Deciphering engraftment of bone marrow human single cells after allogeneic stem cell transplantation

Engraftment of donor cells is a prerequisite to successful allogeneic hematopoietic stem cell transplantation. However, it has been poorly characterized in detail both in experimental models and in human. Bone marrow examination by mass cytometry in 26 patients with myeloid malignancies and 20 healthy donors provides evidence that the proportions of myeloid and B-cell progenitors profoundly differed as compared to healthy controls with a shift toward terminal myeloid differentiation and sharp decrease of myeloid and B-cell progenitors. Principal component analysis clearly separated in 2 recipient groups (R1 and R2) driven by previous GVHD (in R2 patients). We then used single cell CITEseq to decipher donor cell engraftment in 10/26 patients (compared with 4 healthy donors). Forty-one clusters were resolved encompassing all bone marrow cellular components. Projection of patients and R1 and R2 showed drastic segregation of the 2 patient groups which were highly significant different repartition in a compositional analysis. In R2 patients sustained engraftment relied on only 10 clusters that included only committed myeloid progenitors, one stem-cell memory T-cell cluster and activated NK-cell clusters. Gene expression hallmarks disclosed repeatedly similar pathway in transplant recipients, namely, TNFα signaling via NFĸ, interferonƔ response, and allograft rejection. Thus, the cellular composition of the engrafted bone marrow in human is strongly influenced by T-cell mediated processes and inflammation leading to an emergency hematopoiesis. Bone marrow mononuclear cells (BMMNC) from patients and healthy donors were isolated by density gradient centrifugation (Ficoll-Paque) within 3h after bone marrow sampling and cryopreserved in heat-inactivated fetal calf serum (FCS) with 10% dimethyl sulfoxide (DMSO). The cell were stained with CITE-seq antibodies (ADT)