Up-Regulation on Apcs Induces T Cell Proliferation Through CD86

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

This information is current as Kei Ohnuma, Yasuhiko Munakata, Tomonori Ishii, Satoshi of September 23, 2021. Iwata, Seiji Kobayashi, Osamu Hosono, Hiroshi Kawasaki, Nam H. Dang and Chikao Morimoto J Immunol 2001; 167:6745-6755; ; doi: 10.4049/jimmunol.167.12.6745

http://www.jimmunol.org/content/167/12/6745 Downloaded from

References This article cites 51 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/167/12/6745.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on September 23, 2021

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Soluble CD26/Dipeptidyl Peptidase IV Induces T Cell Proliferation Through CD86 Up-Regulation on APCs1

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

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 Downloaded from 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. The Journal of Immunology, 2001, 167: 6745–6755. http://www.jimmunol.org/

he widely distributed 110-kDa cell surface glycoprotein CD26 cDNA (5, 6). In addition, anti-CD26 Ab treatment of T cells CD26 has known dipeptidyl peptidase IV (DPPIV,3 EC leads to a decrease in the surface expression of CD26 via its in- T 3.4.14.5; Ref. 3) activity in its extracellular domain (1, 2). ternalization, and such modulation results in an enhanced prolif- This enzyme is capable of cleaving amino-terminal dipeptides with erative response to anti-CD3 or anti-CD2 stimulation, as well as either L-proline or L-alanine at the penultimate position. CD26/ enhanced tyrosine phosphorylation of signaling molecules such as DPPIV has two physiological forms in human (3). One is the mem- CD3␨ and p56lck (7). Moreover, we have shown that DPPIV en- ϩ brane-bound form preferentially expressed on the CD4 helper/ zyme activity is required for the CD26-mediated T cell costimu- by guest on September 23, 2021 memory T cell population, whereas the other is the soluble form lation (8). More recently, we have shown that internalization of found in serum. Accumulating evidence suggests that DPPIV en- CD26 after cross-linking is mediated in part by the mannose-6- zyme activity plays an essential role in the immune response (4). phosphate (M6P)/insulin-like growth factor II receptor (IGF-IIR), Cross-linking of CD26 and CD3 with solid-phase immobilized and that the interaction of CD26 and M6P/IGFIIR plays a role in mAbs can induce T cell costimulation and IL-2 production by CD26-induced T cell costimulation (9). either human CD4ϩ T cells or Jurkat T cell lines transfected with 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 (10Ð14). CD28-B7 interactions lead to T cell *Division of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan; Departments of †Molecular proliferation, differentiation, and cytokine secretion (14, 15). In Diagnostics and ‡Rheumatology and Hematology, Graduate School of Medicine, To- contrast, engagement of CTLA-4 on activated T cells by B7-1 or ¤ hoku University, Japan; and Department of Lymphoma/Myeloma, M. D. Anderson B7-2 results in an inhibition of T cell responses (16Ð18). However, Cancer Center, Houston, TX 77030 only CD28 is constitutively expressed and, hence, it has an im- Received for publication June 4, 2001. Accepted for publication October 12, 2001. portant role in the generation of an immune response (19Ð24). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance In our previous report, we have shown that recombinant soluble with 18 U.S.C. Section 1734 solely to indicate this fact. CD26 (sCD26) enhanced proliferative responses of PBLs to stim- 1 This study was supported by a grant-in-aid from the Ministry of Education, Science, ulation with the soluble Ag, tetanus toxoid (TT) (25). In addition, Sports and Culture, and from the Ministry of Health, Labor and Welfare, as well as this enhancing effect required DPPIV enzyme activity (8, 25). 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 However, the precise mechanism for enhancement of TT-induced Foundation. T cell proliferation by sCD26 remains unresolved. 2 Address correspondence and reprint requests to Dr. Chikao Morimoto, Division of In the present study, we demonstrated that the target cells of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Sci- sCD26 were the CD14-positive monocytes in the peripheral blood ence, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. E-mail address: [email protected] and that M6P/IGF-IIR played a role in transporting sCD26 into the 3 Abbreviations used in this paper: DPPIV, dipeptidyl peptidase IV; sCD26, soluble monocytes. Importantly, we found that sCD26 could up-regulate CD26; TT, tetanus toxoid; M6P, mannose-6-phosphate; IGF-IIR, insulin-like growth the expression of the CD86 but not CD80 or HLA-DR Ag on factor II receptor; CHO, Chinese hamster ovary; sCD26-OG, Oregon green-conjugated sCD26; M6P/IGF-IIR-red, Texas red-conjugated M6P/IGF-IIR; sCD26/DP- monocytes. These results suggest that sCD26 can be a useful agent PIVϩ, sCD26 with DPPIV; sCD26/DPPIVϪ, sCD26 without DPPIV. in potentiating immune response in selected clinical settings.

Materials and Methods washed three times with PBS. Monocytes (1.0 ϫ 104/well) were then sub- ϫ 5 Isolation and activation of human lymphocyte populations jected to the assay with 1.0 10 /well of purified T cells, which originated from the same donor as the prepared monocytes. Human PBMC, collected from healthy adult volunteers who were immu- nized with TT within two years before donation, were isolated by centri- fugation on Ficoll/Paque (Amersham Pharmacia Biotech, Piscataway, NJ). Preparation of sCD26 PBMC were directly used for the time-course studies. For the reconstitu- ϩ sCD26 with DPPIV (sCD26/DPPIV ) was produced according to the tion experiments, PBMC were further purified into T cell fractions and method described previously (24). Briefly, the expression vector APC fractions. To obtain a highly purified T cell population, PBMC were RcSR␣-26 days 3Ð9, which contains a deletion of the coding sequence for ϩ - separated into an E rosette-positive (E ) population and were used as rest amino acids 3Ð9 of CD26, was transfected into a dihydrofolate reductase- ing T cells as determined by flow cytometric analysis (FACSCalibur; Nip- deficit Chinese hamster ovary (CHO) cell line, DXB-11 by electroporation, pon BD Biosciences, Tokyo, Japan) using an FITC-labeled anti-CD3 mAb together with pMT-2 providing the dihydrofolate reductase gene. Mutant (BD PharMingen, San Diego, CA) with purity being Ͼ95%. To obtain Ϫ sCD26 without DPPIV (sCD26/DPPIV ) was produced in the same APC, an E rosette-negative (EϪ ) population was adhered to plastic plates method except that RcSR␣-26d3-9 was further modified to yield RcSR␣- for4hat37¡C, and adherent cells were used as APC. Monocytes were 26d3-9 S630A, which contains a point mutation at the active site of the either purified by a flow cytometer on the basis of PE-labeled anti-CD14 DPPIV enzyme (Ser630 was replaced by Ala) by site-directed mutagenesis mAb (BD PharMingen)-oriented parameter, or by negative selection using the oligonucleotide. The transfected CHO cells, which produce either through the use of immunomagnetic beads coated with an anti-CD3, CD7, sCD26 or mutant sCD26, were cultured in serum-free CHO-S-SFM II me- CD19, CD45RA, CD56, and IgE mAb (Miltenyi Biotec, Auburn, CA) with dium (Life Technologies) supplemented with 1 ␮M methotrexate (Sigma- purity being Ͼ95%. PBMC were cultured in RPMI 1640 medium (Life Aldrich). The culture supernatant was collected and subjected to affinity Technologies, Grand Island, NY) supplemented with 10% FCS, 100 U/ml chromatography on adenosine deaminase-Sepharose according to the penicillin, and 100 ␮g/ml streptomycin (Life Technologies) and TT (Cal-

methods described previously (9). Downloaded from biochem, 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) mAbs and reagents 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 The source and working concentration of the mAbs used as primary Abs addition of sCD26 in the culture medium at different times. To avoid in- for ﬂow cytometry are as follows: PE-conjugated anti-CD3 (UCHT1, terference by nonspeciﬁc activation of monocytes due to contamination, mouse IgG1; 10 ␮g/ml; BD PharMingen), anti-CD14 (Mo-2, mouse IgM;

polymyxin B sulfate (20 IU/ml; Sigma-Aldrich, St. Louis, MO) was added 10 ␮g/ml; Beckman Coulter, Miami, FL), anti-CD19 (HIB19, mouse IgG1; http://www.jimmunol.org/ to all media and reagents used for APC/monocytes experiments. 10 ␮g/ml; BD PharMingen), and anti-CD56 (NKH1, mouse IgG1; 10 ␮g/ All cells were preincubated in the standard medium for 24 h to minimize ml; Beckman Coulter); FITC-labeled anti-CD80 (BB1, mouse IgM; 10 the risk of potential interference from sCD26 present in human serum (8). ␮g/ml; BD PharMingen), anti-CD86 (IT2.2, mouse IgG2; 10 ␮g/ml; BD In these experiments, cells were incubated in 96-well plates (200 ␮l/well; PharMingen), and anti-HLA-DR (L243, mouse IgG2; 10 ␮g/ml; BD Falcon, Franklin Lakes, NJ). For the time-course study, 1.0 ϫ 105 PBMC PharMingen). Oregon green-conjugated sCD26 (sCD26-OG) was made were incubated with 0.5 ␮g/ml TT for 16 h. Then, 0.5 ␮g/ml sCD26 was with FluoReporter Oregon green protein labeling kit (Molecular Probes, added to the well and was incubated for 0, 6, 12, 24, 48, 72, and 96 h. The Eugene, OR) according to the manufacturer’s instruction. These Oregon day when the ﬁrst sCD26 was added to the culture well was deﬁned as day green-conjugated proteins were used at a concentration of 1 ␮g/ml. Bio- 0 of culture. All assays in proliferation experiments were performed on day tinylated anti-CD14 (Mo-2; IgM; 10 ␮g/ml) was purchased from Beckman

7 of culture after cells were incubated at 37¡Cina5%CO2 humidified Coulter. mAbs for blocking assays were obtained as follows: CD80 (BB- atmosphere. For the reconstitution study, 0.5 ϫ 106 of monocytes were 1), CD86 (IT2.2), and HLA-DR (L243) from were BD PharMingen; the by guest on September 23, 2021 preincubated with TT at a concentration of 0.5 ␮g/ml with different con- chimeric protein of human CTLA-4 and murine Ig (CTLA-4 Ig) and rel- centrations of sCD26 for 24 h. Monocytes were washed with PBS, and then evant control murine Ig were purchased from Ancell (Bayport, MN); and 1 ϫ 104/well of preincubated monocytes were subjected to the assay with mouse anti-human M6P/IGFIIR mAb was kindly provided by Dr. V. 1 ϫ 105/well of purified T cells from the same donor as the prepared Horcjsi (Academy of Science of the Czech Republic, Praha, Czech Re- monocytes. For fixation study, as prefixed stimulation study, after being public). Texas red-conjugated anti-human M6P/IGF-IIR mAb (M6P/IGF- treated with 0.05% glutaraldehyde for 30 s at room temperature, followed IIR-red) was made with FluoReporter Texas red protein labeling kit (Mo- by being washed three times with PBS, 0.5 ϫ 106 of monocytes were lecular Probes) according to the manufacturer’s instructions. In all incubated with TT at a concentration of 0.5 ␮g/ml and with 0.5 ␮g/ml experiments, relevant control mAbs of the same Ig isotype were included sCD26. As postfixed stimulation study, 0.5 ϫ 106 of monocytes were ini- (IgG1 (MOPC-21), IgG2 (G155Ð178), and IgM (G155Ð228) were pur- tially incubated with TT at a concentration of 0.5 ␮g/ml and with 0.5 ␮g/ml chased from BD PharMingen). Egg white lysozyme was purchased from sCD26. Subsequently, the preincubated monocytes were treated with Wako Pure Chemical (Osaka, Japan) and was conjugated with Oregon 0.05% glutaraldehyde for 30 s at room temperature, followed by being green.

FIGURE 1. Proliferative effect on PBMC by sCD26. PBMC (1 ϫ 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 ␮g/ml) was added to the medium to in- cubate for an additional 16 h. sCD26 (0.5 ␮g/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 Ϯ SE calculated from three independently performed experiments. The Journal of Immunology 6747

Table I. sCD26-OG incorporation according to lymphocyte subpopulationsa

PE-Ab CD3 CD14 CD19 CD56

% PE-positive cells among PBMC TTϪ % 73.3 Ϯ 6.5 10.3 Ϯ 4.7 12.4 Ϯ 3.8 4.6 Ϯ 2.3 MFI 779 Ϯ 124.3 985 Ϯ 255.4 610 Ϯ 178.9 523 Ϯ 160.3 TTϩ % 74.8 Ϯ 7.6 9.8 Ϯ 4.6 15.1 Ϯ 4.9 5.9 Ϯ 3.1 MFI 786 Ϯ 144.3 967 Ϯ 233.2 592 Ϯ 171.3 514 Ϯ 152.6

a Freshly isolated PBMC (1 ϫ 106/well) were incubated with or without TT for 16 h after 24-h incubation and were then washed with ice-cold PBS and incubated with various PE-conjugated Abs (CD3, CD14, CD19, and CD56) for 30 min on ice. Analysis was performed by FACSCalibur. The experiments represent mean values Ϯ SE calculated from three independently performed experiments. %, Percent of PE-positive cells cell number among PBMC. MFI, mean ﬂuorescence intensity.

T cell proliferation assay Confocal laser microscopy T cell proliferation induced by APC was measured by [3H]TdR (ICN Ra- For fluorescent microscopy experiments of uptake of sCD26 by monocytes, diochemicals, Irvine, CA) uptake. Seven-day-incubated cells were pulsed purified monocytes were incubated with sCD26-OG (1 ␮g/ml) with or with 1 ␮Ci/well of [3H]TdR 8 h before harvesting onto a glass-fiber filter without preincubation with TT (0.5 ␮g/ml). Cells were then washed in (Wallac, Turk, Finland), and the incorporated radioactivity was quantified ice-cold PBS twice and were incubated in acidic PBS (pH 3.0) to strip any by a liquid scintillation counter (Wallac). sCD26-OG attached to the cell surface. The cells were then attached to Downloaded from microslide glass (Matsunami Glass, Tokyo, Japan) and fixed with 3% para- Flow cytometric analysis formaldehyde in PBS for 15 min at room temperature. Cells were blocked with mouse Ig isotypes (1 ␮g/ml) for 30 min at 4¡C, followed by incuba- Assessment of the PBMC population that takes up sCD26-OG was per- tion with biotinylated anti-CD14 (10 ␮g/ml) for 30 min at 4¡C, then formed with PE-conjugated anti-CD3, anti-CD14, anti-CD19, and anti- ␮ ϫ 6 washed with ice-cold PBS twice and incubated with streptavidin-conju- CD56 mAbs (10 g/ml). For these experiments, 1 10 PBMC/well were gated Texas red conjugate (1/200; Beckman Coulter) for 30 min at 4¡C. In incubated in standard medium containing sCD26-OG for 24 h with or experiments of sCD26 uptake via M6P/IGF-IIR on monocytes, purified http://www.jimmunol.org/ without preincubation with TT. Cells were then washed twice in ice-cold monocytes were incubated with or without TT for 16 h, and sCD26-OG PBS, followed by incubation for 2 min in acidic PBS (pH 3.0) to strip any and M6P/IGF-IIR-red were added to the culture well after being blocked sCD26-OG attached to the cell surface. Cells were subsequently washed in with mouse Ig isotypes (1 ␮g/ml) for 30 min at 4¡C. Cells were then ice-cold PBS and were incubated with mouse Ig isotypes (1 ␮ g/ml) to block incubated for 30 min at 4¡C. For detection of cell surface colocalization, nonspecific binding; this was followed by reaction with PE-conjugated mAbs. cells were washed in ice-cold PBS twice and were attached to microslide In experiments assessing the expression of CD80, CD86, and HLA-DR glass, followed by fixation with 3% paraformaldehyde in PBS for 15 min ϫ 6 - on purified monocytes (0.5 10 monocytes/well), monocytes were pre at room temperature. For detection of intracellular incorporation, cells were incubated with or without TT (0.5 ␮g/ml) for 16 h. Following incubation incubated for 24 and 36 h at 37¡Cina5%CO humidified atmosphere, and with sCD26 (0.5 ␮g/ml) for 24 h, FITC-conjugated CD80, CD86, and 2 ␮ were then washed in ice-cold PBS twice and incubated in acidic PBS (pH HLA-DR mAbs (10 g/ml) were used with PE-conjugated anti-CD14 (10 3.0) to strip any sCD26-OG attached to the cell surface. The cells were then

␮ by guest on September 23, 2021 g/ml) to gate exclusively on the monocyte population. In experiments attached to microslide glass and fixed with 3% paraformaldehyde in PBS assessing the effect of M6P on cellular uptake of sCD26, purified mono- ϫ 6 for 15 min at room temperature. Confocal microscopy was performed with cytes (0.5 10 monocytes/well) were incubated with the appropriate an Olympus IX70 confocal microscope with 60 objective lenses (Olympus, concentration of M6P in the presence or absence of sCD26-OG for 24 h in Tokyo, Japan), using laser excitation at 496 and 568 nm. The widths of standard medium. Cells were then washed twice in ice-cold PBS, followed Oregon green and Texas red emission channels were set to maximize by incubation for 2 min in acidic PBS (pH 3.0) to strip any sCD26-OG specificity. attached to the cell surface. Cells were washed in ice-cold PBS and were incubated with mouse Ig isotypes (1 ␮g/ml) to block nonspecific binding; Relative quantitative RT-PCR assay this was followed by reaction with PE-conjugated anti-CD14 (10 ␮g/ml) to gate exclusively on monocyte population. In experiments assessing expression of CD86 mRNA, purified TT-treated Flow cytometric analysis of 10,000 viable cells was conducted on FACS- monocytes (0.5 ϫ 106 monocytes/well) were incubated with sCD26 (0.5 Calibur (BD Biosciences, Mountain View, CA). Each experiment was re- ␮g/ml) at the appropriate time intervals. RNA was then extracted by the peated at least three times, and the results were provided in the form of a use of TRIzol reagent solution according to the manufacturer’s instruction histogram of a representative experiment, or increased mean percent Ϯ SE (Life Technologies). cDNA was produced by using Thermo-Script II re-

ﬂuorescent intensity compared with control or untreated cells. verse transcriptase (Life Technologies) with oligo(dT)12Ð18 primers. The

Table II. sCD26-OG incorporation according to lymphocyte subpopulationsa

Gated PE-Ab CD3ϩ CD14ϩ CD19ϩ CD56ϩ

% Cells with DPPIVϩ sCD26-OG TTϪ % 14.4 Ϯ 0.5 80.3 Ϯ 5.1 15.4 Ϯ 1.8 5.6 Ϯ 1.0 MFI 47.9 Ϯ 4.8 51.5 Ϯ 2.7 49.2 Ϯ 4.1 48.8 Ϯ 4.8 TTϩ % 13.8 Ϯ 1.6 85.8 Ϯ 5.6 16.1 Ϯ 1.9 4.9 Ϯ 1.1 MFI 44.5 Ϯ 2.9 48.5 Ϯ 3.6 47.4 Ϯ 4.8 40.3 Ϯ 5.2 % Cells with DPPIVϪ sCD26-OG TTϪ % 15.8 Ϯ 1.5 83.1 Ϯ 4.9 14.9 Ϯ 2.8 6.6 Ϯ 1.3 MFI 50.4 Ϯ 4.1 50.6 Ϯ 4.3 49.8 Ϯ 3.2 42.7 Ϯ 5.1 TTϩ % 13.2 Ϯ 1.4 82.4 Ϯ 4.3 17.3 Ϯ 2.9 3.7 Ϯ 1.4 MFI 49.4 Ϯ 3.5 44.1 Ϯ 6.2 47.8 Ϯ 3.7 46.8 Ϯ 6.6

a Freshly isolated PBMC (1 ϫ 106/well) were incubated with or without TT for 16 h after 24-h incubation, and then sCD26/DPPIVϩ-OG was added to the culture wells. After incubation with sCD26-OG for 24 h, cells were washed in ice-cold PBS, and in acidic buffer (pH 3 PBS) for stripping the sCD26-OG on the cell surfaces. Then, cells were incubated with various PE-conjugated Abs (CD3, CD14, CD19, and CD56) for 30 min. Analysis was performed by FACSCalibur. The experiments represent mean values Ϯ SE calculated from three independently performed experiments. %, Percent of sCD26-OG-positive cell number in the various PE-positive cells. Similar experiments were performed using mutant sCD26 of defective DPPIV activity (sCD26/DPPIVϪ-OG). MFI, mean ﬂuorescence intensity. 6748 ENHANCEMENT OF T CELL PROLIFERATION BY sCD26/DPPIV

quantities of mRNA were adjusted equally by using PCR of ␤-actin. For- ward primer was 5Ј-CAAGAGATGGCCACGGCTGCT-3Ј (cDNA position 746Ð766), and reverse primer was 5Ј-TCCTTCTGCATCCTGTCG GCA-3Ј (cDNA-position 1000Ð1020)) as the internal control. CD86 mRNA was amplified with primers designed to amplify the entire coding sequence of CD86 (forward primer was 5Ј-ATGGGACTGAGTAACAT TCTCTTTGTGATGGCCT-3Ј (cDNA position 149Ð181 and linker), and reverse primer was 5Ј-CTCGAGTTAAAAACATGTATCACTTTTGTCG CATGA-3Ј (cDNA position 1090Ð1120 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 Downloaded from 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 Ͻ 0.05 being signiﬁcant).

Results http://www.jimmunol.org/ 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

(21Ð23). However, in the latter stages, direct APC-T cell interac- by guest on September 23, 2021 tion is not always necessary, whereas secreted cytokines such as IL-2 are essential in maintaining the reaction (14, 21). As shown in Fig. 1, 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).

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 I, leukocyte phe- notypes such as CD3, CD14, CD19, and CD56 were not affected by presence of TT. In addition, sCD26 was taken up mainly by FIGURE 2. PBMC subpopulation that takes up sCD26. A, Freshly iso- CD14-positive monocytes (Table II and Fig. 2A). In contrast, flow lated PBMC (1 ϫ 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 monocytes were washed in PBS, and once in acidic buffer (pH 3 PBS) were washed in PBS and in acidic buffer (pH 3 PBS) to strip any to strip any sCD26-OG on the cell surface. The cells were then incubated sCD26-OG on the cell surfaces. The cells were then incubated with the with the biotinylated anti-CD14 Ab and incubated with streptavidin-Texas various PE-conjugated Abs, and the intensity of sCD26-OG was measured red. After incubation with sCD26-OG, sCD26-OG was incorporated in the for flow histograms. Analysis was performed by FACSCalibur. Similar monocytes (B and D, sCD26/DPPIVϩ-OG; C and E, sCD26/DPPIVϪ-OG). experiments were performed with mutant sCD26 of defective DPPIV ac- Although sCD26-OG was detected intracellularly following 24 h of incu- tivity. BÐE, Fluorescent confocal microscopic view of sCD26-OG-incor- bation (B and C), sCD26-OG was not detectable after 36 h of incubation (D porated monocytes. After incubation with sCD26-OG for 24 and 36 h, and E). Bars indicate 10 ␮m. The Journal of Immunology 6749 cytometric analyses showed that T cells (CD3ϩ), B cells (CD19ϩ), and NK cells (CD56ϩ) 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/DPPIVϪ) 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 II and Fig. 2, B and C, show that sCD26/DPPIVϩ, as well as sCD26/DPPIVϪ, was clearly taken up by monocytes. However, as shown in Fig. 2, D (sCD26/DPPIVϩ) and E (sCD26/DPPIVϪ), 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). The target cells of sCD26 are monocytes

Because the main target cells among PBMC were monocytes, as Downloaded from shown in Table II and Fig. 2, AÐC, 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. 3, the enhancing effect of TT-induced T cell proliferation was observed only when mono- http://www.jimmunol.org/ cytes were preincubated with TT and sCD26, but not T cells (Fig. 3, 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. 3A, the degree of TT-induced T cell proliferation was dependent upon the concentration of the exogenously added sCD26. Therefore, these results indicate that the by guest on September 23, 2021 principal target cells of sCD26 are APCs, including monocytes. 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- FIGURE 3. Reconstitution study to determine the target cells of sCD26. ϫ 6 IIR is involved in monocyte uptake of sCD26, we used fluorescent A, Freshly isolated monocytes (0.5 10 /well) were preincubated with 0.5 ␮g/ml TT and with different concentrations of sCD26 for 24 h, then, after confocal microscopy to initially evaluate monocyte expression of washing with PBS, 1.0 ϫ 104/well of the preincubated monocytes were sCD26-OG and M6P/IGF-IIR intracellularly and on the cell sur- subjected to the assay with 1 ϫ 105/well of purified T cells. B, A total of face. For this purpose, fluorescent mouse anti-human M6P/IGF- 1 ϫ 105/well of purified T cells were preincubated with 0.5 ␮g/ml of TT IIR mAb was conjugated with M6P/IGF-IIR-red. Fig. 4A shows and with different concentrations of sCD26 for 24 h. Cells were then that sCD26-OG and M6P/IGF-IIR-red colocalized on the mono- washed with PBS and subjected to the assay. Proliferation of cells was cyte cell surface. Following incubation at 37¡C for 24 h in the monitored in all instances by measuring [3H]TdR incorporation on day 7 of presence of sCD26-OG and M6P/IGF-IIR-red, intracellular colo- culture. Degree of proliferation is indicated as cpm in the ordinate. The calization of these proteins was observed (Fig. 4, B and C). Co- experiments represent mean values Ϯ SE calculated from three indepen- localization of lysozyme and M6P/IGF-IIR was similarly observed dently performed experiments. (Fig. 4D). 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␥R (data not cytes was due to the interaction of sCD26 with its binding protein shown). To further confirm that sCD26 uptake is dependent on its M6P/IGF-IIR. It should be noted that this inhibitory effect of M6P binding to M6P/IGF-IIR, we have performed the inhibition assay on uptake of sCD26 was similarly seen in experiments performed Ϫ to evaluate uptake of sCD26 by monocytes in the presence of with sCD26/DPPIV (data not shown). excess amounts of M6P. As shown in Fig. 4, E and F, FACS analyses showed that the addition of M6P into the culture system Enhancement of TT-induced T cell proliferation by sCD26 is (0, 0.1, 1.0, and 10 ␮M) inhibited the uptake of sCD26 into mono- seen when monocytes are incubated with sCD26 before fixation cytes. The degree of inhibition of sCD26 uptake was dependent Another explanation for the sCD26-induced enhancement of TT- upon the concentration of the exogenously added M6P (Fig. 4E). mediated T cell proliferation following monocyte uptake may be These findings were also observed even in the absence of TT (Fig. the trimming of the MHC class II-bound peptide, hence altering 4F). Thus, these results indicated that uptake of sCD26 into mono- cellular responsiveness to the Ag. Such a phenomenon has been 6750 ENHANCEMENT OF T CELL PROLIFERATION BY sCD26/DPPIV

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. 5, A and B, an enhanced T cell proliferation was seen only when the monocytes were incubated with sCD26/DPPIVϩ 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.

Expression of the costimulatory molecule CD86 is increased on Downloaded from 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 (10Ð15, 21Ð24). For this purpose, freshly isolated monocytes were incubated with sCD26 in the pres-

ence or absence of TT, and the expression of CD80, CD86, and http://www.jimmunol.org/ HLA-DR on monocytes was examined using flow cytometric analysis. As shown in Table III and Fig. 6, 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 II). Therefore, these results showed that uptake of sCD26 into monocytes resulted in an in- by guest on September 23, 2021 crease 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

FIGURE 4. M6P/IGF-IIR plays a role in incorporating sCD26 into monocytes. AÐD, Fluorescent confocal microscopic view of sCD26-incorporated monocytes via M6P/IGF-IIR. The freshly isolated monocytes (0.5 ϫ 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 DPPIVϩ 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/DPPIVϪ conjugated with Oregon green, rather than sCD26/DPPIVϩ. 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 ␮m. E and F, M6P inhibits uptake of sCD26 into monocytes. The freshly isolated monocytes (0.5 ϫ 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 ␮M/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/DPPIVϪ 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. The Journal of Immunology 6751

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 ␮g/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. 7, the increase of surface CD86 expression seen when monocytes were incubated with TT and sCD26/DPPIVϩ 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/DPPIVϪ did not demonstrate enhanced CD86 mRNA levels, again indicating the importance of DPPIV activity in this interaction.

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 Downloaded from with mAbs against CD80, CD86, HLA-DR, or CTLA-4 Ig at a final concentration of 5 ␮g/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. 8A, although mAb against HLA-DR efficiently inhibited T cell proliferation, sCD26 did not have an ef-

fect in the HLA-DR-mediated pathway. Similarly, no effect was http://www.jimmunol.org/ 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. 8A, 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/DPPIVϩ)-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 by guest on September 23, 2021 whether the above costimulatory effect was observed via the in- duction of CD86, CD86 mAb and CTLA-4 Ig were added at different concentrations to TT and/or sCD26 stimulation cultures. As shown in Fig. 8B, TT-mediated T cell proliferation enhanced by FIGURE 5. Enhancing effect of TT-induced T cell proliferation by sCD26/DPPIVϩ was strongly inhibited by CD86 mAb and sCD26 did not result from trimming of the MHC-bound peptide on mono- CTLA-4 Ig in a dose-dependent manner. It should be noted that cytes. Freshly isolated monocytes (0.5 ϫ 106/well) were preincubated with 0.5 ␮g/ml TT for 16 h, followed by a 24-h incubation with sCD26 at CTLA-4 Ig always exerted greater inhibitory effect on TT-mediated T cell proliferation than CD86 mAb. As previously noted in different doses before (A) or after (B) being treated with 0.05% glutaral- ϩ dehyde for 30 s at room temperature. After washing with PBS, 1 ϫ 104/ Fig. 6, TT/sCD26 (DPPIV ) monocytes expressed higher levels of Ϫ well of the preincubated monocytes were then subjected to the assay with CD86 than monocytes incubated with sCD26/DPPIV molecules 1 ϫ 105/well of purified T cells. Similar experiments were performed with in the presence or absence of TT. These data strongly suggested mutant sCD26/DPPIVϪ. Proliferation of cells was monitored in all in- that the increased surface expression of CD86 on monocytes stances by measuring [3H]TdR incorporation on day 7 of culture. Degree of treated with TT and sCD26 (DPPIVϩ) is essential for the enhanc- proliferation is indicated as cpm in the ordinate. The experiments represent ing effect of sCD26 on TT-induced T cell proliferation. mean values Ϯ SE calculated from three independently performed experiments. Discussion In this paper, we report that sCD26 has an enhancing effect on T cell proliferation in the presence of the recall Ags, TT. Impor- immune response to recall Ag. It should be noted that sCD26/ tantly, this enhancement resulted in an increase in the surface ex- Ϫ DPPIV mutant did not affect CD86 expression on monocytes, pression of the costimulatory molecule CD86 on monocytes fol- ϩ consistent with data showing that only sCD26/DPPIV , but not lowing uptake of sCD26. This immune enhancing effect of sCD26 Ϫ sCD26/DPPIV mutant, enhanced TT-induced T cell proliferation was observed within the initial 48 h of treatment with sCD26 in the (8, 24). presence of TT. In the process of T cell proliferative response against a recall Enhanced expression of CD86 on monocytes triggered by TT Ag, several major factors have been shown to contribute to the and sCD26 is dependent on increased synthesis of mRNA maintenance of the biological reaction, including APC, Th cells, To determine whether the enhancement in CD86 expression fol- and selected cytokines (10Ð15, 21Ð24). Initially, an APC-T cell lowing incubation of monocytes with sCD26 in the presence of TT interaction plays a key role in triggering the T cell response, lead- is dependent on increased protein synthesis, levels of mRNA en- ing eventually to expression of the T cell biological program (10, coding for CD86 were quantified by RT-PCR. Freshly isolated 14, 15). Our studies showed that the enhancing effect of sCD26 on 6752 ENHANCEMENT OF T CELL PROLIFERATION BY sCD26/DPPIV

Table III. Increased CD86 expression on monocytes after incubation with sCD26/DPPVIϩ a

TT alone sCD26 alone 0 h 6 h 24 h 48 h 72 h 96 h

% Increase of CD86 expression on monocytes following incubation with sCD26 sCD26/DPPIVϩ 1.2 Ϯ 2 1.5 Ϯ 0.6 4.2 Ϯ 0.8 7.4 Ϯ 1.3 10.5 Ϯ 2.4 12.1 Ϯ 2.1 4.3 Ϯ 1.2 4.1 Ϯ 1.5 sCD26/DPPIVϪ 1.8 Ϯ 1.3 2.4 Ϯ 1.9 1.2 Ϯ 1.1 0.8 Ϯ 1.4 1.6 Ϯ 1.1 1.3 Ϯ 0.8 1.1 Ϯ 0.7 % Increase of CD80 expression on monocytes following incubation with sCD26 sCD26/DPPIVϩ 2.0 Ϯ 0.4 1.7 Ϯ 0.2 1.3 Ϯ 0.1 0.7 Ϯ 0.3 1.6 Ϯ 0.4 0.6 Ϯ 0.2 0.8 Ϯ 0.2 0.5 Ϯ 0.3 sCD26/DPPIVϪ 1.0 Ϯ 0.4 0.3 Ϯ 0.2 0.2 Ϯ 0.1 0.2 Ϯ 0.1 0.1 Ϯ 0.1 0.2 Ϯ 0.1 0.2 Ϯ 0.1 % Increase of HLA-DR expression on monocytes following incubation with sCD26 sCD26/DPPIVϩ 0.3 Ϯ 0.2 1.5 Ϯ 0.6 0.2 Ϯ 0.8 1.4 Ϯ 1.5 3.5 Ϯ 2.6 2.1 Ϯ 1.1 0.4 Ϯ 1.2 1.1 Ϯ 1.5 sCD26/DPPIVϪ 2.8 Ϯ 1.3 1.4 Ϯ 0.7 0.2 Ϯ 1.1 1.4 Ϯ 0.7 0.8 Ϯ 1.2 0.7 Ϯ 0.8 2.3 Ϯ 1.7

a The freshly isolated monocytes (0.5 ϫ 106/well) were incubated with or without TT for 16 h after 24-h incubation, and then sCD26 (0.5 ␮g/ml) was added to the culture wells. After incubation for different time intervals (0, 6, 24, 48, 72, and 96 h), cells were washed in ice-cold PBS, and incubated with FITC-conjugated CD86, CD80, or HLA-DR on ice for 30 min. After washing with PBS, cells were analyzed by FACSCalibur. The experiments represent mean values Ϯ SE calculated from three independently performed experiments. %, Percent increase of FITC intensity compared to FITC intensity of non-sCD26-treated monocytes.

TT-induced T cell proliferation occurred in the early stages of the vation of naive T cells requires strong stimulatory signals provided immune response. Moreover, the cells affected by exogenously by APC (30), and activation of recently activated memory T cells added sCD26 are the CD14-positive monocytes. can be elicited with anti-CD3 mAb alone (31), most memory T Downloaded from CD28 is constitutively expressed on T cells and interacts with cells are still dependent on CD28 triggering for their activation the B7 molecules CD80 and CD86 (20, 21). This interaction leads (22). A stable interaction between APC and T cells is dependent to increased T cell proliferation, IL-2 production, and resistance to not only on the absolute affinity and specificity on the TCR and its apoptosis (14). CD80 and CD86 are type 1 membrane glycopro- ligands, but also on the relative density of molecules available for teins belonging to the Ig superfamily (12, 13). In humans, their contact at the interaction site (32, 33). Therefore, our findings that expression patterns differ according to the nature of the APC. up-regulation of CD86 but not CD80 on monocytes occurs fol- http://www.jimmunol.org/ CD86 expression is constitutive on monocytes and dendritic cells lowing uptake of sCD26 by the same cell population suggest that and is up-regulated by activation (10, 14). In contrast, CD80 is this is one of mechanisms for the enhancement of TT-induced T expressed at low levels on APC and is up-regulated following cell proliferation by sCD26. Although it is presently unclear how activation (14, 15). the relatively subtle up-regulation of CD86 on monocyte surface CD86 has an important role in the priming of naive T cells and resulted in a significantly enhancing effect on TT-mediated T cell activation of memory T cells (10, 14, 19, 27Ð29). Although acti- 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). by guest on September 23, 2021 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-␤, proliferin, and leukemia inhibitory factor, and has been shown to play

FIGURE 6. sCD26/DPPIV induces up-regulation of the costimulatory molecule CD86 on monocytes. Freshly isolated monocytes (0.5 ϫ 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 ␮g/ml) was added to FIGURE 7. Enhanced CD86 mRNA expression following treatment the culture wells. After incubation with sCD26 for different hours, cells with sCD26/DPPIVϩ on TT-treated monocytes. Freshly isolated mono- were washed with PBS, incubated with FITC-conjugated CD80, CD86, or cytes (0.5 ϫ 106/well) were incubated with or without TT for 16 h after a HLA-DR, Abs, and then analysis was performed by FACSCalibur. The 24-h incubation in the standard medium alone, and then sCD26 (0.5 ␮g/ml) histogram shown in this figure is the increased intensity of CD86-FITC on was added to the culture wells. After incubation with sCD26 for 24 h, total monocytes cultured for 24 h with sCD26/PPIVϩ after TT treatment. The RNA was extracted and RT-PCR was performed. Expression of CD86 was single histogram profile of the CD86 expression shown in this figure is one increased in TT-treated monocytes cultured with sCD26/DPPIVϩ. ␤-Actin of three representative experiments. The abscissa represents fluorescence was used as internal control. The different amount of the PCR product of ␤ intensity (log10 scale), and the ordinate represents the respective cell -actin at different PCR cycles showed that the PCR was performed for number. relative quantitation in linear range. The Journal of Immunology 6753

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 ϫ 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 ␮g/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 ␮g/ml) or CTLA-4 Ig (5 ␮g/ ml) for 15 min at 4¡C before onset of culture. Inhibition on T cell proliferation was expressed as the percentage of reactivity of con- Downloaded from trol 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 (0.1Ð20 ␮g/ml) on T cell proliferative response induced by TT/sCD26-treated monocytes. The results were expressed as the percentage of the mean http://www.jimmunol.org/ 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 Ϯ SE of three independently performed experiments. by guest on September 23, 2021

an important role in regulating the activation or degradation of monocytes, despite the uptake of sCD26/DPPIVϪ by the same cell these factors (37Ð41). The present findings indicate that M6P/IGF- population. Previous reports showed that CD26/DPPIV regulated IIR mediates the internalization of sCD26 and that this interaction immune responses by cleaving selected chemokines at the N ter- induces the up-regulation of CD86 on monocytes, resulting in the minus to modify their biological functions (3, 42). Chemokines enhancement of TT-induced T cell proliferation. processed by DPPIV enzyme exhibited lower chemotactic po- Accumulating evidence suggests that DPPIV enzyme activity tency, impaired signaling effects, and altered receptor specificity plays an essential role in CD26-mediated T cell costimulation, as (3, 42). In view of its ability to cleave selected biological factors well as in T cell immune reactions (1). Previous studies suggested as a serine protease, it is possible that CD26/DPPIV exerts its that several potential scenarios may explain the observed effects of effect by cleavage of certain factors that may in turn affect the DPPIV on immune activation (3, 42, 45Ð47). It is possible that up-regulation and/or oligomerization of CD86 on monocytes. Fur- CD26/DPPIV exerts its effect via the membrane-bound form, par- ther studies are required for the isolation and characterization of ticularly in T cells. It is also possible that the CD26/DPPIV exerts CD26/DPPIV-associated factors responsible for regulating the ex- its effect with the soluble form, in view of the fact that CD26/ pression of CD86. DPPIV is actually present in human serum (25). Previous studies Cytokines are critical in maintaining the latter stages of immune with CD26-transfected Jurkat cells showing that DPPIV activity reaction (10, 14, 22). In the initial stages of Ag presentation, the on CD26 augmented cellular responses of CD26-transfected Jurkat capacity of Ag presentation can be modulated by several nonex- cells to external stimuli would support the former possibility (25, clusive mechanisms, including the efficiency of Ag capturing and 48). In contrast, data demonstrating that recombinant sCD26 with loading, MHC molecule density and occupancy, or altered co- DPPIV enzyme activity enhanced proliferative responses of stimulatory molecule expression (32, 33). Various proteins have PBMC to stimulation with the soluble recall Ag TT would support been demonstrated to modulate the steps described above to alter the latter scenario. Our results showed that sCD26/DPPIVϩ had an T cell responsiveness against specific Ags. For example, opsonins enhancing effect on TT-induced T cell proliferation associated augment the ability of Ag capture of APCs, IL-10 increases mac- with an increase in CD86 expression on monocytes. Importantly, ropinocytosis and endocytosis by APC, and IFN-␥ up-regulates the incubation with sCD26/DPPIVϪ did not enhance TT-induced T expression of MHC class II molecules on APC (49Ð51). More- cell proliferation and had no effect on CD86 surface expression on over, recent studies revealed that aminopeptidase N (CD13), one 6754 ENHANCEMENT OF T CELL PROLIFERATION BY sCD26/DPPIV of the ectopeptidases on the cell surface, may trim MHC class 18. Krummel, M. F., and J. P. Allison. 1995. CD28 and CTLA-4 have opposing II-bound peptides on the surface of the APC to modify T cell effects on the response of T cells to stimulation. J. Exp. Med. 182:459. 19. Saito, T. 1998. Negative regulation of T cell activation. Curr. Opin. Immunol. responsiveness (26). However, our findings clearly show that the 10:313. enhancement of TT-induced T cell proliferation by sCD26 did not 20. Caux, C., B. Vanbervliet, C. Massacrier, M. Azuma, K. Okumura, L. L. Lanier, result from trimming of the MHC class II-bound peptide on the and 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: APC surface, in contrast to the findings related to CD13 1841. aminopeptidase. 21. Hathcock, K. S., G. Laszlo, C. Pucillo, P. Linsley, and R. J. Hodes. 1994. Com- It should be noted that although uptake of the sCD26 molecules parative analysis of B7-1 and B7-2 costimulatory ligands: expression and func- by monocytes resulted in an increase in CD86 monocyte surface tion. J. Exp. Med. 180:631. 22. Yi-qun, Z., R. J. Joost van Neerven, A. Kasran, M. de Boer, and J. L. Ceuppens. expression, there is the still possibility that DPPIV enzyme activity 1996. Differential requirements for co-stimulatory signals from B7 family mem- of sCD26 facilitates Ag processing in monocytes, resulting in en- bers by resting versus and recently activated memory T cells towards soluble hanced T cell/monocyte interaction and leading to increased T cell recall antigens. Int. Immunol. 8:37. proliferation. 23. Hakamada-Taguchi, R., T. Kato, H. Ushijima, M. Murakami, T. Uede, and H. Nariuchi. 1998. Expression and co-stimulatory function of B7-2 on murine In summary, we have shown that sCD26 is taken up by mono- CD4ϩ T cells. Eur. J. Immunol. 28:865. 873. cytes and exertes its enhancing effect on T cell proliferation by 24. Manickasingham, S. P., S. M. Anderson, C. Burkhart, and D. C. Wraith. 1998. altering the Ag presenting function of monocytes through the up- Qualitative and quantitative effects of CD28/B7-mediated costimulation on naive T cells in vivo. J. Immunol. 161:3827. regulation of CD86 expression. Our paper also demonstrates that 25. Tanaka, T., J. S. Duke-Cohan, J. Kameoka, A. Yaron, I. Lee, S. F. Schlossman,

sCD26 plays an important role in the immune surveillance process and C. Morimoto. 1994. Enhancement of antigen-induced T-cell proliferation Downloaded from against recall Ags, further elucidating the key role of CD26 in by soluble CD26/dipeptidyl peptidase IV. Proc. Natl. Acad. Sci. USA 91: 3082. immune regulation. 26. Larsen, S. L., L. O. Pedersen, S. Buus, and A. Stryhn. 1996. T cell responses affected by aminopeptidase N (CD13)-mediated trimming of major histocompat- ibility 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, References L. M. Nadler, H. Wakasa, and T. F. Tedder. 1994. The B7-2 (B70) costimulatory

1. Morimoto, C., and S. F. Schlossman. 1998. The structure and function of CD26 molecule expressed by monocytes and activated B lymphocytes is the CD86 http://www.jimmunol.org/ in the T-cell immune response. Immunol. Rev. 161:55. differentiation antigen. Blood 84:1402. 2. von Bonin, A., J. Huhn, and B. Fleischer. 1998. Dipeptidyl-peptidase IV/CD26 28. Vyth-Dreese, F. A., N. T. Dellemijin, D. Majoor, and D. de Jong. 1995. Local- on T cells: analysis of an alternative T-cell activation pathway. Immunol. Rev. ization in situ of the co-stimulatory molecules B7.1, B7.2, CD40 and their ligands 161:43. in normal human lymphoid tissue. Eur. J. Immunol. 25:3023. 3. Oravecz, T., M. Pall, G. Roderiquez, M. D. Gorrell, M. Ditto, N. Y. Nguyen, 29. Yokozeki, H., I. Hatayama, O. Ohki, T. Matsunaga, K. Watanabe, T. Satoh, R. Boykins, E. Unsworth, and M. A. Norcross. 1997. Regulation of the receptor M. Azuma, K. Okumura, and K. Nishioka. 1996. Functional CD86 (B7-2/B70) on specificity and function of the chemokine RANTES (regulated on activation, cultured human Langerhans cells. J. Invest. Dermatol. 106:147. normal T cells expressed and secreted) by dipeptidyl peptidase IV (CD26)-me- 30. Liu, Y., and C. A. Janeway. 1992. Cells that present both specific ligand and diated cleavage. J. Exp. Med. 186:1865. costimulatory activity are the most efficient inducers of clonal expansion of nor- 4. von Bonin, A., J. Huhun, and B. Fleischer. 1998. Dipeptidyl-peptidase IV/CD26 mal CD4 T cells. Proc. Natl. Acad. Sci. USA 89:3845. on T cells: analysis of an alternative T-cell activation pathway. Immunol. Rev. 31. Van de Velde, H., K. Lorre, M. Bakkus, K. Thielemans, J. L. Ceuppens, and by guest on September 23, 2021 161:43. M. de Boer. 1993. CD45ROϩ memory T cells but not CD45RAϩ naive T cells 5. Fleischer, B. 1994. CD26: surface protease involved in T-cell activation. Immu- can be efficiently activated by remote costimulation with B7. Int. Immunol. nol. Today 15:1800. 5:1483. 6. Morimoto, C., and S. F. Schlossman. 1994. CD26: a key costimulatory molecule 32. Prakken, B., M. Wauben, D. Genini, R. Samodal, J. Barnett, A. Mendivil, on CD4 memory T cells. The Immunologist 2:4. L. Leoni, and S. Albani. 2000. Artificial antigen-presenting cells as a tool to 7. Hegen, M., J. Kameoka, R.-P. Dong, S. F. Schlossman, and C. Morimoto. 1997. exploit the immune “synapse.” Nat. Med. 6:1406. Cross-linking of CD26 by antibody induces tyrosine phosphorylation and acti- 33. Grakoui, A., S. K. Bromley, C. Sumen, M. M. Davis, A. S. Shaw, P. M. Aleen, vation of mitogen-activated protein kinase. Immunology 90:257. and M. L. Dustin. 1999. The immunological synapse: a molecular machine con- 8. Tanaka, T., J. Kameoka, A. Yaron, S. F. Schlossman, and C. Morimoto. 1993. trolling T cell activation. Science 285:221. The costimulatory activity of the CD26 antigen requires dipeptidyl peptidase IV 34. Greene, J., L., G. M. Leytze, J. Emswiler, R. Peach, J. Bajorath, W. Cosand, enzymatic activity. Proc. Natl. Acad. Sci. USA 90:4583. and P. Linsley. 1996. Covalent dimerization of CD28/CTLA-4 and oligomer- 9. Ikushima, H., Y. Munakata, T. Ishii, S. Iwata, M. Terashima, H. Tanaka, ization of CD80/CD86 regulate T cell costimulatory interactions. J. Biol. S. F. Schlossman, and C. Morimoto. 2000. Internalization of CD26 by mannose Chem. 271:26762. 6-phosphate/insulin-like growth factor II receptor contributes to T cell activation. 35. Kornfeld, S. 1992. Structure and function of the mannose 6-phosphate/insulin- Proc. Natl. Acad. Sci. USA 97:8439. like growth factor II receptors. Annu. Rev. Biochem. 61:307. 10. Lenschow, D. J., T. L. Walunas, and J. A. Bluestone. 1996. CD28/B7 system of 36. Nykjaer, A., E. I. Christensen, H. Vorum, H. Hager, C. M. Peterson, H. Roigaard, T cell costimulation. Annu. Rev. Immunol. 14:233. H. Y. Min, F. Vilhardt, L. B. Moller, and S. Konfeld. 1998. Mannose 6-phosphate/insulin-like growth factor-II receptor targets the urokinase receptor to ly- 11. Yokochi, T., R. D. Holly, and E. A. Clark. 1982. B lymphoblast antigen (BB-1) sosomes via a novel binding interaction. J. Cell Biol. 141:815. expressed on EBV-activated B cell blasts, B lymphoblastoid cell lines, and Bur- kitt’s lymphomas. J. Immunol. 128:823. 37. Purchio, A. F., J. A. Cooper, A. M. Brunner, M. N. Lioubin, L. E. Gentry, K. S. Kovacina, L. A. Roth, and H. Maruquardt. 1988. Identification of mannose 12. Azuma, M., D. Ito, H. Yagita, K. Okumura, J. H. Phillips, L. L. Lanier, and 6-phosphate in two asparagine-linked sugar chains of recombinant transforming C. Somoza. 1993. B70 antigen is a second ligand for CTLA-4 and CD28. Nature growth factor-␤1 precursor. J. Biol. Chem. 263:14211. 366:76. 38. Dennis, P. A., and D. B. Rifkin. 1991. Cellular activation of latent transform- 13. Freeman, G. J., J. G. Gribben, V. A. Bousiotis, J. W. Ng, V. A. Restivo, ing growth factor-␤ requires binding to the cation-independent mannose L. A. Lombard, G. S. Gray, and L. M. Nadler. 1993. Cloning of B7-2: a CTLA-4 6-phosphate/insulin-like growth factor type II receptor. Proc. Natl. Acad. Sci. counter-receptor that costimulates human T cell proliferation. Science 262:909. USA 88:580. 14. McAdam, A. J., A. N. Schweitzer, and A. H. Sharpe. 1998. The role of B7 ϩ ϩ 39. Lee, S. J., and D. Nathans. 1988. Proliferin secreted by cultured cells binds to co-stimulation in activation and differentiation of CD4 and CD8 T cells. Im- mannose 6-phosphate receptors. J. Biol. Chem. 263:3521. munol. Rev. 165:231. 40. Blanchard, F., S. Raher, L. Duplomb, P. Vusio, V. Pitard, J. L. Taupin, 15. Chambers, C. A. 2001. The expanding world of co-stimulation: the two-signal J. F. Moreau, B. Hoflack, S. Minvielle, and Y. Jacques. 1998. Mannose 6-phos- model revisited. Trends Immunol. 22:217. phate/insulin-like growth factor II receptor mediates internalization and degra- 16. Croft, M., D. D. Duncan, and L. S. Swain. 1992. Response of naive antigen- dation of leukemia inhibitory factor but not signal transduction. J. Biol. Chem. ϩ specific CD4 T cells in vitro: characteristics and antigen-presenting cell require- 273:20886. ments. J. Exp. Med. 176:1431. 41. Blanchard, F., L. Duplomb, S. Raher, P. Vusio, B. Hoflack, Y. Jacques, and 17. Walunas, T. L., D. J. Lenschow, C. Y. Bakker, P. S. Linsley, G. J. Freeman, A. Godard. 1999. Mannose 6-phosphate/insulin-like growth factor II receptor J. M. Green, C. B. Thompson, and J. A. Bluestone. 1994. CTLA-4 can function mediates internalization and degradation of leukemia inhibitory factor but not as a negative regulator of T cell activation. Immunity 1:4405. signal transduction. J. Biol. Chem. 274:24685. The Journal of Immunology 6755

42. Proost, P., I. De Meester, D. Schols, S. Struyf, A.-M. Lambeir, A. Wuyts, 47. Korom, S., I. De Meester, T. H. W. Stadlbauer, A. Chandraker, M. Schaub, G. Opdenakker, E. De Clercq, S. Scharpe, and J. Van Damme. 1998. Amino- M. H. Sayegh, A. Belyaev, A. Haemers, S. Scharpe, and J.W. Kupiec-Weglinski. terminal truncation of chemokines by CD26/dipeptidyl-peptidase IV: conversion 1997. Inhibition of CD26/dipeptidyl peptidase IV activity in vivo prolongs car- of RANTES into a potent inhibitor of monocyte chemotaxis and HIV-1-infection. diac allograft survival in rat recipients. Transplantation 63:1495. J. Biol. Chem. 273:7222. 48. Tanaka, T., J. Kameoka, A. Yaron, S. F. Schlossman, and C. Morimoto. 1990. 43. Dang, N. H., D. A. Hafler, S. F. Schlossman, and J. B. Breitmeyer. 1990. FcR- The costimulatory activity of the CD26 antigen requires dipeptidyl peptidase IV mediated crosslinking of Ta1 (CDw26) induces human T lymphocyte activation. enzyme activity. Proc. Natl. Acad. Sci. USA 90:4586. Cell. Immunol. 125:42. 49. Longoni, D., L. Piemonti, S. Bernasconi, A. Mantovani, and P. Allavena. 1998. 44. Dang, N. H., Y. Torimoto, K. Deusch, S. F. Schlossman, and C. Morimoto. 1990. Interleukin-10 increases mannose receptor expression and endocytic activity in Co-mitogenic effect of solid-phase immobilized anti-1F7 on human CD4 T cell monocyte-derived dendritic cells. Int. J. Clin. Lab. Res. 28:162. activation via CD3 and CD2 pathways. J. Immunol. 144:4092. 50. Morel, A. S., S. Quaratino, D. C. Douek, and M. Londei. 1997. Split activity of 45. Kahne, T., K. Neubert, J. Faust, and S. Ansorge. 1998. Early phosphorylation interleukin-10 on antigen capture and antigen presentation by human dendritic events induced by DPPIV/CD26-specific inhibitors. Cell. Immunol. 189:60. cells: definition of a maturative step. Eur. J. Immunol. 27:26. 46. Kubota, T., G. R. Flentke, W. W. Bachovchin, and B. D. Stollar. 1992. Involve- 51. Lee, Y. J., R. B. Panek, M. Huston, and E. N. Benveniste. 1995. Role of protein ment of dipeptidyl peptidase IV in an in vivo immune response. Clin. Exp. Im- kinase C and tyrosine kinase activity in IFN-␥-induced expression of the class II munol. 89:192. MHC gene. Am. J. Physiol. 268:C127. Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021