The Role of Short Homology Repeats and Tdt in Generation of the Invariant Y6 Antigen Receptor Repertoire in the Fetal Thymus

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Immunity, Vol. 3, 439-447, October, 1995, Copyright 0 1995 by Cell Press The Role of Short Homology Repeats and TdT in Generation of the Invariant y6 Antigen Receptor Repertoire in the Fetal Thymus

Yi Zhang,’ Dragana Cado,’ David M. Asarnow,t In the epithelia of the skin and reproductive tract of mice Toshihisa Komori,* Frederick W. Alt,S there are unique populations of y6 T cells with very re- David H. Raulet,’ and James P. Allison’ stricted TCR repertoire (Asarnow et al., 1988, 1989; Ito- ‘Department of Molecular and Cellular Biology hara et al., 1990; Nandi and Allison, 1991). In the skin, and Cancer Research Laboratory essentially all the T cells express y chains encoded by University of California, Berkeley Vy3-Jyl segments, while those of the reproductive tract Berkeley, California 94720 are encoded by Vy4-Jyl segments. In both cases, the tBerlex, Incorporated junctions are characterized by minimal diversity, and the 15049 San Pablo Avenue absence of nongermline-encoded nucleotides. In these Richmond, California 94804 tissues there is a single and identical predominant 6 chain *Howard Hughes Medical Institute encoded by the VSl-DSP-JS2 segments. In all mouse Children’s Hospital strains examined to date, greater than 90% of the produc- Department of Genetics tively rearranged y and 6 genes in these tissues have the Harvard University Medical School same canonical junctions. These observations raise im- Boston, Massachusetts 02115 portant questions concerning the function and origin of these essentially monomorphic TCR. It has been suggested that these epithelial T cells perform a unique form Summary of immunological surveillance for damage-induced self- antigens (Allison and Havran, 1991; Raulet et al., 1991). Fetal thymic and adult epithelial Vy3 + and Vy4 + T cells The concept of trauma signal surveillance has gained sup- express yS antigen receptors (TCR) with invariant junc- port with the demonstration that skin-derived Vy3+ T cells tions lacking N nucleotides. Using transgenic recombi- can recognize heat-stressed keratinocytes (Havran et al., nation substrates, we show that di- or trinucleotide 1991). repeats, either in the coding region or in P elements, The precursors of epithelial Vy3’ and Vy4+ T cells are have strong effects on the site of recombination. In produced in a series of overlapping waves during fetal other mice bearing a terminal deoxynucleotidyl trans- thymic development (Havran and Allison, 1988; Allison ferase (TdT) transgene under the control of the CD2 and Havran, 1991). Moreover, Vy3’ cells are produced promoter, we found that the frequency of canonical only during fetal development in a process that require junctions was markedly reduced with a concomitant both fetal stem cells and fetal thymic microenvironment increase in in-frame noncanonical junctions with N nu- (Havran and Allison, 1990; lkutaet al., 1990). The potential cleotides. Together, our results show that short ho- TCR repertoires of these fetal precursors is constrained mology repeats direct the site of rearrangement and by the restricted expression and ordered rearrangement thus play a critical role in the generation of y6 T cell of V genes during development (Goldman et al., 1993), receptor canonical junctions. Increased TdT activity as well as by the absence of the diversifying effect of the in Vy3+ T cells has a inhibitory effect on junctional addition of random nucleotides at the junction by terminal homogeneity in these cells. deoxynucleotidyl transferase (TdT) (Landau et al., 1987; Rothenberg and Triglia, 1983). Despite these constraints, there still exists a considerable potential for diversification Introduction either by recombination at different sites or by exonucleo- lytic processing of the coding ends. Diversity in immunoglobulins and T cell antigen receptors It has been suggested that the canonical repertoire is (TCR) is generated through V(D)J rearrangement. The use generated by the effects of cellular selection (Itohara and of different germline gene segments, the imprecise joining Tonegawa, 1990). However, two recent reports suggested of gene segments, the addition of nongermline-encoded that cellular selection may not be required for the formation nucleotides, and the combinatorial association of the pro- of the invariant yS repertoires in Vy3+ and Vy4+ T cells. tein products of the rearranged genes allows potential di- Using a rearrangement-competent transgene with a versity that has been estimated as approaching 10” for frameshift mutation in the Vy3 segment upstream of the immunoglobulins, 10’” for ap TCR, and 10” for $5 TCR joints, it was demonstrated that the transgenic Vy3-Jyl (Davis and Bjorkman, 1988). For ab T cells, it is clear that canonical junctions are still formed even though the the functional repertoire in an individual is shaped by a transgene can not generate functional Vy3 protein (Asar- complex processof negative and positive selection to elim- now et al., 1993). Similarly, it was found that the Vy31Jyl inate self-reactive clones and ensure the survival of clones and VyWJyl canonical junctionswere generated in normal with the capacity to recognize peptide antigens in the con- frequencyinTCR6genemutantmice(Itoharaetal., 1993). text of self-major histocompatibility antigens (Robey and These results give support to the previous suggestion that Fowlkes, 1994). The nature of the forces that shape the the invariant repertoire is a result of directed rearrange- yS TCR repertoire is much less clear, particularly for those ment, rather than cellular selection (Raulet et al., 1991; which reside in the epithelial tissues. Allison and Havran, 1991). However, the exact mecha- Immunity 440

nisms involved in the generation of these canonical junc- repeats are sufficient to direct the V-r3/Jyl rearrangement tions remain to be determined. and formation of the Vy3/Jyl canonical junctions. The in- Previous results have shown a correlation between the fluence of TdT enzyme activity on the formation of the presence of short homology repeats near the ends of the Vy3 invariant repertoire was assessed by generating mice coding segments and the appearance of predominant carrying a transgene that expresses TdT under the CD2 junctional sequences in immunoglobulin H (IgH) gene re- promoter. The junction diversity and the frequency of N arrangement (Gu et al., 1990a, 199Ob). Short homology nucleotide addition in Vy3Nyl junctions were significantly repeats were suggested to be responsible for the limited increased in TdT transgenic mice. These data provide di- junctional diversity of neonatal immunoglobulin gene re- rect evidence that short repeats can direct the site of rearrangement (Feeney, 1991 a, 1992). It has been proposed combination in Vy3Nyl rearrangement, and that TdT ex- that these direct repeats may help to align the broken pression can disrupt the process. Thus, the canonical Vy3 ends during the recombination process (Alt and Baltimore, junctions appear to be the result of rearrangement di- 1982; Roth and Wilson, 1986; Lieber, 1992). ATlATA short rected by short repeats in the absence of TdT. repeats are observed at the Vy3-Jyl , Vy4-Jyl , and V61- DS2 junctions in TCR y and 6 chain rearrangement (Allison Results and Havran, 1991; Raulet et al., 1991). Similarly, AGIGAGI GGA short repeats generated by P element addition ap- Role of AT Short Repeats in Directing pear to be present at the D62-J62 and D62-J61 junctions the Vy3 Rearrangement (Itohara et al., 1993). Since the junctional diversity at all To determine the function of ATshort repeat near the ends these joints is severely restricted, it seemed possible that of the Vy3lJyl coding segments in generating the y6 ca- short homology repeats might promote the formation of nonical sequences, we used a rearrangement substrate certain types of joints and thus contribute to the generation that contains the Vy4, Vy3-Jyl-Cyl coding segments in of canonical junctions. However, there has been no direct the germline configuration (Figure 1A). Termination co- evidence linking the presence of short homology repeats dons have been introduced into the Vy4 and Vy3 coding and the formation of yS canonical junctions. segments to prevent the production of Vy4 and Vy3 protein The level of TdT activity during early fetal thymic devel- products. Previous results showed that the Vy3/Jyl ca- opment may also influence the generation of canonical nonical junctions were generated at the same frequency junctions. TdT is thought to be the major enzyme activity in the transgenic substrate rearrangement as in the endog- responsible for N region addition (Alt and Baltimore, 1982). enous Vy3 gene rearrangement (Asarnow et al., 1993). Mutations in murine TdT gene resulted in total loss of N Point mutations were introduced near the 3’ end of Vy3 nucleotides in the V-D-J recombination junctions of both coding segment in the recombination substrate to destroy I3 and T lymphocytes (Komori et al., 1993; Gilfillan et al., specifically the AT direct repeat or to generate new direct 1993). Loss of TdT enzymatic activity is also considered repeats (Figure 16). Recombination substrates carrying to be responsible for the higher frequency of in-frame ca- the various mutations were injected into fertilized eggs to nonical and out-of-frame predominant Vy3-Jyl junctions generate transgenic mice. Day 17 transgenic fetal thymic in the thymus of adult mice (Komori et al., 1993; Gilfillan DNA was polymerase chain reaction (PCR) amplified us- et al., 1993). A low level of TdT enzyme activity in fetal ing primers designed to amplify specifically either trans- and neonatal spleen and liver B cells has also been sug- genie or endogenous V-J rearrangement junctions. gested to be responsible for the lack of N region diversity in immunoglobulin recombination junctions (Opstelten et al., 1986; Meek, 1990; Feeney, 1990, 1991b, 1993). It was recently shown that presence of TdT activity could dimin- A Ikb ish the effect of homology-directed rearrangement in an - in vitro recombination system (Gerstein and Lieber, 1993). In fetal thymic development, although TdT expression is n I lm---m- weakly detectable in bulk fetal thymocyte populations as vu4 VT3 J-/l w E early as day 15 of ontogeny, its expression is absent in the fetal Vy3+ thymocytes (J. Noble and J. P. A., unpublished s data). These data suggest that lack of TdT enzyme activity VT3 N Jr1 in certain populations of fetal thymocytes may further re- Germline TGTGCCTGCTGGGBTC-T jlT,AGCTCAGGTTIT strict the repertoire diversity and contribute to the formation of canonical Vy3-Jyl and Vy4-Jyl rearrangements. Mutant1 TGTGCCTGCTGGGAACT ATAGCTCAGGTTTT t ckrtroyed tile AT dlmcI Here, we examine the role of an ATdirect repeat present repeat) near the ends of the Vy3 and Jyl coding segments on the Mutam TGTGCCTGCTGGGAAAU ,U,4GCTCAGGTTTT (crated a new ATAdirect generation of Vy3/Jyl canonical junctions using a trans- WX=l) genie recombination substrate containing framshift muta- Mutant3 TGTGCCTGCTGGGAQZl AT,&,XXAGGTTTT tions in the Vy3 and Vy4 coding segments (Asarnow et ccy$ B new AG,AGCTci,recc al., 1993). Point mutations were introduced into this rearrangement substrate near the 3’end of Vy3 coding seg- Figure 1. TCR y Gene Rearrangement Substrates ment to destroy the AT short repeat or introduce new types (A) Schematic diagram of the TCR y gene rearrangement substrate. of short repeats. Our results indicate that ATlATA short (B) Point mutations generated near the B’end of Vy3 coding segment. Site-Specific VDJ Recombination 441

Destruction of the AT Short Repeat Eliminates the marked reduction in the frequency of rearrangements in Generation of Vy3/Jyl Canonical Junction which the V and J segments were juxtaposed in the appro- In mutant 1, the AT dinucleotide near the 3’ end of the priate translational reading frame. While approximately Vy3genesegmentwaschangedtoAA,thusdestroyingthe one-third of rearrangements were in frame in the endoge- direct repeat with the AT at the 5’ end of the Jyl segment nous genes of nontransgenic littermates (See Figure 3C) (Figure 1B). The sequences of Vy31Jyl as well as Vy4/ or in the unmutated recombination substrate (Asarnow et Jyl junctions of the rearranged transgene in the day 17 al., 1993), mutant 1 yielded only 2 of 29 in-frame Vy3/Jyl fetal thymi are shown in Figure 2. Recombination at the rearrrangements. site corresponding to the wild-type canonical junction was Although the elimination of the AT direct repeat pre- not observed among any of the 29 individual Vy3 junctional vented formation of the canonical junction, several types sequences examined in mice carrying mutant 1 substrate. of out-of-frame junctions were repeatedly obtained (Figure This suggests a critical role of the AT direct repeat in gener- 2A). For instance, the junctional sequence indicated as ating the Vy3 canonical junction (Figure 2A). The canoni- type Ill repeated five times. It is notable that this sequence cal Vy4-Jyl sequence was generated in all 8 in-frame Vy4 could be directed by new TA or TAG repeats generated rearrangements of the 15 individual Vy4 rearrangement by addition of the P nucleotides A or AG at the 3’ end junctions examined, indicating that mutation at the Vy3 of the Vy3 segment (Figure 2A). Similarly, the junction junction had no effect on the neighboring Vy4 gene re- indicated as type IV could also be attributed to aTA repeat arrangement in the recombination substrate (Figure 28). generated by P nucleotide addition at the ends of the Vy3 One effect of the destruction of the AT repeats was a and Jyl segments. Short repeats formed by the P nucleotide addition have been previously suggested by several laboratories to direct the VDJ rearrangement in both fetal thymocytes and neonatal B cells (Raulet et al., 1991; Asar- now et al., 1993, ltohara et al., 1993, Komori et al., 1993, Chukwuocha et al., 1995). Our results suggest that even in the absence of an AT direct repeat in the coding segments, short repeats formed by the P element addition can still direct the rearrangement, although the resulting predominant rearrangements are mostly out of frame. These results support a role for the short repeats in directing the IAG,P, TA,P, site of the V-J rearrangement. Other types of junctional sequences were also generated at relatively high frequency (Figure 2A, types V, VI). The mechanisms involved in the generation of these types of junctions are not clear, since they could not be generated through directed rearrangement by either germline direct repeats or repeats generated by P nucleotide addition at the precise ends of the coding sequences. It is generally accepted that P nucleotides are generated by the asymmetrical opening of the hairpin structure, which seals the coding and signal ends immediately after the double-stranded DNAcleavage (Lafaille et al., 1989). How- ever, if the hairpins could be formed not only at the exact junctional breakpoint but also at coding ends where one or two nucleotides are removed from the junctional breakpoint, then the asymmetrical opening of these hairpins could form a new type of P nucleotides, referred to as recessed P nucleotide. The AT short repeats formed Figure 2. Nucleotide Sequences of the Transgenic Vy3 Junctions in Mice Carrying Mutant 1 Rearrangement Substrate by these recessed P nucleotides could explain the generation of these predominant junctional sequences (Figure Junctional sequences of the transgenic Vy3Jyl rearrangement (A) and Vy4Jyl rearrangement (B) in the day 17 fetal thymus of mice 2A, types V, VI). More examples of the AT repeats directed bearing mutant 1 transgenic substrate. The sequences are aligned rearrangements of this type could be found in the junctions with the germline sequences of TCR Vy3 and Vy4 segments (Garman of mutant 2 and 3 transgenic fetuses (Figure 3A, types IV, et al., 1966). The recombination signal sequences are shown in lower- V, VI, VII; see Figure 4, type X). case letters. The AT direct repeats are underlined. The canonical sequence(s) represent the predominant Vy3-JylNy4-Jyl junctional se- Creation of a New ATA Repeat Generates a New quence(s) in both fetal thymus and adult epithelial tissues (Allison Type of Predominant Vy3/JyT Junctional and Havran; 1991). The short repeats that could contribute to the Sequence generation of certain types of junctions are listed in a separate column To determine whether the AT short repeat is sufficient to titled Repeats. Repeats that may have formed after the P nucleotides direct the y6 gene rearrangement, we replaced the CT addition are marked as (P). The junctional nucleotides were assigned following the general order of germline - P nucleotides - N nucleo- dinucleotide sequence at the end of the Vy3 coding se- tides. Junctions are considered to be in-frame if the same open reading quence of mutant 1 with the trinucleotide ATA. This sub- frame as the canonical Vy3Jyl junction is maintained. strate, mutant 2, thus has a 3 nt repeat with the 5’ end of Immunity 442

Figure 4. Nucleotide Sequences of the Transgenic Vy3 in Mice Car- rying Mutant 3 Rearrangement Substrate Junctional sequences of the transgenic Vy3Jyl rearrangements in the day 17 fetal thymus of mice bearing mutant 3 transgenic substrate. The recombination signal sequences are shown in lowercase letters. The AGCT direct repeats are underlined.

transgenic littermates were also sequenced using primers that only amplify the endogenous Vy3/Jyl rearrangement (Figures 3B and 3C). In transgenic fetuses carrying mutant 2 substrate, 10 out of 13 in-frame endogenous Vy3/Jyl rearrangements contained the canonical junction, while in the nontransgenic fetus, 11 out of 13 in-frame endogenous Vy3/Jyl rearrangements were of the canonical sequence (Figures 38 and 3C). The comparable frequency of the Vy3 canonical junction in the rearrangements of endogenous genes in these mice suggests that the presence of the transgenic substrate had no general effect on the rearrangement machinery. Two types of additional predominant junctional sequences were also generated in these mice, type II and Ill (Figure 3A). Again, the high frequency appearance of these two junctional sequences could be attributed to the ATA and AT repeats generated by P nucleotide addition. AG or AGCT Repeats Failed to Direct the Vy3Uyl Rearrangement From the data discussed above, it seems quite clear that AT or ATA repeats are sufficient for the generation of rS canonical sequences. It was of interest to determine whether short repeats with other sequences could also Figure 3. Nucleotide Sequences of Vy3 Junctions in Mice Carrying Mutant 2 Rearrangement Substrate direct rearrangement. The AT repeat near the 3’ end of Junctional sequences of the transgenic (A) and the endogenous (B) the Vy3 coding segment was changed to AG to form AG, Vy3-Jyl rearrangements in the day 17 fetal thymus of mice bearing AGC, or AGCT direct repeats with sequences in the Jyl mutant 2 transgenic substrate. (C) Junctional sequences of the Vy3- coding segment (see Figure 1B). Mice carrying this mu- Jyl rearrangements in day 17fetal thymusof the nontransgeniccontrol tated transgenic substrate, mutant 3, failed to produce any mice. predominant transgenic rearrangement (Figure 4). The resultant transgenic junctions were quite diverse, although the Jyl segment (see Figure lB), a feature shared with two types of out-of-frame junctions could be generated the Vr4 gene. through directed rearrangement by potential TA(G)/TA re- Mice carrying mutant 2 transgenic substrate generated peats formed by P nucleotide addition (Figure 4, types VIII a new type of Vy3Nyl predominant junction (Figure 3A, and IX). These results suggest that AG or AGCT repeats type I). Out of 69 total junctions examined, 29 contain this cannot direct the Vy3gene rearrangement efficiently. Sim- type of VyClike junction. These data suggest that AT short ilarly, a pair of AG direct repeats at the Vy2Nyl junction repeats are sufficient to direct the site of the Vy3/Jyl gene also fail to direct the Vy2 gene rearrangement (Lafaille et rearrangement. The endogenous Vy3Nyl junctions in al., 1989). However, we cannot rule out the possibility that mice carrying the mutant 2 transgene as well as their non- the position of the AG repeat at the Vy3IJyl junction is Site-Specific VDJ Recombination 443

Table 1. Comparison of the Vy3Jyl Junctional Diversity and N Region Insertion in Control and TdT Transgenic Mice Thymus: Fetal Vy3Jyl Junctions in TdT Transgenic and Control Mice Thymus

Mice Frameshift Predominent junctions (percent)’ N nucleotide insertion (percent) Junctional diversityb

Control micec In frame 38148 (80%) 2148 (4%) 8148 (0.16) Out of frame 38172 (53%) 3/72 (4%) 24172 (0.33) TdT transgenic mice In frame 10120 (50%) 6/20 (30%) 11120 (0.55) Out of frame 15129 (52%) 9129 (31%) 16/29 (0.55) a The predominant junctions in the in-frame rearrangements represent the canonical junctions. The predominant junctions in the Vy3 out-of-frame rearrangements represent two types of junctions, which are frequently observed (Figure 3C, types IV and V). b Junctional diversity is defined as the number of different functions in a group of rearrangement junctions divided by the total number of junctions in that group. ’ Vy3 rearrangements in the control mice were obtained from Figures 38 and 3C as well as from TdT nontransgenic littermates (Data unpublished).

also important for its function in directing rearrangement. by day 15 of ontogeny (Duplay et al., 1989; Owen et al., The AG dinucleotide is located 3 nt away from the 5’ end 1988). of the Jyl coding sequence. Since in normal mice the The activity of the CDP-HMTdT construct (Figure 5A) sites of recombination are usually upstream of the AGCT was tested in vitro by transient expression in transfected sequence in Jyl, it is possible that recombination at this EL-4 cells, a mouse T cell lymphoma line. CD2-HMTdT- site is generally precluded by the recombinase (see Fig- transfected T cells contain 5-fold higher TdT activity as ures 2-4; Figure 6; Asarnow et al., 1993, ltohara et al., compared with the untransfected T cells (data not shown). 1993). Cytohistology staining using a polyclonal anti-TdT antibody indicated that TdT enzyme in the transfected cells Formation of the Vy3lJyl Canonical Junction was localized mostly in the cytosol (data not shown). Is Impaired in TdT Transgenic Mice Flow cytometric analysis of thymocytes from TdT Nongermline-encoded nucleotides are not present in ca- transgenic and nontransgenic littermates showed that nonical Vy3 and Vy4 junctions, and are rarely observed Vy3+ cells developed in normal numbers and that the level in any Vy3 and Vy4 junctions in the fetal thymus (Allison of Vy3’ TCR expression was unaffected in the TdT and Havran, 1991). As shown in Figure 3C, we found only transgenic mice (data not shown) two junctions containing nongermline nucleotides in a total Analysis of the Vy3 rearrangements in day 17 fetal thy- of 32 Vy3/Jyl rearrangements. Previous studies have re- mus of the TdT nontransgenic littermates showed typical ported the occurrence of N nucleotides in only about 6% features of the Vy3 rearrangements: dominant presence of out-of-frame Vy3 junctions in the fetal thymus, while of the Vy3 canonical junctions (13 out of 16) in the in-frame in-frame fetal Vy3 junctions contain essentially no N re- rearrangements and absence of the N nucleotides (data gions (Table 2). It is thought that the lack of N nucleotides not shown). However, in the TdT transgenic fetal thymi, may be due to a low level of TdT expression in the early we observed a significant increase in the appearance of fetal thymus. We therefore sought to determine whether nongermline nucleotides in the junctions of both Vy3 in- expression of TdT in the early fetal thymus would affect frame and out-of-frame rearrangements (Figure 56). Ap- the formation of canonical Vy31Jyl junctions. Transgenic proximately 30% of both in-frame and out-of-frame Vy3 mice were produced using aconstruct in which expression junctions contain N nucleotides in these thymi (Table 1). of TdT was regulated by the CD2 promoter (Figure 5A). These N nucleotides contain a high percentage of G/C This promoter was chosen because CD2 is eXpreS8ed by (650/o), a general feature of the TdT involved N nucleotide fetal Vy3+ cells and can be detected on fetal thymocytes addition.

Table 2. Comparison of the Vy3-Jyl Junctional Diversity and N Region Insertion in Control and TdT Transgenic Mice Thymus: Reported Vy3- Jyl Junctions in Fetal, Newborn, and Adult Mice Thymus

Mice Frameshift Predominant junctions (perent) N nucleotide Insertion (percent) Junctional diversity’ Fetal VyS-Jyl junctions In frame 49/54 (91%) o/54 (0%) 4/54 (0.08) Out of frame 31153 (59%) 3/53 (6%) 20/53 (0.36) Newborn VyS-Jyl junctions In frame 14/l 7 (82%) O/l 7 (0%) 4/14 (0.28) Out of frame 15135 (43%) 5135 (14%) 18/35 (0.51) Adult Vy3Jyl junctions In frame 13/32 (41%) 15/32 (47%) 20/32 (0.63) Out of frame 13/55 (24%) 35155 (63%) 43155 (0.78) a The predominant junctions in the in-frame rearrangements represent the canonical junctions. The predominant junctions in the VT3 out-of-frame rearrangements represent two types of junctions, which are frequently observed (Figure 3C, types IV and V). D Junctional diversity is defined as the number of different junctions in a group of rearrangement junctions divided by the total number of junctions in that group. The reported fetal Vy3-Jyl junctions were obtained from Lafaille et al. (1990) and ltohara et al. (1993). Newborn Vy3-Jyl junctions were obtained from Asarnow et al. (1993). Adult VyS-Jyl junctions were obtained from Komori et al. (1993). Immunity 444

Discussion

We have examined two factors that may contribute to the generation of y6 TCR canonical repertoires: the presence of short sequence repeats near the ends of coding segments and TdT activity. The function of the ATshort repeat near the ends of VT3 and Jyl coding segments was examined using mutated rearrangement substrates that contained the Vy4, Vy3, Jyl , Cyl gene segments. Introduction of these substrates into mice allowed us to study the V-J rearrangement in the absence of TCR-mediated positive and negative cellular selections. The rearrangement of these substrates during fetal thymic development occurs independently but closely reflects the endogenous Vy gene rearrangement events. Using this system, we found that AT or ATA direct repeats are sufficient to direct the Vy3lJyl gene rearrangement and to generate canonical junctions. Our results give further support to the idea that positive selection at the cellular level is not required for the generation of the canonical rearrangement. There has been some controversy as to whether short Figure 5. The Effects of TdT on Vy3Jyl Junctional Diversity homology repeats can direct the V-(D)-J rearrangement (A) Schematic diagram of the transgenic construct that expresses the (Lewis, 1994, see discussion). Although several reports mouse-human hybrid terminal deoxynucleotidyl transferase (TdT). indicated that the presence of short direct repeats could (6) The junctional sequences of the Vy3-Jyl rearrangement in day 17 fetal thymus of the TdT transgenic mice. lead to severely restricted junctional diversity, others failed to reach the same conclusion (Gerstein and Lieber, 1993; Chukwuocha et al., 1995; Boubnov et al., 1993). It was also reported that certain predominant neonatal immunoglobulin gene rearrangements are generated through de- The frequency of canonical Vy3Jyl junctions was re- velopmental selection rather than homology-directed reduced from the expected 80%-95% observed in control arrangement (Pandey et al., 1993). However, it is clear mice to about 50% in the TdTtransgenic mice, suggesting that cellular selection is not required for the predominant a partially impaired ability to generate the canonical junc- generation of the canonical Vy3-Jyl and Vy4-Jyl junctions in these mice. The generation of noncanonical in- tions in fetal thymus, since these junctions are observed frame Vy3 junctions could be attributed mostly to the pres- even when TCRs are not expressed on cell surface (Asar- ence of TdT activity, since 6 out of 10 of these junctions now et al., 1993; ltohara et al., 1993). Our results provide contained N nucleotides (Figure 5B). direct evidence that AT/ATA repeats dictate the sites of Our results indicate that introduction of a TdT transgene the Vy3-Jyl rearrangement. Introduction of a ATA repeat into mice increases the junctional diversity of fetal Vy3 at a different position of the Vy3 segment altered the site rearrangement. Junctional diversity can be defined as the of Vy3-Jyl rearrangement and generated a new type of number of different junctions in a group of rearrangement predominant junction (Figure 3A). The position of the AT junctions divided by the total number of junctions in that repeat may also safeguard the rearrangements to main- group. According to this definition, the fetal Vy3 junctions tain proper reading frame, since the elimination of the AT in the TdT transgenic mice have a diversity of 0.55 in repeat drastically reduced the frequency of in-frame Vy3- both in-frame and out-of-frame rearrangements (Table l), Jyl rearrangements (Figure 2A). much higher than that observed in control fetal Vy3 re- Several types of predominant out-of-frame Vy3IJyl rearrangements, which is around 0.08-o. 16 for the in-frame arrangements have been frequently observed (Raulet et rearrangements and 0.33-0.36 for the out-of-frame re- al., 1991; Asarnowet al., 1993, Lafailleet al., 1989; Komori arrangements (Table 1). The junctional diversity of VT3 et al., 1993). It was previously suggested that short repeats rearrangements in fetal TdT transgenic mice is even formed by P nucleotide addition might contribute to the higher than that in the newborn mice thymus but lowerthan formation of these predominant junctional sequences. Our that in the adult thymus (Tables 1 and 2). These results results indicate that repeats generated by P nucleotide indicate that enhanced TdT expression can increase the addition can still direct the rearrangement and form sev- frequency of N nucleotide addition as well as the junctional eral types of out-of-frame predominant junctions even diversity at the Vy31Jyl junction. Thus, lack of TdT activity when the AT repeat in the germline coding region is not during gene rearrangement in the precursors of the VT3’ present (Figure 2A; Figure 4). These results give further and Vy4+ fetal thymocytes may contribute to the genera- support to the idea that short repeats are crucial elements tion of y6 canonical junctions by restricting the repertoire in directing y gene rearrangement. diversity. It is not clear whether short repeats with other se- Site-Specific VDJ Recombination 445

quences can also direct rearrangement. In our system, possibility of these recessed P nucleotides has been pre- AG or AGCT repeats could not effectively direct the Vy3/ viously proposed (M. Lieber, personal communication). Jyl rearrangement (Figure 4). Although AG-rich repeats Short repeats generated by addition of the recessed P have been suggested to direct the D&2/J&2 rearrangement nucleotides could account for the predominant appear- (Itohara et al., 1993) AG direct repeats at the Vy21Jyl ance of several typesof Vy2 junctions (Lafaille et al., 1989). junction did not generate a high frequency of the corre- However, there is no direct evidence so far to support sponding rearrangement (Lafaille et al., 1989). It may be recessed P nucleotide addition. that the sequence content of the short homology is im- Expression of TdT activity is tightly regulated during em- portant in determining whether it is able to direct the re- bryonic thymic development. The absence of N nucleo- arrangement effectively. Another possibility, which we fa- tides in fetal VyBlJyl rearrangement junctions suggested vor, is that the position of the short homology is crucial. that TdT is not expressed at high level at the time when the Thus, if a short repeat is located too far from the ends rearrangement occurs. In the fetal thymi of TdT transgenic of the coding segments, particularly the end of the Jyl mice, we Observed a significant increase in N nucleotide segment, it may not be able to direct the rearrangement. addition and decrease in the frequency of canonical junc- In a particular V-(D)-J recombination system, both the tions (Figure 58; Table 1). These results support the idea sequence content and the position of a short repeat may that lack of TdT activity in the precursors of fetal Vy3+ determine whether it can effectively direct the rearrange- and Vy4+ T cells promotes the generation of the invariant ment. This may also provide an explanation to the discrep- repertories in these cells. Thus, regulation of TdT expres- ancy in the literature regarding whether short homology sion during fetal thymic development may play a major role repeats can direct the rearrangement (reviewed by Lewis, in the programmed development of T cells with invariant 1994). repertoires. It is not known how short homology repeats direct the Our results clearly demonstrate that increased TdT ac- VDJ rearrangement. One of the possible mechanisms tivity has a inhibitory effect on short repeat-directed re- could be the pairing of two single-stranded protruding ends arrangement in vivo. However, the canonical Vy3Jyl through these short homologies (Alt and Baltimore, 1982; junction is still the predominant junction in TdT transgenic Roth and Wilson, 1986; Lieber, 1992). This kind of joining fetal thymus, consisting of 50% of the fetal Vy3-Jyl re- reaction occurs frequently during the DNA breakage and arrangement (Figure 5B). Although this may be due to late rejoining in the nonhomologous recombination in mam- onset or level of expression from the CD2 promoter, it malian cells (reviewed by Roth and Wilson, 1986). The is also possible that the predominance of the canonical existence of P nucleotides suggests that single-stranded junctions reflects the strong effects of the short homology broken ends are intermediates during V-(D)-J recombina- repeats in inhibiting the action of TdT, perhaps by pre- tion (Lafaille et al., 1989; Schuler et al., 1991; Kienker et venting its binding to ends of the rearranging genes. In al., 1991). Although there is no direct experimental evi- adult thymus, 40% of the total in-frame Vy3-Jyl junctions dence for single-stranded pairing of the broken ends dur- are still the canonical junctions (Komori et al., 1993). This ing V-(D)-J recombination, this would provide an explana- suggests that even when TdT activity is high, such as tion for the resultant junctional sequences generated by in the adult thymus, predominant junctions might still be short repeats-directed Vy3-Jyl rearrangement. generated by the directing force of the short homology The presence of sequence-specific or nonspecific DNA repeats and these junctions may be resistant to N nucleo- binding proteins at the recombination joints may also help tide addition. to align the broken joints and promote the pairing of short From the above discussion and previous suggestions repeats. Although several proteins that bind to the recom- (Lewis, 1994) it seems that short repeats-directed re- binant signal sequences have been identified in the past arrangement and N nucleotide addition represent two in- several years, it remains to be proved that these proteins dependent processes in V-(D)-J recombination, which are required for the V-(D)-J recombination (Lewis, 1994). have opposite effects on junctional diversity. Short repeats The recent finding of the Ku80 complex, which binds to direct the rearrangement to form a restricted repertoire, free double-stranded DNA ends gives some support to while TdT increases the repertoire diversity. These two pro- this idea (Taccioli et al., 1994). However, it seems that cesses compete for the available coding joints during the Ku80 has no sequence preference when it binds the bro- rearrangement. The final outcome of this competition will ken DNA ends (Taccioli et al., 1994). be determined by the strength with which the short repeats In this study, we also observed several types of predomi- direct specific rearrangement and the level of TdT activity nant Vy3/Jyl junctions that could not be attributed to the in the cell where recombination is occurring, and will de- presence of germline short repeats or repeats formed by cide the ultimate repertoire diversity in this system. the P nucleotides added precisely at the junctional break- Taken together, these data support the idea that short point (Figure 2A, types V and VI; Figure 3A, types IV, V, homology repeats have a general function in directing re- VI, and VII; Figure 4, type X). However, if P nucleotides arrangements during V-(D)-J recombination. Although can also be added at coding ends where 1 or 2 nt have the presence of short repeats may not change the overall been removed from the double-stranded DNA cleavage efficiency of recombination (Gerstein and Lieber, 1993) site, AT short repeats would be generated that could ac- they certainly can determine the outcome of the re- count forthe high frequency of these rearrangements. The arrangement. In fact, certain types of short repeats may be Immunity 446

evolutionarily selected to ensure the generation of specific was measured according to Koiwai et al. (1986). Polyclonal anti-TdT antibodies against bacterial infection (Feeney, 1991a). antibody was purchased from Life Sciences, Incorporated. A 7.5 kb Sall-Notldigested fragment that contains the entire Cellular selection may also play a role in further enhancing CD2-HMTdT expression cassette was purified and injected into the the junctional homogeneity in certain types of immuno- (C57BU6 x CBA/J)FP fertilized eggs. Two male founders were gener- globulin gene rearrangement; however, it is not required ated and mated with C57BU6 females to establish the transgenic lines. for shaping the TCR repertoires in Vy3+ and, presumably, For timed pregnancies, female mice were examined daily for vaginal Vy4’ T cells. plug, which was considered as day 0. Fetuses were harvested at day 17 and Southern blot analyses of liver genomic DNA were performed Although our results clearly indicate that TCR-mediated to identify TdT transgenic fetuses. cellular selection is not required for the generation of y6 canonical sequences, it is still possible that TCR signaling PCR Amplification and DNA Sequencing might be needed for the maturation of fetal Vy3+ thymo- Fetal thymus was solublized after overnight incubation at 55°C in mouse tail buffer (50 mM Tris [pH 81, 100 mM EDTA, 0.5% SDS, and cytes. It has been recently demonstrated that in the fetal 400 ug/ml proteinase K). DNA was purified by phenol/chloroform plus thymus, Vy31°/HSAhi cells give rise to Vy3”‘/HSA’” cells and chloroformlisoamylalcohol extraction, followed by ethanol precipita- that cyclosporin A (CsA) blocks the appearance of the tion. PCR amplification was performed using the Gene-Amp PCR am- Vy3h’/HSA’o T cells (Leclercq et al., 1993). This suggests plification kit (Perkin Elmer Cetus). Each PCR reaction contained 0.1 a requirement for CsA-sensitive signals, possibly that of pg fetal thymus DNA. Two sets of primers were used for PCR amplification todistinguish transgenicandendogenous rearrangements: Trans- the TCR, for maturation of Vy3+ thymocytes. Thus, it genie Vy3, GAT GAG AAG GAT GAT GCG GAA; Vy4, GAT GCA TAC seems that although TCR-mediated signals are not re- ATA CAC TGC CTC. Endogenous Vy3, GAT GAG AAG GAT GAT quired for shaping the y& T cell repertoires, TCR signaling GGT ACC; Vy4, GAT GCA TAC ATA CAC TGG TAC. The 3’Jyl primer may be needed for the thymic maturation of these T cells was GCG AAG CTT CAG AGG GAA TTA CTA TGA. The PCR products (Allison, 1993). It could be postulated that these signals were gel purified and subcloned into pBluescript II SK(+) T vector (Marchuk et al., 1991). DNA sequence analysis was performed using might be used to survey the expression of TCRs on the Sanger’s dideoxy sequencing methods (Sanger et al., 1977) and cell surface and promote the survival of T cells that have Sequenase purchased from US Biochemical. in-frame rearrangement. It remains to be elucidated how short homology repeats Acknowledgments can direct the rearrangement and whether single-stranded We thank Dr. A. Winoto for the CD2 expression vector and M. Moser DNA pairing is involved. The understanding of the func- for technical assistance. We also thank Dr. C. Chamber for critical tions and mechanisms of the short repeats-directed re- reading of the manuscript and her valuable discussions. This work arrangement will provide valuable insights towards the un- was supported by the National Institute of Health grant Al26942 (to derstanding of the complicated mechanisms of V-(D)-J J. P. A.), Al31650 (to D. H. R.), and Al200047 (to F. W. A.). Y. Z. was supported by National Institutes of Health training grant CA09179 and recombination. a Fellowship from the Leukemia Society of America.

Experimental Procedures Received May 26, 1995; revised August 10, 1995.

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