycles, followed by a final extension at 72°C for 5 min. Amplified DNA was then electrophoresed on a 3% agarose gel and was stained with ethidium bromide.

Blocking assay by mAbs and human CTLA-4 Ig

For blocking experiments, monocytes were incubated with mAbs against CD80, CD86, HLA-DR, or CTLA-4 Ig at various concentrations for 15 min followed by the addition to T cells. mAbs or Ig were left during the entire culture period. Relevant mAbs or control murine Ig were used as isotype controls. Inhibitory effects on T cell proliferation were expressed as the percentage of reactivity of control cultures without the addition of blocking mAbs or Ig, which were performed in parallel.

Statistics

Student’s t test was used to determine whether the difference between control and sample was significant (p < 0.05 being significant).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
sCD26 enhances T cell proliferation in the early stage of immune response to recall Ag

To investigate the mechanism involved in the enhancement of TT-induced T cell proliferation by sCD26, we first performed a time-course analysis by adding sCD26 to the PBMC system stimulated by TT in vitro. In the early stage of the immune response to foreign Ags, direct interaction between APC and T cells is indispensable (21, 22, 23). However, in the latter stages, direct APC-T cell interaction is not always necessary, whereas secreted cytokines such as IL-2 are essential in maintaining the reaction (14, 21). As shown in Fig. 1Go, the addition of sCD26 within first 48 h resulted in an enhancement in T cell proliferation to TT. In contrast, the addition of sCD26 after 48 h did not enhance TT-mediated activation, and an enhancing effect of TT-induced T cell proliferation was not observed in the experiments with the DPPIV-deficient mutant sCD26. These results indicate that sCD26 affected the early stages of immune reaction to TT but did not have an appreciable effect on the latter stages. Moreover, these data are consistent with previous studies showing that the enhancement of TT-induced T cell proliferation requires DPPIV enzyme activity (8).



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 1. Proliferative effect on PBMC by sCD26. PBMC (1 x 105) were incubated in culture medium with or without TT or sCD26. The freshly isolated PBMC was cultured in standard culture medium for 24 h, then TT (0.5 µg/ml) was added to the medium to incubate for an additional 16 h. sCD26 (0.5 µg/ml) was then added to the wells and was incubated for various time periods as shown in the figure. Proliferation of cells was monitored in all instance by measuring [3H]TdR incorporation on day 7 of culture after the first sCD26 was added. Degree of proliferation is indicated as cpm in the ordinate. The experiments represent mean values ± SE calculated from three independently performed experiments.

 
Incubation with sCD26 leads to uptake by monocytes

Because sCD26 affected the early stages of immune response to TT, we next attempted to determine the target cells of sCD26. For this purpose, we incubated PBMC with sCD26-OG for 24 h in the presence or absence of TT. As shown in Table IGo, leukocyte phenotypes such as CD3, CD14, CD19, and CD56 were not affected by presence of TT. In addition, sCD26 was taken up mainly by CD14-positive monocytes (Table IIGo and Fig. 2GoA). In contrast, flow cytometric analyses showed that T cells (CD3+), B cells (CD19+), and NK cells (CD56+) displayed relatively low levels of sCD26. It should be noted that the above findings were observed both in the presence and absence of TT, and that DPPIV-deficient mutant sCD26 (sCD26/DPPIV-) was also preferentially taken up by CD14-positive monocytes. Additional evidence that sCD26 was taken up by monocytes was seen in studies involving confocal microscopy. Concordant data shown in Table IIGo and Fig. 2Go, B and C, show that sCD26/DPPIV+, as well as sCD26/DPPIV-, was clearly taken up by monocytes. However, as shown in Fig. 2Go, D (sCD26/DPPIV+) and E (sCD26/DPPIV-), sCD26 was no longer detectable intracellularly 36 h after incubation of monocytes with sCD26. The disappearance of sCD26 molecules following uptake by monocytes was also observed by flow cytometric analysis (data not shown). It should be noted that this uptake of sCD26 by monocytes was not affected by the presence of TT (data not shown).


View this table:
[in this window]
[in a new window]
  		Table I. sCD26-OG incorporation according to lymphocyte subpopulations1

 

View this table:
[in this window]
[in a new window]
  		Table II. sCD26-OG incorporation according to lymphocyte subpopulations1

 


View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 2. PBMC subpopulation that takes up sCD26. A, Freshly isolated PBMC (1 x 106/well) were incubated with or without TT for 16 h after a 24-h incubation in standard medium alone, and then sCD26-OG was added to the culture wells. After incubation with sCD26-OG for 24 h, cells were washed in PBS and in acidic buffer (pH 3 PBS) to strip any sCD26-OG on the cell surfaces. The cells were then incubated with the various PE-conjugated Abs, and the intensity of sCD26-OG was measured for flow histograms. Analysis was performed by FACSCalibur. Similar experiments were performed with mutant sCD26 of defective DPPIV activity. B–E, Fluorescent confocal microscopic view of sCD26-OG-incorporated monocytes. After incubation with sCD26-OG for 24 and 36 h, monocytes were washed in PBS, and once in acidic buffer (pH 3 PBS) to strip any sCD26-OG on the cell surface. The cells were then incubated with the biotinylated anti-CD14 Ab and incubated with streptavidin-Texas red. After incubation with sCD26-OG, sCD26-OG was incorporated in the monocytes (B and D, sCD26/DPPIV+-OG; C and E, sCD26/DPPIV--OG). Although sCD26-OG was detected intracellularly following 24 h of incubation (B and C), sCD26-OG was not detectable after 36 h of incubation (D and E). Bars indicate 10 µm.

 
The target cells of sCD26 are monocytes

Because the main target cells among PBMC were monocytes, as shown in Table IIGo and Fig. 2Go, A–C, we next attempted to confirm the enhancement of TT-induced T cell proliferation by monocytes that take up sCD26. For this purpose, we performed a reconstitution study by separating T cells and monocytes at the time of incubation with sCD26. As shown in Fig. 3Go, the enhancing effect of TT-induced T cell proliferation was observed only when monocytes were preincubated with TT and sCD26, but not T cells (Fig. 3Go, A and B). Importantly, these studies again confirmed that sCD26-mediated enhancement of TT-induced T cell proliferation required DPPIV enzyme activity. To further confirm that sCD26 uptake by TT-primed monocytes leads to enhancement of T cell proliferation, we performed a reconstitution study at different doses of sCD26. As shown in Fig. 3GoA, the degree of TT-induced T cell proliferation was dependent upon the concentration of the exogenously added sCD26. Therefore, these results indicate that the principal target cells of sCD26 are APCs, including monocytes.



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 3. Reconstitution study to determine the target cells of sCD26. A, Freshly isolated monocytes (0.5 x 106/well) were preincubated with 0.5 µg/ml TT and with different concentrations of sCD26 for 24 h, then, after washing with PBS, 1.0 x 104/well of the preincubated monocytes were subjected to the assay with 1 x 105/well of purified T cells. B, A total of 1 x 105/well of purified T cells were preincubated with 0.5 µg/ml of TT and with different concentrations of sCD26 for 24 h. Cells were then washed with PBS and subjected to the assay. Proliferation of cells was monitored in all instances by measuring [3H]TdR incorporation on day 7 of culture. Degree of proliferation is indicated as cpm in the ordinate. The experiments represent mean values ± SE calculated from three independently performed experiments.

 
Uptake of sCD26 into monocytes occurs via its binding to M6P/IGF-IIR

We recently showed that M6P/IGF-IIR was the binding protein for CD26 and that it played a role in internalizing CD26 molecule into T cells after ligation of CD26 (9). To examine whether M6P/IGF-IIR is involved in monocyte uptake of sCD26, we used fluorescent confocal microscopy to initially evaluate monocyte expression of sCD26-OG and M6P/IGF-IIR intracellularly and on the cell surface. For this purpose, fluorescent mouse anti-human M6P/IGF-IIR mAb was conjugated with M6P/IGF-IIR-red. Fig. 4GoA shows that sCD26-OG and M6P/IGF-IIR-red colocalized on the monocyte cell surface. Following incubation at 37°C for 24 h in the presence of sCD26-OG and M6P/IGF-IIR-red, intracellular colocalization of these proteins was observed (Fig. 4Go, B and C). Colocalization of lysozyme and M6P/IGF-IIR was similarly observed (Fig. 4GoD). In contrast, colocalization was not observed after incubation of sCD26-OG and Texas red-conjugated mouse IgG1 to exclude the possibility of nonspecific binding to Fc{gamma}R (data not shown). To further confirm that sCD26 uptake is dependent on its binding to M6P/IGF-IIR, we have performed the inhibition assay to evaluate uptake of sCD26 by monocytes in the presence of excess amounts of M6P. As shown in Fig. 4Go, E and F, FACS analyses showed that the addition of M6P into the culture system (0, 0.1, 1.0, and 10 µM) inhibited the uptake of sCD26 into monocytes. The degree of inhibition of sCD26 uptake was dependent upon the concentration of the exogenously added M6P (Fig. 4GoE). These findings were also observed even in the absence of TT (Fig. 4GoF). Thus, these results indicated that uptake of sCD26 into monocytes was due to the interaction of sCD26 with its binding protein M6P/IGF-IIR. It should be noted that this inhibitory effect of M6P on uptake of sCD26 was similarly seen in experiments performed with sCD26/DPPIV- (data not shown).



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 4. M6P/IGF-IIR plays a role in incorporating sCD26 into monocytes. A–D, Fluorescent confocal microscopic view of sCD26-incorporated monocytes via M6P/IGF-IIR. The freshly isolated monocytes (0.5 x 106/well) were incubated with or without TT for 16 h after a 24-h incubation in the standard medium alone, and then sCD26-OG and M6P/IGF-IIR mAb were added to the culture wells. A, To detect sCD26 and M6P/IGF-IIR on the cell surface, cells were incubated with sCD26-OG and M6P/IGF-IIR-red. B, After incubation with DPPIV+ sCD26-OG and M6P/IGF-IIR-red, cells were washed in ice-cold PBS and in acidic buffer to strip any sCD26-OG and M6P/IGF-IIR-red on the cell surfaces. Then cells were fixed and prepared for observation. C, Similar experiments as in B were performed using sCD26/DPPIV- conjugated with Oregon green, rather than sCD26/DPPIV+. D, As positive control for M6P/IGF-IIR incorporation, lysozyme was used to bind M6P/IGF-IIR. Single confocal sections show fluorescence in fluorescein and Texas red channels. Bars indicate 10 µm. E and F, M6P inhibits uptake of sCD26 into monocytes. The freshly isolated monocytes (0.5 x 106/well) were incubated with (E) or without (F) TT for 16 h after a 24-h incubation in standard medium alone, and then sCD26 was added to the culture wells in the presence of various concentration of M6P (0, 0.1, 1.0, and 10.0 µM/well). After a 24-h incubation, cells were washed in ice-cold PBS and in acidic PBS to strip any sCD26-OG on the cell surface. Analysis was performed by FACSCalibur. The intensity of sCD26-OG is shown in flow histograms. Similar experiments using mutant sCD26/DPPIV- were performed. The data in this study represent one of three independently performed experiments. The abscissa represents fluorescence intensity (log10 scale), and the ordinate represents the respective cell number.

 
Enhancement of TT-induced T cell proliferation by sCD26 is seen when monocytes are incubated with sCD26 before fixation

Another explanation for the sCD26-induced enhancement of TT-mediated T cell proliferation following monocyte uptake may be the trimming of the MHC class II-bound peptide, hence altering cellular responsiveness to the Ag. Such a phenomenon has been described previously with CD13 aminopeptidase N (25). CD13 contributes to Ag processing by trimming the MHC class II-bound peptide on the APC surface. Because CD26, like CD13, is also an ectopeptidase, it may have the effect of trimming MHC class II-bound peptide on the surface of APC. To evaluate this issue, we fixed TT-pulsed monocytes before and after incubation with sCD26, and subsequently examined whether the enhancing effect of sCD26 on T cell activation was observed. As shown in Fig. 5Go, A and B, an enhanced T cell proliferation was seen only when the monocytes were incubated with sCD26/DPPIV+ before fixation. In contrast, monocytes incubated with sCD26 after fixation did not alter the immune response occurring after the Ag peptide bound to MHC molecule. Therefore, these results suggested that enhancement of TT-induced T cell proliferation by sCD26 does not result from trimming of the MHC class II-bound peptide on the surface of APC. Rather, it is likely that internalization of sCD26 into monocytes affects the interaction of monocytes and T cells.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 5. Enhancing effect of TT-induced T cell proliferation by sCD26 did not result from trimming of the MHC-bound peptide on monocytes. Freshly isolated monocytes (0.5 x 106/well) were preincubated with 0.5 µg/ml TT for 16 h, followed by a 24-h incubation with sCD26 at different doses before (A) or after (B) being treated with 0.05% glutaraldehyde for 30 s at room temperature. After washing with PBS, 1 x 104/well of the preincubated monocytes were then subjected to the assay with 1 x 105/well of purified T cells. Similar experiments were performed with mutant sCD26/DPPIV-. Proliferation of cells was monitored in all instances by measuring [3H]TdR incorporation on day 7 of culture. Degree of proliferation is indicated as cpm in the ordinate. The experiments represent mean values ± SE calculated from three independently performed experiments.

 
Expression of the costimulatory molecule CD86 is increased on monocytes following exposure to sCD26

We next analyzed the expression of several surface molecules on monocytes that have been previously described to play a role in T cell/monocyte interaction (10, 11, 12, 13, 14, 15, 21, 22, 23, 24). For this purpose, freshly isolated monocytes were incubated with sCD26 in the presence or absence of TT, and the expression of CD80, CD86, and HLA-DR on monocytes was examined using flow cytometric analysis. As shown in Table IIIGo and Fig. 6Go, CD86 molecule expression on monocytes was increased within the first 48 h after TT and sCD26 stimulation. However, the increase in CD86 expression was no longer observed after 48 h of sCD26 incubation. In contrast, expression of CD80 and HLA-DR was not affected by stimulation of TT and sCD26 (Table IIGo). Therefore, these results showed that uptake of sCD26 into monocytes resulted in an increase in CD86 expression when monocytes were pulsed with TT and sCD26. Furthermore, these findings suggested that the enhanced CD86 expression on sCD26-treated monocytes contributed to trigger TT-induced T cell proliferation in the early stages of the immune response to recall Ag. It should be noted that sCD26/DPPIV- mutant did not affect CD86 expression on monocytes, consistent with data showing that only sCD26/DPPIV+, but not sCD26/DPPIV- mutant, enhanced TT-induced T cell proliferation (8, 24).


View this table:
[in this window]
[in a new window]
  		Table III. Increased CD86 expression on monocytes after incubation with sCD26/DPPVI+ 1

 


View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 6. sCD26/DPPIV induces up-regulation of the costimulatory molecule CD86 on monocytes. Freshly isolated monocytes (0.5 x 106/well) were incubated with or without TT for 16 h after a 24-h incubation in the standard medium alone, and then sCD26 (0.5 µg/ml) was added to the culture wells. After incubation with sCD26 for different hours, cells were washed with PBS, incubated with FITC-conjugated CD80, CD86, or HLA-DR, Abs, and then analysis was performed by FACSCalibur. The histogram shown in this figure is the increased intensity of CD86-FITC on monocytes cultured for 24 h with sCD26/PPIV+ after TT treatment. The single histogram profile of the CD86 expression shown in this figure is one of three representative experiments. The abscissa represents fluorescence intensity (log10 scale), and the ordinate represents the respective cell number.

 
Enhanced expression of CD86 on monocytes triggered by TT and sCD26 is dependent on increased synthesis of mRNA

To determine whether the enhancement in CD86 expression following incubation of monocytes with sCD26 in the presence of TT is dependent on increased protein synthesis, levels of mRNA encoding for CD86 were quantified by RT-PCR. Freshly isolated monocytes were incubated with or without TT for 16 h after a 24-h incubation in the standard medium alone, and then sCD26 (0.5 µg/ml) was added to the culture wells. After incubation with sCD26 for 24 h, cells were processed for RNA isolation as described in Materials and Methods. As demonstrated in Fig. 7Go, the increase of surface CD86 expression seen when monocytes were incubated with TT and sCD26/DPPIV+ is also associated with increased mRNA levels, suggesting that enhanced protein synthesis is one potential mechanism for enhanced CD86 surface expression. Of importance is the fact that monocytes incubated with TT and sCD26/DPPIV- did not demonstrate enhanced CD86 mRNA levels, again indicating the importance of DPPIV activity in this interaction.



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 7. Enhanced CD86 mRNA expression following treatment with sCD26/DPPIV+ on TT-treated monocytes. Freshly isolated monocytes (0.5 x 106/well) were incubated with or without TT for 16 h after a 24-h incubation in the standard medium alone, and then sCD26 (0.5 µg/ml) was added to the culture wells. After incubation with sCD26 for 24 h, total RNA was extracted and RT-PCR was performed. Expression of CD86 was increased in TT-treated monocytes cultured with sCD26/DPPIV+. {beta}-Actin was used as internal control. The different amount of the PCR product of {beta}-actin at different PCR cycles showed that the PCR was performed for relative quantitation in linear range.

 
Inhibitory effects of mAbs and CTLA-4 Ig on T cell proliferation by monocytes triggered by TT and sCD26

To further define the role of various surface molecules on T cell proliferation induced by TT-treated monocytes, monocytes were first treated with TT and/or sCD26, and then they were incubated with mAbs against CD80, CD86, HLA-DR, or CTLA-4 Ig at a final concentration of 5 µg/ml for 15 min at 4°C before onset of culture. Cells were then incubated at 37°C for the entire culture period. As shown in Fig. 8GoA, although mAb against HLA-DR efficiently inhibited T cell proliferation, sCD26 did not have an effect in the HLA-DR-mediated pathway. Similarly, no effect was observed in the TT-induced T cell proliferation after treatment of monocytes with anti-CD80 in the presence or absence of sCD26. In contrast, as shown in the lower panel of Fig. 8GoA, whereas T cell proliferation was inhibited by the presence of mAb against CD86 molecule and CTLA-4 Ig, this inhibition was significantly stronger in the culture with TT/sCD26 (sCD26/DPPIV+)-treated monocytes. The blocking effect of anti-CD86 Ab and CTLA-4 Ig was not exerted through the inhibition of DPPIV activity of sCD26, as examined in liquid phase by ELISA (data not shown). To confirm whether the above costimulatory effect was observed via the induction of CD86, CD86 mAb and CTLA-4 Ig were added at different concentrations to TT and/or sCD26 stimulation cultures. As shown in Fig. 8GoB, TT-mediated T cell proliferation enhanced by sCD26/DPPIV+ was strongly inhibited by CD86 mAb and CTLA-4 Ig in a dose-dependent manner. It should be noted that CTLA-4 Ig always exerted greater inhibitory effect on TT-mediated T cell proliferation than CD86 mAb. As previously noted in Fig. 6Go, TT/sCD26 (DPPIV+) monocytes expressed higher levels of CD86 than monocytes incubated with sCD26/DPPIV- molecules in the presence or absence of TT. These data strongly suggested that the increased surface expression of CD86 on monocytes treated with TT and sCD26 (DPPIV+) is essential for the enhancing effect of sCD26 on TT-induced T cell proliferation.



View larger version (34K):
[in this window]
[in a new window]
  		FIGURE 8. Inhibitory effect of CD86 mAb and CTLA-4 Ig on T cell proliferation induced by TT/sCD26-treated monocytes. A, Freshly isolated monocytes (0.5 x 106/well) were incubated with or without TT for 16 h after a 24-h incubation in the standard medium alone, and then sCD26 (0.5 µg/ml) was added to the culture wells (the presence or absence of TT/sCD26 is indicated in the box to the right). After incubation with or without sCD26 for 24 h, monocytes were incubated with the indicated mAbs (5 µg/ml) or CTLA-4 Ig (5 µg/ml) for 15 min at 4°C before onset of culture. Inhibition on T cell proliferation was expressed as the percentage of reactivity of control cultures without addition of mAbs or control human Ig performed in parallel. B, Dose-dependent inhibitory effects of anti-CD86 mAb and human CTLA-4 Ig (0.1–20 µg/ml) on T cell proliferative response induced by TT/sCD26-treated monocytes. The results were expressed as the percentage of the mean values obtained in the presence of the isotype-matched negative control or control murine Ig. Proliferation of T cells was monitored in all instances by measuring [3H]TdR incorporation on day 7 of culture. Bars are representative of mean values of percentge of inhibition or proliferation ± SE of three independently performed experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this paper, we report that sCD26 has an enhancing effect on T cell proliferation in the presence of the recall Ags, TT. Importantly, this enhancement resulted in an increase in the surface expression of the costimulatory molecule CD86 on monocytes following uptake of sCD26. This immune enhancing effect of sCD26 was observed within the initial 48 h of treatment with sCD26 in the presence of TT.

In the process of T cell proliferative response against a recall Ag, several major factors have been shown to contribute to the maintenance of the biological reaction, including APC, Th cells, and selected cytokines (10, 11, 12, 13, 14, 15, 21, 22, 23, 24). Initially, an APC-T cell interaction plays a key role in triggering the T cell response, leading eventually to expression of the T cell biological program (10, 14, 15). Our studies showed that the enhancing effect of sCD26 on TT-induced T cell proliferation occurred in the early stages of the immune response. Moreover, the cells affected by exogenously added sCD26 are the CD14-positive monocytes.

CD28 is constitutively expressed on T cells and interacts with the B7 molecules CD80 and CD86 (20, 21). This interaction leads to increased T cell proliferation, IL-2 production, and resistance to apoptosis (14). CD80 and CD86 are type 1 membrane glycoproteins belonging to the Ig superfamily (12, 13). In humans, their expression patterns differ according to the nature of the APC. CD86 expression is constitutive on monocytes and dendritic cells and is up-regulated by activation (10, 14). In contrast, CD80 is expressed at low levels on APC and is up-regulated following activation (14, 15).

CD86 has an important role in the priming of naive T cells and activation of memory T cells (10, 14, 19, 27, 28, 29). Although activation of naive T cells requires strong stimulatory signals provided by APC (30), and activation of recently activated memory T cells can be elicited with anti-CD3 mAb alone (31), most memory T cells are still dependent on CD28 triggering for their activation (22). A stable interaction between APC and T cells is dependent not only on the absolute affinity and specificity on the TCR and its ligands, but also on the relative density of molecules available for contact at the interaction site (32, 33). Therefore, our findings that up-regulation of CD86 but not CD80 on monocytes occurs following uptake of sCD26 by the same cell population suggest that this is one of mechanisms for the enhancement of TT-induced T cell proliferation by sCD26. Although it is presently unclear how the relatively subtle up-regulation of CD86 on monocyte surface resulted in a significantly enhancing effect on TT-mediated T cell proliferation, available data support the notion that oligomerization or clustering of CD80/CD86 molecules on APC leads to prolonged CD28/CTLA-4 receptor occupancy (34).

Recently, we reported that sCD26 specifically bound to M6P/IGF-IIR present on the cell surface of the K562 cell line (9). The M6P/IGF-IIR is located mainly in the intracellular compartments, particularly in the trans-Golgi network and endosomes, with a small portion of the receptor present on the cell surface (35). The primary function of M6P/IGF-IIR is the cellular trafficking of M6P-containing glycoproteins from the trans-Golgi network or the cell surface to endosomes/lysosomes (35, 36). The receptor also binds to a mannose-6-phosphated precursor form of TGF-{beta}, proliferin, and leukemia inhibitory factor, and has been shown to play an important role in regulating the activation or degradation of these factors (37, 38, 39, 40, 41). The present findings indicate that M6P/IGF-IIR mediates the internalization of sCD26 and that this interaction induces the up-regulation of CD86 on monocytes, resulting in the enhancement of TT-induced T cell proliferation.

Accumulating evidence suggests that DPPIV enzyme activity plays an essential role in CD26-mediated T cell costimulation, as well as in T cell immune reactions (1). Previous studies suggested that several potential scenarios may explain the observed effects of DPPIV on immune activation (3, 42, 45, 46, 47). It is possible that CD26/DPPIV exerts its effect via the membrane-bound form, particularly in T cells. It is also possible that the CD26/DPPIV exerts its effect with the soluble form, in view of the fact that CD26/DPPIV is actually present in human serum (25). Previous studies with CD26-transfected Jurkat cells showing that DPPIV activity on CD26 augmented cellular responses of CD26-transfected Jurkat cells to external stimuli would support the former possibility (25, 48). In contrast, data demonstrating that recombinant sCD26 with DPPIV enzyme activity enhanced proliferative responses of PBMC to stimulation with the soluble recall Ag TT would support the latter scenario. Our results showed that sCD26/DPPIV+ had an enhancing effect on TT-induced T cell proliferation associated with an increase in CD86 expression on monocytes. Importantly, incubation with sCD26/DPPIV- did not enhance TT-induced T cell proliferation and had no effect on CD86 surface expression on monocytes, despite the uptake of sCD26/DPPIV- by the same cell population. Previous reports showed that CD26/DPPIV regulated immune responses by cleaving selected chemokines at the N terminus to modify their biological functions (3, 42). Chemokines processed by DPPIV enzyme exhibited lower chemotactic potency, impaired signaling effects, and altered receptor specificity (3, 42). In view of its ability to cleave selected biological factors as a serine protease, it is possible that CD26/DPPIV exerts its effect by cleavage of certain factors that may in turn affect the up-regulation and/or oligomerization of CD86 on monocytes. Further studies are required for the isolation and characterization of CD26/DPPIV-associated factors responsible for regulating the expression of CD86.

Cytokines are critical in maintaining the latter stages of immune reaction (10, 14, 22). In the initial stages of Ag presentation, the capacity of Ag presentation can be modulated by several nonexclusive mechanisms, including the efficiency of Ag capturing and loading, MHC molecule density and occupancy, or altered costimulatory molecule expression (32, 33). Various proteins have been demonstrated to modulate the steps described above to alter T cell responsiveness against specific Ags. For example, opsonins augment the ability of Ag capture of APCs, IL-10 increases macropinocytosis and endocytosis by APC, and IFN-{gamma} up-regulates the expression of MHC class II molecules on APC (49, 50, 51). Moreover, recent studies revealed that aminopeptidase N (CD13), one of the ectopeptidases on the cell surface, may trim MHC class II-bound peptides on the surface of the APC to modify T cell responsiveness (26). However, our findings clearly show that the enhancement of TT-induced T cell proliferation by sCD26 did not result from trimming of the MHC class II-bound peptide on the APC surface, in contrast to the findings related to CD13 aminopeptidase.

It should be noted that although uptake of the sCD26 molecules by monocytes resulted in an increase in CD86 monocyte surface expression, there is the still possibility that DPPIV enzyme activity of sCD26 facilitates Ag processing in monocytes, resulting in enhanced T cell/monocyte interaction and leading to increased T cell proliferation.

In summary, we have shown that sCD26 is taken up by monocytes and exertes its enhancing effect on T cell proliferation by altering the Ag presenting function of monocytes through the up-regulation of CD86 expression. Our paper also demonstrates that sCD26 plays an important role in the immune surveillance process against recall Ags, further elucidating the key role of CD26 in immune regulation.


   Footnotes
 
1 This study was supported by a grant-in-aid from the Ministry of Education, Science, Sports and Culture, and from the Ministry of Health, Labor and Welfare, as well as by National Institutes of Health Grant AR33713. N.H.D. is the recipient of a grant from the M. D. Anderson Cancer Center Physician-Scientist Program and the V Foundation. Back

2 Address correspondence and reprint requests to Dr. Chikao 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 address: morimoto{at}ims.u-tokyo.ac.jp Back

3 Abbreviations used in this paper: DPPIV, dipeptidyl peptidase IV; sCD26, soluble CD26; TT, tetanus toxoid; M6P, mannose-6-phosphate; IGF-IIR, insulin-like growth factor II receptor; CHO, Chinese hamster ovary; sCD26-OG, Oregon green-conjugated sCD26; M6P/IGF-IIR-red, Texas red-conjugated M6P/IGF-IIR; sCD26/DPPIV+, sCD26 with DPPIV; sCD26/DPPIV-, sCD26 without DPPIV. Back

Received for publication June 4, 2001. Accepted for publication October 12, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Morimoto, C., S. F. Schlossman. 1998. The structure and function of CD26 in the T-cell immune response. Immunol. Rev. 161:55.[Medline]
  2. von Bonin, A., J. Huhn, B. Fleischer. 1998. Dipeptidyl-peptidase IV/CD26 on T cells: analysis of an alternative T-cell activation pathway. Immunol. Rev. 161:43.[Medline]
  3. Oravecz, T., M. Pall, G. Roderiquez, M. D. Gorrell, M. Ditto, N. Y. Nguyen, R. Boykins, E. Unsworth, M. A. Norcross. 1997. Regulation of the receptor specificity and function of the chemokine RANTES (regulated on activation, normal T cells expressed and secreted) by dipeptidyl peptidase IV (CD26)-mediated cleavage. J. Exp. Med. 186:1865.[Abstract/Free Full Text]
  4. von Bonin, A., J. Huhun, B. Fleischer. 1998. Dipeptidyl-peptidase IV/CD26 on T cells: analysis of an alternative T-cell activation pathway. Immunol. Rev. 161:43.
  5. Fleischer, B.. 1994. CD26: surface protease involved in T-cell activation. Immunol. Today 15:1800.
  6. Morimoto, C., S. F. Schlossman. 1994. CD26: a key costimulatory molecule on CD4 memory T cells. The Immunologist 2:4.
  7. Hegen, M., J. Kameoka, R.-P. Dong, S. F. Schlossman, C. Morimoto. 1997. Cross-linking of CD26 by antibody induces tyrosine phosphorylation and activation of mitogen-activated protein kinase. Immunology 90:257.[Medline]
  8. Tanaka, T., J. Kameoka, A. Yaron, S. F. Schlossman, C. Morimoto. 1993. The costimulatory activity of the CD26 antigen requires dipeptidyl peptidase IV enzymatic activity. Proc. Natl. Acad. Sci. USA 90:4583.
  9. Ikushima, H., Y. Munakata, T. Ishii, S. Iwata, M. Terashima, H. Tanaka, S. F. Schlossman, C. Morimoto. 2000. Internalization of CD26 by mannose 6-phosphate/insulin-like growth factor II receptor contributes to T cell activation. Proc. Natl. Acad. Sci. USA 97:8439.[Abstract/Free Full Text]
  10. Lenschow, D. J., T. L. Walunas, J. A. Bluestone. 1996. CD28/B7 system of T cell costimulation. Annu. Rev. Immunol. 14:233.[Medline]
  11. Yokochi, T., R. D. Holly, E. A. Clark. 1982. B lymphoblast antigen (BB-1) expressed on EBV-activated B cell blasts, B lymphoblastoid cell lines, and Burkitt’s lymphomas. J. Immunol. 128:823.[Abstract]
  12. Azuma, M., D. Ito, H. Yagita, K. Okumura, J. H. Phillips, L. L. Lanier, C. Somoza. 1993. B70 antigen is a second ligand for CTLA-4 and CD28. Nature 366:76.[Medline]
  13. Freeman, G. J., J. G. Gribben, V. A. Bousiotis, J. W. Ng, V. A. Restivo, L. A. Lombard, G. S. Gray, L. M. Nadler. 1993. Cloning of B7-2: a CTLA-4 counter-receptor that costimulates human T cell proliferation. Science 262:909.[Abstract/Free Full Text]
  14. McAdam, A. J., A. N. Schweitzer, A. H. Sharpe. 1998. The role of B7 co-stimulation in activation and differentiation of CD4+ and CD8+ T cells. Immunol. Rev. 165:231.[Medline]
  15. Chambers, C. A.. 2001. The expanding world of co-stimulation: the two-signal model revisited. Trends Immunol. 22:217.[Medline]
  16. Croft, M., D. D. Duncan, L. S. Swain. 1992. Response of naive antigen-specific CD4+ T cells in vitro: characteristics and antigen-presenting cell requirements. J. Exp. Med. 176:1431.[Abstract/Free Full Text]
  17. Walunas, T. L., D. J. Lenschow, C. Y. Bakker, P. S. Linsley, G. J. Freeman, J. M. Green, C. B. Thompson, J. A. Bluestone. 1994. CTLA-4 can function as a negative regulator of T cell activation. Immunity 1:4405.
  18. Krummel, M. F., J. P. Allison. 1995. CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation. J. Exp. Med. 182:459.[Abstract/Free Full Text]
  19. Saito, T.. 1998. Negative regulation of T cell activation. Curr. Opin. Immunol. 10:313.[Medline]
  20. Caux, C., B. Vanbervliet, C. Massacrier, M. Azuma, K. Okumura, L. L. Lanier, J. Banchereau. 1994. B70/B7-2 is identical to CD86 and is the major functional ligand for CD28 expressed on human dendritic cells. J. Exp. Med. 180:1841.[Abstract/Free Full Text]
  21. Hathcock, K. S., G. Laszlo, C. Pucillo, P. Linsley, R. J. Hodes. 1994. Comparative analysis of B7-1 and B7-2 costimulatory ligands: expression and function. J. Exp. Med. 180:631.[Abstract/Free Full Text]
  22. Yi-qun, Z., R. J. Joost van Neerven, A. Kasran, M. de Boer, J. L. Ceuppens. 1996. Differential requirements for co-stimulatory signals from B7 family members by resting versus and recently activated memory T cells towards soluble recall antigens. Int. Immunol. 8:37.[Abstract/Free Full Text]
  23. Hakamada-Taguchi, R., T. Kato, H. Ushijima, M. Murakami, T. Uede, H. Nariuchi. 1998. Expression and co-stimulatory function of B7-2 on murine CD4+ T cells. Eur. J. Immunol. 28:873.
  24. Manickasingham, S. P., S. M. Anderson, C. Burkhart, D. C. Wraith. 1998. Qualitative and quantitative effects of CD28/B7-mediated costimulation on naive T cells in vivo. J. Immunol. 161:3827.[Abstract/Free Full Text]
  25. Tanaka, T., J. S. Duke-Cohan, J. Kameoka, A. Yaron, I. Lee, S. F. Schlossman, C. Morimoto. 1994. Enhancement of antigen-induced T-cell proliferation by soluble CD26/dipeptidyl peptidase IV. Proc. Natl. Acad. Sci. USA 91:3082.[Abstract/Free Full Text]
  26. Larsen, S. L., L. O. Pedersen, S. Buus, A. Stryhn. 1996. T cell responses affected by aminopeptidase N (CD13)-mediated trimming of major histocompatibility complex class II-bound peptides. J. Exp. Med. 84:183.
  27. Engel, P., J. G. Gribben, G. J. Freeman, L. J. Zhou, Y. Nozawa, M. Abe, L. M. Nadler, H. Wakasa, T. F. Tedder. 1994. The B7-2 (B70) costimulatory molecule expressed by monocytes and activated B lymphocytes is the CD86 differentiation antigen. Blood 84:1402.[Abstract/Free Full Text]
  28. Vyth-Dreese, F. A., N. T. Dellemijin, D. Majoor, D. de Jong. 1995. Localization in situ of the co-stimulatory molecules B7.1, B7.2, CD40 and their ligands in normal human lymphoid tissue. Eur. J. Immunol. 25:3023.[Medline]
  29. Yokozeki, H., I. Hatayama, O. Ohki, T. Matsunaga, K. Watanabe, T. Satoh, M. Azuma, K. Okumura, K. Nishioka. 1996. Functional CD86 (B7-2/B70) on cultured human Langerhans cells. J. Invest. Dermatol. 106:147.[Medline]
  30. Liu, Y., C. A. Janeway. 1992. Cells that present both specific ligand and costimulatory activity are the most efficient inducers of clonal expansion of normal CD4 T cells. Proc. Natl. Acad. Sci. USA 89:3845.[Abstract/Free Full Text]
  31. Van de Velde, H., K. Lorre, M. Bakkus, K. Thielemans, J. L. Ceuppens, M. de Boer. 1993. CD45RO+ memory T cells but not CD45RA+ naive T cells can be efficiently activated by remote costimulation with B7. Int. Immunol. 5:1483.[Abstract/Free Full Text]
  32. Prakken, B., M. Wauben, D. Genini, R. Samodal, J. Barnett, A. Mendivil, L. Leoni, S. Albani. 2000. Artificial antigen-presenting cells as a tool to exploit the immune "synapse.". Nat. Med. 6:1406.[Medline]
  33. Grakoui, A., S. K. Bromley, C. Sumen, M. M. Davis, A. S. Shaw, P. M. Aleen, M. L. Dustin. 1999. The immunological synapse: a molecular machine controlling T cell activation. Science 285:221.[Abstract/Free Full Text]
  34. Greene, J., G. M. L., J. Leytze, R. Emswiler, J. Peach, W. Bajorath, W. Cosand, P. Linsley. 1996. Covalent dimerization of CD28/CTLA-4 and oligomerization of CD80/CD86 regulate T cell costimulatory interactions. J. Biol. Chem. 271:26762.[Abstract/Free Full Text]
  35. Kornfeld, S.. 1992. Structure and function of the mannose 6-phosphate/insulin-like growth factor II receptors. Annu. Rev. Biochem. 61:307.[Medline]
  36. Nykjaer, A., E. I. Christensen, H. Vorum, H. Hager, C. M. Peterson, H. Roigaard, H. Y. Min, F. Vilhardt, L. B. Moller, S. Konfeld. 1998. Mannose 6-phosphate/insulin-like growth factor-II receptor targets the urokinase receptor to lysosomes via a novel binding interaction. J. Cell Biol. 141:815.[Abstract/Free Full Text]
  37. Purchio, A. F., J. A. Cooper, A. M. Brunner, M. N. Lioubin, L. E. Gentry, K. S. Kovacina, L. A. Roth, H. Maruquardt. 1988. Identification of mannose 6-phosphate in two asparagine-linked sugar chains of recombinant transforming growth factor-{beta}1 precursor. J. Biol. Chem. 263:14211.[Abstract/Free Full Text]
  38. Dennis, P. A., D. B. Rifkin. 1991. Cellular activation of latent transforming growth factor-{beta} requires binding to the cation-independent mannose 6-phosphate/insulin-like growth factor type II receptor. Proc. Natl. Acad. Sci. USA 88:580.[Abstract/Free Full Text]
  39. Lee, S. J., D. Nathans. 1988. Proliferin secreted by cultured cells binds to mannose 6-phosphate receptors. J. Biol. Chem. 263:3521.[Abstract/Free Full Text]
  40. Blanchard, F., S. Raher, L. Duplomb, P. Vusio, V. Pitard, J. L. Taupin, J. F. Moreau, B. Hoflack, S. Minvielle, Y. Jacques. 1998. Mannose 6-phosphate/insulin-like growth factor II receptor mediates internalization and degradation of leukemia inhibitory factor but not signal transduction. J. Biol. Chem. 273:20886.[Abstract/Free Full Text]
  41. Blanchard, F., L. Duplomb, S. Raher, P. Vusio, B. Hoflack, Y. Jacques, A. Godard. 1999. Mannose 6-phosphate/insulin-like growth factor II receptor mediates internalization and degradation of leukemia inhibitory factor but not signal transduction. J. Biol. Chem. 274:24685.[Abstract/Free Full Text]
  42. Proost, P., I. De Meester, D. Schols, S. Struyf, A.-M. Lambeir, A. Wuyts, G. Opdenakker, E. De Clercq, S. Scharpe, J. Van Damme. 1998. Amino-terminal truncation of chemokines by CD26/dipeptidyl-peptidase IV: conversion of RANTES into a potent inhibitor of monocyte chemotaxis and HIV-1-infection. J. Biol. Chem. 273:7222.[Abstract/Free Full Text]
  43. Dang, N. H., D. A. Hafler, S. F. Schlossman, J. B. Breitmeyer. 1990. FcR-mediated crosslinking of Ta1 (CDw26) induces human T lymphocyte activation. Cell. Immunol. 125:42.[Medline]
  44. Dang, N. H., Y. Torimoto, K. Deusch, S. F. Schlossman, C. Morimoto. 1990. Co-mitogenic effect of solid-phase immobilized anti-1F7 on human CD4 T cell activation via CD3 and CD2 pathways. J. Immunol. 144:4092.[Abstract]
  45. Kahne, T., K. Neubert, J. Faust, S. Ansorge. 1998. Early phosphorylation events induced by DPPIV/CD26-specific inhibitors. Cell. Immunol. 189:60.[Medline]
  46. Kubota, T., G. R. Flentke, W. W. Bachovchin, B. D. Stollar. 1992. Involvement of dipeptidyl peptidase IV in an in vivo immune response. Clin. Exp. Immunol. 89:192.[Medline]
  47. Korom, S., I. De Meester, T. H. W. Stadlbauer, A. Chandraker, M. Schaub, M. H. Sayegh, A. Belyaev, A. Haemers, S. Scharpe, J.W. Kupiec-Weglinski. 1997. Inhibition of CD26/dipeptidyl peptidase IV activity in vivo prolongs cardiac allograft survival in rat recipients. Transplantation 63:1495.[Medline]
  48. Tanaka, T., J. Kameoka, A. Yaron, S. F. Schlossman, C. Morimoto. 1990. The costimulatory activity of the CD26 antigen requires dipeptidyl peptidase IV enzyme activity. Proc. Natl. Acad. Sci. USA 90:4586.[Abstract/Free Full Text]
  49. Longoni, D., L. Piemonti, S. Bernasconi, A. Mantovani, P. Allavena. 1998. Interleukin-10 increases mannose receptor expression and endocytic activity in monocyte-derived dendritic cells. Int. J. Clin. Lab. Res. 28:162.[Medline]
  50. Morel, A. S., S. Quaratino, D. C. Douek, M. Londei. 1997. Split activity of interleukin-10 on antigen capture and antigen presentation by human dendritic cells: definition of a maturative step. Eur. J. Immunol. 27:26.[Medline]
  51. Lee, Y. J., R. B. Panek, M. Huston, E. N. Benveniste. 1995. Role of protein kinase C and tyrosine kinase activity in IFN-{gamma}-induced expression of the class II MHC gene. Am. J. Physiol. 268:C127.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
K. Ohnuma, M. Uchiyama, T. Yamochi, K. Nishibashi, O. Hosono, N. Takahashi, S. Kina, H. Tanaka, X. Lin, N. H. Dang, et al.
Caveolin-1 Triggers T-cell Activation via CD26 in Association with CARMA1
J. Biol. Chem., March 30, 2007; 282(13): 10117 - 10131.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
K. Ohnuma-Ishikawa, T. Morio, T. Yamada, Y. Sugawara, M. Ono, M. Nagasawa, A. Yasuda, C. Morimoto, K. Ohnuma, N. H. Dang, et al.
Knockdown of XAB2 Enhances All-Trans Retinoic Acid-Induced Cellular Differentiation in All-Trans Retinoic Acid-Sensitive and -Resistant Cancer Cells
Cancer Res., February 1, 2007; 67(3): 1019 - 1029.
[Abstract] [Full Text] [PDF]

Home page