Inhibition Of CD26 Research Articles

CD26 Deficiency Controls Macrophage Polarization Markers and Signal Transducers during Colitis Development and Resolution.

Ulcerative colitis (UC) is a multifactorial condition characterized by a destructive immune response that failed to be attenuated by common regulatory mechanisms which reduce inflammation and promote mucosa healing. The inhibition of CD26, a multifunctional glycoprotein that controls the immune response via its dipeptidyl peptidase (DP) 4 enzyme activity, was proven to have beneficial effects in various autoimmune inflammatory diseases. The polarization of macrophages into either pro-inflammatory M1 or anti-inflammatory M2 subclass is a key intersection that mediates the immune-inflammatory process in UC. Hence, we hypothesized that the deficiency of CD26 affects that process in the dextran sulfate sodium (DSS)-induced model of UC. We found that mRNA expression of M2 markers arginase 1 and Fizz were increased, while the expression of M1 marker inducible NO synthase was downregulated in CD26−/− mice. Decreased STAT1 mRNA, as well as upregulated pSTAT6 and pSTAT3, additionally support the demonstrated activation of M2 macrophages under CD26 deficiency. Finally, we investigated DP8 and DP9, proteins with DP4-like activity, and found that CD26 deficiency is not a key factor for the noted upregulation of their expression in UC. In conclusion, we demonstrate that CD26 deficiency regulates macrophage polarization toward the anti-inflammatory M2 phenotype, which is driven by STAT6/STAT3 signaling pathways. This process is additionally enhanced by the reduction of M1 differentiation via the suppression of proinflammatory STAT1. Therefore, further studies should investigate the clinical potential of CD26 inhibitors in the treatment of UC.

CD26 Inhibition Potentiates the Therapeutic Effects of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells by Delaying Cellular Senescence.

Mesenchymal stem cells (MSCs) are recognized as potential treatments for multiple degenerative and inflammatory disorders as a number of animal and human studies have indicated their therapeutic effects. There are also several clinically approved medicinal products that are manufactured using these cells. For such large-scale manufacturing requirements, the in vitro expansion of harvested MSCs is essential. Multiple subculturing of MSCs, however, provokes cellular senescence processes which is known to deteriorate the therapeutic efficacy of the cells. Strategies to rejuvenate or selectively remove senescent MSCs are therefore highly desirable for fostering future clinical applications of these cells. In this present study, we investigated gene expression changes related to cellular senescence of MSCs derived from umbilical cord blood and found that CD26, also known as DPP4, is significantly upregulated upon cellular aging. We further observed that the inhibition of CD26 by genetic or pharmacologic means delayed the cellular aging of MSCs with their multiple passaging in culture. Moreover, the sorting and exclusion of CD26-positive MSCs from heterogenous cell population enhanced in vitro cell attachment and reduced senescence-associated cytokine secretion. CD26-negative MSCs also showed superior therapeutic efficacy in mouse lung emphysema model. Our present results collectively suggest CD26 is a potential novel target for the rejuvenation of senescent MSCs for their use in manufacturing MSC-based applications.

Combination of a CD26 Inhibitor, G-CSF, and Short-term Immunosuppressants Modulates Allotransplant Survival and Immunoregulation in a Rodent Hindlimb Allotransplant Model.

Recent studies have demonstrated that inhibition of CD26 potentiates stromal cell-derived factor-1α (SDF-1α), promotes tissue regeneration, and suppresses the rejection of organ transplants. This study investigated whether the combination of a CD26 inhibitor (CD26i) with granulocyte colony-stimulating factor (G-CSF) and short-term immunosuppressants modulates vascularized composite tissue allotransplant survival in a rodent orthotopic hindlimb allotransplant model. The hindlimb allotransplantation from Brown-Norway to Lewis rats was divided into 4 groups. Group 1 (controls) did not receive any treatment. Group 2 was treated with short-term antilymphocyte serum (ALS) and cyclosporine-A (CsA). Group 3 was administrated CD26i and G-CSF. Group 4 received a combination of CD26i/G-CSF/ALS/CsA. Each subgroup comprised 10 rats. Peripheral blood and sampling of transplanted tissues were collected for immunological and histological analysis. The results revealed that allotransplant survival was found to be significantly prolonged in group 4 with CD26i/G-CSF/ALS/CsA treatment compared with those in the other groups. The interleukin-10 and transforming growth factor-βl levels, the percentage of CD4+/CD25+/FoxP3+ T cells, as well as the levels of SDF-1α expressions were significantly increased in group 4 compared with those in the other groups. Group 4 revealed a statistical increase in the percentage of donor cells (RT1n) expression in the recipient peripheral blood, and the mixed lymphocyte reaction showed hyporesponsiveness of the T cells to donor alloantigens. The combination of CD26i/G-CSF and short-term immunosuppressants prolongs allotransplant survival by inducing immunoregulatory effects and enhancing the percentage of SDF-1α expression. This immunomodulatory approach has great potential as a strategy to increase vascularized composite allotransplantation survival.

Functional Dissection of CD26 and Its Pharmacological Inhibition by Sitagliptin During Skin Wound Healing.

BackgroundSkin fibroblasts are primary mediators underlying wound healing and therapeutic targets in scar prevention and treatment. CD26 is a molecular marker to distinguish fibroblast subpopulations and plays an important role in modulating the biological behaviors of dermal fibroblasts and influencing skin wound repair. Therapeutic targeting of specific fibroblast subsets is expected to reduce skin scar formation more efficiently.Material/MethodsSkin burn and excisional wound healing models were surgically established in mice. The expression patterns of CD26 during wound healing were determined by immunohistochemical staining, real-time RT-PCR, and western blot assays. Normal fibroblasts from intact skin (NFs) and fibroblasts in wounds (WFs) were isolated and sorted by fluorescence-activated cell sorting (FACS) into 4 subgroups – CD26+ NFs, CD26− NFs, CD26+ WFs, and CD26− WFs – for comparisons of their capacities of proliferation, migration, and collagen synthesis. Pharmacological inhibition of CD26 by sitagliptin in skin fibroblasts and during wound healing were further assessed both in vitro and in vivo.ResultsIncreased CD26 expression was observed during skin wound healing in both models. The CD26+ fibroblasts isolated from wounds had significantly stronger abilities to proliferate, migrate, and synthesize collagen than other fibroblast subsets. Sitagliptin treatment potently diminished CD26 expression, impaired the proliferation, migration, and collagen synthesis of fibroblasts in vitro, and diminished scar formation in vivo.ConclusionsOur data reveal that CD26 is functionally involved in skin wound healing by regulating cell proliferation, migration, and collagen synthesis in fibroblasts. Pharmacological inhibition of CD26 by sitagliptin might be a viable strategy to reduce skin scar formation.

Abstract 103: Cd26 Knockout And Inhibition Promotes Dorsal Wound Healing Via Modulation Of Engrailed-1 Positive Fibroblasts

Purpose: Engrailed-1 (En1) positive fibroblasts are responsible for scar formation in the dorsal skin of adult mice during the postnatal period. The cell surface marker CD26, also known as dipeptidyl-peptidase-4 (DPP4), is expressed by the vast majority of En1 positive fibroblasts and can be used to isolate this lineage of scar-forming fibroblasts. We have previously shown that inhibition of CD26 with Diprotin results in decreased cutaneous scarring during wound healing. To further interrogate this biology, we hypothesized that inhibition of CD26 with a small molecule CD26/DPP4 inhibitor (MK0626) or CD26 knockout mice, might improve the rate of wound healing, decrease scar fibrosis, and achieve a more regenerative phenotype in the context of wound healing. Methods: The effects of MK0626 were initially evaluated on NIH 3T3 fibroblasts in vitro. Migration, measured by scratch assay, and proliferation were assessed on treatment and control specimens at 12 and 24 hours. Results were analyzed using ImageJ. To test the effects of MK0262 in vivo, bilateral full thickness wounds were created in the dorsal dermis of 10-week old C57Bl/6 (wild-type) mice. Equivalent wounds were also made in the dorsal dermis of En1mTmG CD26 knockout mice (CD26-/+;En1Cre;R26mTmG). A stented-wound healing model developed in our laboratory, which better mimics human wound healing kinetics, was used. The wild-type mice received oral MK0626 at a high dose (30 mg/kg), low dose (3 mg/kg), or saline control every other day over the course of wound healing. Wounds were harvested at post-operative day 14. Results:In vitro scratch assay showed significantly increased fibroblast migration and increased proliferation with treatment of MK0626 compared with vehicle control. In vivo, orally-administered MK0626 significantly improved the rate of wound closure in wild-type mice. Dermal scar thickness (as well as En-1 expression) was decreased in En1mTmG CD26 knockout mice when compared with En1mTmG control mice. Conclusions: Modulation of CD26 expression shows therapeutic potential to encourage regenerative wound healing. Specifically, our in vivo experiments show that with oral CD26 inhibition or CD26 knockout, the wound closure rate is increased and the dermal scar is thinner, the latter of which correlates with decreased presence of the En1-lineage-positive fibroblasts. Accelerated wound closure with decreased scarring is immensely beneficial from a clinical perspective.

Treatment of Patients with Steroid Refractory Acute Graft Vs Host Disease (SR-GvHD): A Matched Paired Analysis of Anti-CD26 (Begelomab) Compared to Other Treatment

Background. CD26 is a multifunctional glycoprotein expressed both as a soluble form and on the cell surface of various tissues, including activated T lymphocytes and intestinal epithelial cells. CD26 has enzymatic activity (DPP IV) and cleaves N-terminal dipeptides of hormones, cytokines and chemokines . Inhibition of CD26 impairs T cells migration across the endothelial barrier (Trends in Immunology 2008; 29:295). We have used a monoclonal antibody directed against CD26 , to treat SR-GvHD, and showed response rates and long term survival in 50% of patients (Acta Haematologica 1985; 73:185). This anti-CD26 antibody has been recently made available for clinical use (Begelomab) and we have tested the antibody in patients with SR-GvHD .Begelomab. Begelomab is a murine IgG2B monoclonal antibody against CD26 , produced by a P3X63 hybridoma (ADIENNE SA, Lugano, Switzerland). Twenty eight patients with SR-GvHD, over 18 years of age, were enrolled in two prospective trials testing safety and efficacy of Begelomab at different doses : the first 12 patients, in a pilot study, received 2 mg/day for 5 days. The second group of 16 patients entered a dose finding study with 2 mg/m^2/day x5, 3 mg/dayx5, 4.5 mg/m^2day5 , or 3 mg/m^2/dayx5 followed by 6 additional biweekly doses.There were no adverse events attributable to Begelomab, and several consecutive infusions, including the additional 6 doses in 3 weeks, were gien without any allergic reaction,Controls. 82 patients were randomly matched to the 28 Begelomab patients (maximum possible matches, Optimal Match, NCSS 10), from a group of 91 SR-aGvHD patients treated in Genova (n=36), Seattle, USA (n=22) and Salamanca ,Spain (n=33). Matching was performed on GvHD grading, age, disease phase and donor type. The 82 matched Controls were composed of 36 patients from Genova , 17 from Seattle and 27 from Salamanca. Controls received best available treatment, which included anti-thymocyte globulin, extracorporeal photopheresis , mycophenolate , sirolimus , etanercept , high dose steroids high dose cyclophosphamide , alfa1 anti-tripsin.Clinical characteristics of the patients. The median age of the 2 groups was 44 vs 42 years in Controls vs Begelomab (p=0.2); proportion of donors other than HLA identicall sibling 65% vs 79% (p=0.2), patients with active disease at transplant 51% vs 46% (p=0.6); day of acute GvHD from transplant 17 vs 22 days (p=0.04). Overall grading of acute GvHD , at the time the patient was considered steroid refractory, was comparable in the 2 groups : grade III-IV was 77% vs 74% in Controls and Begelomab respectively (p=0.7); stage 3-4 skin was 50% vs 57% (p=0.5), stage 3-4 liver was 5% vs 21% in Begelomab (p=0.008) and stage 3-4 gut was seen in 30% and 28% respectively (p=0.8).Response to Begelomab. Response data were available for 55 Controls and 28 Begelomab patients: day 28 responders were 41% in controls vs 75% for Begelomab (p=0.004). In patients with grade 2-4 gut GvHD it was 34% vs 82% (p=0.001).Outcome. Survival data was available for all 110 patients. The cumulative incidence of non relapse mortality (NRM) at 180 days, was 50% in Controls and 25% in Begelomab (p=0.008) (Fig.1), and the cumulative incidence of relapse related mortality (RRD) at 3 years was 15% vs 25% respectively (p=0.1) The overall survival at 1 year was 31% vs 50% in Controls and Begelomab patients (p=0.06).Conclusions. Begelomab induces a high remission rate on day+28, in patients with steroid refractory acute GvHD, including a proportion of severe gut and liver GvHD. Non relapse mortality at 6 months was significantly lower in Begelomab patients, as compared to a group of 82 matched controls: this was seen despite a significantly greater proportion of patients with liver stage 3-4 GvHD in the Begelomab group. There was a borderline survival advantage at 1 year for Begelomab patients. To confirm these results, a multicenter prospective randomized trial is ongoing, in patients with SR-aGvHD , comparing Begelomab versus best standard therapy DisclosuresNo relevant conflicts of interest to declare.

Dipeptidylpeptidase IV (CD26) defines leukemic stem cells (LSC) in chronic myeloid leukemia

AbstractChronic myeloid leukemia (CML) is a stem cell (SC) neoplasm characterized by the BCR/ABL1 oncogene. Although mechanisms of BCR/ABL1-induced transformation are well-defined, little is known about effector-molecules contributing to malignant expansion and the extramedullary spread of leukemic SC (LSC) in CML. We have identified the cytokine-targeting surface enzyme dipeptidylpeptidase-IV (DPPIV/CD26) as a novel, specific and pathogenetically relevant biomarker of CD34+/CD38─ CML LSC. In functional assays, CD26 was identified as target enzyme disrupting the SDF-1-CXCR4-axis by cleaving SDF-1, a chemotaxin recruiting CXCR4+ SC. CD26 was not detected on normal SC or LSC in other hematopoietic malignancies. Correspondingly, CD26+ LSC decreased to low or undetectable levels during successful treatment with imatinib. CD26+ CML LSC engrafted NOD-SCID-IL-2Rγ−/− (NSG) mice with BCR/ABL1+ cells, whereas CD26─ SC from the same patients produced multilineage BCR/ABL1– engraftment. Finally, targeting of CD26 by gliptins suppressed the expansion of BCR/ABL1+ cells. Together, CD26 is a new biomarker and target of CML LSC. CD26 expression may explain the abnormal extramedullary spread of CML LSC, and inhibition of CD26 may revert abnormal LSC function and support curative treatment approaches in this malignancy.

Glypican-3–mediated inhibition of CD26 by TFPI: a novel mechanism in hematopoietic stem cell homing and maintenance

AbstractDirectional migration determines hematopoietic stem/progenitor cell (HSPC) homing, which depends upon the interaction between the chemokine CXCL12 and its receptor CXCR4. CD26 is a widely expressed membrane-bound ectopeptidase that cleaves CXCL12 thereby depleting its chemokine activity. We identified tissue-factor pathway inhibitor (TFPI) as a biological inhibitor of CD26 in murine and human HSPCs. We observed low-level TFPI expression in endothelial cells in the bone marrow (BM), which did not increase following radiation injury. Treatment of HSPCs with TFPI in vitro led to enhanced HSPC migration toward CXCL12, as well as homing and engraftment in the BM upon transplantation. We found that Glypican-3 (GPC3), a heparan sulfate proteoglycan expressed on murine as well as human HSPCs, mediated this effect. TFPI did not affect CD26 activity, migration, or homing of GPC3−/− HSPCs, while it affected GPC1−/− HSPCs similar to wild-type HSPCs. Moreover, proliferation of GPC3−/− but not GPC1−/− BM HSPCs was significantly increased, which was associated with a decrease in the primitive HSC pool in BM and an increase in proportion of the circulating HSPCs in the peripheral blood. Hence, we present a novel role for TFPI and GPC3 in regulating HSC homing as well as retention in the BM.

Engraftment of retrovirally transduced Bet v 1-GFP expressing bone marrow cells leads to allergen-specific tolerance

Molecular chimerism is a promising strategy to induce tolerance to disease-causing antigens expressed on genetically modified haematopoietic stem cells. The approach was employed successfully in models of autoimmunity and organ transplantation. Recently, we demonstrated that molecular chimerism induces robust and lasting tolerance towards the major grass pollen allergen Phl p 5.Since allergens are a group of antigens differing widely in their function, origin and structure we further examined the effectiveness of molecular chimerism using the Phl p 5-unrelated major birch pollen allergen Bet v 1, co-expressed with the reporter GFP. Besides, inhibition of CD26 was used to promote engraftment of modified stem cells.Retrovirus VSV-Betv1-GFP was generated to transduce 5-FU-mobilized BALB/c hematopoietic cells to express membrane-bound Bet v 1 (VSV-GFP virus was used as control). Myeloablated BALB/c mice received Betv1-GFP or GFP expressing bone marrow cells, pre-treated with a CD26 inhibitor. Chimerism was followed by flow cytometry. Tolerance was assessed by measuring allergen-specific isotype levels in sera, RBL assays and T-cell proliferation assays.Mice transplanted with transduced BMC developed multi-lineage molecular chimerism which remained stable long-term (>8 months). After repeated immunizations with Bet v 1 and Phl p 5 serum levels of Bet v 1-specific antibodies (IgE, IgG1, IgG2a, IgG3 and IgA) remained undetectable in Betv1-GFP chimeras while high levels of Phl p 5-specific antibodies developed. Likewise, basophil degranulation was induced in response to Phl p 5 but not to Bet v 1 and specific non-responsiveness to Bet v 1 was observed in proliferation assays.These data demonstrate successful tolerization towards Bet v 1 by molecular chimerism. Stable long-term chimerism was achieved under inhibition of CD26. These results provide evidence for the broad applicability of molecular chimerism as tolerance strategy in allergy.

Upregulation of CD26 expression in epithelial cells and stromal cells during wound-induced skin tumour formation

We have previously described InvEE transgenic mice in which non-dividing, differentiating epidermal cells express oncogenically activated MAPK kinase 1 (MEK1). Skin wounding triggers tumour formation in InvEE mice via a mechanism that involves epidermal release of IL-1α and attraction of a pro-tumorigenic inflammatory infiltrate. To look for potential effects on the underlying connective tissue, we screened InvEE and wild-type epidermis for differential expression of cytokines and immune modulators. We identified a single protein, CD26 (dipeptidyl peptidase-4). CD26 serum levels were not increased in InvEE mice. In contrast, CD26 was upregulated in keratinocytes expressing mutant MEK1 and in the epithelial compartment of InvEE tumours, where it accumulated at cell-cell borders. CD26 expression was increased in dermal fibroblasts following skin wounding but was downregulated in tumour stroma. CD26 activity was stimulated by calcium-induced intercellular adhesion in keratinocytes, suggesting that the upregulation of CD26 in InvEE epidermis is due to expansion of the differentiated cell layers. IL-1α treatment of dermal fibroblasts stimulated CD26 activity, and therefore epidermal IL-1α release may contribute to the upregulation of CD26 expression in wounded dermis. Pharmacological blockade of CD26, via Sitagliptin, reduced growth of InvEE tumours, while combined inhibition of IL-1α and CD26 delayed tumour onset and reduced tumour incidence. Our results demonstrate that inappropriate activation of MEK1 in the epidermis leads to changes in dermal fibroblasts that, like the skin inflammatory infiltrate, contribute to tumour formation.

Effective mobilization of hematopoietic progenitor cells in G-CSF mobilization defective CD26 −/− mice through AMD3100-induced disruption of the CXCL12-CXCR4 axis

We previously reported that inhibition or loss of CD26 (DPPIV/dipeptidylpeptidase IV) results in a defect in normal mobilization of hematopoietic stem and progenitor cells induced by granulocyte-colony stimulating factor (G-CSF). This suggests that CD26 is a necessary component of the mobilization pathway. Our goal in this study was to determine whether mobilization can be induced by the CXCR4 antagonist AMD3100 in mice lacking CD26 (CD26(-/-)). Ten week old CD26(-/-) and C57BL/6 mice received a subcutaneous injection of AMD3100. One hour post-injection the mice were euthanized and peripheral blood and bone marrow were collected and evaluated. AMD3100 mobilizes hematopoietic progenitors into the peripheral blood of CD26(-/-) and mice. Our finding that AMD3100 rapidly mobilizes hematopoietic progenitor cells from the bone marrow into the periphery in CD26-deficient transgenic mice that otherwise exhibit a mobilization defect in response to G-CSF suggests that: (1) CD26 is downstream of G-CSF but upstream of the CXCL12-CXCR4 axis and (2) AMD3100 can be used as a single agent to mobilize hematopoietic stem and progenitor cells in normal donors or patients that have an intrinsic defect in their response to G-CSF treatment. Stem cell transplants are often the only curative treatment in some cancer patients. The ability to perform the transplantation and its success is dependent on the ability to mobilize adequate numbers of hematopoietic progenitor cells. The use of AMD3100 as a single agent would give patients or donors an additional option for a successful stem cell transplant.

CD26 Inhibition Preferentially Enhances In Vitro Migration of G-CSF + Plerixafor (AMD3100) Mobilized PB as Compared to G-CSF Mobilized PB In Multiple Myeloma Autografts.

AbstractAbstract 3714Hematopoietic stem and progenitor cell (HSC/HPC) trafficking into the bone marrow during hematopoietic stem cell transplantation (HSCT) is thought to be an important biological step that can be targeted to improve transplant efficiency and subsequent immune reconstitution. It has been reported that loss/inhibition of the peptidase CD26, also known as DPPIV (dipeptidylpeptidase IV), increases the transplant efficiency of mouse HSC/HPC (Christopherson, KW 2nd, et al. Science 2004). In a similar manner, inhibition of CD26 on CD34+ or lin− cord blood (CB) cells improves in vitro cell migration and in vivo engraftment into immunodeficient mice (Christopherson, KW 2nd, et al. Stem Cells and Dev 2007). There is currently controversy as to whether inhibition of CD26 on mobilized peripheral blood (mobPB) has benefit since it has been reported that inhibition of CD26 does not improve migration of G-CSF mobPB CD34+ cells in response to SDF-1 (Kawai K, et al. Stem Cells and Dev 2007. 16:361–70) but does improve the migration of G-CSF mobPB mononuclear cells (MNC) (Frank R, et al. ASH Abstract #372, 2009). Additionally, it has been reported that AMD3100 mobPB c-kit+ mouse cells have high CD26 expression similar to BM in contrast to G-CSF mobPB c-kit+ mouse cells which have very little CD26 expression (Bonig H, Exp Hem, 2009). The combination G-CSF + AMD3100 (a CXCR4 antagonist also known as plerixafor or Mozobil ®) has been recently introduced into the clinic for use in obtaining mobPB from Multiple Myeloma patients. We therefore investigated whether CD26 on human mobPB MNC obtained from Multiple Myeloma autografts mobilized with either G-CSF or G-CSF + plerixafor would have an effect on SDF-1 induced cell migration. To do this, non-adherent MNC from mobPB were obtained from discarded bags following the completion of stem cell infusion of previously frozen autografts. CD26 expression was detected by flow cytometery. CD26 activity was determined using the substrate Gly-Pro P-nitroanilide (Gly-Pro-pNA). To assess migration, chemotaxis assays were performed by loading 5×104 cells into the upper chamber of a 96 well transwell plate, adding human SDF-1α (0–400 ng/mL) in the lower chamber, and incubating for 2 hours at 37°C, 5% CO2. By flow cytometry, CD26 expression was 21.61±2.19% and 21.65±3.68% of the CD45+ cell population for G-CSF and G-CSF + plerixafor mobPB MNCs respectively. CD26 activity was 15.4±1.2 pmol pNA/minute and 16.3±0.4 pmol pNA/minute for G-CSF and G-CSF + AMD3100 MNCs. During functional in vitro assays, cells were cultured with or without a 5mM concentration of the CD26 inhibitor Diprotin A (DpA). DpA treatment of G-CSF + plerixafor mobPB resulted in a significant increase in cell migration as compared to untreated cells. Specifically, the percent migration at 400ng/mL SDF-1α was 21.3±2.4% and 31.6±3.0% for untreated and DpA treated G-CSF + plerixafor mobPB cells respectively (p=<0.01, n=10). The percent migration in response to 400ng/mL SDF-1α was 19.8±2.0% and 25.3±2.6% for untreated and DpA treated G-CSF mobPB cells respectively (p=0.21, n=7). These data indicate that CD26 inhibition, by treatment with DpA, results in a preferential increase in SDF-1 induced migration of G-CSF + plerixafor mobPB cells as compared to G-CSF mobPB cells. The preferential response does not appear to correlate to any change in CD26 expression or activity since both types of mobPB have equivalent expression and activity prior to the migration assay. Our data suggest that un-fractionated mobPB collected following mobilization using the combination of G-CSF + plerixafor (AMD3100) may have inherently different cell trafficking regulatory properties when compared to mobPB collected following G-CSF alone and that additional regulatory mechanisms allow the G-CSF + plerixafor mobPB to have an enhanced response to SDF-1 in the absence of CD26 suppression. Additionally, our data suggest that inhibition of CD26 peptidase activity may be more clinically relevant in the G-CSF + plerixafor mobilized patient population with regard to achieving improved stem cell trafficking. Disclosures:Fung:Genzyme: Honoraria, Speakers Bureau.

Glycosylation Engineering Combined with CD26 Inhibitor Enhances Umbilical Blood Cells Engraftment In the NOD/SCID Mouse

AbstractAbstract 4687Given the tremendous need for and potential of umbilical cord blood (UCB) to be utilized as a donor source for hematopoietic stem cell (HSC) transplantation in adults, there is a strong push to overcome the constraints created by the limited volumes and subsequent limited HSC and hematopoietic progenitor cell (HPC) numbers available for HSC transplantation from a single collection. Hematopoietic stem cell homing is believed to be critical for the development of methodologies to improve transplant efficiency and subsequently immune reconstitution during hematopoietic stem cell transplantation in clinical setting. We have previously described the functional defect of partial homing receptor in cord blood stem cell (the higher activity of CD26 and the lower expression of P/E-selectin ligand) in vitro. Respectively, we had confirmed that using of glycosylation engineering and CD26 inhibitor can fix the defect of stem cell homing. However, there is no evidence can prove whether the combination would improve the ability of homing. In present study, to explore the use of glycosylation combined with CD26 inhibitor as a method of improving the transplant efficiency of CB CD34+ hematopoietic stem cells (expressing CD45), we utilized the xenotransplanted nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model in vivo. 6–8 weeks age of NOD/SCID mice were divided into 5 groups (8 mice per group): blank control group (without stem cells infusion); control group (CD34+ cells infusion alone); glycosylation engineering group; CD26 inhibitor group and combination group (glycosylation engineering combined CD26 inhibitor group). Purified CD34+ stem cells (5×105 per mice) derived from fresh UCB by immunomagnetic beads were injected into lethally irradiated NOD/SCID mice intravenously. To investigate the effect of early engraftment in vivo, we monitored the expression of CD45 in peripheral blood and the overall viability rate weekly until the fourth week by flow cytometry. One week after transplantation, there is no expression of CD45 in peripheral blood in any group, but we observed 2.52±1.41% and 4.8% expression in bone marrow of recipient mice respectively in control and glycosylation engineering groups. All mice in the blank control group were dead and overall viability rate were almost the same for the other four groups (80% in control group,80% in glycosylation engineering group, 100% in CD26 inhibitor group and 100% in combination group). The second week, there is only expression of CD45 in recipient mice for combination group 0.63±0.37%. The overall survival rate among control were significantly lower than the other four groups (40% in control group,80% in glycosylation engineering group, 100% in CD26 inhibitor group and 100% in combination group, P<0.05). In the third week, all groups except the blank control group expressed CD45 in peripheral blood, we observed 0.81±0.12%, 2.81±0.14%, 3.12±0.53% and 7.05±0.87% in peripheral blood of recipient mice for control, glycosylation engineering, CD26 inhibitor treated and combination group. The overall survival rate among CD26 inhibitor treated and combination groups were significantly higher than the other three groups. (40% in control group,40% in glycosylation engineering group, 80% in CD26 inhibitor group and 80% in combination group, P<0.05). In the fourth week, we observed 15.87±0.21%, 30.51±0.62%, 34.33±0.82% and 40.22±1.62% in peripheral blood of recipient mice for control, glycosylation engineering, CD26 inhibitor treated and combination group. The overall survival rate among CD26 inhibitor treated and combination groups were significantly higher than the other three groups. (40% in control group,40% in glycosylation engineering group, 80% in CD26 inhibitor group and 80% in combination group, P<0.05). These results provide the evidence that the combination of two methods have better effect on early implantation than use them respectively. Our results also supported the potential use of CD26 inhibitor combined with glycosylation engineering to treat the umbilical blood which the number of stem cell is less than bone marrow and peripheral blood during hematopoietic stem cell transplantation as a method to improve the transplant efficiency. Furthermore, we aimed to investigate the long-term implantation effect of combination of glycosylation engineering and inhibition of CD26 in vivo. Disclosures:Sun:International Cooperation Research Fund of Anhui Provincial Scientific and Technologic Committee (08080703026): Research Funding; Fund of the Key Medical Project of Anhui Provincial healthy department (2010A005): Research Funding; Anhui Provincial “115” Industrial Innovation Program: Research Funding.

Dreams for Type 1 Diabetes: Shutting Off Autoimmunity and Stimulating β-Cell Regeneration

Although not the first study to show that inhibitors of dipeptidyl peptidase IV (DPP-IV) can reverse newly diagnosed diabetes in nonobese diabetic (NOD) mice (1), a study by Tian et al. (2) in this issue of Endocrinology confirms this finding with a different DPP-IV inhibitor (NVP-DPP728) and provides explanations for the reversal by finding reduced islet inflammation, increases in CD4 CD25 FoxP3 regulatory T cells, and -cell regeneration. Impressively, 6 wk of treatment with this agent reversed diabetes in 73% of the mice. DPP-IV inhibitors are usedclinically to treatdiabetesbecause they slow the breakdown of the incretin hormone glucagon-like peptide 1 (GLP-1), which can stimulate insulin secretion, inhibit -cell apoptosis, and promote -cell replication (3). However, these inhibitors have long been known to have effects on the immune system, which presumably contributed to the marked reduction in islet inflammation. In particular, lymphocytes have a membrane-associated protein with DPP-IV activity, CD26, which has been shown to have important effects upon T-cell development, migration, and cytokine production (4). It has been previously shown that inhibition of DPP-IV can slow the progression of experimental allergic encephalitis and increase the production of TGF, which is known to suppress autoimmunity (5). In the present study, the reversal of diabetes was accompanied by a reduction of insulitis, an increase in CD4 CD25 FoxP3 regulatory T cells, and an increase in circulating TGF. Interestingly, the regulatory T cells were particularly plentiful in the thymus and pancreatic lymph nodes. It is possible that the therapeutic benefit of the present study is not completely due to the effects of DPP-IV inhibition on the immune system but in part a result of increased circulating levels of GLP-1. Inhibition of CD26 is an attractive mechanism, but the actual contribution from this specific mechanism remains to be defined. It is even possible that GLP-1 can somehow ameliorate immune killing of -cells independently of DPP-IV. We certainly have much more to learn about the effects of GLP-1 on the immune system (3). It is noteworthy that the combination of GLP-1 and gastrin has been shown to reverse diabetes in NOD mice, and this reversal was even seen in some mice with just GLP-1 treatment alone (6). The effect of GLP-1 on the target of the immune attack, the -cell, may be very important. GLP-1 is known to have an antiapoptotic effect upon -cells, probably by bolstering the insulin receptor substrate 2 signaling pathway (7, 8). Therefore, it is possible that a reduction of -cell death and less release of antigen could somehow influence the balance of T-cell regulation and slow the rate of immune destruction. Clearly, more work is needed to sort out the relative contributions of DPP-IV inhibition and GLP-1 agonism. Another addition to the equation is gastrin, the circulating levels of which can be increased with either injections of the peptide or the use of proton pump inhibitors. We know that gastrin given in combination with either epidermal growth factor or GLP-1 agonism can stimulate -cell regeneration (6, 9). Now we must ask how gastrin might contribute to reduced autoimmune destruction. The demonstration of -cell regeneration, accompanied by increased -cell replication, is similar to what has been found with some other but not all interventions that reverse diabetes in NOD mice. It seems likely that increases in -cell mass result from replication of residual -cells, although there is a provocative recent report sug-

Use of the CD26 Inhibitor Diprotin A (Ile-Pro-Ile) to Increase Mobilized Peripheral Blood Mononuclear Cell Adhesion and Migration.

Abstract 372▪▪This icon denotes an abstract that is clinically relevant.A firm understanding of the biology of hematopoietic stem and progenitor cell (HSC/HPC) trafficking is believed to be critical for the development of methodologies to improve transplant efficiency and subsequent immune reconstitution during hematopoietic stem cell transplantation (HSCT) in the clinical setting. We have previously reported that suppression of the peptidase CD26/DPPIV (dipeptidylpeptidase IV) on mouse HSC/HPC as a method of increasing transplant efficiency (Christopherson, KW 2nd, et al. Science 2004. 305:1000-3). We have also described that inhibition of CD26 on CD34+ or lin- cord blood (CB) cells improves engraftment into immunodeficient mice (Christopherson, KW 2nd, et al. Stem Cells and Dev 2007. 16:355-60). However, it has been reported that inhibition of CD26 on CD34+ G-CSF mobilized peripheral blood (PB) cells does not improve migration in response to SDF-1 or engraftment into NOD/SCID mice (Kawai K, et al, et al. Stem Cells and Dev 2007. 16:361-70). Given the widespread usage of un-fractionated mobilized PB as a source of cells for transplantation in both the autologous and allogeneic setting, the implications of also studying un-fractionated mobilized PB should not to be overlooked. We therefore investigated whether inhibition of CD26 activity on mobilized PB (mobPB) mononuclear cells (MNC) would have an effect on CXCL12/SDF-1 mediated adhesion and migration. These results were compared directly with data from CB MNC. Non-adherent MNCs from mobPB and CB were obtained by density centrifugation followed by adhesion to plastic for 90 minutes. By flow cytometry, CD26 expression was approximately 48% and 20% of the CD45+ cell population with Mean Fluorescent Intensity (MFI) of 1,200 and 1,000 for mobPB and CB MNCs respectively. Expression of CD26 was confirmed by Western Blot in total cell lysates from mobPB and CB MNCs. CD26 activity, as determined using the substrate Gly-Pro P-nitroanilide (Gly-Pro-pNA), was 36.7±2.8 pmol pNA/minute for mobPB MNCs and 30.9±2.8 pmol pNA/minute for CB MNCs (n=16). Prior to use in functional in vitro assays, cells were first cultured with or without a 5mM concentration of the CD26 inhibitor Diprotin A (DpA) for 15 minutes in DPBS+0.2%BSA at 37oC, 5% CO2. To perform static adhesion assay, cells were treated with SDF-1α (0-800 ng/mL) for 30 minutes, loaded onto human fibronectin coated plates, and then allowed to incubate an additional 30 minutes at 37oC, 5% CO2. DpA pre-treatment of mobPB and CB MNC was observed to result in an increase in adhesion as compared to untreated cells. Specifically, the percent adherence to fibronectin in response to 400ng/mL SDF-1α increased from 50.1±4.5% for untreated to 70.1±2.3% for DpA treated mobPB MNCs (p≤0.05, n=6). The percent adherence for CB MNCs increased from 48.2%±5.1 for untreated to 65.4±5.9% for DpA treated cells in response to 400ng/mL SDF-1α (p≤0.05, n=6). To assess migration, chemotaxis assays were performed by loading 2×105 cells into the upper chamber of a transwell plate, adding SDF-1α (0-400 ng/mL) in the lower chamber, and incubating for 2 hours at 37oC, 5% CO2. DpA pre-treatment of mobPB and CB cells was observed to result in an increased migration as compared to untreated cells. Specifically, the percent migration at 400ng/mL SDF-1α was 20.0±1.7% and 27.8±1.9% for untreated and DpA treated mobPB MNCs respectively (p≤0.01, n=7). The percent migration in response to 400ng/mL SDF-1α was 58.8±1.4% for untreated CB MNCs and 70.6 ± 2.9% for DpA treated CB MNCs (p≤0.01, n=4). These data indicate that a substantial portion of G-CSF mobPB cells express CD26 and exhibit CD26 peptidase activity similar to that seen on CB cells. In addition, CD26 inhibition on these cells results in an increase in functional response to SDF-1 as quantitated by adhesion and migration. Taken in the context of other studies examining sorted CD34+ mobPB our data suggest that un-fractionated mobPB may have inherently different regulatory properties due to the presence of accessory cells. Additionally, our data suggest that suppression of functional response of HSC/HPC to the chemokines SDF-1 by CD26 activity may be an endogenous regulatory mechanism that extends beyond CB to also include mobPB. Future studies are needed to pursue whether inhibition of CD26 activity can serve as a technique by which the trafficking of hematopoietic stem and progenitor cells from un-fractionated mobilized peripheral blood can be manipulated for possible clinical benefit. Disclosures:No relevant conflicts of interest to declare.

Chemokines in hematopoiesis

Understanding the regulation of hematopoiesis is important for enhanced efficacy of hematopoietic stem and progenitor cell transplantation. Chemokines influence migration, survival, and other actions of hematopoietic stem and progenitor cells. This article summarizes recent progress in understanding the production and actions of chemokines and chemokine receptors, with an emphasis on the SDF-1/CXCL12-CXCR4 axis. The literature from 2006 to the present is replete with information on SDF-1/CXCL12 activity, including induced intracellular signaling in hematopoietic progenitor cells, lymphocytes, other innate immune cells, breast cancer, and other tumor cells, and on production of SDF-1/CXCL12, and CXCR4, as well as on actions/production of other chemokines. Studies describing these intense research areas are discussed. Chemokine-chemokine receptor interactions are important to hematopoiesis and immune cell function, two highly interactive processes. Recent studies have clarified the role of chemokines and their receptors in regulating hematopoiesis, and agents modulating chemokines are being evaluated in preclinical and clinical studies. Examples of such efforts include inhibition of CD26 for enhanced homing and engraftment of hematopoietic stem and progenitor cells, and the use of the SDF-1/CXCL12-CXCR4 antagonist, AMD3100 for mobilization of hematopoietic stem and progenitor cells and their use for stem cell transplantation.

Inhibition/Depletion of CD26/DPPIV Enhances Survival Promoting Activity of SDF-1/CXCL12 and Myelosuppressive Activities of Inhibitory Chemokines on Hematopoietic Progenitor Cells.

Hematopoiesis is regulated by an interacting network of cells, cell surface proteins, and cytokines/chemokines. CD26 is a cell surface peptidase (Dipeptidylpeptidase IV) that cleaves the N-terminal dipeptide from various substrates, such as selected members of the chemokine family, at a penultimate proline or alanine residue. CD26 is expressed by hematopoietic stem (HSCs) and progenitor (HPCs) cells, as well as a number of other immature and mature cell types. By truncating the chemokine stromal derived factor-1 (SDF-1/CXCL12), CD26 has been implicated in modulation of chemotaxis, homing and mobilization of HSCs/HPCs. Inhibition of CD26 by small peptides such as Diprotin A, or deletion of CD26 (e.g. cells from CD26 −/− mice or siRNA induced decreases in CD26) manifests in enhanced chemotaxis of HSCs/HPCs to SDF-1/CXCL12, and enhanced homing/engraftment of HSCs in mice. We hypothesized that inhibition of CD26 would manifest in enhanced activities of other functions of SDF-1/CXCL12. Moreover, since CD26 truncates other chemokines, we reasoned that inhibition of CD26 would enhance hematopoietic activities of these other chemokines. SDF-1/CXCL12 enhances survival/anti-apoptosis of HSCs/HPCs. We now report that pretreating mouse bone marrow cells (BMCs) with Diprotin A, whether or not the cells were washed prior to plating, or use of CD26 −/− mouse BMCs in a delayed addition of growth factor setting resulted in enhanced survival of HPCs at concentrations of SDF-1/CXCL12 at least 100-fold less than that which would normally manifest survival enhancing activities. We also assessed the influence of CD26 inhibition or deletion on the myelosuppressive effects of chemokines with known inhibitory activity and those that had not previously shown this activity. Suppression/deletion of CD26 on mouse BMCs greatly enhanced the activity of myelosuppressive chemokines such as is MIP-1α/CCL3 and GCP-2/CXCL6, such that these chemokines demonstrated inhibition of colony formation by multi-cytokine stimulated CFU-GM, BFU-E, and CFU-GEMM, at concentrations of these chemokines at least 100-fold less than that possible without inhibition/deletion of CD26. However, CD26 inhibition/deletion did not change the activity of non-myelosuppressive chemokines such as MIP-1β/CCL4, RANTES/CCL5, or SDF-1/CXCL12 into suppressive molecules. Since survival of HPCs is related to the cell cycle status of HPCs, such that slow or non-cycling HPCs are less sensitive to stresses leading to apoptosis, the enhanced activity of SDF-1/CXCL12 and the myelosuppressive chemokines (which are specifically inhibitory to HPCs in S-phase of the cell cycle) may act as coordinating separate signals to protect HPCs from stress-induced apoptosis. These results demonstrate that CD26 plays a modulating role on different hematopoietic activities mediated by chemokines, and this role for CD26 should be taken into account when describing models of chemokine effects on hematopoiesis, and attempts at modifying these effects.

CD26 Inhibition on CD34+ or Lineage− Human Umbilical Cord Blood Donor HSC/HPC Improves Long-Term Engraftment into NOD/SCID/Beta2null Immunodeficient Mice.

Given the tremendous need for and potential of umbilical cord blood to be utilized as a donor source for hematopoietic stem cell transplantation in adults, there is a strong push to overcome the constraints created by the limited volumes and subsequent limited hematopoietic stem and progenitor cell numbers available for hematopoietic stem cell transplantation from a single collection. We have previously described the use of CD26 inhibitor treatment of donor cells as a method to increase the transplant efficiency of mouse hematopoietic stem and progenitor cells into a mouse recipient (Christopherson, KW 2nd, et al, Science 2004. 305:1000–1003). We therefore hypothesized that inhibition of CD26 on human donor cord blood prior to transplant would result in an increase in long-term engraftment potential. To test this hypothesis, we isolated CD34 enriched (CD34+) or lineage depleted (lin−) pooled cord blood cells, evaluated their level of CD26 expression and activity, and then tested their ability to engraft into the NOD/SCID/B2mnull immunodeficient mouse model of hematopoietic stem cell transplantation with or without prior CD26 inhibitor treatment. We observed that long-term engraftment into the recipient mouse bone marrow at twelve weeks post sub-lethal irradiation (350cGy), followed by transplantation of 1x105 pooled donor cells was 13.4±2.0% and 55.5±4.0% CD45+ human cells in the untreated, and CD26 inhibitor (Diprotin A) treated CD34+ donor cells respectively. We also observed engraftment levels in the mouse recipient bone marrow of 6.2±0.7% and 47.0±2.8% CD45+ human cells for untreated and CD26 inhibitor treated lin− cells respectively. These measurements represent approximately a 4-fold and 7.5-fold improvement in the engraftment of long-term repopulating cells resulting from CD26 inhibitor treatment of either CD34+ or lin− donor cells respectively. These pre-clinical results establish a basis on which to propose the use of CD26 inhibitor treatment of donor cord blood units prior to transplantation for the purpose of improving transplant efficiency and subsequently patient outcome.

Inhibition of CD26 in Human Cord Blood CD34+Cells Enhances Their Engraftment of Nonobese Diabetic/Severe Combined Immunodeficiency Mice

CD26, a surface serine dipeptidylpeptidase IV (DPPIV) expressed on different cell types, cleaves the amino-terminal dipeptide from some chemokines, including stromal-derived factor-1 (SDF-1/CXCL12). SDF-1/CXCL12 plays important roles in hematopoietic stem cell (HSC) homing, engraftment, and mobilization. Inhibition of CD26 peptidase activity enhances homing, engraftment, and competitive repopulation in congenic mouse bone marrow cell transplants. Our studies evaluated a role for CD26 in in vivo engraftment of HSCs from human umbilical cord blood (CB) into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Pretreating purified CD34(+) human CB cells with Diprotin A, a DPPIV inhibitor, for 15 min significantly enhanced engraftment. Treatment did not affect differentiation of CD34(+) cells in vivo, as measured phenotypically by human markers CD33, CD38, CD19, and CD34. We found that the percentage of CD26(+) cells within the more immature cells (CD34(+)CD38()) was significantly higher than in the more mature population (CD34(+)CD38(+)). These results suggest that inhibition of CD26 may be one way to enhance engraftment of limiting numbers of stem cells during CB transplantation.

The Simpson–Golabi–Behmel syndrome causative Glypican‐3, binds to and inhibits the dipeptidyl peptidase activity of CD26

Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked condition shown to be the result of deletions of the glypican-3 (GPC3) gene. GPC3 is a proteoglycan localized to the cell membrane via a glycosylphosphatidyl-inositol (GPI) anchor. To further elucidate the GPC3 function(s), we have screened various cell lines for proteins that interact with GPC3, resulting in the isolation of a 115 kDa protein, identified as CD26. The interaction occurred with both the glycosylated and unglycosylated forms of GPC3 and led to the inhibition of CD26 peptidase activity. Moreover, introduction of CD26 into Cos-1 cells was accompanied by the up-regulation of cell growth, while inclusion of recombinant GPC3 in the media reduced the growth of CD26 transfected Cos-1 cells, drastically. Furthermore, HepG2 C3A cells containing CD26 underwent apoptosis in the presence of recombinant GPC3 in both concentration and time-dependant manner. In light of the fact that inhibition of CD26 reduces the rate of cell proliferation, we propose that a number of physical findings observed in SGBS patients may be a consequence of a direct interaction of GPC3 with CD26. Furthermore, GPC3 without the GPI anchor is capable of inducing apoptosis indicating that neither the GPI anchor nor the membrane attachment is required for apoptosis induction.