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Both CD4 + and CD8 + Tregs play a critical role in the control of immune responses and immune tolerance; however, our understanding of CD8 + Tregs is limited while they are particularly promising for therapeutic application. We report here existence of highly suppressive human CD8 + CD45RC low/- Tregs expressing Foxp3 and producing IFNγ, IL-10, IL-34, and TGFβ to mediate their suppressive activity. We demonstrate that total CD8 + CD45RC low/- Tregs can be efficiently expanded in the presence of anti-CD3/28 mAbs, high-dose IL-2 and IL-15 and that such expanded Tregs efficiently delay GVHD and human skin transplantation rejection in immune humanized mice. Robustly expanded CD8 + Tregs displayed a specific gene signature, upregulated cytokines and expansion in the presence of rapamycin greatly improved proliferation and suppression. We show that CD8 + CD45RC low/- Tregs are equivalent to canonical CD4 + CD25 high CD127 low/- Tregs for suppression of allogeneic immune responses in vitro . Altogether, our results open new perspectives to tolerogenic strategies in human solid organ transplantation and GVHD.

The generation of gene-edited animals using the CRISPRs/Cas9 system is based on microinjection into zygotes which is inefficient, time consuming and demands high technical skills. We report the optimization of an electroporation method for intact rat zygotes using sgRNAs and Cas9 protein in combination or not with ssODNs (~100 nt). This resulted in high frequency of knockouts, between 15 and 50% of analyzed animals. Importantly, using ssODNs as donor template resulted in precise knock-in mutations in 25-100% of analyzed animals, comparable to microinjection. Electroporation of long ssDNA or dsDNA donors successfully used in microinjection in the past did not allow generation of genome-edited animals despite dsDNA visualization within zygotes. Thus, simultaneous electroporation of a large number of intact rat zygotes is a rapid, simple, and efficient method for the generation of a variety of genome-edited rats.

Developing protocols aimed at inhibiting effector T cells would be key for the treatment of T cell-dependent autoimmune diseases including type 1 autoimmune diabetes (T1D) and multiple sclerosis (MS). While heme oxygenase-1 (HO-1) inducers are clinically approved drugs for non-immune-related diseases, they do have immunosuppressive properties when administered systemically in rodents. Here we show that HO-1 inducers inhibit antigen-specific effector T cells when injected intradermally together with the T cell cognate antigens in mice. This phenomenon was observed in both a CD8 + T cell-mediated model of T1D and in a CD4 + T cell-dependent MS model. Intradermal injection of HO-1 inducers induced the recruitment of HO-1 + monocyte-derived dendritic cell (MoDCs) exclusively to the lymph nodes (LN) draining the site of intradermal injection. After encountering HO-1 + MoDCs, effector T-cells exhibited a lower velocity and a reduced ability to migrate towards chemokine gradients resulting in impaired accumulation to the inflamed organ. Intradermal co-injection of a clinically approved HO-1 inducer and a specific antigen to non-human primates also induced HO-1 + MoDCs to accumulate in dermal draining LN and to suppress delayed-type hypersensitivity. Therefore, in both mice and non-human primates, HO-1 inducers delivered locally inhibited effector T-cells in an antigen-specific manner, paving the way for repositioning these drugs for the treatment of immune-mediated diseases., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Human respiratory syncytial virus (hRSV) is the leading cause of severe lower respiratory tract infections in children. The development of novel prophylactic and therapeutic antiviral drugs against hRSV is imperative to control the burden of disease in the susceptible population. In this study, we examined the effects of inducing the activity of the host enzyme heme oxygenase-1 (HO-1) on hRSV replication and pathogenesis on lung inflammation induced by this virus. Our results show that after hRSV infection, HO-1 induction with metalloporphyrin cobalt protoporphyrin IX significantly reduces the loss of body weight due to hRSV-induced disease. Further, HO-1 induction also decreased viral replication and lung inflammation, as evidenced by a reduced neutrophil infiltration into the airways, with diminished cytokine and chemokine production and reduced T cell function. Concomitantly, upon cobalt protoporphyrin IX treatment, there is a significant upregulation in the production of IFN-α/β mRNAs in the lungs. Furthermore, similar antiviral and protective effects occur by inducing the expression of human HO-1 in MHC class II + cells in transgenic mice. Finally, in vitro data suggest that HO-1 induction can modulate the susceptibility of cells, especially the airway epithelial cells, to hRSV infection., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Interleukin-34 is a cytokine with only partially understood functions, described for the first time in 2008. Although IL-34 shares very little homology with CSF-1 (CSF1, M-CSF), they share a common receptor CSF-1R (CSF-1R) and IL-34 has also two distinct receptors (PTP-ζ) and CD138 (syndecan-1). To make the situation more complex, IL-34 has also been shown as pairing with CSF-1 to form a heterodimer. Until now, studies have demonstrated that this cytokine is released by some tissues that differ to those where CSF-1 is expressed and is involved in the differentiation and survival of macrophages, monocytes, and dendritic cells in response to inflammation. The involvement of IL-34 has been shown in areas as diverse as neuronal protection, autoimmune diseases, infection, cancer, and transplantation. Our recent work has demonstrated a new and possible therapeutic role for IL-34 as a Foxp3 + Treg-secreted cytokine mediator of transplant tolerance. In this review, we recapitulate most recent findings on IL-34 and its controversial effects on immune responses and address its immunoregulatory properties and the potential of targeting this cytokine in human.

Dendritic cells (DCs) represent essential antigen-presenting cells that are critical for linking innate and adaptive immunity, and influencing T-cell responses. Among pattern recognition receptors, DCs express C-type lectin receptors triggered by both exogenous and endogenous ligands, therefore dictating pathogen response, and also shaping T-cell immunity. We previously described in rat, the expression of the orphan C-type lectin-like receptor-1 (CLEC-1) by DCs and demonstrated in vitro its inhibitory role in downstream T helper 17 (Th17) activation. In this study, we examined the expression and functionality of CLEC-1 in human DCs, and show a cell-surface expression on the CD16 - subpopulation of blood DCs and on monocyte-derived DCs (moDCs). CLEC-1 expression on moDCs is downregulated by inflammatory stimuli and enhanced by transforming growth factor β. Moreover, we demonstrate that CLEC-1 is a functional receptor on human moDCs and that although not modulating the spleen tyrosine kinase-dependent canonical nuclear factor-κB pathway, represses subsequent Th17 responses. Interestingly, a decreased expression of CLEC1A in human lung transplants is predictive of the development of chronic rejection and is associated with a higher level of interleukin 17A ( IL17A ). Importantly, using CLEC-1-deficient rats, we showed that disruption of CLEC-1 signaling led to an enhanced Il12p40 subunit expression in DCs, and to an exacerbation of downstream in vitro and in vivo CD4 + Th1 and Th17 responses. Collectively, our results establish a role for CLEC-1 as an inhibitory receptor in DCs able to dampen activation and downstream effector Th responses. As a cell-surface receptor, CLEC-1 may represent a useful therapeutic target for modulating T-cell immune responses in a clinical setting., Competing Interests: Conflict-of-interest disclosure: The authors declare no competing financial interests.

Rat and human CD4 + and CD8 + Tregs expressing low levels of CD45RC have strong immunoregulatory properties. We describe here that human CD45 isoforms are nonredundant and identify distinct subsets of cells. We show that CD45RC is not expressed by CD4 + and CD8 + Foxp3 + Tregs, while CD45RA/RB/RO are. Transient administration of a monoclonal antibody (mAb) targeting CD45RC in a rat cardiac allotransplantation model induced transplant tolerance associated with inhibition of allogeneic humoral responses but maintained primary and memory responses against cognate antigens. Anti-CD45RC mAb induced rapid death of CD45RC high T cells through intrinsic cell signaling but preserved and potentiated CD4 + and CD8 + CD45RC low/- Tregs, which are able to adoptively transfer donor-specific tolerance to grafted recipients. Anti-CD45RC treatment results in distinct transcriptional signature of CD4 + and CD8 + CD45RC low/- Tregs. Finally, we demonstrate that anti-human CD45RC treatment inhibited graft-versus-host disease (GVHD) in immune-humanized NSG mice. Thus, short-term anti-CD45RC is a potent therapeutic candidate to induce transplantation tolerance in human., Competing Interests: CG and IA have submitted a patent (WO2016016442) that is pending.

Background: There is an ever-increasing need of monoclonal antibodies (mAbs) for biomedical applications and fully human binders are particularly desirable due to their reduced immunogenicity in patients. We have applied a strategy for the isolation of antigen-specific B cells using tetramerized proteins and single-cell sorting followed by reconstruction of human mAbs by RT-PCR and expression cloning., Results: This strategy, using human peripheral blood B cells, enabled the production of low affinity human mAbs against major histocompatibility complex molecules loaded with peptides (pMHC). We then implemented this technology using human immunoglobulin transgenic rats, which after immunization with an antigen of interest express high affinity-matured antibodies with human idiotypes. Using rapid immunization, followed by tetramer-based B-cell sorting and expression cloning, we generated several fully humanized mAbs with strong affinities, which could discriminate between highly homologous proteins (eg. different pMHC complexes)., Conclusions: Therefore, we describe a versatile and more effective approach as compared to hybridoma generation or phage or yeast display technologies for the generation of highly specific and discriminative fully human mAbs that could be useful both for basic research and immunotherapeutic purposes.

BAC transgenic mammalian systems offer an important platform for recapitulating human gene expression and disease modeling. While the larger body mass, and greater genetic and physiologic similarity to humans render rats well suited for reproducing human immune diseases and evaluating therapeutic strategies, difficulties of generating BAC transgenic rats have hindered progress. Thus, an efficient method for BAC transgenesis in rats would be valuable. Immunodeficient mice carrying a human SIRPA transgene have previously been shown to support improved human cell hematopoiesis. Here, we have generated for the first time, human SIRPA BAC transgenic rats, for which the gene is faithfully expressed, functionally active, and germline transmissible. To do this, human SIRPA BAC was modified with elements to work in coordination with genome engineering technologies-piggyBac, CRISPR/Cas9 or TALEN. Our findings show that piggyBac transposition is a more efficient approach than the classical BAC transgenesis, resulting in complete BAC integration with predictable end sequences, thereby permitting precise assessment of the integration site. Neither CRISPR/Cas9 nor TALEN increased BAC transgenesis. Therefore, an efficient generation of human SIRPA transgenic rats using piggyBac opens opportunities for expansion of humanized transgenic rat models in the future to advance biomedical research and therapeutic applications.

The recent emergence and application of engineered endonucleases have led to the development of genome editing tools capable of rapidly implementing various targeted genome editions in a wide range of species. Moreover, these novel tools have become easier to use and have resulted in a great increase of applications. Whilst gene knockout (KO) or knockin (KI) animal models are relatively easy to achieve, there is a bottleneck in the detection and analysis of these mutations. Although several methods exist to detect these targeted mutations, we developed a heteroduplex mobility assay on an automated microfluidic capillary electrophoresis system named HMA-CE in order to accelerate the genotyping process. The HMA-CE method uses a simple PCR amplification of genomic DNA (gDNA) followed by an automated capillary electrophoresis step which reveals a heteroduplexes (HD) signature for each mutation. This allows efficient discrimination of wild-type and genome-edited animals down to the single base pair level., (Copyright © 2016 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

Genome editing has now been reported in many systems using TALEN and CRISPR-Cas9 nucleases. Precise mutations can be introduced during homology-directed repair with donor DNA carrying the wanted sequence edit, but efficiency is usually lower than for gene knockout and optimal strategies have not been extensively investigated. Here, we show that using phosphorothioate-modified oligonucleotides strongly enhances genome editing efficiency of single-stranded oligonucleotide donors in cultured cells. In addition, it provides better design flexibility, allowing insertions more than 100 bp long. Despite previous reports of phosphorothioate-modified oligonucleotide toxicity, clones of edited cells are readily isolated and targeted sequence insertions are achieved in rats and mice with very high frequency, allowing for homozygous loxP site insertion at the mouse ROSA locus in particular. Finally, when detected, imprecise knockin events exhibit indels that are asymmetrically positioned, consistent with genome editing taking place by two steps of single-strand annealing., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The rat is an important animal model to understand gene function and model human diseases. Since recent years, the development of gene-specific nucleases has become important for generating new rat models of human diseases, to analyze the role of genes and to generate human antibodies. Transcription activator-like (TALE) nucleases efficiently create gene-specific knockout rats and lead to the possibility of gene targeting by homology-directed recombination (HDR) and generating knock-in rats. We describe a detailed protocol for generating knockout and knock-in rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models.

In transplantation tolerance, numerous regulatory populations have the capacity to inhibit allograft rejection; however, their compensatory capacities have never been clearly evidenced. We have previously demonstrated that the tolerogenic effect mediated by CD8(+)CD45RC(low) regulatory T cells (Tregs) in a model of organ transplantation with CD40Ig could be abrogated by permanent depletion of CD8(+) cells that resulted in allograft rejection in half of the recipients. This result demonstrated that CD8(+) Tregs were essential, but also that half of the recipients still survived indefinitely. We also demonstrated that no other regulatory populations, besides CD8(+) Tregs, could induce and maintain allograft tolerance in CD40Ig-treated tolerant animals. In the current study, we analyzed the mechanisms that arose following CD8(+) Treg depletion and allowed establishment of networks of new regulatory cells to maintain allograft survival. We identified regulatory B cells (Bregs) and regulatory myeloid cells (RegMCs) as being responsible of the maintenance of the long-term allograft survival. We demonstrated that both regulatory cell subsets efficiently inhibited antidonor immune responses in adoptively transferred recipients. Although Bregs were induced, they were not essential for the maintenance of the graft as demonstrated in IgM-deficient recipients. In addition, we showed that RegMCs were the most suppressive and acted alone, whereas Bregs activity was associated with increased suppressive activity of other subsets in adoptively transferred recipients. Altogether, to our knowledge, we demonstrated in this study for the first time the emergence of both Bregs and RegMCs following Tregs depletion and highlighted the importance of regulatory cell networks and their synergistic potential in transplantation., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Heme-oxygenase 1 (HO-1) prevents T cell-mediated inflammatory disease by producing carbon monoxide (CO) and impairing DC immunogenicity. However, the cellular mechanisms causing this inhibition are unknown. Here, we show that CO impairs mitochondrial function in DCs by reducing both the mitochondrial membrane potential and ATP production, and resembling the effect of a nonlethal dose of a classical mitochondria uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Moreover, both CO and CCCP reduced cargo transport, endosome-to-lysosome fusion, and antigen processing, dampening the production of peptide-MHC complexes on the surface of DCs. As a result, the inhibition of naive CD4(+) T-cell priming was observed. Furthermore, mitochondrial dysfunction in DCs also significantly reduced CD8(+) T cell-dependent type 1 diabetes onset in vivo. These results showed for the first time that CO interferes with T-cell priming by blocking an unknown mitochondria-dependent antigen-processing pathway in mature DC. Interestingly, other immune functions in DCs such as antigen capture, cytokine secretion, costimulation, and cell survival relied on glycolysis, suggesting that oxidative phosphorylation might only play a key role for the maturation of antigen-containing endosomes. In conclusion, CO produced by HO-1 impairs antigen-dependent inflammation by regulating DC immunogenicity by a mitochondria-dependent mechanism., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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-β1-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., (Copyright © 2015 by The American Association of Immunologists, Inc.)

The generation of genetically-modified organisms has been revolutionized by the development of new genome editing technologies based on the use of gene-specific nucleases, such as meganucleases, ZFNs, TALENs and CRISPRs-Cas9 systems. The most rapid and cost-effective way to generate genetically-modified animals is by microinjection of the nucleic acids encoding gene-specific nucleases into zygotes. However, the efficiency of the procedure can still be improved. In this work we aim to increase the efficiency of CRISPRs-Cas9 and TALENs homology-directed repair by using TALENs and Cas9 proteins, instead of mRNA, microinjected into rat and mouse zygotes along with long or short donor DNAs. We observed that Cas9 protein was more efficient at homology-directed repair than mRNA, while TALEN protein was less efficient than mRNA at inducing homology-directed repair. Our results indicate that the use of Cas9 protein could represent a simple and practical methodological alternative to Cas9 mRNA in the generation of genetically-modified rats and mice as well as probably some other mammals.

Cytokines and metabolic pathway-controlling enzymes regulate immune responses and have potential as powerful tools to mediate immune tolerance. Blockade of the interaction between CD40 and CD40L induces long-term cardiac allograft survival in rats through a CD8+CD45RClo Treg potentiation. Here, we have shown that the cytokine IL-34, the immunoregulatory properties of which have not been previously studied in transplantation or T cell biology, is expressed by rodent CD8+CD45RClo Tregs and human FOXP3+CD45RCloCD8+ and CD4+ Tregs. IL-34 was involved in the suppressive function of both CD8+ and CD4+ Tregs and markedly inhibited alloreactive immune responses. Additionally, in a rat cardiac allograft model, IL-34 potently induced transplant tolerance that was associated with a total inhibition of alloantibody production. Treatment of rats with IL-34 promoted allograft tolerance that was mediated by induction of CD8+ and CD4+ Tregs. Moreover, these Tregs were capable of serial tolerance induction through modulation of macrophages that migrate early to the graft. Finally, we demonstrated that human macrophages cultured in the presence of IL-34 greatly expanded CD8+ and CD4+ FOXP3+ Tregs, with a superior suppressive potential of antidonor immune responses compared with non-IL-34-expanded Tregs. In conclusion, we reveal that IL-34 serves as a suppressive Treg-specific cytokine and as a tolerogenic cytokine that efficiently inhibits alloreactive immune responses and mediates transplant tolerance.

Background: The vascular remodeling responsible for pulmonary arterial hypertension (PAH) involves predominantly the accumulation of α-smooth muscle actin-expressing mesenchymal-like cells in obstructive pulmonary vascular lesions. Endothelial-to-mesenchymal transition (EndoMT) may be a source of those α-smooth muscle actin-expressing cells., Methods and Results: In situ evidence of EndoMT in human PAH was obtained by using confocal microscopy of multiple fluorescent stainings at the arterial level, and by using transmission electron microscopy and correlative light and electron microscopy at the ultrastructural level. Findings were confirmed by in vitro analyses of human PAH and control cultured pulmonary artery endothelial cells. In addition, the mRNA and protein signature of EndoMT was recognized at the arterial and lung level by quantitative real-time polymerase chain reaction and Western blot analyses. We confirmed our human observations in established animal models of pulmonary hypertension (monocrotaline and SuHx). After establishing the first genetically modified rat model linked to BMPR2 mutations (BMPR2(Δ140Ex1/+) rats), we demonstrated that EndoMT is linked to alterations in signaling of BMPR2, a gene that is mutated in 70% of cases of familial PAH and in 10% to 40% of cases of idiopathic PAH. We identified molecular actors of this pathological transition, including twist overexpression and vimentin phosphorylation. We demonstrated that rapamycin partially reversed the protein expression patterns of EndoMT, improved experimental PAH, and decreased the migration of human pulmonary artery endothelial cells, providing the proof of concept that EndoMT is druggable., Conclusions: EndoMT is linked to alterations in BPMR2 signaling and is involved in the occlusive vas cular remodeling of PAH, findings that may have therapeutic implications., (© 2015 American Heart Association, Inc.)

We previously described that in a rat model of heart transplantation tolerance was dependent on CD8+CD45RClow Tregs that over-expressed fibrinogen-like protein 2 (FGL2)/fibroleukin. Little is known on the immunoregulatory properties of FGL2. Here we analyzed the transplantation tolerance mechanisms that are present in Lewis 1A rats treated with FGL2. Over-expression of FGL2 in vivo through adenovirus associated virus -mediated gene transfer without any further treatment resulted in inhibition of cardiac allograft rejection. Adoptive cell transfer of splenocytes from FGL2-treated rats with long-term graft survival (> 80 days) in animals that were transplanted with cardiac allografts inhibited acute and chronic organ rejection in a donor-specific and transferable tolerance manner, since iterative adoptive transfer up to a sixth consecutive recipient resulted in transplantation tolerance. Adoptive cell transfer also efficiently inhibited anti-donor antibody production. Analysis of all possible cell populations among splenocytes revealed that B lymphocytes were sufficient for this adoptive cell tolerance. These B cells were also capable of inhibiting the proliferation of CD4+ T cells in response to allogeneic stimuli. Moreover, gene transfer of FGL2 in B cell deficient rats did not prolong graft survival. Thus, this is the first description of FGL2 resulting in long-term allograft survival. Furthermore, allograft tolerance was transferable and B cells were the main cells responsible for this effect.

Background: Polarized airway epithelial cell cultures modelling Cystic Fibrosis Transmembrane conductance Regulator (CFTR) defect are crucial for CF and biomedical research. RNA interference has proven its value to generate knockdown models for various pathologies. More recently, genome editing using CRISPR-Cas9 artificial endonuclease was a valuable addition to the toolbox of gene inactivation., Methods: Calu-3 cells and primary HAECs were transduced with HIV-1-derived lentiviral vectors (LVV) encoding small hairpin RNA (shRNA) sequence or CRISPR-Cas9 components targeting CFTR alongside GFP. After sorting of GFP-positive cells, CFTR expression was measured by RT-qPCR and Western blot in polarized or differentiated cells. CFTR channel function was assessed in Ussing chambers. Il-8 secretion, proliferation and cell migration were also studied in transduced cells., Results: shRNA interference and CRISPRCas9 strategies efficiently decreased CFTR expression in Calu-3 cells. Strong CFTR knockdown was confirmed at the functional level in CRISPR-Cas9-modified cells. CFTR-specific shRNA sequences did not reduce gene expression in primary HAECs, whereas CRISPR-Cas9-mediated gene modification activity was correlated with a reduction of transepithelial secretion and response to a CFTR inhibitor. CFTR inactivation in the CRISPR-Cas9-modified Calu-3 cells did not affect migration and proliferation but slightly increased basal interleukin-8 secretion., Conclusion: We generated CFTR inactivated cell lines and demonstrated that CRISPR-Cas9 vectorised in a single LVV efficiently promotes CFTR inactivation in primary HAECs. These results provide a new protocol to engineer CF primary epithelia with their isogenic controls and pave the way for manipulation of CFTR expression in these cultures.

A few animal models of Duchenne muscular dystrophy (DMD) are available, large ones such as pigs or dogs being expensive and difficult to handle. Mdx (X-linked muscular dystrophy) mice only partially mimic the human disease, with limited chronic muscular lesions and muscle weakness. Their small size also imposes limitations on analyses. A rat model could represent a useful alternative since rats are small animals but 10 times bigger than mice and could better reflect the lesions and functional abnormalities observed in DMD patients. Two lines of Dmd mutated-rats (Dmdmdx) were generated using TALENs targeting exon 23. Muscles of animals of both lines showed undetectable levels of dystrophin by western blot and less than 5% of dystrophin positive fibers by immunohistochemistry. At 3 months, limb and diaphragm muscles from Dmdmdx rats displayed severe necrosis and regeneration. At 7 months, these muscles also showed severe fibrosis and some adipose tissue infiltration. Dmdmdx rats showed significant reduction in muscle strength and a decrease in spontaneous motor activity. Furthermore, heart morphology was indicative of dilated cardiomyopathy associated histologically with necrotic and fibrotic changes. Echocardiography showed significant concentric remodeling and alteration of diastolic function. In conclusion, Dmdmdx rats represent a new faithful small animal model of DMD.

The rat is a model of choice to understanding gene function and modeling human diseases. Since recent years, successful engineering technologies using gene-specific nucleases have been developed to gene edit the genome of different species, including the rat. This development has become important for the creation of new rat animals models of human diseases, analyze the role of genes and express recombinant proteins. Transcription activator-like (TALE) nucleases are designed nucleases consist of a DNA binding domain fused to a nuclease domain capable of cleaving the targeted DNA. We describe a detailed protocol for generating knockout rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner., (© 2014 Remy et al.; Published by Cold Spring Harbor Laboratory Press.)

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.

In a rat heart allograft model, preventing T cell costimulation with CD40Ig leads to indefinite allograft survival, which is mediated by the induction of CD8+CD45RClo regulatory T cells (CD8+CD40Ig Tregs) interacting with plasmacytoid dendritic cells (pDCs). The role of TCR-MHC-peptide interaction in regulating Treg activity remains a topic of debate. Here, we identified a donor MHC class II-derived peptide (Du51) that is recognized by TCR-biased CD8+CD40Ig Tregs and activating CD8+CD40Ig Tregs in both its phenotype and suppression of antidonor alloreactive T cell responses. We generated a labeled tetramer (MHC-I RT1.Aa/Du51) to localize and quantify Du51-specific T cells within rat cardiac allografts and spleen. RT1.Aa/Du51-specific CD8+CD40Ig Tregs were the most suppressive subset of the total Treg population, were essential for in vivo tolerance induction, and expressed a biased, restricted Vβ11-TCR repertoire in the spleen and the graft. Finally, we demonstrated that treatment of transplant recipients with the Du51 peptide resulted in indefinite prolongation of allograft survival. These results show that CD8+CD40Ig Tregs recognize a dominant donor antigen, resulting in TCR repertoire alterations in the graft and periphery. Furthermore, this allopeptide has strong therapeutic activity and highlights the importance of TCR-peptide-MHC interaction for Treg generation and function.

One of the major goals in the research of autoimmune diseases is to develop specific therapies to regulate the expression and function of gene products that could contribute to restoring tolerance to self-constituents and replace conventional systemic immunosuppression, which is associated with important undesired side effects. Although significant progress has been made on the understanding of the pathogenesis of autoimmunity, therapies for these ailments have not seen a change. During the last decade, different strategies such as pharmacologic or gene therapy modulation of heme oxygenase-1 (HO-1) and the administration of its metabolic product, carbon monoxide (CO), have been shown to display beneficial immunoregulatory and cytoprotective properties. In different experimental autoimmune conditions, such as Experimental autoimmune encephalomyelitis, type-1 diabetes and systemic lupus erythematosus, genetic or pharmacological modulation of HO-1, as well as delivery of CO have shown to ameliorate disease progression. Furthermore, it has been demonstrated that dendritic cell and monocyte function can be modulated by HO-1 and/or CO. In this article, recent data related to the immunoregulatory properties of HO-1/CO will be discussed, focusing on their potential therapeutic use to treat autoimmune diseases.

Induced pluripotent stem cells (iPSCs) offer certain advantages over embryonic stem cells in cell replacement therapy for a variety of neurological disorders. However, reliable procedures, whereby transplanted iPSCs can survive and differentiate into functional neurons, without forming tumors, have yet to be devised. Currently, retroviral or lentiviral reprogramming methods are often used to reprogram somatic cells. Although the use of these viruses has proven to be effective, formation of tumors often results following in vivo transplantation, possibly due to the integration of the reprogramming genes. The goal of the current study was to develop a new approach, using an adenovirus for reprogramming cells, characterize the iPSCs in vitro, and test their safety, survivability, and ability to differentiate into region-appropriate neurons following transplantation into the rat brain. To this end, iPSCs were derived from bone marrow-derived mesenchymal stem cells and tail-tip fibroblasts using a single cassette lentivirus or a combination of adenoviruses. The reprogramming efficiency and levels of pluripotency were compared using immunocytochemistry, flow cytometry, and real-time polymerase chain reaction. Our data indicate that adenovirus-generated iPSCs from tail-tip fibroblasts are as efficient as the method we used for lentiviral reprogramming. All generated iPSCs were also capable of differentiating into neuronal-like cells in vitro. To test the in vivo survivability and the ability to differentiate into region-specific neurons in the absence of tumor formation, 400,000 of the iPSCs derived from tail-tip fibroblasts that were transfected with the adenovirus pair were transplanted into the striatum of adult, immune-competent rats. We observed that these iPSCs produced region-specific neuronal phenotypes, in the absence of tumor formation, at 90 days posttransplantation. These results suggest that adenovirus-generated iPSCs may provide a safe and viable means for neuronal replacement therapies.

Background: Zinc finger nucleases (ZFNs) are promising tools for genome editing for biotechnological as well as therapeutic purposes. Delivery remains a major issue impeding targeted genome modification. Lentiviral vectors are highly efficient for delivering transgenes into cell lines, primary cells and into organs, such as the liver. However, the reverse transcription of lentiviral vectors leads to recombination of homologous sequences, as found between and within ZFN monomers., Methods: We used a codon swapping strategy to both drastically disrupt sequence identity between ZFN monomers and to reduce sequence repeats within a monomer sequence. We constructed lentiviral vectors encoding codon-swapped ZFNs or unmodified ZFNs from a single mRNA transcript. Cell lines, primary hepatocytes and newborn rats were used to evaluate the efficacy of integrative-competent (ICLV) and integrative-deficient (IDLV) lentiviral vectors to deliver ZFNs into target cells., Results: We reduced total identity between ZFN monomers from 90.9% to 61.4% and showed that a single ICLV allowed efficient expression of functional ZFNs targeting the rat UGT1A1 gene after codon-swapping, leading to much higher ZFN activity in cell lines (up to 7-fold increase compared to unmodified ZFNs and 60% activity in C6 cells), as compared to plasmid transfection or a single ICLV encoding unmodified ZFN monomers. Off-target analysis located several active sites for the 5-finger UGT1A1-ZFNs. Furthermore, we reported for the first time successful ZFN-induced targeted DNA double-strand breaks in primary cells (hepatocytes) and in vivo (liver) after delivery of a single IDLV encoding two ZFNs., Conclusion: These results demonstrate that a codon-swapping approach allowed a single lentiviral vector to efficiently express ZFNs and should stimulate the use of this viral platform for ZFN-mediated genome editing of primary cells, for both ex vivo or in vivo applications.

Expression of human antibody repertoires in transgenic animals has been accomplished by introducing large human Ig loci into mice and, more recently, a chimeric IgH locus into rats. With human VH, D and JH genes linked to the rat C-region antibody expression was significantly increased, similar to wild-type levels not found with fully human constructs. Here we compare four rat-lines containing the same human VH-region (comprising 22 VHs, all Ds and all JHs in natural configuration) but linked to different rat CH-genes and regulatory sequences. The endogenous IgH locus was silenced by zinc-finger nucleases. After breeding, all lines produced exclusively chimeric human H-chain with near normal IgM levels. However, in two lines poor IgG expression and inefficient immune responses were observed, implying that high expression, class-switching and hypermutation are linked to optimal enhancer function provided by the large regulatory region at the 3' end of the IgH locus. Furthermore, exclusion of Cδ and its downstream interval region may assist recombination. Highly diverse IgG and immune responses similar to normal rats were identified in two strains carrying diverse and differently spaced C-genes., (© 2013. Published by Elsevier B.V. All rights reserved.)

Despite the immunosuppressive, homing, and regenerative capabilities of mesenchymal stem cells (MSCs), their ability to migrate to arthritic joints and influence the course of arthritis in vivo remains poorly understood. The objective of this study was to determine if allogeneic MSCs migrate to inflamed joints in vivo and to determine if MSCs expressing the costimulation blocker cytotoxic T lymphocyte associated antigen-4 coupled to immunoglobulin-G (CTLA4Ig) could be used to ameliorate collagen induced arthritis (CIA). The migration of systemically delivered inbred mouse strain (FVB) MSCs to migrate to inflamed joints in CIA was studied using real-time quantitative polymerase chain reaction. Furthermore, the effect of BALB/c MSCs modified with an adenoviral vector to express CTLA4Ig, on T cell function in vitro and on CIA in vivo was assessed. After systemic delivery of FVB MSCs, eGFP DNA was detectable in the joints of mice with CIA confirming that some MSCs had reached to inflamed joints. BALB/c MSCs suppressed the secretion of both TNFα and IFNγ, and reduced the ratio of Th1:Th2 cytokine expression, by DBA/1 T cells in vitro irrespective of viral modification. The expression of CTLA4Ig did not augment this effect. Despite a worsening of disease scores after infusion of BALB/c MSCs in vivo, BALB/c MSCs expressing CTLA4Ig significantly delayed the onset of inflammatory arthritis in CIA. These data demonstrate that allogeneic MSCs can migrate to the inflamed joints of CIA in vivo and that genetically modified allogeneic MSCs may be considered for development of gene therapy strategies for inflammatory arthritis.

Purpose: Autologous bone grafting (BG) remains the standard reconstruction strategy for large craniofacial defects. Calcium phosphate (CaP) biomaterials, such as biphasic calcium phosphate (BCP), do not yield consistent results when used alone and must then be combined with cells through bone tissue engineering (BTE). In this context, total bone marrow (TBM) and bone marrow-derived mesenchymal stem cells (MSC) are the primary sources of cellular material used with biomaterials. However, several other BTE strategies exist, including the use of growth factors, various scaffolds, and MSC isolated from different tissues. Thus, clinicians might be unsure as to which method offers patients the most benefit. For this reason, the aim of this study was to compare eight clinically relevant BTE methods in an "all-in-one" study., Methods: We used a transgenic rat strain expressing green fluorescent protein (GFP), from which BG, TBM, and MSC were harvested. Progenitor cells were then mixed with CaP materials and implanted subcutaneously into nude mice. After eight weeks, bone formation was evaluated by histology and scanning electron microscopy, and GFP-expressing cells were tracked with photon fluorescence microscopy., Results/conclusions: Bone formation was observed in only four groups. These included CaP materials mixed with BG or TBM, in which abundant de novo bone was formed, and BCP mixed with committed cells grown in two- and three-dimensions, which yielded limited bone formation. Fluorescence microscopy revealed that only the TBM and BG groups were positive for GFP expressing-cells, suggesting that these donor cells were still present in the host and contributed to the formation of bone. Since the TBM-based procedure does not require bone harvest or cell culture techniques, but provides abundant de novo bone formation, we recommend consideration of this strategy for clinical applications.

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.

Heme oxygenase-1 (HO-1) inhibits immune responses and inflammatory reactions via the catabolism of heme into carbon monoxide (CO), Fe(2+) , and biliverdin. We have previously shown that either induction of HO-1 or treatment with exogenous CO inhibits LPS-induced maturation of dendritic cells (DCs) and protects in vivo and in vitro antigen-specific inflammation. Here, we evaluated the capacity of HO-1 and CO to regulate antigen presentation on MHC class I and MHC class II molecules by LPS-treated DCs. We observed that HO-1 and CO treatment significantly inhibited the capacity of DCs to present soluble antigens to T cells. Inhibition was restricted to soluble OVA protein, as no inhibition was observed for antigenic OVA-derived peptides, bead-bound OVA protein, or OVA as an endogenous antigen. Inhibition of soluble antigen presentation was not due to reduced antigen uptake by DCs, as endocytosis remained functional after HO-1 induction and CO treatment. On the contrary, CO significantly reduced the efficiency of fusion between late endosomes and lysosomes and not by phagosomes and lysosomes. These data suggest that HO-1 and CO can inhibit the ability of LPS-treated DCs to present exogenous soluble antigens to naïve T cells by blocking antigen trafficking at the level of late endosome-lysosome fusion., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multiple alterations affecting the normal function of immune cells, such as lymphocytes, dendritic cells (DCs) and monocytes. Although the understanding of autoimmunity has significantly increased, the breakthrough in effective therapies has been modest, making necessary the development of new therapeutic strategies. Here we propose that a new potential target for therapy is haem oxygenase-1 (HO-1), an enzyme that catalyses the degradation of the haem group into biliverdin, carbon monoxide (CO) and Fe(2+) . These products exhibit immunosuppressive and anti-inflammatory effects, which can contribute to improving tolerance during organ transplantation. Because HO-1 is highly expressed by immune cells involved in SLE pathogenesis, such as monocytes and DCs, we evaluated whether induction of HO-1 expression or the administration of CO could ameliorate disease in the FcγRIIb knockout (KO) mouse model for SLE. We found that CO administration decreased the expansion of CD11b(+) cells, prevented the decline of regulatory T cells and reduced anti-histone antibodies observed in untreated FcγRIIb KO mice. Furthermore, CO-treated animals and HO-1 induction showed less kidney damage compared with untreated mice. These data suggest that HO-1 modulation and CO administration can ameliorate autoimmunity and prevent the lupus symptoms shown by FcγRIIb KO mice, highlighting HO-1 as a potential new target for autoimmune therapy., (© 2013 John Wiley & Sons Ltd.)