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The Journal of Immunology, 1998, 161: 228-233.
Copyright © 1998 by The American Association of Immunologists

Development of Human Peripheral TCRBJ Gene Repertoire1

Toshihiro Nanki, Hitoshi Kohsaka2 and Nobuyuki Miyasaka

First Department of Internal Medicine, School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies of TCRBJ gene repertoires of human peripheral T lymphocytes showed that all TCRBV family transcripts had some common features in BJ gene usage, and nevertheless, transcripts of each BV family gene had a distinct pattern. To discern how the development of the peripheral BJ repertoire is controlled, the effects of preferential BJ gene rearrangement, thymic selection, and peripheral stimulation on the repertoire formation were investigated. A PCR-ELISA technique was used to examine the immature CD3-CD4+CD8-, and mature CD3+CD4+CD8- and CD3+CD4-CD8+ thymocytes, and peripheral CD4+ and CD8+ T lymphocytes for their BJ gene repertoires. Analogous to the peripheral repertoire, the BJ gene repertoires of the immature thymocytes displayed common features, and each BV transcript had a distinct pattern. All features were conserved well by those of mature thymocytes and peripheral T lymphocytes. In addition, the BJ gene repertoires of mature CD4 and CD8 thymocytes and peripheral lymphocytes with the same coreceptors were apparently different in a few BV-BJ combinations. The results showed that the overall BJ gene repertoire pattern was developed before antigenic selection. Thus, the preferential BJ gene expression, primarily based on preferential use of certain BJ gene rearrangements, dictates the peripheral BJ gene repertoire, which is then further modified by thymic selection and peripheral stimulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Most peripheral T lymphocytes express TCR-{alpha}ß on the cell surface. The TCR V regions are encoded by rearranged V, D (ß- and {delta}-chains only), and J gene segments with additional noncoding (N) sequences (1, 2). The expressed TCRBV gene repertoire of peripheral T lymphocytes in humans is controlled by genetic factors (3), predominantly influenced by HLA (4, 5). Our previous studies revealed that genetic factors regulate the peripheral TCRBJ gene repertoire, and that all of the TCRBV transcripts studied had some common features in BJ gene usage (6). Nevertheless, TCR transcripts from each TCRBV family displayed a distinct BJ gene profile, which was better defined in CD4 than in CD8 lymphocytes.

Recombination of the TCR gene segments takes place during maturation of T cells in the thymus (7, 8). The products of productively rearranged TCRB and pre-TCR-{alpha} (pT{alpha}3) form pre-TCR complexes on the surface of thymocytes, which then maturate to CD4+CD8+ thymocytes (9, 10). On the surface of the double-positive cells, the recombined TCRA and TCRB genes are expressed as complete TCR. Subsequently, the cells undergo positive and negative selections to select useful cells and to eliminate potentially harmful cells. The selection processes are mediated by the interaction of TCR, HLA, and HLA-binding Ags. The mature T cells enter the periphery, and undergo clonal expansion or deletion by antigenic stimulation. Thus, factors that could contribute to formation of peripheral TCRBJ gene repertoire include TCR gene rearrangement, thymic selections, and peripheral stimulation.

To delineate major factors contributing to the peripheral TCRBJ gene repertoire formation, we have analyzed BJ gene repertoires of immature thymocytes, which had not undergone thymic selections (11), mature single-positive thymocytes, and peripheral single-positive lymphocytes. The results demonstrated that TCRBV-BJ recombination is biased before antigenic stimulation, and this biased recombination is a major factor regulating the peripheral BJ gene repertoire. Thymic selections and peripheral stimulations also contribute to modify the repertoire.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Samples

Normal thymic fragments and peripheral blood were collected from 11 children (A to K) during heart surgery to correct congenital heart abnormalities. Consent forms were obtained before the operation. They were free of immunologic or hematologic disorders. Their ages ranged from 1 to 13 yr (mean, 5.6 yr).

Separation of cells

Thymocytes were minced and pressed through metal mesh to make single cell suspension in RPMI 1640 medium (Life Technologies, Gaithersburg, MD). The PBL were prepared with Ficoll-Hypaque gradient centrifugation. The thymocytes (1–1.5 x 108) were stained with phycoerythrin (PE)-cyanine 5-conjugated anti-CD3 mAb (UCHT-1; Coulter, Miami, FL), FITC-conjugated anti-CD4 mAb (T4; Coulter), and PE-conjugated anti-CD8 mAb (T8; Coulter). The CD3-CD4+CD8-, CD3+CD4+CD8-, and CD3+CD4-CD8+ thymocytes were collected with FACS (EPICS Elite Flow Cytometer; Coulter). The frequencies of CD3-CD4+CD8-, CD3+CD4+CD8-, and CD3+CD4-CD8+ thymocytes were 5.1 ± 2.6%, 11.5 ± 3.7%, and 3.5 ± 1.7%, respectively. The PBL (5–10 x 106 cells) were stained with FITC-conjugated anti-CD4 mAb (T4) and PE-conjugated anti-CD8 mAb (T8), and CD4+CD8- and CD4-CD8+ T cell subsets were isolated with the same sorter. Purity of the separated cells was more than 94%. The CD3-CD4+CD8- thymocytes were sorted twice to collect 3.8 ± 1.9 x 105 cells with final purity of more than 99%.

Analyses of TCRBJ gene repertoire

TCRBJ gene repertoires of TCRBV5, BV13, and BV17 gene family transcripts of the separated cells were analyzed by a PCR-ELISA method. The method was described in detail elsewhere (6, 12). In brief, first-strand cDNA, derived from the separated cells, was amplified by PCR using a TCRBV-specific sense primer and a biotinylated TCRBC-specific antisense primer.

To test the specificity of the 13 BJ gene-specific probes, 13 different TCR clones, each of which contained a different BJ recombination, were ligated with pT7Blue T-Vector (Novagen, Madison, WI). The BJ gene regions were amplified with corresponding BV gene-specific primers (13) and a biotinylated TCRBC-specific antisense primer.

The PCR products were immobilized onto streptavidin-coated 96-well ELISA plates. Thirteen BJ-specific probes labeled with digoxigenin were placed into the wells. The presence of 3 M tetramethylammonium chloride in the hybridization solution ensured equal hybridization efficacy of the probes (14). The plates were incubated with anti-digoxigenin Abs labeled with peroxidase, and reacted with tetramethylbenzidine microwell peroxidase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD). The absorbance values were recorded at 450 nm with a microplate reader. According to the standard curve, the absorbance values were transformed to concentrations. The frequency of each BJ gene usage was calculated by dividing the concentration of each BJ gene by the total concentration of BJ genes.

Statistical analyses

Multivariate cluster analyses of the TCRBJ gene repertoire data were performed by Ward’s method and city-block (Manhattan) distances, using Statistica 3.0 from Statsoft (Tulsa, OK).

To compare the BJ gene usage by different BV transcripts and by different T cell subsets, Wilcoxon signed rank test was used with assistance of StatView 4.11J (Abacus Concepts, Berkeley, CA) software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Validity of the TCRBJ gene repertoire analysis

The specificities of the 13 BJ-specific probes were first examined by probing 13 different TCR gene clones, each of which contained a different BJ gene. Each of the designed probes hybridized specifically to the corresponding BJ gene, and no cross-hybridization was observed (Fig. 1Go).



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  		FIGURE 1. Specificity of the TCRBJ probes. Each of the 13 specific BJ probes was hybridized with PCR products derived from 13 different TCRBJ genes.

 
The reproducibility of the method was confirmed by repeat analyses. The BJ gene repertoires of TCRBV13 transcripts derived from PBL of two individuals were analyzed in two independent experiments. As shown in Figure 2Go, the two BJ repertoires of the same individual were almost identical.



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  		FIGURE 2. Reproducibility of the PCR-ELISA method. The TCRBJ gene repertoires of the TCRBV13 transcripts from two cDNA samples were analyzed in two independent experiments. {circ}, I-1 and {circ}, I-2 are from a donor (I), and {blacksquare}, II-1 and {blacksquare}, II-2 are from another donor (II).

 
Common TCRBJ gene usage shared by different BV family transcripts

To analyze the effects of preferential TCR gene rearrangement, thymic selections, and peripheral stimulations on development of the peripheral BJ gene repertoire, we compared BJ gene usage of immature and mature thymocytes, and peripheral T lymphocytes. Using flow cytometry, the CD3+CD4+CD8- and CD3+CD4- CD8+ thymocytes as mature CD4 and CD8 thymocytes (7, 8, 11), and peripheral CD4 and CD8 T lymphocytes as mature peripheral T lymphocytes were separated from 11 donors (A to K). As immature thymocytes, CD3-CD4+CD8- cells in the thymus were isolated. They were sorted twice to eliminate CD3+ cells, and the final purity was more than 99%. The cells from six subjects (A to F) were sufficient for complete TCRBJ repertoire analysis.

It is known that the CD3-CD4+CD8- thymocyte population does not express complete TCR on the cell surface, and has not undergone thymic selections (11). Thus, the BV-BJ gene usage by this population should reflect preferential TCR gene rearrangement, and possibly preferential TCR-ß paring with pT{alpha}. Single-positive thymocytes and peripheral T lymphocytes were studied to discern the effects of thymic selections and peripheral stimulation. The BJ gene repertoires of TCRBV5, 13, and 17 gene family transcripts were chosen, because previous studies of the repertoires of the BV transcripts in PBL showed that they have distinct BJ gene usage patterns, depending on the combined BV genes (6).

The TCRBJ gene frequencies of BV5, 13, and 17 family transcripts from the five T cell subsets are depicted in Figures 3Go, 4, and 5. The BJ gene usage of immature thymocytes (panels A in Figs. 3Go, 4Go, and 5) had common features shared by all BV transcripts. The BJ2 cluster genes were used more frequently than BJ1 cluster genes. The BJ2S1 gene was used most frequently, and the BJ1S4, BJ1S6, BJ2S4, and BJ2S6 genes were rarely used. These common usage profiles displayed by the immature thymocytes were well conserved by those of mature CD4 and CD8 thymocytes (panels B and C in Figs. 3Go, 4Go, and 5Go) and peripheral CD4 and CD8 T lymphocytes (panels D and E in Figs. 3Go, 4Go, and 5Go).



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  		FIGURE 3. TCRBJ gene repertoires of TCRBV5 family transcripts. Repertoires of immature CD3-CD4+CD8- thymocytes (A) from six donors, and mature CD3+CD4+CD8- thymocytes (B), mature CD3+CD4-CD8+ thymocytes (C), peripheral CD4 lymphocytes (D), and peripheral CD8 lymphocytes (E) from 11 donors were analyzed. Symbols represent individual donors.

 


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  		FIGURE 4. TCRBJ gene repertoires of TCRBV13 family transcripts.

 


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  		FIGURE 5. TCRBJ gene repertoires of TCRBV17 family transcripts.

 
Distinct BJ gene usage by TCRBV family transcripts

The repertoires in panels A in Figures 3Go, 4Go, and 5Go also showed that each TCRBV family transcript from immature thymocytes had a unique pattern in the BJ gene usage. Compared with the other BV genes, the TCRBV5 gene used the BJ2S3 gene less frequently (p < 0.05). The BV13 gene combined with BJ2S5 gene more frequently than the other BV genes (p < 0.05). The BJ1S5 gene was used most frequently by the BV17 transcripts (p < 0.05). To assess the similarity of the BJ gene repertoires in each BV family, a multivariate analysis, cluster analysis (Ward’s method and city-block (Manhattan) distances) of the total of 18 different BJ gene repertoires of immature thymocyte was performed. Based on similarity measured by the method, all of the repertoires segregated into three groups. All of six BV5 repertoires fell into one group, as did four of six BV13 repertoires, and all of six BV17 repertoires. The dendrogram is shown in Figure 6Go. Thus, each BV family transcript from immature thymocytes had a distinct BJ gene usage pattern.



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  		FIGURE 6. Cluster analysis of TCRBJ gene repertoires of the immature thymocytes. From 18 BJ gene repertoire data, the distance of every combination of 2 BJ gene repertoires was calculated by Ward’s method and city-block (Manhattan) distance. All calculated distances, divided by the maximum distance, are shown in a dendrogram. 5, the BJ gene repertoires of TCRBV5 transcripts; 13, those of BV13 transcripts; and 17, those of BV17 transcripts. Listed below the alphabets are the identifications of the donors.

 
The distinct usage of immature thymocytes was conserved by those of mature thymocytes (panels B and C in Figs. 3Go, 4Go, and 5Go). The same cluster analysis grouped the total of 33 different BJ gene repertoires of mature CD4 thymocytes into three. Six of 11 BV5 repertoires fell into one group, as did all of 11 BV13 repertoires, and 10 of 11 BV17 repertoires. The BJ gene repertoires of mature CD8 thymocytes also segregated into three groups. Six of 11 BV5 repertoires fell into one group, as did 9 of 11 BV13 repertoires, and 9 of 11 BV17 repertoires.

The distinct BV-specific BJ gene usage was also conserved by peripheral T lymphocytes (panels D and E in Figs. 3Go, 4Go, and 5Go). Cluster analysis of the different BJ gene repertoires of peripheral CD4 or CD8 subsets showed that they segregated into three groups. All of 11 BV5 repertoires of CD4 T cells fell into one group, as did 10 of 11 BV13 repertoires, and all of 11 BV17 repertoires. Seven of 11 BV5 repertoires of CD8 T cells fell into one group, as did 9 of 11 BV13 repertoires, and 5 of 11 BV17 repertoires.

Differences of TCRBJ gene repertoire between mature CD4 and CD8 thymocytes, and between mature thymocytes and peripheral lymphocytes

As described above, mature CD4 and CD8 thymocytes, and peripheral CD4 and CD8 lymphocytes shared gross features of the BV-specific BJ gene usage with immature thymocytes. Nevertheless, the BJ gene repertoires of mature CD4 and CD8 thymocytes were different in individual BV-BJ combinations. As is shown in Table IGo, 4 BJ gene segments of 13 BJ genes were used more frequently by TCRBV5 transcripts in either of the two thymocyte populations. Two and five BJ genes were used with biases in TCRBV13 and BV17 transcripts, respectively.


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  		Table I. Differential usage of TCRBV-BJ gene combinations by mature CD4 and CD8 thymocytes

 
Moreover, the BJ repertoires of mature thymocytes and peripheral lymphocytes with the same coreceptor molecules were also different in individual BV-BJ gene usage. TCRBV5-BJ1S5 and TCRBV13-BJ1S1 gene combinations were used more frequently by the mature CD4 thymocytes, and TCRBV13-BJ2S3, BJ2S5, and TCRBV17-BJ2S5 combinations were used more frequently by peripheral CD4 lymphocytes (Table IIGo). TCRBV13-BJ1S5 combination was used more frequently, and TCRBV5-BJ2S1 and TCRBV13-BJ2S1 were used less frequently by mature CD8 thymocytes than peripheral CD8 lymphocytes (Table IIIGo).


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  		Table II. Differential usage of TCRBV-BJ gene combinations by mature CD4 thymocytes and peripheral CD4 lymphocytes

 

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  		Table III. Differential usage of TCRBV-BJ gene combinations by mature CD8 thymocytes and peripheral CD8 lymphocytes

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have studied the development of human TCRBJ gene repertoire by analyzing the repertoires of immature thymocytes, mature CD4 and CD8 thymocytes, and peripheral CD4 and CD8 lymphocytes. The results showed that the BJ gene usage by three BV transcripts of immature thymocytes had common features. At the same time, they displayed a distinct BJ gene usage profile depending on the BV genes used. The same features were conserved by the BJ repertoires of mature thymocytes and peripheral lymphocytes. Thus, preferential expression of TCRBV-BJ genes by the thymocytes before antigenic selection is a major factor dictating the peripheral BJ gene repertoire formation.

Jeddi-Tehrani et al. (15) previously analyzed BJ gene repertoire of double-positive (CD4+CD8+) thymocytes with low CD3 expression. They showed that the thymocyte population did not use BJ genes randomly, and the overall usage of the BJ gene was conserved by single-positive mature thymocytes. Since the double-positive thymocytes include the cells that have undergone thymic selections (16, 17, 18, 19, 20), the antigenic selection might influence the biased BJ usage. In contrast, the CD3-CD4+CD8- thymocyte population we examined expresses recombined TCR gene, but has not been selected by TCR ligands, and therefore, not influenced by Ags (11).

The present observation suggested that TCRBV genes do not recombine with all of the BJ genes by equal chance. The common BJ gene repertoire profiles displayed by all of the BV transcripts might result from preferential BD-BJ gene recombination, which precedes BV-BD recombination. TCR gene polymorphisms in the spacer region or noncoding regions of the recombination signal sequences may affect the efficiency of BJ rearrangement, as was reported in the BV genes (21, 22). Recently, it was reported that the nucleotide sequences at coding ends also affect the efficiency of VDJ recombination (23, 24).

It is of interest that even the immature thymocytes have the BV-specific BJ gene repertoires. Because BJ genes recombine with BD genes, and subsequently with the BV genes (25), it is unlikely that BV gene directly affects the BJ gene rearrangement. However, specific TCRBD-BJ recombinants with particular gene conformation might be favored for further recombination with limited BV genes.

The CD3-CD4+CD8- immature thymocytes in humans do not express pre-TCR complexes (pT{alpha}/TCR-ß) on the cell surface (11). However, in theory, the VDJ repertoire formation does not necessarily require surface expression of pT{alpha}. The interaction within the cell could influence subsequent T cell development. Nevertheless, pT{alpha} has no domains corresponding to V domains of other TCR (26), and the immature thymocytes from mice with TCR transgene lacking a V domain gene can maturate to CD4+CD8+ double-positive thymocytes (27). They all suggest that the selections at the protein level do not play a significant role in the BJ repertoire formation of the immature thymocytes.

The method used in the present study involved PCR amplification of TCRBJ genes with primers specific to the flanking gene components. Thus, amplification efficiency did not depend on BJ gene sequences. It was followed by PCR-ELISA format quantitation. This technique enabled us to amplify all BJ genes with equal efficiency and to analyze a large number of transcripts. Both were essential for accurate statistical interpretation.

Another approach to obtain the data would be to analyze the VJ frequencies of nonproductively rearranged alleles that could elucidate the contribution at the protein level more clearly. However, PCR amplification of genomic TCR requires amplification with BJ-specific primers, and subsequent DNA sequencing of multiple clones. The results generated by this set of experiments would inevitably be derived from a limited number of TCR clones that are amplified by potentially biased PCR.

The BJ gene repertoires of mature CD4 and CD8 thymocytes were different. Moreover, there were several distinctions in the BJ gene repertoires of mature thymocytes and peripheral lymphocytes with the same coreceptor molecules. Thus, the BJ repertoire differences were formed by thymic antigenic selections and peripheral antigenic stimulation. This selection of the BJ gene repertoire of peripheral T lymphocytes has been reported to be regulated in part by HLA (12, 28). As a platform to present Ags to the thymocytes or a source of antigenic peptides, HLA could modulate the BJ gene usage in the thymus and the periphery.

In the present study, we have found that peripheral CD8 T cells display distinct BJ gene repertoires, depending on the BV gene family. It was not clear in our previous studies of adult TCRBJ gene repertoire because oligoclonal expansions were often observed in the peripheral CD8 lymphocyte subsets. The peripheral CD8 T cell expansions were reported in patients with virus infection (29, 30, 31), rheumatoid arthritis (32, 33, 34, 35), and multiple myeloma (36), and even in healthy donors, especially in the elderly population (37, 38, 39). Since we limited our evaluation to young children, clonal expression had only a minor influence on the BJ repertoires of CD8 T cells.

The cluster analysis, which does not estimate p values, was a useful tool to reveal TCRBV-specific BJ gene usage. This proved to be a sensitive method to analyze the TCRBJ repertoire of immature thymocytes. However, it does not necessarily have enough power to handle a large amount of data with small differences and/or under the influence of multiple factors. Indeed, a comprehensive analysis of all of the BJ repertoires from the three BV transcripts of the CD4 and CD8 T cell subsets could not segregate into six small groups. This is probably due to an increased number of factors contributing to these repertoires, including genetics and especially HLA (6, 12, 28).

In conclusion, preferential BJ gene expression, primarily based on preferential rearrangement, dictates development of peripheral TCRBJ gene repertoire, which is further modulated by thymic selections and peripheral stimulation.


   Acknowledgments
 
We thank Drs. Shuichi Hoshino and Kenji Hiramatsu for providing the thymus samples and PBL, Dr. Jiong Fan for his technical contribution, and Dr. Jeanne F. Attrep for her assistance with the manuscript.


   Footnotes
 
1 This work was supported in part by grants-in-aid from Ministry of Health and Welfare and from Ministry of Education, Science, Sports, and Culture, Japan. Back

2 Address correspondence and reprint requests to Dr. Hitoshi Kohsaka, First Department of Internal Medicine, School of Medicine, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8519 Japan. E-mail address: Back

3 Abbreviations used in this paper: pT{alpha}, pre-T cell receptor-{alpha}; PE, phycoerythrin. Back

Received for publication August 28, 1997. Accepted for publication March 4, 1998.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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