Somatic polyploidization and characterization of induced polyploids of Dioscorea rotundata and Dioscorea cayenensis

Genetic improvement of major food yams is constrained by a number of factors, such as the scarcity of flowers and lack of synchronization between male and female flowering. Consequently, somaclonal variation including somatic polyploidization has been considered as a useful tool in yam breeding. Somatic polyploidization and its effect on phenotypic traits of Dioscorea species such as D. alata, D. japonica and D. zingiberensis has been reported; however, optimization of this method in two major yam species, D. rotundata and D. cayenensis, is yet to be achieved and the effect of polyploidization on phenotypic traits of this species yet to be elucidated. In the present study, a high rate of somaclonal polyploid variation was successfully achieved by in vitro colchicine treatment of D. rotundata and D. cayenensis. In most cases, except TDc 3704, the highest rate of polyploid induction appeared after 0.1% colchicine treatment. However, in triploid yellow yam accessions the induction rate was relatively low. Tetraploid variants of D. rotundata tended to display somewhat rounder leaves than their diploid parents. The size and shape of D. rotundata stoma were also affected by levels of ploidy, with tetraploid variants exhibiting larger stomata at alower density compared to their diploid parents. The efficient method of in vitro polyploidy induction reported here is therefore a highly useful tool for obtaining polyploid variants for use as genetic resources in D. rotundata breeding.Keywords: Leaf, nodal segment culture, ploidy level, somaclonal variation, stoma, white guinea yam, yellow Guinea ya

TCRBV and BJ usage of NY-ESO-1-specific CD8⁺ T cell clones.

<p>TCRB CDR3 sequence analyses of NY-ESO-1-specific CD8⁺ T cell clones were performed. (A) HLA-B52:01-restricted clone 6-8L has the dominant V-J pair TCRBV11-03*01 and TCRBJ02-01*01. Only a few V-J pairs other than this dominant pair were observed in clone 6-8L. (B) HLA-B35:01-restricted clone 2F6 has a dominant V-J pair TCRBV05-08*01 and TCRBJ02-04*01.</p

Clinical and immunological response to NY-ESO-1f peptide vaccine in patient TK-f01.

<p><b>(A) Clinical response of patient TK-f01.</b> A right hilar lymph node (white arrow) and a small metastatic nodule in the left lobe (red arrow) were monitored on CT images as target lesions. The sum of diameters of tumor lesions (black line) and serum CEA levels (red line) are plotted. Vaccination (arrow) and CT scan (triangle) are indicated. <b>(B) NY-ESO-1-specific CD8</b>^<b>+</b><b>T response.</b> PBMCs were obtained at the indicated time points during vaccination. CD8⁺ T cells were purified from PBMCs using magnetic beads (Miltenyi Biotec) and stimulated with NY-ESO-1 overlapping peptide pools in vitro for 12 days. The IFN-γ capture assay was then performed 4 hr after stimulation with PFA-treated CD4- and CD8-depleted PBMC pulsed with the peptides. The net percentage of IFN-γ-producing cells within total CD8⁺ T cells was determined.</p

TCRB CDR3 repertoire analysis of PBMCs over the course of NY-ESO-1f peptide vaccination.

<p>Differential usages of TCRBV and BJ genes in the patient’s PBMCs at days 0, 106, 127 and 337 are shown. Read numbers of V-J gene pair are shown in the 3D graph.</p

TCRB repertoire analysis for tracking NY-ESO-1-specific CD8⁺ T cell clones.

<p>The frequencies of the 15 most frequent TCRB CDR3 sequences in pretreatment PBMCs (Day 0) and from the NY-ESO-1-specific clones 6-8L and 2F6 were tracked over time during the course of treatment. The known TCRB CDR3 sequences for HLA-B*35-restricted EBV- and HLA-A*24-restricted CMV-specific T cells were also detected in PBMCs. The frequency of each CDR3 sequence is shown. The CDR3 sequence of clone 6-8L was the second most frequent sequence and remained present at high frequency from the initiation of the treatment to three months after the last vaccination. The CDR3 sequence of 2F6 was not detected in PBMCs on day 0; however, it was detected at low frequency on day 106 and day 337.</p

Detection and Tracking of NY-ESO-1-Specific CD8⁺ T Cells by High-Throughput T Cell Receptor β (TCRB) Gene Rearrangements Sequencing in a Peptide-Vaccinated Patient

<div><p>Comprehensive immunological evaluation is crucial for monitoring patients undergoing antigen-specific cancer immunotherapy. The identification and quantification of T cell responses is most important for the further development of such therapies. Using well-characterized clinical samples from a high responder patient (TK-f01) in an NY-ESO-1f peptide vaccine study, we performed high-throughput T cell receptor β-chain (TCRB) gene next generation sequencing (NGS) to monitor the frequency of NY-ESO-1-specific CD8⁺ T cells. We compared these results with those of conventional immunological assays, such as IFN-γ capture, tetramer binding and limiting dilution clonality assays. We sequenced human TCRB complementarity-determining region 3 (CDR3) rearrangements of two NY-ESO-1f-specific CD8⁺ T cell clones, 6-8L and 2F6, as well as PBMCs over the course of peptide vaccination. Clone 6-8L possessed the TCRB CDR3 gene TCRBV11-03*01 and BJ02-01*01 with amino acid sequence CASSLRGNEQFF, whereas 2F6 possessed TCRBV05-08*01 and BJ02-04*01 (CASSLVGTNIQYF). Using these two sequences as models, we evaluated the frequency of NY-ESO-1-specific CD8⁺ T cells in PBMCs ex vivo. The 6-8L CDR3 sequence was the second most frequent in PBMC and was present at high frequency (0.7133%) even prior to vaccination, and sustained over the course of vaccination. Despite a marked expansion of NY-ESO-1-specific CD8⁺ T cells detected from the first through 6th vaccination by tetramer staining and IFN-γ capture assays, as evaluated by CDR3 sequencing the frequency did not increase with increasing rounds of peptide vaccination. By clonal analysis using 12 day in vitro stimulation, the frequency of B*52:01-restricted NY-ESO-1f peptide-specific CD8⁺ T cells in PBMCs was estimated as only 0.0023%, far below the 0.7133% by NGS sequencing. Thus, assays requiring in vitro stimulation might be underestimating the frequency of clones with lower proliferation potential. High-throughput TCRB sequencing using NGS can potentially better estimate the actual frequency of antigen-specific T cells and thus provide more accurate patient monitoring.</p></div

The 15 most frequent CDR3 sequences in the pretreatment PBMCs and dominant CDR3 sequences of NY-ESO-1-specific CD8 T cell clones.

<p>Bold: CDR3 sequence of NY-ESO-1-specific CTL clones</p><p>The 15 most frequent CDR3 sequences in the pretreatment PBMCs and dominant CDR3 sequences of NY-ESO-1-specific CD8 T cell clones.</p