ABSTRACT: Here we investigated bone marrow (BM) features in a large cohort of 97 patients with autoimmune hemolytic anemia (AIHA) and observed a high prevalence of hypercellularity, dyserythropoiesis, reticulin fibrosis, and T-cell infiltration (65%, 29%, 76%, and 69% of patients, respectively). These findings were associated with inadequate bone marrow compensation, more severe anemia at onset, and need of multiple treatments. In a subset of warm type AIHA patients we investigated BM microenvironment by single-cell RNA sequencing. We found distinct immune cell profiles across disease stages (diagnosis, remission, relapse). In particular, upregulation of inflammatory response pathways was noted in CD8+, CD4+, and monocyte subsets during relapse compared to diagnosis and remission. Moreover, by single-cell TCR sequencing, we found small T cell clones at diagnosis that may either disappeared or expanded at remission. Disappearing clones exhibited a naive CD8+ phenotype and were more likely to respond to glucocorticoid treatment. Expanding clones showed upregulation of cytotoxic T cell markers and may play a role in the transition to a chronic/relapsing phase. Finally, cytokine gene expression differed across disease phases. At relapse, pro-inflammatory cytokines such as TNF-alpha, IL-1, and IL-6 were upregulated in CD4+ and CD8+ T cells, while TGF-beta was downregulated, potentially counteracting the transition to chronic phase. This is the largest study evaluating BM histology and clinical characteristics, and the first evaluation of BM microenvironment by single-cell RNA sequencing. We showed a complex scenario encompassing T-cell infiltration, clonality, and up/down-regulation of cytokine genes, associated with a more severe and relapsing disease.
