Protecting Stem Cell Derived Pancreatic Beta-Like Cells From Diabetogenic T Cell Recognition

Abstract

Type 1 diabetes results from an autoimmune attack directed at pancreatic beta cells predominantly mediated by T cells. Transplantation of stem cell derived beta-like cells (sBC) have been shown to rescue diabetes in preclinical animal models. However, how sBC will respond to an inflammatory environment with diabetogenic T cells in a strict human setting has not been determined. This is due to the lack of model systems that closely recapitulates human T1D. Here, we present a reliable in vitro assay to measure autologous CD8 T cell stimulation against sBC in a human setting. Our data shows that upon pro-inflammatory cytokine exposure, sBC upregulate Human Leukocyte Antigen (HLA) class I molecules which allows for their recognition by diabetogenic CD8 T cells. To protect sBC from this immune recognition, we utilized genome engineering to delete surface expression of HLA class I molecules and to integrate an inducible overexpression system for the immune checkpoint inhibitor Programmed Death Ligand 1 (PD-L1). Genetically engineered sBC that lack HLA surface expression or overexpress PD-L1 showed reduced stimulation of diabetogenic CD8 T cells when compared to unmodified cells. Here, we present evidence that manipulation of HLA class I and PD-L1 receptors on sBC can provide protection from diabetes-specific immune recognition in a human setting.

Secreted cytokines provide local immune tolerance for human stem cell-derived islets

“…The utility of an immune-evasive/tolerogenic islet cell replacement therapy relies on the ability to maintain transgene expression throughout cell differentiation and after transplantation. Thus, to analyze the effect of various gene-editing strategies for the immune protection of SC-islet cells, we engineered SC-islet cells to constitutively express tolerogenic molecules including PD-L1 (Castro-Gutierrez et al, 2021; Yoshihara et al, 2020) and the HLA-E long-chain fusion (Gornalusse et al, 2017) from the GAPDH locus, both in a HLA-competent and HLA-deficient background. We report the lack of a xeno-protective effect of PD-L1 over-expression in SC-islet cells.…”

“…This could be explained by the presence of other immune cell subsets such as macrophage, monocytes and dendritic cells that may play a role in indirect allograft rejection (Oberbarnscheidt et al, 2014; Wyburn et al, 2005; Zhuang et al, 2016). Ultimately, this highlights the limitations of exclusively using in vitro immune cell co-culture assays to assess the effect of genetic modification on the protection SC-islet cells from immune destruction (Castro-Gutierrez et al, 2021; Leite et al, 2022).…”

“…Knockout of the beta 2 microglobulin (B2M) gene effectively eliminated HLA class I expression on B2M -/- , B2P and BEC SC-islet cells. After IFN-γ stimulation, HLA class I and PD-L1 were upregulated in WT SC-islet cells (Castro-Gutierrez et al, 2021), whereas B2P SC-islet cells constitutively over-expressed PD-L1 and lacked HLA class I expression ( Figure 1C ). A similar expression profile of HLA-E and HLA class I expression was observed on WT and BEC SC-islet cells ( Figure 1D ) (Gornalusse et al, 2017).…”

“…Current strategies to protect allografted islet cells include encapsulation (Alagpulinsa et al, 2019; Bochenek et al, 2018), modifying the patient’s immune system by co-administration of biologicals such as low dose IL-2 and anti-CD3 (Tepluzimab) (Hartemann et al, 2013; Herold et al, 2019), and/or genetically modifying the SC-islets. Since the main contributors of immune recognition and rejection are the human leukocyte antigens (HLAs), targeting the HLAs has been performed in iPSCs to reduce or eliminate the immune response against foreign cells (Castro-Gutierrez et al, 2021; Deuse et al, 2019; Gornalusse et al, 2017; Han et al, 2019; Harding et al, 2019; Riolobos et al, 2013; Xu et al, 2019; Yoshihara et al, 2020). To date, the engineering strategies demonstrating some immune-protection of SC-islet cells have employed lentiviral over-expression of PD-L1 and the selective retention of a single HLA-A2 allele in HLA-B/C deficient cells (Parent et al, 2021; Yoshihara et al, 2020).…”

Emerging diabetes therapies: Bringing back the β-cells

“…Using genomic engineering [ [44] , [45] , [46] , [47] ] or epigenetic modification [ 20 ], several groups have reported the successful generation of hypoimmune PSCs and cells derived therefrom [ 42 ]. These “immune cloaking” strategies usually work either by removing receptors important for immune cell recognition or by artificially elevating immune suppressive protein (e.g., immune checkpoint inhibitors) levels on the surface of the cells.…”