Stem-Cell-Derived β-Like Cells with a Functional PTPN2 Knockout Display Increased Immunogenicity

Abstract

Type 1 diabetes is a polygenic disease that results in an autoimmune response directed against insulin-producing beta cells. PTPN2 is a known high-risk type 1 diabetes associated gene expressed in both immune- and pancreatic beta cells, but how genes affect the development of autoimmune diabetes is largely unknown. We employed CRISPR/Cas9 technology to generate a functional knockout of PTPN2 in human pluripotent stem cells (hPSC) followed by differentiating stem-cell-derived beta-like cells (sBC) and detailed phenotypical analyses. The differentiation efficiency of PTPN2 knockout (PTPN2 KO) sBC is comparable to wild-type (WT) control sBC. Global transcriptomics and protein assays revealed the increased expression of HLA Class I molecules in PTPN2 KO sBC at a steady state and upon exposure to proinflammatory culture conditions, indicating a potential for the increased immune recognition of human beta cells upon differential PTPN2 expression. sBC co-culture with autoreactive preproinsulin-reactive T cell transductants confirmed increased immune stimulations by PTPN2 KO sBC compared to WT sBC. Taken together, our results suggest that the dysregulation of PTPN2 expression in human beta cell may prime autoimmune T cell reactivity and thereby contribute to the development of type 1 diabetes.

Keywords: CRISPR/Cas9 knockout; PTPN2; autoimmunity; autoreactive TCR transductants; co-culture; direct differentiation; genetic risk; stem-cell-derived pancreatic beta cells; type 1 diabetes.

Figure 1

Generation of a CRISPR-mediated PTPN2 knockout in human pluripotent stem cells. (A) Schematic of CRISPR-mediated disruption of PTPN2 in hPSCs at the functional domain between exons 5 and 7. Four gRNAs targeted the functional domain of PTPN2, with two at exon 5 and two at exon 7, resulting in a large excision upon successful targeting. (B) Representative gDNA PCR amplification of clonal WT and PTPN2 KO hPSC lines employing primers that will only amplify intact, unedited DNA of exon 5. A 639 bp band amplified in WT and no band amplified in the PTPN2 KO (n = 2, independent analyses). (C) Representative gDNA PCR amplification of clonal WT and PTPN2 KO hPSC lines employing primers that will only amplify after the successful deletion of the DNA segment spanning exon 5–7 with the forward primer in exon 5 and the reverse primer in exon 7. A 1050 bp band amplified in PTPN2 KO but not in WT (n = 2, independent analysis). (D) Representative immunofluorescence images of WT and PTPN2 KO hPSC colonies. Samples were stained for OCT4 (red), NANOG (green), and SOX2 (purple) (n = 2, independent analysis). Scale bar represents 20 μm. (E) Representative Western blot analysis of PTPN2 protein expression in WT and PTPN2 KO hPSC lines indicating expression in WT hPSCs at 48 kDa and no expression in PTPN2 KO hPSCs (n = 3, independent analysis).

Figure 2

Direct differentiation of WT and PTPN2 KO to generate stem-cell-derived beta-like cells. (A) Schematic of stepwise suspension culture based direct differentiation of WT and PTPN2 KO sBC clusters. Critical developmental factors at key stages are shown. Human pluripotent stem cells (hPSC), definitive endoderm (DE), primitive gut tube (PGT), pancreatic progenitor (PP), and stem-cell-derived beta-like cells (sBC). (B) Representative bright field images of WT and PTPN2 KO differentiating clusters at key developmental stages. hPSC clusters, DE, and sBC clusters with live fluorescence imaging of insulin promoter-driven GFP expression. Scale bars represent 200 μm for the hPSC and DE images and 600 μm for sBC and GFP+ sBC images. (C) Flow cytometric quantification of pluripotency markers at day 0 (SOX2 and TRA160) and DE markers at day 3 (SOX17 and FOXA2) for WT (gray) and PTPN2 KO (blue) clusters. (n = 7 WT, n = 9 KO) ns = no significant difference, (D) Representative immunofluorescence images of WT and PTPN2 KO sBC clusters. Samples were stained for DAPI (blue), PDX1 (red), C-peptide (green), glucagon (red), somatostatin (violet), and insulin (green). Scale bar represents 20 μm. (n = 3 WT, n = 2 PTPN2 KO). (E) Representative immunofluorescence images of WT and PTPN2 KO sBC clusters. Samples were stained for insulin (green). Scale bar represents 20 μm (n = 3 for WT and PTPN2 KO). (F) Quantification of GFP expression in WT and PTPN2 KO sBC. Data are presented as mean +/− SE percentage of GFP+ cells from 4 independent differentiations. Analyzed with unpaired Student’s t test with no significant difference denoted as ns. (G) Total proinsulin content, insulin content, and proinsulin-to-insulin content ratios per 1000 FACS-sorted GFP+ sBC (n = 7 WT and n = 4 PTPN2 KO independent differentiation experiments with 3 × 1000 cells collected per experiment). Error bars are representative of the mean ± SE. Analyzed with unpaired Student’s t test with no significant differences denoted as ns. (H) Representative Western blot analysis for the PTPN2 protein or endogenous control protein Cyclophilin B of sorted day 23 GFP+ sBCs from WT and PTPN2 KO (n = 3 independent differentiations).

Figure 3

PTPN2 KO sBC displayed increased HLA class I expression. (A) Volcano plot of bulk RNAseq differential gene expression of GFP+ WT and PTPN2 KO sBCs. An adjusted p-value of 0.01 was used as the threshold for statistical significance with 308 differentially expressed genes. (B–D) Gene set enrichment from the bulk RNA seq of GFP+ WT and PTPN2 KO sBCs of differentially expressed genes reveals hallmark IFNγ response (B), oxidative phosphorylation (C), and IL6 JAK/STAT3 signaling (D). Ranked genes are shown along the x-axis with the vertical ticks representing the location of the genes in this gene set. The heatmap displays the expression of the genes: right showing those more expressed in the first group (PTPN2 KO) and left showing those more expressed in the second group (WT). The blue line shows the enrichment score. (E,F) Representative flow cytometry histogram and quantification (G,H) for the detection of surface HLA-ABC expression in WT (black) and PTPN2 KO sBC (blue) at steady state (n = 7) or treated with IFNγ for 48 h with 100 ng/mL (n = 3) shown with sBC clusters and gated for GFP+ sBCs. (* p ≤ 0.03, ** p ≤ 0.002). (I) Representative immunofluorescence image (n = 2) of WT (top) and PTPN2 KO (bottom) sBCs expressing insulin (green) HLA-ABC (pink) and DAPI (blue). Scale bar represents 20 μm. ns = no significant difference (J) Western blot of proteins extracted from GFP+ WT and PTPN2 KO sBCs expressing HLA Class 1 (n = 1).

Figure 4

Activated autoreactive T-cell transductants produce increased levels of IL-2 when stimulated by PTPN2 KO sBCs. (A) Schematic depicting the sBC and CD8 T cell receptor transductant (5KC-1.E6 or 5KC-1.C8) co-culture experiment. (B,C) Representative stimulation assay (of 3 technical repeats) of WT (gray) and PTPN2 KO (blue) sBC co-cultured with 5KC-1.C8 (TCR #1 PPI: 1-11) (n = 1) (B) or with 5KC-1.E6 (TCR #10 PPI: 15-24) (n = 4) transductants cells. T cells of type 5KC alone or treated with anti-CD3 antibody served as negative and positive controls, respectively. Data are presented as mean secreted mIL-2 concentration +/− SEM. Unpaired t-test was performed with * p ≤ 0.0332 and ** p ≤ 0.0021.