Single-cell analysis highlights a population of Th17-polarized CD4+ naïve T cells showing IL6/JAK3/STAT3 activation in pediatric severe aplastic anemia

Acquired aplastic anemia (AA) has been long ascribed to immune-mediated hematopoietic failure, with up to 65%–70% of cases showing hematologic recovery following immunosuppressive therapies (ISTs) such as antithymocyte globulin (ATG). Furthermore, long-standing clinical experience has demonstrated the efficacy of calcineurin inhibitors (usually cyclosporine) as maintenance therapy [1, 2]. Modern insights into the heterogeneous molecular evidence have shifted the paradigm of AA frontline management from restoration of dysregulated immune to stimulation of functional hematopoiesis [[3], [4], [5]]. However, the mechanistic cues of immune attack causing bone marrow (BM) failure remain to be elucidated [6].

The hypervariable complementarity-determining region 3 (CDR3) found in T cell receptors (TCRs) is responsible for unique antigen recognition and T cell activation in healthy BM. Skewed oligoclonality of TCR has been identified by CDR3 spectratyping as indirect evidence of the role of activated cytotoxic T cells in the BM failure underlying AA [[7], [8], [9]]. Nonetheless, skewed oligoclonal cytotoxic CD8+ T cell expansion accompanied by the release of proinflammatory cytokines and induction of apoptosis seems unlikely to fully explain the immunologic mechanisms of BM impairment in AA [10, 11]. Indeed, CD4+ T cell-induced autoimmune responses have also been considered crucial in the pathophysiology of AA, contributing indispensably to the development of BM failure. There is mounting speculation that imbalanced IFN-γ–producing CD4+ T helper cells (Th1 cells), IL-4–producing CD4+ T helper cells (Th2 cells), regulatory T cells (Tregs), and IL-17–producing CD4+ T helper cells (Th17 cells)—all of which have been identified as dysfunctional in different autoimmune disorders [[12], [13], [14], [15]]—play pivotal roles in the consecutive immunopathogenesis of severe and very severe aplastic anemia (SAA/VSAA); these cells are also likely to be associated with disease severity [16, 17]. Aside from T lymphocyte imbalance and clonality, analyses thus far have not dissected the molecular mechanisms underlying the immune pathogenicity correlated with BM failure; the intrinsic heterogeneity of the CD4+ and CD8+ T cell compartments is likely to be key in this regard. Furthermore, the identification of functionally critical determinants closely associated with clinical outcomes in SAA due to hypocellular BM has previously been limited by analytical resolution.

Recently, single-cell transcriptomic sequencing has been used to attain insights into the sustained abnormal transcriptional status of hematopoietic stem and progenitor cells (HSPCs) attacked by engaged T cells, yielding potential therapeutic opportunities for AA. However, the regulatory mechanisms and immune signatures governing cellular fate are possibly posttranscriptional and therefore beyond the scope of genetic landscape analysis or transcriptomic profiling. In contrast, mass cytometry utilizing more than 40 metal-tagged antibodies has allowed the characterization of integrated functional cellular behavior tightly associated with protein expression, involving signaling cues [18] and fate programming checkpoints [19]. Previous attempts utilizing mass cytometry have elucidated the unique immune signatures of Treg subpopulations from AA patients using annotations from an antibody panel based on cell surface markers, transcription factors, and cytokines [17]. These explorations provided insight into the functional categorization of rare, complex cell populations with distinct immune signatures at single-cell resolution.

To understand how the remodeling of BM cellular composition underlies autoimmune responses in SAA and VSAA, as well as to interrogate the interplay of immune cells and BM failure, we adopted a single-cell mass cytometry (also known as ‘cytometry by time-of-flight’, or CyTOF) approach. Comparison of bone marrow mononuclear cells (BMMCs) from pediatric SAA/VSAA patients at pre- or post-IST stages with healthy BMMCs enabled us to functionally define critical BM cellular compartments correlated with disease onset and therapeutic responses. Building on the functional characterization of the T lineage compartment, we used single-cell RNA sequencing to deeply validate potential regulatory mechanisms underlying immune-based hematopoietic sabotage in AA and pinpoint indicative biomarkers correlated with clinical outcomes. Our results highlight potential therapeutic targets for autoimmune diseases with similar underlying mechanisms.

留言 (0)

沒有登入
gif