Enhancement of Endothelial Progenitor Cell Numbers and Migration by H1152, a Rho Kinase Specific Inhibitor

Endothelial progenitor cells (EPCs) are applied in the treatment of ischemic diseases. In ex vivo culture of human cord-blood derived EPCs, H1152, (S)-(+)-2-methyl-1-[(4-methyl-5-iso-quinolinyl) sulfonyl]-homopiperazine, markedly increased the number of EPCs. It also induced EPC migration, stimulated the phosphorylation of AKT, and reduced the expression of p27 in the EPCs. Thus H1152 can be used effectively in ex vivo expansion of EPCs.     

Human somatic cell gene therapy

The prelude to successful human somatic gene therapy, i.e. the efficient transfer and expression of a variety of human genes into target cells, has already been accomplished in several systems. Safe methods have been devised to do this using non-viral and viral vectors. Potentially therapeutic genes have been transferred into many accessible cell types, including hematopoietic cells, hepatocytes and cancer cells, in several different approaches to ex vivo gene therapy. Successful in vivo gene therapy requires improvements in tissuetargeting and new vector design, which are already being sought. Gene-transfer protocols have been approved for human use in inherited diseases, cancer and acquired disorders. Althouth the results of these trials to date have been somewhat disappointing, human somatic cell gene therapy promises to be an effective addition to the arsenal of approaches to the therapy of many human diseases in the 21st century if not sooner.     

A comparative study of ectonucleotidase and P2 receptor mRNA profiles in C6 cell line cultures and C6 <Emphasis Type="Italic">ex vivo</Emphasis> glioma model

Glioblastoma multiforme is the most common type of primary brain tumour and has the worst clinical outcome. Nucleotides represent an important class of extracellular molecules involved in cell proliferation, differentiation and apoptosis. Alterations in purinergic signalling have been implicated in pathological processes, such as cancer, and glioma cell lines are widely employed as a model to study the biology of brain tumours. Increasing evidence, however, suggests that glioma cell lines may not present all the phenotypic and genetic characteristics of the primary tumours. We have compared the biological characteristics of C6 rat glioma cells in culture and the same cells after their implantation in the rat brain and growth in culture (denominated as the C6 ex vivo culture model). Parameters evaluated included cell morphology, differentiation, angiogenic markers, purinergic receptors and ecto-nucleotidase mRNA profile/enzymatic activity. Analysis of the C6 glioma cell line and C6 ex vivo glioma cultures revealed distinct cell morphologies, although cell differentiation and angiogenic marker expressions were similar. Both glioma models co-expressed multiple P2X and P2Y receptor subtypes with some differences. In addition, the C6 glioma cell line and C6 ex vivo glioma cultures exhibited similar extracellular ATP metabolism and cell proliferation behaviour when exposed to cytotoxic ATP concentrations. Thus, the disruption of purinergic signalling is a feature shown not only by glioma cell lineages, but also by primary glioma cultures. Our results therefore suggest the participation of the purinergic system in glioma malignancy. This study was supported by grants from the Brazilian agencies CNPq, FAPERGS and Fundo de Incentivo à Pesquisa e Eventos (HCPA). E. Braganhol and D. Huppes were recipients of Brazilian CNPq fellowships; A. Bernardi was the recipient of a CAPES fellowship.     

Cytoprotective effect of dieckol on human endothelial progenitor cells (hEPCs) from oxidative stress-induced apoptosis

Abstract

Although endothelial progenitor cells (EPCs) have been used to promote revascularization after peripheral or myocardial ischemia, excess amounts of reactive oxygen species (ROS) are often involved in senescence and apoptosis of EPCs, thereby causing defective neovascularization and reduced or failed recovery. Here, we examined the cytoprotective effect of Ecklonia cava-derived antioxidant dieckol (DK) on oxidative stress-induced apoptosis in EPCs to improve EPC bioactivity for vessel repair. Although H₂O₂ (10 ^{? 3} M) increased the intracellular ROS level in EPCs, DK (10ug/ml) pretreatment suppressed the H₂O₂-induced ROS increase and drastically reduced the ratios of apoptotic cells. H₂O₂-induced ROS increased the phosphorylation of p38 MAPK and JNK; this was inhibited by DK pretreatment. H₂O₂ treatment increased the phosphorylation of NF-κB, which was blocked by pretreatment with SB 203580, a p38 MAPK inhibitor, or SP 600125, a JNK inhibitor. H₂O₂ decreased the cellular levels of Bcl-2 and c-IAPs, cellular inhibitors of apoptosis proteins, but increased caspase-3 activation. However, all these effects were inhibited by pretreatment with DK. Injection of DK-mixed EPCs (DK + EPCs) into myocardial ischemic sites in vivo induced cellular proliferation and survival of cells at the ischemic sites and, thereby, enhanced the secretion of angiogenic cytokines at the ischemic sites. These results show that DK + EPC exhibit markedly enhanced anti-apoptotic and antioxidative capabilities, unlike that shown by EPCs alone; thus, they contribute to improved repair of ischemic myocardial injury through cell survival and angiogenic cytokine production.     

Rosuvastatin stimulates clonogenic potential and anti‐inflammatory properties of endothelial progenitor cells

EPCs (endothelial progenitor cells) exert vasculoprotective effects and can be used for regenerative therapies. However, several isolation protocols have been described, with inconsistent results. Statins are among the most effective compounds that stimulate EPC numbers in vivo and ex vivo. We aim to describe the effects of rosuvastatin on different subtypes of putative EPCs. EPCs were cultured from mononuclear cells of blood donors and isolated according to three protocols: CFU‐EC (colony forming units‐endothelial cells), early (or ‘monocytic’) EPCs and late outgrown EPCs. Rosuvastatin (0.1–100 nM) was added at the beginning of culture (T0) or after the initial adhesion step (T1). Polarization of monocytic EPCs was assessed as expression of proinflammatory M1 markers (CD68 and CCR2) or anti‐inflammatory M2 markers (CX3CR1, CD163, CD206). We found that 1 nM rosuvastatin increased the number of CFU‐EC and late EPCs by about 3‐fold, while lower concentrations had no significant effects. Rosuvastatin (0.1 nM) increased AcLDL⁺Lectin⁺ early EPCs by about 60%, while higher concentrations exerted inhibitory effects on early EPCs. Addition of rosuvastatin at T0 was more effective in stimulating CFU‐EC and early EPCs, while addition at T1 was more effective in stimulating late EPCs. Rosuvastatin had no effects on proliferation rate of CFU‐EC, early EPCs and late EPCs. We also found that 0.1 nM rosuvastatin reduced the M1/M2 ratio in early EPCs, which retain monocytic features. In conclusion, we show that rosuvastatin had significant stimulatory effects on EPCs irrespective of the culture protocol. Rosuvastatin also induced anti‐inflammatory polarization of monocytic EPCs.     

Overview of a quality assurance/quality control compliance program consistent with FDA regulations and policies for somatic cell and gene therapies: a four year experience

Somatic cell and gene therapy involve the application of biological technologies to an individual patient through the use of living cells which provide a therapeutic benefit (Aliski, 1991). Various forms of cellular and gene therapies are being developed and evaluated in an increasing number of clinical trials for congential and acquired disorders. The potential and progress of these therapeutic applications have resulted in an increasing effort by the Food and Drug Administration (FDA) to develop the regulatory framework under which these therapeutic approaches would insure safety and efficacy, the primary mandate of the FDA.Over five years ago Cellcor began to define the parameters, specifications, and conditions relevant to a Quality Assurance/Quality Control (QA/QC) program that has evolved to insure safety and maximize the efficacy of applications of the company'sex vivo technology, autolymphocyte therapy. Autolymphocyte therapy is an outpatient form of somatic cell immunotherapy based upon the infusion of T cells that have been activatedex vivo using a combination of previously generated autologous cytokines and an anti-CD3 monoclonal antibody.We have been able to demonstrate the feasibility for the safe, controlled, and consistent preparation and delivery of a cellular therapy by application of relevant GMP regulations. This presentation reviews aspects of this program and chronicles our experience which at present amounts to over 4400 infusions for over 700 patients. This program provides a high degree of assurance that a cellular therapy program can be carried out in a multisite mode involving hundreds of patients through the strict adherence to cGMP as set forth in existing regulations. It would be prudent that developers of cellular andex vivo gene therapies establish a similar cell processing and QA/QC infrastructure at an early developmental stage to optimize safety and reproducibility and facilitate regulatory review.     

Endogenous and induced angiogenic characteristics of human chorion‐derived stem cells

Cell‐based therapy using stem cells has emerged as one of the pro‐angiogenic methods to enhance blood vessel growth and sprouting in ischaemic conditions. This study investigated the endogenous and induced angiogenic characteristics of hCDSC (human chorion‐derived stem cell) using QPCR (quantitative PCR) method, immunocytochemistry and fibrin‐matrigel migration assay. The results showed that cultured hCDSC endogenously expressed angiogenic–endogenic‐associated genes (VEGF, bFGF, PGF, HGF, Ang‐1, PECAM‐1, eNOS, Ve‐cad, CD34, VEGFR‐2 and vWF), with significant increase in mRNA levels of PGF, HGF, Ang‐1, eNOS, VEGFR‐2 and vWF following induction by bFGF (basic fibroblast growth factor) and VEGF (vascular endothelial growth factor). These enhanced angiogenic properties suggest that induced hCDSC provides a stronger angiogenic effect for the treatment of ischaemia. After angiogenic induction, hCDSC showed no reduction in the expression of the stemness genes, but had significantly higher levels of mRNA of Oct‐4, Nanog (3), FZD9, ABCG‐2 and BST‐1. The induced cells were positive for PECAM‐1 (platelet/endothelial cell adhesion molecule 1) and vWF (von Willebrand factor) with immunocytochemistry staining. hCDSC also showed endothelial migration behaviour when cultured in fibrin‐matrigel construct and were capable of forming vessels in vivo after implanting into nude mice. These data suggest that hCDSC could be the cells of choice in the cell‐based therapy for pro‐angiogenic purpose.     

Profiling of T‐cell receptor signaling complex assembly in human CD4 T‐lymphocytes using RP protein arrays

The RP protein (RPP) array approach immobilizes minute amounts of cell lysates or tissue protein extracts as distinct microspots on NC‐coated slide. Subsequent detection with specific antibodies allows multiplexed quantification of proteins and their modifications at a scale that is beyond what traditional techniques can achieve. Cellular functions are the result of the coordinated action of signaling proteins assembled in macromolecular complexes. These signaling complexes are highly dynamic structures that change their composition with time and space to adapt to cell environment. Their comprehensive analysis requires until now relatively large amounts of cells (>5×10⁷) due to their low abundance and breakdown during isolation procedure. In this study, we combined small scale affinity capture of the T‐cell receptor (TCR) and RPP arrays to follow TCR signaling complex assembly in human ex vivo isolated CD4 T‐cells. Using this strategy, we report specific recruitment of signaling components to the TCR complex upon T‐cell activation in as few as 0.5 million of cells. Second‐ to fourth‐order TCR interacting proteins were accurately quantified, making this strategy specially well‐suited to the analysis of membrane‐associated signaling complexes in limited amounts of cells or tissues, e.g., ex vivo isolated cells or clinical specimens.     

Sitagliptin‐mediated preservation of endothelial progenitor cell function via augmenting autophagy enhances ischaemic angiogenesis in diabetes

Recently, the dipeptidyl peptidase‐4 (DPP‐4) inhibitor sitagliptin, a major anti‐hyperglycaemic agent, has received substantial attention as a therapeutic target for cardiovascular diseases via enhancing the number of circulating endothelial progenitor cells (EPCs). However, the direct effects of sitagliptin on EPC function remain elusive. In this study, we evaluated the proangiogenic effects of sitagliptin on a diabetic hind limb ischaemia (HLI) model in vivo and on EPC culture in vitro. Treatment of db/db mice with sitagliptin (Januvia) after HLI surgery efficiently enhanced ischaemic angiogenesis and blood perfusion, which was accompanied by significant increases in circulating EPC numbers. EPCs derived from the bone marrow of normal mice were treated with high glucose to mimic diabetic hyperglycaemia. We found that high glucose treatment induced EPC apoptosis and tube formation impairment, which were significantly prevented by sitagliptin pretreatment. A mechanistic study found that high glucose treatment of EPCs induced dramatic increases in oxidative stress and apoptosis; pretreatment of EPCs with sitagliptin significantly attenuated high glucose‐induced apoptosis, tube formation impairment and oxidative stress. Furthermore, we found that sitagliptin restored the basal autophagy of EPCs that was impaired by high glucose via activating the AMP‐activated protein kinase/unc‐51‐like autophagy activating kinase 1 signalling pathway, although an autophagy inhibitor abolished the protective effects of sitagliptin on EPCs. Altogether, the results indicate that sitagliptin‐induced preservation of EPC angiogenic function results in an improvement of diabetic ischaemia angiogenesis and blood perfusion, which are most likely mediated by sitagliptin‐induced prevention of EPC apoptosis via augmenting autophagy.     

Spontaneous transformation of adult mesenchymal stem cells from cynomolgus macaques in vitro

Mesenchymal stem cells (MSCs) have shown potential clinical utility in cell therapy and tissue engineering, due to their ability to proliferate as well as to differentiate into multiple lineages, including osteogenic, adipogenic, and chondrogenic specifications. Therefore, it is crucial to assess the safety of MSCs while extensive expansion ex vivo is a prerequisite to obtain the cell numbers for cell transplantation. Here we show that MSCs derived from adult cynomolgus monkey can undergo spontaneous transformation following in vitro culture. In comparison with MSCs, the spontaneously transformed mesenchymal cells (TMCs) display significantly different growth pattern and morphology, reminiscent of the characteristics of tumor cells. Importantly, TMCs are highly tumorigenic, causing subcutaneous tumors when injected into NOD/SCID mice. Moreover, no multiple differentiation potential of TMCs is observed in vitro or in vivo, suggesting that spontaneously transformed adult stem cells may not necessarily turn into cancer stem cells. These data indicate a direct transformation of cynomolgus monkey MSCs into tumor cells following long-term expansion in vitro. The spontaneous transformation of the cultured cynomolgus monkey MSCs may have important implications for ongoing clinical trials and for models of oncogenesis, thus warranting a more strict assessment of MSCs prior to cell therapy.     

Thrombin bound to a fibrin clot confers angiogenic and haemostatic properties on endothelial progenitor cells

Recent data suggest that endothelial progenitor cells (EPCs) are involved in recanalizing venous thrombi. We examined the impact of a fibrin network, and particularly of adsorbed thrombin, on EPCs derived from cord blood CD34(+) cells. Fibrin networks generated in microplates by adding CaCl(2) to platelet-depleted plasma retained adsorbed thrombin at the average concentration of 4.2 nM per well. EPCs expressed high levels of endothelial cell protein C receptor and thrombomodulin, allowing the generation of activated protein C on the fibrin matrix in the presence of exogenous human protein C. The fibrin matrix induced significant EPC proliferation and, when placed in the lower chamber of a Boyden device, strongly enhanced EPC migration. These effects were partly inhibited by hirudin by 41% and 66%, respectively), which suggests that fibrin-adsorbed thrombin interacts with EPCs via the thrombin receptor PAR-1. Finally, spontaneous lysis of the fibrin network, studied by measuring D-dimer release into the supernatant, was inhibited by EPCs but not by control mononuclear cells. Such an effect was associated with a 10-fold increase in plasminogen activator inhibitor-1 (PAI-1) secretion by EPCs cultivated in fibrin matrix. Overall, our data show that EPCs, in addition to their angiogenic potential, have both anticoagulant and antifibrinolytic properties. Thrombin may modulate these properties and contribute to thrombus recanalization by EPCs.     

Endothelial reconstitution by CD34+ progenitors derived from baboon embryonic stem cells

In this study, we used a large non‐human primate model, the baboon, to establish a step‐wise protocol to generate CD34+ endothelial progenitor cells (EPCs) from embryonic stem cells (ESCs) and to demonstrate their reparative effects. Baboon ESCs were sequentially differentiated from embryoid body cultures for 9 days and then were specified into EPCs by culturing them in monolayer for 12 days. The resulting EPCs expressed CD34, CXCR4 and UEA‐1, but neither CD31 nor CD117. The EPCs were able to form intact lumen structures when seeded on Matrigel, took up Dil‐LDL, and responded to TNF‐α. Angioblasts specified in EGM‐2 medium and ECGS medium had 6.41 ± 1.16% (n = 3) and 9.32 ± 3.73% CD34+ cells (n = 3). The efficiency of generating CD34+ EPCs did not differ significantly from ECGS to EGM‐2 culture media, however, angioblasts specified in ECGS medium expressed a higher percentage of CD34+/CXCR4+ cells (3.49 ± 1.32%, n = 3) than those specified in EGM‐2 medium (0.49 ± 0.52%, n = 3). To observe their reparative capacity, we purified CD34+ progenitors after specification by EGM‐2 medium; inoculated fluorescently labelled CD34+ EPCs into an arterial segment denuded of endothelium in an ex vivo system. After 14 days of ex vivo culture, the grafted cells had attached and integrated to the denuded surface; in addition, they had matured further and expressed terminally differentiated endothelial markers including CD31 and CD146. In conclusion, we have proved that specified CD34+ EPCs are promising therapeutic agents for repairing damaged vasculature.     

Pro‐angiogenic potential of human chorion‐derived stem cells: in vitro and in vivo evaluation

Human chorion‐derived stem cells (hCDSC) were previously shown to demonstrate multipotent properties with promising angiogenic characteristics in monolayer‐cell culture system. In our study, we investigated the angiogenic capability of hCDSC in 3‐dimensional (3D) in vitro and in vivo angiogenic models for the purpose of future application in the treatment of ischaemic diseases. Human CDSC were evaluated for angiogenic and endogenic genes expressions by quantitative PCR. Growth factors secretions were quantified using ELISA. In vitro and in vivo vascular formations were evaluated by histological analysis and confocal microscopic imaging. PECAM‐1⁺ and vWF⁺ vascular‐like structures were observed in both in vitro and in vivo angiogenesis models. High secretions of VEGF and bFGF by hCDSC with increased expressions of angiogenic and endogenic genes suggested the possible angiogenic promoting mechanisms by hCDSC. The cooperation of hCDSC with HUVECS to generate vessel‐like structures in our systems is an indication that there will be positive interactions of hCDSC with existing endothelial cells when injected into ischaemic tissues. Hence, hCDSC is suggested as the novel approach in the future treatment of ischaemic diseases.     

In vivo inhibition of epidermal growth factor receptor autophosphorylation prevents receptor internalization

The question whether epidermal growth factor (EGF)-induced receptor endocytosis requires the prior autophosphorylation via the EGF receptor (EGFR) kinase domain has been a matter of long-standing debate. In the airway epithelial cell line NCI-H292, the EGFR kinase domain inhibitor BIBW 2948 BS was found to inhibit both autophosphorylation and subsequent internalization of the endogenous EGFR with similar IC₅₀ values. Applying an ex vivo EGFR internalization assay in a clinical study, the in vivo effect of inhalatively administered BIBW 2948 BS was determined directly at the targeted receptor in airway tissues from COPD patients. In these experiments, the in vivo inhibition of the EGFR kinase domain prevented the EGF-induced internalization of EGFR.     

A Novel CXCR4 antagonist enhances angiogenesis via modifying the ischaemic tissue environment

Endothelial progenitor cells (EPCs) play a capital role in angiogenesis via directly participating in neo‐vessel formation and secreting pro‐angiogenic factors. Stromal cell‐derived factor 1 (SDF‐1) and its receptor CXCR4 play a critical role in the retention and quiescence of EPCs within its niche in the bone marrow. Disturbing the interaction between SDF‐1 and CXCR4 is an effective strategy for EPC mobilization. We developed a novel CXCR4 antagonist P2G, a mutant protein of SDF‐1β with high antagonistic activity against CXCR4 and high potency in enhancing ischaemic angiogenesis and blood perfusion. However, its direct effects on ischaemic tissue remain largely unknown. In this study, P2G was found to possess a robust capability to promote EPC infiltration and incorporation in neo‐vessels, enhance the expression and function of pro‐angiogenic factors, such as SDF‐1, vascular endothelial growth factor and matrix metalloprotein‐9, and activate cell signals involved in angiogenesis, such as proliferating cell nuclear antigen, protein kinase B (Akt), extracellular regulated protein kinases and mammalian target of rapamycin, in ischaemic tissue. Moreover, P2G can attenuate fibrotic remodelling to facilitate the recovery of ischaemic tissue. The capability of P2G in direct augmenting ischaemic environment for angiogenesis suggests that it is a potential candidate for the therapy of ischaemia diseases.     

Engineered endothelial progenitor cells that overexpress prostacyclin protect vascular cells

Prostacyclin (PGI2) is a potent vasodilator and important mediator of vascular homeostasis; however, its clinical use is limited because of its short (<2‐min) half‐life. Thus, we hypothesize that the use of engineered endothelial progenitor cells (EPCs) that constitutively secrete high levels of PGI2 may overcome this limitation of PGI2 therapy. A cDNA encoding COX‐1‐10aa‐PGIS, which links human cyclooxygenase‐1 (COX‐1) to prostacyclin synthase (PGIS), was delivered via nucleofection into outgrowth EPCs derived from rat bone marrow mononuclear cells. PGI2‐secreting strains (PGI2‐EPCs) were established by continuous subculturing of transfected cells under G418 selection. Genomic PCR, RT‐PCR, and Western blot analyses confirmed the overexpression of COX‐1‐10aa‐PGIS in PGI2‐EPCs. PGI2‐EPCs secreted significantly higher levels of PGI2 in vitro than native EPCs (P < 0.05) and showed higher intrinsic angiogenic capability; conditioned medium (CM) from PGI2‐EPCs promoted better tube formation than CM from native EPCs (P < 0.05). Cell‐ and paracrine‐mediated in vitro angiogenesis was attenuated when COX‐1‐10aa‐PGIS protein expression was knocked down. Whole‐cell patch‐clamp studies showed that 4‐aminopyridine‐sensitive K⁺ current density was increased significantly in rat smooth muscle cells (rSMCs) cocultured under hypoxia with PGI2‐EPCs (7.50 ± 1.59 pA/pF; P < 0.05) compared with rSMCs cocultured with native EPCs (3.99 ± 1.26 pA/pF). In conclusion, we successfully created EPC strains that overexpress an active novel enzyme resulting in consistent secretion of PGI2. PGI2‐EPCs showed enhanced intrinsic proangiogenic properties and provided favorable paracrine‐mediated cellular protections, including promoting in vitro angiogenesis of native EPCs and hyperpolarization of SMCs under hypoxia. J. Cell. Physiol. 227: 2907–2916, 2012. © 2011 Wiley Periodicals, Inc.     

Generating natural killer cells for adoptive transfer: expanding horizons

Natural killer (NK) cells are unique innate lymphoid cells that have therapeutic potential in adoptive cell transfer-based cancer immunotherapy that has been established across a range of early-phase clinical trials. NK cells for use in adoptive transfer therapies are obtained from various sources, including primary NK cells from peripheral blood or apheresis products (autologous or allogeneic) and umbilical cord blood. NK cells have also been generated from CD34+ hematopoietic progenitors, induced pluripotent stem cells, embryonic stem cells and malignant cell lines. Apheresis-derived NK cell products are often administered after brief cytokine-based ex vivo activation, ideally aiming for in vivo expansion and proliferation. NK cells from other sources or from smaller volumes of blood require a longer period of expansion prior to therapeutic use. Although ex vivo NK cell expansion introduces a concern for senescence and exhaustion, there is also an opportunity to achieve higher NK cell doses, modulate NK cell activation characteristics and apply genetic engineering approaches, ultimately generating potent effector cells from small volumes of readily available starting materials. Herein the authors review the field of clinical-grade NK cell expansion, explore the desirable features of an idealized NK cell expansion approach and focus on techniques used in recently published clinical trials.     

Use of HLA-B27 tetramers to identify low-frequency antigen-specific T cells in <Emphasis Type="Italic">Chlamydia</Emphasis>-triggered reactive arthritis

Reports of the use of HLA-B27/peptide tetrameric complexes to study peptide-specific CD8⁺ T cells in HLA-B27⁺-related diseases are rare. To establish HLA-B27 tetramers we first compared the function of HLA-B27 tetramers with HLA-A2 tetramers by using viral epitopes. HLA-B27 and HLA-A2 tetramers loaded with immunodominant peptides from Epstein–Barr virus were generated with comparable yields and both molecules detected antigen-specific CD8⁺ T cells. The application of HLA-B27 tetramers in HLA-B27-related diseases was performed with nine recently described Chlamydia-derived peptides in synovial fluid and peripheral blood, to examine the CD8⁺ T cell response against Chlamydia trachomatis antigens in nine patients with Chlamydia-triggered reactive arthritis (Ct-ReA). Four of six HLA-B27⁺ Ct-ReA patients had specific synovial T cell binding to at least one HLA-B27/Chlamydia peptide tetramer. The HLA-B27/Chlamydia peptide 195 tetramer bound to synovial T cells from three of six patients and HLA-B27/Chlamydia peptide 133 tetramer to synovial T cells from two patients. However, the frequency of these cells was low (0.02–0.09%). Moreover, we demonstrate two methods to generate HLA-B27-restricted T cell lines. First, HLA-B27 tetramers and magnetic beads were used to sort antigen-specific CD8⁺ T cells. Second, Chlamydia-infected dendritic cells were used to stimulate CD8⁺ T cells ex vivo. Highly pure CD8 T cell lines could be generated ex vivo by magnetic sorting by using HLA-B27 tetramers loaded with an EBV peptide. The frequency of Chlamydia-specific, HLA-B27 tetramer-binding CD8⁺ T cells could be increased by stimulating CD8⁺ T cells ex vivo with Chlamydia-infected dendritic cells. We conclude that HLA-B27 tetramers are a useful tool for the detection and expansion of HLA-B27-restricted CD8⁺ T cells. T cells specific for one or more of three Chlamydia-derived peptides were found at low frequency in synovial fluid from HLA-B27⁺ patients with Ct-ReA. These cells can be expanded ex vivo, suggesting that they are immunologically functional.