Clonality Analysis Of γδT Cell In Patients With Pure Red Cell Aplasia

Objective To explore if there is any clonal γδT cell in patients with acquired pure red cell aplasia (PRCA) through rearrangement analysis of TCR-γ gene of the γδT cell extracted from peripheral blood of patients with PRCA.Material and methods The complementary determining region 3 (CDR3) of TCR-γ gene of γδT cells in 14 PRCA patients and 10 normal individuals were amplified by one-step reverse transcription-polyerase chain reaction (RT-PCR) with specific primers. The RT-PCR products were then analyzed by agarose electrophoresis and polyacrylamide gel electrophoresis (PAGE) plus gel image analysis software in order to determine the CDR3 region length spectrum. Two oligoclonal results were confirmed by means of direct sequencing.Results 1. Positive bands of 230bp (Ⅵ subfamily) or 240bp (Ⅶ subfamily) were observed in all 14 patients with PRCA and 10 normal controls by 2% agarose electrophoresis. 2. In 8% PAGE analysis, single dense band was observed in 4 (VI subfamily in P01 and P03 and Ⅶ subfamily in P04 and P08) of 14 PRCA patients, which showed a dominant sharp peak with a few short peaks in gel image analysissoftware implicating oligoclonality. Patterns of a smear or multiple bands over a smear were observed in the other 10 PRCA patients and 10 normal controls, which showed a broad peak or multiple peaks with gray-scale values of Gaussian distribution in gel image analysis software implicating polyclonality. 3. (1) As far as clinical state was concerned, the 4 PRCA patients with oligoclonal y<5T cells include 1 in 6 remitted PRCA patients, 1 in 3 incipient PRCA patients and 2 in 5 replased PRCA patients. (2)As far as the efficacy of Cyclosprine A (CsA) was concerned, the 4 patients with oligoclonal y5T cells were all susceptible to CsA. 4. Two RT-PCR products (VI subfamily in P01 and VII subfamily in P04) were sequenced, resulting in confirmation of the existence of oligoclonal y<5T cells in these two patients. Conclusion 1. Polyclonal y6T cells were observed in 10 out of the 14 PRCA patients, suggesting a reactive proliferation of y5T cells in PRCA. 2. Oligoclonal yST cells were only observed in 4 out of 10 PRCA patients who were responsive to CsA. There seemed no exact relationship between clonal proliferation of y8T cells and clinical situation of PRCA, suggesting that more studies are needed to explore the real role of the cloanl y8T cells and the nonclonal y<5T cells in the pathogenesis of PRCA.