Genetic Modification of Limbal Stem Cells to Decrease Allogeneic Immune Responses

Abstract

Limbal stem cell (LSC) transplantation is the only efficient treatment for patients affected by LSC deficiency (LSCD). Allogeneic LSC transplantation is one of the most successful alternative for patients with bilateral LSCD. Nevertheless, the high variability of the human leukocyte antigens (HLA) remains a relevant obstacle to long-term allogeneic graft survival. This study characterized the immunologic properties of LSCs and proposed a genetic engineering strategy to reduce the immunogenicity of LSCs and of their derivatives. Hence, LSC HLA expression was silenced using lentiviral vectors encoding for short hairpin (sh) RNAs targeting β2-microglobulin (β2M) or class II major histocompatibility complex transactivator (CIITA) to silence HLA class I and II respectively. Beside the constitutive expression of HLA class I, LSCs showed the capability to upregulate HLA class II expression under inflammatory conditions. Furthermore, LSCs demonstrated the capability to induce T-cell mediated immune responses. LSCs phenotypical and functional characteristics are not disturbed after genetic modification. However, HLA silenced LSC showed to prevent T cell activation, proliferation and cytotoxicity in comparison to fully HLA-expressing LSCs. Additionally; HLA-silenced LSCs were protected against antibody-mediated cellular-dependent cytotoxicity. Our data is a proof-of-concept of the feasibility to generate low immunogenic human LSCs without affecting their typical features. The use of low immunogenic LSCs may support for long-term survival of LSCs and their derivatives after allogeneic transplantation.

Downregulation of Swine Leukocyte Antigen Expression Decreases the Strength of Xenogeneic Immune Responses towards Renal Proximal Tubular Epithelial Cells

“…One of the strategies proposed to reduce xenograft immunogenicity is the downregulation of swine leukocyte antigen (SLA) class I and class II expression [ 16 ]. A robust silencing of SLA class I and class II expression may decrease the strength of allo- or xenogeneic immune responses by generating an immunologically invisible status of tissues and organs supporting graft survival [ 17 , 18 , 19 ]. The use of PTECs isolated from genetically modified pigs or in vitro genetically modified PTECs may contribute to evaluate the effect of specific genetic modifications in human immune response towards porcine-derived kidneys and to monitor the status and strength of xenogeneic immune response after transplantation.…”

“…In an allogeneic setting, we have previously demonstrated the possibility of stably silencing the expression of MHC class I and II on complete organs such as the rat kidney, limb, or pig lung. These studies showed the possibility to silence MHC expression on complex 3D structures consisting of a variety of cell types and with different immunogenic potentials [ 16 , 18 , 19 ]. Despite the value of the possibility to evaluate the strength of immune responses towards a specific organ cell type, which may contribute to further clarifying mechanisms of rejection in xenotransplantation, the use of more complex systems may contribute to assessing such responses in a more physiological setting.…”

“…In the allogeneic transplantation model, silencing MHC expression on different cell types, tissues, and organs was shown to be feasible and contributed to decreasing the susceptibility to antibody-mediated cellular and complement-dependent cytotoxicity. Furthermore, silencing MHC expression was shown to induce weaker T-cell allogeneic immune responses and contribute to the significantly lower secretion of pro-inflammatory cytokines of IL-6 and IFN-γ [ 18 , 19 , 38 , 39 ]. IL-6 is involved in innate immune responses as well as in adaptive immunity.…”