Figure 1.
Amplification of TCRα/β chains from single CD8+ T-cells.
(A) Experimental strategy for amplifying and sequencing TCRα/β chains by integrated 5′-RACE (RACE) and multiplex PCR (Multiplex). (B) Visualization of TCRα/β chain transcripts by agarose gel electrophoresis. The results were obtained from 96 cells in the naive subset in donor 1 and are shown as a representative result. R: 5′-RACE, M: mutitiplex PCR.
Figure 2.
TCRα/β-chain transcripts expressed in CD8+ T-cells. For characterization of TCRα/β chain transcripts, 1,250 and 1,661 sequence reads were used for TCRα and TCRβ chains, respectively. (A) Frequency of in-frame, out-of-frame, and germline transcripts for TCRα chains. Out-of-frame transcripts represent a sequence that contains either a stop codon or a frame-shift, whereas germline transcripts are defined by the absence of a variable segment and evidence that the transcript was started from a genomic region located in the upstream of either a joining or diversity segment. (B) Frequency of in-frame, out-of-frame, and germline transcripts for TCRβ chains. (C) Frequency of dual TCRα chain transcripts. The samples that had 2 transcripts were defined as dual TCRs. P: productive TCRs representing in-frame transcripts. UP: unproductive TCRs representing out-of-frame transcripts. (D) Frequency of dual TCRβ-chain transcripts. (E) Size distribution of CDR3α and CDR3β lengths. Samples representing productive TCRs (1,011 and 1,444 reads for TCRα and TCRβ chains, respectively) were extracted from the whole data set. The CDR3 lengths were identified by the IMGT/V-Quest tool at the amino acid (aa) level. (F) Paired analysis of CDR3α and CDR3β lengths. By use of 736 samples, the most frequent pair was identified at the position intersecting 11 and 13 aa of CDR3α and CDR3β lengths, respectively, as shown in red.
Table 1.
Success rate of single-cell PCR for TCRα/β chains in CD8+ T-cell subsets.
Figure 3.
Identification of transcription initiation sites (TISs) in TCRα/β chain.
(A) Distribution of TISs in TCRα/β chains. Samples containing a translation initiation site (773 and 930 reads for TCRα and TCRβ chains, respectively) were used for the analysis, and the initiation site was defined as zero. (B) Distribution of TISs in TRAVs of TCRα chains. Using the same data set, TISs were determined for individual TRAVs. (C) Distribution of TISs in TRBVs of TCRβ chains. A single dot represents a position of TIS obtained from a single sequence read.
Figure 4.
TRAV and TRBV usage in CD8+ T-cell subsets among 3 unrelated donors.
(A) Frequency of TRAV usage in the 3 donors. (B) Frequency of TRBV usage in the 3 donors. (C-D) TRAV (C) and TRBV (D) usage in CD8+ T-cell subsets among the 3 donors. The same data set used for TIS analysis was used for all analysis of TRAV and TRBV usage. The frequency is proportional to the density of colors: white (low) to black (high). The pseudogenes of TRAV and TRBV are shown in red. N: naive, CM: central memory, EEM: early effector memory, LEM: late effector memory, E: effector. Statistical analysis was performed using the χ2 and Student' s t test. *: P<0.05, EEM>N, EEM>LEM, EEM>E, **: P<0.05, N>CM, N>EEM, N>LEM, ***: P<0.05, LEM>EEM, ****: P<0.05, N>E.
Figure 5.
TRAJ, TRBJ, and TRBD usage in CD8+ T-cell subsets among the 3 unrelated donors.
(A) Frequency of TRAJ usage in the 3 donors. (B) Frequency of TRBJ usage in the same donors. (C) Frequency of TRBD usage in the 3 donors. (D) TRAJ usage in CD8+ T-cell subsets among the 3 donors. (E) TRBJ usage in CD8+ T-cell subsets among the same donors. The frequency is proportional to the density of colors from white (low) to black (high). The pseudotypes of TRAJ and TRBJ are shown in red. Statistical analysis was performed using the χ2 and Student's t test. *: P<0.05, N>EEM, N>E.
Figure 6.
Identity and clonotype of TCRα/β chains in CD8+ T-cell subsets among the 3 unrelated donors.
A data set not including samples showing germline transcripts was used for the analysis (1,207 and 1,540 reads for TCRα and TCRβ chains, respectively). (A) Frequency of TCRα chain identity in CD8+ T-cell subsets. The identity was defined as more than 2 appearances of samples with the identical usage of TRAV, TRAJ, and CDR3α. (B) Frequency of identical TCRα and β chains in CD8+ T-cell subsets. The identity was defined as more than 2 appearances of samples with the identical usage of TRBV, TRBD, TRBJ, and CDR3β. (C) Frequency of paired TCRα/β clonotype in CD8+ T-cell subsets. The percentage was measured by using a data set obtained from 901 paired TCRα/β sequence reads. The clonotype was defined by the more than 2 appearances of samples showing identical TCRα/β chains. (D) Proportion of TCRα/β clonotypes in CD8+ T-cell subsets among the 3 unrelated donors. The TCRα/β clonotypes identified in “C” were discriminated by type and proportion (see Table S4).