CRISPR/Cas9-Mediated Disruption of PD-L1 Reduces the T Cell Suppressive Effect of Wharton's Jelly Mesenchymal Stromal Cells and Their Extracellular Vesicles
Authors: Dunavin Neil , Li Meizhang , Atay Safinur , Mitchell Jim , Soder Rupal , Abhyankar Sunil , McGuirk Joseph P. , Godwin Andrew K , Dawn Buddhadeb 
Published: October 2019

Abstract
Background
Mesenchymal stem/stromal cells (MSCs) are a promising treatment for acute graft-versus-host disease and other inflammatory diseases. MSCs express programmed death-ligand 1 (PD-L1) that has been reported to contribute to MSC-mediated immune suppression through T-cell receptor (TCR)-mediated inhibitory signaling. MSCs actively secrete extracellular vesicles (EVs) that have immune modulatory properties resembling their cell of origin. Although the release of EVs by MSCs has been extensively reported, the presence and functional impact of immune inhibitory checkpoint molecules in MSC-derived EVs is still being determined. We sought to determine if PD-L1 and other immune inhibitory checkpoint molecules are enriched in MSC-derived EVs. In this study we report that PD-L1 is enriched in MSC-EVs, and that PD-L1(+) EVs and PD-L1(-) EVs differ in their T cell immune suppressive capacity.
Methods
Umbilical cords and healthy donor blood samples were collected on separate IRB-approved protocols after written informed consent (HSC#1546/HSC#5929). Umbilical cord Wharton's jelly-MSCs (WJMSC) were isolated from umbilical cords under GMP conditions and characterized by morphology, multiparameter flow cytometry (MPFC), and differentiation capacity. Three clinical-grade WJMSC cell lines were cultured at low passage (2-5) in EV-depleted media. EVs were isolated from the media by differential ultracentrifugation and the 100K pellets enriched with exosomes were characterized by Western blot (WB), nanoparticle tracking analysis (NTA), and electron microscopy (EM). To further identify EV-enriched immune proteins, the WJMSC and EV proteomes were characterized by Tandem Mass Tag (TMT) labeling and LC MS/MS, using isobaric labels to compare protein quantities between samples. Gene disruption of PD-L1 was performed through site-specific CRISPR/Cas9 introducing a single cysteine nucleotide deletion in exon 3; the genetic mutation was confirmed by Sanger sequencing. The absence of PD-L1 transcript was confirmed by RT-PCR, and the absence of PD-L1 protein was confirmed by WB, immunofluorescence, MPFC, and IFNγ-response. PBMC were isolated from healthy donor blood samples; T cells were activated by CD3/CD28 beads and induced CD154 was measured on CD4(+) T cells by MPFC. Alternatively, CMV-experienced T cells were activated with CMV-pp65 peptide and induced IFNγ was measured on CD8(+) T cells by MPFC. Activated T cells were treated with PD-L1(+) and PD-L1(-) WJMSCs or their derived EVs. Phospho-Zap-70 (Tyr319)/Syk (Tyr352) MPFC was used to analyze T-cell receptor (TCR)-mediated signaling.
Results
WJMSCs were CD73(+), CD90(+), CD105(+), and CD34(-), CD45(-), CD11b(-), CD19(-) and HLA-DR(-); and inhibited proliferation in a mixed lymphocyte reaction. WJMSCs-derived EVs were cup-shaped 100-200nm nanoparticles by EM and were CD9(+), CD81(+), CD73(+), CD90(+), and CD105(+) by WB. Quantitative proteomic profiling revealed a total of 1,634 proteins across WJMSC-derived EV preparations. Proteins that were highly enriched in MSC-derived EVs and that were classified under "negative regulation of lymphocyte activation" (Gene Ontology Biological Processes; GOBP) included PDL1, CD276, DLG1, TNR6, ERBB2, TGFβ1, LYN, and HMGB3. Both WJMSCs and EVs suppressed CD4(+) T cell activation by CD3/CD28 beads and CD8(+) T cell activation by CMV peptide. CRISPR/Cas9 edited WJMSC exhibited normal morphology and differentiation characteristics. PD-L1 gene disruption efficiently reduced PD-L1 protein expression on WJMSC and their derived EVs, and reduced the ability of both to suppress T cell activation. Similarly, blocking PD-L1 with a specific internalization antibody decreased WJMSC and EV-mediated T cell activation.
Conclusion
Proteomic analyses reveal that PD-L1 is enriched in WJMSC-derived EVs. CRISPR/Cas9-mediated disruption of PD-L1 in WJMSCs alone did not affect the release of EVs in vitro. Gene-edited WJMSCs stably generate PD-L1(-) EVs. The PD-L1(-) EVs generated from PD-L1(-) WJMSC did not suppress T cell activation in co-culture. These findings indicate that PD-L1 is enriched in WJMSC EVs and is biologically active. WJMSC-EVs can deliver membrane-bound PD-L1 to T cells, resulting in attenuated TCR signaling.

Disclosures
McGuirk: Fresenius Biotech: Research Funding; Bellicum Pharmaceuticals: Research Funding; Astellas Pharma: Research Funding; Pluristem Ltd: Research Funding; Gamida Cell: Research Funding; Kite Pharma: Honoraria, Other: travel accommodations, expenses, speaker ; Novartis Pharmaceuticals Corporation: Honoraria, Other: speaker, Research Funding.