T Cell Transcriptomes from Paroxysmal Nocturnal Hemoglobinuria Patients Reveal Novel Signaling Pathways

Background. Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired blood disease, characterized by hemolytic anemia, bone marrow (BM) failure, and venous thrombosis. The etiology of PNH is a somatic mutation in the phosphatidylinositol glycan class A gene (PIG-A) on the X chromosome, which causes deficiency in glycosyl phosphatidylinositol-anchored proteins (GPI-APs). The involvement of T cells in PNH is strongly supported by clinical overlap between PNH and aplastic anemia (AA); the presence of GPI-AP deficient cells in AA associated with favorable response to immunosuppressive therapy; and an oligoclonal T cell repertoire in PNH patients. However, the molecular mechanisms responsible for the aberrant immune responses in PNH patients are not well understood. To identify aberrant molecular mechanisms involved in immune targeting of hematopoietic stem cells in BM, RNA sequencing (RNA-seq) was applied to examine the transcriptome of T cell subsets from PNH patients and healthy controls.

Method. Blood samples were obtained after informed consent from 15 PNH patients and 15 age-matched healthy controls. For RNA extraction, freshly isolated peripheral blood mononuclear cells were sorted on the same day of blood draw to obtain four different T cell (CD3⁺ CD14^- CD19^- ViViD^-) populations [CD4⁺ naïve (CD45RA⁺ CD45RO^-), CD4⁺ memory (CD45RA^- CD45RO⁺), CD8⁺ naïve (CD45RA⁺ CD45RO^-), and CD8⁺ memory (CD45RA^- CD45RO⁺) T cells] by fluorescence-activated cell sorter . RNA-Seq analysis from three PNH and three healthy controls was performed using the Illumina HiSeq™ 2000 platform. The Ingenuity® Pathway Analysis and Gene set enrichment analysis (GSEA) were employed to elucidate transcriptional pathways. RNA-seq data were validated by flow cytometry and quantitative real-time RT-PCR (RT-qPCR).

Results and Discussion. Differentially expressed gene analysis of four T cell subsets showed distinct gene expression signatures in individual T cell subsets. In CD4⁺ naïve T cells, 11 gene expression levels were significantly different: five upregulated (including SRRM2 and TNFSF8) and six downregulated genes (including GIMAP6) (> 2 fold change, false discovery rate [FDR] < 0.05). In CD4⁺ memory T cells, 25 gene expression levels were significantly different: 15 upregulated (including JUND and TOB1) and 10 downregulated genes (including GIMAP4). In CD8⁺ naive T cells, only two gene expression levels were significantly different: upregulated CTSW and downregulated RPL9. In CD8⁺ memory T cells, seven gene expression levels were significantly different: two upregulated (CTSW and DPP4) and five downregulated genes (including SLC12A7). Further, differentially expressed gene analysis was performed by combining CD4⁺ naïve, CD4⁺ memory, CD8⁺ naïve, and CD8⁺ memory T cells from PNH or healthy controls, respectively. Out of 55 gene expression levels that were significantly different, 41 were upregulated (including TNFAIP3, JUN, JUND, TOB1, TNFSF8, and CD69) and 14 downregulated (including GIMAP4). By canonical pathways analysis, putative gene network interactions of differentially expressed genes were significantly enriched for canonical pathways of TNFR1, TNFR2, IL-17A, and CD27 signaling. By GSEA, the most significantly upregulated gene sets in CD4⁺ naïve, CD4⁺ memory, CD8⁺ naïve, and CD8⁺ memory T cells from PNH patients displayed gene signatures related to the "IGF1 pathway", "Pre-NOTCH expression and processing", "AP-1 pathway", and "ATF2 pathway", respectively. For validation of the RNA-seq data, we chose seven genes (TNFAIP3, JUN, JUND, TOB1, TNFSF8, CD69, and CTSW) because these are important mediators involved in regulation for T cells and dysregulation of these genes is associated with autoimmune diseases. Differential expression levels of TNFAIP3, JUN, and TOB1 were validated by RT-qPCR. By flow cytometry, higher expression of CD69 and TNFSF8 was confirmed in CD4⁺ and CD8⁺T cells from PNH compared to healthy controls.

Conclusion. Using RNA-seq, we identified novel molecular mechanisms and pathways which may underlie the aberrant T cell immune status in PNH. Specific dysregulation of T cell intracellular signaling may contribute to BM failure and the inflammatory environment in PNH. Understanding these pathways may provide new therapeutic strategies to modulate T cell immune responses in BM failure.