Chronic idiopathic neutropenia (CIN) is an acquired disorder of granulopoiesis characterized by hypoplastic and left-shifted granulocytic series in the bone marrow (BM). It has been shown that CIN patients display increased number of activated T-lymphocytes with myelosuppressive properties in the BM and peripheral blood (PB) that induce Fas-mediated apoptotic death of the BM myeloid progenitor cells by producing interferon-γ and Fas-ligand. Epidemiological data have also shown female predominance and HLA-DRB1*1302 genetic background in CIN patients. The aim of the present study was to characterize T-cell receptor (TCR) Vβ repertoire of CD3+ cells in patients with CIN and explore the presence of dominant T-cell expansions with possible pathogenetic significance. Eighty patients with CIN were studied after informed consent. All patients had PB neutrophil counts below 1800/μL (mean 1411±380 neutrophils/μL, range 100-1799 neutrophils/μL), displayed negative anti-neutrophil antibody activity and were satisfying the previously reported diagnostic criteria for the disease. PB and BM samples from the patients were subjected to flow-cytometric analysis for the quantification of the TCR Vβ repertoire of the CD3+ cells (IOTest Beta Mark kit, Beckman-Coulter). Vβ family expansions were defined as above of 2SD (standard deviation) from the mean values of 85 healthy controls. DNA samples from immunomagnetically sorted PB and BM CD3+ cells were also subjected to multiplex polymerase chain reaction (PCR) analysis using the BIOMED2 protocol that covers all Vβ TCR gene rearrangements. We found that 54 patients, i.e. a proportion of 67.5% displayed one or more prominent clones in PB CD3+ cells. The Vβ16, Vβ12 and, in a lower proportion, the Vβ7.2 were the most frequently expanded subsets in PB, identified in 53.75%, 15% and 6.25% of CIN patients, respectively. Furthermore, CIN patients as a group displayed statistically increased proportion of Vβ16, Vβ12 and Vβ7.2 subsets within the CD3+ PB cells (1.96±1.30, 2.49±2.56 and 2.97±1.72 respectively) compared to controls (0.90±0.29, 1.66±0.54 and 1.47±1.03, respectively) (P<0.001, P<0.01 and P<0.001, respectively). In 20 patients, BM cells were also studied. Sixteen of 20 patients (80%) showed one or more prominent clones in CD3+ BM cells. In accordance with the PB data, the Vβ16, Vβ12 and Vβ7.2 were also the most frequently expanded subsets in the BM, identified in 65%, 10% and 10% of CIN patients, respectively. Further analysis showed that although more than one T-cell subset might be expanded in each patient, an identical expansion pattern was noted individually in BM and PB. Interestingly, a proportion of patients displayed prominent clones only in BM and not in PB but not vice versa, suggesting selective expansion or persistence of these subsets within the BM. None of the patients displayed monoclonal TCR Vβ expansion on multiplex PCR and heteroduplex analysis of BM and PB CD3+ lymphocyte subsets. In conclusion, in vivo dominant T-cell responses are identified in the majority of patients with CIN. These data substantiate further our hypothesis for the T-cell mediated immune nature of CIN and provide novel insights in the pathophysiology of the disease with possible therapeutic implications.

Author notes

Disclosure: No relevant conflicts of interest to declare.