CD4 and CD8 regulatory T cell characterization in the rat using a unique transgenic Foxp3-EGFP model.

Background: Regulatory T cells (Treg) in diverse species include CD4 and CD8 T cells. In all species, CD8 Treg have been only partially characterized and there is no rat model in which CD4 and CD8 FOXP3 Treg are genetically tagged.

Results: We generated a Foxp3-EGFP rat transgenic line in which FOXP3 gene was expressed and controlled EGFP.

CRISPR/Cas9-mediated inactivation of the phosphatase activity of soluble epoxide hydrolase prevents obesity and cardiac ischemic injury.

Introduction: Although the physiological role of the C-terminal hydrolase domain of the soluble epoxide hydrolase (sEH-H) is well investigated, the function of its N-terminal phosphatase activity (sEH-P) remains unknown.

Objectives: This study aimed to assess in vivo the physiological role of sEH-P.

Methods: CRISPR/Cas9 was used to generate a novel knock-in (KI) rat line lacking the sEH-P activity.

IL-34 deficiency impairs FOXP3 Treg function in a model of autoimmune colitis and decreases immune tolerance homeostasis.

Background: Immune homeostasis requires fully functional Tregs with a stable phenotype to control autoimmunity. Although IL-34 is a cytokine first described as mainly involved in monocyte cell survival and differentiation, we recently described its expression by CD8 Tregs in a rat model of transplantation tolerance and by activated FOXP3 CD4 and CD8 Tregs in human healthy individuals. However, its role in autoimmunity and potential in human diseases remains to be determined.

Generation of a Human Deafness Sheep Model Using the CRISPR/Cas System.

CRISPR/Cas9 system is a promising method for the generation of human disease models by genome editing in non-conventional experimental animals. Medium/large-sized animals like sheep have several advantages to study human diseases and medicine. Here, we present a protocol that describes the generation of an otoferlin edited sheep model via CRISPR-assisted single-stranded oligodinucleotide-mediated Homology-Directed Repair (HDR), through direct cytoplasmic microinjection in in vitro produced zygotes.

SIRPγ-CD47 Interaction Positively Regulates the Activation of Human T Cells in Situation of Chronic Stimulation.

A numerous number of positive and negative signals various molecules modulate T-cell activation. Within the various transmembrane proteins, SIRPγ is of interest since it is not expressed in rodents. SIRPγ interaction with CD47 is reevaluated in this study.

In vitro recovery of FIX clotting activity as a marker of highly functional hepatocytes in a hemophilia B iPSC model.

Background And Aims: Pluripotent stem cell-derived hepatocytes differentiated in monolayer culture are known to have more fetal than adult hepatocyte characteristics. If numerous studies tend to show that this immature phenotype might not necessarily be an obstacle to their use in transplantation, other applications such as drug screening, toxicological studies, or bioartificial livers are reliant on hepatocyte functionality and require full differentiation of hepatocytes. New technologies have been used to improve the differentiation process in recent years, usually evaluated by measuring the albumin production and CYP450 activity.

The impact of CD160 deficiency on alloreactive CD8 T cell responses and allograft rejection.

CD160 is a member of the immunoglobulin superfamily with a pattern of expression mainly restricted to cytotoxic cells. To assess the functional relevance of the HVEM/CD160 signaling pathway in allogeneic cytotoxic responses, exon 2 of the CD160 gene was targeted by CRISPR/Cas9 to generate CD160 deficient mice. Next, we evaluated the impact of CD160 deficiency in the course of an alloreactive response.

Toward a better definition of hematopoietic progenitors suitable for B cell differentiation.

The success of inducing human pluripotent stem cells (hIPSC) offers new opportunities for cell-based therapy. Since B cells exert roles as effector and as regulator of immune responses in different clinical settings, we were interested in generating B cells from hIPSC. We differentiated human embryonic stem cells (hESC) and hIPSC into B cells onto OP9 and MS-5 stromal cells successively.

Increased antitumor efficacy of PD-1-deficient melanoma-specific human lymphocytes.

Background: Genome editing offers unique perspectives for optimizing the functional properties of T cells for adoptive cell transfer purposes. So far, editing has been successfully tested mainly in chimeric antigen receptor T (CAR-T) cells and human primary T cells. Nonetheless, for patients with solid tumors, the adoptive transfer of effector memory T cells specific for tumor antigens remains a relevant option, and the use of high avidity T cells deficient for programmed cell death-1 (PD-1) expression is susceptible to improve the therapeutic benefit of these treatments.

Generation of gene-edited rats by delivery of CRISPR/Cas9 protein and donor DNA into intact zygotes using electroporation.

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.

A Rapid and Cost-Effective Method for Genotyping Genome-Edited Animals: A Heteroduplex Mobility Assay Using Microfluidic Capillary Electrophoresis.

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.

Implication of matrix metalloproteinase 7 and the noncanonical wingless-type signaling pathway in a model of kidney allograft tolerance induced by the administration of anti-donor class II antibodies.

In rats, tolerance to MHC-incompatible renal allografts can be induced by the administration of anti-donor class II Abs on the day of transplantation. In this study we explored the mechanisms involved in the maintenance phase of this tolerance by analyzing intragraft gene expression profiles by microarray in long-term accepted kidneys. Comparison of the gene expression patterns of tolerated to syngeneic kidneys revealed 5,954 differentially expressed genes (p < 0.

TLR9 ligand enhances proliferation of rat CD4+ T cell and modulates suppressive activity mediated by CD4+ CD25+ T cell.

Toll-like receptors (TLRs) play a crucial role in the initiation of innate responses following microbial infection and also in adaptive immune responses by orchestrating the activation of different cell populations. TLRs are expressed at high levels in antigen-presenting cells and recent studies have demonstrated the expression and biological role of TLRs in mouse and human CD4(+) T cells. In this study, we analyzed TLR mRNA expression in rat CD4(+) T cells using stringent quantitative reverse transcription-PCR conditions enabling a direct comparison of the levels of each TLR.

New evidence for a role of allograft accommodation in long-term tolerance.

Background: Progressively better therapies have largely prevented or at least effectively treated acute allograft rejection. Consequently, the long-term survival of solid organ transplants has increasingly become limited primarily by the development of chronic allograft rejection. The mechanisms of chronic rejection remain largely unknown and the induction of specific tolerance would be the ultimate achievement in transplant immunology.

Differential pattern recognition receptor expression but stereotyped responsiveness in rat spleen dendritic cell subsets.

Dendritic cells (DC) are a heterogeneous population of APC endowed with specific functions. The nature of the DC subset involved in the course of an immune response to a specific pathogen might be important for inducing the appropriate effectors. In addition, each DC subset might also exhibit intrinsic functional plasticity.

LF 15-0195 treatment protects against central nervous system autoimmunity by favoring the development of Foxp3-expressing regulatory CD4 T cells.

Experimental autoimmune encephalomyelitis (EAE) is an instructive model for the human demyelinating disease multiple sclerosis. Lewis (LEW) rats immunized with myelin-basic protein (MBP) develop EAE characterized by a single episode of paralysis, from which they recover spontaneously and become refractory to a second induction of disease. LF 15-0195 is a novel molecule that has potent immunosuppressive effects in several immune-mediated pathological manifestations, including EAE.

Accumulation of T cells with potent regulatory properties and restricted Vbeta7-TCR rearrangements in tolerated allografts.

Background: We have previously demonstrated that a short-course treatment with LF15-0195, a 15-deoxyspergualin analogue, induces donor-specific tolerance of cardiac allografts in rats and expansion of splenic CD4CD25 regulatory T cells.

Methods: To further characterize long-term tolerance in this model, we have analyzed the phenotype, regulatory properties and TCR-Vbeta usage of the T cells infiltrating the tolerated allografts.

Results: We demonstrate that the tolerated allografts express high levels of FoxP3 transcripts and contain a large number of CD4 T cells, half of which express CD25.

Identification of a new member of the CD20/FcepsilonRIbeta family overexpressed in tolerated allografts.

We identified a novel rat gene specifically overexpressed in tolerated heart allografts in a model of tolerance induced by donor-specific blood transfusion (DST). We named this gene TORID, for tolerance-related and induced transcript. We show that TORID expression can be attributed to non-T cells infiltrating tolerated grafts.

Induction of Fractalkine and CX3CR1 mediated by host CD8+ T cells in allograft tolerance induced by donor specific blood transfusion.

Background: Donor-specific tolerance to heart allografts in the rat can be achieved by donor-specific blood transfusions (DST) before transplantation. This tolerance induction requires the presence of host CD8 T cells and is characterized by the infiltration of numerous leukocytes.

Methods: To identify new mediators involved in tolerance induction, gene searching was performed and resulted in the identification of the Fractalkine receptor, CX3CR1, as being highly expressed in tolerated allografts.

Different patterns of TCR beta chain regulation following allo- and xeno-transplantation.

Background: In the concordant hamster-to-rat cardiac xenograft model, recipients treated with cobra venom factor for the first 10 days following transplantation and daily with Cyclosporine A (CsA) do not reject their grafts. However, when CsA is withdrawn on day 40, an acute cellular rejection occurs within 4 +/- 1 days. Allografts performed in the same conditions are rejected within 18 +/- 4 days.

The role of TNF-related activation-induced cytokine-receptor activating NF-kappa B interaction in acute allograft rejection and CD40L-independent chronic allograft rejection.

We analyzed the role of TNF-related activation-induced cytokine (TRANCE), a member of the TNF family expressed on activated T cells that shares functional properties with CD40L, and its receptor-activating NF-kappaB (RANK) which is mostly expressed on mature dendritic cells, during allogenic responses in vivo using a rodent heart allograft model. TRANCE mRNA was strongly up-regulated in acutely rejected allografts on days 4 and 5 posttransplantation whereas RANK was detected as early as day 1 but did not show further up-regulation during the first week. Immunofluoresence analyses of heart allografts showed that 80 and 100% of TRANCE and RANK-expressing cells were T cells and APCs, respectively.

Rapid selection of differentially expressed genes in TNF[alpha]-activated endothelial cells.

Background: RNA differential display (DD) RT-PCR is a useful method to identify and clone differentially expressed genes. However, the rate of false positives and redundancy associated with this PCR-based method as well as laborious downstream screening steps constitute major limitations. Here we present DD RT-PCR and reverse northern (RN) protocols allowing rapid and acurate identification of genes upregulated in porcine endothelial cells (EC) in response to TNFalpha.

Temporal analysis of cytokine gene expression during infiltration of porcine neuronal grafts implanted into the rat brain.

A large array of evidence supports the involvement of infiltrating T lymphocytes in the rejection process of intracerebral neuronal xenografts. Little is known, however, about the molecular mechanisms that drive the recruitment of this cell type. In the present work, we used real-time RT-PCR methodology to investigate the kinetics of cytokine gene expression during the infiltration of fetal porcine neurons (PNEU) implanted into the striatum of LEW.

Importance, mechanisms and limitations of the distant bystander effect in cancer gene therapy of experimental liver tumors.

GCV-ablation of transplanted TK-positive liver tumors or the application of syngenic and allogenic HSV-TK/GCV oncolysates significantly reduced the size of synchronously growing untreated sister tumors in the liver. These TK-negative liver tumors constantly showed an increased infiltration by mononuclears (x4). The relative abundance of CD 4/8, NK and monocyte subtypes remained constant.