Antigenic stimulation induces recombination activating gene 1 and terminal deoxynucleotidyl transferase expression in a murine T-cell hybridoma

Cellular Immunology 274 (2012) 19–25

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Antigenic stimulation induces recombination activating gene 1 and terminal deoxynucleotidyl transferase expression in a murine T-cell hybridoma Yuan Zhang a, Min Shi b, Qian Wen a, Wei Luo a, Zhi Yang a, Mingqian Zhou a, Li Ma a,⇑ a b

Institute of Molecular Immunology, School of Biotechnology, Southern Medical University, Guangzhou 510515, China Department of Oncology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China

a r t i c l e

i n f o

Article history: Received 11 November 2011 Accepted 28 February 2012 Available online 14 March 2012 Keywords: T cell receptor Secondary rearrangement Recombinase genes Mature T cell clone

a b s t r a c t Secondary rearrangements of the T cell receptor (TCR) represent a genetic correction mechanism which changes T cell specificity by re-activating V(D)J recombination in peripheral T cells. Murine T-cell hybridoma A1.1 was employed to investigate whether antigenic stimulation induced re-expression of recombinase genes and altered TCR Vb expression. Following repeated antigenic stimulation, A1.1 cells were induced to re-express recombination activating gene (RAG)1 and terminal deoxynucleotidyl transferase (TdT) which are generally considered prerequisite to TCR gene rearrangement. Accompanied with the significant changes in TCR mRNA levels over time, it is suggested that secondary rearrangements may be induced in A1.1 cells, which represent a mature T cell clone capable of re-expressing RAG genes and possesses the prerequisite for secondary V(D)J rearrangement. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction T cells employ antigen-specific receptors (TCR) to recognize ‘self’ or ‘non-self’ antigen presented in the context of major histocompatibility complexes (MHC) expressed on the surface of antigen presenting cells (APC). The TCR is a heterodimer composed of somatically rearranged a and b (in some cases c and d) chains [1]. The generation of a functional TCR variable region that defines the antigen-recognition specificity of respective TCRs requires the assembly of variable (V), diversity (D) and joining (J) gene segments [2] which confer each TCR with unique specificity. In mouse T lymphocyte linages, the Vb gene segments are grouped into 20 different subfamilies, including 24 members that represent various antigen recognition specificities [3]. A large number of different V, D and J recombination events containing various numbers of nucleotide insertions or deletions at junctional sites contribute to the enormous diversity of T cell repertoire, allowing T cells to respond to thousands of different antigenic peptide sequences [4]. Abbreviations: RAG, recombination activating gene; TCR, T cell receptor; TdT, terminal deoxynucleotidyl transferase; MHC, major histocompatibility complex; APCm, antigen presenting cells; V, variable region; D, diversity region; J, joining region; RSS, recombination signal sequences; NOD, non-obese diabetic; DCs, dendritic cells; HCA, hepatocellular carcinoma-associated; rmGM-CSF, recombination mouse granulocyte macrophage colonystimulating factor; rmIL-4, recombination mouse interleukin-4; TNF-a, tumor necrosis factor-a; M-MuLV, Moloney murine leukemia virus; qPCR, quantitative real-time PCR; mGAPDH, mouse glyceraldehyde-3-phosphate dehydrogenase; CDR3, complementarity-determining region 3; mAb, monoclonal antibody; bp, base pairs. ⇑ Corresponding author. Fax: +86 20 61648322. E-mail address: [email protected] (L. Ma). 0008-8749/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellimm.2012.02.008

During early stages of T cell development, V(D)J recombination is initiated by the lymphocyte-specific recombination activating gene (RAG)1 and 2 which cooperate to direct site-specific cleavage after recognizing and synapsing the target recombination signal sequences (RSS) that flank each V(D)J gene segment and composed of highly conserved heptamer and nonamer sequences separated by relatively nonconserved 12 or 23 base pair (bp) spacers (12/23 RSS) [5]. The cleaved gene segments are then joined by constitutively expressed DNA-repair enzymes, such as the terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase catalyzing nonhomologous end-joining reactions [6]. Immunological dogma states that RAG genes and TdT are permanently down-regulated following T cell maturation and migration out of the thymus [7]. However, expression of a functional ab TCR alone is not sufficient to silence RAG genes and TdT expression since re-activation of RAG genes has been reported in peripheral T cells. For example, RAG re-expression and secondary V(D)J rearrangement were induced in mature T cells when treated with anti-CD3 and IL-7 [8]. As a consequence of RAG-mediated V(D)J recombination events, these T cells modified their receptors, resulting in changes in the specificity of antigen recognition and the generation of a new TCR [9–11], thereby providing an additional mechanism for generating additional diversity among TCR genes. The exact mechanism resulting in secondary rearrangement was not clear. For example, whether there is a rule for altered specificities to the original TCR post-rearrangement, whether T cells can be forced to change its original antigen specificity to a specific direction as the relationship of lock and key, following antigenic stimulation. Studies examining the recombinase genes

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RAG and TdT re-expression and secondary rearrangement events have been primarily carried out on complex T lymphocyte populations treated by superantigen (SAg) and some cytokines [12,13]. However, few reports described secondary rearrangement events in a T cell clone following antigenic stimulation, which arises the need for more detailed analysis of the mechanisms associated with secondary rearrangements resulting in altered antigen specificities in peripheral T cells. Murine T-cell hybridoma A1.1 is a T cell clone that possesses a relatively simple TCR gene background. It displays a mature T-cell phenotype i.e., TCRa/b+, CD3+, CD4+ and expresses MHC (I-Ad) and is specific to a defined synthetic polypeptide antigen, poly-18 [14]. In this study, a protein hepatocellular carcinoma-associated antigen 59 (HCA59) [15] was employed to stimulate A1.1 cells to examine if a tumor protein can induce secondary V(D)J rearrangement and result in changes in the original antigen specificity of A1.1 cells. 2. Materials and methods 2.1. Animals and cells culture procedures Male Balb/c mice (6–8 weeks of age) were purchased from the Laboratory Animal Center of Southern Medical University (Guangzhou, China). The murine T-cell hybridoma A1.1 was kindly provided by Yufang Shi (Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ, USA) and constructed by fusing poly-18-reactive T cell blasts with the BW5147 thymoma (negative for TCR expression). Dendritic cells were prepared by isolating bone marrow cells from Balb/c mice and culturing them in complete RPMI 1640, i.e., RPMI 1640 supplemented with 10% FCS (fetal calf serum) and 2 mM L-glutamine (HyClone Laboratories, Logan, UT) in the presence of 1000 U/ml recombinant mouse granulocyte macrophage colony stimulating factor (rmGM-CSF) and 500 U/ml recombinant murine interleukin-4 (rmIL-4) (PeproTech, Rocky Hill, NJ) for 7 days. On the eighth day, DCs were pulsed with 10 ng/ml HCA59 antigen (provided by Beijing Genomics Institute, Chinese Academy of Sciences, Beijing, China) and 20 ng/ml tumor necrosis factor-a (TNF-a) (PeproTech) for 24 h. Murine splenic T cells were isolated by Ficoll–Hypaque (Shanghai Second Chemistry Factory, Shanghai, China) and maintained in complete RPMI 1640. A1.1 cells were cloned by limiting dilution in culture media containing feeder cells harvested from murine peritoneal fluid and three colonies with a probability of monoclonality above 95% were selected for further analysis. A1.1 cells (105 per well) were cultured with antigen HCA59pulsed DCs (2  104 per well) in 24-well plates and maintained in culture medium for 7 days and harvested at 24 h intervals. A1.1 cells cultured alone or with unloaded DCs were used as controls. All groups were tested in triplicate. 2.2. cDNA amplification by PCR Total RNA was extracted from 2  106 cells using a total RNA extraction kit (Omega Biotech Company, Norcross, GA) according to manufacturer’s instructions and quantified by spectrophotometry (Biophotometer Plus, Eppendorf, Hamburg, Germany). Following treatment with DNase I (MBI Fermentas, St. Leonrod, Germany), 1 lg RNA was transcribed with a cDNA synthesis kit (MBI Fermentas, St. Leonrod, Germany) according to the manufacturer’s instructions. Total RNA was incubated at 42 °C for 1 h with 250 pM oligo-(dT) primer, 200 U Moloney murine leukemia virus (M-MuLV) reverse transcriptase and 250 lM of each dNTP in 20 ll final volume. Expression analysis of mouse TCR Vb gene families was performed by semi-nested PCR as described previously [16]. Briefly, following a initial denaturing step at 95 °C for 3 min, the first round PCR was carried out at 95 °C for 1 min, 55 °C for

1.5 min and 72 °C for 2 min for 40 cycles followed by a final extension at 72 °C for 10 min. A second-round of PCR amplifications corresponding to each mouse TCR Vb gene family examined were performed with 4 ll of the first-round products as templates using conditions at 95 °C for 60 s, 60 °C for 45 s and 72 °C for 60 s for 35 cycles followed by a final extension at 72 °C for 10 min. Each PCR product was visualized on ethidium bromide-stained agarose gels [17]. Primers used for TCR Vb gene family-specific amplification were designed as described by Pannetier et al. [18], except for Cb1 and Cb2 that were described by Yoshida et al. [19]. The nomenclature for murine TCR Vb gene families recommended by Arden et al. [3] was used. Quantitative real-time PCR (qPCR) was performed in triplicate using a Mastercycler ep realplex (Eppendorf, Hamburg, Germany) and the FastStart Universal SYBR Green Master (ROX) kit (Roche Applied Science, Mannheim, Germany) to analyze expression levels of RAG1, RAG2, TdTS, TdTL [6], the TCR Vb10–Vb1 fusion, and TCR Vb1 mRNAs. All gene-expression results were expressed as arbitrary units relative to the expression of mouse glyceraldehyde-3phosphate dehydrogenase (mGAPDH) using previously described primers [20] (Table 1). 2.3. CDR3 spectratype analysis using GeneScan and sequencing Analysis of the CDR3 spectratype was performed as previously described [16]. Briefly, fluorescent PCR products were mixed with formamide and loading dye (25 mM ethylenediamine tetraacetic acid, 50 ng/ml dextran blue). After denatured at 94 °C for 2 min, 2 ll of the mixture was loaded onto pre-warmed 6% acrylamide sequencing gels and run for 2 h on a 50-lane Applied Biosystems model 373A DNA sequencer (Applied Biosystems, Foster City, CA). The data were analyzed using GeneScan software version 672 [21]. DNA sequencing of the amplified products was determined using an ABI 377 DNA sequencer. 2.4. Flow cytometry Cells were resuspended in PBS/0.1% BSA at a density of 2  106/ ml and 100 ll of cell suspension was incubated with 10 lg/ml FITC-labeled monoclonal antibody (mAb) anti-Vb1 (BL37.2) (Immunotech Beckman–Coulter, Marseille, France) in dark at 4 °C for 30 min. After several washes, the cells were resuspended in 300 ll of PBS/0.1% BSA and analyzed by FACScan (Becton Dickinson, Franklin Lakes, NJ, USA). 2.5. Western blot analysis Total cellular proteins were extracted using RIPA lysis buffer (ShangHai Biocolor BioScience Technology Company, Shanghai, China) containing PhosSTOP Phosphatase inhibitor Cocktail (Roche). Respective protein extracts were subjected to 12% (w/v) Table 1 Sequences of primer pairs used for Quantitative real-time PCR. Gene

Primer pairs

Accession Nos.

RAG1

F = 50 -TCCCAAGGAAAGTGACCGTG-30 R = 50 -AATGCAGATCCTACAGAATAGA-30 F = 50 -TACAAAGGCAGCATAGACT-30 R = 50 -CAAGTTAGCAGGGCGTAT-30 F = 50 -GAAGGCCATCCGTGTAGATC-30 R = 50 -GGTTCAATGTAGTCCAGTC-30 F = 50 -GAAGGCCATCCGTGTAGATC-30 R = 50 -GACACTTTCCCATCCAAAGGTG-30 F = 50 -AGCTGCAGGCTTCTCCTCTATGT-30 R = 50 -TGGATCCACGGCAGATATATGTAA-30 F = 50 -TTTGTGCCAGCAGCCAGATA-30 R = 50 -CTCCACGTGGTCAGGGAAGA-30

NM_009019.2

RAG2 TdTS TdTL Vb1 Vb10

NM_009020.3 AF316015.1 AF316014.1 AE000663.1 AE000663.1

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Fig. 1. Background expression of T cell receptor (TCR) Vb families in murine T-cell hybridoma A1.1. (A) PCR products of 20 TCR Vb families from mouse splenic T cells (positive control) were analyzed on a 1.5% agarose gel and visualized by ethidium bromide staining. Each TCR Vb family has a common specific band of approximate 250– 500 bp. M, DL2000 DNA marker. (B) The CDR3 spectratype of mouse splenic T cells showed approximately eight bands for each TCR Vb gene family. (C) The PCR products of 20 TCR Vb families from A1.1 cells subjected to 1.5% agarose gels. Only TCR Vb1 and Vb10 were detected. M, DL2000 DNA marker. (D) CDR3 size and fluorescence intensity analysis of Vb1 and Vb10 from A1.1 cells.

sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS– PAGE) and then transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA). Membranes were then probed with an anti-RAG1 monoclonal antibody (T-20, Santa Cruz Biotechnology, Santa Cruz, CA). Binding was visualized using an ECL Plus Kit (Amersham Biosciences, Piscataway, NJ) followed by exposure of respective blots to Kodak X-ray film. 2.6. Statistical analysis One-Way ANOVA was used to determine significant differences between the expression levels of TCR Vb1 and the Vb10–Vb1 fusion at each time point tested between groups. Least Significant Difference or Dunnett’s T3 was performed for post hoc multiple comparisons. The Kruskal–Wallis H test was used to determine significant differences in the expression levels between RAG1, TdTL and TdTS at each time point examined compared to levels observed on day 1 post stimulation. A value of P < 0.05 was considered statistically significant. SPSS statistical software version 16.0 (SPSS, Chicago, IL) was used to perform all statistical analysis. 3. Results 3.1. TCR expression analysis of the murine T-cell hybridoma A1.1 Secondary rearrangement of TCR genes was carried out by first defining background expression of TCR Vb families in the T cell hybridoma A1.1 using mouse splenic T cells as a positive control. RT-PCR assays using a panel of TCR b chain-specific oligonucleotides were performed and each TCR Vb gene family expressed by splenic cells had specific bands of approximately 250– 500 bp, demonstrating the complexity of the splenic T cell populations (Fig. 1A). These specific bands were actually comprised of sev-

eral bands, respectively, differing in length by 3 bp that could be further separated by the sequencing gels subjected to GeneScan analysis. TCR CDR3 spectratypes analyzed by GeneScan showed that in mouse splenic T cells, approximately eight bands could be seen for each TCR Vb gene family (Fig. 1B). However, a similar analysis carried out using the murine T-cell hybridoma A1.1 and three subclones identified only the Vb1 and Vb10 family members. It was noteworthy that there was a difference in the size of their PCR products i.e., the band corresponding to Vb1 was approximately 300 bp and that of Vb10 was of an unexpected 700 bp size (Fig. 1C). GeneScan analysis confirmed a single peak corresponding to TCR Vb1 and Vb10, respectively, as expected (Fig. 1D). Sequence analysis confirmed that each TCR Vb1 gene transcript corresponded to TCR Vb1–Jb2.5–Cb1 junctions according to sequence data reported by GeneBank (Accession Nos. AE000663 for Vb1 and Vb10 and AE000665 for Jb2.5 and Cb1). Surprisingly, a V–V segment fusion of unexpected size was detected in the Vb10 gene transcripts that appeared to be a Vb10–Vb1–Jb2.5–Cb1 rearrangement product. The complete nucleotide sequence of Vb10 includes a 54 bp nucleotide sequence of the 30 -termini of Vb10 variable gene segment fused to a 291 bp nucleotide sequence of the 30 -termini of the Vb1 variable gene segment. Located between them was one intron of 159 bp that contained a 23RSS followed by sequences corresponding to the entire Jb and Cb regions. However, the Vb10 and Vb1 variable gene segments were not in the same reading frame (Fig. 2). These data indicated that the murine T-cell hybridoma A1.1 stably expressed both the TCR Vb1 and a Vb10–Vb1 fusion product.

3.2. Detection of RAG and TdT in A1.1 cells following antigen stimulation The surprising identification of a V–V junction in the TCR sequence of A1.1 cells suggested that secondary rearrangement

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Fig. 2. Nucleotide and deduced amino acid sequences of TCR Vb10 transcripts from murine T-cell hybridoma A1.1. The deduced amino acid sequence is shown below the nucleotide sequence. The V–V segment fusion sequence includes a 30 -terminal partial nucleotide sequence of the Vb10 variable gene segment of about 54 bp and a partial Vb1 variable gene segment of about 291 bp with one intron of 159 bp containing a 23RSS followed by complete Jb and Cb gene region sequences.

events might occur. This observation prompted experiments designed to analyze the expression of RAG1, RAG2 and two alternatively spliced TdT transcripts (TdTS and TdTL) [6] and the incidence of secondary V(D)J rearrangements in A1.1 cells following antigenic stimulation. Cells were stimulated with the tumor antigen HCA59 in the presence of APCs. As expected, no expression of recombinases was detected in A1.1 cultured alone or with DCs (data not shown). However, RT-PCR results demonstrated that RAG1, TdTL and TdTS (but not RAG2 mRNA) could be detected in HCA59-stimulated A1.1 cells. Cloning and sequencing of PCR products matched these sequences to published data, respectively (GeneBank Accession Nos. NM_009019.2, AF316014.1, and AF316015.1). To ascertain whether HCA59 antigen treatment affected RAG1 and TdT mRNA expression over time, qPCR was performed at different time points post antigen stimulation. RAG1 mRNA was detected as early as day 1 post stimulation, reaching maximum expression levels by day 2 and dropped significantly at day 3. Quantification of RAG1 mRNA levels revealed a 2–3-fold difference in expression levels between days 2 and 3. Thereafter, expression rapidly decreased to barely detectable levels at day 4 (Fig. 3A). Western blot analysis confirmed the expression of RAG1 in the nuclear fraction of stimulated A1.1 cells (Fig. 3B). Kinetic analysis of TdTL and TdTS mRNA demonstrated that their expression levels were similar (Fig. 3C) and detectable by day 1 post stimulation with levels increasing significantly by day 4 and reaching maximal levels by day 6. By day 7, expression levels dropped to slightly above baseline. These data demonstrated that antigenic stimulation provided signals resulting in RAG1 and TdT re-expression in T cell populations residing outside the thymus. 3.3. Temporal TCR expression in A1.1 cells following HCA59 stimulation To determine whether re-expression of recombinase genes in A1.1 cells induced by antigenic stimulation resulted in altered TCR

Vb expression, TCR Vb gene usage was further analyzed by RTPCR. Unexpectedly, no new Vb gene family usage was found i.e., only the original TCR Vb1 and the Vb10–Vb1 fusion were detected. We compared the CDR3 spectratype and nucleotide sequences corresponding to Vb1 and the Vb10–Vb1 fusion identified in A1.1 cells before and after antigen-stimulation, and no changes were found (data not shown). Moreover, flow cytometry analysis showed that only Vb1 could be detected on A1.1 cell surfaces regardless of antigenic stimulation, confirming that treatment with HCA59 did not induce altered TCR Vb expression in A1.1 cells (Fig. 4A). Re-expression of recombinase RAG and TdT prompted us to explore whether TCR Vb1 or fusion Vb10–Vb1 mRNA expression was affected by HCA59 stimulation over time, quantitative PCR was performed at different time points post antigenic stimulation. To ensure that the Vb1 gene segment assayed expressed TCR Vb1– J2.5–Cb1 but not the TCR Vb10–Vb1 fusion, we designed a new Vb1 forward primer which was located within the 50 termini of Vb1 missing in the Vb10–Vb1 fusion. Interestingly, TCR mRNA levels in antigen-stimulated A1.1 cells significantly changed over time (Fig. 4). Vb10–Vb1 fusion mRNA levels increased beginning on day 4 post stimulation and these expression levels were maintained over the next 2 days (Fig. 4B) compared to the Vb1 mRNA levels that increased on day 5 post stimulation and remained elevated through day 6 (Fig. 4C). Expression levels of both Vb1 and the Vb10–Vb1 fusion mRNAs dropped to hardly detectable levels on day 7. 4. Discussion In this study, we observed an unexpected Vb10–Vb1 fusion in A1.1 cells in addition to the normal expression of TCR Vb1, as the V–V fusion has been reported in B cells and were considered to be one kind of atypical secondary rearrangements, the unexpected Vb10–Vb1 fusion in A1.1 cells suggested that secondary rearrangements were possible. Following stimulation with antigen HCA59, we found that A1.1 could be induced to re-express RAG and TdT

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Fig. 3. Analysis of RAG and TdT gene expression in T-cell hybridoma A1.1. A1.1 cells were repeatedly stimulated with HCA59 antigen presented by DCs for 7 days. (A) RNA was extracted at 24 h intervals and RAG1 mRNA expression was assayed by qPCR. Expression of RAG1 mRNA was only detected during the first 3 days post stimulation. (B) Western blot analysis of RAG1 expression in A1.1 cells 2 days post stimulation. Lane 1–3: nuclear fractions from A1.1 maintained alone, A1.1 cultured with DCs and A1.1 treated with DC-pulsed antigen HCA59, respectively. (C) Quantitative PCR analysis of TdTL and TdTS mRNA expression. The expression levels of both TdT mRNAs accumulated beginning on day 1 and peaked at day 6. Error bars represent the SD of the mean.

accompanied with significant changes to TCR mRNA levels over time following repeated antigenic stimulation. These findings confirmed that a tumor protein could induce the recombinase genes RAG and TdT re-expression which were considered prerequisite to TCR gene secondary rearrangement, the T cell clone A1.1 may have undergone secondary rearrangements even though these rearrangements may be considered ‘invalid’ since changes to surface TCR expression were not observed. Data presented this report demonstrated that the A1.1 cell line represents a mature T cell clone capable of re-expressing RAG genes and possesses the prerequisite for secondary V(D)J rearrangement. Studies focused on secondary rearrangements have been reported primarily in B cells, with few studies documenting similar events in T cells following sequence analysis of TCR genes. Through a series of different genotyping assays, previous studies demonstrated that a series of atypical secondary rearrangements could be performed to rescue B cells with nonproductive VDJ complexes from being ‘‘dead-end’’ products of the B cell developmental pathway, resulting in B cell receptors with altered specificities. These rearrangements included DH–DH, VH replacements, secondary DJH rearrangements and VH to VHDJ joining [22,23]. However, fusion of a V segment to VDJ sequences in T cells has not ever been reported.

Fig. 4. Differential TCR expression over time in murine T-cell hybridoma A1.1 following repeated antigen stimulation. (A) During antigen stimulation, A1.1 cells were examined by flow cytometry for the expression of TCR Vb1 daily. Similar results were obtained and a representative experiment shown. Only Vb1+ cells were detected. Quantitative PCR was performed to quantify TCR Vb10–Vb1 fusion (B) and Vb1 (C) mRNA levels at indicated time points. Error bars represent the SD of the mean.

The exact mechanisms resulting in the generation of aberrant V–VDJ joints observed in T cells have not been defined. Among the approximately 20 functional Vb families described for the mouse TCR-b locus, Vb10 localizes adjacent to Vb1 [24] (Fig. 5). According to the ‘‘regulated’’ model of allelic exclusion, productive Vb to DJb rearrangements on one allele prevents recombination on another TCR b allele. By contrast, an initial nonproductive VDJb

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gested that a tumor antigen can induce RAG and TdT re-expression, and may subsequently mediate secondary V(D)J rearrangement in peripheral T lymphocytes, thus provide a foundation for defining the precise mechanisms for TCR gene rearrangement. Fig. 5. Genomic organization of the murine germline TCR b locus. TCR segments are diagrammed to show the relative positions of the Vb segments in the TCR b complex according to GeneBank sequence (Accession No.: AE000663, AE000664, and AE000665). Vb10 and Vb1 are adjacent in the germline locus. Cb1 and Cb2 are constant regions. D and J segments are not shown.

rearrangement on one allele can be followed by efficient utilization of the alternate allele assembling into a complete TCR b chain variable region gene [25]. Accordingly, the non-productive Vb10– Vb1–DJ rearrangement detected in A1.1 may be followed by activating Vb1 to DJ rearrangement on the alternate allele resulting in the expression of a single TCR Vb1 product on the cell surface. Co-expression of RAG and TdT is considered prerequisite to secondary V(D)J rearrangement. RAG1 alone can bind to DNA and recognize an RSS, but displays a significantly weaker specificity and stability than association of RAG1 and RAG2 [26]. Expression of TdT has been considered an indirect marker for VDJ rearrangement during T cell development in the thymus [6]. However, in this study, RAG1 and TdT co-expression was induced in A1.1 cells following treatment with antigen, confirming that their expression could be induced in mature T cells and may subsequently mediate secondary V(D)J gene rearrangements. RAG1 expression could be detected only during the first 3 days post stimulation, consistent with its transient re-expression in response to foreign antigen stimulation demonstrated by Serra et al. [27]. However, continuous expression of TdT mRNA was observed with increased level over time during the entire stimulation period except for day 7. The different expression profiles of RAG1 and TdT suggested that their expressions are not interrelated [28] and regulated by different mechanisms. This is supported by data showing that TdT expression could be detected in RAG1-negative cells [29]. By contrast, RAG2 induction was difficult to detect, probably due to its extremely low expression levels in A1.1 cells. The lack of alteration to TCR expression or to the TCR sequences observed in A1.1 cells after antigenic stimulation indicated that T cell specificity had not been affected. However, expression levels of the Vb1 and the Vb10–Vb1 fusion mRNA were found to change significantly over time, suggesting that A1.1 cells may have been induced to undergo RAG1- and TdT-mediated secondary rearrangements. The reason for the failure in inducing alterations to TCR Vb gene usage in A1.1 cells by secondary rearrangement events may be due to the absence of RAG2 expression that limited secondary rearrangements to the TCR. RAG expression is a critical and tightly controlled progress that occurs during T cell development and it has been demonstrated that complete V(D)J recombination requires the cooperation of both RAG1 and RAG2 [5]. RAG1 or RAG2 mRNA, but rarely both, were detected in mature T cells undergoing secondary rearrangement [27], mirroring their complex regulation at the transcriptional level [30]. Therefore, absence of RAG2 expression in A1.1 cells suggested that poor recombinase activity was likely since only RAG1 could be detected and whose presence alone was insufficient to mediate efficient secondary rearrangement events with the potential of resulting in changes to TCR Vb gene usage. Alternatively, co-expression of RAG1 and RAG2 may have occurred in a few cells that underwent secondary rearrangement resulting in altered TCR Vb expression. However, neither the RAG2 nor the alterations to TCR Vb gene usage was detectable with our detection methods. Although mechanisms in the atypical V–VDJ rearrangements and the possibility of alteration in TCR specificities in A1.1 cells induced by other antigens remain to be elucidated, our results sug-

Conflict of interest statement The authors alone are responsible for the content and writing of the paper. The authors reported no potential conflicts of interest. Acknowledgments This work was supported financially by the National Natural Science Foundation of China (30972680 and 81171539), Natural Science Foundation of Guangdong Province (S2011020003154), and The State Key Laboratory for Molecular Virology and Genetic Engineering (2011KF03). We thank Yufang Shi for kindly providing the murine T-cell hybridoma A1.1. References [1] M.M. Davis, P.J. Bjorkman, T-cell antigen receptor genes and T-cell recognition, Nature 334 (6181) (1988) 395–402. [2] G.M. Vaitaitis, M. Poulin, R.J. Sanderson, K. Haskins, D.H. Wagner Jr, Cutting edge: CD40-induced expression of recombination activating gene (RAG) 1 and RAG2: a mechanism for the generation of autoaggressive T cells in the periphery, J. Immunol. 170 (7) (2003) 3455–3459. [3] B. Arden, S.P. Clark, D. Kabelitz, T.W. Mak, Mouse T-cell receptor variable gene segment families, Immunogenetics 42 (6) (1995) 501–530. [4] A. Jackson, H.D. Kondilis, B. Khor, B.P. Sleckman, M.S. Krangel, Regulation of T cell receptor beta allelic exclusion at a level beyond accessibility, Nat. Immunol. 6 (2) (2005) 189–197. [5] J.D. Curry, M.S. Schlissel, RAG2’s non-core domain contributes to the ordered regulation of V(D)J recombination, Nucleic Acids Res. 36 (18) (2008) 5750– 5762. [6] L.A. Bentolila, M. Fanton d’Andon, Q.T. Nguyen, O. Martinez, F. Rougeon, N. Doyen, The two isoforms of mouse terminal deoxynucleotidyl transferase differ in both the ability to add N regions and subcellular localization, EMBO J. 14 (17) (1995) 4221–4229. [7] P.J. Fink, C.J. McMahan, Lymphocytes rearrange, edit and revise their antigen receptors to be useful yet safe, Immunol. Today 21 (11) (2000) 561–566. [8] E. Lantelme, L. Orlando, P. Porcedda, V. Turinetto, M. De Marchi, A. Amoroso, S. Mantovani, C. Giachino, An in vitro model of T cell receptor revision in mature human CD8+ T cells, Mol. Immunol. 45 (2) (2008) 328–337. [9] M.A. McGargill, J.M. Derbinski, K.A. Hogquist, Receptor editing in developing T cells, Nat. Immunol. 4 (2000) 336–341. [10] D. Mayerova, K.A. Hogquist, Central tolerance to self-antigen expressed by cortical epithelial cells, J. Immunol. 172 (2) (2004) 851–856. [11] J. Sprent, H. Kishimoto, The thymus and negative selection, Immunol. Rev. 185 (2002) 126–135. [12] C.Y. Huang, R. Golub, G.E. Wu, O. Kanagawa, Superantigen-induced TCR alpha locus secondary rearrangement: role in tolerance induction, J. Immunol. 168 (7) (2002) 3259–3265. [13] J. Huang, S.K. Durum, K. Muegge, Cutting edge: histone acetylation and recombination at the TCR gamma locus follows IL-7 induction, J. Immunol. 167 (11) (2001) 6073–6077. [14] R. Bissonnette, H.G. Zheng, R.T. Kubo, B. Singh, D.R. Green, A T helper cell hybridoma produces an antigen-specific regulatory activity, Relationship to the T cell receptor by serology and antigenic fine specificity, J. Immunol. 146 (9) (1991) 2898–2907. [15] Y. Wang, K.J. Han, X.W. Pang, H.A. Vaughan, W. Qu, X.Y. Dong, J.R. Peng, H.T. Zhao, J.A. Rui, X.S. Leng, J. Cebon, A.W. Burgess, W.F. Chen, Large scale identification of human hepatocellular carcinoma-associated antigens by autoantibodies, J. Immunol. 169 (2) (2002) 1102–1109. [16] W. Luo, L. Ma, Q. Wen, N. Wang, M.Q. Zhou, X.N. Wang, Analysis of the interindividual conservation of T cell receptor alpha- and beta-chain variable regions gene in the peripheral blood of patients with systemic lupus erythematosus, Clin. Exp. Immunol. 154 (3) (2008) 316–324. [17] X.S. Yao, G.W. Zhang, L. Ma, Q. Wen, J.L. Hou, M.J. Meng, S.W. Ma, M.F. Liang, Y. Lin, Z.Q. Wu, X.W. He, J.F. Wang, X.N. Wang, Analysis of the CDR3 length of TCR ab T cells in the peripheral blood of patients with chronic hepatitis B, Hepatol. Res. 35 (1) (2006) 10–18. [18] C. Pannetier, M. Cochet, S. Darche, A. Casrouge, M. Zöller, P. Kourilsky, The sizes of the CDR3 hypervariable regions of the murine T-cell receptor b chains vary as a function of the recombined germ-line segments, Proc. Natl. Acad. Sci. USA 90 (9) (1993) 4319–4323. [19] R. Yoshida, T. Yoshioka, S. Yamane, T. Matsutani, T. Toyosaki-Maeda, Y. Tsuruta, R. Suzuki, A new method for quantitative analysis of the mouse T-cell

Y. Zhang et al. / Cellular Immunology 274 (2012) 19–25

[20] [21]

[22]

[23]

[24]

receptor V region repertoires: comparison of repertoires among strains, Immunogenetics 52 (1–2) (2000) 35–45. S.K. Dessain, H. Yu, R.R. Reddel, R.L. Beijersbergen, R.A. Weinberg, Methylation of the human telomerase gene CpG island, Cancer Res. 60 (3) (2000) 537–541. C. Assaf, M. Hummel, E. Dippel, S. Goerdt, H.H. Müller, I. Anagnostopoulos, C.E. Orfanos, H. Stein, High detection rate of T-cell receptor beta chain rearrangements in T-cell lymphoproliferations by family specific polymerase chain reaction in combination with the GeneScan technique and DNA sequencing, Blood 96 (2) (2000) 640–646. M. Reth, P. Gehrmann, E. Petrac, P. Wiese, A novel VH to VHDJH joining mechanism in heavy-chain-negative(null) pre-B cells results in heavy-chain production, Nature 322 (28) (1986) 840–842. E.T. Luning Prak, M. Monestier, R.A. Eisenberg, B cell receptor editing in tolerance and autoimmunity, Ann. N. Y. Acad. Sci. 1217 (2011) 96– 121. F. Chen, L. Rowen, L. Hood, E.V. Rothenberg, Differential transcriptional regulation of individual TCR Vb segments before gene rearrangement, J. Immunol. 166 (3) (2001) 1771–1780.

25

[25] B. Khor, B.P. Sleckman, Intra- and inter-allelic ordering of T cell receptor b chain gene assembly, Eur. J. Immunol. 35 (3) (2005) 964–970. [26] Y. Akamatsu, M.A. Oettinger, Distinct roles of RAG1 and RAG2 in binding the V(D)J recombination signal sequences, Mol. Cell. Biol. 18 (8) (1998) 4670– 4678. [27] P. Serra, A. Amrani, B. Han, J. Yamanouchi, S.J. Thiessen, P. Santamaria, RAGdependent peripheral T cell receptor diversification in CD8+ T lymphocytes, Proc. Natl. Acad. Sci. USA 99 (24) (2002) 15566–15571. [28] Z. Zhang, X. Wu, B.H. Limbaugh, S.L. Bridges Jr, Expression of recombinationactivating genes and terminal deoxynucleotidyl transferase and secondary rearrangement of immunoglobulin kappa light chains in rheumatoid arthritis synovial tissue, Arthritis Rheum. 44 (10) (2001) 2275–2284. [29] T. Umiel, P. Pattengale, K. Weinberg, Recombination activating gene-1 (RAG-1) expression in all differentiation stages of B-lineage precursor acute lymphoblastic leukemia, Leukemia 7 (3) (1993) (140–435). [30] N. Yannoutsos, V. Barreto, Z. Misulovin, A cis element in the recombination activating gene locus regulates gene expression by counteracting a distant silencer, Nat. Immunol. 5 (4) (2004) 443–450.