Abstract Background: Immune Aplastic Anemia (AA) is an autoimmune disease where hematopoietic stem and progenitor cells (HSPC) are destroyed by cytotoxic CD8+ T cells, resulting in peripheral cytopenias, i.e. anemia, thrombocytopenia and neutropenia. Most compelling evidence that HSPC destruction is immune-mediated is that two-thirds of severe AA patients demonstrate hematologic improvement in response to immunosuppressive therapy consisting of anti-thymocyte globulin and cyclosporin A. Interferon γ (IFNγ) and tumor necrosis factor α (TNFα) have both been implicated as critical effector molecules involved in the destruction of bone marrow HSPCs in AA. Furthermore, mRNA expression of the inflammatory chemokine receptor CXCR3 and the chemokine CCL20, a ligand of CCR6, has been shown to be elevated in peripheral blood and bone marrow mononuclear cells, suggesting a role for T helper/cytotoxic (Th/c) 1 and Th/c17 cells in the pathobiology of AA. Methods To further elucidate the pathobiology of AA, we conducted a comprehensive multiproteomic analysis of adult AA patients eligible for bone marrow transplant (BMT; n=20; samples pre-BMT) and matched healthy controls (n=20). We performed plasma proteomic analyses using Olink and conducted high-dimensional immunophenotyping by flow cytometry using peripheral blood mononuclear cells (PBMCs). Results Using Olink proteomic analysis, we observed that, relative to healthy controls, plasma from AA patients exhibited significantly higher levels of several hematopoietic growth factors, including thrombopoietin (TPO), erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF) and Fms-like tyrosine kinase 3 ligand (FLT3LG), indicating that an AA disease signature is reflected in circulation. Furthermore, we observed that AA patients had significant (Bonferroni corrected) elevations in interleukin 15 (IL15), a cytokine that plays a crucial role in T cell activation and effector functions. Thus, we looked at other T cell-associated molecules and observed trends towards elevations in several other cytokines in plasma of AA patients, including IFNγ, IL17A, IL17F, and interleukin 2 receptor α (IL2Rα). Interestingly, there was also a trend towards an increase in plasma CCL20 in AA patients, as previously reported. Using high-dimensional flow cytometry immunophenotyping on PBMCs, we observed an increase in the percentage of circulating T cells, both CD4+ and CD8+ T cells, in PBMCs from AA patients. These T cells exhibited an activated phenotype with increased surface expression of the costimulatory receptor 4-1BB. Profiling of both CD4+ and CD8+ T cell subsets revealed a T helper 17 (Th17) and T cytotoxic 1 (Tc1) cell enrichment with increased proportions in overall Th17 (both Th1.17 and Th17.22 cells) and Tc1 cells (both Tc1 and Tc1.17 cells). While antigen presentation plays a critical role in initiating immune responses in autoimmune diseases, little is known about the role of antigen presenting cells (APCs) in AA pathobiology. Here, we showed that, while the percentage of professional APCs, i.e., B cells, monocytes and dendritic cells, was reduced in blood of AA patients, these cells presented an activated phenotype characterized by significant upregulation of the costimulatory receptor 4-1BB at their surface. In addition, B cells from AA patients exhibited higher surface expression of the inflammatory chemokine receptor CCR6 and the antigen presentation molecule HLA-DR, while monocytes expressed higher level of CCR7, a chemokine receptor essential for cell migration to lymph nodes.Conclusion: In this study, we confirmed that AA is characterized by significant increases in several hematopoietic growth factors in the periphery, which may reflect compensatory mechanisms that counterbalance the lack of blood cell production in the bone marrow. Furthermore, we demonstrated that AA is an autoimmune disease characterized by an inflammatory phenotype biased towards a Th/c1.17 immune response and increased circulating Th1 and Th17 cytokines (e.g. IFNγ and IL17A/F). Finally, we showed that multiple APCs may participate in antigen presentation in AA, as both B cells and monocytes/DCs exhibit an activated phenotype with increased expression of chemokine receptors and major histocompatibility complex class II molecules. Overall, our data suggest that broad APC activation and pathogenic Th/c1.17 immune responses contribute to AA pathobiology.
Floch-Ramondou et al. (Mon,) studied this question.