Dysfunctional Tregs with Absence of Cytokine-Secreting CD4⁺ T-Cell Subsets and Marked Expansion of Th1 Cells Is a Hallmark of Idiopathic Aplastic Anaemia (AA)

Autoimmunity is an important contributor in the aetiology of AA. Although the expansion of oligoclonal CD8⁺ T-cells and their correlation with response to immunosuppressive therapy (IST) has been reported previously, the role of CD4⁺ in the pathogenesis is not elucidated. The focus of this study was to investigate the role of different CD4⁺ T-cell subsets, including regulatory T-cells (Tregs) and T helpers (Th1, Th2 and Th17) in the pathobiology of idiopathic AA.

The percentage and absolute numbers of CD4⁺ and CD8⁺ T-cell subsets, NK & B cells and dendritic cells (DCs) in peripheral blood were assessed in 42 patients with idiopathic AA prior to any IST and 8 healthy age matched controls. T-cells were stimulated first and stained intracellularly for IFN-γ and TNF-a (Th1), IL-4 (Th2) and IL-17 (Th17). Serum levels of 30 cytokines were measured by 30 Plex bead analysis (Luminex). NK cells were defined as CD3^– CD56⁺. B cells were defined as CD3^-CD19⁺. CD3⁺ CD4⁺.T-cell subsets were defined as CD45RO^–CD27⁺ naïve, CD45RO⁺ CD27⁺ CD62L⁺ central memory, CD45RO⁺ CD27⁺ CD62L^– effector memory, CD45RO⁺CD27^– effectors and CD45RO^– CD27^– terminal effectors. DCs were defined based on their BDCA 1,2, 3 & CD16 expression. CD4 Tregs were defined as CD3⁺CD4⁺ CD25^high CD27⁺Foxp3⁺. Treg subsets were defined as (1) CD45RA⁺CD25^lo resting Tregs, (2) CD45RA^-CD25^hi activated Tregs, and (3) cytokine-secreting CD45RA^-CD25^lo non-Tregs¹. Treg function was evaluated by cytokine secretion of T effector cells (Te) with and without Tregs. IFN-γ secreting CD4⁺ T-cells (Th1) were enriched by magnetic beads followed by FACS sorting. The clonality of Th1 cells was evaluated based on the diversity of T-cell receptors by spectratyping as well as sequencing. Transcription factor expression was measured by qPCR.

There were no significant differences in the number or percentage of different CD8 T-cells compared to healthy controls. Surprisingly, despite a borderline decrease in the absolute number of naïve (p=0.19) and central memory (p=0.20) CD4⁺T-cells the number and percentage of Tregs were no different from healthy controls (1.36×10⁷/L v 1.34×10⁷/L, p=0.57). Although the ratio of Tregs to CD4⁺ T-effectors (Te) was higher than in healthy controls, the difference was not significant (0.49 v 0.12, p=0.86). The absolute numbers and percentages of Th1 cells and TNF-α ⁺ CD4⁺ T-cells were significantly higher in AA patients compared to healthy controls (4.2 × 10⁷/L v 0.9 × 10⁷/L & 2.44 × 10⁸ v 1.26 × 10⁸(p=0.001, p=0.004)). The diversity of T-cell receptor on Th1 cells was significantly lower compared to healthy age matched controls (on average 21 & 52 peaks).

Amongst AA patients, the numbers of Th2, Th17, NK and B cells were not significantly different from healthy controls, whereas the absolute numbers of all DCs were reduced(p<0.01). The serum levels of proliferative cytokines, EGF (p=0.01), HGF (p=0.01), VEGF (p=0.01) and pro-inflammatory cytokines IL-13 (p=0.02), IL-8 (p<0.001) were significantly higher in AA patients. The percentage of cytokine secreting CD4⁺ CD25⁺ T-cells was markedly decreased in AA patients and the activated Treg subsets were predominantly of CD45RA⁺ phenotype, which was significantly different from healthy controls. Sorted Tregs from AA patients were unable to suppress cytokine secretion by Te cells in a 1:1 co-culture. However, IL-2, IFN-γ and TNF-α secretion of Te from AA patients was suppressible by allogeneic Tregs from healthy controls (on average 11 time suppression), whereas Tregs from AA patients were unable to suppress healthy Te cells. However, dysfunctional Tregs were not associated with abnormality of transcription factors, as judged by the levels of STAT1, 3, 4, 5 & 6, FoxP3 & T-bet of Tregs that were not significantly different from healthy age matched controls.

Our data show that although FoxP3⁺ Tregs are normal in AA, a subset of these cells is markedly reduced and the activated Tregs aberrantly express CD45RA. Furthermore, unlike normal Tregs, the Tregs from AA patients do not suppress the inflammatory cytokine secretion by Te cells. The absence of DCs in the peripheral blood suggests their immigration to the inflammation site (e.g. bone marrow), which may play a role in the polarisation of T helpers toward a Th1 phenotype. Clonal expansion of Th1 cells may suggest potential antigen specificity that may lead to AA phenotype.

1. Miyara M, et al. Immunity. 2009.

No relevant conflicts of interest to declare.