OC 18.3 - New profile of immune embryonic-like megakaryocytes: Role on RUNX1

Sunday, June 23, 2024

10:00 – 10:15 ICT

Room: 211 A-D

Presenting Author(s)

Cecilia P. Marin Oyarzun, PhD

Postdoctoral researcher
Inserm UMR1287, Gustave Roussy, Université Paris Saclay, Équipe labelisée LNCC, Villejuif, France
Villejuif, Ile-de-France, France

Co-Author(s)

BA

Brahim Arkoun

Postdoctoral researcher
Inserm UMR1287, Gustave Roussy, Université Paris Saclay, Équipe labelisée LNCC, Villejuif, France
Villejuif, Ile-de-France, France
NB

Nathalie Balayn

Assistant engineer
Inserm UMR1287, Gustave Roussy, Université Paris Saclay, Équipe labelisée LNCC, Villejuif, France
Villejuif, Ile-de-France, France
VS

Vithuzane Selvarasa

Master student
Sorbonne Université, Paris, France
Villejuif, Ile-de-France, France
AG

Ana Glembtosky

Researcher
Universidad de Buenos Aires, Facultad de Medicina, Instituto de Investigaciones Médicas "Alfredo Lanari", IDIM UBA-CONICET, Departamento de Hematologia Investigacion, Buenos Aires, Argentina
Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina
MA

Marine Aglave

Bioinformatician
Gustave Roussy, Plateforme de Bio-informatique, Villejuif, France
Villejuif, Ile-de-France, France
WV

William Vainchenker

Emeritus Researcher
Inserm UMR1287, Gustave Roussy, Université Paris Saclay, Équipe labelisée LNCC, Villejuif, France
Villejuif, Ile-de-France, France
IP

Isabelle Plo

Researcher
Inserm UMR1287, Gustave Roussy, Université Paris Saclay, Équipe labelisée LNCC, Villejuif, France
Villejuif, Ile-de-France, France
Paula Heller

Researcher
Universidad de Buenos Aires, Facultad de Medicina, Instituto de Investigaciones Médicas "Alfredo Lanari", IDIM UBA-CONICET, Departamento de Hematologia Investigacion, Buenos Aires, Argentina
Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina
HR

Hana Raslova

Researcher
INSERM U1287, Gustave Roussy, Université Paris Saclay, Villejuif, France
Villejuif, Ile-de-France, France

Background: RUNX1 familial platelet disorder (RUNX1-FPD) is characterized by germline RUNX1 mutations, thrombocytopenia, leukemic predisposition and in some cases eczema or autoimmune disorders. The main function of megakaryocyte is thrombopoiesis, but recently subpopulations of immune MKs were described. The role of RUNX1 in their development is presently unknown.

Aims: Study whether “immune-like” human MKs can be derived from hiPSC and the role of RUNX1 in their emergence and function.

Methods: WT and RUNX1-FPD (p.Thr246Argfs*8) hiPSC were differentiated to MKs, sorted on CD41+CD42+ at day 16 of culture and analyzed by single cell RNA sequencing (scRNAseq) 10X Genomics. Immunofluorescence, qPCR and FACS analysis were used to study RNA and protein exchange.

Results: scRNAseq analysis revealed six MKs clusters, two of them presenting signatures of immune pathways (fig1). The small cluster (MKC5), expressing lower levels of MK markers, was related to neutrophils. We demonstrated RNA and protein exchange between myeloid cells and MKs in vitro explaining this cluster´s molecular signature. We also showed that CD41+CD42+CD14+ cells were in fact macrophages phagocyting MKs and not really a cluster of immune MKs.
The most important immune cluster (MKC1) was characterized at molecular level by lower MPL expression, “Interferon type I response” and activated NFkB pathways and higher MHCI, suggesting its involvement in antigen presentation and inflammation. RUNX1-FPD MKs presented downregulation of MPL and MHCI in all clusters except MKC1, in which up-regulation of NFkB-pathway genes was detected (fig2).

Conclusion(s): We described two “immune-like” MKs clusters: MKC5, a culture artifact; and MKC1, with increased MHCI expression and NFKB activation, with still unknown function in immunity. In RUNX1-FPD, the NFkB pathway is hyperactivated in the MKC1 cluster, probably due to loss of its negative regulation by RUNX1. In contrast, in the other clusters a decrease in MHCI was associated with MPL down-regulation suggesting the TPO/MPL axis is involved in MHCI expression.