Antonio M. Risitano

In-vivo dominant immune responses in aplastic anaemia: Molecular tracking of putatively pathogenetic T-cell clones by TCR β-CDR3 sequencing

BACKGROUNDnAplastic anaemia is a bone-marrow-failure syndrome characterised by low blood-cell counts and fatty bone marrow. In most cases, no obvious aetiological factor can be identified. However, clinical responses to immunosuppression strongly suggest an immune pathophysiology.nnnMETHODSnTo test the hypothesis that aplastic anaemia results from antigen-specific lymphocyte attack against haemopoietic tissue, we analysed effector immunity, seeking especially dominant specific T-cell responses. Blood samples from 54 patients with aplastic anaemia were subjected to flow cytometry to define T-cell-receptor Vbeta-chain usage and expansion of particular Vbeta subsets. We measured the size distribution of the complementarity-determining region 3 (CDR3) for expanded Vbeta subsets, then cloned and sequenced skewed, oligoclonal, or monoclonal peaks.nnnFINDINGSnExpanded Vbeta subsets were identified in almost all the patients. Over-represented Vbeta subsets from CD8-positive cells showed oligoclonal or monoclonal CDR3 size patterns. The CDR3 sequence repertoire in aplastic anaemia showed much redundancy compared with healthy donors. We identified patient-specific putative pathogenetic clonotypes that were not detectable in controls. In selected patients who were assessed longitudinally, these clonotypes were quantitatively related to disease activity. Selective killing of autologous haemopoietic progenitors by the Vbeta-specific lymphocyte population was shown in one patient. These apparently pathogenetic CDR3 sequences showed homology between individuals, suggesting a role for a semi-public immune response in the pathophysiology of aplastic anaemia.nnnINTERPRETATIONnIn-vivo dominant clonal immune response can be identified in many patients with aplastic anaemia, which is evidence for an underlying antigen-driven immune process. Longitudinal tracking by molecular techniques could inform individual clinical decisions and the development of new treatments in autoimmune diseases.nnnRELEVANCE TO PRACTICEnAlthough the target of the aberrant immune response is the haemopoietic stem cell, the triggering antigens remain unknown. We combined cell phenotypic, molecular biology, and functional analyses to study the effector arm of immunity in an attempt to establish an immune pathophysiology. Clinical application of such a model could broadly extend to other autoimmune diseases.

CD34+ cells from paroxysmal nocturnal hemoglobinuria (PNH) patients are deficient in surface expression of cellular prion protein (PrP c)

Cellular prion protein (PrP(c)) is a glycosylphosphatidylinositol (GPI)-anchored protein (GPI-AP) constitutively expressed by neurons but also in hematopoietic cells. In trasmissible spongiform encephalopathies, the protease-resistant form of prion (PrP (s c)) converts the host PrP(c) into the pathologic form. We have investigated PrP(c) expression in hematopoietic cells from paroxysmal nocturnal hemoglobinuria (PNH). In this disease, due to somatic mutations in PIG-A gene, biosynthesis of the (GPI)-anchor is impaired and affected cells lack membrane expression of all GPI-AP. Normal and PNH hematopoietic progenitors and paired wild-type (WT) and PIG-A mutant cell lines were used for analysis of intracellular and surface PrP(c) expression using flow cytometry and Western blot.By flow cytometry, PrP(c) was constitutively present on normal CD34(+) cells, including more immature CD38(dim) cells, as well as hematopoietic cell lines. Similar results were obtained in purified CD34(+). Phospholipase C treatment confirmed that PrP(c) was expressed on the membrane via the GPI-anchor. In PNH patients, GPI-AP-deficient CD34(+) cells lacked PrP(c) membrane expression. PIG-A-mutated cell lines (Jurkat, K562, C(EBV), A(EBV)), in contrast to their normal counterparts, did not express surface PrP(c). However, we detected intracellular PrP(c) at approximately equivalent levels in both normal and PIG-A-mutated cells using intracellular flow cytometry and Western blotting. Cells and cell lines with PNH phenotype together with their normal counterparts may be a suitable system to explore the function of membrane PrP(c) in the hematopoietic system. Conversely, PrP(c) is a good model to elucidate the fate of GPI-AP in PIG-A-deficient cells.