Identification of T Cell Receptors Specific to Antigens Presented By HLA-B5401 on IPS Cell-Derived Hematopoietic Stem Cells in a Patient with Acquired Aplastic Anemia Carrying B5401-Lacking Leukocytes

Abstract

[Background] Cytotoxic T lymphocytes (CTLs) specific to hematopoietic stem cells (HSCs) are thought to play an important role in the development of acquired aplastic anemia (AA). The presence of HLA class I allele-lacking leukocytes as a result of 6pLOH or allelic mutations in about 25% of AA patients strongly suggests that CTLs specific to antigens presented by the specific HLA alleles are involved in the destruction of normal HSCs as well as in the escape of HLA-lacking HSCs. Establishing CTLs from these patients by culturing the patients' CD8+ cells with targeted HSCs may be useful for identifying autoantigens of AA. However, it is generally impossible to obtain sufficient numbers of HSCs from the patients for functional assays and in vitro stimulation of patient's T cells with HSCs may induce clonal expansion of CTLs irrelevant to pathogenesis of AA. To solve these problems, we generated iPS cells (iPSCs) from an untreated AA patient who possessed HLA-B5401-lacking leukocytes for inducing HSCs and attempted to identify cDNAs of T-cell receptor (TCR) α and β chains of predominant T cells in the patient's bone marrow (BM) using a single T-cell analysis, which may specifically recognize autologous HSCs. [Methods] iPSCs were generated from the peripheral blood mononuclear cells of an AA patient (56-year-old man), whose moderate AA later responded to cyclosporine therapy, using Yamanaka's method. The patient's monocytes accounted for 20% wild-type, 20% 6pLOH(+) and 60% B5401-lacking cells at the time of sampling. CD34+ cells were induced from these iPSCs and injected into the BM of C57BL/6.Rag2 mice harboring NOD-Sirpa (BRGS). CD8+ T cells in the BM of the patient were subjected to a single cell sorting followed by cDNA amplification of TCRVβ chains. TCRα chains of five T cells that showed the five most recurrent TCRVβs were determined, and the TCRβ cDNAs were retrovirally transfected to Jurkat cells that lacked TCRβ chain and were transfected with IFN-γ cDNA. Specific responses of each TCR transfectant to iPSC-derived CD34+ cells were determined using ELISA for detecting IFN-γ. [Results] Five iPSC clones, including wild type (n=2), 6pLOH(+) (n=1), and B5401-lacking (B5401[-]) (n=2) clones, that had different mutations in B*54:01 were generated. Three-week culture of the iPSCs in the presence of various growth factors produced hematopoietic cells consisting of 50% CD34+ cells of each phenotype, which were able to reconstitute of BRGS mice with 3.5% to 8.9% human CD45+ cells in the peripheral blood at 9-12 weeks after injection. When the four TCR transfectants were tested for their response to wild-type and B5401-lacking CD34+ cells, all four transfectants secreted higher amounts of IFN-γ in response to wild-type cells than to B5401(-) CD34+ cells. [Conclusions] HSCs with original genotypes capable of reconstituting hematopoiesis in immunodeficient mice were successfully induced from iPSCs that had been generated from an AA patient's monocytes. The TCR transfectants showing a specific response to B5401(+) wild-type CD34+ cells will be useful for identifying the ligand peptides presented by B5401. DisclosuresNo relevant conflicts of interest to declare.