Bone marrow failure syndromes are not only serious and debilitating diseases but they can also progress toward leukemia. Identifying the specific factors contributing into this malignant transition is extremely important. Our group focus in past few years has been the role of the immune system in bone marrow failure and we showed the importance of specific subtypes of immune cells in both pathogenesis and malignant transformation of these diseases. The AAMDSIF research grant will allow us to focus on differences in immune changes in aplastic anemia and paroxysmal nocturnal hemoglobinuria (PNH) and will enable us to utilize the state of the art technologies (such as multidimensional mass cytometry and high throughput DNA sequencing) to further understand the role of immune system in these diseases as well as identifying new important biological markers for diagnosis and classification of aplastic anemia and PNH.
The aim of this grant is to identify the specific immune dysregulation in paroxysmal nocturnal haemoglobinuria (PNH) and aplastic anaemia (AA)/PNH, and also correlate the interplay between the PNH cells and specific T-cell subsets called NKT-cells in a sequential longitudinal study. We will also identify ways that different therapies modulate the immune response and utilise it for the therapeutics of marrow failures. Cutting edge technologies such as mass-cytometry (CyTOF) and high precision DNA sequencing (deep sequencing) are being used to address these questions.
Progress so far Patient recruitment:
We have identified 89 patients with PNH through the King’s College London (KCL) tissue bank which are consented to participate in research projects (10 patients were recruited since last year). Among these patients, we have access to peripheral blood mononuclear cells (PBMCs) of 26 patients at time of diagnosis and following therapy with C5 inhibitor (Eculizumab). PBMCs from these patients are stored as viable cells and being used for this study. We have also recruited 16 AA patients with a small to moderate PNH clone (with no cases of hemolytic PNH) and 8 age matched healthy volunteers (HDs) as control groups thus far.
Identifying the specific immune signature(s) for PNH and AA/PNH
The first aim of this research was to identify a specific signature which identifies AA/PNH patients and could potentially predict their response to immunosuppressive therapy (IST). We have used CyTOF technology and multiple bioinformatics packages to identify potential immune signature(s), which predicts response to immunosuppression in AA/PNH. This enabled us to distinguish two distinct subpopulations of human T-cells (known as T regulatory cells (Tregs)), in AA and HDs and to demonstrate clear differences in AA that predicted response to treatment. The results from this study are under revision for publication and the AAMDSIF has been acknowledged in this work. Up to now, blood samples from 5 PNH patients have been stained and analysed pre and post Eculizumab by the same method (10 samples) and the preliminary data suggest that type of Treg cells could also predict response to therapy in PNH. We have successfully optimized the CyTOF panel for identification of another important immune cells in PNH (CD8+ T-cell). We were able to identify the classic CD8+ T-cell subpopulations as well as novel clusters of cells in PNH patients. We will continue to analyse more samples by CyTOF to confirm the persistence and biological relevance of the identified clusters in PNH, AA/PNH.
Plans for the second year
We would like to continue this work by accomplishing the following tasks:
1. Finalise the CyTOF run for all collected samples from PNH patients pre and post Eculizumab and finalise the data analysis.
2. Once the CyTOF data analysis has been completed, and the set of markers which accurately define the identified cells in PNH, these markers will be used to isolate these cells and perform the additional molecular studies as outlined in our application.
The ultimate goal of the second part is to identify NKT-cells within the expanded population of cells and see whether these cells can distinguish PNH from AA/PNH and how they would affect the response to Eculizumab.