Comparison of T-cell receptor β chain variable region 23-1 open reading frame and 3-1 F CDR3 repertoire in cDNA and gDNA from peripheral blood mononuclear cells of healthy volunteers by high-throughput sequencing

Introduction

T-cell receptors (TCRs) are either TCR αβ or TCR γδ heterodimers that are formed by four types of peptide chains α, β, γ, and δ. Each polypeptide chain in the thymus rearranges from the TCR germline gene to form a diverse CDR3 repertoire. According to the definition and annotation of the IMGT and EMBL, the TCR germline gene entity can be defined according to three types: open reading frame (ORF), functional gene (F) and pseudogene (P). An open reading frame is a portion of a gene’s sequence that contains a sequence of bases, uninterrupted by stop sequences, that could potentially encode a protein. A functional gene’s coding region has an ORF without a stop codon, and for which there is no described defect in the splicing sites, recombination signals, or regulatory elements. Pseudogenes are the remnants of genomic sequences of genes which are no longer functional; they are frequent in most eukaryotic genomes, and an important resource for comparative genomics [1-4].
In the process of TCR creation, according to the principle of allelic exclusion, the β chain is rearranged earlier than the α chain, so detecting a β chain could reflect the character of TCR. According to the homology of the human TRBV gene, 65 TRBV genes can be divided into 24 conservative families, which can cover more than 85% of human TCR β chain genes. The complementarity-determining region 3 (CDR3) of a specific TCR β chain could be amplified through designing sense primers and TRBC antisense primers. According to the definition and annotation of the IMGT, the human TCR β locus consists of 48 TRBV functional genes, 19 TRBV pseudogenes, and 7 ORFs. However, some TRBV genes are difficult to classify. For example, TRBV7-3 is classified into either a functional gene or an ORF, namely, TRBV7-3*01, TRBV7-3*04 and TRBV7-3*05 (accession numbers X61440, X74843 and M13550, respectively), whereas TRBV7-3*02 and TRBV7-3*03 are classified into ORFs (accession numbers M97943 and AF009660). TRBV16 is classified into either a functional gene or a pseudogene: TRBV16*01 and TRBV16*03 are classified into a functional gene (accession numbers L26231 and L26054), whereas TRBV16*02 is classified into a pseudogene (accession number U03115) [3, 4]. Identification of the functions of these TRBVs is highly complex.
There is currently a debate on how to define and classify human TRBV19S1 (TRBV23-1), and the human T-cell receptor β chain variable region (clone HVB10.1) was initially defined as a pseudogene, then later described as an ORF because nucleotide bases GT were replaced by AT in the donor splice site, whereas only a portion of the V-gene has been identified [3]. In 1996, Currie et al. [5] discovered a single extra base in the TRBV19S1 leader sequence, which leads to reading frame changes after mRNA splicing, thereby resulting in premature termination of mRNA translation. Therefore, the TRBV19S1 would be non-functional after rearrangement. However, in 1999, while conducting a research study involving children infected with HIV, Than et al. [6] discovered that TRBV19S1 had dominant usage expression, thus prompting him to suggest that these T-cells participate in the immune response. In 1999, Manfras et al. [7] sequenced more than 500 TRBV20S1 and TRBV19S1 genes. Their study identified different CDR3 lengths between the productive sequences (TRBV20S1) and in-frame nonproductive sequences (TRBV19S1), although no significant differences in TRBD and TRBJ usage and nucleotide additions in junctional sequences were observed.
These analyses were based on the intrinsic defects in gene sequences or by cloning a specific CDR3 region to indirectly demonstrate the in-frame and out-of-frame rearrangements of TRBV23-1. The mechanism underlying the participation of TRBV23-1 in the VDJ rearrangement, and the characteristics of CDR3 repertoires rearranged by TRBV23-1 as well as differences in CDR3 repertoires rearranged by a TRBV functional gene have not been elucidated to date. In this study, we compared the characteristics of the CDR3 repertoire that has been rearranged by TRBV23-1 (ORF) with that by TRBV3-1 (F) in the cDNA samples from four healthy volunteers by laser capillary electrophoresis scanning and 454 GS FLX high-throughput sequencing (HTS). We also compared the characteristics of the CDR3 repertoire that has been rearranged by TRBV23-1 with that by TRBV3-1 in the gDNA samples from six healthy volunteers by Illumina sequencing. Our results show that the frequencies of in-frame sequences in the TRBV23-1 CDR3 repertoire were significantly lower than those in the TRBV3-1 CDR3 repertoire. The TRBV23-1 CDR3 repertoire, which differed from the TRBV3-1 in-frame and out-of-frame CDR3 repertoire, consisted of 1/3 in-frame sequences and 2/3 out-of-frame sequences. The usage of TRBD1 was higher than that of TRBD2 in the TRBV23-1 in-frame and out-of-frame CDR3 repertoire. In four cDNA samples from PBMCs, the TRBV23-1 in-frame and out-of-frame CDR3 repertoire showed a longer N2 relative to that of N1. The mechanisms of rearrangement in the TRBV23-1 (ORF) and TRBV3-1 (F) CDR3 repertoire were different.

Material and methods

The CDR3 repertoire of TRBV23-1 and TRBV3-1 in the cDNA samples from PBMCs of four healthy volunteers

Ten-milliliter peripheral blood samples were collected from each of four healthy volunteers (H-1: male, 14 years old; H-2: female, 40 years old; H-3: male, 13 years old; H-4: male, 13 years old). H-3 and H-4 were a pair of twins. H-2 was a mother of twins. H-1 was the same age as the twins. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Hypaque density gradient centrifugation. The study protocol was approved by the local ethics committee. The volunteers had signed the informed consent, and in the case of the younger subjects under the age of 15, they also had the signed informed consent from their parents. Total RNA was extracted from each of the four PBMCs samples, and cDNA was prepared. Using the TRBC downstream primer with the FAM label and the individual TRBV upstream primers, the TRBV CDR3 regions were amplified by PCR (1 cycle of 94°C for 3 min, 1 cycle; 35 cycles each of 94°C for 60 s, 55°C for 60 s, and 72°C for 60 s; 1 cycle of 72°C for 10 min). The PCR products were stored at –20°C.
Laser capillary electrophoresis DNA scan (ABI3730) was used to analyze each PCR product of the TRBV gene CDR3. Peak Scanner Software v1.0 was used to analyze the CDR3 spectratyping. The TRBV23-1 and TRBV3-1 CDR3 PCR products of 4 healthy volunteers were chosen for cloning and sequence analysis of in-frame or out-of-frame rearrangements that consisted of the CDR3-region (Fig. 1).
Using the four sets of upstream and downstream primers with specific base labels (Table 1), the CDR3 regions of the TRBV genes were separately amplified by polymerase chain reaction (PCR; using the same conditions as earlier described). Roche 454 GS FLX high-throughput sequencing was used to determine the genomic sequences of the CDR3 repertoire (the PCR products) of each sample by Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China). IMGT High V-QUEST was used to screen the original HTS data (Fig. 1). First, “Unproductive”, “Warnings”, “Unknown functionality”, and “No results” sequences were removed. Then, the in-frame and out-of-frame CDR3 sequences with a V-gene consistency rate of > 90% were selected for analysis [8-11].

The CDR3 repertoire of TRBV23-1 and TRBV3-1 in the gDNA samples from PBMCs of six healthy volunteers

Ten-milliliter samples of peripheral blood were collected from each of six healthy volunteers (H-5: male, 46 years old; H-6: male, 46 years old; H-7: male, 35 years old; H-8: male, 35 years old; H-9: male, 22 years old; H-10: female, 22 years old). The study protocol was approved by the local ethics committee. The subjects had signed the informed consent. PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation and genomic DNA was extracted from the six samples, yielding the following individual total amounts: H-5, 1,800 ng; H-6, 1,990 ng; H-7, 3,350 ng; H-8, 5,090 ng; H-9, 2,380 ng; and H-10, 1,730 ng.
The CDR3 repertoire of human TCR beta chain preparation and Illumina HTS sequencing of the gDNA samples were completed by Adaptive Biotechnologies Corp [12]. Adaptive Biotechnologies has developed a novel method that amplifies rearranged TCR CDR3 sequences and exploits the capacity of high-throughput sequencing technology to sequence tens of thousands of TCR CDR3 chains simultaneously (Fig. 1). Because the technology utilizes gDNA, the frequency of sequenced CDR3 chains is representative of the relative frequency of each CDR3 sequence in the starting population of T-cells [13-15]. The CDR3 sequences were screened and analyzed using the ImmuneSEQ assay [16] and the IMGT High V-QUEST [3]. The CDR3 sequences of TRBV3-1 and TRBV23-1 were selected from all the CDR3 repertoire sequences for analysis. First, ‘Unproductive’, ‘Warnings’, ‘Unknown functionality’, and ‘No results’ sequences were removed. Then, the in-frame and out-of-frame CDR3 sequences with a V-gene consistency rate of > 90% were selected for analysis [14, 15].

Experimental reagents and statistical analysis

Total RNA and total DNA extraction reagent kits, the gel extraction kit, the PCR purification kit, the Taq PCR master mix, and the DNA extraction and freeze-dry kit were obtained from Qiagen (Germany). DNA marker, DNA loading buffer, and cDNA synthesis kits were obtained from MBI Fermentas (Canada).
TRBD gene and TRBJ gene usage were compared using the 2 test; N1 and N2 lengths of nucleotides were compared using ANOVA. p < 0.05 was considered statistically significant. All statistically significant differences are indicated; *p < 0.05, **p < 0.01, ***p < 0.001 [12, 17-19].

Results

CDR3 spectratyping of TRBV23-1 and TRBV3-1 in cDNA samples from PBMCs of four healthy volunteers

CDR3 spectratyping of TRBV23-1 showed a 1-bp or 2-bp (out-of-frame) and 3-bp (in-frame) interval length and peak. On the other hand, the CDR3 spectratyping of TRBV3-1 showed a 3-bp (in-frame) interval length and peak (Fig. 2, Table 2). In the nine cloned sequencing results of the TRBV23-1 CDR3 region PCR products that were randomly collected from H-4, four were in-frame with 3-bp insertions, three were out-of-frame with a 1-bp insertion, and two were out-of-frame with 2-bp insertions (Table 3).

CDR3 repertoire characterization of TRBV23-1 and TRBV3-1 in healthy volunteers by HTS

The cDNA samples were extracted from PBMCs of four healthy volunteers. A total of 509 reads of the TRBV23-1 CDR3 complete sequences of the four samples, including 142 in-frame and 367 out-of-frame sequences, were identified. A total of 2,678 reads of the TRBV3-1 CDR3 complete sequences were identified, which included 1,322 in-frame and 1,356 out-of-fame sequences (Table 4). The gDNA samples were extracted from PBMCs of six healthy volunteers. A total of 1,091 CDR3 complete sequence reads were detected in TRBV23-1, which included 350 in-frame reads and 741 out-of-frame reads. A total of 5,043 CDR3 complete sequence reads were detected in TRBV3-1, which included 4,254 in-frame reads and 789 out-of-frame reads (Table 5).
For both cDNA and gDNA, the CDR3 total sequences of TRBV23-1 were significantly smaller than those of the CDR3 sequences of TRBV3-1 (Tables 4 and 5, Fig. 3); the total number of CDR3 sequences of TRBV23-1 out-of-frame was significantly higher than that of the TRBV23-1 in-frame sequences (Tables 4 and 5, Fig. 4).

Comparative analysis of the CDR3 repertoire with TRBD and TRBJ usage of TRBV23-1 and TRBV3-1 by HTS

For both cDNA and gDNA, the usage of TRBD1 in the CDR3 repertoire of TRBV3-1 in-frame sequences was higher than that of TRBD2 in terms of mean percentage; however, it was not statistically significant (p > 0.05) (Fig. 5). The usage of TRBD1 in the CDR3 repertoire of TRBV23-1 in-frame and out-of frame sequences was higher than that of TRBD2 and was statistically significant (p < 0.001) (Fig. 5). The usage of TRBJ2 was significantly higher than that of TRBJ1 in terms of the CDR3 repertoire of all TRBV23-1 and TRBV3-1 (p < 0.001) (Fig. 6).
The usage of TRBD1 in the CDR3 repertoire of TRBV3-1 out-of-frame sequences was higher than that of TRBD2 and was statistically significant (p < 0.01) in cDNA samples (Fig. 5A). The usage of TRBD1 in the CDR3 repertoire of TRBV3-1 out-of-frame sequences was lower than that of TRBD2 and was statistically significant (p < 0.001) for the gDNA samples (Fig. 5B).

Comparative analysis of the CDR3 repertoire lengths with N1 an N2 of TRBV23-1 and TRBV3-1 by HTS

For the cDNA samples, the CDR3 repertoire of TRBV3-1 in-frame sequences showed that N2 was longer than N1 in terms of mean length; however, this was not statistically significant (p > 0.05). The CDR3 repertoire of TRBV23-1 in-frame sequences showed that N2 was longer than N1 and was statistically significant (p < 0.05). The CDR3 repertoire of TRBV3-1 and TRBV23-1 out-of-frame sequences consistently showed that N2 was longer than N1 and was statistically significant (p < 0.001) (Fig. 7A). However, the CDR3 repertoire of all TRBV23-1 and TRBV3-1 sequences showed no differences in the lengths of N1 and N2 for the gDNA samples (p > 0.05) (Fig. 7B).

Discussion

Several studies have shown that human TRBV19S1 (TRBV23-1) is a pseudogene [5, 7]; however, it is also possible that this gene may have undergone rearrangements as a functional TCR [6]. The IMGT and EMBL initially defined the T-cell receptor beta chain variable region (clone HVB10.1) as a pseudogene, which was later described as an ORF because the nucleotide bases GT were replaced by AT in the donor splice site, whereas only a portion of the V-GENE has been identified [3, 4]. The characteristics of CDR3 repertoire rearrangement by TRBV23-1 and the differences in the function of TRBV genes require further analysis.
We compared the CDR3 repertoire rearranged by TRBV23-1 to those rearranged by a random selecting functional gene (TRBV3-1) in cDNA and gDNA samples from PBMCs of healthy volunteers. We determined that: (1) the CDR3 spectratyping of TRBV23-1 (cDNA samples from four volunteers) showed with 1-bp or 2-bp (out-of-frame) and 3-bp (in-frame) interval lengths and peaks (Fig. 2, Table 2). However, the CDR3 spectratyping of TRBV3-1 showed with 3-bp (in-frame) interval length and peak (Fig. 2, Table 2); (2) the TRBV23-1 CDR3 region PCR products were randomly selected from H-4 (cDNA samples), four sequences in the CDR3 region were in-frame with 3-bp insertions, three sequences in the CDR3 region were out-of-frame with a 1-bp insertion, and two were out-of-frame with 2-bp insertions (Table 3); (3) the CDR3 repertoire of TRBV23-1 was expressed at significantly lower levels than that of TRBV3-1 (Tables 4 and 5); (4) the CDR3 repertoire of TRBV23-1 consisted of 2/3 out-of-frame sequences and 1/3 in-frame sequences, which was exactly the opposite to that observed of the TRBV3-1 in-frame and out-of-frame CDR3 repertoire. These findings suggest that the CDR3 repertoire that was rearranged by TRBV23-1 mainly emerged from random rearrangements (2/3 sequences were 1-bp and 2-bp insertions, whereas 1/3 of the sequences harbored 3-bp insertions), and the CDR3 repertoire rearranged by TRBV23-1 may have rendered non-functionality, as well as in the absence of positive and negative selection. The CDR3 repertoire rearranged by TRBV3-1 was mainly composed of an in-frame CDR3, and a few out-of-frame CDR3 regions. In the gDNA samples, the percent of in-frame CDR3 regions was > 2/3, and the percentage of in-frame CDR3 regions was < 2/3, which may be attributable to cDNA samples that were not amplified by multiplex PCR. In our other experiments on functional TCR repertoires, the percentage of in-frame CDR3 in both human and mice was > 2/3 (cDNA samples and gDNA samples). The results from these massive CDR3 repertoire sequences from TRBV23-1 and TRBV3-1 are in agreement with those of Burkhard’s research, which used a small amount of cloning sequencing data to compare and analyze TRBV19 and TRBV20 [7].
The maintenance of TRBV functional gene and ORF (or pseudogene) out-of-frame CDR3 sequences at a certain proportion in peripheral blood might be possibly due to the TCR β chain undergoing rearrangements, with the V-D-J combination rearrangement occurring first in one chromosome. Theoretically, the rearrangement of the TRBV functional gene, pseudogene and ORF is random [20]. In the CDR3 repertoire formed by rearrangements, the CDR3 region insertion/splicing is random, where the ratio of 1-bp, 2-bp, and 3-bp change is 1/3, respectively. Because TRBV ORFs or pseudogenes do not have functional protein expression after rearrangement, the cells that undergo TRBV ORF or pseudogene rearrangements would either die or initiate a second V-D-J rearrangement in another chromosome. If the second rearrangement were successful, the cells would enter the subsequent selection and maturation stages. If not, then the cells would eventually die. Hence, because TRBV ORFs or pseudogenes are not functionally expressed after rearrangement, these will not be subjected to positive and negative selections to mature. In theory, the total CDR3 repertoire of the TRBV ORF or pseudogene is eventually formed after initiation of rearrangements in another chromosome and successful functional selections. Therefore, the total CDR3 repertoire of the TRBV ORF or pseudogene follows the rearrangement rules of base insertion/deletion, thereby yielding the in-frame ratio of approximately 1/3 (3-bp insertion/deletion) and the out-of-frame ratio of approximately 2/3 (1-bp and 2-bp insertion/deletion). At the same time, because TRBV ORF or pseudogenes have no functionality after rearrangement, the formation of their peripheral CDR3 repertoire depends on successful rearrangements involving the TRBV functional gene in another chromosome. Consequently, the number of CDR3 sequences of the TRBV ORF or pseudogene with no functionality should be much lower than that of TRBV functional genes. However, TRBV functional genes would only mature after random rearrangement and positive and negative selections. Theoretically, the ratio of TRBV functional gene in-frame CDR3 and out-of-frame CDR3 sequences is extremely complex and closely related to individualized selection [5, 21]. The pattern of rearrangement and selection of TRBV23-1 CDR3 repertoire detected by HTS is consistent with the theory, and these results suggest that we can detect the CDR3 repertoires of the ORF or pseudogene by HTS as a control to analyze the mechanism underlying rearrangements and positive/negative selection of functional genes.
We then compared the usage of TRBD and TRBJ in TRBV23-1 and TRBV3-1 CDR3 repertoires, and the CDR3 repertoire with TRBD1 usage in the TRBV23-1 in-frame and out-of-frame was higher than that of TRBD2, whereas TRBV3-1 in-frame and out-of-frame CDR3 repertoires differed. These findings suggest that the mechanism underlying TRBD rearrangement with TRBV3-1 (F) and TRBV23-1 (ORF) were different, and subsequently yielded variable CDR3 repertoires. However, the usage of TRBJ was similar among these repertoires, and that of TRBJ2 was significantly higher than that of TRBJ1 in the TRBV23-1 and TRBV3-1 CDR3 repertoire in all the cDNA and gDNA samples from PBMCs of volunteers. Meanwhile, at the TCR loci, the 3’ end of TRBV or TRBD in TCR β chains is a heptamer (CACAGTG)-23 base pair (bp)-nonamer (ACAAAAACC) rearrangement of signal sequences (3’ TRBV 23 RSS and 3’ TRBD 23 RSS), whereas the 5’ end of TRBD or TRBJ is a nonamer-12bp-heptamer rearrangement of signal sequences (5’ TRBD 12 RSS and 5’ TRBJ 12 RSS). V-D-J recombination occurs only between segments with the 23 RSS terminal and segments with the 12 RSS terminal, and this restriction is called the 12/23 rule. V-D-J rearrangement of the TCR-beta chain follows the 12/23 rule and the beyond 12/23 restriction. The mechanism of differential gene rearrangement in the TRBV23-1 and TRBV3-1 CDR3 repertoire may be related to the 12/23 rule and the beyond 12/23 restriction, but that needs further study.
The diversity of the TCR CDR3 repertoire is the result of random combinations, insertions, and splicing of the germline VDJ gene fragment. The random nucleotide insertion (N region) was very important for composition of TCR β CDR3 diversity during the TCR β gene combination process. The region in which the nucleotide fragments randomly added into the Vβ-Dβ junction was called the N1 region. The N2 region was where the nucleotide fragments randomly added into the Dβ-Jβ junction. We also compared the length of N1 and N2 in TRBV23-1 and TRBV3-1 CDR3 repertoires. In the four cDNA samples from PBMCs, the TRBV23-1 in-frame and out-of-frame CDR3 repertoire showed that N2 was longer than N1 and similar to the TRBV3-1 out-of-frame CDR3 repertoire, whereas in the gDNA samples from PBMC of six volunteers, the TRBV23-1 and TRBV3-1 in-frame CDR3 and out-of-frame CDR3 repertoire showed no difference in the lengths of N1 and N2. These results show that the insertions during rearrangement of TRBV23-1 and TRBV3-1 (gDNA samples) were similar, whereas in the cDNA samples, the length of N in unproductive repertoires, namely, TRBV3-1 out-of-frame, TRBV23-1 in-frame and TRBV23-1 out-of-frame, showed the same N2 > N1, while the length of N2 and N1 in the productive repertoire TRBV3-1 was similar, thereby suggesting that the long N2 was closely related to the unproductive TCR receptor.
Taken together, the results of the present study generate the following conclusions: (1) The TRBV23-1 (ORF) CDR3 repertoire expression frequency is lower than that of the TRBV3-1 (F) CDR3 repertoire, indicating that the non-functional rearrangements of TRBV23-1 limit the probabilities of being selected for taking part in rearrangement and development-maturation. (2) Due to the lack of functional maturation, the ratio of TRBV23-1 (ORF) in-frame CDR3 and out-of-frame CDR3 completely follows the theoretical rules of random rearrangement. In contrast, the TRBV3-1 (F) in-frame CDR3 and out-of-frame CDR3 ratio, although following the theoretical rules of random rearrangement, must reach maturity through individual negative and positive selection, thereby resulting in complicated and variable ratio characteristics. (3) In the cDNA of the CDR3 repertoire, due to the lack of selection by functional maturation of TRBD gene usage, N-region insertion/splicing of TRBV23-1 (ORF) in-frame and out-of-frame CDR3 present characteristics that are similar to the TRBV3-1 (F) out-of-frame CDR3 repertoire (non-functional).
In summary, these results indicate that analysis of the composition and characteristics of the TRBV23-1 (ORF) CDR3 repertoire could provide the basis for research on the rules and mechanisms of TCR random rearrangement and the positive or negative selection process. At the same time, it could provide experimental quality control and research references for the analysis of the TRBV functional gene CDR3 repertoire.

Acknowledgements

We are grateful to the ten healthy volunteers for supporting this study. We thank Adaptive Biotechnologies Corporation (Seattle, WA, US) and Majorbio Bio-pharm Technology Co., Ltd (Shanghai, China) for help with human TCR β chain CDR3 repertoire sequencing and analysis.

The work was supported by grants from the National Prophase Project on Basic Research of China (973 pre-Program, 2008CB517310), the Natural Science and International Cooperation Program of Guizhou Province (2008-700105 & 2007-2122) and the National Natural Science Foundation of China (31160195 and 81441048).
The authors declare no conflict of interest.

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