Single-Cell Analysis of T-Cell Receptor Repertoire of HTLV-1 Tax-Specific Cytotoxic T Cells in Allogeneic Transplant Recipients with Adult T-Cell Leukemia/Lymphoma

Abstract

Adult T-cell leukemia (ATL) is a lymphoproliferative malignancy associated with human T-cell lymphotropic virus type 1 (HTLV-1) infection. Recently, it has been shown that allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective treatment for ATL, and that HTLV-1 Tax-specific CD8 + cytotoxic T cells (CTL) contribute to the graft-versus-ATL effect. In the present study, we, for the first time, analyzed the T-cell receptor (TCR) repertoire of isolated Tax 301-309 (SFHSLHLLF)-specific CTLs in HLA-A*2402 + ATL patients before and after allo-HSCT by single-cell reverse transcription-PCR. The Tax 301-309 -specific CTLs in bone marrow and peripheral blood showed highly restricted oligoclonal diversity. In addition, a unique conserved amino acid motif of "P-D/P-R" in TCR-β complementarity-determining region 3 in either BV7-or BV18-expressing CTLs was observed not only in all of the samples from ATL patients, but also in samples from the same patient before and after HSCT. Furthermore, the P-D/P-R motif-bearing CTL clones established from peripheral blood samples after HSCT exhibited strong killing activity against the HTLV-1-infected T cells of the patient. CTL clones were not established in vitro from samples prior to allo-HSCT. In addition, CTL clones with a strong killing activity were enriched in vivo after HSCT in the patient. Hence, Tax 301-309 -specific CTLs in ATL patients might have a preference for TCR construction and induce strong immune responses against the HTLV-1-infected T cells of patients, which contribute to the graft-versus-ATL effects after allo-HSCT. However, further analyses with a larger number of patients and more frequent sampling after allo-HSCT is required to confirm these findings. Cancer Res; 70(15);

Unique Features of Fish Immune Repertoires: Particularities of Adaptive Immunity Within the Largest Group of Vertebrates

“…However, advances in high-throughput sequencing of VH:VL pairs (DeKosky et al 2013; Tan et al 2014) likely will allow a comprehensive analysis of VH:VL gene usage and somatic hypermutation in FACS-sorted Ag-specific B-cell populations. Such methods will certainly be more powerful than the current complex and expensive methods based on single sorting and RT-PCR (Tanaka et al 2010). …”

Single-cell T-cell receptor-β analysis of HLA-A*2402-restricted CMV- pp65-specific cytotoxic T-cells in allogeneic hematopoietic SCT

“…[7][8][9][10] Recently, we and others have developed a direct single-cell RT-PCR analysis that enables the simultaneous identification and quantification of CTL clones without the effects of in vitro expansion. 11,12 To the best of our knowledge, the in vivo dynamics and monitoring of CMV-CTL clones have not been assessed in Allo-HCT. In addition, the clonotypes in HLA-A*2402-restricted CMV-CTLs have not been clarified, even though HLA-A*2402 is the most common HLA-A allele in the Japanese population (B60%).…”

“…The aminoacid (AA) sequences in CDR3 of TCR-b for sorted cells at a single-cell level were directly analyzed and determined after reverse transcript (RT)-PCR for TCR-b gene amplification as described previously. 11,12 In brief, individual CMV-pp65 tetramer þ T cells were sorted at a single-cell level into PCR tubes. After direct cell lyses, cDNAs of TCR-b were synthesized by RT, with a TCR-b constant region gene-specific primer.…”

High-Throughput Sequencing of the Paired Human Immunoglobulin Heavy and Light Chain Repertoire

“…However, existing immune repertoire sequencing technologies yield data on only one of the two chains of immune receptors and thus cannot provide information about the identity of immune receptor pairs encoded by individual B or T lymphocytes 2,7,8 . Sequence analysis of V H :V L pairs is currently done by microtiterwell sorting of individual B cells followed by single-cell RT-PCR (scRT-PCR) and Sanger sequencing [8][9][10][11][12][13][14][15] ; however, at most a few hundred V H :V L pairs (a number dwarfed by the enormous size of the human antibody repertoire) are identified by means of scRT-PCR [11][12][13][14] . Microfluidic methods for RT-PCR and the sequencing of two or more genes (e.g., using the Fluidigm platform 16 ) have so far been limited to only 96 wells per run and require complex, proprietary instrumentation.…”