Analysis of T‐cell clonality in bone marrow of patients with acquired aplastic anaemia

Abstract

Summary. Acquired aplastic anaemia (AA) represents a state of bone marrow (BM) failure which is characterized by BM hypocellularity and pancytopenia. It has been hypothesized that in some AA patients, bone marrow failure is secondary to the targeted destruction of haemopoietic stem cells by autoreactive T cells. The response of T cells to antigenic stimulation has been shown, in a number of animal models and in autoimmune diseases, to result in the (oligo)clonal expansion of positively reacting T cells. For this reason, we studied the utilization of 24 T-cell receptor-variable gene segments (TCRBV) and the clonality in BM aspirates and peripheral blood (PB) of seven AA patients. BM from transplant donors served as controls. Determination of TCRBV gene segment usage revealed no significant differences between patients and controls.Clonality within each family was analysed by singlestrand conformation polymorphism (SSCP) analysis. Clonal and clonally predominant bands were seen in BM of three AA patients in five to eight TCRBV families. Clonal rearrangements were encountered less often in BM of control subjects. In conclusion, our results suggest an antigen-driven T-cell response in the BM of predominantly AA patients resulting in oligoclonal T-cell outgrowth.Keywords: aplastic anaemia, clonality, T-cell receptor-, SSCP, V usage.Aplastic anaemia (AA) is an acquired disorder affecting haemopoietic stem cells and is characterized by a reduced bone marrow (BM) cellularity and pancytopenia. Previously, we have studied the clonality of haemopoiesis in peripheral blood (PB) cells of female patients, who entered remission after anti-thymocyte globulin (ATG) therapy. Employing a PCR-based X-chromosome inactivation analysis, it was shown that about half of the patients exhibited clonal haemopoiesis (Van Kamp et al, 1991;Melenhorst et al, 1996). Analysis of the rearrangement patterns at the T-cell receptor-and immunoglobulin heavy chain loci by PCR revealed a polyclonal character of gene rearrangements, irrespective of the X-chromosome inactivation status of the lymphocyte populations.Clonal haemopoiesis in AA may result from a reduced stem cell pool to a clonal composition. It has been hypothesized that in a subgroup of AA patients this reduction is caused by autoreactive T lymphocytes (Young, 1992), recognizing an autoantigen expressed on haemopoietic progenitor cells. Evidence for the T-cellmediated pathogenesis of AA is found in the response of 70% of the patients treated with immunosuppressives, i.e. anti-lymphocyte globulin (ALG)/anti-thymocyte globulin (ATG) and cyclosporin A (Young & Barrett, 1995;Frickhofen et al, 1991). An increased percentage of CD8 HLA-DR T cells in PB and BM has been found (Zoumbos et al, 1985;Maciejewski et al, 1994) as well as a significant association of AA with the HLA-DR2 allele (Nimer et al, 1994;Nakao et al, 1994). The association of an HLA-allele with autoimmune disease predisposition is one of the most clearly established factors, and is related to the MHC-restricted T-cell response (T...

Intracellular interferon-γ in circulating and marrow T cells detected by flow cytometry and the response to immunosuppressive therapy in patients with aplastic anemia

“…24 As in other autoimmune states, activated T cells appear to localize in the target tissue. 7,25,26 While circulating cells may reflect BM activity, in hematologic disease intracellular cytokine detection of BM T cells may be preferable to testing PB, as these cells scored positive in some patients in whom IFN-␥ was not detected in blood cells (see below); other measurements, 7 such as flow cytometric determination of lymphocyte cell surface expression of activation markers and gene amplification to detect IFN-␥ mRNA, 12 are also consistent with marrow localization of immune system activity. The finding of substantial numbers of IFN-␥-containing T cells in AA is consistent with results of other types of analysis of lymphocyte function in this disease, especially recent data indicating that some patients, especially those dependent on continued cyclosporine administration to maintain blood counts, show marked skewing of V ␤ chain use, 27 suggestive of a specific antigenic response.…”

Changes in T-cell receptor VB repertoire in aplastic anemia: effects of different immunosuppressive regimens

“…30 In one study of severe AA, a broadly normal VB distribution pattern with overexpression of a few VB types indicative of selective CDR3 usage was observed, but skewing was not limited to specific VB families, perhaps due to different HLA backgrounds, and analysis of the J regions excluded monoclonality. 12 In cyclosporine A (CsA)-dependent AA, in contrast to refractory cases, a significantly skewed CDR3 size pattern was found and all patients shared the HLA-DRB1*1501 genotype 13 ; in addition, VB15 CDR3 sequencing showed dominant clones in 5 patients. An abnormal T-cell repertoire has also been found in PNH, which often accompanies typical AA and may share with it some aspects of an autoimmune pathophysiology.…”

“…10 Recently, polymorphisms within the complementarity-determining region 3 (CDR3) of the variable beta (VB) chain of the T-cell receptor (TCR) have been utilized for the study of immune mechanisms in AA. [11][12][13] TCR VB CDR3 is a nongermline encoded hypervariable region directly related to T-cell recognition of specific peptides in the appropriate human leukocyte antigen (HLA) context. CDR3 thus defines a unique clonotype generated by a somatic rearrangement, resulting in discrete amino acid differences within each VB chain.…”

High Dose Cyclophosphamide Treatment for Autoimmune Disorders

“…Although not originally considered an autoimmune disease, ample clinical and laboratory data support the hypothesis that in most cases of aplastic anemia autoreactive cytotoxic T lymphocytes are directly responsible for the bone marrow destruction [16,17,18,19,20,21]. Aplastic anemia is classified as severe (SAA) when the bone marrow cellularity is <25% and there is marked peripheral cytopenias in at least two hematopoietic lineages (neutrophil count <0.5 × 10 9 /l, platelet count <20 × 10 9 /l and absolute reticulocyte count of <60,000) [22].…”