Ignacio Anegon - Academia.edu (original) (raw)

Papers by Ignacio Anegon

Scientific Reports

Different mutations of the OTOF gene, encoding for otoferlin protein expressed in the cochlear in... more Different mutations of the OTOF gene, encoding for otoferlin protein expressed in the cochlear inner hair cells, induces a form of deafness that is the major cause of nonsyndromic recessive auditory neuropathy spectrum disorder in humans. We report the generation of the first large animal model of OTOF mutations using the CRISPR system associated with different Cas9 components (mRNA or protein) assisted by single strand oligodeoxynucleotides (ssODN) to induce homology-directed repair (HDR). Zygote microinjection was performed with two sgRNA targeting exon 5 and 6 associated to Cas9 mRNA or protein (RNP) at different concentrations in a mix with an ssODN template targeting HDR in exon 5 containing two STOP sequences. A total of 73 lambs were born, 13 showing indel mutations (17.8%), 8 of which (61.5%) had knock-in mutations by HDR. Higher concentrations of Cas9-RNP induced targeted mutations more effectively, but negatively affected embryo survival and pregnancy rate. This study repo...

JCI insight, 2017

Rat and human CD4+ and CD8+ Tregs expressing low levels of CD45RC have strong immunoregulatory pr... more 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 CD45RChigh T cells through intrinsic cell signaling but preserved and potentiated CD4+ and CD8+ CD45RClow/- 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+ CD45RClow/- Tregs. Finally, we demonstrate that anti-human CD45RC treatment inhibited graft-versus-h...

Previous work on blockade of CD40-CD40 ligand interaction in mice and primates with anti-CD40 lig... more Previous work on blockade of CD40-CD40 ligand interaction in mice and primates with anti-CD40 ligand mAbs has resulted in a moderate prolongation of allograft survival without the development of true allograft tolerance. In this study, we show in rats that adenovirus-mediated gene transfer of CD40Ig sequences into the graft resulted in prolonged (>200 days) expression of CD40Ig and in long-term (>300 days) survival. Recipients expressing CD40Ig displayed strongly (>90%) inhibited mixed leukocyte reactions and alloantibody production at early (days 5 and 17) and late time points (>100 day) after transplantation, but showed limited inhibition of leukocyte infiltration and cytokine production as evaluated by immunohistology at early time points (day 5). Recipients of long-surviving hearts showed donor-specific hyporesponsiveness since acceptance of second cardiac allografts was donor specific. Nevertheless, long-term allografts (>100 days) displayed signs of chronic rejection vasculopathy. Occluded vessels showed leukocyte infiltration, mainly composed of CD4 ؉ and CD8 ؉ cells, macrophages, and mast cells. These recipients also showed antidonor CTL activity. Recipients expressing CD40Ig did not show nonspecific immunosuppression, as they were able to mount anticognate immune responses that were partially inhibited at early time points and were normal thereafter. We conclude that gene transfer-mediated expression of CD40Ig resulted in a highly efficient inhibition of acute heart allograft rejection in rats. This treatment induced donor-specific inhibition of certain alloreactive mechanisms in the short-, but not the long-term, which resulted in long-term survival of allografts concomitant with the development of chronic rejection.

Journal of Leukocyte Biology

Although IL‐34 and CSF‐1 share actions as key mediators of monocytes/macrophages survival and dif... more Although IL‐34 and CSF‐1 share actions as key mediators of monocytes/macrophages survival and differentiation, they also display differences that should be identified to better define their respective roles in health and diseases. IL‐34 displays low sequence homology with CSF‐1 but has a similar general structure and they both bind to a common receptor CSF‐1R, although binding and subsequent intracellular signaling shows differences. CSF‐1R expression has been until now mainly described at a steady state in monocytes/macrophages and myeloid dendritic cells, as well as in some cancers. IL‐34 has also 2 other receptors, protein‐tyrosine phosphatase zeta (PTPζ) and CD138 (Syndecan‐1), expressed in some epithelium, cells of the central nervous system (CNS), as well as in numerous cancers. While most, if not all, of CSF‐1 actions are mediated through monocyte/macrophages, IL‐34 has also other potential actions through PTPζ and CD138. Additionally, IL‐34 and CSF‐1 are produced by different cells in different tissues. This review describes and discusses similarities and differences between IL‐34 and CSF‐1 at steady state and in pathological situations and identifies possible ways to target IL‐34, CSF‐1, and its receptors.

Frontiers in Immunology

Regulatory Tcells (Treg) are essential components of peripheral immune homeostasis. Adoptive Treg... more Regulatory Tcells (Treg) are essential components of peripheral immune homeostasis. Adoptive Treg cell therapy has shown efficacy in a variety of immune-mediated diseases in preclinical studies and is now moving from phase I/IIa to larger phase II studies aiming to demonstrate efficacy. However, hurdles such as in vivo stability and efficacy remain to be addressed. Nevertheless, preclinical models have shown that Treg function and specificity can be increased by pharmacological substances or gene modifications, and even that conventional T cells can be converted to Treg potentially providing new sources of Treg and facilitating Treg cell therapy. The exponential growth in genetic engineering techniques and their application to T cells coupled to a large body of knowledge on Treg open numerous opportunities to generate Treg with “superpowers”. This review summarizes the genetic engineering techniques available and their applications for the next-generation of Super-Treg with increase...

Frontiers in Genetics

The rat has been extensively used as a small animal model. Many genetically engineered rat models... more The rat has been extensively used as a small animal model. Many genetically engineered rat models have emerged in the last two decades, and the advent of gene-specific nucleases has accelerated their generation in recent years. This review covers the techniques and advances used to generate genetically engineered rat lines and their application to the development of rat models more broadly, such as conditional knockouts and reporter gene strains. In addition, genome-editing techniques that remain to be explored in the rat are discussed. The review also focuses more particularly on two areas in which extensive work has been done: human genetic diseases and immune system analysis. Models are thoroughly described in these two areas and highlight the competitive advantages of rat models over available corresponding mouse versions. The objective of this review is to provide a comprehensive description of the advantages and potential of rat models for addressing specific scientific questi...

Transplantation

In vivo analysis of human immune responses in immunodeficient rats.

International Journal of Molecular Sciences

Antigen-presenting cells (APCs) including dendritic cells (DCs) play a critical role in the devel... more Antigen-presenting cells (APCs) including dendritic cells (DCs) play a critical role in the development of autoimmune diseases by presenting self-antigen to T-cells. Different signals modulate the ability of APCs to activate or tolerize autoreactive T-cells. Since the expression of heme oxygenase-1 (HO-1) by APCs has been associated with the tolerization of autoreactive T-cells, we hypothesized that HO-1 expression might be altered in APCs from autoimmune-prone non-obese diabetic (NOD) mice. We found that, compared to control mice, NOD mice exhibited a lower percentage of HO-1-expressing cells among the splenic DCs, suggesting an impairment of their tolerogenic functions. To investigate whether restored expression of HO-1 in APCs could alter the development of diabetes in NOD mice, we generated a transgenic mouse strain in which HO-1 expression can be specifically induced in DCs using a tetracycline-controlled transcriptional activation system. Mice in which HO-1 expression was indu...

Frontiers in Immunology

Corticosteroids (CS) are standard therapy for the treatment of Duchenne's muscular dystrophy (DMD... more Corticosteroids (CS) are standard therapy for the treatment of Duchenne's muscular dystrophy (DMD). Even though they decrease inflammation, they have limited efficacy and are associated with significant side effects. There is therefore the need for new protolerogenic treatments to replace CS. Dystrophin-deficient rats (Dmd mdx) closely resemble the pathological phenotype of DMD patients. We performed the first Immunophenotyping of Dmd mdx rats and showed leukocyte infiltration in skeletal and cardiac muscles, which consisted mostly of macrophages and T cells including CD45RC high T cells. Muscles of DMD patients also contain elevated CD45RC high T cells. We treated Dmd mdx rats with an anti-CD45RC MAb used in previous studies to deplete CD45RC high T cells and induce immune tolerance in models of organ transplantation. Treatment of young Dmd mdx rats with anti-CD45RC MAb corrected skeletal muscle strength and was associated with depletion of CD45RC high T cells with no side effects. Treatment of young Dmd mdx rats with prednisolone resulted in increase in skeletal muscle strength but also severe growth retardation. In conclusion, anti-CD45RC MAb treatment has potential in the treatment of DMD and might eventually result in reduction or elimination of CS use.

Transplantation

Although cluster of differentiation (CD)8 regulatory T (Treg) cells have been in the last 20 year... more Although cluster of differentiation (CD)8 regulatory T (Treg) cells have been in the last 20 years more studied since evidences of their role in tolerance as been demonstrated in transplantation, autoimmune diseases and cancer, their characteristics are still controversial. In this review, we will focus on recent advances on CD8 Treg cells and description of a role for CD8 Treg cells in tolerance in both solid organ transplantation and graft-versus-host disease and their potential for clinical trials.

Transplantation, Jan 24, 2018

Immunodeficient mice are invaluable tools to analyze the long-term effects of potentially immunog... more Immunodeficient mice are invaluable tools to analyze the long-term effects of potentially immunogenic molecules in the absence of adaptive immune responses. Nevertheless, there are models and experimental situations that would beneficiate of larger immunodeficient recipients. Rats are ideally suited to perform experiments in which larger size is needed and are still a small animal model suitable for rodent facilities. Additionally, rats reproduce certain human diseases better than mice, such as ankylosing spondylitis and Duchenne disease and these disease models would greatly benefit of immunodeficient rats to test different immunogenic treatments. We describe the generation of Il2rg-deficient rats and their crossing with previously described Rag1-deficient rats to generate double-mutant RRG animals. As compared to Rag1-deficient rats, Il2rg-deficient rats were more immunodeficient since partially lacked not only T and B cells but also NK cells. RRG animals showed a more profound im...

PloS one, 2018

Anti-HCMV treatments used in immunosuppressed patients reduce viral replication, but resistant vi... more Anti-HCMV treatments used in immunosuppressed patients reduce viral replication, but resistant viral strains can emerge. Moreover, these drugs do not target latently infected cells. We designed two anti-viral CRISPR/Cas9 strategies to target the UL122/123 gene, a key regulator of lytic replication and reactivation from latency. The singleplex strategy contains one gRNA to target the start codon. The multiplex strategy contains three gRNAs to excise the complete UL122/123 gene. Primary fibroblasts and U-251 MG cells were transduced with lentiviral vectors encoding Cas9 and one or three gRNAs. Both strategies induced mutations in the target gene and a concomitant reduction of immediate early (IE) protein expression in primary fibroblasts. Further detailed analysis in U-251 MG cells showed that the singleplex strategy induced 50% of indels in the viral genome, leading to a reduction in IE protein expression. The multiplex strategy excised the IE gene in 90% of all viral genomes and thu...

Scientific reports, Jan 29, 2017

The generation of gene-edited animals using the CRISPRs/Cas9 system is based on microinjection in... more 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.

Blood Advances

Key Points CLEC-1 is restricted to CD16− myeloid DCs in human blood and acts as an inhibitory rec... more Key Points CLEC-1 is restricted to CD16− myeloid DCs in human blood and acts as an inhibitory receptor to restrain downstream Th17 activation. CLEC-1–deficient rats highlight an in vivo function for CLEC-1 in preventing excessive T-cell priming and effector Th responses.

Cellular and Molecular Life Sciences

72% between human, rat, or mouse [1]. Since 2008, studies have identified roles for IL-34 in area... more 72% between human, rat, or mouse [1]. Since 2008, studies have identified roles for IL-34 in areas as remote as neuronal protection, bone-degenerative diseases, delayedtype hypersensitivity, infection, cancer, and more recently transplantation.

Immunotherapy

This 22nd edition of the Nantes Actualités Transplantation annual meeting was co-organized for th... more This 22nd edition of the Nantes Actualités Transplantation annual meeting was co-organized for the second time with the LabEx Immuno-Graft Oncology network. This international meeting was held on 1 and 2 June 2017 in Nantes (western France). The topic of this 2-day meeting was ‘Immunotherapies in transplantation and cancer’. This meeting brought together 17 international invited speakers, young researchers and 220 attendees mainly from Europe and North America. It was a unique opportunity to bring together the pioneers and leading immunologists in the fields of transplantation and cancer, focusing on shared mechanisms that control immune responses in organ or bone marrow transplantation and in cancer.

Transgenic Research

On May 11th and 12th 2017 was held in Nantes, France, the international meeting ''Advances in tra... more On May 11th and 12th 2017 was held in Nantes, France, the international meeting ''Advances in transgenic animal models and techniques'' (http:// www.trm.univ-nantes.fr/). This biennial meeting is the fifth one of its kind to be organized by the Transgenic Rats ImmunoPhenomic (TRIP) Nantes facility (http:// www.tgr.nantes.inserm.fr/). The meeting was supported by private companies (SONIDEL, Scionics computer innovation, New England Biolabs, MERCK, genOway, Journal Disease Models and Mechanisms) and by public institutions (

Transplantation

Cells of the myeloid lineage, including suppressor cells (MDSCs), macrophages (Mφ) and dendritic ... more Cells of the myeloid lineage, including suppressor cells (MDSCs), macrophages (Mφ) and dendritic cells (DCs), are very important players in organ transplant rejection and graft-versus-host disease in unmanipulated recipients. Myeloid-derived cells have also been involved in rodent models of tolerance induction using costimulation blockade and through the administration of tolerogenic cytokines. Cell therapy approaches using in vitro–cultured Mφ and monocyte-derived DCs in organ transplantation have been developed in recent years, including clinical trials, such as The ONE Study consortium (www.onestudy.org/) and efforts have been launched to standardize cell culture and phenotyping methods, such as the Action to Focus and Accelerate Cell-based Tolerance-inducing Therapies consortium (www. afactt.eu). Because myeloid-derived cells are heterogeneous and plastic in their function, it is important to better define these cells and compare their phenotype, genetic program, and function. As an example of efforts in this direction, a recent publication made side-by-side comparisons of mouse MDSCs, regulatory Mφ (Mregs) and monocyte-derived Tolerogenic-DCs (TolDCs). These cell types had different phenotypes, could suppress T cell proliferation in vitro, and prolong skin allograft survival through different mechanisms. Mregs are generated from peripheral blood monocytes cultured in vitro with low doses of M-CSF in the presence of human serum and finally activated with IFN-γ. Human Mregs and TolDCs are the monocyte-derived cells of the panel of tolerogenic cells used in TheONE Study consortium. With the objective of discovering novel markers of human Mregs, Riquelme et al describe in this issue of Transplantation that combining the generation of a monoclonal antibody and transcriptomic analyses of newly and previously generated data that dehydrogenase/reductase 9 (DHRS9)

Immunotherapy, Sep 1, 2017

FOCIS goes South: Advances in Translational and Clinical Immunology was the first Federation of C... more FOCIS goes South: Advances in Translational and Clinical Immunology was the first Federation of Clinical Immunology Societies (FOCIS) ( www.focisnet.org ) meeting held in Latin America (May 15-17, 2017, Santiago de Chile, Chile). The meeting was organized as a 3-day workshop and was fostered by the Millennium Institute on Immunology and Immunotherapy, a recently nominated FOCIS Center of Excellence. The workshop brought together FOCIS associates, such as members of the FOCIS Board of Directors, Directors of different Centers of Excellence, regional speakers and 350 attendees. The Meeting covered aspects of immune regulation and modulation, as well as immunotherapy in areas of autoimmunity, transplantation, cancer and infectious diseases, among others. The activity also had a full-day immunology course and a day-long flow cytometry course.

Frontiers in immunology, 2017

Fasciola hepatica, also known as the liver fluke, is a trematode that infects livestock and human... more Fasciola hepatica, also known as the liver fluke, is a trematode that infects livestock and humans causing fasciolosis, a zoonotic disease of increasing importance due to its worldwide distribution and high economic losses. This parasite immunoregulates the host immune system by inducing a strong Th2 and regulatory T immune response by immunomodulating dendritic cell (DC) maturation and alternative activation of macrophages. In this paper, we show that F. hepatica infection in mice induces the upregulation of heme-oxygenase-1 (HO-1), the rate-limiting enzyme in the catabolism of free heme that regulates the host inflammatory response. We show and characterize two different populations of antigen presenting cells that express HO-1 during infection in the peritoneum of infected animals. Cells that expressed high levels of HO-1 expressed intermediate levels of F4/80 but high expression of CD11c, CD38, TGFβ, and IL-10 suggesting that they correspond to regulatory DCs. On the other hand,...

Scientific Reports

Different mutations of the OTOF gene, encoding for otoferlin protein expressed in the cochlear in... more Different mutations of the OTOF gene, encoding for otoferlin protein expressed in the cochlear inner hair cells, induces a form of deafness that is the major cause of nonsyndromic recessive auditory neuropathy spectrum disorder in humans. We report the generation of the first large animal model of OTOF mutations using the CRISPR system associated with different Cas9 components (mRNA or protein) assisted by single strand oligodeoxynucleotides (ssODN) to induce homology-directed repair (HDR). Zygote microinjection was performed with two sgRNA targeting exon 5 and 6 associated to Cas9 mRNA or protein (RNP) at different concentrations in a mix with an ssODN template targeting HDR in exon 5 containing two STOP sequences. A total of 73 lambs were born, 13 showing indel mutations (17.8%), 8 of which (61.5%) had knock-in mutations by HDR. Higher concentrations of Cas9-RNP induced targeted mutations more effectively, but negatively affected embryo survival and pregnancy rate. This study repo...

JCI insight, 2017

Rat and human CD4+ and CD8+ Tregs expressing low levels of CD45RC have strong immunoregulatory pr... more 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 CD45RChigh T cells through intrinsic cell signaling but preserved and potentiated CD4+ and CD8+ CD45RClow/- 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+ CD45RClow/- Tregs. Finally, we demonstrate that anti-human CD45RC treatment inhibited graft-versus-h...

Previous work on blockade of CD40-CD40 ligand interaction in mice and primates with anti-CD40 lig... more Previous work on blockade of CD40-CD40 ligand interaction in mice and primates with anti-CD40 ligand mAbs has resulted in a moderate prolongation of allograft survival without the development of true allograft tolerance. In this study, we show in rats that adenovirus-mediated gene transfer of CD40Ig sequences into the graft resulted in prolonged (>200 days) expression of CD40Ig and in long-term (>300 days) survival. Recipients expressing CD40Ig displayed strongly (>90%) inhibited mixed leukocyte reactions and alloantibody production at early (days 5 and 17) and late time points (>100 day) after transplantation, but showed limited inhibition of leukocyte infiltration and cytokine production as evaluated by immunohistology at early time points (day 5). Recipients of long-surviving hearts showed donor-specific hyporesponsiveness since acceptance of second cardiac allografts was donor specific. Nevertheless, long-term allografts (>100 days) displayed signs of chronic rejection vasculopathy. Occluded vessels showed leukocyte infiltration, mainly composed of CD4 ؉ and CD8 ؉ cells, macrophages, and mast cells. These recipients also showed antidonor CTL activity. Recipients expressing CD40Ig did not show nonspecific immunosuppression, as they were able to mount anticognate immune responses that were partially inhibited at early time points and were normal thereafter. We conclude that gene transfer-mediated expression of CD40Ig resulted in a highly efficient inhibition of acute heart allograft rejection in rats. This treatment induced donor-specific inhibition of certain alloreactive mechanisms in the short-, but not the long-term, which resulted in long-term survival of allografts concomitant with the development of chronic rejection.

Journal of Leukocyte Biology

Although IL‐34 and CSF‐1 share actions as key mediators of monocytes/macrophages survival and dif... more Although IL‐34 and CSF‐1 share actions as key mediators of monocytes/macrophages survival and differentiation, they also display differences that should be identified to better define their respective roles in health and diseases. IL‐34 displays low sequence homology with CSF‐1 but has a similar general structure and they both bind to a common receptor CSF‐1R, although binding and subsequent intracellular signaling shows differences. CSF‐1R expression has been until now mainly described at a steady state in monocytes/macrophages and myeloid dendritic cells, as well as in some cancers. IL‐34 has also 2 other receptors, protein‐tyrosine phosphatase zeta (PTPζ) and CD138 (Syndecan‐1), expressed in some epithelium, cells of the central nervous system (CNS), as well as in numerous cancers. While most, if not all, of CSF‐1 actions are mediated through monocyte/macrophages, IL‐34 has also other potential actions through PTPζ and CD138. Additionally, IL‐34 and CSF‐1 are produced by different cells in different tissues. This review describes and discusses similarities and differences between IL‐34 and CSF‐1 at steady state and in pathological situations and identifies possible ways to target IL‐34, CSF‐1, and its receptors.

Frontiers in Immunology

Regulatory Tcells (Treg) are essential components of peripheral immune homeostasis. Adoptive Treg... more Regulatory Tcells (Treg) are essential components of peripheral immune homeostasis. Adoptive Treg cell therapy has shown efficacy in a variety of immune-mediated diseases in preclinical studies and is now moving from phase I/IIa to larger phase II studies aiming to demonstrate efficacy. However, hurdles such as in vivo stability and efficacy remain to be addressed. Nevertheless, preclinical models have shown that Treg function and specificity can be increased by pharmacological substances or gene modifications, and even that conventional T cells can be converted to Treg potentially providing new sources of Treg and facilitating Treg cell therapy. The exponential growth in genetic engineering techniques and their application to T cells coupled to a large body of knowledge on Treg open numerous opportunities to generate Treg with “superpowers”. This review summarizes the genetic engineering techniques available and their applications for the next-generation of Super-Treg with increase...

Frontiers in Genetics

The rat has been extensively used as a small animal model. Many genetically engineered rat models... more The rat has been extensively used as a small animal model. Many genetically engineered rat models have emerged in the last two decades, and the advent of gene-specific nucleases has accelerated their generation in recent years. This review covers the techniques and advances used to generate genetically engineered rat lines and their application to the development of rat models more broadly, such as conditional knockouts and reporter gene strains. In addition, genome-editing techniques that remain to be explored in the rat are discussed. The review also focuses more particularly on two areas in which extensive work has been done: human genetic diseases and immune system analysis. Models are thoroughly described in these two areas and highlight the competitive advantages of rat models over available corresponding mouse versions. The objective of this review is to provide a comprehensive description of the advantages and potential of rat models for addressing specific scientific questi...

Transplantation

In vivo analysis of human immune responses in immunodeficient rats.

International Journal of Molecular Sciences

Antigen-presenting cells (APCs) including dendritic cells (DCs) play a critical role in the devel... more Antigen-presenting cells (APCs) including dendritic cells (DCs) play a critical role in the development of autoimmune diseases by presenting self-antigen to T-cells. Different signals modulate the ability of APCs to activate or tolerize autoreactive T-cells. Since the expression of heme oxygenase-1 (HO-1) by APCs has been associated with the tolerization of autoreactive T-cells, we hypothesized that HO-1 expression might be altered in APCs from autoimmune-prone non-obese diabetic (NOD) mice. We found that, compared to control mice, NOD mice exhibited a lower percentage of HO-1-expressing cells among the splenic DCs, suggesting an impairment of their tolerogenic functions. To investigate whether restored expression of HO-1 in APCs could alter the development of diabetes in NOD mice, we generated a transgenic mouse strain in which HO-1 expression can be specifically induced in DCs using a tetracycline-controlled transcriptional activation system. Mice in which HO-1 expression was indu...

Frontiers in Immunology

Corticosteroids (CS) are standard therapy for the treatment of Duchenne's muscular dystrophy (DMD... more Corticosteroids (CS) are standard therapy for the treatment of Duchenne's muscular dystrophy (DMD). Even though they decrease inflammation, they have limited efficacy and are associated with significant side effects. There is therefore the need for new protolerogenic treatments to replace CS. Dystrophin-deficient rats (Dmd mdx) closely resemble the pathological phenotype of DMD patients. We performed the first Immunophenotyping of Dmd mdx rats and showed leukocyte infiltration in skeletal and cardiac muscles, which consisted mostly of macrophages and T cells including CD45RC high T cells. Muscles of DMD patients also contain elevated CD45RC high T cells. We treated Dmd mdx rats with an anti-CD45RC MAb used in previous studies to deplete CD45RC high T cells and induce immune tolerance in models of organ transplantation. Treatment of young Dmd mdx rats with anti-CD45RC MAb corrected skeletal muscle strength and was associated with depletion of CD45RC high T cells with no side effects. Treatment of young Dmd mdx rats with prednisolone resulted in increase in skeletal muscle strength but also severe growth retardation. In conclusion, anti-CD45RC MAb treatment has potential in the treatment of DMD and might eventually result in reduction or elimination of CS use.

Transplantation

Although cluster of differentiation (CD)8 regulatory T (Treg) cells have been in the last 20 year... more Although cluster of differentiation (CD)8 regulatory T (Treg) cells have been in the last 20 years more studied since evidences of their role in tolerance as been demonstrated in transplantation, autoimmune diseases and cancer, their characteristics are still controversial. In this review, we will focus on recent advances on CD8 Treg cells and description of a role for CD8 Treg cells in tolerance in both solid organ transplantation and graft-versus-host disease and their potential for clinical trials.

Transplantation, Jan 24, 2018

Immunodeficient mice are invaluable tools to analyze the long-term effects of potentially immunog... more Immunodeficient mice are invaluable tools to analyze the long-term effects of potentially immunogenic molecules in the absence of adaptive immune responses. Nevertheless, there are models and experimental situations that would beneficiate of larger immunodeficient recipients. Rats are ideally suited to perform experiments in which larger size is needed and are still a small animal model suitable for rodent facilities. Additionally, rats reproduce certain human diseases better than mice, such as ankylosing spondylitis and Duchenne disease and these disease models would greatly benefit of immunodeficient rats to test different immunogenic treatments. We describe the generation of Il2rg-deficient rats and their crossing with previously described Rag1-deficient rats to generate double-mutant RRG animals. As compared to Rag1-deficient rats, Il2rg-deficient rats were more immunodeficient since partially lacked not only T and B cells but also NK cells. RRG animals showed a more profound im...

PloS one, 2018

Anti-HCMV treatments used in immunosuppressed patients reduce viral replication, but resistant vi... more Anti-HCMV treatments used in immunosuppressed patients reduce viral replication, but resistant viral strains can emerge. Moreover, these drugs do not target latently infected cells. We designed two anti-viral CRISPR/Cas9 strategies to target the UL122/123 gene, a key regulator of lytic replication and reactivation from latency. The singleplex strategy contains one gRNA to target the start codon. The multiplex strategy contains three gRNAs to excise the complete UL122/123 gene. Primary fibroblasts and U-251 MG cells were transduced with lentiviral vectors encoding Cas9 and one or three gRNAs. Both strategies induced mutations in the target gene and a concomitant reduction of immediate early (IE) protein expression in primary fibroblasts. Further detailed analysis in U-251 MG cells showed that the singleplex strategy induced 50% of indels in the viral genome, leading to a reduction in IE protein expression. The multiplex strategy excised the IE gene in 90% of all viral genomes and thu...

Scientific reports, Jan 29, 2017

The generation of gene-edited animals using the CRISPRs/Cas9 system is based on microinjection in... more 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.

Blood Advances

Key Points CLEC-1 is restricted to CD16− myeloid DCs in human blood and acts as an inhibitory rec... more Key Points CLEC-1 is restricted to CD16− myeloid DCs in human blood and acts as an inhibitory receptor to restrain downstream Th17 activation. CLEC-1–deficient rats highlight an in vivo function for CLEC-1 in preventing excessive T-cell priming and effector Th responses.

Cellular and Molecular Life Sciences

72% between human, rat, or mouse [1]. Since 2008, studies have identified roles for IL-34 in area... more 72% between human, rat, or mouse [1]. Since 2008, studies have identified roles for IL-34 in areas as remote as neuronal protection, bone-degenerative diseases, delayedtype hypersensitivity, infection, cancer, and more recently transplantation.

Immunotherapy

This 22nd edition of the Nantes Actualités Transplantation annual meeting was co-organized for th... more This 22nd edition of the Nantes Actualités Transplantation annual meeting was co-organized for the second time with the LabEx Immuno-Graft Oncology network. This international meeting was held on 1 and 2 June 2017 in Nantes (western France). The topic of this 2-day meeting was ‘Immunotherapies in transplantation and cancer’. This meeting brought together 17 international invited speakers, young researchers and 220 attendees mainly from Europe and North America. It was a unique opportunity to bring together the pioneers and leading immunologists in the fields of transplantation and cancer, focusing on shared mechanisms that control immune responses in organ or bone marrow transplantation and in cancer.

Transgenic Research

On May 11th and 12th 2017 was held in Nantes, France, the international meeting ''Advances in tra... more On May 11th and 12th 2017 was held in Nantes, France, the international meeting ''Advances in transgenic animal models and techniques'' (http:// www.trm.univ-nantes.fr/). This biennial meeting is the fifth one of its kind to be organized by the Transgenic Rats ImmunoPhenomic (TRIP) Nantes facility (http:// www.tgr.nantes.inserm.fr/). The meeting was supported by private companies (SONIDEL, Scionics computer innovation, New England Biolabs, MERCK, genOway, Journal Disease Models and Mechanisms) and by public institutions (

Transplantation

Cells of the myeloid lineage, including suppressor cells (MDSCs), macrophages (Mφ) and dendritic ... more Cells of the myeloid lineage, including suppressor cells (MDSCs), macrophages (Mφ) and dendritic cells (DCs), are very important players in organ transplant rejection and graft-versus-host disease in unmanipulated recipients. Myeloid-derived cells have also been involved in rodent models of tolerance induction using costimulation blockade and through the administration of tolerogenic cytokines. Cell therapy approaches using in vitro–cultured Mφ and monocyte-derived DCs in organ transplantation have been developed in recent years, including clinical trials, such as The ONE Study consortium (www.onestudy.org/) and efforts have been launched to standardize cell culture and phenotyping methods, such as the Action to Focus and Accelerate Cell-based Tolerance-inducing Therapies consortium (www. afactt.eu). Because myeloid-derived cells are heterogeneous and plastic in their function, it is important to better define these cells and compare their phenotype, genetic program, and function. As an example of efforts in this direction, a recent publication made side-by-side comparisons of mouse MDSCs, regulatory Mφ (Mregs) and monocyte-derived Tolerogenic-DCs (TolDCs). These cell types had different phenotypes, could suppress T cell proliferation in vitro, and prolong skin allograft survival through different mechanisms. Mregs are generated from peripheral blood monocytes cultured in vitro with low doses of M-CSF in the presence of human serum and finally activated with IFN-γ. Human Mregs and TolDCs are the monocyte-derived cells of the panel of tolerogenic cells used in TheONE Study consortium. With the objective of discovering novel markers of human Mregs, Riquelme et al describe in this issue of Transplantation that combining the generation of a monoclonal antibody and transcriptomic analyses of newly and previously generated data that dehydrogenase/reductase 9 (DHRS9)

Immunotherapy, Sep 1, 2017

FOCIS goes South: Advances in Translational and Clinical Immunology was the first Federation of C... more FOCIS goes South: Advances in Translational and Clinical Immunology was the first Federation of Clinical Immunology Societies (FOCIS) ( www.focisnet.org ) meeting held in Latin America (May 15-17, 2017, Santiago de Chile, Chile). The meeting was organized as a 3-day workshop and was fostered by the Millennium Institute on Immunology and Immunotherapy, a recently nominated FOCIS Center of Excellence. The workshop brought together FOCIS associates, such as members of the FOCIS Board of Directors, Directors of different Centers of Excellence, regional speakers and 350 attendees. The Meeting covered aspects of immune regulation and modulation, as well as immunotherapy in areas of autoimmunity, transplantation, cancer and infectious diseases, among others. The activity also had a full-day immunology course and a day-long flow cytometry course.

Frontiers in immunology, 2017

Fasciola hepatica, also known as the liver fluke, is a trematode that infects livestock and human... more Fasciola hepatica, also known as the liver fluke, is a trematode that infects livestock and humans causing fasciolosis, a zoonotic disease of increasing importance due to its worldwide distribution and high economic losses. This parasite immunoregulates the host immune system by inducing a strong Th2 and regulatory T immune response by immunomodulating dendritic cell (DC) maturation and alternative activation of macrophages. In this paper, we show that F. hepatica infection in mice induces the upregulation of heme-oxygenase-1 (HO-1), the rate-limiting enzyme in the catabolism of free heme that regulates the host inflammatory response. We show and characterize two different populations of antigen presenting cells that express HO-1 during infection in the peritoneum of infected animals. Cells that expressed high levels of HO-1 expressed intermediate levels of F4/80 but high expression of CD11c, CD38, TGFβ, and IL-10 suggesting that they correspond to regulatory DCs. On the other hand,...