Analysis of autoantigens in immune-mediated aplastic anemia-identification of an epitope of a CD4^+ T-cell clone

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To determine an epitope of a CD4^+ T-cell clone, ZN1, that appears to have an essential role in the development of aplastic anemia, we attempted to establish a system to screen a peptide capable of stimulating this T-cell clone using CLIP-replacement Ii-chain gene expression vectors. ZN1 was immortalized using infection with Herpesvirus saimiri. Stimulation with cultured bone marrow mononuclear cells (BMMCs) in the presence of IL-3 and GM-CSF induced DNA synthesis by ZN1. When monoclonal antibodies (mAbs) against HLA-DR, HLA-DR2 or HLA-DQ were added to the culture, both anti-DR and anti-DR2 mAbs inhibited DNA synthesis by ZN1 in response to cultured BMMCs, suggesting restriction ** antigen recognition by HLA-DR2. Then, we transfected COS7 cells with a DR2 b gene plasmid and a DR a chain plasmid. Flow cytometry confirmed expression of DR2 by the COS7 transfectant. We are now testing interferon-γ excretion by ZN1 stimulated by the HLA-DR2-COS7 that was transfected with CLIP-replacement Ii chain gene vectors.In relation to this project, we screened cDNA library derived from the patient's bone marrow mononuclear cells using serological identification of antigens by recombinant expression cloning to identify autoantigens in AA. IgG antibodies recognizing α globin (residue 1-101, α globin^<1-101>) was detected in the patient's serum. Immunoblotting using recombinant α globin^<1-101> detected the specific antibodies in 21 of 25 (84.0%) AA patients. When the patient's lymphocytes were stimulated with α globin^<1-11>, a low percentage of CD8* T cells reactive to this peptide were generated. The cultured T cells showed cytotoxicity against HLA-A*0201^+JY cells that were pulsed with this peptide. Addition of T cells stimulated by α globin^<1-11> to autologous CD34^+ cells reduced the number of colonies derived from BFU-E and CFU-GM to nearly a half of the control, α globin may serve as a target of immune system attack in AA patients possessing HLA-A-*0201.