Murine Bone Marrow-Derived Megakaryocytes Are Capable of Antigen Cross-Presentation on Major Histocompatibility Class (MHC) I Molecules

Major Histocompatibility complex (MHC) class I is a complex composed of two proteins, the MHC-encoded alpha heavy chain and the beta2-microglobulin light chain. In the endoplasmic reticulum, it receives an antigen peptide of usually eight to eleven residues in its groove and is exported to the plasma membrane. In classical MHC class I presentation, endogenous antigens are presented to CD8+ T cells. On the other hand, cross-presentation consists in the presentation of exogenously-derived proteins in the context of MHC class I molecules to CD8+ T lymphocytes, inducing a cytotoxic response against the antigen presenting cell (APC). Platelets have been previously shown to be able to cross-present immunogenic ovalbumin (OVA) peptides, as well as bacterial peptides and to trigger CD8+ T lymphocyte activation.  It is thought that the antigen cross presenting function of platelets is transmitted from megakaryocytes (MK) during thrombopoiesis, but whether megakaryocytes can cross-present is still unknown. To address this question, we developed an in vitro endocytosis model of murine bone marrow-derived megakaryocytes.  C57BL/6 mouse megakaryocytes are enriched by gradient sedimentation and pulsed with fluorescent ovalbumin (OVA) for various times. In addition, we used DQ-OVA, a BODIPY FL-conjugated OVA, which becomes fluorescent upon proteolysis. Cells were then analyzed by flow cytometry or confocal microscopy to assess the OVA megakaryocyte internalization, its proteolytic degradation and its loading onto MHC class I molecules. We can show the endocytosis of exogenous OVA by megakaryocytes, in a pathway involving lysosomes. The endocytosed OVA were subsequently proteolytically cleaved into the immunogenic OVA peptide SIINFEKL and the peptide is inserted into the antigen binding groove of MHC class I molecules. The MHC class I-OVA peptide complexes are stored in the MK alpha-granules and can be translocated to the plasma membrane.  Thus, we have shown that MKs can effectively process and present exogenous antigens on MHC class I molecules.  This opens a new avenue toward a functional characterization of this pathway and its modulation, leading to new therapeutic approaches in autoimmune diseases targeting megakaryocytes, such as immune thrombocytopenia (ITP).