Dipeptidyl peptidase IV (CD26) activity in human alloreactive T cell subsets varies with the stage of differentiation and activation status

Transplant Immunology 1997; 5: 152-161

Dipeptidyl peptidase IV (CD26) activity in human alloreactive T cell subsets varies with the stage of differentiation and activation status Phillip Ruizax, Lei Haoa, Keith ZuckerC, Natalia Zacharievicha, AnaL Vicianaa, Mark Shenkind and Joshua MillerC Departments of “Pathology,bA4icrobiology/Immunology and %.ugery, Universityof Miami School of Medicine, Miami, and dCoulter Corporation, Miami, Florida Received 11 March; accepted for publication 8 April 1997

Abstract: Dipeptidyl peptidase IV (DPP IV), also known as CD26, is a transmembrane serine aminopeptidase which has an ontogenically related expression on T cells and participates in several immunological functions. CD26 appears to play an important role in alloimmunity during host T cell activation subsequent to alloantigen encounter and is a way by which effector T cells traverse graft endothelial barriers. In order to help to elucidate the role of the CD26 molecule in alloimmune responses, DPP IV activity and CD26 antigenic expression were assessed during the initial phases of completely MHCdisparate human mixed lymphocyte reactions (MLRs) and in several long-term alloreactive T cell clones. Our methods involved the use of a rhodamine-110~conjugated dipeptide substrate specific for DPP IV in two-colour cytofluorographic analysis that allowed stimultaneous lineage marker evaluation. Polyclonal populations of alloreactive CD4 and CD8 T cells contained DPP IV activity at 1 and 10 min of incubation that was variably elevated from resting T cells with the enzyme activity confined to CD26+ cells. T cell clones derived from MLRs were established with IL-2 supplementation and alloantigen restimulation and had reduced CD62L expression with functional specificity to the stimulating MHC. While CD26 expression remained stable, DPP IV activity was variable in the alloreactive T cell clones, with enzyme function in the latter appearing to coincide with the timing of alloantigen restimulation. These studies demonstrate that DPP IV activity varies among phenotypically distinct alloreactive T cell subsets and appears to be altered with the activation status of the effector cells. These findings raise the potential of a role for CD26/DPP IV in the generation of specific alloimmunity. With this methodology, it may be possible to reveal whether specific alterations in the activity of this molecule in T cell populations promote graft acceptance and to determine the molecular requirements for these changes.

Introduction The nonintegrin rece&tor CD26, also known as dipeptidyl peptidase IV (DPP IV) is one of a heterogeneous and multifunctional group of surface molecules which also possess Address of correspondence: PhiUip Ruix, MD, PhD, University of Miami School of Medicine, Department of Pathology (D-33), PO Box 016960, Miami, FL 33101, USA. 0 Arnold 1997

enzymatic activity and can be present on a broad spectrum of cell types, including haematopoietic cells.3 The gene encoding CD26, a 110-120 kDa surface glycoprotein, has been sequenced and is located on chromosome 2,4 the protein possesses serine aminopeptidase activity (DPP IV) capable of cleaving polypeptides at locations containing amino-terminal dipeptides that have either L-alanine or L-proline in position 2; this enzymatic activity can be distributed intracellularly or on the cell surface6 This molecule also has a binding affinity for 0966_3274(97)TI179OA

Dipeptidyl peptdase N (CD26) activity

extracellular matrix via collagen’,’ as well as the capacity to interact with adenosine deaminase and CD45,9.1othe latter molecules being involved in cellular signal transduction pathways. As with several other surface enzyme molecules, CD26 is widely distributed on a variety of tissues4 and cell types, including lymphocytes of T,‘l B,12or NK13 lineages. The level of CD26 expression varies on different T cell subsets, with mature14 and memory (CD45RO+)” T cells having higher amounts, and there can be an upregulation of this molecule when cells are undergoing antigen-mediated or nonspecific cell activation.16 In this regard, CD26 appear8 to be involved in a spectrum of immunological functions. For example, CD26 has been described in different systems as a costimulatory molecule during T cell activation, purportedly providing a ‘second signal’ that, among other changes, allows IL-2 production and IL-2 receptor upregulation with an increase in T cell proliferation.17’18 Direct binding and crosslinking of CD26 serves to activate T cells whereas an interference with the molecule’s activity using specific tripeptides or antibody ha8 been reported to have an immunosuppressive effect. Wer9 and others” have described CD26 to be involved in the ontogenetic development of T cells, with this molecule probably participating in programmed cell death (apoptosis) occurring in the thymus. Several studies have implied that enzymatic activity or binding to adenosine deaminase is involved in or necessary for CD26 to participate in immunological functions,21 although some studies have questioned this issue.22 CD26 has been proposed to be involved in the pathogenetic mechanisms of several disease processes including rheumatoid arthritis,23 leprosy and other granulomatous di8eases,24 lupu? and HIV cellular transport,26 althou the latter issue has been highly controversial and disputed.“, g There is also speculation that CD26 is involved in alloimmune responses in several ways. For example, CD26 may participate as a direct stimulator or costimulator for the initiation of T cell responses to alloantigens and could serve as a means by which alloactivated immune effector cells traverse the vasculature into the interstitial regions of the allograft.29 Soluble and alternative forms of CD26 exi.@ in relation to this, it has been reported that DPP IV activity appear8 elevated in the serum of patients undergoing transplant rejection.31 However, in lieu of the information available, relatively little is understood regarding the function and/or role(s) that DPP IV/CD26 activity plays in the development of alloimmunity. In this regard, these studies were designed to characterize levels and changes in DPP IV activity of T lymphocytes during the generation of alloimmunity to complete MHC-disparate cells. Previous studies determining DPP IV activity have typically required extensively purified cell populations that were ultimately disrupted to measure enzyme activity;20 among other drawbacks, this approach prevents accurate phenotypic analysis and requires extremely large numbers of cells. lb circumvent these problems, a flow cytometric technique was developed which utilizes a rhodamine-llO-conjugated dipeptide substrate specific” for DPP IV to examine directly the intracellular enzyme activity among subsets of T cells responding to alloantigens. This method allows simultaneous measurements of lineage markers and physical characteristics of the cells, which remain undisrupted. Our results demonstrate fluctuating levels of DPP IV activity among the different T cell subset8 during the course of an alloimmune response, with the variations in DPP IV activity often associated with re-exposure to the original stimulating alloantigens. These findings evoke the possibility that Transplant Immunology 1997; 5: 152-161

CD26 plays a role in the attainment alloactivated T cells.

of immunocompetence

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Hypothesis CD26 is involved in the acquisition of alloimmune capacity in T cells and this may be expressed by changes in the enzymatic activity associated with this molecule.

Materials and Methods Rmgellts Rhodamine-110~conjugated glycine-proline (Gly-Pro) and rhodamine-llO-conjugated alanine-alanine (Ala-Ala) dipeptides (CellProbe”) were synthesized3”3 and generously provided by Coulter Corporation (Miami, FL, USA). These two dipeptide compounds serve as substrates for DPP IV and parallel each other in terms of detection of enzyme activity. All of the results presented were obtained with Gly-Pro but were often substantiated with Ala-Ala. We have previously reported data demonstrating the specificity of these substrates.l’ Other reagents included phycoerythrin (PE)-conjugated antibodies directed to CD4 (T4), CD8 (T8) and CD26 (Coulter) and a three-colour antibody cocktail combination directed to CD4 (PE), CD8 (Per-CP) and CD62L (L-selectin/LeuS) (fluorescein isothiocyanate, FITC, generously provided by Noel Warner, Becton Dickinson, San Jose, CA, USA). Purified and unconjugated anti-CD3, anti-Vg5.2 and anti-Vl36.0 were used for the antibody coating experiments (ImmunotecNCoulter). Cell preparation Human EDTA anticoagulated peripheral blood or buf@ coat leucopacks were aseptically obtained from normal volunteer8 and mononuclear cell populations were isolated by Ficollhypaque (Sigma, St Louis, MO, USA) density gradient centrifugation as described previously.” The cells were washed twice with serum-free RPMI-1640 (Media Facility, University of Miami) supplemented with 500 U penicillin/streptomycin/ml (Media Facility, University of Miami), counted and used as described below. Bulk mixed lymphocyte reactions (MLR) and establishment of loneterm alloreactive T cell lines

Bulk MLRs were prepared by adding 10 x 106/ml mononuclear cells (i.e. responder) with 10 x lO?ml irradiated, mononuclear cells (2500 rad) or EBV-transformed (5000 rad) (see below) B cells (i.e. stimulator) in the presence of 45 ml of ‘complete’ medium (RPMI-1640 with 10% complement-inactivated AB human serum; North American Biologicals, Miami, FL, USA) plus penicillin/streptomycin, 0.02 M HEPES buffer] in T-75 cm2 tissue culture flasks (Sarstedt, Newton, NC, USA); the flasks were incubated at 37°C 5% CO2 for 7-10 days, washed, then transferred to T-25 cm2 tissue culture flasks (Sarstedt) in a 1:5 ratio with irradiated stimulator cells. These cultures were maintained for up to 90 days in culture with periodic restimulation with irradiated donor cells (every 10 days) and complete medium that contained 10 r&ml of recombinant human IL-2 (R&D, Minneapolis, MN, USA) (every 5 days). Samples of cells were obtained at various time-points from these cultures for analysis in the assays described below. The alloreactive cells from these cultures were also taken at several timepoints and cloned using

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a limiting dilution technique?5 Propagation of these T cells was performed using IL-Zsupplemented complete medium and schedules of restimulation with donor-specific cells analogous to the times used for the bulk cultures. Clonality was confirmed by flow cytometry and TCRBV analysis by reverse transcription - polymerase chain reaction (RT-PCR) as described below. Clones were used in assays both before and following restimulation with alloantigen whenever feasible. Flow cytometry Fresh unstimulated mononuclear cells, cells from bulk cultures and cloned T cell lines were evaluated for DPP IV activity usin a flow cytometry-based method as reported previously. 1$ Essentially, cell populations were resuspended in 50 pl of complete Dulbecco’s modified Eagle medium (DMEM) and incubated with 5 pl of a 3 x lo-4 M solution of rhodamine-llOconjugated dipeptides (Gly-Pro or Ala-Ala) for 1 and 10 min at 37°C; these periods were established in previous studies to be the optimal incubation times to show DPP IV activity with this substrate (unpublished data). There were no significant differences between the substrates insofar as activity levels that could be detected. At the end of the incubation period, enzymatic activity was stopped by placing the tubes in an ice bath. The cells were then washed twice with phosphate-buffered saline (PBS) containing 0.1% sodium azide at 4°C (5000 rpm, 5 min) and then fixed with 2% paraformaldehyde. In the majority of experiments, two-c&our staining was utilized to evaluate concomitant antigen expression of other markers along with the DPP IV activity; PE-conjugated anti-CD4 (T4), PE-conjugated anti-CD8 (T8) or PE-conjugated anti-CD26 (Coulter) were added to the cells after the dipeptide incubation and washing steps and incubated at 4°C for 30 min, then washed twice. Samples were flxed and analysed in less than 2 h after staining on an XL flow cytometer (Coulter). The instrument was calibrated before experiments for electronic stability and fluoresence compensation using unlabelled and directly labelled FITC- and PE-conjugated latex beads (Immunobrite, Coulter). A backgated scattergram of propidium iodide (PI)-stained cells was sometimes used to exclude dead cells (PI-positive cells) from analysis. Fluorescence emission from a 488 nm 15 MW argon ion air cooled laser was detected at 530 nm (FL-l for FITC and Rhodamine), 585 nm (FL-2 for PI and phycoerythrin) and 650 nm (FL-3) for PE-CYCHROME. Listmode data on 10 000 gated cells were collected with 1024 channel resolution and later analysed by using System II software, version 1.0 software (Coulter). For the dipeptides, fluorescence intensity was calculated as the arithmetic mean channel fluorescence of log amplified data measured on 10 000 cells. Functional assays

MLR and ‘iCr-release cytotoxicity functional assays were performed with bulk MLR cultures and cell lines by techniques described previously.36,37 HLA Typing

HLA typing for class I and class II loci was performed with donor and stimulator cells by standard serological typing methods (One Lambda, Canoga Park, CA, USA).

blood or huffy coat leucopacks were treated with magnetic beads coated with mouse anti-human CD2 (Dynal, Lake Success, NY, USA) at 4°C for 30 min with rotation. Thereafter, the cells were exposed to a magnet and the negatively selected cells (i.e. B cell-enriched) were pooled, washed and counted. Then, 2 x lo6 cells were incubated with supernatant collected from the CRL-1612 cell line (ATCC, Rockville, MD, USA) which is infected with EBV and sheds the virus when growing in vitm. The B cell-enriched fraction of cells in RPM&1640 with 10% fetal calf serum (FCS) medium (2.5 ml) and the EBV-contaming supematant were incubated in T-flasks (25 cm’, 5 ml) at 37“C, 5% C02, for 21 days, after which the cultures were monitored to determine whether cell transformation had occurred. Bansformation was accompanied by an increased proliferative fraction (DNA analysis) and abnormal cellular morphology. Transformed cells were then grown in RPMI-1640 complete medium and used as 51Cr-labelled targets for cytotoxicity assays or as stimulators when testing alloproliferation in MLRs. Amplification of lCR V beta genes by RT-R

RT-PCR was performed according to the method of Panzara et aL3’ In brief, total RNA was extracted from l-3 x lo6 Ficollhypaque purified peripheral blood lymphocyte (PBL) or allogeneic reactive T cell clones by an acid guanidium phenol 1.7). Thereafter, 0.1-0.2 ug of total RNA was reverse-transcribed into cDNA in a 10 pl reaction containing 1 ull0 x PCR buffer II (Perkin Elmer, Norwalk, CN, USA), 0.6 pl(15 mM) MgC12, 1 pl of 10 mM deoxyribonucleoside triphosphates (dNTPs A, T, C, G), 0.125 U random hexamers (Pharmacia LKB Biotechnology, Piscataway, NJ, USA), 0.5 p1 RNAse inhibitor and 0.5 ul MuLV reverse transcriptase. The reaction mixture was incubated at room temperature for 10 min, followed by incubation at 42°C for 45 min and 95°C for 5 min. The mixture was then quickly chilled on ice. Variable regions of the P-chain gene of the human T cell receptor (TCRBV)-specihc PCR products were generated using oligonucleotide primers specific for TCRBV l-20, along with a constant pregion primer?’ The samples were amplified for 35 cycles (1 min at 95°C 1 min at 55°C and 1 min at 72°C) in a DNA thermal cycler (Perkin Elmer), followed by 72°C for 7 min and then a 10°C hold. Amplified products were separated and visualized on 2% agarose gels containing ethidium bromide. Specific TCRBV PCR products were confirmed by dot blotting as follows: the PCR products were spotted on to a nylon membrane (Qiagen, Chatswarth, CA, USA) using a dot-blot apparatus (Bio Rad Laboratoria, Hercules, CA, USA) and then hybridized for 1 h at 42°C with an internal Cj3 region oligonucleotide probe directly labelled with biotin. After an initial wash to remove unhybridiied probe, a horseradish peroxidase (HRP)-avidin conjugate (Sigma) was added and incubation was continued for an additional 10 min at room temperature. Blots were washed at a final stringency of 2.5 x SSPE/O.l% sodium dodecyl sulfate (SDS) at 50°C for 12 mm followed by colour development with a solution of 3,3’,5,5’-tetramethyl-benzidine prior to photography.

Epstein-Barr virus (EBV) transformation of B cells

Antibody-induced cell proliferation The methods for solid-phase antibody cross-linking were used

EBV transformation of stimulator cells was performed using a modification of a previously published method38 Essentially, mononuclear cell populations isolated from the peripheral

with modification.40*41Initially, goat anti-mouse IgG-Fc antibody (Sigma) was bound to 96-well round-bottomed microtitre plates (10 @ml at pH 9.6 overnight, at 4°C). Free antibody was

Transplant Immunology 1997; 5: 152-161

Dipeptidyl Rzptidase lV (CD26) activity

then removed by washing twice with cold PBS. Allogeneic reactive cells were harvested from the culture and washed twice with RPMI-1640, then resuspended at a concentration of 1 x lo6 cells/ml in RPMI-1640 with 10% human AI3 serum and 1.0 @ml IL-2 100 ul of the cell suspension was then added per well. At this point, the anti-TCRBV or anti-CD3 antibodies were also added to the wells to achieve a final concentration of 10 @ml. The plate was incubated in 5% COz, 37°C for 48 h. The cells were then harvested and tested by flow cytometry for the presence of DPP IV and other markers as described above. The presence of a proliferative response was confirmed by measuring [3H]thymidine incorporation over the final 18 h. of culture in duplicate cultures.41

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and CD8+ T cell subsets using three-colour flow cytometry since we4*and others43 have previously associated a diminution or loss of CD62L on these T cells with the generation of memory funo tion. Following an initial increase, there was a reduction in the percentage of CD4+ and CD8+ T cells which coexpressed CD62L over the first 45 days of the bulk MLRs (Figure 1). Thereafter, the proportion of CD4+ T cells expressing CD62L slightly increased, while the fraction of CD8+ T cells coexpressing CD62L continued to decrease. These results implied that

Results Generation of alloimmune T cells Bulk MLRs were performed as described

in Materials and Methods, with PBL serving as sources of responders and stimulators from a variety of normal individuals. HIA typing was carried out on all of the PBL serving as sources for the responders and stimulators and complete MHC disparity was observed for all of the combinations tested with the exception of combination no. 5, which showed partial identity at the HLAA locus (Table 1). On several occasions, the responder-stimulator pairs were repeated several times and bulk cultures in these combinations were established in replicate so that multiple, sequential samples could be obtained and tested for the phenotypic and functional parameters described below. In addition, RT-PCR for TCRBV families was performed on the responder populations prior to the initiation of the alloimmune response as well as on polyclonal and cloned alloreactive cells established during the course of the MLR. Our results with TCRBV analysis revealed a normal distribution of TCRBV families in resting, unstimulated populations while predominant TCRBV clones were evident during the course of the MLR and single TCRBV families were seen in the cloned populations. No preferential pattern of TCRBV usage was evident among the polyclonal alloreactive T cells or in the clones (data not shown). Serial phenotypic measurements of several surface molecules were evaluated during the course of the MLR and, ultimately, of the clones which were isolated and propagated. Figure 1 shows the relative changes in the CD4+ and CD8+ T cell subsets through the first 45 days of the bulk MLR, revealing the eventual increase in the proportion of CD4+ cells as compared to CD8+ cells. CD62L (L-selectin) was also measured on the CD4+

Table 1 Responder and stimulator MLR combinations? Combinationb 1 2 3 4 5 6 7 8

0

lo-20

21-44

AS

MLRDay Figure 1 Serial evaluations of the mononuclear cell populations present

in bulk MLRs to determine phenotypically (by flow cytometry) the relative percentages of CD4+ and CD8+ cell populations, including those coexpressing CD62L (bottom graph). Each point represents the mean value of multiple separate experiments (a = 14) 2 SD.

HL4 disparities

Responder HLA typeC A2, A28, BW50, BW56, DRll, DR12, DQ3, DQW6 Al, A26, B8, B17, DR7, DR9, DQ2, DQ3 A3, A23, B7, BW59, DR15, DQl, DQW6 A2, A25, BW71, BW76, CW7, DR7, DRlO, DQWl, DQW2 A2, A24, B44, B41, CW2, CW7, DRl, DQl, DQ3 A2, AW69, B8, BW60, CW3, CW7, DR8, DR52, DQ2, DQ4 A31, AW69, B7810, B1508, CW3, CW7, DR8, DR53 A3, A26, B62, CW3, DR4, DR53

aBulk MLRs were established as described in Materials and Methods. bMultiple combinations were performed, with some carried out many times. %%A typing was performed as described in Materials and Methods.

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l-5

Stimulator HLA type A23, A26, B38, B63, DR6, DR4, CW7/BW4 A23, A26, B38, B63, DR6, DR4, CW7lBW4 A2, AW36, B44, BW60, DR13, DR13, DR15, DQl, DQ4 A23, A26, B38, B63, DR6, DR4, CW7iBW4 A2, AW36, B44, BW60, DR13, DR15, DQl, DQ4 A23, A26, B38, B63, DR6, DR4, CW7lBW4 A3, A26, B62, BW6, CW3, DR4, DR53 A31, AW69, B7801, B1508, CW3, CW7, DR8, DR53

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concordant with the development of alloreactivity there was phenotypic evidence of a higher proportion of memory cells. The presence of memory function was further supported by the presence of specific functional responses (see below). Functional measurements were obtained at various timepoints in the course of the MLR. All of the cultures tested (from 2 10 days after initiation) demonstrated specific proliferative responses in MLRs (96-well assay) to irradiated donor-specific stimulators, but not to allogeneically distinct third-party control cells (data not shown). Likewise, “Cr-release assays for the determination of specific cell-mediated cytoxicity generated in the MLR were performed (data not shown). These latter data also confirmed the presence of specific cytotoxic immune effector cells in the MLR, with specific lysis of donor-specific targets, but no significant lysis of third-party targets.

Dipeptidyl peptidaee IV activity in polyclonal alloreactive cells The goal of these experiments was to determine whether DPP

IV activity and CD26 expression on T cells changed during the course of the generation of an alloimmune response. In order to assess the activity of DPP IV, specific dipeptide probes specific for DPP IV were utilized which were conjugated to rhodamine-110. These probes rapidly enter the cells whereupon the dipeptide bond is cleared by intracellular enzymes, and fluorescence is emitted and measured by a cytofluorographic technique as reported previously.” As noted before, this method allows concurrent measurement of molecules on the cell surface (i.e. using antibodies conjugated to other fluorochromes) which can simultaneously be distinguished by the flow cytometer. Using this technique we were able to measure DPP IV activity after 1 and 10 min of incubation at 37°C on CD4+, CD8+ and

2

Fii 2 Representative flow cytometry two-colour histograms for the measurement of DPP IV activity (x-axis) at 1 min (left column graphs) and at 10 min (right column graphs) in unstimulate.d, naive peripheral blood mononuclear cells prior to the initiation of a mixed lymphocyte reaction. ‘Rvo-colour staining was performed to determine the enzyme’s activity on CD4+ (top graphs), CD@ (middle graphs) and CD26+ (bottom graphs) cells and the technique for this staining procedure is described in Materials and Methods.

Transplant Immunology 1997; 5: 152-161

Figure 3 Representative flow cytometry two-colour histograms for the measurement of DPP Iv activity (x-axis) at 1 min (left column graphs) and at 10 mm (right column graphs) in stimulated, responding peripheral blood mononuclear cells following 25 days in culture during a bulk MLR in the presence of irradiated, completely MHC-disparate stimulator cells. Iko-colour staining was performed to determine the enzyme’s activity on CD4+ (top graphs), CD8+ (middle graphs) and CD26+ (bottom graphs) cells and the technique for this staining procedure and the bulk MLR is described in Materials and Methods.

Dipeptidyl Fkptidase N (CD26) activity

CD26+ cells from the bulk MLRs (and clones, see below). Figure 2 shows a representative set of two-colour flow cytometry histograms for peripheral blood mononuclear cells prior to the initiation of a MLR. Normal CD4+ and CD8+ T cell subsets displayed a baseline level of DPP IV activity that was higher after 10 min incubation and tended to be higher in CD4+ cells than in CD8+ cells. A majority of the CD26+ cells contained DPP IV activity after 10 min, but the level of expression of CD26 in resting, unstimulated cells was typically low in intensity (Figure 2). Sequential measurements of DPP IV activity were performed on cells isolated from the bulk MLRs at several time-points following initiation of the cultures. Figure 3 shows an example of the DPP IV activity associated with CD4+, CD8+ or CD26+ cells from a bulk MLR after 25 days of culture and restimulation with alloantigen. CD4+ and CD8+ T cell subsets had fluctuations in their levels of DPP IV activity over the course of the MLR and DPP IV activity increased at certain time-points. Figure 4 shows the mean DPP IV activity values for CD4+ and CD8+ T cells taken from multiple MLRs, with heightened DPP

100

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IV activity generally being seen in CD4+ cells over CD8+ cells. In addition, both CD4+ and CD8+ cells tended to have higher activity after certain periods of culture (e.g. 5 days and 45 days), with overall activity curves between the two T cell subsets that resembled each other (Figure 4). Antigen expression of CD26 was also increased (e.g. Figure 3) on the alloactivated cells, although no definitive association could be established with DPP IV enzyme activity. Among other variables examined, a possible relationship was found between an increase in DPP IV activity and the timing of restimulation with the specilic alloantigen, and the degree of proliferation present in the MLR tended to be associated with elevated DPP IV activity (data not shown). Dipeptidyl peptidaee IV activity in alloreactive T cell clone8 In an effort to examine further the activity of DPP IV in allore

active cells, measurement of this enzyme was performed on purified T cell clones from our bulk MLR cultures. As described in Materials and Methods, proliferating effector cells were isolated and cloned from the MLRs and propagated using IL-2 and repeated cycles of restimulation with specific allogeneic stimulator cells. The clonal nature of the lines was contirmed by demonstrating the existence of a sole TCRBV in the line by RT-PCR (data not shown). Phenotypic analysis of the cell lines used in these experiments showed a slight predominance in the percentage of CD8+ T cell clones established over CD4+ lines (Figure 5).

40 20 0

1

4 Serialcytofluorographicevaluationsof DPP IV activity at 1 min and at 10 min in the mononuclear cell populations present in bulk MLRs at various time-points during the culture period. lko-colour staining was performed to determine the enzyme’s activity on CD4+ (top graph) and CD@ (middle graph) cells and the technique for this staining procedure and the bulk MLR is described in Materials and Methods. Each point represents the mean value of multiple separate experiments (n = 14) + SD. The bottom graph provides a comparison of the DPP IV activity at 10 min between CD4+ and CD@ cells. * Statistically significant differences (p < 0.05) between resting cells (i.e. MLR day 0) and the cells at different points of the MLR culture period.

2

3

4

5

6

7

8

9

Figure

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ClOWNpmber

FIgwe 5 Cytofluorographic evaluations of DPP IV activity at 1 mm and at 10 mm in cloned alloreactive T cell lines. ‘Rvo-colour staining was performed to determine the enzyme’s activity on CDS+ (top graph) and CD4+ (bottom graph) clones and the technique for this staining procedure is described in Materials and Methods.

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Functional analysis of the lines confirmed specificity to the stimulating alloantigen by either specific proliferation or cell-mediated cytotoxicity (micro-MLR and %-release assays, respectively) by the alloreactive lines to stimulators or targets MHC identical to the original stimulating alloantigen used in the MLR (data not shown). The cell lines were grown for an average period of 3 months following cloning with varying efficiencies of proliferation, although some of the cell lines had a much longer lifespan. DPP IV activity was measured on the T cell lines using the cytofluorographic method described above, in combination with staining for either CD4, CD8 or CD26. Figure 5 shows DPP IV activity for the cell lines that were tested: depending on the availability of sufficient numbers of cells, some lines were evaluated more than once. The data demonstrate that there were variable amounts of DPP IV activity among the cell lines. As a group, CD8+ T cell lines tended to have elevated activity with rapid kinetics (e.g. high activity at 1 min) compared with other cell populations. However, some of the CD8+ lines showed only minimal levels of DPP IV CD4+ T cell clones had more rapid kinetics of DPP IV activity than did resting cells but, in general, had lower total activity than several of the CD8+ clones. CD26 expression on the lines was typically elevated but was not directly correlated to the DPP IV activity, whereas the cell lines with the highest level of enzyme activity generally exhibited the greatest proliferation to specific alloantigens in the MLR (data not shown).

No Stimulation

CD3 Ab

In an effort to determine whether DPP IV could be modulated in these cells, several (n = 7) clones which had exhibited vigorous growth were tested for changes in enzymatic activity after specific and nonspecific stimulation. Figure 6 is an example of the results with one cell line (TCRBV5.2+) showing how DPP IV activity was upregulated from the levels evident in resting cells following exposure to specific alloantigen as well as crosslinking of the cells with plate-bound anti-CD3 or antiTCRBV5.2. Figure 7 shows the serial measurements of DPP IV activity in a separate cell line over its lifetime, including the polyclonal phase and the subsequent period when the cells were cloned and the cell lines were established. These results typified other experiments which revealed that DPP IV activity was upregulated following activation of the cells, including after reexposure to the original alloantigen. From these data we concluded that alloactivation and the subsequent proliferation of the effector T cells is associated with an increase in the enzymatic activity, the latter which can be sustained for an extended duration following the encounter with alloantigen (Figure 7).

Discussion The capacity of a host’s T lymphocytes to respond and propagate effectively upon confrontation with allogeneic cells from a transplanted organ is under a considerable number of

Vb 5.2

Ab

ARTx

Figure 6 Representative experiment showing cytofluorographic evaluations of DPP IV activity at 10 min in a cloned CD4+ alloreactive T cell line, BHO2-ART. ‘Rvo-colour staining was performed to determine the enzyme’s activity along with CD26 expression as described in Materials and Methods. The cell line, which had been previously found by R’I-PCR analysis to be solely TCRBV5.2 positive, was evaluated for DPP IV(CD26) without stimulation (upper left) and following a 2-day exposure to plates coated with either anti-VP.2 (upper right) or anti-CD3 (lower left). The cell line was also evaluated after a 3-day exposure to irradiated, stimulator cells (AKfx, lower right) identical to the original stimulating alloantigen. Controls included exposure to an antibody not directed to the cell line’s T cell receptor (anti-Vw.0) and incubation with nonspecific stimulator cells. In both cases (not shown) there was no increase in DPP IV activity and the cells displayed a histogram essentially identical to the upper left (no stimulation) panel shown above.

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Dipeptidyl Peptidase IV (CD26) activity

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Figure 7 Graph depicting serial cytofluorographic evaluations of DPP IV activity at 10 min over several time-points from a bulk MLR and in a CDS+ alloreactive T cell line, KYOO-JAS, which was cloned from this MLR. The clone was established after approximately 60 days of the bulk MLR culture. The arrows designate points in the history of the MLR culture or the cell line when allostimulation was performed.

influences, including restriction and regulation by cellularbound molecules and soluble biologically active mediators. Although a formidable list of factors has been implicated in the control of alloreactivity (e.g. MHC and cytokines)@,” there still exist many potential candidates which could provide at least partial regulation of a host’s immunological recognition and ultimate destruction of an allograft. In this regard, membrane-associated proteolytic enzymes are poorly understood with respect to their role(s) in alloimmunity. These enzymes are a ubiquitous group of molecules which are broadly distributed among many cell types and displa notably diverse functions, including cellular differentiation, Z conversion of inactive to active forms of peptides e.g. cytokines, complement and coagulation cascades),47,4! dynamic cell membrane activities such as exocytosis and fusion,49 and regulation of protein kinase activities5’ Among the most principal of these membrane-associated enzymes is DPP IV (CD26). Mature T and B cells and thymocytes are among the cells of the haematopoietic system which possess DPP IV (CD26) and we and others have previously reported studies implicating this serine aminopeptidase to play a role in T lymphocyte ontogeny,‘9’20 as a costimulato molecule in antigen-specific activation,“‘18 cytokine activi J ‘J’ and antibody production:3 In addition, DPP IV is noteworthy in that it facilitates the binding by cells of connective tissue elements and viral envelope proteins, asJ4 the former interaction is conceivably important as a means for T lymphocytes to traffic and reside in the extracellular matrix of organs (e.g. interstitium). DPP IV has a preferential substrate affinity for the glycine-proline motif,5 underscoring a possible way by which this enzyme activates or deactivates other molecules. We have recently reported our development of a cytoenzymatic assay that utilizes a glycine-proline membrane-permeable fluorogenic substrate and which was previously demonstrated to be specifically measuring intracellular DPP IV activity.” The interaction of DPP IV with this substrate is at least partially an Transplant Immunology 1997; 5: 152-161

energy-dependent process and since CD26 is associated with clathrin-coated vesicles,55 the assay represents a rapid intemalization of dipeptide or conjugated amino acid. Among the attractive features of this assay is that DPP IV activity can be measured precisely without disruption of the cellular architecture and the method used for preserving the cells allows simultaneous staining with antibody reagents that are directly conjugated to fluorochromes which spectrally are nonoverlapping with the fluorescence emission of the rhodaminethat is linked to the glycine-proline dipeptide. Although one previous report has described increased DPP IV activity in the serum of patients experiencing allograft rejection,31 there is essentially no understanding of the potentially important roles that CD26 may play in alloreactive immune responses, not only as a molecule involved in cellular activation but also as one of the ways by which alloreactive T cells span graft endothelial barriers. Since it appears that enzymatic activity may be integral to the capacity of CD26 to behave as a costimulatory molecule,21,56 the cytofluorogenic assay was used to measure DPP IV activity at 1 and 10 min in T cell subpopulations that were reacting to MHC-disparate allogeneic stimulator cells. %o-colour flow cytometry was used to phenotype the T cells and the other studies performed simultaneously were useful to help to characterize the populations in question. For example, RT-PCR for TCRBV analysis performed at the initiation of the cultures demonstrated a normally distributed T cell repertoire among the normal controls, and analysis of the MLRs after several weeks revealed (as previously published by others5’) the existence of predominant TCRBV subtypes within the T cell clones (data not shown). The presence of solitary TCRBV types in the cloned populations confirmed the single cell origin of the lines and was instrumental in determining which monoclonal antibody to TCRBV would be used for stimulating the cell lines for an examination of T cell activation and the relationship with enzyme activity. Overall, we did not appreciate any

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significant differences in the DPP IV activity of T cells among individuals in the initial phases of the MLR or among clones as related to the TCRRV phenotype. In general, DPP IV activity and CD26 expression became elevated as T cells underwent activation and acquired memory cell status upon encounter with alloantigens. CD4+ and CD8+ T cells both had heightened DPP IV activity from resting, unstimulated cells after the cells became activated during the course of the MLR. However, activity levels were not static and showed variations over the culture period. Periodic examination of the enzyme activity levels revealed that the timing of restimulation of the responding T cells with alloantigen specific to the initiating stimulator in the MLR resulted in increases in the DPP IV activity. This was confirmed by examining the levels of activity in the alloreactive T cell clones before and after allostimulation. Our conclusion from these experiments was that allospecific, memory T cells undergoing a burst of activity when confronted with specific alloantigen have a concomitant increase in DPP IV levels. In this regard, important issues remain unresolved regarding this relationship between DPP IV and alloactivation. Previous work has shown roles for CD26 in T cell activation”,r8 and, based on the present data, we likewise speculate that CD26 (with DPP IV activation) is a potentially important component in the achievement of immunocompetance to alloantigens. DPP IV may be primarily involved in the initial phases of alloactivation but this enzyme may also participate in alloreactivity by several mechanisms that optimize conditions for the responding population to evolve into competent effector cells. For example, DPP IV maintains the potential to cleave several cytokines which could change the character of the local inflammatory response in the allograft by affecting the differentiation pathways of responding host T lymphocytes into particular subsets of helper, cytotoxic and regulatory cell populations. Additionally, effector cells bearing higher levels of CD26/DPP IV conceivably have an increased capacity to traffic in the interstitial compartment of the allograft, based on previous reports of the binding affinity of CD26 for collagen. Ultimately, this would enhance the ability of these cells to approach and injure donor cell structures (e.g. tubules). Interestingly, as reported by others in a separate model,” the level of enzyme activity did not appear to be directly related to the intensity of CD26 expression on the effector T cells. This latter point illustrates that the inherent level of enzyme activity is separable from the amount of CD26 epitope expressed on the cell surface, raising the possibility that enzymatic activity may be one of the more crucial components of CD26 in alloreactivity. There may be varying ratios of intracellular to surface-associated CD26 as the cells differentiate and progress through activation steps. Experiments are planned to attempt to dissect out the latter issue as well as to determine at what point CD26/DPP IV affects the molecular chain of events associated with T cell activation, particularly following an encounter with an alloantigen. Finally, we are in the process of exploring the effectiveness of different stimulatory populations (e.g. endothelial cells) to induce and regulate DPP IV activity in immune effector cells. These results support previous findings that memory cells generally possess higher levels of DPP IV activity,15 but a considerable degree of heterogeneity was also found among T cells in the level of the enzyme which was measurable. For instance, alloreactive CD4+ cells tended to have higher DPP IV activity during the initial phases of the MLR (i.e. when polyclonal populations were being measured) than CD8+ cells, whereas cloned Trnnsplant Immunology

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CD4 T cell lines overall had lower activity than CD8+ lines. Among the clones of either phenotype were ones which had consistently low DPP IV activity, irrespective of the timing of alloantigen restimulation. Our preliminary evidence suggests that the lines with lower enzymatic activity also tended to have reduced effector cell function and proliferative capacity. This could imply that T cells completely or partially incapable of responding to alloantigens (e.g. anergic) may have associated changes in DPP IV (CD26), and studies are in progress to address this question. In conclusion, we have provided evidence suggesting that a multifunctional cellular protease such as DPP IV has, by several potential means, an important participatory role in the achievement of immunocompetence in T cells encountering alloantigen and in determining the spectrum of T cells responding to an allograft in a host. Acknowledgements The authors wish to thank Dr V. Esquenazi for assistance with

HLA typing and Rosanna Lam for excellent clerical support.

References 1 Ulmer A, Mattem T Feller A, Heymann E, Flad HD. CD26 antigen is a surface dipeptidyl peptidase IV (DPP IV) as characterized by monoclonal antibodies clone TII-19-4-7 and 4ELlC7. Scund J Immunol1990; 31: 429-35. 2 De Meester I, Vanhoof G, Hendriks D, Demuth HU, Yaron A, Scarpe S. Characterization of dipeptidyl peptidase IV (CD26) from human lymphocytes. Clin Chim Acta 1992; 210: 23-34. 3 Shipp MA, Look AL. Hematopoietic differentiation antigens that are membrane associated enzymes: cutting is the key! Blood 1993; 82: 1052-70. 4 Abbott CA, Baker E, Sutherland GR, McCaughan GW. Genomic organization, exact localization, and tissue expression of the human CD26 (dipeptidyl peptidase IV) gene. Immunogenetics 1994; 40: 331-38. 5 Walter R, Simmons WI-I, Yoshimoto T Proline specific endo-and exopeptidases. Mel Cell Biochem 1980; 30: 111-27. 6 Mattem T Reich C, Duchrow M, Ansorge S, Ulmer AJ, Flad HD. Antibody-induced modulation of CD26 surface expression. Immunology 1995; 84: 595-600. 7 Naquet P Vivier I, Gorvel JP et al. Activation of mouse T lymphocytes by a monoclonal antibody to a developmentally regulated surface aminopeptidase. Immunol Rev 1989; 111: 177-93. 8 Shin&u Y, Shaw S. Lymphocyte interactions with extracellular matrix. FASEB J 1991; 5: 2292-99. 9 Kameoka J, Tanaka T, Nojima Y, Schlossman SF, Morimoto C. Direct association of adenosine deaminase with a T cell activation antigen CD26. Science 1993; 261: 46669. 10 Torimoto Y, Dang NH, Vivier E, Tanaka T, Schlossman SE Morimoto C. Coassociation of CD26 (dipeptidyl peptidase IV) with CD45 on the surface of human T lymphocytes. JZmmunoll991; 147: 2514-17. 11 Schon E, Jahn S, Kiessig I-IU et al. The role of dipeptidyl peptidase IV in human T lymphocyte activation inhibitors and antibodies against dipeptidyl peptidase IV suppress lymphocyte proliferation and immunoglobulin synthesis in vitro. Eur J Immunol 1987, 17: 1821-26. 12 Buhling F, Junker U, Reinhold D, Neubert K, Jager L, Ansorge S. Functional role of CD26 on human B lymphocytes. Zmmunol Lett 1995; 4s: 47-51. 13 Buhling F, Dunx D, Reinhold D er al. Expression and functional role of dipeptidyl peptidase IV on human natural killer cells. Nat Immun 1994; 13: 270-79. 14 Kameoka J, Sato T Torimoto Yet al. Differential CD26-mediated activation of the CD3 and CD2 pathways after CD6-depleted allogeneic bone marrow transplantation. Blood 1995; 85: 1132-37.

DipeptidylPeptidase N (CD26) activity

15 Vanham G, Kestems L, De Meester L el al. Decreased expression of the memory marker CD26 on both CD4(+) and CDS(+) T lymphocytes of HIV infected subjects. JAcquir Immune De& Syndr 1993; 6: 749-57. 16 Martin M, Huguet J, Centelles JJ, France R. Expression of EctoAdenosine deaminase and CD26 in human T cells triggered by the TCR-CD3 complex. J bnmunol1995; 155:463W3. 17 Dang NH, lbrimoto Y, Deusch K, Schlossman SE Morimoto C. Comitogenic effect of solid phase immunobiiid anti-IF7 on human CD4 T cell activation via CD3 and CD2 pathways. J Immunol1990; 144:4092-100. 18 Bednarczyk J, Carroll SM, Marin C, McIntyre B. Triggering of the proteinase dipeptidyl peptidase IV (CD26) amplifies human T lymphocyte proliferation. J Cell Biochem 1991; 46: 206-X 19 Ruiz P, Nassiri M, Steele B, Viciana AL. Cytofluorographic evidence that thymocyte dipeptidyl peptidase IV (CD26) activity is altered with stage of ontogeny and apoptotic status. Cytometry 1996; 23: 322-29. 20 Bauvois B. Murine thymocytes possess specific cell surface-associated exoaminopeptidase activities: preferential expression by immature CD4CD8-subpopulation. Eur J bnmuno 11990; 20: 459-68. 21 Tanaka ‘I; Kameoka J, Yaron A, Schlossman SE Morimoto C. The costimulatory activity of the CD26 antigen requires dipeptidyl peptidase IV enzymatic activity. Proc NatlAcad Sci USA 1993;90: 4586-90. 22 De Meester IA, Kestens LL, Vanham GL et al. &stimulation of CD4+ and CD8+ T cells through CD26: the ADA-binding epitope is not essential for complete signaling. JLeulroc Biol1995; 58: 325-30. 23 Muscat C, Bertotto A, Agea E et al. Expression and functional role of lF7 (CD26) antigen on peripheral blood and synovial fluid T cells in rheumatoid arthritis patients. Clin Exp Immunol1994,98: 252-56. 24 Scheel-Toellner D, Richter E, Toellner KM et al. CD26 expression in leprosy and other granulomatous diseases correlates with the production of interferon-y. Lab Invest 1995; 73: 685-90. 25 Van Leer EHG, Bruijin JA, Prins FA, Hoedemaeker PJ, De Heer E Redistribution of glomerular dipeptidyl peptidase type IV in experimental lupus nephritis. Lab Invest 1993; 68: 550-56. 26 Callebaut C, Krust B, Jacotot E, Hovanessian AG. T cell activation antigen, CD26 as a cofactor for entry of HIV in CD4+ cells. Science 1993; 262: 2045-50. 27 West WHL, Stott EJ. Cell surface expression of CD26 does not correlate with susceptibility to immunodeficiency viruses. AIDS 1994; 8: 1349-51. 28 Werner A, Mattem T, Ulmer AJ, Flad HD, Kurth R, Maier M. CD26 is not required for infection of the lymphoma cell line C8166 with HIV-l. AIDS 1994; 8: 1348-49. 29 Masuyama JI, Berman JS, Cruikshank Ww, Morimoto C, Center DM. Evidence for recent as well as long term activation of T ceils migrating through endothelial cell monolayers in vitro. J Immunol 1992; 148: 1367-74. 30 Duke-Cohan JS, Morimoto C, Rocker JA, Schlossman SE A novel form of dipeptidyl peptidase IV found in human serum: isolation, characterization, and comparison with T lymphocyte membrane dipeptidyl peptidase IV (CD26). J Biol Chem 1995; 270: 14107-14. 31 Sanda MG, Pierson R, Smith C, Reemtsma K, Rose E. Serum Dipeptidyl Peptidase IV in cardiac transplant recipients. Tmnsplant Proc 1989; 21: 2525-26. 32 Leytus SP, Patterson WL, Mange1 WE New class of sensitive and selective fluorogenic substrates for serine proteinases. Biochem J 1983; 215: 253-60. 33 Assfalg-Machleidt I, Rothe G, Klingel S et al. Membrane permeable fluorogenic rhodamine substrates for selective determination of cathepsin L. Biol Chem Hoppe-Styler 1992; 373: 433-40. 34 Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Stand J Lab Clin Invest 1968; 21: 77-89. 35 Rosen-Bronson S, Johnson AH, Hartzman RJ, Eckels DD. Human allospecific TLCs generated against HLA antigens associated with DRl through DRw8: growth and specificity analysis. Immunogenetics 1986; 24: 368-78. Transplant Immunology

1997; 5: 152-161

161

36 Ruiz P, Streilein JW. Evidence that I-E-negative mice resistant to neonatal H-2 tolerance induction display ubiquitous thymic cional deletion of donor-reactive T cells. Transplantation 1993; 55: 321-28. 37 Ruiz P Hoffman TM, Howell DN et al. Evidence that pretransplant donor blood transfusion prevents rat renal allograft dysfunction but not the in situ cellular alloimmune or morphologic manifestations of rejection. Tmnsplantation 1988; 45: 1-7. 38 Nilsson K, Klein G. Phenotypic and cytogenetic characteristics of human B lymphoid cell lines and their relevance for the etiology of Burkitt’s lymphoma. Adv Cancer Bes 1982; 37: 319-80. 39 Panzara MA, Gussoni E, Steinman L, Oksenberg JR. Analysis of the T cell repertoire using the PCR and specific oligonucleotide primers. Biotechniques 1992; 12: 728-35. 40 Charley M, McCoy JP, Deng JS, Jegesothy B. Anti-V region antibodies as &Almost Clonotypic’ reagents for the study of cutaneous T cell lymphomas and leukemias. JInvest Dermutoll995; 95: 614-17. 41 Nassiri M, Viciana A, Streilein JW, Ruiz F! Donor-specific skin transplants activate allodestructive T cells in mice resistant to neonatal H-2 tolerance induction. Transpluntation 1993; 56: 1460-67. 42 Ruiz P, Nassiri M, Viciana AL, Padmanabhan J, Streilein JW. Characterization of donor chimerism, alloreactive host T cells and memory cell development in thymi from mice resistant to neonatal transplantation tolerance. J Immunol1995; 154:633-43. 43 Budd RC, Cerottini JC, Horvath C et al. Distinction of virgin and memory T lymphocytes: stable acquisition of the Pgp-1 glycoprotein concomitant with antigenic stimulation. J Immunol 1987; 138: 3120-29. 44 Lechler RI, Lombardi G, Batchelor JR, Reinsmoen N, Bach FH. The molecular basis of alloreactivity. Immunol T+ 1990; 11: 83-89. 45 Hutchinson IV Cellular mechanisms of allograft rejection. Curr Opin Immunoll991; 3: 722-28. 46 Di Stefano JE Beck G, Lane B, Zucker S. Role of tumor cell membrane-bound serine proteases in tumor-induced target cytolysis. Cancer Res 1982; 42: 207-18. 47 Frank, MM, Fries LE The role of complement in inflammation and phagocytosis. Immunol Today 1991; 12: 321-26. 48 Scholz W, Mentlein R, Heymann E, Feller AC, Ulmer AJ, Flad HD. Interleukin 2 production by human T lymphocytes identified by antibodies to Dipeptidyl Peptidase IV Cell Immunol 1985; 93: 199-211. 49 Mundy DI, Strittmatter WJ. Requirement for metalloendoprotease in exocytosis. Evidence in mast cells and adrenal chromaffin cells. Cell 1985; 40: 645-56. 50 Alhanaty E, Shaltiel S. Limited proteolysis of the catalytic subunit of CAMP-dependent protein kinase: A membranal regulatory device656. Biochem Biophys Bes Commun 1979; 89: 323-32. 51 Morimoto C, Schlossman SF. CD26: a key costimulatory molecule on CD4 memory T cells. Immunologist 1994; 2: &7. 52 Fukiwara H, Fukuoka M, Yasuda K et al. Qtokines stimulate dipeptidyl peptidase-IV expression on human luteinizing granulosa cells. J Clin Endocrinol Metab 1994,79: 1007-11. 53 Morimoto C, Torimoto Y, Levison Get al. lF7, a novel cell surface molecule involved in helper function of CD4 cells. J Immunol1989; 143:3430-39. 54 Dang NH, Torimoto Y, Schlossman SF, Morimoto C. Human CD4 helper T cell activation: functional involvement of two distinct collagen receptor IF7 and VLA integrin family. Jm Med 1990; 172: 649-52. 55 Droz D, Zachar D, Charbit L, Gogusev Chretien JY, Iris L. Expression of human nephron differentiation molecules in renal cell carcinoma. Am J Puthol1990; 137: 895-905. 56 Reinhold D, Bank U, Buhling F et al. Inhibitors of Dipeptidyl Peptidase IV (DP IV, CD26) specifically suppress proliferation and modulate cytokine production of strongly CD26 expressing U937 cells. Immunobiology 1994; 192: 121-36. 57 Hu W, Weyand CM, Goronzy JJ. The T-cell receptor VP6 gene usage in alloreactive T-cell responses. Human Immunol 1995; 42: 72-80.