Reduction of Tregs with Expansion of Th1 Cells and Lack of IFN-γ Secretion by GPI Negative T-Cells In PNH

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haematopoietic stem cell disorder characterized by intravascular haemolysis and thrombosis. The pathogenetic link with bone marrow failure syndromes is well recognized, however the process of clonal expansion of the glycosylphosphatidylinositol (GPI)-deficient cells over normal haemotopoiesis remains unclear.

To further elucidate mechanisms leading to clonal expansion in PNH, we investigated the immunological profile and performed high-resolution genome-wide karyotyping using Affymetrix SNP6 microarrays.

The percentage and absolute numbers of CD4⁺ and CD8⁺ T-cell subsets, NK cells and B cells in peripheral blood were assessed in 8 patients with PNH prior to any therapy and 8 healthy age matched controls. High resolution SNP6 karyotyping was performed on bone marrow (n=15) and peripheral blood (n=12) of these patients. Bone marrow from an additional 8 patients was enriched for CD34⁺59^- and CD34⁺59⁺ cell fractions for SNP array karyotyping. Abberations that overlapped by >50% with variations found in the Database of Genomic Variants, as well as an internal series of 91 normal subjects were excluded from further analysis.

T-cells were stimulated and then stained intracellularly for TNF-α and IFN-γ (Th1), IL-4 (Th2) and IL-17 (Th17). 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. CD4⁺ Tregs were defined as CD3⁺CD4⁺ CD25^high CD27⁺Foxp3⁺.

There were no significant differences in the number or percentage of different CD8⁺ and CD4⁺ T-cells compared to healthy controls except for the number of Tregs and Th1 cells. In our cohort of patients, the number of Th1 cells was significantly higher than healthy controls (4.1×10⁷/L v 0.93 × 10⁷/L, p=0.039), whereas the number of Tregs cells was lower (0.75 × 10⁷/L v 1.36 × 10⁷/L, p=0.028). There was no significant difference in the number of Th2 and Th17 cells between patient and healthy subjects.

Within CD4⁺ T-cells two distinct CD59⁺ and CD59^- populations were identified, of which the CD59^- cells were unable to secrete IFN-γ in response to stimulation compared to CD59⁺ population. On average 48% of CD4⁺ CD59⁺ T-cells secrete IFN-γ compared to 2% in the CD59^- population. There was no significant difference in IL17 and IL4 secretion between CD59⁺ and CD59^- T-cells.

SNP karyotyping revealed three regions of uniparental disomy (UPD); UPD1p26.11-p34.3, UPD1p13.3-p13.1in one peripheral blood sample and UPD7q32.1-q34 in one bone marrow sample. There were no additional somatic genomic aberrations detected in any of the samples. Of note, purified CD34⁺59^- cells did not reveal any clonal copy number changes or regions of UPD.

Specific analysis of Xp22.1 did not reveal any aberrations of the PIGA gene, suggesting aberrations of the PIGA gene may be restricted to mutations or epigenetic abnormalities. Our immunological profiling revealed an expansion of Th1 cells and diminished Tregs in the peripheral blood, which is in contrast to our published data from both MDS and AA patients. The lack of IFN-γ secretion by GPI deficient T-cells also suggests an additional immunological defect in these patients, which may contribute in disease pathogenesis.

No relevant conflicts of interest to declare.