Skewing of the T Cell Receptor Gene Repertoire and Public Clonotypes in Cytotoxic T Cells of Patients with Chronic Idiopathic Neutropenia: A Role for Antigen Selection in Disease Development

Abstract 831

Chronic idiopathic neutropenia (CIN) is a disorder of neutrophil production usually characterized by benign and asymptomatic course and female predominance. The defective hematopoiesis in CIN can be mainly attributed to accelerated Fas-mediated death of the CD34⁺/CD33⁺ granulocytic progenitor cells, secondary to an inflammatory bone marrow (BM) microenvironment. Crucial to CIN pathogenesis are the increased numbers of activated T cells identified in both peripheral blood (PB) and BM of CIN patients. Recently, using flow cytometry and CDR3 spectratyping, we obtained for the first time evidence for expanded T cell populations alluding to repertore skewing in both PB and BM of patients with CIN, but more prominent in the BM CD8⁺ subset. Prompted by these fndings, here we significantly extended our studies of the cytotoxic T cell responses in CIN in order to obtain a comprehensive view of the role of antigen selection in CIN pathogenesis. The study included 18 patients with CIN, 17 females and 1 male, diagnosed according to the established criterial for CIN. TRBV-TRBD-TRBJ gene rearrangements were amplified on either genomic DNA or cDNA isolated from CD8+ cells of PB (n=6) or BM (n=12) samples. PCR products were subcloned by transformation into E.Coli/TOP10F' competent bacteria and individual colonies were chosen randomly and subjected to Sanger sequencing. Sequence data were analyzed using the International imMunoGenetics information system and more particularly the IMGT/V-QUEST tool. Overall, 507 TRBV-TRBD-TRBJ gene rearrangements were analyzed (19-30/case) of which 466 were productive since they used functional TRBV genes and also carried in-frame CDR3s. A polyclonal profile was seen in only 3/18 cases (16.7%). The remaining cases were found to carry clusters of identical rearrangements corresponding to distinct immunodominant clonotypes. The frequency of each clonotype was determined by dividing the number of identical sequences by the total number of subcloned sequences analyzed. In 10/18 cases (55.5%), the dominant clonotype accounted for 12.8–22.6% of the total, whereas in 5/18 cases (27.8%) it accounted for 34.2–68.4% of the total. The TRBV28 gene was used by the dominant clonotype of three different CIN cases; the TRBV10-2, TRBV10-3, TRB19, and TRBV7-8 genes were identified in the major clonotypes of two cases each. Importantly, cluster analysis of the CDR3 sequences of all CIN cases of the present study identified 4 different rearrangements that were shared by different patients (public clonotypes). In conclusion, the present study strongly suggests that CIN may result from an autoimmune reaction directed against granulocytic progenitors triggered by a restricted range of, as yet, unidentified antigen(s). The finding of public clonotypes may indicate that public antigenic stimuli and/or shared immune processes underlie CIN development, at least for a proportion of cases. Overall, our results further support the concept that CIN may share common pathophysiologic mechanisms with other disorders characterized by T-cell and cytokine mediated suppression of hematopoiesis, perhaps representing a milder extreme within the spectrum of these acquired immune-mediated BM failure syndromes.