T-Cell Receptor Complementarity Determining Region Analysis of Peripheral Blood and Bone Marrow T-Lymphocyte Subsets and Quantitative Evaluation of T-Regulatory Cells in Patients with Chronic Idiopathic Neutropenia

Clonal/oligoclonal expansions of cytotoxic T-lymphocytes (CTLs) and quantitative/qualitative abnormalities of T-regulatory cells (Tregs) have been implicated in the pathophysiology of acquired bone marrow (BM) failure syndromes such as aplastic anemia, myelodysplastic syndromes and large granular lymphocyte proliferative disease. Chronic Idiopathic Neutropenia (CIN) is an acquired disorder of granulopoiesis characterised by increased apoptosis of BM CD34⁺/CD33⁺ granulocytic progenitor cells. We have previously provided evidence for oligoclonal T-cell expansions with possible pathogenetic significance in CIN. The aim of the current study is (a) to define and compare the magnitude of CD4⁺ and CD8⁺ T-cell responses and (b) to evaluate the number of Tregs, in the peripheral blood (PB) and BM of CIN patients. Eighty five CIN patients and 85 healthy controls, age- and sex-matched to the patients, were studied after informed consent. All patients had PB neutrophil counts below 1800/μL (mean 1410 ± 330 neutrophils/μL, range 100–1799 neutrophils/μL), displayed negative anti-neutrophil antibody activity and were satisfying the previously reported diagnostic criteria for the disease.The T-cell receptor (TCR)-Vβ repertoire was analysed by flow cytometry (Vβ spectratyping) in the PB and BM CD3⁺, CD4⁺ and CD8⁺ cells. The size distribution of the TCR-Vβ complementarity determining region 3 (CDR3) was analysed in immunomagnetically sorted PB and BM CD4⁺ and CD8⁺ cells by PCR according to the -2 protocol using a fluorescence-based DNA sequencer (CDR3 spectratyping). Vβ family expansions were defined as above of 2 standard deviations from the mean values of the 85 healthy controls. CDR3 oligoclonal/monoclonal patterns were defined upon comparison with the normal Gaussian-type size distribution. The Treg cell frequency was defined as the proportion of FOXP3⁺ cells within the CD4⁺/CD25^high PB and BM cell fraction by flow cytometry. We found that 69.41% and 82.61% of CIN patients displayed one or more predominant TCR-Vβ family expansions within the CD3⁺ cell fraction of PB and BM, respectively. None of the controls displayed clonal/oligoclonal expansions in either PB or BM (P<0.001 and P<0.001, respectively). Further analysis showed that 58.62% and 74.19% of CIN patients displayed a skewed TCR-Vβ repertoire in PB CD4⁺ and CD8⁺ cells, respectively. A higher proportion of patients i.e. 93.75% and 75.0% displayed a skewed TCR-Vβ repertoire in BM CD4+ and CD8⁺ cells, respectively. The total sum of predominant TCR-Vβ clones was statistically significant increased in patients’ BM compared to PB in all subpopulations studied, namely the CD3⁺, CD4⁺ and CD8⁺ cells (P<0.05, P<0.05, P<0.05, respectively). Interestingly, CDR3 spectratyping identified a skewed (oligoclonal) pattern in PB and/or BM CD8⁺ cells in 100% of patients. PCR analysis of PB and BM CD4⁺ cells revealed a normal Gaussian-like CDR3 size distribution in all but three patients. None of the controls displayed a skewed CDR3 profile in either CD4⁺ or CD8⁺ cells. The percentage of FOXP3⁺ cells within the PB CD4⁺CD25^high cell fraction was significantly decreased in CIN patients (57.77%±15.77%) compared to controls (72.95±12.23%; P=0.0004). A parallel analysis of Treg cell proportion in patients’ PB and BM showed statistically significant increased percentage of FOX3⁺ cells within the CD4⁺/CD25^high T-cells of BM (68.51%±11.88%) compared to PB (52.20%±12.77%) (P=0.0005). No statistically significant difference was found in the percentage of FOX3⁺ cells within the CD4⁺/CD25^high T-cells of BM compared to PB in healthy controls. These data suggest that oligoclonal CTL expansions are uniformly identified in CIN patients. The polarized cell subsets are more frequent in BM than the PB suggesting the possible existence of “target” within the BM. Whether, however, the CD34⁺/CD33⁺ BM cells represent the target or the innocent bystanders of this immune process, remains elusive. The low frequency of Tregs in the PB and the accumulation of these cells in patients’ BM represents probably a compensatory control mechanism aiming to suppress the local immune reactions. All these data substantiate further the concept that CIN belongs to the spectrum of T-cell mediated BM failure syndromes.