Prime suspects: cross-presenting platelet EVs

In this issue of Blood, Marcoux et al¹ identified platelet-derived extracellular vesicles (PEVs) that harbor proteasomes and can process and present antigens on class I molecules to CD8⁺ T cells in lymphoid organs. Their findings have implications for numerous pathologic conditions that are caused by or involve platelet activation.

The role of platelets and megakaryocytes as innate immune regulators has gained support from numerous studies over the years.² Platelets and megakaryocytes contain functional proteasomes for antigen processing and peptide loading onto the major histocompatibility complex class I molecules (MHC-I) that can cross-present antigens to CD8⁺ T cells.^3,4 When activated, platelets release extracellular vesicles (EVs) from their plasma membrane or endosomal compartments. Unlike platelets, which are mainly confined to the blood circulation, the smaller-sized PEVs readily traffic to organs and tissues where they could potentially deliver platelet-derived molecules and engage target cells. However, it was not known whether PEVs harbor antigen-presentation machinery and can contribute to adaptive immunity in lymphoid tissues that are inaccessible to platelets.

Marcoux et al now show, using high-sensitivity flow cytometry, the presence of PEVs containing a functional proteasome along with a functional antigen-presentation machinery and T-cell costimulatory surface molecules. Proteasome-containing PEVs were detected in the blood of healthy individuals, constituting roughly 2.6% of total PEVs, equivalent to 2 million PEVs per milliliter of blood. Proteasome-containing PEVs increased, in vitro and in a mouse model, following stimulation of human platelets with immune complex injections. PEVs transfused into mice disseminated within 15 minutes into lymphoid organs, primarily the spleen and lymph nodes, and were detected in the lymph, with roughly 14% of PEVs containing an active proteasome. Approximately 1% to 2% of the lymph PEV⁺ proteasomes also expressed MHC-I (amounting to as much as 5 × 10⁵ proteasome⁺ MHC-I⁺ PEVs). More importantly, when pulsed in vitro with the ovalbumin (OVA)-derived peptide, the MHC-I proteasomes were able to present the peptide in complex with MHC-I on their surface. Impressively, when pulsed with the native OVA protein, MHC-I/OVA peptide complexes were detected on PEVs, indicating that PEVs can proteolytically process OVA into smaller peptides for presentation via MHC-I. Finally, PEVs pulsed with OVA peptide or native OVA induced antigen-specific OT-1 CD8⁺ T-cell proliferation and increased cytokine expression, confirming that PEVs can crosspresent antigens to T cells. The actual role of PEVs in the stimulation of T cells in vivo including with less immunodominant antigens that OVA remains to be determined. Nevertheless, these findings raise the exciting possibility that PEVs may actively participate in adaptive immunity, including the generation of antiplatelet (eg, CD41 or CD61) immunity and may even inadvertently contribute to pathologic disorders, such as autoimmune diseases, similar to mitochondria-containing PEVs.⁵ Given that EVs from megakaryocytes⁶ are also detected in blood under healthy conditions, future studies are needed to examine the role of megakaryocyte proteasome+ EVs in crosspresentation.

As emphasized by the investigators, packaging of crosspriming machinery into PEVs by platelets suggests that the T-cell–modulating properties of platelets may also extend outside of the confines of the blood. Platelets and megakaryocytes can be infected and/or induce immunity against pathogens, but they lack the ability to enter the lymphatic system. Analogous to released exosomes from dendritic cells containing phagocytosed pathogens,⁷ it may be that platelet-derived cytosolic microbial proteins transferred to PEVs could be processed by their proteasome and contribute to the presentation of microbial antigens within lymph tissues. Given that EVs derived from other immune cells, such as dendritic cells, can crosspresent in vivo,⁸ it is important, as the investigators propose, to determine the relative impact of PEVs, which are among the most abundant EVs in blood, as antigen-presenting elements. Equally important is determine how efficient and sustained the activation is, because there are relatively small fractions of PEVs that coexpress proteasome and MHC-I. Compared with PEVs harboring proteasomes, PEV-containing mitochondria are rare in lymph, but unlike mitochondria+ PEVs which were elevated in platelet concentrates (PCs) that caused adverse transfusion reactions (ATRs),^9,10 no significant increase was found in the concentrations of proteasome-containing PEVs in the ATR-associated PCs. This highlights the functional heterogeneity in PEVs, which appears to be linked to their content and tissue distribution, underscoring the role of platelet-sorting mechanisms that determine which organelles are transferred from the platelet to PEVs.

In summary, the present study has uncovered a novel regulator of adaptive immunity, namely PEVs harboring proteasomes that enable antigen processing and presentation on the plasma membrane. Along with megakaryocytes and platelets, PEVs can be added to the list of nonprofessional antigen-presenting entities with potential for cross-presentation to CD8⁺ T lymphocytes. Future studies are needed to define their protective vs pathologic roles in infection/tumor immunity and autoimmunity.