BRIEF DEFINITIVE REPORT:
The V-J Recombination of  T Cell Receptor-gamma Genes Is Blocked in Interleukin-7 Receptor-deficient Mice

By Kazushige Maki,* Shinji Sunaga,* and Koichi Ikuta*Dagger

From the * Department of Disease-related Gene Regulation Research (Sandoz), Faculty of Medicine, The University of  Tokyo, Tokyo 113, Japan; and the Dagger  Department of Medical Chemistry, Faculty of Medicine, Kyoto University, Kyoto 606, Japan

Summary
Materials and Methods
Results
Discussion
Footnotes
Acknowledgements
References


Summary

IL-7R-deficient mice have severely impaired expansion of early lymphocytes and lack gamma delta T cells. To elucidate the role of IL-7R on gamma delta T cell development, we analyzed the rearrangements of TCR-alpha , beta , gamma , and delta  genes in the thymus of the IL-7R-deficient mice. Southern blot analysis with a Jgamma 1 probe revealed that more than 70% of Jgamma 1 and Jgamma 2 alleles are recombined to form distinct Vgamma 1.2-Jgamma 2 and Vgamma 2-Jgamma 1 fragments in control mice. On the contrary, no such recombination was detected in the mutant mice. The rearrangements in the TCR-alpha , beta , and delta  loci were comparably observed in control and mutant mice. PCR analysis indicated that the V-J recombination of all the Vgamma genes is severely hampered in the mutant mice. The mRNA of RAG-1, RAG-2, Ku-80, and terminal deoxynucleotidyl transferase (TdT) genes was equally detected between control and mutant thymi, suggesting that the expression of common recombination machinery is not affected. These data demonstrated that the V-J recombination of the TCR gamma  genes is specifically blocked in the IL-7R-deficient mice and suggested the presence of highly specific regulation for TCR gamma  gene rearrangement.


IL-7 is a growth factor for early B and T cell precursors. It was first characterized by its ability to support the growth of pre-B cells. Subsequently, it has been shown to support survival and growth of early thymocytes and promote rearrangement of TCR beta  and gamma  chains in fetal thymus and fetal liver cultures (1, 2). In vivo administration of neutralizing antibodies to IL-7 and IL-7R resulted in the inhibition of both B and T lymphopoiesis (3, 4). Finally, IL-7- and IL-7Rdeficient mice have severely impaired expansion of early lymphocytes (5, 6).

gamma delta T cells have unique features in contrast with alpha beta T cells (7, 8). gamma delta T cells expressing specific Vgamma chain appear as several successive waves in the developing thymus and each of them shows specific tissue distribution in the adult mouse. However, little is known about the mechanism of gamma delta T cell development. In fetal thymic organ culture, addition of IL-7 promotes expansion of mature gamma delta T cells but prevents generation of mature alpha beta T cells (9). The epithelial cells in the skin and the gut produce IL-7 (10, 11), and dendritic epidermal T cells proliferate in response to IL-7 (10). Additionally, IL-7 induced rearrangement of Vgamma 2 and Vgamma 4, but not Vgamma 3 or Vgamma 5 genes, and sustained expression of RAG-1 and RAG-2 genes (1, 2). Collectively, these results suggested that IL-7 may be involved in development and maintenance of gamma delta T cells in the thymus and the periphery.

Although T and B lymphopoiesis is severely hampered, decreased but certain numbers of alpha beta T cells and B cells exist in the periphery of the IL-7R-deficient mice, and they normally respond to mitogenic stimuli such as Con A and LPS (12). In contrast, gamma delta T cells are completely absent in the IL-7R-deficient mice as well as in IL-2R-gamma - and Jak3-deficient mice: no gamma delta T cells were detected in fetal and adult thymus, spleen, skin, small intestine, and liver of IL-7Rdeficient mice (12). Two possibilities can be considered to explain the lack of gamma delta T cells in the IL-7R-deficient mice. The one is that gamma delta T cell precursors may completely depend on IL-7 for their survival and/or proliferation. The other is that IL-7 may be a key factor for the induction of the TCR gamma  gene rearrangements in T cell precursors.

To test the latter hypothesis, we analyzed the recombination status of TCR loci in the alpha beta T cells remaining in IL-7R-deficient mice. The V-J recombination was almost completely blocked in the TCR gamma  locus in the mutant thymus, whereas the TCR alpha , beta , and delta loci were rearranged at comparable levels with control thymus. These results clearly demonstrated that the signal from IL-7R plays an indispensable role on the induction of TCR gamma  gene rearrangement. Thus, the mouse TCR gamma  locus will provide a unique system to analyze the mechanism of cytokine-induced gene rearrangements.


Materials and Methods

Mice. IL-7R-deficient mice were established by replacing the exon 2 with a PGK-neo cassette as described (12). Animals heterozygous (+/-) and homozygous (-/-) for the IL-7R mutation were on the (129/Ola × C57BL/6)F3 hybrid background. The age of fetuses was determined by scoring for the appearance of a vaginal plug and taking as day 0 the morning on which the mating plug was observed. All mice were maintained under the specific pathogen-free conditions in the Animal Center for Biomedical Research, Faculty of Medicine (The University of Tokyo).

Southern Blot Analysis. Thymocyte genomic DNA was digested with HindIII or EcoRI restriction enzyme and electrophoresed through 0.7% agarose gel. The DNA was transferred to polyvinylidene difluoride filters (Immobilon; Millipore, Bedford, MA) and hybridized with 32P-labeled probes. The following fragments were used as probes: Jgamma 1, a 1.1 kb StyI-HindIII fragment containing the Jgamma 1 segment and its 3' flanking region of genomic DNA from KN6 (15); Jalpha 1, a 3.5 kb EcoRI-HindIII fragment of TA28.1 (16); Jdelta 1, a 2.5 kb SacI fragment of pCDS17 (17); Jbeta 2, a 2.3 kb EcoRI fragment of mouse genomic Jbeta region (18). To confirm equal loading of genomic DNA, the membranes were hybridized with a 1.3 kb KpnI fragment of mouse RAG-2 cDNA (19). Southern blots were analyzed and radioactivity was quantitated using a Bio-image Analyzer (Fujix BAS2000; Fuji Film, Tokyo, Japan). The percentage of rearranged alleles was calculated by normalizing with the radioactivity of the RAG-2 probe.

PCR Analysis. Thymocyte DNA was prepared from fetuses at day 17 of gestation and mice at 4 wk old. PCR was carried out in a 25 µl reaction mixture containing 0.5 ng template DNA (0.5 µg for TCR beta  genes), 50 pmol each primer, 200 mM each dNTP, and 2.5 U Taq DNA polymerase. For TCR gamma  genes, samples were amplified for 30 cycles of 45 s at 94°C, 2 min at 50°C, and 1 min at 72°C. For TCR delta  and beta  genes, PCR was performed as described previously (20). The PCR products were electrophoresed in 3% agarose gel, blotted onto nylon membranes, and hybridized with 32P-labeled oligonucleotide probes. PCR primers are as follows: Vgamma 1.1 and Vgamma 1.2, 5'-CTTCCATATTTCTCCAACACAGC-3'; Jgamma 2, 5'-ACTATGAGCTTTGTTCCTTCTG-3'; Jgamma 4, 5'-ACTACGAGCTTTGTCCCTTTGG-3'; 5' RAG-2, 5'-CACATCCACAAGCAGGAAGTACAC-3'; 3' RAG-2, 5'-GGTTCAGGGACATCTCCTACTAAG-3'. Vgamma 2, Vgamma 3, Vgamma 4, Vgamma 5, Jgamma 1, Vdelta 1, Vdelta 4, Vdelta 5, Jdelta 1, Vbeta 8.2, Dbeta 2, and Jbeta 2 primers were described previously (20). Oligonucleotide probes used are as follows: Jgamma 2, 5'-CAAATACCTTGTGAAAGCCCGAGC-3'; Jgamma 4, 5'-CAAATATCTTGACCCATGATGTGC-3'. Jgamma 1, Jdelta 1, and Jbeta 2 oligonucleotide probes were described previously (20, 22). Radioactivity was analyzed using the Bio-image Analyzer.

RT-PCR Analysis. Total RNA was isolated using the AGPC method as described (24). RNA samples were treated with RQ-1 RNase-free DNase (Promega Corp., Madison, WI) to remove contaminating genomic DNA. Oligo (dT)-primed cDNA was prepared by Molony murine leukemia virus RNase H- reverse transcriptase (GIBCO BRL, Gaithersburg, MD) at 37°C for 1 h. PCR was carried out for 25 cycles of 1 min at 94°C, 1 min at 50°C for hypoxanthine phosphoribosyl transferase (HPRT) or at 55°C for others, and 1 min at 72°C. The PCR products were electrophoresed in 3% agarose gel, blotted onto nylon membranes, and hybridized with 32P-labeled probes. The following DNA fragments were used as probes: RAG-1, a 1.4-kb EcoRI fragment of mouse RAG-1 cDNA (19); terminal deoxynucleotidyl transferase (TdT), a 1.3-kb EcoRI-EcoRV fragment of mouse TdT cDNA, M16-1b (25); Ku-80, a 540-bp PCR fragment of Ku-80 cDNA; HPRT, a 350-bp PCR fragment of HPRT cDNA. The RAG-2 probe was described above. The following PCR primers are used: 5' RAG-1, 5'-GCAATGAGGAAGTGAGTCTGGA-3'; 3' RAG-1, 5'-CTGAGGAAGGTATTGACACGGA-3'; 5' Ku-80, 5'-AGAGGACACTATTCAAGGGTAC-3'; 3' Ku-80, 5'-AGACACTGGTACAATCGCTGAA-3'; 5' TdT, 5'-ACTGCGACATCTTAGAGTCA-3'; 3' TdT, 5'-CTTCCCCTTAGTCCTGTCAT-3'; 5' HPRT, 5'-CTCGAAGTGTTGGATACAGG-3'; 3' HPRT, 5'-TGGCCTATAGGCTCATAGTG-3'. Radioactivity was analyzed using the Bio-image Analyzer.


Results

V-J Recombination of TCR gamma  Genes Is Blocked in IL-7Rdeficient Mice.

To examine whether the signal from IL-7R affects the V-J recombination, we compared the rearrangement of the TCR gamma  genes between the thymocytes of IL-7R +/- and -/- mice. The thymocyte DNA from 4-wk-old mice was digested with HindIII or EcoRI, and a Southern blot was hybridized with the Jgamma 1 probe (Fig. 1 A, left). The Jgamma 1 probe allows the analysis of DNA rearrangements involving not only Jgamma 1 but also Jgamma 2 and Jgamma 3 gene segments (15). The ES cell DNA showed a 6.6-kb Jgamma 1, a 9.0-kb Jgamma 3, and a 11.7-kb Jgamma 2 germline fragment. The thymocyte DNA from IL-7R +/- mice showed decreased intensity of Jgamma 1 and Jgamma 2 germline fragments compared with embryonic stem (ES) cell DNA. Furthermore, a 3.6-kb Vgamma 1.2-Jgamma 2 and a 1.4-kb Vgamma 2-Jgamma 1 fragment was clearly detected in IL-7R +/- mice. Quantification of the radioactivity revealed that 71% and 74% of Jgamma 1 and Jgamma 2 alleles, respectively, were rearranged in thymocytes. Because gamma delta T cells are only 0.3% of total thymocytes (12), the majority of the Vgamma 1.2-Jgamma 2 and Vgamma 2-Jgamma 1 recombined fragments are derived from alpha beta T cells or precursor cells. On the other hand, no fragment derived from Vgamma 1.2-Jgamma 2 or Vgamma 2-Jgamma 1 recombination was detected in IL-7R -/- mice (Fig. 1 A). This result demonstrates that Vgamma 1.2-Jgamma 2 and Vgamma 2-Jgamma 1 rearrangements are almost completely blocked in alpha beta T cells in IL-7R-deficient mice.



Fig. 1. TCR gene rearrangements in the thymus of IL-7R-deficient mice. Lane 1, thymocytes from IL-7R +/- mice; lane 2, thymocytes from IL-7R -/- mice; lane 3, E14.1 ES cells. The position of HindIIIdigested phage lambda  DNA fragments was shown on the right. (A) Thymocyte DNA was digested with HindIII. A Southern blot was sequentially hybridized with the Jgamma 1 (left), the Jbeta 2 (middle), and the RAG-2 (right) probes. (B) Thymocyte DNA was digested with EcoRI. A Southern blot was sequentially hybridized with the Jdelta 1 (left), the Jalpha 1 (middle), and the RAG-2 (right) probes.
[View Larger Versions of these Images (72 + 75K GIF file)]

We next examined adult and fetal thymus DNA by PCR with Vgamma 1.1+1.2, Vgamma 2, Vgamma 3, Vgamma 4, Vgamma 5, Jgamma 1, Jgamma 2, and Jgamma 4 primers. Thymus DNA revealed large amounts of PCR products with all the Vgamma -Jgamma primer pairs in IL-7R +/- mice. On the other hand, V-J rearrangement was greatly reduced in all the TCR gamma  genes in IL-7R -/- thymus; the signal of Vgamma 5-Jgamma 1 product was 150-fold reduced relative to IL-7R +/- mice, and those of Vgamma 1.1- Jgamma 4, Vgamma 1.2-Jgamma 2, Vgamma 2-Jgamma 1, Vgamma 3-Jgamma 1, and Vgamma 4-Jgamma 1 products were undetectable in IL-7R -/- mice (Fig. 2 A). Amplification with RAG-2 primers produced roughly the equal amount of PCR products in both IL-7R +/- and -/- thymus, suggesting that approximately the same amount of DNA was used in this analysis. These results support the data of Southern blot analysis and suggest that the V-J recombination is almost completely blocked in IL-7R-deficient mice not only in Vgamma 1.2 and Vgamma 2 genes but also in all the other Vgamma genes.


Fig. 2. TCR gene rearrangements in the thymocytes detected by PCR. Rearrangement of TCR gamma  (A), delta  (B), and beta  (C) genes. The DNA from thymocytes of fetuses at day 17 of gestation (for Vgamma 3-Jgamma 1, Vgamma 4- Jgamma 1, and Vdelta 1-Jdelta 1 rearrangements) and at 4 wk old (for Vgamma 1.1-Jgamma 4, Vgamma 1.2-Jgamma 2, Vgamma 2- Jgamma 1, Vgamma 5-Jgamma 1, Vdelta 4-Jdelta 1, Vdelta 5- Jdelta 1, and all the beta gene rearrangements) was amplified by PCR, and the Southern blots of the products were hybridized with oligonucleotide probes. Combination of primers used are shown on the left side (A and B). Dbeta -Jbeta 2 and Vbeta 8.2-Dbeta -Jbeta 2 rearranged fragments are shown on the left side (C).
[View Larger Version of this Image (39K GIF file)]

Rearrangements of TCR alpha , beta , and delta  Genes Take Place Normally in IL-7R-deficient Mice.

We next analyzed the rearrangement of other TCR genes by Southern blot analysis. First, the Southern blot was hybridized with Jbeta 2 probe (see Fig. 1 A, middle). The ES cell DNA showed a 4.8-kb germline Jbeta 2 fragment. Thymocyte DNA showed decreased intensity of the Jbeta 2 germline fragment and smear patterns of Jbeta 2 recombined fragments in both IL-7R +/- and -/- mice. Quantification revealed that 81% and 69% of Jbeta 2 alleles are rearranged in IL-7R +/- and -/- thymus, respectively. Thus, the frequency of Jbeta 2 rearrangement is slightly decreased in IL-7R -/- mice compared with IL-7R +/- mice. Next, we hybridized a Southern blot with Jdelta 1 and Jalpha 1 probes (see Fig. 1 B). A 6.8-kb Jdelta 1 and a 7.7-kb Jalpha 1 germline fragment was detected in ES cell DNA. These were greatly reduced in the thymus because of deletion of the delta  locus by Valpha -Jalpha recombination in alpha beta T cells. Extra faint fragments and a smear pattern of Jdelta recombined fragments were detected in IL-7R -/- mice as well as in IL-7R +/- mice. Thymocyte DNA from IL-7R -/- mice showed several Vdelta -Ddelta -Jdelta and Ddelta -Jdelta recombined fragments at the comparable intensity with that from IL-7R +/- mice.

The rearrangement of TCR delta  genes was further examined by PCR with Vdelta 1, Vdelta 4, Vdelta 5, and Jdelta 1 primers. In contrast with TCR gamma  genes, the signals of Vdelta 1-Ddelta -Jdelta 1, Vdelta 4- Ddelta -Jdelta 4, and Vdelta 5-Ddelta -Jdelta 1 fragments were only slightly diminished (two- to eightfold reduction) in IL-7R -/- mice relative to IL-7R +/- mice (Fig. 2 B). Because IL-7R -/- thymus lacks gamma delta T cells (12), the Vdelta -Jdelta recombined fragments are probably derived from alpha beta T cells and precursor cells. Thus, the difference in the amounts of TCR delta  products may be attributed to the presence and absence of gamma delta T cells in the thymus of IL-7R +/- and -/- mice, respectively. Collectively, TCR delta  gene rearrangement seems not to be severely hampered in the IL-7R-deficient mice, supporting the data of Southern blot analysis.

Next, PCR amplification with Vbeta 8.2, Dbeta 2, and Jbeta 2 primers revealed six Dbeta -Jbeta and six Vbeta -Jbeta recombined fragments in both the IL-7R +/- and -/- thymus DNA (Fig. 2 C). These results demonstrate that IL-7R is not essential for both Dbeta -Jbeta and Vbeta -Dbeta -Jbeta recombinations. It is recently reported that IL-7 supported Dbeta to Jbeta rearrangements but not Vbeta to Dbeta Jbeta rearrangement in fetal thymic organ culture of fetal liver precursor cells (26). However, our results do not support the notion that IL-7 may play some specific role on Dbeta -Jbeta recombination. All these results suggested that the rearrangements of TCR alpha , beta , and delta  genes take place normally in IL-7R-deficient mice.

Expression of Common Recombination Machinery in IL-7Rdeficient Mice.

RAG-1 and RAG-2 are indispensable for V-D-J recombination, and several other gene products such as TdT, Ku p70/80 and DNA-dependent protein kinase catalytic subunit are also involved in V-D-J recombination (27). To examine whether the signal from IL-7R affects the expression of these genes, we amplified cDNA prepared from adult thymocytes of IL-7R +/- and -/- mice by PCR with RAG-1, RAG-2, TdT, and Ku-80 primers, and hybridized with each cDNA probes (Fig. 3). The levels of RAG-1, RAG-2, TdT, and Ku-80 transcripts in IL-7R -/- mice were almost comparable to those of IL-7R +/- mice. These results suggest that the mutation of IL-7R did not inhibit the expression of RAG-1, RAG-2, TdT, and Ku-80 genes.


Fig. 3. Expression of V-(D)-J recombination-associating genes in the thymus of the IL-7R-deficient mice. cDNA prepared from 4-wk-old adult thymocytes was amplified by PCR using RAG-1, RAG-2, TdT, Ku-80, and HPRT primers, and the Southern blots of PCR products were hybridized with each probe.
[View Larger Version of this Image (64K GIF file)]


Discussion

TCR gamma  genes are frequently recombined in alpha beta T cells (28). More than 70% of Vgamma 1.2 and Vgamma 2 alleles are recombined in total thymocytes. In this study, we used this phenomenon to examine whether TCR gamma  recombination is blocked in alpha beta T cell precursors of IL-7R-deficient mice. IL-7R-deficient mice had no detectable TCR gamma  recombination by Southern blot analysis. Furthermore, the recombination of all the Vgamma genes was undetectable in fetal and adult thymi by PCR analysis. Thus, we demonstrated that the signal from IL-7R is indispensable for the V-J recombination of TCR gamma genes in alpha beta T cell precursors. And it is highly possible that the TCR gamma  recombination is also blocked in gamma delta T cell precursors as well as in alpha beta T cell precursors. This would be certainly one reason why IL-7Rdeficient mice lack gamma delta T cells.

There are three significant features in our observation. First, this blockade is specific for TCR gamma  genes. The recombination of TCR alpha , beta , and delta  genes are not affected. In addition, the recombination of IgH and L chain genes is probably not hampered by the mutation, because the IL-7Rdeficient mice have decreased but certain numbers of surface IgM+ B cells in the periphery (12). Second, the recombination of all the Vgamma genes is blocked. In a previous report, IL-7 induced the rearrangement of Vgamma 2 and Vgamma 4, but not Vgamma 3 or Vgamma 5 genes in fetal thymic organ culture of fetal liver precursors (2). In contrast, not only Vgamma 2 and Vgamma 4 but also all the other Vgamma genes in the TCR gamma 1, gamma 2, and gamma 4 clusters are hampered to recombine in the mutant mice. Third, TCR gamma  gene recombination is blocked not only in gamma delta but also in alpha beta T cell precursors. These features suggest the presence of highly specific regulation for TCR gamma  gene rearrangement.

To explain the specific inhibition of TCR gamma  recombination in the IL-7R-deficient mice, one possibility can be considered. It is to suppose that the TCR gamma  locus may contain a specific cis-control element(s). One possible candidate for this element is TCR gamma  enhancers. Recently, it was reported that IL-7 induces the phosphorylation and DNA binding activity of Stat5 protein in T cells (29). Because each TCR gamma  enhancer contains a Stat5 binding sequence (30, 31), Stat5 may play a role on the regulation of TCR gamma  recombination. It is also possible that some unknown factor(s) other than Stat5 may specifically regulate the recombination of TCR gamma  locus.


Footnotes

Address correspondence to Koichi Ikuta, Department of Medical Chemistry, Faculty of Medicine, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606, Japan.

Received for publication 9 August 1996

   This work was supported by a grant-in-aid from the Ministry of Education, Science, and Culture of Japan, and by the grant provided by the Ichiro Kanehara Foundation.

We thank Drs. Y. Takagaki, H. Yamagishi, O. Koiwai, and Y. Hashimoto for probes and discussion; Ms. M. Sugimori and M. Iidaka for technical assistance; Dr. S. Takeda for critically reading the manuscript; and Drs. M. Fujiwara and T. Honjo for encouragement.


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Copyright © 1996 by The Rockefeller University Press.