While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and livestock.

Treatment with CD40Ig results in indefinite allograft survival in a complete MHC-mismatched heart allograft model in the rat. Here we show that serial second, third, and fourth adoptive transfers of total splenocytes from CD40Ig-treated recipients into secondary recipients led to indefinite donor-specific allograft acceptance. Purification of splenocyte subpopulations from CD40Ig-treated recipients demonstrated that only the adoptively transferred CD8⁺CD45RC^low subset resulted in donor-specific long-term survival, whereas CD8⁺CD45RC^low T cells from naive animals did not. Accepted grafts displayed increased indoleamine 2,3-dioxygenase (IDO) expression restricted in the graft to ECs. Coculture of donor ECs with CD8⁺CD45RC^low T cells purified from CD40Ig-treated animals resulted in donor-specific IDO expression dependent on IFN-γ. Neutralization of IFN-γ or IDO triggered acute allograft rejection in both CD40Ig-treated and adoptively transferred recipients. This study demonstrates for what we believe to be the first time that interference in CD40–CD40 ligand (CD40-CD40L) interactions induces allospecific CD8⁺ Tregs that maintain allograft survival. CD8⁺CD45RC^low T cells act through IFN-γ production, which in turn induces IDO expression by graft ECs. Thus, donor alloantigen-specific CD8⁺ Tregs may promote local graft immune privilege through IDO expression.

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.

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⁺CD45RC^lo 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⁺CD45RC^lo Tregs and human FOXP3⁺CD45RC^loCD8⁺ 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.

We have previously shown that human monocyte‐derived dendritic cells (DC) express indoleamine 2,3‐dioxygenase (IDO), as well as several other enzymes of the kynurenine pathway at the mRNA level upon maturation. The tolerogenic mechanisms of this pathway remain unclear. Here we show that LPS‐treated DC metabolize tryptophan as far as quinolinate. We found that IDO contributes to LPS and TNF‐α + poly(I:C)‐induced DC maturation since IDO inhibition using two different inhibitors impairs DC maturation. IDO knock‐down using short‐hairpin RNA also led to diminished LPS‐induced maturation. In line with these results, the tryptophan‐derived catabolites 3‐hydroxyanthranilic acid and 3‐hydroxykynurenine increased maturation of LPS‐treated DC. Concerning the molecular mechanisms of this effect, IDO acts as an intermediate pathway in LPS‐induced production of reactive oxygen species and NF‐κB activation, two processes that lead to DC maturation. Finally, we show that mature DC expand CD4⁺CD25^high regulatory T cells in an IDO‐dependent manner. In conclusion, we show that IDO constitutes an intermediate pathway in DC maturation leading to expansion of CD4⁺CD25^high regulatory T cells.

The hallmark of chronic rejection is the occlusion of the artery lumen by intima hyperplasia as a consequence of leukocyte infiltration and vascular smooth muscle cell (VSMC) migration and proliferat...