Bioengineered Glycoprotein-Deficient iPSC-Derived Megakaryocytes for the Sensitive Detection and Characterization of Patient Antibodies in Autoimmune Thrombocytopenia

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by a persistently low platelet count. Many patients have only mild bleeding symptoms, but severe and life-threatening bleeding may occur that can require extensive therapy for many years and reduce one's health-related quality of life. ITP remains a diagnosis of exclusion due to the lack of reliable standard tests or biomarkers to confirm its diagnosis. Although platelet autoantibodies targeting platelet surface glycoproteins (GP) are the major underlying cause of ITP, diagnostic testing for platelet autoantibody detection is not routinely employed, mainly due to the low sensitivity of currently available methods. There is a significant need, therefore, for alternative, practical diagnostic reagents and methods that can both detect and characterize anti-platelet autoantibodies with high specificity and high sensitivity. To fulfill this unmet need, we generated a series of Class I HLA-negative, blood group O, glycoprotein-deficient, human induced pluripotent stem cell (iPSC) lines, differentiated them into megakaryocytes, and used the resulting cells to develop a rapid, whole-cell flow cytometric assay that can simultaneously detect and characterize the specificities of anti-platelet autoantibodies. Since GPIIb-IIIa (= the integrin αIIbβ3) and GPIb-IX are the two most commonly observed autoimmune targets in ITP, we used CRISPR technology to ablate either the gene encoding GPIIb or the gene encoding GPIbα to generate iPSC lines. GPIIb-deficient iPSC-derived megakaryocytes showed complete loss of surface GPIIb-IIIa expression, but maintained normal αVβ3 expression, as expected. GPIbα-deficient iPSC-derived megakaryocytes completely lost GPIbα expression, along with reduced expression of the other components of the GPIb-IX-V complex. We then examined the ability of these iPSC-derived megakaryocyte cell lines to react with four different patient ITP sera. Multiple control normal human sera showed extremely low background binding to all WT and GP-deficient megakaryocytes by flow cytometry, benefiting from the unique design of the lack of ABO and Class I HLA antigens on the cell surface. In contrast, all four ITP patient sera demonstrated strong antibody binding to wild-type and GPIbα-deficient megakaryocytes. Two of these failed to bind to GPIIb-deficient megakaryocytes, demonstrating that their autoantibodies are specific for epitopes present on the GPIIb-IIIa complex. The other two ITP sera showed significantly reduced, but not negative, binding to GPIIb-deficient megakaryocytes, strongly suggesting that the autoantibodies are likely specific for the integrin β3 subunit common to both GPIIb-IIIa (αIIbβ3) and αVβ3. The availability of novel bioengineered wild-type megakaryocyte cell lines expressing intact platelet glycoprotein antigens on their surface, together with select glycoprotein-deficient megakaryocytes serving as internal controls, should facilitate autoantibody detection and characterization, and significantly benefit clinical diagnosis and treatment of ITP.