Vα gene replacement in a TCRα knock-in mouse

0014-2980/01/1010-2919$17.50+.50/0© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001

V > gene replacement in a TCR > knock-in mouse

Rachel Golub1, 2, Ching-Yu Huang3, Osami Kanagawa3 and Gillian E. Wu1

1 Departments of Immunology and Medical Biophysics, University of Toronto, Toronto, Canada2 Institut Pasteur, Unite du developpement des lymphocytes, Paris, France3 Center for Immunology, Department of Pathology and Medicine, Washington University School

of Medicine, St. Louis, USA

Using a TCR § chain knock-in mouse, we demonstrate that V-gene replacement can operatein the T cell receptor § locus. Functional TCR § chain transcripts generated by V § -genereplacement at the site of the V § -embedded heptamer were identified in splenic T cells. Thisfinding shows that V § -gene replacement can likely be used to shape the peripheral T cellrepertoire. The conservation of the embedded heptamer in most V § segments adds supportto the notion that V-gene replacement is a mechanism maintained to diversify the immunesystem and that argues that it is common to B and T cells.

Key words: T cell receptor / Repertoire development / V gene replacement / Generation of diver-sity / Transgenic / Knockout

Received 8/3/01Revised 25/6/01Accepted 17/7/01

[I 21829]

Abbreviations: KI: Knocked in RAG: Recombination acti-vating genes LM-PCR: Ligation-mediated-PCR DP: Dou-ble positive RSS: Recombination signal sequences

1 Introduction

In developing T and B cells, a series of ordered generearrangements, termed V(D)J recombination, generatesa repertoire of TCR and BCR with vast specificities (e. g.[1]). Although most aspects of V(D)J recombination arecommon to T and B cells [2], mechanisms that generatefurther diversity differ. Differences include: TCR recog-nize antigenic peptides presented by MHC moleculeswhereas immunoglobulins (Ig) bind antigen directly[3–6]. There is little evidence for affinity maturation in Tcells in the periphery [7–10], whereas such events arecommon in B cells. T cells can have more than one dis-tinct TCR on its surface – the TCR § locus can continueto rearrange after a functional § chain has been formed –whereas mature B cells with more than one receptorspecificity have not been detected in normal mice [11,12], or rarely [13].

V-gene replacement can diversify the Ig heavy chaingenes after the formation of a VDJH unit [14–20]. It con-tributes to the BCR repertoire by replacing, via a V(D)J-type recombination, an already rearranged VH segmentwith an upstream germline VH gene segment. The recom-bination is mediated by the embedded heptamerencoded by the rearranged VH gene and the RSS of theincoming germline VH gene [21]. When this event resultsin the formation of a new in-frame VDJH rearrangement,it (most likely) creates a new BCR specificity. The

embedded heptamer is located at the 3' end of most Vgene segments. In its orientation shown in Fig. 1, theembedded heptamer has the GTG motif required forV(D)J recombination, and usually at least two other of theheptamer consensus nucleotides (CACTGTG) [22]. TheGTG motif is strikingly conserved in this positionthroughout the Ig superfamily members. The GTG motifis present in more than 80% of the mouse and human VH

genes, and in all V Q segments of the mouse, chicken andrabbit [22, 23]. It is similarly conserved in the TCR lociwith ˚ 90% of V § segments and ˚ 95% of V + segmentsencoding an embedded heptamer [23]. This conserva-tion suggested to us that it could be used for V-genereplacement in V § (Fig. 1) and V g segments, and hencefor generating additional TCR diversity.

To investigate whether V § -gene replacement occurs, wetook an approach that has revealed its occurrence inthe B lineage; namely the use of mice with targeted in-sertions of antigen receptors [17, 20]. Here, we used amouse with a knocked-in (KI) functionally rearrangedTCR § (V § 2B4J § 47) from a cytochrome c specific T cellhybridoma 2B4 [24]. We previously reported that newTCR specificities can be generated in this strain, usuallyby a leap-frog type of rearrangement whereby a germlineV § joins to a germline J § , deleting the knocked-in rear-rangement [24, 25]. Here, we examined the joining of agermline V § to the inserted rearranged V § J § . Using 5'-RACE PCR, we identified V § J § replacement rearrange-ments in splenic T cells. Using LM-PCR, we identified V §signal ends cleaved at the embedded heptamer in thethymus, but failed to identify them in the periphery.These results demonstrate that V § -gene replacementcan be used to diversify the thymic TCR repertoire andcan be maintained in the periphery.

Eur. J. Immunol. 2001. 31: 2919–2925 Mouse V § gene replacement 2919

Fig. 1. (A) Diagram of the construction of the V § 2B4J § 47knock-in segment in the “KI” mouse. (B) Diagram of embed-ded heptamer V § -gene replacement. Signal- and coding-end intermediates are generated by RAG mediated V(D)Jrecombination using the embedded heptamer of theknocked-in V § 2B4 and the RSS of an incoming germline V §gene segment. The resolution of the V § and J § coding endsgenerates a VJ § gene-replaced structure with a new V § andthe old 2B4J § 47 segment. It would be usual to observe pro-cessing in the new V § and J § coding joint.

2 Results

2.1 A role for V > -gene replacement in shapingthe mature TCR repertoire

We reasoned that if V § replacement were used to diver-sify the TCR repertoire, replaced V § J § rearrangementsshould be present in the periphery. As we had found thata leap-frog type of rearrangement product was commonin the KI V § 2B4J § 47 allele [24], we devised an assay thatwould detect transcripts containing the 2B4 J § , but lack-ing the 2B4 V § , and hence likely to be V § -gene replace-ments: cDNA were generated using a 5’ anchor and a3’ primer complementary to the C § region to enrich forTCR § chain RNA. Because the strong expression of theKI transgene precluded the use of the same anchor asthe 5’ primer for the following RT-PCR, three V § familyspecific primers (V § 8, V § 20, V § Y) were used for the 5’forward primers, with a 2B4J § 47 primer for the 3’ backprimer (see Sect. 4). For the KI/KI mice, we cloned andsequenced over 60 cDNA and found six V § -replacedrearrangements (Fig. 2) (the other clones were thesequence of the KI V § 2B4J § 47 cDNA). Three of the

six V § -replacements possessed N-nucleotides at thereplacement junction, one sequence (# 4, Fig. 2) was iso-lated twice. There was a striking similarity in the process-ing of the J § border of five of the clones, resulting in ageneral maintainance of the CDR3 region length. A simi-lar restriction has been observed for other TCR § chainsspecific for cytochrome c [26]. Clone # 4 lacks the con-served YXC motif. This absence due to a single nucleo-tide change may be a PCR error. The terminal three cod-ing nucleotides of the incoming V § gene in clones 1 and3 are GTG. GTG are the three nucleotides of the RSSheptamer required for V(D)J recombination, and theirpresence in this position, abutting the CAC of the V §RSS, is intriguing.

For the KI/wt mice, we analyzed 50 cDNA sequencesand found three V § -replaced, in frame, rearrangements.Although we could not rule out the possibility that thesethree clones came from the wild type allelic locus, wethink it unlikely because the V § -J § border and the result-ing CDR3 regions are identical to the V § 2B4J § 47 hybrid-oma. In all cases the CDR3 length is remarkably main-tained, perhaps reflecting structural constraints of part-nering the pre-selected TCR g chain.

2.2 V > gene replacement intermediates arepresent in CD4+CD3lo thymocytes

Finding evidence of V § -gene replacement in the periph-ery with restricted CD3 structure led us to examine thy-mocytes and lymph node T cells of the KI/KI mice todetermine where and when the replacement events weretaking place. CD4+/CD3lo and CD4+/CD3hi thymocytepopulations and CD4+/CD3hi and CD4–/CD3hi lymphnode populations were isolated by flow cytometry sort-ing. Sorted cells were lysed, the ligation-mediated (LM)-PCR assay performed to detect signal-end recombina-tion intermediates (Fig. 3) and the products analyzed bySouthern blotting, cloning and sequencing. After optimi-zation, three independent LM-PCR assays were per-formed on each sorted sample. Bands could be visual-ized after one round of LM-PCR, but in order to clone theproducts, a second round with one nested primer had tobe used. Since we expected that these products wouldbe difficult to detect – they are intermediates in thecleavage stage of V(D)J recombination and hence areends of cleaved chromosomes (Fig. 1) – we used condi-tions that generated multiple bands on the first round(relinquishing specificity for quantity), but a specific bandon the second [20, 27]. Sequencing the products allowedus to determine whether cleavage occurred at theembedded heptamer and, thus, was RAG mediated. Ascan be seen in Fig. 3, such cleavage products were diffi-cult to identify and only by sequencing all products were

2920 R. Golub et al. Eur. J. Immunol. 2001. 31: 2919–2925

Fig. 2. Nucleotide sequence of V § 2B4J § 47 replacements. Shown are the sequences of clones amplified by 5' RACE-PCR fromsplenocytes of KI/KI (samples 1–4 and 8) and KI/wt (samples 28, 34, 36) mice. Sequences shown contain, and flank, the CDR3region as marked. The first row is the V § 2B4J § 47 sequence. Based on a GenBank homology search [42], each incoming V § genesegment was attributed to a V § family indicated at the left of the sequence after the reference of the clone (#). The sign (+) indi-cates an in-frame rearrangement and the sign (−), an out-of-frame rearrangement. Clones 8, 28, and 34 have “unprocessed”2B4J § 47 coding ends in that the sequences 3’ of the V § 2B4 embedded heptamer remain. The other clones have processing atboth coding ends.

Fig. 3. (A). LM-PCR strategy. The signal-end intermediates are amplified by nested PCR with 5' primers specific for V § 2B4, V § 1and V § 2 and a 3' primer, specific for the BW linker BW1HR (14). (B). Analysis of V § -replacement intermediates by the LM-PCRassay in thymocytes (THY) and lymph nodes (LN). Ethidium bromide stained agarose gel of V § -replacement intermediates fromsorted populations from the thymus: CD4+/CD3hi (lane 1) and CD4+/CD3lo (lane 2) and from sorted populations from the lymphnodes: CD4+/CD3hi (lane 3) and CD4–/CD3hi (lane 4). The Southern blot was probed with V § 3 (5'-GAGCGCTACAGC-ACCCTGCACA-3'), a primer specific for V § 2B4 segments. The amplification products of 233 bp corresponds to the V § 2B4 seg-ment cleaved at the embedded heptamer. (C). Signal-end intermediate sequences obtained from the LM-PCR assay in the differ-ent subset of cells are detailed. Only the 233 bp band in lane 2 was V § 2B4 sequences cleaved at the embedded heptamer.

we able to identify products specifically cleaved at theembedded heptamer. In KI/KI thymocytes, only theCD4+/CD3lo cells contained V § 2B4 segments cleaved atthe embedded heptamer (Fig. 3). CD4+/CD3lo thymo-cytes are at the DP stage which is characterized by sev-eral major differentiation events including successiveTCR § gene rearrangements, positive selection, negativeselection and transition to the single positive stage [3, 4,28]. Absence of V § -gene replacement intermediates inthe CD3hi thymocytes correlates with the down-regulation of RAG proteins after positive selection[29–31]. In KI/KI lymph node cells, we were unable to

detect any intermediates of V § -gene replacement ineither the CD4+/CD3hi or in the CD4–/CD3hi populations(Fig. 3).

2.3 Positive selection of T cells does not preventV > gene replacement in the thymus

The finding that V § gene placement initiated in the thy-mocytes suggested to us that it occurred simply as aresult of RAG expression concurrent with accessible V §alleles [32–34]. Examination of V § replacement in a thy-


Fig. 4. FACS Analysis of thymocytes from KI TCR § /TCR g(kxb) and KI/KI mice. CD4+/CD8+/TCRhi (R1) and CD4+/CD8+/TCRlo (R2) populations were isolated by sorting. Sam-ples from R1 and R2 were then reanalyzed with the samemachine to inspect the purity of the TCRhi and TCRlo popula-tions.

Fig. 5. (A) Analysis of V § -replacement intermediates by LM-PCR in thymocytes of KI/KI (1) and KI TCR § /TCR g (kxb) (2) mice atdifferent stages of T cell development. Ethidium bromide stained agarose gel of V § amplification products. For both strains, twopopulations were analyzed: CD4+/CD8+/TCRlo and CD4+/CD8+/TCRhi. Below is the Southern blot of the gel probed with V § 3. (B)Description of sequences obtained from the LM-PCR assay of the CD4–/CD8–/TCRlo population.

mic environment that positively selects for the KI rear-rangement, allowed us to investigate this hypothesis. Toexamine the effect of a positively selecting MHC, weused mice referred to as KI/2B4 TCR g (kxb) which were

derived by crossing the KI/wt mice (H-2b) to the 2B4TCR g chain transgenic mice (H-2k) [24]. Thymocytesfrom KI/KI mice and KI/2B4 TCR g (kxb) mice weresorted in parallel according to the level of surfaceexpression of the TCR complex (Fig. 4) and LM-PCRassays performed (Fig. 5). As before, three independentassays were performed on each sorted sample. Theamplification products obtained were similar in the neu-tral and positively selecting backgrounds. V § 2B4 signal-end intermediates cleaved at the embedded heptamerwere found in both strains, and only in the TCRlo popula-tion. Previously, we had found that the frequency of dele-tion of the KI gene by leap-frog replacement was greatlyreduced in the positively selecting thymus with 30% ofDP thymocytes being rescued from deletion [24]. Thefinding that a positively selecting MHC background hasno demonstrable effect on the presence of V § -genereplacement intermediates supports the concept thatTCR gene assembly, including replacement, can occuras long as RAG is present. Thus, the V § gene replace-ment intermediates we observe in DP CD3lo thymocytescould be a consequence of on-going V § gene rearrange-ments as seen by the absence of an effect of positiveselection. When some event, such as selection, takesplace, the cell progresses to a more mature stage whereRAG is not expressed and gene assembly, includingreplacement, ceases.

3 Discussion

In summary, the data reveal, for the first time in T cells,that V-gene replacement can occur at the § chain geneof a TCR § g . The finding of transcripts for the § chainresulting from V § -replacement in the splenocytes of KI/KI and KI/wt mice indicates that this mechanism can beused to shape the peripheral T cell repertoire. As in Bcells, V-gene replacement in T cells generated different


receptor diversities. The absence of V § LM-PCR inter-mediates in these same tissues argues that the replace-ment took place elsewhere, likely in the thymus duringthymocyte development.

Products that were not V § -replacement intermediateswere identified in all LM-PCR assays (Figs. 3, 5). Theseamplification products were products from the V § 11family (of which V § 2B4 is a member) cleaved at variouspositions including the recombination signal sequences’(RSS) heptamer. As only live cells were used in the assay,these DNA degradation products were likely generatedduring the cell sorting stress or other manipulations. Butbecause they are not cleaved specifically at the embed-ded heptamer, it is very unlikely that they could beassembled into functional VJ § gene segments and soare, by definition, not V § -replacement intermediates. Thequantity and quality of the “nonspecific” PCR bandsyielded no clue as to their significance.

The absence of detectable intermediates of V § -genereplacement in KI/KI lymph node cells is unlike otherresults examining secondary V(D)J rearrangements atthe TCR g locus [35, 36]. Using a mouse transgenic forthe g chain, McMahan and Fink found secondary g chainrearrangements in the CD4+ peripheral population [35].Lantelme and colleagues, examining CD4+CD3lo acti-vated T lymphocytes from human peripheral blood,found both RAG transcripts and TCR g recombinationintermediates [36]. Our inability to detect § locus embed-ded heptamer V-gene replacements in the periphery maybe, in part, because of the predominance of the com-mon, V § J § leap-frog secondary replacement rearrange-ments that delete the knocked in TCR § gene, excludingV § 2B4 embedded heptamer-mediated replacements.Our assay, which detects intermediates, may not be sen-sitive enough to detect rare products, if they were there.In addition, our analysis examined “conventional”peripheral T cells in non-immunized mice. Immunizationor other stimulation may enhance RAG re-expression,leading to an enhanced ability to undergo V § -genereplacement. Alternatively, there may be fundamentaldifferences in the regulation of secondary rearrange-ments for the TCR § and g chains [25, 37, 38].

It is not possible yet to identify a replaced VJ § in normalmice as no identifying nucleotides remain, and no tech-niques are sensitive enough to identify replacementintermediates in the diverse V § locus. Thus the fre-quency of replacement in unmanipulated mice remains aconjecture. Evidence that the frequency is at least30–40-fold less than secondary V § J § rearrangementcomes from our examination of 38 individual hybridomasfrom the KI mouse which were all negative for the surfaceexpression of the KI/TCR § chain. All 38 had undergone

V § J § leap-frog rearrangement to create their new TCR §chain (unpublished). In the KI mouse, T cells attemptingto generate new TCR § chains have more than 40 J § seg-ments they can use sequentially for secondary rear-rangements and may carry on until they get positivelyselected. In contrast, there is only one V § J § target for V §replacement. This difference in target number coupledwith the “poorer” RSS of the embedded heptamer wouldbe expected to result in fewer replacements than V § J §leap-frog events [22].

The conservation of the embedded heptamer in the V gsegments suggests that V g gene replacement could alsobe an additional mechanism whereby the g chain of theTCR § g is diversified. In the V § and V g segments, no realconsensus nonamer could be identified. However, it iswell established that contrary to the heptamer, the nona-mer is not absolutely required in each RSS for V(D)Jrecombination [39, 40]. The conservation of an embed-ded heptamer at the end of most V gene segments is notrandom. In the GTG motif, GT are position two and threeof the codon for Cys present in the g F strand. The con-servation of this Cys in Ig superfamily proteins under-scores the importance of this residue in the proper fold-ing of the Ig domain. However, the codon usage for Cysis biased at this precise position in V segments: ˚ 80%of these Cys residues are encoded by TGT [22] althoughthe codon usage of Cys in the mouse genome is aboutequal for TGT and TGC [41]. The conservation of the lastG of the GTG motif is not linked to the folding of theimmunoglobulin domain as different amino acids can befound in this position. Conservation of this GTG impliesthat the mechanism of V-gene replacement has beensubjected to a strong selective pressure to be conservedin T cells as well as in B cells.

4 Materials and methods

4.1 Mice

Mice referred to as KI/KI are homologous at the TCR § chainlocus where a rearranged V § 2B4J § 47 segment is insertedinto the 5' region of the J § locus (described in [24]). The 2B4segment derives from a cytochrome c specific T cell hybrid-oma. KI/wt mice are heterologous at the TCR § chain locus.Mice referred to as KI/2B4 TCR g (kxb) were obtained bycrossing the KI/wt mice (H-2b) to the 2B4 TCR g chain trans-genic mice (H-2k).

4.2 Preparation of cells and cell sorting

Thymocytes and lymph node cells were isolated from 4–8-week old mice. Cells were stained with PE-conjugated anti-CD3, and FITC conjugated anti-CD4. The populations ana-


lyzed in the lymph nodes were CD4+/CD3hi and CD4–/CD3hi.For the thymus, the populations analyzed were CD4+/CD3lo

and CD4+/CD3hi. For the analyses investigating the effect ofa positive selecting mouse background, thymocytes werestained with a combination of three antibodies: FITC-conjugated anti-CD4, FITC-conjugated anti-CD8, and PE-conjugated anti-TCR. Two populations were analyzed for theKI/KI and the KI TCR § /TCR g (kxb) mice: CD4+/CD8+/TCRlo

and CD4+/CD8+/TCRhi. Sorted samples were reanalyzedwith the same machine to evaluate purity. Populations ofstained cells were sorted with a FACStarPlus equipped withTurbo sort using Lyses2 software (Becton Dickinson).

4.3 RT-PCR

RNA was purified with Tri-Reagent (Molecular ResearchCenter, OH). Anchored cDNA were synthesized using a 3'C § primer. PCR was performed using 5' primers for V § fami-lies: V § 20 (5'-GATCCTGAATTGTGAYTACGAGAATGAT-3'),V § 8 (5'-CCTGTGATGCTGAACTGCACCTATCAG-3'), V § Y(5'-TCTYTCAACTGCACTTCAAGTGATCGT-3'), and a 3'primer specific for 2B4J § 47 (5'-GTATAACACTCAGAA-CGGTTCCTTGA-3'). The V § primers were chosen to be in aregion far enough from the embedded heptamer for easyamplification and detection by agarose gel electrophoresis.We aligned the sequences of V § genes from GenBank anddesigned the primers by inspection. Our criteria were oligo-mers that would less commonly amplify the inserted V § , andamplify other V § genes from different V § families. They alsohad to work with our J back primer, and give single bandswhen the conditions were optimized. The final set of primerswe used were named V § 20, V § 8, and V § Y. While notexhaustive, we reasoned that finding replacement with thesethree would indicate whether replacement occurred or not.cDNA amplifications were done twice on RNA from each oftwo mice of each genotype, on different days. The RT-PCRconditions were: 30 cycles of 30 s at 95 °C, 30 s at (55 °C for10 cycles, 54 °C for 10 cycles, and 53 °C for 10 cycles),1 min at 72 °C.

4.4 Ligation-mediated PCR

DNA from cell lysates (1.5 ? g) was ligated to the BW linker(0.2 ? M final) with 2 units T4 ligase. The first round of PCRwas performed with 300–400 ng ligated DNA. A TouchDown PCR program was used: 5 cycles of 30 s at 94 °C,30 s at 58 °C, and 1 min at 72 °C, then 10 cycles in which theannealing temperature was 57 °C and another 15 cycles at55 °C. The second round of PCR was done under the sameconditions with 1 ? l of 1/50 dilution of the first PCR and 8 ngeach of BW and nested V § 2 primer. Primers: V § 1 (5'-GGAGATCAGGTGGAGCAGAGTC-3'), V § 2 (5'-GACCGGT-TCTGCTCTGAGATG-3'), BW1HR (5'-CCGGGAGATCTGA-ATTCGTG-3').

4.5 Analysis and cloning of PCR products

Southern blots were probed with V § 3 (5'-GAGCGCTACAGCACCCTGCACA-3'), specific for V § 2B4segments. The PCR products were cloned using the TA-cloning method (Clontech, CA), and sequenced in bothdirections using the T7 sequencing kit (Pharmacia, CA).NCBI BLAST was used to identify gene sequences [42].

Acknowledgements: We are grateful to Ana Cumano,Antonio Bandeira, Alexandre Hassanin and Vasco Barretofor helpful critical comments. We thank Queenie Lam andStacy Hirano for excellent technical assistance. This workwas supported by the Medical Research Council of Canada(MRC), the Terry Fox Marathon of Hope, the Leukemia Soci-ety of Canada, the Cancer Research Society of Canada, andthe National Institutes of Health (NIH).

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Correspondence: Gillian E. Wu, Ontario Cancer Institute,610 University Ave, Room 8-113, Toronto, Ontario, CanadaM5G 2M9Fax: +1 416-946-2086e-mail: gillian.wu — utoronto.ca


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Vα gene replacement in a TCRα knock-in mouse