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International Immunology, Vol. 15, No. 3, pp. 393-402, March 2003
© 2003 Japanese Society for Immunology

FEATURED ARTICLE OF THE MONTH

Expression of recombination-activating gene in mature peripheral T cells in Peyer’s patch

Eisuke Kondo1, Hiroshi Wakao1, Haruhiko Koseki2, Toshitada Takemori3, Satoshi Kojo1, Michishige Harada1, Minako Takahashi1, Sakura Sakata1, Chiori Shimizu1, Toshihiro Ito1, Toshinori Nakayama1 and Masaru Taniguchi1

1 Laboratory for Immune Regulation, RIKEN Research Center for Allergy and Immunology, and Department of Molecular Immunology, and 2 Department of Molecular Embryology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan 3 Department of Immunology, National Institute of Infectious Diseases Tokyo 162-8640, Japan

Correspondence to: M. Taniguchi, Department of Molecular Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail: taniguti{at}med.m.chiba-u.ac.jp
Transmitting editor: M. Miyasaka


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Recombination-activating gene (RAG) 1 and 2 are essential for the gene rearrangement of antigen receptors of both T and B cells. To investigate RAG gene expression in peripheral lymphoid organs other than the thymus and bone marrow, we established mice in which a green fluorescent protein (GFP) gene is knocked-in the RAG2 gene locus (RAG2-GFP mice). In the thymus and bone marrow of heterozygous RAG2-GFP mice, as expected, GFP expression was detected in the appropriate stages of developing T and B cells. Interestingly, only a fraction of Thy-1.2+ cells in the Peyer’s patch were found to be GFP+ amongst the peripheral lymphoid organs. The GFP+ cells expressed high levels of surface TCRß and CD3, suggesting mature T cells with rearranged TCR{alpha}ß. However, they showed activated/memory phenotypes, i.e. CD45RBlow, CD69high, CD44high and CD62Llow, and belonged to a CD4+CD8+ population expressing c-kit, IL-7R and pT{alpha} characteristic of immature developing lymphocytes. Moreover, RAG+ Peyer’s patch T cells seem to be of thymic origin as judged by their expression of CD8{alpha}ß. These results show that there exists a fraction of mature T cells expressing RAG genes in the Peyer’s patch, implying a potential for a secondary rearrangement of TCR in extrathymic tissues.

Keywords: green fluorescent protein, knock-in mice, RAG2, TCR rearrangement


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The development of T and B cells is controlled by rearrangement of the genes coding for the variable region of the BCR or TCR, which are composed of variable (V), diversity (D) and joining (J) gene segments (1). The lymphocyte-specific proteins, recombination-activating gene (RAG) 1 and 2, initiate V(D)J recombination events. RAG genes have long been considered to be expressed only in immature lymphocytes in bone marrow and thymus, and are readily down-regulated in mature lymphocytes. In developing thymocytes, RAG1 and 2 are expressed first in CD4–CD8– double-negative (DN) thymocytes, which can be further subdivided based on CD44 and CD25 expression. RAG expression initiates at the CD44+CD25+ stage and continues to be detected at the next CD44–CD25+ stage in which TCRß, {gamma} and {delta} rearrangement commences (2,3). Functional V(D)J rearrangements lead to the cell-surface expression of pre-TCR composed of TCRß and pT{alpha} (4), and down-regulate RAG gene expression at the CD44–CD25– stage. Subsequently, during differentiation into CD4+CD8+ double-positive (DP) thymocytes, TCR{alpha} gene rearrangement occurs with high expression of RAG gene products (2,5). RAG expression is then down-regulated again during the maturation of DP thymocytes into CD4 or CD8 single-positive (SP) mature thymocytes (6).

Similarly, RAG gene expression in immature B cells is developmentally controlled. Two periods of B cell development express RAG genes. First, RAG is detected in pro-B cells that undergo IgH chain rearrangement (7). Functional IgH chain expression leads to pre-BCR expression and the down-regulation of RAG gene expression (8). Subsequently, RAG gene expression is up-regulated in later pre-B cells, coincident with the onset of IgL chain rearrangement (8). Then functional light chains lead to the expression of surface BCR and down-regulate RAG gene expression.

The developmental regulation of RAG gene expression described above has been confirmed by the establishment of RAG-green fluorescent protein (GFP) transgenic mice or RAG2:GFP fusion gene knock-in mice, in which GFP expression is under the control of endogenous RAG promoters in bone marrow and thymus (9,10). The majority of GFP+ cells were found to be pro-B cells, pre-B cells and immature B cells in bone marrow and DP immature T cells in the thymus, while no mature B and T cells were found to be GFP+ in the RAG gene-manipulated mice, suggesting the physiological expression of GFP in immature lymphocytes. It has been previously reported that the expression of RAG genes was demonstrated by PCR and immunofluorescence in germinal center (GC) B cells that phenotypically resembled immature bone marrow B cells (11–15). From these results, it has been hypothesized that the GC, but not other tissues, is the site in which mature B cells induce RAG and mediate the secondary rearrangement, responsible for an additional means of somatic receptor diversification. However, analysis of reporter mice in which a RAG2:GFP or RAG1:GFP fusion gene replaces the endogenous RAG locus suggest that RAG+ immature B cells from bone marrow accumulate in the spleen by effects of adjuvant and infectious agents, which could be the source of ‘RAG+ GC B cells’ (10,16,17).

In the case of mature T cells, RAG gene re-expression in peripheral CD4+ T cell has been described in a murine TCRß chain transgenic model, in which tolerogen-mediated chronic peripheral selection against cells expressing the transgenic TCR led to the down-regulation of TCR expression and the up-regulation of RAG gene expression, and resulted in the surface expression of endogenous TCRß chains (18). Similarly, RAG gene expression has been demonstrated in human CD4+CD3low peripheral T cells with low TCR expression (19). These T cells showed unusual characteristics of altered TCR expression. However, RAG gene expression in peripheral mature T cells under physiological conditions remains unclear. It has also not been determined which organ is responsible for the induction of RAG gene expression in the peripheral lymphoid tissues. Here, we established RAG2-GFP knock-in mice to investigate RAG gene expression in mature T cells in peripheral lymphoid tissues under physiological conditions.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Establishment of RAG2 knockout, GFP knock-in mice
A 4.0-kb SalI–XbaI 129/sv genomic DNA fragment containing the RAG2 gene was modified (20). A 0.85-kb PstI fragment containing the RAG2 open reading frame was replaced by a 0.65-kb enhanced GFP (EGFP) coding region, generated by PCR with template EGFP-C1 (Clontech, Palo Alto, CA). The EGFP fragment was ligated in-frame at the RAG2 start codon. A 1.2-kb pMC1 neo cassette (Stratagene, La Jolla, CA) cloned between two flanked 34-bp lox P signal sequences (Invitrogen, Carlsbad, CA) was inserted at the 3'-end of the EGFP fragment. The resulting RAG2 mutated fragment was subcloned into a pPNT TK vector (targeting vector, Fig. 1A). Electroporation of the targeting vector to R1 embryonic stem (ES) cells was performed as described (21). Seven homologous mutant clones were identified by Southern blot analysis with 32P-labeled 0.5-kb 3'-external probe (XhoI–SalI 0.5-kb 5'-RAG2 intron fragment). ES clones containing a mutated RAG2-GFP allele were aggregated with BDF1 female mouse morula in aggregation drops as described (21). After culturing at 37°C for 24 h in a CO2 incubator, the aggregated blasto cysts were transplanted into the uteri of foster mothers. Southern blot analysis of tail DNA using a specific probe for the mutant allele was performed. The expected 7.1-kb fragments in the XbaI digest and 3.7-kb fragment in the XbaI/EcoRI digest were detected in mice carrying the mutant allele (Fig. 1B). The heterozygous mutant (RAG2-GFP hetero) mice were subsequently crossed to generate homozygous mutant (RAG2-GFP homo) mice.



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  		Fig. 1. Generation of RAG2 knockout, GFP knock-in (RAG2-GFP) mice. (A) Design of the RAG2 knockout, GFP knock-in targeting construct. The genomic restriction endonuclease map of the 6.0-kb XbaI fragment of the RAG2 locus clone. XhoI (X), SalI (S), PstI (P) and EcoRI (E) sites are indicated. The vector was constructed as described in Methods. The 0.85-kb PstI fragment of the RAG2 coding region was replaced by a 0.65-kb EGFP-coding fragment and pMC1 neo SalI–XhoI 1.2-kb cassette within two flanked lox P signals. The predicted structure of the RAG2-EGFP mutant allele before pMC1 neo cassette deletion is shown at the bottom. SalI–XhoI 0.5-kb 5'-RAG2 external probe (Ex) is indicated. (B) Southern blot analysis with a 5'-external probe on XbaI and XbaI/EcoRI digestion isolated from wild-type ES cells (wild-type) and ES cells targeted with the RAG2 knockout, GFP knock-in construct (RAG-GFP hetero). This yielded a 7.1-kb fragment by XbaI digestion and a 3.7-kb fragment by XbaI/EcoRI digestion for the targeted allele.

 
Lymphocyte preparation
Freshly prepared thymocytes, spleen cells, peritoneal exudate cells (PEC), mesenteric lymph node cells, liver lymphocytes and Peyer’s patch cells were suspended in PBS supplemented with 2% FCS and 0.1% sodium azide. Intestinal intraepitherial lymphocytes (iIEL) were prepared with Percoll (Amersham-Pharmacia, Little Chalfont, UK). Briefly, the intestine was put inside out after washing, cut into four pieces and incubated in 1 x HBSS supplemented with 5% FCS under vigorous agitation at 37°C. The resulting suspensions were passed through the column filled with glass wool and pelleted through 30% Percoll solution. Cells were resuspended in 44% Percoll solution and overlaid onto 70% Percoll solution. After centrifugation, the interphase containing iIEL was recovered and subjected to further analysis.

Flow cytometry analysis
In general, 106 cells were incubated on ice for 30 min with FITC-, phycoerythrin (PE)-, CyChrome-, allophycocyanin (APC)-, Cy5- and biotin-conjugated mAb as described (22). The following mAb were purchased from PharMingen (San Jose, CA): CD4–PE, CD8–Cy5, CD69–PE, NK1.1–PE, CD25–PE, HAS (CD24)–PE, CD90–PE, Thy-1.2–PE, TCR{alpha}ß–Cy5, CD4–Cy5, CD8–Cy5, B220–APC, CD44–biotin, IgM–biotin, Mac-1–biotin, Gr-1–biotin, CD4–biotin, CD8–biotin, c-kit–biotin, IL-7R{alpha}–biotin, CD3{epsilon}–biotin, CD45RB–biotin, CD62L–biotin and CD69–biotin. In order to visualize biotin-conjugated mAb, Texas red–streptavidin, Red613–streptavidin or CyChrome–streptavidin was used (PharMingen). For direct staining, cells were first incubated with an anti-Fc{gamma}RII/III (2.4G2; PharMingen) to prevent the non-specific binding of mAb via FcR interactions. Dead cells were excluded by staining with propidium iodide (PI). Stained cells were analyzed on a FACS Vantage (Becton Dickinson, San Jose, CA) and an Epics XL (Coulter, Palo Alto, CA). Data were analyzed with CellQuest (Becton Dickinson) and Flow Jo software (Tree Star, San Jose, CA).

For B cell analysis, single-cell suspensions were prepared from bone marrows and the red blood cells depleted by incubation in 0.83% NH4Cl. Cells were blocked with 20 µg/ml anti-Fc{gamma}RII/III (2.4G2), and incubated with a mixture of biotinylated antibodies against IgM, CD23, CD90, CD5, TER119 and NK1.1 (PharMingen, San Jose, CA), and IgD, Mac-1 and Gr-1 (Southern Biotechnology Associates, Birmingham, AL). After washing, the cells were incubated for 30 min with a mixture of antibodies [APC-conjugated anti-B220 (anti-B220APC), PE-conjugated anti-CD43 (anti-CD43PE) and Texas Red (TX)-labeled anti-CD24 (anti-CD24TX)] and with TriColor (TC)-conjugated streptavidin (streptavidinTC; Caltag, Burlingame, CA) and propidium iodide. Cells recognized by the biotinylated mAb and dead cells were excluded by gating, and viable B220+/dull cells were then selected under a lymphocyte gate on forward with side light scatter. In some experiments, the cells were incubated for 30 min with a mixture of biotinylated antibodies against anti-IgM, IgD and CD23 and anti-B220APC followed by incubation with PE-conjugated streptavidin and PI.

RNA preparation and RT-PCR
Approximately 45,000 Thy-1.2+(TCRß+) GFP– and 25,000 Thy-1.2+(TCRß+) GFP+ cells from the Peyer’s patches were resuspended with TRIzol solution (Invitrogen, Carlsbad, CA). Between 1000 and 100,000 thymocytes were also resuspended with TRIzol solution. RNA extraction was performed with chloroform followed by isopropanol precipitation with glycogen. Total RNA prepared from sorted cells or from kidney and thymocytes was subjected to reverse transcription into cDNA with oligo(dT) primer using SuperScript II reverse transcriptase (Invitrogen). PCR reaction with ß-actin primers was performed to ensure the equal amounts of cDNA. The primers used for each PCR reaction was described previously (23,24).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
GFP expression in developing thymocytes of heterozygous and homozygous RAG2-GFP mice
GFP expression was studied on developing thymocytes. The expression of RAG2 gene is strictly regulated developmentally (3,6). The cell number, CD4/CD8 and TCR{alpha}ß/CD69 profiles, and CD44/CD25 profiles of CD4–CD8– (DN) thymocytes were similar between wild-type and heterozygous RAG2-GFP mice (Fig. 2A, C and E, left and middle panels). The number of thymocytes in homozygous RAG2-GFP mice was ~100-fold lower and only DN thymocytes were detected (Fig. 2A). The developmental block appears to be at the CD44–CD25+ DN thymocyte stage, similar to that of RAG2-deficient mice (Fig. 2C) (20).



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  		Fig. 2. GFP expression in the developing thymocytes of RAG2-GFP mice. Flow cytometric analysis was performed on thymocytes isolated from RAG2-GFP heterozygous mice, homozygous mice and wild-type mice after staining for the indicated cell-surface markers. Cell yield is indicated by the boxed numbers and the percentages of cells are shown in the respective quadrants. A wild-type histogram (no GFP expression, shaded area) was overlaid with that of RAG2-GFP heterozygous (solid line) and homozygous (bold line) mice. (A and B) Thymocytes were stained with anti-CD4–PE and anti-CD8–Cy5. CD4–CD8– DN, CD4+CD8+ DP, CD4 SP and CD8 SP subsets were electronically gated and analyzed for GFP expression. (C and D) Thymocytes were stained with anti-CD4–APC, anti-CD8–APC, anti-CD25–PE, anti-CD44–biotin and avidin–Red613. APCbright cells were gated out electronically. CD44+CD25–, CD44+CD25+, CD44–CD25+ and CD44–CD25– subsets of CD4–CD8– DN thymocytes were gated and analyzed for GFP expression. (E and F) Thymocytes were stained with anti-CD69–PE and anti-H57 (TCRß)–Cy5. CD69–TCR– (gate #1), CD69intTCRint (gate #2), CD69+TCR+ (gate #3) and CD69–TCR+ (gate #4) subsets were gated and analyzed for GFP expression.

 
Representative GFP expression profiles of electronically gated DN, CD4+CD8+ (DP), CD4+CD8– (CD4 SP) and CD4–CD8+ (CD8 SP) subsets are shown in Fig. 2B. As expected, GFP expression was detected in DN and DP subsets, but not in the CD4 SP or CD8 SP subset. The expression levels of GFP in DN cells in homozygous RAG2-GFP mice were significantly higher than in heterozygous RAG2-GFP mice. In the DN subset, GFP expression was observed in the CD44+CD25+, CD44–CD25+ and CD44–CD25– stages, suggesting a normal expression pattern of the RAG gene (Fig. 2D). These results are consistent with those of a previous report (10). Furthermore, GFP expression was down-regulated between small resting and large cycling cells in the CD44–CD25+ subset (data not shown). GFP expression was also analyzed in subsets defined by CD69 and TCR{alpha}ß expression (Fig. 2E and F). The level of both CD69 and TCR{alpha}ß appears to increase concomitantly (gate #1 -> gate #2 -> gate #3) during the transition between DP and SP thymocytes (23). As shown in Fig. 2F, GFP expression was shut off in the late DP subset, CD69highTCRhigh cells (gate #3).

GFP expression in developing B cells in bone marrow
Similar to T cells, the development of B cells in heterozygous RAG2-GFP mice was normal as determined by cell number, IgM/B220 profiles and HSA/B220 profiles (data not shown). Again, as we expected, GFP+ cells were detected in HSA+ and HSAdull B220+ bone marrow subsets containing pre-B and pro-B cells (Fig. 3A, d and e) and in IgM–B220+cells (Fig. 3B, c). GFP expression seemed inversely correlated with the surface IgM level. In homozygous RAG2-GFP mice, absolute numbers of bone marrow cells decreased by about half and B cell development was blocked at the B220+IgM– pro-/pre-B cell stage (data not shown). These results are consistent with those of RAG2-deficient and RAG2:GFP fusion gene knock-in mice (10,20).



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  		Fig. 3. GFP expression in developing B cells in bone marrow of RAG2-GFP mice. (A) Bone marrow cells were obtained from RAG2-GFP (c) and littermate mice (a and b). The cells were incubated with biotinylated mAb against IgM, IgD, CD23, CD90, CD5, Gr-1, Mac-1, NK1.1 and TER119, followed by staining with B220APC, HSATX and CD43PE mAb. These cells were stained with avidin–TriColor and PI. Cells recognized by the biotinylated mAb and PI+ dead cells were excluded from analysis by gating. Viable cells were analyzed under a lymphocyte gate on forwarded and side light scatters. B220+/dull cells were gated (G1 in a) and separated into HSAhigh (G2), HSAdull (G3) and HSA– (G4) in (b) and (c). Histograms represent the relative number of GFP+ cells in the HSAhighB220+ (d), HSAdullB220+ (e) and HSA–B220dull (f) populations of RAG2-GFP mice (solid line) and littermates (dotted line). (B) Bone marrow cells from heterozygous RAG2-GFP mice were incubated with biotinylated mAb against IgM, IgD and CD23, followed by staining with B220APC and with avidin–PE and PI. Viable and B220+/dull cells were analyzed under a lymphocyte gate (a), as described in (A). Ig+ cells, including immature B cells and recirculating B cells, and Ig– B cells were gated in G1 and G2 respectively. Histograms show the expression of GFP in Ig+ (b) and Ig– B cells (c).

 
GFP expression in a Thy-1.2+ subset of the Peyer’s patch
The results obtained thus far suggest that GFP expression faithfully reflects the expression pattern of the endogenous RAG2 gene products. Therefore, heterozygous RAG2-GFP mice would be useful animals for identifying T cell populations with RAG gene expression in peripheral lymphoid organs. Consequently, we sought GFP-expressing T cells in peripheral lymphoid organs including spleen, inguinal lymph nodes (ILN), iIEL, liver, PEC and Peyer’s patch in heterozygous RAG2-GFP mice. Among the samples tested, we found significant GFP expression in the Peyer’s patch, but not in other secondary lymphoid organs (Fig. 4A). Since it has been reported that RAG is expressed in a small CD3– population of iIEL (5,25,26), we further examined GFP expression in iIEL after staining with anti-CD3 (Fig. 4B, upper panel). However, we did not detect GFP+ cells in the CD3– iIEL populations (Fig. 4B, lower panel).



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  		Fig. 4. GFP expression in lymphocytes from secondary lymphoid tissues. (A) Lymphocytes in the thymus, spleen, ILN, iIEL, liver, PEC and Peyer’s patch were prepared, and GFP expression was determined by flow cytometric analysis. Shaded areas represent background fluorescence in wild-type mice. (B) GFP expression in CD3– fractions in iIEL. After staining with anti-CD3{epsilon}, gated CD3{epsilon}– fractions were measured for GFP expression (upper panel). Histograms of cells from RAG2-GFP heterozygous mice (line) from gate #1 plus #2 or from gate #2 are overlaid with histograms of wild-type cells (shaded).

 
The GFP+ cells in the Peyer’s patch were Thy-1.2+ and represented ~0.1–3% of Peyer’s patch Thy-1.2+ cells (Fig. 5A). To examine further the characteristics of these cells, the expression of various cell-surface markers was compared between Thy-1.2+GFP+ (gate #3) and Thy-1.2+GFP– (gate #2) cells (Fig. 5B and C). The GFP+ Peyer’s patch T cells showed a mature T cell phenotype with rearranged TCR{alpha}ß receptors on the cell surface, as they expressed very high levels of CD3{epsilon} and TCRß (Fig. 5B). They also expressed molecules characteristic of developing immature lymphocytes, such as IL-7R{alpha}, c-kit and CD44. An activation marker antigen, CD69, was also positive. In addition, they did not express CD62L, which is positive in the majority of CD4+CD8+ thymocytes. The unique phenotypic features indicate that GFP+ Peyer’s patch T cells are not immature thymocyte emigrants, although the majority of the GFP+ T cells were revealed to be of a CD4+CD8+ DP phenotype (Fig. 5E). As shown in Fig. 5C, GFP+ DP Peyer’s patch T cells expressed CD8ß, suggesting a subset developed in the thymus (CD8{alpha}ß-expressing cells), but not in the gut (27–30). Interestingly, low but significant levels of IL-7R{alpha}-expressing cells were also detected in Peyer’s patch T cells from wild-type mice (Fig. 5F), suggesting physiological consequences of these cells.



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  		Fig. 5. GFP expression in Peyer’s patch T cells. Flow cytometric analysis was performed with 30 x 106 Peyer’s patch cells from pooled RAG2-GFP heterozygous and wild-type mice. (A) The gates used for multicolor analysis (#1, #2 and #3) are shown. The percentages of cells in gate #3 are depicted. (B and C) Peyer’s patch lymphocytes were stained with anti-Thy-1.2–PE and several biotinylated antibodies. Thy-1.2+GFP+ (gate #3 shown in A) and Thy-1.2+GFP– (gate #2 shown in A) subsets were electronically gated, and the expression of CD3{epsilon}, TCR{alpha}ß, IL-7R{alpha}, c-kit, CD69, CD45RB, CD44, CD62L, CD8ß and NK1.1 was evaluated. The background staining histogram (shaded) was overlaid on those showing the Thy-1.2+GFP– (solid line) and Thy-1.2+GFP+ (bold line) populations. In the panels showing CD8ß and NK1.1 staining in (C), staining profiles of Thy-1.2+GFP– (shaded) and Thy-1.2+GFP+ (open) cells were overlaid. (D and E) Peyer’s patch lymphocytes were stained with anti-CD4–PE and anti-CD8–Cy5. CD4–CD8– DN, CD4+CD8+ DP, CD4 SP and CD8 SP subsets were gated and analyzed for GFP expression. Shaded areas represent background fluorescence in wild-type mice. (F) IL-7R{alpha} expression on Thy-1.2+ Peyer’s patch T cells (using gate #1 shown in A) from wild-type and RAG2-GFP heterozygous mice. The shaded area represents background staining with an isotype-matched control mAb.

 
GFP signal correlates with the endogenous RAG expression in GFP-RAG2 heterozygous mice
Finally, we have performed RT-PCR to confirm that the observed GFP signal indeed corresponded to the endogenous RAG expression (Fig. 6). Both Thy-1.2+GFP+ and Thy-1.2+GFP– cells were sorted from Peyer’s patches of RAG2-GFP heterozygous mice. After RT-PCR, PCR products were subjected to the Southern blot analysis (Fig. 6). As anticipated, both RAG1 and RAG2 mRNA were detected in Thy-1.2+GFP+ cells, but not in Thy-1.2+GFP– cells (Fig. 6, lanes 2 and 3). The expression level of RAG1 and RAG2 in Thy-1.2+GFP+ population was equivalent to that of 1000 thymocytes respectively (Fig. 6, cf. lane 3 and 6). Further analysis demonstrated that there was mRNA for the surrogate TCR{alpha} chain (pT{alpha}) in the Thy-1.2+GFP+ and Thy-1.2+GFP– cells (Fig. 6). Intriguingly, pT{alpha}b mRNA was almost absent in the Thy-1.2+GFP– populations (Fig. 6, lane 2). As for pT{alpha}a expression, a higher level of the mRNA was present in Thy-1.2+GFP– populations than Thy-1.2+GFP+ cells (Fig. 6, cf. lane 2 and 3). This pT{alpha} expression profile was in contrast to that of thymus (31). There was less CD3{epsilon} in Thy-1.2+GFP+ cells than in Thy-1.2+GFP– cells (Fig. 6, cf. lane 2 and 5). The level of CD3{epsilon} in Thy-1.2+GFP+ cells was almost equivalent to that in 1000 thymocytes (Fig.6, cf. lane 3 and 6). These data clearly demonstrated that the mRNA expression profile in Thy-1.2+GFP+ cells was distinct from that of thymocytes.



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  		Fig. 6. RAG1, RAG2, pT{alpha} and CD3{epsilon} mRNA expression in Thy-1.2+ Peyer’s patch cells. After sorting, 1.0 x 104 cells from the Thy-1.2+GFP– and Thy-1.2+GFP+ fractions from RAG2-GFP heterozygous mice were used for RT-PCR analysis. For comparison, equal numbers of cells from kidney and 1–100,000 thymocytes were subjected to RT-PCR analysis. ß-Actin was used as a control to ensure the equal amount of mRNA recovered from these fractions. PCR products were electrophoresed on agarose gel and detected by probes specific for each transcript.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
RAG2-GFP knock-in mice (RAG2:GFP mice) were established in order to investigate the presence of lymphocytes expressing RAG2 gene, particularly in the secondary lymphoid tissues. In homozygous RAG2-GFP mice, T and B cell development was completely arrested in a manner similar to that described previously in RAG2-deficient mice (20). In heterozygous RAG2-GFP mice, both T and B cell development appeared to be normal, and GFP expression was found in the expected subsets of developing lymphocytes (Figs 2 and 3).

Three lines of similar gene-manipulated GFP mice have been reported (9,10,32), and all focus on the development of B cells and editing of BCR molecules. Monroe et al. established mice in which a functional GFP:RAG2 fusion gene is knocked-in the endogenous RAG2 locus. In these mice, GFP-expressing B cells are B220+CD43high pro-B cells and B220+CD43low pre-B cells in bone marrow, and B220+pB130-140high immature B cells in spleen (10). Yu et al. established transgenic mice carrying an artificial bacterial chromosome that encodes GFP instead of RAG2. In these mice, GFP is expressed in all immature B cells, including B220+CD43+ pro-B cells, B220lowCD43–IgM– pre-B cells in bone marrow and B220+493+ transitional B cells in spleen, while B220+CD43–, B220+HSA+ mature B cells are negative for GFP expression (9). Igarashi et al. described heterozygous RAG1-GFP/neo knock-in mice in which the neo gene is present in the forward orientation upstream of the RAG gene. GFP+ cells were found to be B220+ immature B cells in the GC of lymphoid follicles in the spleen, axillary and mesenteric lymph nodes, and Peyer’s patch (33). As for T cell development in these gene-manipulated animals, GFP or RAG gene expression was confined to the thymus and was restricted to immature populations, including CD44–CD25+, CD44+CD25+ and CD44–CD25– DN subsets. No GFP+ cells were reported in the peripheral T cell populations.

Unlike these reports, we found that small but significant GFP+ and Thy-1.2+ populations are present in the Peyer’s patch, but not in other secondary lymphoid tissues so far analyzed (Fig. 4). The GFP+ cells in the Peyer’s patch are clearly categorized as mature T cells because of their high level Thy-1.2, CD3{epsilon} and TCRß expression (Fig. 5). Interestingly, GFP+ Peyer’s patch T cells show unique phenotypes such as being CD4+CD8+ and CD44+, being reminiscent of developing immature thymocytes (Fig. 5). In addition, this population also possesses an activation marker, CD69, making the cells resemble activated peripheral T cells or thymocytes undergoing selection events (34). The most intriguing finding is that GFP+ Peyer’s patch T cells express characteristic molecules in developing lymphocytes, such as IL-7R, c-kit and pT{alpha}, despite their high expression of TCRß and CD3{epsilon} (Fig. 5B and C).

In addition to these unique features, the expression profile of pT{alpha}a and pT{alpha}b in Thy-1.2+ Peyer’s patch cells is worthy of note (Fig. 6). Given that pT{alpha}a retains TCRß chain within the cells, while pT{alpha}b allows its cell-surface expression (31), abundance of pT{alpha}b in GFP+ cells may permit high level of TCRß expression (Fig. 5B). The dominant expression of pT{alpha}b over pT{alpha}a in GFP+ cells suggests that these cells are similar to mature T cells (31,35). Together, these data support the notion that Thy-1.2+GFP+ Peyer’s patch cells are not immigrants from thymus that represent merely the immature T cells, but rather indicate that Thy-1.2+ GFP+ cells in the Peyer’s patch constitute a novel T cell subset.

In RAG2-transgenic mice, GFP is not synthesized as a fusion protein, but this strain carries a 165-kb bacterial artificial chromosome, and fluorescence levels are much higher with a dynamic range of 3–4 log (9). However, the half-life of GFP in this strain is 2–3 days, probably much longer than that of the endogenous RAG2 protein (16). In contrast, the dynamic range of GFP in RAG2-GFP knock-in mice is ~1 log over background (Figs 2 and 3) (10). Although the GFP signal may not truly reflect the expression of RAG2 in RAG2-GFP knock-in mice, we detected RAG1 and RAG2 mRNA by RT-PCR in the GFP+Thy-1.2+, but not in the GFP–Thy-1.2+ fraction in the Peyer’s patch of RAG2-GFP knock-in mice (Fig. 6). This unequivocally demonstrates that GFP expression observed in our RAG2-GFP mice directly mirrors the expression of RAG2.

Some particular microenvironments, such as that in Peyer’s patch, are thus likely to induce signals or provide cellular scaffolding to promote the re-expression of the recombination machinery in mature T cells. Some T cells with memory or immature phenotypes are present in normal Peyer’s patch (Fig. 5F) and anti-ovalbumin TCR transgenic mice (36).

As well as our present data, messages for the RAG1 and/or 2 genes have been found to be present in TCRß chain transgenic mouse (18) and in human CD4+ T cell clones with altered surface expression of TCR, such as low or defective expression of CD3{epsilon} and TCR{alpha}ß (19). Although the level of RAG expression is low (103- to 104-fold less than in thymocytes), RAG1/RAG2+ clones undergo secondary TCR rearrangements (19). Furthermore, mature T cells with altered TCR and RAG expression are present in increased numbers in patients with defective responses to DNA damage (37). Thus, RAG expression in these cells may correlate with the cellular requirement to rescue them from defective phenotypes. However, this is not the case in the present study, since RAG expression in T cells in Peyer’s patch was observed under normal physiological conditions. Because of the high level of TCRß and CD3{epsilon} expression, these GFP+ T cells are distinct from those with altered TCR expression (Fig. 5B). Thus, RAG2-GFP mice may be interesting models to show the presence of receptor revision in mature T cells and to address its physiological relevance.

In line with this expectation, we have detected a high frequency of rearrangement of TCR{alpha} and ß loci in GFP+, but not in GFP–, Peyer’s patch T cells as judged by the presence of DNA double-strand breaks and circular DNA associated with recombination events. In addition, the profile of DNA double-strand breaks in GFP+ Peyer’s patch T cells was different from that in TCRß+ CD4+CD8+ DP cells in thymus, indicating that these cells were not migrants from thymus harboring a residual GFP signal without RAG expression and recruited into the Peyer’s patch (H. Wakao et al., submitted for publication).

Finally, it is worth noting that GFP+ T cells in Peyer’s patch are not related to recently identified RAG+ immature extrathymic T cells, cryptopatches in the small intestine (38). Immature cryptopatch T cells are CD8{alpha}{alpha} T cells. In contrast, GFP+ Peyer’s patch mature T cells express CD8{alpha}ß, but not CD8{alpha}{alpha}, indicating that they are thymus-derived and not gut-derived T cells. Although previous studies demonstrated that there exists RAG transcripts in a CD3– fraction of iIEL, we did not detect GFP+ cells in the CD3– fraction of iIEL of RAG2-GFP knock-in mice (Fig. 4B) (5,25,26). This is most likely due to the difference in sensitivity between FACS and other analysis such as in situ hybridization and PCR.

In conclusion, we have identified a novel subset of T cells in the Peyer’s patch, whose physiological role is yet to be elucidated.


   Acknowledgements
 
The authors thank Ms Hiroko Tanabe and Kaoru Sugaya for preparation of this manuscript. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology (Japan) (Grants-in-Aid for Scientific Research, Priority Areas Research 13218016 and 12051203, Scientific Research A 13307011, B 14370107 and C 12670293, and Special Coordination Funds for Promoting Science and Technology), the Ministry of Health, Labor and Welfare (Japan) (the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research), and the Human Frontier Science Program Research Grant (RG00168/2000-M206).


   Abbreviations
 
APC—allophycocyanin

B6—C57BL/6

DN—double negative

DP—double positive

EGFP—enhanced green fluorescent protein

ES—embryonic stem

FCM—flow cytometry

GC—germinal center

GFP—green fluorescent protein

iIEL—intestinal intraepitherial lymphocytes

ILN—inguinal lymph nodes

PE—phycoerythrin

PEC—peritoneal exudate cell

PI—propidium iodide

RAG—recombination-activating gene

SP—single positive


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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