Macrophage CD38 overexpression drives primary platelet engraftment failure via paracrine suppression following allogeneic hematopoietic stem cell transplantation

Abstract

Abstract Background: Post-transplant thrombocytopenia (PT) is a severe complication following hematopoietic stem cell transplantation (HSCT), characterized by persistent thrombocytopenia and increased bleeding risk. Dysregulated CD38 expression in monocytes/macrophages is implicated in PT, but the mechanisms linking CD38 to inflammatory imbalance, impaired megakaryocyte differentiation, and delayed platelet recovery remain unclear. Aims: This study aimed to define the pathological role of macrophage CD38 in PT, investigating its effects on megakaryocyte dysfunction and platelet regeneration, and evaluating CD38 inhibition as a therapeutic intervention. Methods : Proportions of CD38+ monocytes/macrophages in PT patients and controls were quantified by flow cytometry. Cytokine profiles were assessed using MSD U-PLEX and ELISA, and megakaryocyte transcription factors were analyzed by qRT-PCR. Myeloid-specific CD38-knockout mice (Lyz2-Cre;CD38fl/fl) were validated using electrophoresis, RT-qPCR, flow cytometry, and Western blotting. Paracrine effects were examined in Transwell co-cultures with bone marrow-derived macrophages and hematopoietic stem cells. Allogeneic transplantation models utilized irradiated mice, monitored for hematologic recovery. Anti-CD38 monoclonal antibody (αCD38) therapy was tested in murine models. Mechanistic analyses included single-cell RNA sequencing (scRNA-seq) to resolve hematopoietic heterogeneity and bulk RNA sequencing to profile myeloid transcriptional changes. Ligand-receptor interactions were mapped using CellChat, and kinase signaling (p-ERK/p-AKT) was validated by Western blotting. Results : PT patients exhibited elevated proportions of CD38+ monocyte/macrophage versus controls (84.12% [60.42–87.58%] vs. 55.31% [50.48–62.70%], P<0.001), correlating inversely with platelet counts (r=-0.693, P=0.004) and megakaryocyte numbers (r=-0.720, P=0.003). Pro-inflammatory cytokines increased, whereas anti-inflammatory mediators and megakaryocyte transcription factors decreased. In co-culture systems, myeloid-specific CD38-knockoutmice (Lyz2-Cre; CD38fl/fl) demonstrated significant expansion of megakaryocyte progenitors and enhanced megakaryocyte differentiation compared to genetic controls (CD38fl/fl). Cytokine shifts in bone marrow supernatants were characterized by decreased levels of pro-inflammatory cytokines and elevated levels of immunomodulatory cytokines. Mechanistically, CD38 deficiency activated the IGF-1R–ERK/AKT signaling axis, significantly increasing the phosphorylated/total protein ratios of both ERK and AKT , an effect reversed by IGF-1R inhibition. Transplantation models demonstrated accelerated platelet recovery in myeloid-specific CD38-knockout mice versus genetic controls, with significantly higher platelet counts. In transplantation models, mice with myeloid-specificCD38-knockout exhibited accelerated platelet recovery and significantly higher platelet counts compared to genetic controls. αCD38 therapy induced transient suppression followed by expedited regeneration. Conclusions : Macrophage CD38 overexpression drives PT pathogenesis by promoting inflammatory dysregulation and impairing megakaryopoiesis via IGF-1-dependent ERK/AKT signaling. CD38 inhibition accelerates platelet recovery, supporting its therapeutic potential for PT.