Megakaryocytes Direct CD4 + T Cell Responses Via Major Histocompatibility Complex Class II Antigen Presentation

Abstract

Megakaryocytes (MKs) are rare, large, hematopoietic cells that canonically have the role of producing platelets. Advances in single-cell RNA-sequencing (scRNA-seq) have revealed a previously under-appreciated degree of MK heterogeneity suggesting that both murine and human MKs may perform functions beyond platelet production. Among other inflammatory molecules, these studies identified that select MK subsets express major histocompatibility complex (MHC II) antigen presentation-associated transcripts and display an upregulation of antigen processing pathways. Together, these findings suggest a potential immunoregulatory function of select MK subsets. However, functional validation substantiating these transcriptional signatures and the proposed MK immunoregulatory capacities has lagged, and the expression of MHC II in bone marrow MKs remains controversial. In our studies, we aimed to both quantify and functionally assess immunomodulatory bone marrow MKs. Using flow cytometry and high-resolution confocal imaging, we showed that MHC class II is expressed on ~20% of MKs at various stages of differentiation. Of the cell populations identified, MHC class II expression was observed in CD41+ and vWF+ platelet-biased hematopoietic stem cells (HSCs), c-Kit+CD150+CD41+ MK progenitors, immature CD41+ MKs and mature CD41+ CD42d+ MKs. Intriguingly, we also observed that MHC class II expression was dramatically upregulated, (2.5-fold increase in expression) in mature MKs and MK progenitors of aged mice. Additionally, MHCII expression was significantly correlated with vWF and CD41 expression in long-term HSCs, with 60-70% of MHCII+ HSCs co-expressing both of these markers. To further validate MHC class II expression in the MK lineage, we utilized two unique murine MHCII transgenic reporter models. The first was an MHCII-EGFP knock-in mouse in which endogenous class II product is replaced by GFP, and the second was a Cd74tdT reporter mouse in which the invariant chain (CD74) can be visualized via tdTomato upon MHC class II pathway activation. In both models, approximately 20-30% of MKs were MHCII positive, consistent with antibody staining in wild-type mice. To substantiate the immunoregulatory function of primary murine MKs, we assayed antigen presentation capacity and ability to stimulate T-cells in vitro. We observed that bone marrow derived MKs express basal levels of multiple co-stimulatory receptors including CD80, CD83, CD86, and CD40 in addition to MHC class II. We also observed significant upregulated of these receptors upon TLR stimulation (TLR3, TLR4 TLR9) in a dose dependent manner. Notably, co-culture of MKs with CD4+ T cells resulted in naive T cell activation and proliferation. To verify that these results extended to human MKs, we examined HLA class II expression in MKs differentiated from CD34+ cells obtained from human bone marrow. Comparable with the murine studies, we found that human MKs also upregulated class II pathway molecules, suggesting that human MKs may also engage with the adaptive immune system. Together, our results support the hypothesis that there is an immunomodulatory sub-population of MKs that can be distinguished by MHCII surface expression, engages in class II antigen presentation, and can activate CD4+ T cells to prime adaptive immune responses. Future studies will investigate the in vivo role of these immunomodulatory functions of MK and how they may be exploited to alter T cell differentiation in the context of inflammatory, infectious, or age-associated thrombotic diseases.