Potential role of B- and NK-cells in the pathogenesis of pediatric aplastic anemia through deep phenotyping
Aplastic anemia (AA) represents a remnant group in pediatric patients with bone marrow failure (BMF) after exclusion of identifiable causes of BMF, such as inherited bone marrow failure syndromes (IBMFS). The current Dutch consensus diagnostic guideline for pediatric BMF patient results in approximately 60% identifiable causes of BMF and the remaining 40% is classified as AA. Therefore, in spite of the widely accepted hypothesis of an auto-immune mechanism underlying AA, the pediatric AA cohort represents a heterogeneous group of unidentified events causing BMF. This might also partly explain poor response to immune suppressive therapy (IST). Due to the lack of diagnostic tools to prove immune AA and markers to predict IST response, together with the unequaled curative potential of hematopoietic stem cell transplantation (HSCT), most pediatric severe AA patients are momentarily treated by HSCT.
Our overall aim is to dissect the heterogeneous group of pediatric AA patients into subgroups based on disease etiology with or without immune involvement. In this study, we aimed to investigate the role of NK- and B-cells in a pediatric AA cohort.
Seven pediatric AA patients were included. The diagnosis of AA was based on the combination of peripheral cytopenia and hypocellular bone marrow and the exclusion of all other known causes of BMF by extensive diagnostics towards IBMFS, predisposition syndromes and secondary BMF. As controls, seven age-matched healthy bone marrow donors were included. Paired peripheral blood and bone marrow samples were collected prior to treatment. Peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) were isolated with Ficoll density gradient centrifugation and analyzed by high-dimensional spectral flow cytometry.
We observed a reduced absolute number of NK-cells in peripheral blood of AA patients with a skewed distribution towards CD56bright NK-cells in a subgroup of patients. Interestingly, the enriched CD56bright NK-cells had a lower expression of CD45RA and TIGIT and a higher expression of CD16, compared to healthy donors.
Also, B-cell counts were lower in AA patients. Subset analysis revealed that transitional B-cells were significantly reduced in both absolute numbers and relative values compared to healthy controls. As these cells were previously hypothesized as regulatory cells in AA, decreased numbers might be involved in defective inhibition of auto-reactive T-cells. Interestingly, even in patients with normal distribution of earlier stages of B-cells within the bone marrow, the transitional compartment was reduced, indicating selective differentiation failure from immature to transitional B-cells or a selective loss.
Our findings provide a base for future studies to unravel the role of transitional B-cells and CD56bright NK-cells in larger cohorts of pediatric AA patients as a diagnostic marker for a subgroup of immune AA.