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In a unique model of fluorescent based mapping of EV recipient cells, RBC-EVs were found to signal to cardiac cells and regulate gene expression in a model of ischemic heart failure., Extracellular vesicles (EVs) mediate intercellular signaling by transferring their cargo to recipient cells, but the functional consequences of signaling are not fully appreciated. RBC-derived EVs are abundant in circulation and have been implicated in regulating immune responses. Here, we use a transgenic mouse model for fluorescence-based mapping of RBC-EV recipient cells to assess the role of this intercellular signaling mechanism in heart disease. Using fluorescent-based mapping, we detected an increase in RBC-EV–targeted cardiomyocytes in a murine model of ischemic heart failure. Single cell nuclear RNA sequencing of the heart revealed a complex landscape of cardiac cells targeted by RBC-EVs, with enrichment of genes implicated in cell proliferation and stress signaling pathways compared with non-targeted cells. Correspondingly, cardiomyocytes targeted by RBC-EVs more frequently express cellular markers of DNA synthesis, suggesting the functional significance of EV-mediated signaling. In conclusion, our mouse model for mapping of EV-recipient cells reveals a complex cellular network of RBC-EV–mediated intercellular communication in ischemic heart failure and suggests a functional role for this mode of intercellular signaling.

Single cell sorting is commonly used for ensuring monoclonality and producing homogenous target cell populations. Current single cell verification methods involve manually confirming the existence of single cells or colonies in a well using a standard light microscope. However, the manual verification method is time-consuming and highly tedious, which prompts a need for an accurate and rapid detection method for verifying single cell sorting capability. Here, we demonstrate a rapid single cell sorting verification method using the Celigo Image Cytometer. Calcein AM-stained Jurkat cells and fluorescent beads are sorted into 96-well half area microplates using the MoFlo Astrios EQ. Whole well bright field and fluorescent images are acquired and analyzed using the image cytometer in less than 8 min. The proposed single cell verification detection method in multi-well microplates can allow for quick optimization of FACS instruments at flow core laboratories, as well as improvement of downstream biological assays by accurately confirming the presence of single cells in each well.

Author(s): Das, Avash; Valkov, Nedyalka; Salvador, Ane; Kur, Ivan; Ziegler, Olivia; Yeri, Ashish; Garcia, Fernando Camacho; Lu, Shulin; Khamesra, Aushee; Xiao, Chunyang; Rodosthenous, Rodosthenis; Li, Guoping; Srinivasan, Srimeenakshi; Toxavidis, Vasilis; Tigges, John; Laurent, Louise; Momma, Stefan; Ghiran, Ionita; Das, Saumya | Abstract: Summary Extracellular vesicles (EV) mediate intercellular signaling by transferring their cargo to recipient cells. Red blood cell (RBC)-derived EVs constitute a significant proportion of circulating EVs and have been implicated in regulating immune responses. Here, we describe a transgenic mouse model for fluorescent-based mapping of RBC-EV target cells based on the functional transfer of EV-contained Cre-recombinase to target cells. In a murine model of ischemic heart failure, we detect an increase in RBC-EV-targeted cardiomyocytes in the hearts and microglial cells in the brains. Cells targeted by RBC-EVs present an enrichment of genes implicated in cell proliferation and metabolism pathways compared to non-recombined (non-targeted) cells. Cardiomyocytes targeted by RBC-EVs are more likely to demonstrate cellular markers of DNA synthesis and proliferation, suggesting functional significance of EV-mediated signaling. In conclusion, we leverage our mouse model for mapping of RBC-EV targets in murine ischemic heart failure to demonstrate quantitative and qualitative changes in RBC-EV recipients.

Single cell sorting is commonly used for ensuring monoclonality and producing homogenous target cell populations. Current single cell verification methods involve manually confirming the existence of single cells or colonies in a well using a standard light microscope. However, the manual verification method is time-consuming and highly tedious, which prompts a need for an accurate and rapid detection method for verifying single cell sorting capability. Here, we demonstrate a rapid single cell sorting verification method using the Celigo Image Cytometer. Calcein AM-stained Jurkat cells and fluorescent beads are sorted into 96-well half area microplates using the MoFlo Astrios EQ. Whole well bright field and fluorescent images are acquired and analyzed using the image cytometer in less than 8 min. The proposed single cell verification detection method in multi-well microplates can allow for quick optimization of FACS instruments at flow core laboratories, as well as improvement of downstream biological assays by accurately confirming the presence of single cells in each well.

Here, we describe a comprehensive methodology for the setup and standardization of EV analysis using nanoscale flow cytometry. Controls of different size ranges, fluorescent intensities, and materials can be used to set up distribution curves that are then used for instrument optimization and as a reference guide. Using these controls, flow cytometry instruments can be primed for the detection, analysis, and sorting of specific EV populations. This allows for cross platform comparison and the ability to monitor both quality control (QC) and quality assurance (QA). The method here describes the use of nanoparticles to optimize a flow cytometer for small particle detection. It also outlines the procedures necessary to recover EVs for downstream applications.

Introduction: Extracellular vesicles (EVs) function as novel mediators of intercellular communication. Here, we describe a novel, fluorescence switch-based, experimental model to study EV-mediated communication between RBCs and the heart that permits characterization of cross-talk between RBCs and cardiomyocytes at homeostasis and after myocardial infarction. Methods: Mice with RBC-specific expression of cre (Erythropoietin Receptor (EpoR) Cre) were crossed with reporter mTmG Rosa26 mice to yield EpoRCre/mTmG off-springs with membrane GFP expression in RBCs and RBC-derived EVs. Cultured dermal fibroblasts from mTmG mice and a mT/floxed/mGFP HEK 293 reporter cell line were used to assess transfer of functional cre in RBC-derived EVs. To determine targets of RBC-EVs, organs from i)EpoRCre/mTmG (n=3), ii) mTmG (n=3) or iii) mTmG mice transfused with RBC-EVs from EpoR-cre mice and targets of RBC-EVs (determined by mGFP expression due to cre-recombination) were assessed by confocal microscopy. Finally, ischemia-reperfusion-infarction (30 min. LAD ligation) was done in EpoRCre/mGmT mice (n=3) and their blood and organs harvested after a span of 4 weeks to analyze changes in quality and quantity of RBC-EV targets following MI. Results: 1. RBC-EVs (mGFP positive) in plasma accounted for about 9% of total fluorescent EVs as detected by nano-flow cytometry and microscopy. 2. RBC-EVs contained cre protein by EM, and in vitro dermal fibroblasts from mTmG mice or mT/floxed/mGFP HEK 293 reporter cells showed mGFP expression with EpoRCre RBC-EVs, suggesting EV-mediated transfer of functional cre. 3. Cre-mediated recombination was noted in diverse organs in EpoRCre/mTmG mice and mTmTG mice transfused with EpoRCre- EVs with the bone marrow, heart, lungs, kidney and spleen showing the largest degree of recombined cells. 4. Target profile of RBC-EVs demonstrates a distinct pattern of EV-mediated communication among the organs at baseline that may be altered in different disease models. Conclusion: We show proof-of-concept for a novel model to study origin and targets of EV-mediated intercellular communication with significant EV-mediated communication between RBCs and cardiomyocytes under homeostatic conditions.

Extracellular vesicles (EVs) serve an import ant function as mediators of intercellular communication. Exercise is protective for the heart, although the signaling mechanisms that mediate this cardioprotection have not been fully elucidated. Here using a nano-flow cytometry, we found a rapid increase in plasma EVs in human subjects undergoing exercise stress testing. We subsequently identified that serum EVs were increased by ~1.85 fold in mice after 3-week swimming. Intramyocardial injection of equivalent quantities of EVs from exercised mice and non-exercised controls provided similar protective effects against acute ischemia/reperfusion (I/R) injury in mice. However, injection of exercise-induced EVs in a quantity equivalent to the increase seen with exercise (1.85 Swim group) significantly enhanced the protective effect. Similarly, treatment with exercise-induced increased EVs provided additional anti-apoptotic effect in H2O2-treated H9C2 cardiomyocytes mediated by the activation of ERK1/2 and HSP27 signaling. Finally, by treating H9C2 cells with insulin-like growth factor-1 (IGF-1) to mimic exercise stimulus in vitro, we found an increased release of EVs from cardiomyocytes associated with ALIX and RAB35 activation. Collectively, our results show that exercise-induced increase in circulating EVs enhances the protective effects of endogenous EVs against cardiac I/R injury. Exercise-derived EVs might serve as a potent therapy for myocardial injury in the future.

Lineage (CD3e, CD11b, GR1, B220 and Ly-76) negative hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) infiltrate islet allografts within 24 h posttransplantation. In fact, lineage(negative) Sca-1(+) cKit(+) ("LSK") cells, a classic signature for HSCs, were also detected among these graft infiltrating cells. Lineage negative graft infiltrating cells are functionally multi-potential as determined by a standard competitive bone marrow transplant (BMT) assay. By 3 months post-BMT, both CD45.1 congenic, lineage negative HSCs/HPCs and classic "LSK" HSCs purified from islet allograft infiltrating cells, differentiate and repopulate multiple mature blood cell phenotypes in peripheral blood, lymph nodes, spleen, bone marrow and thymus of CD45.2 hosts. Interestingly, "LSK" HSCs also rapidly infiltrate syngeneic islet transplants as well as allogeneic cardiac transplants and sham surgery sites. It seems likely that an inflammatory response, not an adaptive immune response to allo-antigen, is responsible for the rapid infiltration of islet and cardiac transplants by biologically active HSCs/HPCs. The pattern of hematopoietic differentiation obtained from graft infiltrating HSCs/HPCs, cells that are recovered from inflammatory sites, as noted in the competitive BMT assay, is not precisely the same as that of intramedullary HSCs. This does not refute the obvious multi-lineage potential of graft infiltrating HSCs/HPCs.

THE opportunity for low cost, high quality training and supervision is an attractive one to most scientists in the cytometry field. Many have had little if any training, no expertise based locally, or may not have the funds to attend regional or international training courses. In addition, rather than general cytometry they may require assistance with a particular application, which could be accomplished in a number of hours through support from an expert. Separately, shared resource scientists, also known as core facility staff, are generally responsible for the performance and upkeep of multiple complex instruments, as well as monitoring, training etc. Some acquisitions or cell sorts may take hours for a single run, and with limited staff the scientist is usually multitasking on an hourly basis. Also, if users of the core facility are having problems and staff is unavailable, valuable samples or time may be lost. There is potential for error at many steps of flow cytometry, including sample preparation, experimental design and fluorochrome selection, instrument set up, compensation, gating, analysis, and interpretation. External/offsite tracking may improve monitoring, and perhaps limit such errors. Also interand intra-instrumentation variations can lead to different results for the same sample. A means of unifying performance, setup, and interpretation is vital, in particular with multicenter collaborations. Peer-review of results and analysis, as well as hierarchical approval systems, provides some solutions, but further stream-lining is clearly beneficial. Remote access technology for cytometry (or remote cytometry) may offer solutions to many such issues and can be performed at very low costs to the institution. There is potential for such systems to facilitate instrumentation monitoring, control equipment in containment areas, speed up vendor service, improve training courses, increase collaborations, and further promote cytometry globally.

To study molecular mechanisms involved in hematopoietic stem cell (HSC) mobilization after liver resection and determine impacts on liver regeneration.Extracellular nucleotide-mediated cell signaling has been shown to boost liver regeneration. Ectonucleotidases of the CD39 family are expressed by bone marrow-derived cells, and purinergic mechanisms might also impact mobilization and functions of HSC after liver injury.Partial hepatectomy was performed in C57BL/6 wild-type, Cd39 ectonucleotidase-null mice and in chimeric mice after transplantation of wild-type or Cd39-null bone marrow. Bone marrow-derived HSCs were purified by fluorescence-activated cell sorting and administered after hepatectomy. Chemotactic studies were performed to examine effects of purinergic receptor agonists and antagonists in vitro. Mobilization of human HSCs and expression of CD39 were examined and linked to the extent of resection and liver tests.Subsets of HSCs expressing Cd39 are preferentially mobilized after partial hepatectomy. Chemotactic responses of HSCs are increased by CD39-dependent adenosine triphosphate hydrolysis and adenosine signaling via A2A receptors in vitro. Mobilized Cd39 HSCs boost liver regeneration, potentially limiting interleukin 1β signaling. In clinical studies, mobilized human HSCs also express CD39 at high levels. Mobilization of HSCs correlates directly with the restoration of liver volume and function after partial hepatectomy.We demonstrate CD39 to be a novel HSC marker that defines a functionally distinct stem cell subset in mice and humans. HSCs are mobilized after liver resection, limit inflammation, and boost regeneration in a CD39-dependent manner. These observations have implications for monitoring and indicate future therapeutic avenues.

During their lifetime, like all other cell types, red blood cells (RBCs) release both exosomes and plasma membrane derived EVs (ectosomes). RBC exosomes are formed only during the development of RBCs in bone marrow, and are released following the fusion of microvesicular bodies (MVB) with the plasma membrane. On the other hand, RBC EVs are generated during normal aging of RBCs in circulation by budding of the plasma membrane due to complement -mediated calcium influx, followed by vesicle shedding. This makes red blood cells and stored red cells a reliable source of EVs for basic and clinical research.

Here, we describe a comprehensive methodology for the setup and standardization of EV analysis using nanoscale flow cytometry. Controls of different size ranges, fluorescent intensities, and materials can be used to set up distribution curves that are then used for instrument optimization and as a reference guide. Using these controls, flow cytometry instruments can be primed for the detection, analysis, and sorting of specific EV populations. This allows for cross platform comparison and the ability to monitor both quality control (QC) and quality assurance (QA). The method here describes the use of nanoparticles to optimize a flow cytometer for small particle detection. It also outlines the procedures necessary to recover EVs for downstream applications.

BACKGROUND:We have previously demonstrated that CD133 and CD39 are expressed by hematopoietic stem cells (HSC), which are mobilized after liver injury and target sites of injury, limit vascular inflammation, and boost hepatic regeneration. Plasma microparticles (MP) expressing CD39 can block endothelial activation. Here, we tested whether CD133 MP might be shed in a CD39-dependent manner in a model of liver injury and could potentially serve as biomarkers of liver failure in the clinic.METHODS:Wild-type and Cd39-null mice were subjected to acetaminophen-induced liver injury. Mice were sacrificed and plasma MP were isolated by ultracentrifugation. HSC and CD133 MP levels were analyzed by fluorescence-activated cell sorting. Patients were enrolled with acute (n=5) and acute on chronic (n=5) liver injury with matched controls (n=7). Blood was collected at admission and plasma CD133 and CD39 MP subsets were analyzed by fluorescence-activated cell sorting.RESULTS:HSC and CD133 MP levels were significantly increased only in the plasma of wild-type mice with acetaminophen hepatotoxicity (PCONCLUSIONS:HSC and plasma CD133 MP levels increase in a CD39-dependent manner during experimental acute liver injury. Increased levels of CD39 MP are differentially noted in patients with liver injury. Further research is needed to determine whether MP fluxes are secondary to pathophysiologic insults to the liver or might reflect compensatory responses.

Here we present methods to longitudinally track islet allograft–infiltrating T cells in live mice by endoscopic confocal microscopy and to analyze circulating T cells by in vivo flow cytometry. We developed a new reporter mouse whose T cell subsets express distinct, ‘color-coded’ proteins enabling in vivo detection and identification of effector T cells (Teff cells) and discrimination between natural and induced regulatory T cells (nT reg and iT reg cells). Using these tools, we observed marked differences in the T cell response in recipients receiving tolerance-inducing therapy (CD154-specific monoclonal antibody plus rapamycin) compared to untreated controls. These results establish real-time cell tracking as a powerful means to probe the dynamic cellular interplay mediating immunologic rejection or transplant tolerance. Understanding the cellular immune response to transplanted tissue has a key role in developing strategies to improve transplant outcomes. Tissue biopsy is the standard method for accessing immune cell infiltration in the graft, but the method is both invasive and inadequate when temporal information is needed to characterize an immune reaction that progresses dynamically over time. Advances in molecular imaging techniques that combine cell labeling with the use of whole-body imaging modalities such as positron emission tomography, magnetic resonance imaging and bioluminescence imaging have led to promising approaches for tracking immune cells noninvasively in vivo 1–4 , but these methods lack the sensitivity and spatial resolution to quantify events at the single-cell level. In addition, there is need to track multiple cell populations using labels that are not diluted by cell division. Here we describe a real-time optical method for simultaneous tracking of multiple T cell subsets that are color-coded with distinct fluorescent reporters in a mouse model in which pancreatic islet transplants are placed beneath the renal capsule. As rejection occurs at 2 weeks in control untreated hosts, we track the T cell response for a period of 2 weeks after transplantation in both the circulatory compartment (by in vivo flow cytometry) 5 and within the allograft (by endoscopic confocal microscopy) 6 . In vivo flow cytometry allows noninvasive, continuous detection and quantification of fluorescently labeled cells in the circulation without the need to draw blood samples 5 . Endoscopic confocal microscopy enables minimally invasive imaging of internal organs with cellular definition by inserting a narrow-diameter endomicroscope through a small incision in the skin 6 . We show that repeated imaging of the islet allograft just beneath the renal capsule can be accomplished in the same mouse over the two-week period.

The identification of extracellular vesicles (EVs) as intercellular conveyors of biological information has recently emerged as a novel paradigm in signaling, leading to the exploitation of EVs and their contents as biomarkers of various diseases. However, whether there are diurnal variations in the size, number, and tissue of origin of blood EVs is currently not known, and could have significant implications when using EVs as biomarkers for disease progression. Currently available technologies for the measurement of EV size and number are either time consuming, require specialized equipment, or lack sufficient accuracy across a range of EV sizes. Flow cytometry represents an attractive alternative to these methods; however, traditional flow cytometers are only capable of measuring particles down to 500 nm, which is significantly larger than the average and median sizes of plasma EVs. Utilizing a Beckman Coulter MoFlo XDP flow cytometer with NanoView module, we employed nanoscale flow cytometry (termed nanoFCM) to examine the relative number and scatter distribution of plasma EVs at three different time points during the day in 6 healthy adults. Analysis of liposomes and plasma EVs proved that nanoFCM is capable of detecting biologically-relevant vesicles down to 100 nm in size. With this high resolution configuration, we observed variations in the relative size (FSC/SSC distributions) and concentration (proportions) of EVs in healthy adult plasma across the course of a day, suggesting that there are diurnal variations in the number and size distribution of circulating EV populations. The use of nanoFCM provides a valuable tool for the study of EVs in both health and disease; however, additional refinement of nanoscale flow cytometric methods is needed for use of these instruments for quantitative particle counting and sizing. Furthermore, larger scale studies are necessary to more clearly define the diurnal variations in circulating EVs, and thus further inform their use as biomarkers for disease.

Producción Científica, CD4(+) T cells are involved in the development of autoimmunity, including multiple sclerosis (MS). Here we show that nicotinamide adenine dinucleotide (NAD(+)) blocks experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, by inducing immune homeostasis through CD4(+)IFNγ(+)IL-10(+) T cells and reverses disease progression by restoring tissue integrity via remyelination and neuroregeneration. We show that NAD(+) regulates CD4(+) T-cell differentiation through tryptophan hydroxylase-1 (Tph1), independently of well-established transcription factors. In the presence of NAD(+), the frequency of T-bet(-/-) CD4(+)IFNγ(+) T cells was twofold higher than wild-type CD4(+) T cells cultured in conventional T helper 1 polarizing conditions. Our findings unravel a new pathway orchestrating CD4(+) T-cell differentiation and demonstrate that NAD(+) may serve as a powerful therapeutic agent for the treatment of autoimmune and other diseases.

Phosphohydrolysis of extracellular ATP and ADP is an essential step in purinergic signaling that regulates key pathophysiological processes, such as those linked to inflammation. Classically, this reaction has been known to occur in the pericellular milieu catalyzed by membrane bound cellular ecto-nucleotidases, which can be released in the form of both soluble ecto-enzymes as well as being associated with exosomes. Circulating ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1/CD39) and adenylate kinase 1 (AK1) activities have been shown to be present in plasma. However, other ecto-nucleotidases have not been characterized in depth. An in vitro ADPase assay was developed to probe the ecto-enzymes responsible for the ecto-nucleotidase activity in human platelet-free plasma, in combination with various specific biochemical inhibitors. Identities of ecto-nucleotidases were further characterized by chromatography, immunoblotting, and flow cytometry of circulating exosomes. We noted that microparticle-bound E-NTPDases and soluble AK1 constitute the highest levels of ecto-nucleotidase activity in human plasma. All four cell membrane expressed E-NTPDases are also found in circulating microparticles in human plasma, inclusive of: CD39, NTPDase 2 (CD39L1), NTPDase 3 (CD39L3), and NTPDase 8. CD39 family members and other ecto-nucleotidases are found on distinct microparticle populations. A significant proportion of the microparticle-associated ecto-nucleotidase activity is sensitive to POM6, inferring the presence of NTPDases, either −2 or/and −3. We have refined ADPase assays of human plasma from healthy volunteers and have found that CD39, NTPDases 2, 3, and 8 to be associated with circulating microparticles, whereas soluble AK1 is present in human plasma. These ecto-enzymes constitute the bulk circulating ADPase activity, suggesting a broader implication of CD39 family and other ecto-enzymes in the regulation of extracellular nucleotide metabolism.

To evaluate changes in endothelial progenitor cells (EPCs) and cytokines in patients with diabetic foot ulceration (DFU) in association with wound healing. METHODS: We studied healthy subjects, diabetic patients not at risk of DFU, at risk of DFU and with active DFU. We prospectively followed the DFU patients over a 12-week period. We also investigated similar changes in diabetic rabbit and mouse models of wound healing. RESULTS: All EPC phenotypes except the kinase insert domain receptor (KDR)(+)CD133(+) were reduced in the at risk and the DFU groups compared to the controls. There were no major EPC differences between the control and not at risk group, and between the at risk and DFU groups. Serum stromal-cell derived factor-1 (SDF-1) and stem cell factor (SCF) were increased in DFU patients. DFU patients who healed their ulcers had lower CD34(+)KDR(+) count at visits 3 and 4, serum c-reactive protein (CRP) and granulocyte-macrophage colony-stimulating factor (GM-CSF) at visit 1, interleukin-1 (IL-1) at visits 1 and 4. EPCs tended to be higher in both diabetic animal models when compared to their non-diabetic counterparts both before and ten days after wounding. CONCLUSIONS: Uncomplicated diabetes does not affect EPCs. EPCs are reduced in patients at risk or with DFU while complete wound healing is associated with CD34(+)KDR(+) reduction, suggesting possible increased homing. Low baseline CRP, IL-1α and GM-CSF serum levels were associated with complete wound healing and may potentially serve as prognostic markers of DFU healing. No animal model alone is representative of the human condition, indicating the need for multiple experimental models

2A Peptide sequences are now being widely used to construct multicistronic expression vectors. It is suggested that when only the first 2A-linked protein bears a signal sequence, the signal-less protein(s) downstream of 2A can also be translocated into the mammalian endoplasmic reticulum system through a "slipstreaming" mechanism. By using flow cytometry and cell surface CD90 as a localization indicator, we show here that slipstreaming translocation does not occur in mammalian cells; that is, the second protein downstream of 2A still requires signal sequence for secretary or membrane-anchored expression.

Foxp3(+) regulatory T cells (Tregs) express both ectoenzymes CD39 and CD73, which in tandem hydrolyze pericellular ATP into adenosine, an immunoinhibitory molecule that contributes to Treg suppressive function. Using Foxp3GFP knockin mice, we noted that the mouse CD4(+)CD39(+) T-cell pool contains two roughly equal size Foxp3(+) and Foxp3(-) populations. While Foxp3(+)CD39(+) cells are CD73(bright) and are the bone fide Tregs, Foxp3(-)CD39(+) cells do not have suppressive activity and are CD44(+)CD62L(-)CD25(-)CD73(dim/-), exhibiting memory cell phenotype. Functionally, CD39 expression on memory and Treg cells confers protection against ATP-induced apoptosis. Compared with Foxp3(-)CD39(-) naïve T cells, Foxp3(-)CD39(+) cells freshly isolated from non-immunized mice express at rest significantly higher levels of mRNA for T-helper lineage-specific cytokines IFN-gamma (Th1), IL-4/IL-10 (Th2), IL-17A/F (Th17), as well as pro-inflammatory cytokines, and rapidly secrete these cytokines upon stimulation. Moreover, the presence of Foxp3(-)CD39(+) cells inhibits TGF-beta induction of Foxp3 in Foxp3(-)CD39(-) cells. Furthermore, when transferred in vivo, Foxp3(-)CD39(+) cells rejected MHC-mismatched skin allografts in a much faster tempo than Foxp3(-)CD39(-) cells. Thus, besides Tregs, CD39 is also expressed on pre-existing memory T cells of Th1-, Th2- and Th17-types with heightened alloreactivity.