PD-1/PD-L1 blockade abrogates a dysfunctional innate-adaptive immune axis in critical ß-coronavirus disease.

Severe COVID-19 is associated with hyperinflammation and weak T cell responses against SARS-CoV-2. However, the links between those processes remain partially characterized. Moreover, whether and how therapeutically manipulating T cells may benefit patients are unknown. Our genetic and pharmacological evidence demonstrates that the ion channel TMEM176B inhibited inflammasome activation triggered by SARS-CoV-2 and SARS-CoV-2-related murine ß-coronavirus. Tmem176b-/- mice infected with murine ß-coronavirus developed inflammasome-dependent T cell dysfunction and critical disease, which was controlled by modulating dysfunctional T cells with PD-1 blockers. In critical COVID-19, inflammasome activation correlated with dysfunctional T cells and low monocytic TMEM176B expression, whereas PD-L1 blockade rescued T cell functionality. Here, we mechanistically link T cell dysfunction and inflammation, supporting a cancer immunotherapy to reinforce T cell immunity in critical ß-coronavirus disease.

TNB-738, a biparatopic antibody, boosts intracellular NAD+ by inhibiting CD38 ecto-enzyme activity.

Cluster of differentiation 38 (CD38) is an ecto-enzyme expressed primarily on immune cells that metabolize nicotinamide adenine dinucleotide (NAD+) to adenosine diphosphate ribose or cyclic ADP-ribose and nicotinamide. Other substrates of CD38 include nicotinamide adenine dinucleotide phosphate and nicotinamide mononucleotide, a critical NAD+ precursor in the salvage pathway. NAD+ is an important coenzyme involved in several metabolic pathways and is a required cofactor for the function of sirtuins (SIRTs) and poly (adenosine diphosphate-ribose) polymerases. Declines in NAD+ levels are associated with metabolic and inflammatory diseases, aging, and neurodegenerative disorders. To inhibit CD38 enzyme activity and boost NAD+ levels, we developed TNB-738, an anti-CD38 biparatopic antibody that pairs two non-competing heavy chain-only antibodies in a bispecific format. By simultaneously binding two distinct epitopes on CD38, TNB-738 potently inhibited its enzymatic activity, which in turn boosted intracellular NAD+ levels and SIRT activities. Due to its silenced IgG4 Fc, TNB-738 did not deplete CD38-expressing cells, in contrast to the clinically available anti-CD38 antibodies, daratumumab, and isatuximab. TNB-738 offers numerous advantages compared to other NAD-boosting therapeutics, including small molecules, and supplements, due to its long half-life, specificity, safety profile, and activity. Overall, TNB-738 represents a novel treatment with broad therapeutic potential for metabolic and inflammatory diseases associated with NAD+ deficiencies.Abbreviations: 7-AAD: 7-aminoactinomycin D; ADCC: antibody dependent cell-mediated cytotoxicity; ADCP: antibody dependent cell-mediated phagocytosis; ADPR: adenosine diphosphate ribose; APC: allophycocyanin; cADPR: cyclic ADP-ribose; cDNA: complementary DNA; BSA: bovine serum albumin; CD38: cluster of differentiation 38; CDC: complement dependent cytotoxicity; CFA: Freund's complete adjuvant; CHO: Chinese hamster ovary; CCP4: collaborative computational project, number 4; COOT: crystallographic object-oriented toolkit; DAPI: 4',6-diamidino-2-phenylindole; DNA: deoxyribonucleic acid; DSC: differential scanning calorimetry; 3D: three dimensional; εNAD+: nicotinamide 1,N6-ethenoadenine dinucleotide; ECD: extracellular domain; EGF: epidermal growth factor; FACS: fluorescence activated cell sorting; FcγR: Fc gamma receptors; FITC: fluorescein isothiocyanate; HEK: human embryonic kidney; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IgG: immunoglobulin; IFA: incomplete Freund's adjuvant; IFNγ: Interferon gamma; KB: kinetic buffer; kDa: kilodalton; KEGG: kyoto encyclopedia of genes and genomes; LDH: lactate dehydrogenase; M: molar; mM: millimolar; MFI: mean fluorescent intensity; NA: nicotinic acid; NAD: nicotinamide adenine dinucleotide; NADP: nicotinamide adenine dinucleotide phosphate; NAM: nicotinamide; NGS: next-generation sequencing; NHS/EDC: N-Hydroxysuccinimide/ ethyl (dimethylamino propyl) carbodiimide; Ni-NTA: nickel-nitrilotriacetic acid; nL: nanoliter; NK: natural killer; NMN: nicotinamide mononucleotide; OD: optical density; PARP: poly (adenosine diphosphate-ribose) polymerase; PBS: phosphate-buffered saline; PBMC: peripheral blood mononuclear cell; PDB: protein data bank; PE: phycoerythrin; PISA: protein interfaces, surfaces, and assemblies: PK: pharmacokinetics; mol: picomolar; RNA: ribonucleic acid; RLU: relative luminescence units; rpm: rotations per minute; RU: resonance unit; SEC: size exclusion chromatography; SEM: standard error of the mean; SIRT: sirtuins; SPR: surface plasmon resonance; µg: microgram; µM: micromolar; µL: microliter.

A bispecific antibody agonist of the IL-2 heterodimeric receptor preferentially promotes in vivo expansion of CD8 and NK cells.

The use of recombinant interleukin-2 (IL-2) as a therapeutic protein has been limited by significant toxicities despite its demonstrated ability to induce durable tumor-regression in cancer patients. The adverse events and limited efficacy of IL-2 treatment are due to the preferential binding of IL-2 to cells that express the high-affinity, trimeric receptor, IL-2Rαßγ such as endothelial cells and T-regulatory cells, respectively. Here, we describe a novel bispecific heavy-chain only antibody which binds to and activates signaling through the heterodimeric IL-2Rßγ receptor complex that is expressed on resting T-cells and NK cells. By avoiding binding to IL-2Rα, this molecule circumvents the preferential T-reg activation of native IL-2, while maintaining the robust stimulatory effects on T-cells and NK-cells in vitro. In vivo studies in both mice and cynomolgus monkeys confirm the molecule's in vivo biological activity, extended pharmacodynamics due to the Fc portion of the molecule, and enhanced safety profile. Together, these results demonstrate that the bispecific antibody is a safe and effective IL-2R agonist that harnesses the benefits of the IL-2 signaling pathway as a potential anti-cancer therapy.

Preclinical Assessment of Autologous Tolerogenic Dendritic Cells From End-stage Renal Disease Patients.

BACKGROUND: Kidney transplantation is the therapeutic of choice for patients with kidney failure. While immunosuppressive drugs can control graft rejection, their use is associated with increased infections and cancer, and they do not effectively control chronic graft rejection. Cell therapy is an attractive strategy to minimize the use of pharmacological drugs. METHODS: We recently developed a protocol to generate human monocyte-derived autologous tolerogenic dendritic cells (ATDCs) from healthy volunteers. Herein, we transferred the ATDC manufacturing protocol to a Good Manufacturing Practice (GMP)-compliant facility. Furthermore, we compared the phenotype and in vitro functions of ATDCs generated from patients with end-stage renal disease to those generated from healthy volunteers. RESULTS: We describe the critical steps for GMP-compliant production of ATDCs and define the quality criteria required to allow release of the cell products. Furthermore, we showed that ATDCs generated from healthy volunteers and patients with kidney failure display the same tolerogenic profile based on their phenotype, resistance to maturation, and ability to modulate T-cell responses. CONCLUSIONS: Together, these results allowed us to define the production process and the quality criteria for the release of ATDCs before their administration in patients receiving a kidney transplant.

Targeting TMEM176B Enhances Antitumor Immunity and Augments the Efficacy of Immune Checkpoint Blockers by Unleashing Inflammasome Activation.

Although immune checkpoint blockers have yielded significant clinical benefits in patients with different malignancies, the efficacy of these therapies is still limited. Here, we show that disruption of transmembrane protein 176B (TMEM176B) contributes to CD8+ T cell-mediated tumor growth inhibition by unleashing inflammasome activation. Lack of Tmem176b enhances the antitumor activity of anti-CTLA-4 antibodies through mechanisms involving caspase-1/IL-1ß activation. Accordingly, patients responding to checkpoint blockade therapies display an activated inflammasome signature. Finally, we identify BayK8644 as a potent TMEM176B inhibitor that promotes CD8+ T cell-mediated tumor control and reinforces the antitumor activity of both anti-CTLA-4 and anti-PD-1 antibodies. Thus, pharmacologic de-repression of the inflammasome by targeting TMEM176B may enhance the therapeutic efficacy of immune checkpoint blockers.

Role of myeloid regulatory cells (MRCs) in maintaining tissue homeostasis and promoting tolerance in autoimmunity, inflammatory disease and transplantation.

Myeloid cells play a pivotal role in regulating innate and adaptive immune responses. In inflammation, autoimmunity, and after transplantation, myeloid cells have contrasting roles: on the one hand they initiate the immune response, promoting activation and expansion of effector T-cells, and on the other, they counter-regulate inflammation, maintain tissue homeostasis, and promote tolerance. The latter activities are mediated by several myeloid cells including polymorphonuclear neutrophils, macrophages, myeloid-derived suppressor cells, and dendritic cells. Since these cells have been associated with immune suppression and tolerance, they will be further referred to as myeloid regulatory cells (MRCs). In recent years, MRCs have emerged as a therapeutic target or have been regarded as a potential cellular therapeutic product for tolerance induction. However, several open questions must be addressed to enable the therapeutic application of MRCs including: how do they function at the site of inflammation, how to best target these cells to modulate their activities, and how to isolate or to generate pure populations for adoptive cell therapies. In this review, we will give an overview of the current knowledge on MRCs in inflammation, autoimmunity, and transplantation. We will discuss current strategies to target MRCs and to exploit their tolerogenic potential as a cell-based therapy.

Tolerogenic Dendritic Cells in Solid Organ Transplantation: Where Do We Stand?

Over the past century, solid organ transplantation has been improved both at a surgical and postoperative level. However, despite the improvement in efficiency, safety, and survival, we are still far from obtaining full acceptance of all kinds of allograft in the absence of concomitant treatments. Today, transplanted patients are treated with immunosuppressive drugs (IS) to minimize immunological response in order to prevent graft rejection. Nevertheless, the lack of specificity of IS leads to an increase in the risk of cancer and infections. At this point, cell therapies have been shown as a novel promising resource to minimize the use of IS in transplantation. The main strength of cell therapy is the opportunity to generate allograft-specific tolerance, promoting in this way long-term allograft survival. Among several other regulatory cell types, tolerogenic monocyte-derived dendritic cells (Tol-MoDCs) appear to be an interesting candidate for cell therapy due to their ability to perform specific antigen presentation and to polarize immune response to immunotolerance. In this review, we describe the characteristics and the mechanisms of action of both human Tol-MoDCs and rodent tolerogenic bone marrow-derived DCs (Tol-BMDCs). Furthermore, studies performed in transplantation models in rodents and non-human primates corroborate the potential of Tol-BMDCs for immunoregulation. In consequence, Tol-MoDCs have been recently evaluated in sundry clinical trials in autoimmune diseases and shown to be safe. In addition to autoimmune diseases clinical trials, Tol-MoDC is currently used in the first phase I/II clinical trials in transplantation. Translation of Tol-MoDCs to clinical application in transplantation will also be discussed in this review.

Comparative Study of the Immunoregulatory Capacity of In Vitro Generated Tolerogenic Dendritic Cells, Suppressor Macrophages, and Myeloid-Derived Suppressor Cells.

BACKGROUND: Regulatory myeloid cell (RMC) therapy is a promising strategy for the treatment of immunological disorders such as autoimmune disease and allograft transplant rejection. Various RMC subsets can be derived from total bone marrow using different protocols, but their phenotypes often overlap, raising questions about whether they are truly distinct. METHODS: In this study, we directly compared the phenotype and function of 3 types of RMCs, tolerogenic dendritic cells, suppressor macrophages, and myeloid-derived suppressor cells, generated in vitro from the same mouse strain in a single laboratory. RESULTS: We show that the 3 RMC subsets tested in this study share some phenotypic markers, suppress T cell proliferation in vitro and were all able to prolong allograft survival in a model of skin transplantation. However, our results highlight distinct mechanisms of action that are specific to each cell population. CONCLUSIONS: This study shows for the first time a side-by-side comparison of 3 types of RMCs using the same phenotypic and functional assays, thus providing a robust analysis of their similarities and differences.

Generation and Characterization of Mouse Regulatory Macrophages.

In the last years, cell therapy has become a promising approach to therapeutically manipulate immune responses in autoimmunity, cancer, and transplantation. Several types of lymphoid and myeloid cells origin have been generated in vitro and tested in animal models. Their efficacy to decrease pharmacological treatment has successfully been established. Macrophages play an important role in physiological and pathological processes. They represent an interesting cell population due to their high plasticity in vivo and in vitro. Here, we describe a protocol to differentiate murine regulatory macrophages in vitro from bone marrow precursors. We also describe several methods to assess macrophage classical functions, as their bacterial killing capacity and antigen endocytosis and degradation. Importantly, regulatory macrophages also display suppressive characteristics, which are addressed by the study of their hypostimulatory T lymphocyte capacity and polyclonal T lymphocyte activation suppression.

Regulatory B Cells with a Partial Defect in CD40 Signaling and Overexpressing Granzyme B Transfer Allograft Tolerance in Rodents.

Emerging knowledge regarding B cells in organ transplantation has demonstrated that these cells can no longer be taken as mere generators of deleterious Abs but can also act as beneficial players. We previously demonstrated in a rat model of cardiac allograft tolerance induced by short-term immunosuppression an accumulation in the blood of B cells overexpressing inhibitory molecules, a phenotype also observed in the blood of patients that spontaneously develop graft tolerance. In this study, we demonstrated the presence in the spleen of regulatory B cells enriched in the CD24(int)CD38(+)CD27(+)IgD(-)IgM(+/low) subpopulation, which are able to transfer donor-specific tolerance via IL-10 and TGF-ß1-dependent mechanisms and to suppress in vitro TNF-α secretion. Following anti-CD40 stimulation, IgD(-)IgM(+/low) B cells were blocked in their plasma cell differentiation pathway, maintained high expression of the inhibitory molecules CD23 and Bank1, and upregulated Granzyme B and Irf4, two molecules described as highly expressed by regulatory B cells. Interestingly, these B cells recognized specifically a dominant donor Ag, suggesting restricted specificity that could lead to a particular B cell response. Regulatory B cells were not required for induction of tolerance and appeared following Foxp3(+)CD4(+)CD25(+) regulatory T cells, suggesting cooperation with regulatory T cells for their expansion. Nevertheless, following transfer to new recipients, these B cells migrated to the allograft, kept their regulatory profile, and promoted local accumulation of Foxp3(+)CD4(+)CD25(+) regulatory T cells. Mechanisms of regulatory B cells and their cell therapy potential are important to decipher in experimental models to pave the way for future developments in the clinic.

Evaluation of the therapeutic potential of bone marrow-derived myeloid suppressor cell (MDSC) adoptive transfer in mouse models of autoimmunity and allograft rejection.

Therapeutic use of immunoregulatory cells represents a promising approach for the treatment of uncontrolled immunity. During the last decade, myeloid-derived suppressor cells (MDSC) have emerged as novel key regulatory players in the context of tumor growth, inflammation, transplantation or autoimmunity. Recently, MDSC have been successfully generated in vitro from naive mouse bone marrow cells or healthy human PBMCs using minimal cytokine combinations. In this study, we aimed to evaluate the potential of adoptive transfer of such cells to control auto- and allo-immunity in the mouse. Culture of bone marrow cells with GM-CSF and IL-6 consistently yielded a majority of CD11b+Gr1hi/lo cells exhibiting strong inhibition of CD8+ T cell proliferation in vitro. However, adoptive transfer of these cells failed to alter antigen-specific CD8+ T cell proliferation and cytotoxicity in vivo. Furthermore, MDSC could not prevent the development of autoimmunity in a stringent model of type 1 diabetes. Rather, loading the cells prior to injection with a pancreatic neo-antigen peptide accelerated the development of the disease. Contrastingly, in a model of skin transplantation, repeated injection of MDSC or single injection of LPS-activated MDSC resulted in a significant prolongation of allograft survival. The beneficial effect of MDSC infusions on skin graft survival was paradoxically not explained by a decrease of donor-specific T cell response but associated with a systemic over-activation of T cells and antigen presenting cells, prominently in the spleen. Taken together, our results indicate that in vitro generated MDSC bear therapeutic potential but will require additional in vitro factors or adjunct immunosuppressive treatments to achieve safe and more robust immunomodulation upon adoptive transfer.

Generation and in vivo evaluation of IL10-treated dendritic cells in a nonhuman primate model of AAV-based gene transfer.

Preventing untoward immune responses against a specific antigen is a major challenge in different clinical settings such as gene therapy, transplantation, or autoimmunity. Following intramuscular delivery of recombinant adeno-associated virus (rAAV)-derived vectors, transgene rejection can be a roadblock to successful clinical translation. Specific immunomodulation strategies potentially leading to sustained transgene expression while minimizing pharmacological immunosuppression are desirable. Tolerogenic dendritic cells (TolDC) are potential candidates but have not yet been evaluated in the context of gene therapy, to our knowledge. Following intramuscular delivery of rAAV-derived vectors expressing an immunogenic protein in the nonhuman primate model, we assessed the immunomodulating potential of autologous bone marrow-derived TolDC generated in the presence of IL10 and pulsed with the transgene product. TolDC administered either intradermally or intravenously were safe and well tolerated. While the intravenous route showed a modest ability to modulate host immunity against the transgene product, intradermally delivery resulted in a robust vaccination of the macaques when associated to intramuscular rAAV-derived vectors-based gene transfer. These findings demonstrate the critical role of TolDC mode of injection in modulating host immunity. This study also provides the first evidence of the potential of TolDC-based immunomodulation in gene therapy.

Effector mechanisms of rejection.

Organ transplantation appears today to be the best alternative to replace the loss of vital organs induced by various diseases. Transplants can, however, also be rejected by the recipient. In this review, we provide an overview of the mechanisms and the cells/molecules involved in acute and chronic rejections. T cells and B cells mainly control the antigen-specific rejection and act either as effector, regulatory, or memory cells. On the other hand, nonspecific cells such as endothelial cells, NK cells, macrophages, or polymorphonuclear cells are also crucial actors of transplant rejection. Last, beyond cells, the high contribution of antibodies, chemokines, and complement molecules in graft rejection is discussed in this article. The understanding of the different components involved in graft rejection is essential as some of them are used in the clinic as biomarkers to detect and quantify the level of rejection.

Immunoregulatory function of IL-27 and TGF-ß1 in cardiac allograft transplantation.

BACKGROUND: Deciphering the mechanisms of tolerance represents a crucial aim of research in transplantation. We previously identified by DNA chip interleukin (IL)-27 p28 and transforming growth factor (TGF)-ß1 as overexpressed in a model of rat cardiac allograft tolerance mediated by regulatory CD4CD25 T cells. The role of these two molecules on the control of the inflammatory response remains controversial. However, both are involved in the regulation of the T helper 17/Treg axis, suggesting their involvement in tolerance. METHODS: We analyzed regulation of IL-27 and TGF-ß1 expression in allograft response and their role in tolerance by using blocking anti-TGF-ß antibody and by generating an adeno-associated virus encoding IL-27. RESULTS: Here, we confirmed the overexpression of IL-27 and TGF-ß1 in tolerated cardiac allografts in two different rodent models. We observed that their expression correlates with inhibition of T helper 17 differentiation and with expansion of regulatory CD4CD25 T cells. We showed in a rat model that anti-TGF-ß treatment abrogates infectious tolerance mediated by the transfer of regulatory CD4CD25 T cells. Moreover, overexpression of IL-27 by adeno-associated virus administration in combination with a short-term immunosuppression allows prolongation of cardiac allograft survival and one tolerant recipient. We found that IL-27 overexpression did not induce Foxp3CD4CD25 T-cell expansion but rather IL-10-expressing CD4 T cells in the tolerant recipient. CONCLUSIONS: Taken together, these data suggest that both TGF-ß1 and IL-27 play a role in the mechanisms of tolerance. However, in contrast to TGF-ß1, IL-27 seems not to be involved in regulatory CD4CD25 T-cell expansion but rather in their mode of action.

Preparation of mouse bone marrow-derived dendritic cells with immunoregulatory properties.

Tolerogenic dendritic cells (Tol-DCs) are critical players in physiological tolerance. Moreover, they also play a role in immune regulation both in a pathophysiological context and when used as therapeutic tools in cell therapy strategies. Here, we describe a protocol to differentiate murine Tol-DCs from bone marrow precursors in vitro. Importantly, Tol-DCs actively suppress T cells stimulated with immunogenic allogeneic DCs. Indeed, Tol-DCs generated using this approach can be useful in studying and characterising the immunoregulatory pathways of Tol-DC and in developing Tol-DC-based cell therapy protocols using in vivo models.

Mechanism and localization of CD8 regulatory T cells in a heart transplant model of tolerance.

Despite accumulating evidence for the importance of allospecific CD8(+) regulatory T cells (Tregs) in tolerant rodents and free immunosuppression transplant recipients, mechanisms underlying CD8(+) Treg-mediated tolerance remain unclear. By using a model of transplantation tolerance mediated by CD8(+) Tregs following CD40Ig treatment in rats, in this study, we show that the accumulation of tolerogenic CD8(+) Tregs and plasmacytoid dendritic cells (pDCs) in allograft and spleen but not lymph nodes was associated with tolerance induction in vascularized allograft recipients. pDCs preferentially induced tolerogenic CD8(+) Tregs to suppress CD4(+) effector cells responses to first-donor Ags in vitro. When tolerogenic CD8(+) Tregs were not in contact with CD4(+) effector cells, suppression was mediated by IDO. Contact with CD4(+) effector cells resulted in alternative suppressive mechanisms implicating IFN-gamma and fibroleukin-2. In vivo, both IDO and IFN-gamma were involved in tolerance induction, suggesting that contact with CD4(+) effector cells is crucial to modulate CD8(+) Tregs function in vivo. In conclusion, CD8(+) Tregs and pDCs interactions were necessary for suppression of CD4(+) T cells and involved different mechanisms modulated by the presence of cell contact between CD8(+) Tregs, pDCs, and CD4(+) effector cells.

Tmem176B and Tmem176A are associated with the immature state of dendritic cells.

DCs play a central role in the development of innate and adaptive immunity but also in the induction and maintenance of immune tolerance. Identification of factors that govern DC activation, their maturation state, and their capacity to induce proinflammatory or tolerogeneic responses therefore represents a crucial aim of research. We previously identified a new molecule, Tmem176B (which we named TORID initially), as highly expressed in a model of allograft tolerance in the rat. We showed that its overexpression in rat DCs blocked their maturation, suggesting a role for this molecule in the maturation process. To characterize the function of Tmem176B further, we used a split-ubiquitin yeast, two-hybrid system to identify interacting partners and found that Tmem176B associated with itself but also with Tmem176A, a membrane protein similar to Tmem176B. Interestingly, these two molecules showed similar mRNA expression patterns among various murine tissues and immune cells and were both down-regulated following DC maturation. In addition, we showed that in using RNAi, these molecules are both involved in the maintenance of the immature state of the DCs. Taken together, these data suggest that Tmem176B and Tmem176A associate to form multimers and restrain DC maturation. Therefore, these two molecules may represent valid targets to regulate DC function.

Characterization of Schlafen-3 expression in effector and regulatory T cells.

Members of the Slfn protein family have been implicated in the regulation of cell growth, hematopoietic cell differentiation, and T cell development/differentiation in the thymus. Ten members of this family have been described in the mouse, and they have been divided into three subgroups based on the overall sequence homology and the size of the encoded proteins. We have identified Slfn3, a member of Subgroup II, as an overexpressed gene in CD4(+) CD25(+) T cells in the periphery. Interestingly, we demonstrate that upon activation and proliferation, Slfn3 mRNA is down-regulated in CD4(+) CD25(+) Tregs and up-regulated in CD4(+) CD25(-) Teffs. Moreover, TGF-beta inhibits the expression of Slfn3 in anti-CD3/CD28-activated CD4+ T cells, and the same conditions induce FoxP3 mRNA. Our results suggest that Slfn3 could have a role in T cell differentiation and activation.

The C-type lectin-like receptor CLEC-1, expressed by myeloid cells and endothelial cells, is up-regulated by immunoregulatory mediators and moderates T cell activation.

C-type lectin receptors have recently been described as playing crucial roles in immunity and homeostasis since these proteins are able to recognize pathogens as well as self-Ags. We identified the C-type lectin-like receptor-1, CLEC-1, as being overexpressed in a model of rat allograft tolerance. We previously described in this model the expression of numerous cytoprotective molecules by graft endothelial cells and their interplay with regulatory CD4(+)CD25(+) T cells. In this study, we demonstrate that CLEC-1 is expressed by myeloid cells and specifically by endothelial cells in tolerated allografts and that CLEC-1 expression can be induced in endothelial cells by alloantigen-specific regulatory CD4(+)CD25(+) T cells. Analysis of CLEC-1 expression in naive rats demonstrates that CLEC-1 is highly expressed by myeloid cells and at a lower level by endothelial cells, and that its expression is down-regulated by inflammatory stimuli but increased by the immunoregulators IL-10 or TGFbeta. Interestingly, we demonstrate in vitro that inhibition of CLEC-1 expression in rat dendritic cells increases the subsequent differentiation of allogeneic Th17 T cells and decreases the regulatory Foxp3(+) T cell pool. Additionally, in chronically rejected allograft, the decreased expression of CLEC-1 is associated with a higher production of IL-17. Taken together, our data suggest that CLEC-1, expressed by myeloid cells and endothelial cells, is enhanced by regulatory mediators and moderates Th17 differentiation. Therefore, CLEC-1 may represent a new therapeutic agent to modulate the immune response in transplantation, autoimmunity, or cancer settings.

Superiority of bone marrow-derived dendritic cells over monocyte-derived ones for the expansion of regulatory T cells in the macaque.

Regulatory T cells (Treg) have been identified as playing a pivotal role in the control of tolerance and in the suppression of pathologic immune responses in autoimmune diseases, transplantation, and graft-versus-host disease. Treg expanded ex vivo by dendritic cells could be potential reagents to promote antigen-specific tolerance in vivo. However, in vivo studies have been carried out mostly in rodents and will need validation in primates before clinical application. We characterized macaque dendritic cell derived either from bone marrow with and without prior CD34+ cell selection (BMDC), or from CD14+ peripheral blood mononuclear cells (Mo-DC). We demonstrate that with a semi-mature phenotype, BMDC are superior to Mo-DC in their capacity to expand freshly isolated allogeneic macaque CD4+ CD25+ CD127- Foxp3+ Treg in vitro in the presence of interleukin-2. Moreover, the expanded Treg maintain their phenotype and suppressive activity. These data provide a step toward the use of macaque dendritic cell to expand Treg for future preclinical testing.