T-Cell Receptor Amino Acid Sequence Selected By Cytotoxic T Cells Specific to HTLV-1 in HLA-A02:01-Positive ATL Patients

Purpose: Adult T cell leukemia/lymphoma (ATL) is an aggressive peripheral malignancy of T cells caused by human T cell lymphotropic virus type-1 (HTLV-1). Its prognosis remains very poor. Tax is the most important regulatory protein for HTLV-1, which is associated with the proliferation of host cells. Tax has been considered as an important target antigen for CD8⁺cytotoxic T cells (CTLs). We aimed to reveal such a unique sequence of complementarity-determining region 3 (CDR3) of the T cell receptor (TCR)-α and-β chain of HLA-A*02:01-restricted Tax_11-19-specific CTLs (Tax-CTL).

Methods: We serially analyzed Tax-CTLs from ATL patients with HLA-A*02:01 who had received allogeneic hematopoietic cell transplantation (all-HCT), chemotherapies alone, or had not ever received any treatment. Tax-CTLs were defined as a living CD3⁺CD8⁺Tax-tetramer⁺cells. After sorting Tax-CTLs directly from an actual patient sample into a PCR tube, TCR-α and -β chains of individual CTL clones were determined by the next-generation sequence (NGS) method with SMARTer technology. In addition, we cultured and established individual Tax-CTL clones from a sorted single cell, and finally evaluated the cytotoxicity of Tax-CTLs by calcein-acetoxymethyl (AM)-based cytotoxicity assay.

Results: Peripheral blood samples were obtained from 7 patients (Pt-1,-2,-3,-4,-5,-6 and -7). Pt-1 had received allo-HCT and the samples were available 270 days, 1 year, 3 years, 5 years and 7 years after allo-HCT. Samples before treatment were available in the other 5 patient (acute ATL Pt-2,-3, and -6; chronic ATL, Pt-4; HTLV-1 asymptomatic charrier, Pt-5), while the samples after treatment were available in 1 patient (acute ATL Pt-7). Tax-specific CTLs were detected in 0.03-0.58% of total peripheral blood mononuclear cells, and 144-1000 cells per sample (median; 250 cells) were sorted and analyzed for TCR determination. Of them, 2 to 30 Tax-specific CTL clones were identified for each patient. Tax-specific CTLs seemed oligoclonal, and their usage of BV and BJ genes were restricted in TCR-BV28(30.5%), -BV6-5(33.3%), and -BJ2-1(30.5%), while their usage of AV and AJ genes were restricted in TCR-AV12-2(54.5%) and -AJ24(24.2%) gene families. The length of amino acids (AA) in TCR-α and TCR-β CDR3 of all Tax-CTLs clones seemed to be symmetrically distributed, and the most frequently observed AA length was 13 in TCR-α, and 15 in TCR-β. The unique motifs of "DSWGK" in TCR-α and "LAGG" in TCR-β at CDR3 were observed in almost all ATL patients. The 'DSWGK' motif was observed in 0-81.7% (median; 55.5%), and the 'LAGG' motif was observed in 5.2%-80.3% (median; 48%). Pt-2 didn't have any clones with 'DSWGK' motif in TCR-α, whereas regarding 'LAGG' motif in TCR-β, all patients had it in this study. We established Tax_11-19-specific CTL clones from a single cell of Pt-4, which showed killing activities against Tax-peptide-pulsed HLA-A2⁺ T2 cell lines.

Conclusion: These methods and results can help us to better understand immunity against ATL, leading to future studies on the clinical application of adoptive T-cell therapies in the future.

Kanda:Mundipharma Pharmaceuticals: Research Funding.