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.

Highlights

  • Limbal stem cells (LSCs) constantly maintain the homeostasis of corneal epithelium

  • LSCs were stimulated with IFNg to evaluate their capacity to upregulate human leukocyte antigens (HLA) class I and class II levels. b2M, CIITA and HLADR transcripts as well as surface expression of HLA class I and II proteins were analyzed on stimulated and non-stimulated LSCs (Figures 1A–C)

  • These data correlated with an increase of HLA class I and II surface expression compared to unstimulated LSCs

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Summary

Introduction

Limbal stem cells (LSCs) constantly maintain the homeostasis of corneal epithelium. The main causes leading to LSCD are chemical and thermal burns, ultraviolet exposure, ionizing radiation, chemotherapeutic agents, Low Immunogenic Limbal Stem Cells viral, bacterial or fungal infections or genetic disorders [3]. CLET is one of the most effective showing 70 to 80% success rate in the regeneration of the corneal epithelium [9]. Pellegrini et al first described autologous CLET in which limbal epithelial cells from the patients healthy eye were collected, cultivated and expanded in vitro prior transplantation into the diseased eye [10]. Allo-CLET is associated with continuous systemic immunosuppression to prevent allograft loss [11]. Adverse effects related to immunosuppression regimes are always present and demand closed monitoring [12]

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