Heterologous Expression of Mutated HLA-G1 Reduces Alloreactivity of Human Dermal Fibroblasts

Abstract

Aim: To engineer a stable HLA-G molecule and evaluate its immunomodulatory properties in transgenic human dermal fibroblasts (HDFs). Materials & methods: A mutated HLA-G1 (mHLA-G1) molecule was generated by modifying the endoplasmic reticulum retrieval motif and 3′-untranslated region miRNA-binding sites of HLA-G1. Immunomodulatory properties of transgenic HDF-mHLA-G1 were evaluated in vitro. Results: Stable mHLA-G1 expressing HDF cells were successfully generated and flow cytometry analysis revealed that mHLA-G1 efficiently localized to the cell surface. Natural killer cell-mediated cytolysis of HDF-mHLA-G1/green fluorescent protein (GFP) was reduced by 73% compared with HDF-GDP. HDF-mHLA-G1/GFP decreased phytohemagglutinin-activated peripheral blood mononuclear cell proliferation by 30% versus HDF-GFP. Conclusion: We are the first to successfully create a human fibroblast source with reduced alloreactivity using a novel mHLA-G1 construct. This approach may be extended to other cell types including human embryonic stem cells for use in allogeneic transplantation for cell-based regenerative medicine applications.

Keywords::

• alloreactivity
• cytotoxicity
• HLA-G
• immune tolerance
• nucleofection
• PiggyBac
• PBMC

Financial & competing interests disclosure

All three authors were employees of, held stock or stock options in, and/or were inventors on issued or pending patents assigned to Escape Therapeutics, Inc. BM Hantash and co-authors were supported by a Stem Cell Transplantation Immunology Award (RM1–01711) from California Institute for Regenerative Medicine. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Additional information

Funding

All three authors were employees of, held stock or stock options in, and/or were inventors on issued or pending patents assigned to Escape Therapeutics, Inc. BM Hantash and co-authors were supported by a Stem Cell Transplantation Immunology Award (RM1–01711) from California Institute for Regenerative Medicine. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.