R E V I E W
`I M M U N O L O G Y T O D AY
`
`CD26, let it cut or cut it down
`
`Ingrid De Meester, Stephan Korom, Jo Van Damme and Simon Scharpé
`
`T
`
`he lymphocyte surface glyco-
`protein CD26 is characterized
`by an array of diverse func-
`tional properties. Scientists in
`several disciplines have independently in-
`vestigated various aspects of CD26 for over
`three decades, and complementary research
`in clinical chemistry, enzymology, immunol-
`ogy, virology and medicine was necessary to
`disclose the complex nature of this intrigu-
`ing protein1. Like CD13 (Ref. 2), CD26
`belongs to a unique class of membrane-
`associated peptidases, and is identical to
`dipeptidyl-peptidase IV (DPP IV, EC 3.4.14.5).
`It has a high selectivity for peptides with a
`proline or alanine at the second position3
`and cleaves off dipeptides at the N-terminus
`of such peptides.
`
`Structure of CD26
`The complete cDNA and derived amino acid sequence for human
`CD26 was first published in 1992 (Ref. 4). The CD26 gene5 (Box 1) en-
`codes a type II transmembrane protein of 766 amino acids, which is
`anchored to the lipid bilayer by a single hydrophobic segment lo-
`cated at the N-terminus (Fig. 1), and has a short cytoplasmic tail of
`six amino acids. A flexible stalk links the membrane anchor to a large
`glycosylated region, a cysteine-rich region and a C-terminal catalytic
`domain. Alignment of the amino acid sequences reveals a high de-
`gree of conservation between different species, with the C-terminal
`segment showing the highest level of identity.
`The active site Ser630 of human CD26 is surrounded by Gly-Trp-
`Ser630-Tyr-Gly-Gly-Tyr-Val, which corresponds to the motif Gly-X-
`Ser-X-Gly proposed for serine-type peptidases. A more extended con-
`sensus motif around the catalytic serine is shared by all members of
`the prolyl oligopeptidase family, to which CD26 belongs. The linear
`order of the catalytic triad in this family of peptidases is nucleophile-
`acid-base (Ser-Asp-His), an arrangement not common for ‘classic’
`serine-type peptidases (trypsin or chymotrypsin-like enzymes) but
`characteristic of the a/b hydrolase fold. This protein fold (Fig. 1),
`which is shared by a number of enzymes of widely different catalytic
`function (for example, lipases, cholinesterases and proteases), consists
`of a core a/b sheet of eight b strands connected by a helices and loops
`containing the three catalytic residues. Peptidases whose secondary
`structure has been predicted or characterized as an a/b hydrolase fold
`form a separate clan, named clan SC (Ref. 7). Recently, the crystal
`structures of two members of this peptidase clan have been revealed.
`Both the bacterial prolyl aminopeptidase (EC 3.4.11.5) from
`
`PII: S0167-5699(99)01486-3
`
`The costimulatory properties of
`CD26 have been studied extensively
`and significant progress has been
`made in unravelling the complex
`nature of this molecule. Here, we
`summarize recent findings on
`molecular and functional
`characteristics of CD26. We argue
`that a multidisciplinary approach
`might reveal the molecular events
`underlying the role of CD26 in HIV
`infection and immune,
`inflammatory and endocrine
`responses.
`
`Xanthomonas campestris8 and the mammalian
`prolyl oligopeptidase (EC 3.4.21.26) from
`porcine muscle9 consist of two domains. The
`core of the three-dimensional structures of the
`peptidase domain corresponds to the pre-
`dicted a/b hydrolase fold10. The non-catalytic
`domain of prolyl oligopeptidase is an unusual
`b propeller that excludes larger peptides from
`entering the active site, which is situated at
`the interface between the two domains.
`Although compelling experimental evi-
`dence indicates that the extracellular part of
`CD26 consists of three separate domains11,
`only the high-resolution crystal structure
`will permit significant progress in the de-
`tailed understanding of the selective ligand-
`binding and substrate hydrolysis by CD26.
`At this moment, several questions concern-
`ing the structure of this highly stable, large, intrinsic membrane
`protein remain unanswered, for example: (1) How do the different
`domains of one monomer interact with each other? (2) How do the
`subunits dimerize12? (3) Which of the different domains/epitopes on
`the cell surface are accessible? (4) What is the relationship with other
`integral membrane serine-type proteases, such as fibroblast activ-
`ation protein a (FAP-a)13.
`
`Multiple alliances but no known partner
`In addition to its exopeptidase activity (discussed below), CD26
`specifically binds several proteins outside its substrate-binding site.
`In contrast to other costimulatory molecules on the T cell, such as
`CD28, no counter-receptor has been identified for CD26.
`
`Extracellular matrix interactions
`More than a decade ago, a role for CD26 in cell interactions with the
`extracellular matrix (ECM) was described. These interactions were
`specified to involve fibronectin and collagen: collagen binds to a re-
`gion in the cysteine domain14 between residues 238 and 495, whereas
`fibronectin binds residues 469 to 479 (Ref. 15). Although the 1F7 anti-
`gen – later identified as CD26 – was reported to be a functional col-
`lagen receptor, the role of CD26 as a cell adhesion molecule is still
`unclear16,17. The low affinities most probably cause experimental dif-
`ficulties in studying these phenomena, although they might have a
`physiological function. CD26 expressed on rat lung capillary en-
`dothelia serves as an adhesion receptor for breast cancer cells and me-
`diates lung colonization by these cells. Fibronectin associated with
`the tumor cell surface is the principal CD26 ligand in this process18.
`
`0167-5699/99/$ – see front matter © 1999 Elsevier Science Ltd. All rights reserved.
`
`A U G U S T
`V o l . 2 0
`N o . 8
`
`1 9 9 9
`3 6 7
`
`Page 1 of 9
`
`AstraZeneca Exhibit 2070
`Mylan v. AstraZeneca
`IPR2015-01340
`
`

`
`R E V I E W
`I M M U N O L O G Y T O D AY
`
`Box 1. CD26 gene5,6
`
`• About 90 kb
`• 26 exons, ranging from 45 b to 1.4 kb
`• Same exon–intron organization in mice and humans
`• No TATA box or CAAT box in 59untranslated region
`• GC-rich region contains binding site for transcription factors
`• Hepatocyte nuclear factor 1 causes transcriptional activation
`• Localized on the long arm of chromosome 2 (2q24)
`
`ADA binding
`In 1993, CD26 was identified as the binding protein for adenosine
`deaminase (ADA)19. ADA catalyzes the irreversible deamination of
`adenosine and 29deoxyadenosine to inosine and 29deoxyinosine, re-
`spectively. The enzyme is well known because the hereditary lack of
`ADA activity causes severe impairment of cellular and humoral im-
`munity. In most tissues, its catalytic moiety (a single polypeptide of
`~40 kDa) interacts with an ADA-binding protein (ADAbp), now rec-
`ognized to be CD26. Complex formation between ADA and CD26
`preserves the individual enzymatic activities of both molecules. On
`the T-cell surface, CD26 serves as a receptor for ADA and this obser-
`vation triggered research on its functional implications in immune
`regulation20. Recently, Dong et al. determined the binding site for
`ADA on CD26: using truncated human–rat swap mutants and cross-
`blocking studies, five epitope groups were defined on CD26 and one
`of them (aa 359 to 449) appeared to encompass the ADA-complexing
`domain. The residues 340–343 on the CD26 molecule proved to be
`essential for ADA binding21. Although the ADA binding site is situ-
`ated in the cysteine-rich region, most of the currently available mono-
`clonal antibodies (mAbs) against CD26 are directed towards the
`glycosylation region22. A model for immunoregulation by
`CD26–ADA–adenosine has been proposed16, in which CD26 is as-
`sumed to modulate the concentration of local extracellular adeno-
`sine, which provides negative signals to T cells. More work is needed
`to verify this hypothesis in physiological and pathological condi-
`tions, such as chronic inflammatory diseases. The observation that
`ADA–CD26 binding occurs in several but not all mammals
`(ADA–CD26 interaction was not seen in rodents23) suggests that this
`interaction is a ‘coincidence’ without biological significance, or is a
`new example of interspecies variation in the regulation of a number
`of physiological systems.
`
`CD26 expression
`CD26 is strongly expressed on epithelial cells (kidney proximal
`tubulus, intestine, bile duct) and on several types of endothelial cells
`and fibroblasts as well as leukocyte subsets24 (see below). This rather
`wide expression of the gene encoding CD26/DPP IV is in contrast to
`the restricted expression of the related FAP-a gene. A detailed
`characterization of the promotor regions of both genes might help in
`understanding the regulation of CD26 synthesis. An interesting fea-
`ture of CD26 traffic is its ability to enter into an endocytosis/exocy-
`tosis cycle, which involves re-entry into the Golgi apparatus and re-
`sults in glycosylation changes12. This might explain the presence of
`different forms of CD26 during T-cell activation25. A similar molecular
`
`A U G U S T
`3 6 8
`V o l . 2 0
`
`1 9 9 9
`N o . 8
`
`heterogeneity of CD26 in different tissues or in different individuals
`points to post-transcriptional processing. Sialylation of CD26 in-
`creases significantly with age and extreme hypersialylation is found
`in HIV-infected individuals26.
`The soluble form of CD26 (sCD26) lacks the intracellular tail and
`transmembrane regions; it is found at high levels in seminal fluid,
`whereas moderate and low amounts are detected in plasma and
`cerebrospinal fluid, respectively.
`
`CD26 expression in the hematopoetic system
`Originally described as a T-cell activation molecule, CD26 is now re-
`garded as a non-lineage antigen, whose expression is regulated by
`the differentiation and activation status of immune cells. Although
`CD26 is absent on resting B and NK cells, it is induced on their sur-
`face upon stimulation24,27. In resting peripheral blood mononuclear
`cells (PBMCs), a small subpopulation of T cells expresses CD26 at
`high density on the surface28 (CD26bright T cells) and displays a num-
`ber of defined phenotypic and functional characteristics, which are
`summarized in Fig. 2.
`
`CD26bright T cells
`The CD26bright subpopulation, which belongs to the CD45R0+ popu-
`lation of T cells and exhibits a ‘late-memory’ phenotype, is respon-
`sible for most of the interleukin 2 (IL-2) production. This small sub-
`population of lymphocytes is necessary for the proliferative response
`to recall antigens and allogeneic cells as well as for cytotoxic T lym-
`phocyte (CTL) activity against alloantigens16. Helper functions for
`T-cell-dependent antibody responses are also confined to CD26bright
`cells and CD26 expression has been associated with T helper 1 (Th1)-
`type cells29. The finding that only IL-12 [and not other cytokines
`secreted after IL-12 stimulation, such as IL-1b, interferon g (IFN-g), or
`tumor necrosis factor a (TNF-a)] upregulates CD26 surface expres-
`sion in phytohemmaglutinin-activated PBMCs supports the in vivo
`observations that CD26 expression is a marker for Th1 cells30.
`The number of CD26bright cells and/or CD26 antigen density is
`higher during active phases of autoimmune diseases and decreases
`during immunosuppression (Box 2)28,31–39. In autoimmune diseases,
`high numbers of CD26+ cells are found at inflammation sites40. The
`capacity of CD26bright T cells to migrate transendothelially and to in-
`filtrate inflammatory sites40,41 might be related to the coexpression of
`the CC chemokine receptor CCR5 on T cells42. Although probably
`not all features of CD26bright cells (highlighted in Fig. 2) are function-
`ally related to the expression of this antigen, a number of immune
`functions can be influenced by targeting CD26.
`
`(Co)stimulation via CD26
`In general, the activation of T cells requires at least two signals. The
`first is provided by stimulation of the T-cell receptor (TCR) complex
`by specific peptide antigen or mAb. The second signal can be deliv-
`ered by triggering costimulatory surface molecules that, similar to
`adhesion molecules, belong to the group of ‘accessory molecules’
`
`Page 2 of 9
`
`

`
`R E V I E W
`I M M U N O L O G Y T O D AY
`
`Asp
`His
`
`Ser
`
`Interactions
`
`"
`
`.....
`
`Fibronectin
`
`aa 469-479
`
`aa 340-343
`
`ADA
`
`TA5.9
`22C3
`
`1F7
`BA5
`TA1
`
`CB1
`
`Collagen
`
`Out
`
`In
`
`CD45
`
`Fig. 1. Schematic representation of the CD26 dimer on the T-cell surface. The left half of the picture
`shows the main structural features of this type II integral membrane protein. The vertical bar represents
`the primary structure and shows the positions of the active site residues as well as the putative borders
`of the extracellullar domains. There is experimental evidence for three extracellular domains within one
`monomer. To date, their structure and position towards one another is unresolved; for the catalytic
`domain, an a/bprotein fold is hypothesized (top): a core a/bsheet comprising eight strands (arrows),
`linked by helices (cylinders) and loops (lines). The dotted lines indicate possible insertions and the cata-
`lytic residues are situated on loops. On the T-cell surface, CD26 binds ADA in its cysteine-rich
`domain. The residues (aa) on CD26 involved in the interactions are indicated. Antibodies known to
`recognize closely related epitopes are shown in dotted circles. The peptide substrates interact with the
`catalytic domain to allow the active site residues to cleave off the N-terminal dipeptide from peptides
`with susceptible sequences. Abbreviations: ADA, adenosine deaminase; aa, amino acid residues.
`
`adaptor protein43. A study on signal transduction in the human
`hepatocarcinoma cell line, PLC/PRF15, indicates that a tyrosine
`phosphorylation pathway is activated after CD26 triggering even in
`the absence of the TCR/CD3 complex45.
`
`A U G U S T
`V o l . 2 0
`N o . 8
`
`1 9 9 9
`3 6 9
`
`that are involved in a series of antigen
`non-specific interactions between antigen-
`presenting cells (APCs) and T cells during a
`physiological activation process. The costimu-
`latory properties of CD26 have been studied
`extensively in vitro16. Although different ex-
`perimental conditions sometimes provide
`conflicting results, it is accepted that several
`distinct anti-CD26 mAbs have costimulatory
`activities in anti-CD3-driven activation of
`pure T-cell subsets (either CD4+ or CD8+ T
`cells), and that the extent and kinetics of the
`response differs between mAbs recognizing
`different epitopes.
`In addition to PBMCs, Jurkat T cells are
`frequently used to study signaling through
`CD26. Because parental Jurkat cells do not
`express detectable amounts of CD26 mRNA,
`cell clones transfected with wild-type CD26
`or enzymatically inactive CD26 mutants are
`interesting tools for stimulation and co-
`stimulation assays. Upon CD26-mediated
`costimulation, IL-2 production is higher in
`cells expressing wild-type CD26, suggesting
`that the DPP IV enzymatic activity of CD26
`might contribute to, but is not essential for,
`signal transduction16. However, using a
`mouse TCR+ T-cell hybridoma, other authors
`have shown that the enzymatic activity of
`CD26 is not required for its stimulatory and
`costimulatory activity43. Together, the avail-
`able in vitro data do not permit the role of the
`enzymatic activity of CD26 in the in vivo
`immune response to be predicted.
`Biochemical and immunomodulation
`studies (reviewed in Ref. 16) provide evi-
`dence that CD26 on the T-cell surface inter-
`acts directly with CD45, a protein tyrosine
`phosphatase. However, the regions in each
`of these large integral membrane enzymes
`that are involved in the physical associ-
`ations, as well as the importance for signal-
`ing via CD26, are still unknown.
`Crosslinking of CD26 by antibody causes
`tyrosine phosphorylation of an array of in-
`tracellular proteins involved in TCR/CD3-
`mediated signal transduction44, including
`the z chain, p56lck, p59fyn, ZAP-70, mitogen-
`activated protein kinase (MAPK), c-Cbl and
`phospholipase Cg. The expression of the
`TCR on the cell surface has proved to be essential for effective sig-
`naling via CD26 and the z chain is necessary but not sufficient for
`CD26-mediated triggering. This suggests that CD26 does not di-
`rectly associate with the z chain but requires a hitherto undefined
`
`Structure
`
`COOH
`
`His
`Asp
`Ser
`
`Catalytic domain
`
`Cysteine-rich domain
`
`Glycosylation domain
`
`Flexible stalk
`Transmembrane region
`Intracellular tail
`
`NH2
`
`766
`
`740
`708
`630
`
`492
`
`290
`
`49
`29
`
`6 1
`
`Page 3 of 9
`
`

`
`R E V I E W
`I M M U N O L O G Y T O D AY
`
`CD45
`
`TCR
`
`CCR5
`
`CD26
`
`LFA1
`
`CD26bright T cell
`
`(a) T-cell help for B-cell Ig synthesis
`
`CD26bright T cell
`
`B cell
`
`(b) Proliferation upon sAg and allogeneic cells
`
`CD26bright T cell
`
`(c) Secretion of Th1 type cytokines
`
`IFN-g
`
`CD26bright T cell
`
`IL-2
`
`(d) Transendothelial migration
`
`CD26bright T cell
`
`Fig. 2. Phenotypic and functional characteristics of peripheral blood T cells
`expressing high levels of CD26 on their surface (CD26bright). The CD26bright
`T cells constitute a subpopulation of CD45R0+ T cells, and CD26 occurs on
`subpopulations of CD4+, as well as CD8+ T cells; it is coexpressed with LFA-
`1 and CCR5. CD26bright T cells are important in (a) recruiting T-cell help for
`B-cell Ig synthesis; (b) proliferation in response to sAgs and allogeneic cells;
`(c) secretion of Th1-type cytokines; and (d) transendothelial migration ca-
`pacity. Abbreviations: CCR5, CC chemokine receptor 5; IFN-g, interferong;
`Ig, immunoglobulin; IL-2, interleukin 2; LFA-1, leukocyte function-associated
`molecule 1; sAgs, soluble antigens; TCR, T-cell receptor; Th1, T helper 1.
`
`Cutting down CD26 enzymatic activity
`Another approach to studying the role of the enzymatic activity of
`CD26 in immune regulation involves the development and intro-
`duction of specific inhibitors in appropriate in vitro and in vivo
`settings (Ref. 46 and reviewed in Ref. 47). However, their use re-
`quires monitoring for possible toxic effects as well as inclusion of ap-
`propriate controls. As more specific inhibitors become available that
`
`A U G U S T
`3 7 0
`V o l . 2 0
`
`1 9 9 9
`N o . 8
`
`significantly differ in chemical structure or mechanism of action,
`comparative studies are feasible, facilitating interpretation of results.
`Cutting down the enzymatic activity of CD26 by specific inhibitors
`suppresses T-cell proliferation in vitro and decreases antibody pro-
`duction in mice immunized with bovine serum albumin48. Whether
`the origin of the depressive effect on immunoglobulin secretion is T-
`cell mediated or can be attributed solely to B cells is not yet clear. In
`mice of the autoimmune MLR/Mp-lpr/lpr (MLR:l) strain, serum DPP
`IV activity increased markedly in parallel with lymph node swelling
`and anti-nDNA antibody production49. No results of long-term in
`vivo administration of specific inhibitors in this disease model have
`been reported to date.
`The putative role of CD26 after alloantigen exposure in vivo was
`studied by analyzing the effect of the low-molecular weight inhibitor
`pro-pro-diphenylphosphonate in the immune cascade triggered by
`organ transplantation. The treatment abrogated acute rejection, re-
`sulting in prolonged allograft survival in a rat cardiac transplantation
`model. The suppression of systemic CD26 enzyme activity was asso-
`ciated with severely impaired host CTL responses in vitro50. Moreover,
`treated animals lacked the allospecific IgM response and had greatly
`decreased IgG serum titers51. Therefore, inhibition of the enzymatic
`activity of CD26 is thought to interfere with the early steps in the
`humoral response to alloantigen after organ transplantation.
`Although we are far from a complete understanding52, there are
`some indications of the cytokines involved in the immune alter-
`ations caused by specific CD26 inhibitors. Using a series of related
`competitive inhibitors, Reinhold et al. demonstrated a significant
`suppression of IL-2, IL-10, IL-12 and IFN-g production in pokeweed
`mitogen-stimulated T cells. Under the same conditions, these in-
`hibitors induce an increase in secretion of the latent form of trans-
`forming growth factor b1 (TGF-b1), which suggests that the ‘immuno-
`inhibitory’ TGF-b plays a role in the immunosuppression induced
`by CD26 inhibitors53. Together, these results support a role for CD26
`in immune regulation; in addition to in vivo targeting of CD26 with
`antibodies and/or specific inhibitors, knockout experiments and
`overexpression of CD26 will provide additional insights into the
`underlying molecular mechanisms.
`
`Natural substrates of CD26: cutting makes
`the difference
`The immunosuppressive effect of CD26 enzyme inhibitors, as well as
`the observation that soluble wild-type CD26 enhances impaired re-
`sponses to recall antigen in vitro54,55, indicate that a further search for
`its possible biological substrates would be useful. Several cytokines,
`hematopoietic growth factors, neuropeptides and hormones share
`the X-Pro or X-Ala motif at their N-terminus. As with N-terminal cy-
`clization, the presence of a proline near the N-terminus serves as
`structural protection against non-specific proteolytic degradation56.
`Bauvois has shown the importance of the cell-surface peptidases,
`cathepsin G, DPP IV and g-glutamyl transpeptidase, in the control of
`certain cytokine activities57. The failure of CD26 to process intact ma-
`ture IL-1a, IL-1b and IL-2 in contrast to N-terminal synthetic pep-
`tides of these cytokines, suggests that only small molecules serve as
`
`Page 4 of 9
`
`

`
`R E V I E W
`I M M U N O L O G Y T O D AY
`
`Box 2. Alteration in the number of CD26bright T cells during diseases
`
`Increase
`Rheumatoid arthritis31
`Multiple sclerosis32
`Graves’ disease33
`Hashimoto’s thyroiditis34
`Sarcoidosis35
`Psychological stress36
`
`natural substrates58. Table 1 lists the peptides
`that are attacked by CD26, grouped accord-
`ing to the result of the truncation on their
`biological activity. Substance P is an example
`of a short and well-studied natural substrate
`that, by a stepwise release of Arg-Pro and
`Lys-Pro, is cleaved into the active hepta-
`peptide substance P(5–11) (Ref. 59). The pen-
`tapeptide enterostatin, formed by cleavage of secreted pancreatic
`procolipase, is also cut by CD26/DPP IV (Ref. 60). Its structure–
`activity relationship is not clear yet. CD26/DPP IV might also play a
`role in the in vivo inactivation of endomorphin 2, an endogenous
`tetrapeptide with high affinity for the m opioid receptor61.
`
`Decrease
`AIDS28,37
`Down’s syndrome38
`Common variable hypogammaglobulinemia39
`
`Members of the pancreatic polypeptide family62, including neuro-
`peptide Y and peptide YY, form another important and remarkably
`conserved group of substrates. Truncation by CD26 generates a shift
`in their receptor selectivity and alters their biological specificity from
`vasoconstriction to growth factor activity63.
`
`Table 1. Catalytic activities of CD26
`
`Peptide
`
`N-terminus
`
`Amino acids
`
`Assay system
`
`Refs
`
`Alteration receptor specificity
`Pancreatic polypeptide family
`Peptide YY
`Neuropeptide Y
`
`Chemokines
`RANTES
`SDF-1a
`Eotaxin
`MDC
`
`Neuropeptides
`Substance P
`
`Inactivation
`Glucagon/VIP family
`GRF(1–44) amide
`GIP
`GLP-1(7–36) amide
`GLP2
`PHM
`
`Neuropeptides
`Endomorphin 2
`
`Tyr-Pro-Ile-Lys-
`Tyr-Pro-Ser-Lys-
`
`Ser-Pro-Tyr-Ser-
`Lys-Pro-Val-Ser-
`Gly-Pro-Ala-Ser-
`Gly-Pro-Tyr-Gly-
`
`Arg-Pro-Lys-Pro-
`
`Tyr-Ala-Asp-Ala-
`Tyr-Ala-Glu-Gly-
`His-Ala-Glu-Gly-
`His-Ala-Asp-Gly-
`His-Ala-Asp-Gly-
`
`Tyr-Pro-Phe-PheNH2
`
`No effect observed on biological activity
`Chemokines
`GCP-2
`
`Gly-Pro-Val-Ser-
`
`Biological effect unknown to date
`Enterostatin
`Val-Pro-Asp-Pro-Arg
`
`Chemokines
`IP-10
`
`Val-Pro-Leu-Ser-
`
`36
`36
`
`68
`68
`74
`69
`
`11
`
`44
`42
`30
`33
`27
`
`4
`
`75
`
`5
`
`77
`
`In vitro
`In vitro
`
`In vitro
`In vitro
`In vitro
`In vitro
`
`62
`62
`
`69, 70
`72, 73
`67, 71
`74
`
`In vitro/in vivo
`
`59
`
`In vitro
`In vitro/in vivo
`In vitro/in vivo
`In vivo
`In vitro
`
`In vivo
`
`In vitro
`
`In vitro
`
`In vitro
`
`64
`65, 66
`66, 67
`68
`65
`
`61
`
`70
`
`60
`
`69
`
`Abbreviations: GCP-2, granulocyte chemotactic protein 2; GIP, glucose-dependent insulinotropic polypeptide; GLP-1, glucagon-like peptide 1; GRF, growth
`hormone-releasing factor; IP-10, interferon-inducible protein 10; MDC, macrophage-derived chemokine; PHM, peptide histidine methionine; SDF-1, stromal
`cell-derived factor 1; VIP, vasoactive intestinal peptide.
`
`A U G U S T
`V o l . 2 0
`N o . 8
`
`1 9 9 9
`3 7 1
`
`Page 5 of 9
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`

`
`R E V I E W
`I M M U N O L O G Y T O D AY
`
`(a)
`
`M-tropic virus
`
`CCR5
`
`(b)
`
`T-tropic virus
`
`CXCR4
`
`CD4+ cell
`
`CD4+ cell
`
`RANTES (1–68)
`
`CCR5
`
`RANTES (3–68)
`
`CCR5
`
`CD4+ cell
`
`CD26
`
`"
`
`CD4+ cell
`
`SDF1a (1–67)
`
`CXCR4
`
`CD4+ cell
`
`"
`
`SDF1a (3–67)
`
`CXCR4
`
`CD4+ cell
`
`CD26 = Protection
`"
`
`CD26 = Loss of protection
`"
`
`Fig. 3. Involvement of CD26 in HIV-1 entry. (a) M-tropic viruses use CCR5 as a cofactor for entry into the cell together with the CD4 antigen. A highly
`significant increase in the antiviral activity of RANTES was noted upon removal of the two N-terminal amino acids by CD26. (b) T-tropic viruses use
`CXCR4 as the coreceptor for entry into the CD4+ host cell and the CXCR4 ligand SDF-1aprotects against entry of T-tropic viruses. The release of the N-
`terminal dipeptide Lys-Pro from SDF-1aby CD26 diminishes its antiviral activity. Therefore, processing of chemokines by CD26 enzyme activity in the
`circulation and on the surface of mononuclear cells might play a dual role in HIV-1 entry. Abbreviations: CCR5, CC chemokine receptor 5; CXCR4, CXC
`chemokine receptor 4; M-tropic virus, macrophage-tropic virus; T-tropic virus, T-cell-tropic HIV-1 virus; SDF-1a, stromal cell-derived factor 1a.
`
`The CD26-mediated processing of a number of secretins, mem-
`bers of the glucagon peptide superfamily, constitutes a crucial deter-
`minant of their biological functions64–68. In vivo, glucagon-like pep-
`tide-1(7–36)amide (GLP-1) is truncated rapidly by CD26/DPP IV,
`resulting in the formation of GLP-1(9–36), which might act as an
`antagonist at the GLP-1 receptor. Inhibition of DPP IV activity en-
`hances insulin secretion and improves glucose tolerance in several
`animal models for type II diabetes, pointing to the therapeutic po-
`tential of CD26/DPP IV-specific inhibitors67. The chain length of
`these natural substrates suggests that peptides of up to 40 amino
`acids can be processed not only in vitro but also in vivo.
`
`Chemokines as targets for CD26 activity
`Based on the above evidence, it seemed reasonable that chemokines,
`a family of relatively small cytokines that induce leukocyte mi-
`gration (chemotaxis), might be substrates for this highly selective
`ectopeptidase. The importance of the N-terminal region of
`chemokines for signaling through binding to their receptors, as well
`
`A U G U S T
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`V o l . 2 0
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`1 9 9 9
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`as the natural occurrence of N-terminally truncated chemokines,
`prompted a study of chemokine processing by CD26. Along with
`others, we have reported that several chemokines can be processed
`by CD26/DPP IV (Refs 69–74). For some chemokines, such as granu-
`locyte chemotactic protein 2 (GCP-2), no difference in biological ac-
`tivity or signaling (Ca2+ mobilization) is observed between the intact
`and the CD26-truncated molecule70. By contrast, CD26-processed
`RANTES(3–68) has a more than ten times lower chemotactic potency
`for monocytes and eosinophils. RANTES(3–68) also has impaired
`binding and signaling properties through CCR1 and CCR3 but re-
`mains fully active on CCR5. Thus, truncation of RANTES by CD26
`introduces higher receptor selectivity69,70. Another CC chemokine,
`eotaxin, attracts eosinophils to sites of parasitic infection and allergic
`inflammation. Its chemotactic potency for blood eosinophils and sig-
`naling capacity through CCR3 are reduced 30 times upon truncation
`by CD26 (Ref. 71). Stromal cell-derived factor 1a (SDF-1a), a consti-
`tutively expressed CXC chemokine involved in the maturation of B
`cells, also loses the Lys-Pro dipeptide from its N-terminus upon
`incubation with CD26/DPP IV (Refs 72, 73). SDF-1a(3–68) lacks
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`chemotactic and CXCR4 (CXC receptor 4) signaling properties but
`still desensitizes SDF-1a(1–68)-induced Ca2+ responses through CXCR4.
`Under the conditions used for removing the N-terminal dipep-
`tide from the above-mentioned chemokines, a double truncation of
`macrophage-derived chemokine (MDC) is observed. MDC loses not
`only Gly1-Pro2 but also Tyr3-Gly4, suggesting that the substrate
`specificity might be less restricted than is generally accepted. Com-
`pared with the intact chemokine, MDC(5–69) has a reduced chemo-
`tactic activity for lymphocytes and monocyte-derived dendritic
`cells, but it remains as chemotactic as intact MDC(1–69) for mono-
`cytes74.
`Most probably, the accessibility of the CD26/DPP IV active center
`for a number of chemokines is facilitated by the great flexibility of
`the N-terminus. The data above show that truncation by CD26/DPP
`IV often has drastic effects on the biological activity of individual
`chemokines and confirm the importance of their N-terminal residues
`for receptor activation. Further studies are needed to define the pre-
`ferred targets in vivo under various physiological and pathological
`circumstances.
`
`HIV and CD26: dual role?
`Since the identification of HIV-1, the relation between CD26 expres-
`sion and AIDS has been studied with varying intensity. It is now
`well recognized that HIV-infected individuals have significantly
`lower percentages of CD26bright cells than non-infected controls28,37.
`Because CD26bright T cells play a crucial role in the immune response
`upon recall antigen challenge, the early and selective loss of CD26+
`cells has been related to the progressive impairment of the immune
`function in these patients28,75. This view is supported by the finding
`that the defective recall antigen response in HIV-1-infected individ-
`uals can be restored in vitro by the addition of soluble CD26 (Ref. 55).
`The identification of the chemokine receptors CXCR4 and CCR5 as
`coreceptors for HIV (reviewed in Ref. 76) boosted earlier interest77 in
`the involvement of CD26 in HIV entry. The fact that some of the
`chemokines that can be truncated N-terminally by CD26 are soluble
`HIV-suppressive factors (such as RANTES and SDF-1a) provides the
`opportunity to investigate the role of CD26 in HIV infection.
`When intact and CD26-truncated RANTES molecules were com-
`pared for their ability to inhibit HIV-1 infection of PBMCs with
`macrophage-tropic (M-tropic) strains, truncated RANTES(3–68) was
`found to be a much more potent HIV-1 inhibitor than intact RANTES
`(Ref. 78) (Fig. 3). This difference in antiviral potency between intact
`and CD26-truncated RANTES is even more pronounced in CCR5-
`transfected cell lines. Therefore, CD26 can contribute to the protec-
`tion against infection with M-tropic HIV-1 in vivo by producing a far
`more potent anti-HIV-1 derivative of RANTES. By contrast, N-ter-
`minal processing of SDF-1a by soluble CD26/DPP IV (Ref. 73) or
`cell-surface CD26/DPP IV (Ref. 72) significantly diminished the
`anti-HIV-1 potency of the chemokine. CD26/DPP IV is able to
`process RANTES and SDF-1a, with different effects on their anti-
`HIV activity. High levels of CD26/DPP IV may be beneficial during
`early stages of HIV infection where M-tropic CCR5-using HIV-1
`strains predominate. Later on, when T-tropic (T-cell-tropic) viruses
`
`are found, CD26/DPP IV can facilitate HIV dissemination by dimin-
`ishing the protective effects of SDF-1a. An increased rate of HIV
`entry in CD4+/CXCR4+ T cells expressing relatively high levels of
`CD26 has been observed79.
`The HIV-1 transactivating protein TAT plays a role in viral repli-
`cation, and also exerts immunosuppressive properties in vitro, which
`some authors have attributed to its interaction with CD26 (Ref. 80),
`although others have not been able to confirm this22,81. Inhibition by
`TAT and other cationic peptides depends on hypersialylation of
`CD26/DPP IV (Ref. 26). The binding of such cationic peptides might
`result in inhibition of DPP IV activity by partly blocking the access of
`substrate to the active site. These recent observations on the in-
`volvement of CD26 in HIV-1 binding/entry, together with the well-
`recognized induction of conformational changes and de novo lateral
`interactions at the cell surface upon binding of an HIV particle82, em-
`phasize the complexity of HIV-1 entry into the host cell.
`
`Conclusion
`CD26/DPP IV is a large, multifunctional glycoprotein, whose role in
`immunology has mainly been studied in T-cell biology. Recently,
`progress has been made in defining different epitopes, but no three-
`dimensional structure is yet available. CD26/DPP IV-mediated trun-
`cation of natural substrates can alter their function, stressing the
`necessity of measuring bioactive forms instead of immunoreactive
`‘pools’ of modified peptides. The nature of its substrates, together
`with its regulated expression and non-enzymatic interactions char-
`acterize active participation of CD26 in the immune, nerve and
`endocrin