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Distinct Requirements for IL-7 in Development of TCR Cells During Fetal and Adult Life¹

Karen Laky^2,*, Julia M. Lewis, Robert E. Tigelaar and Lynn Puddington^3,*

^* Department of Medicine, Division of Immunology, University of Connecticut Health Center, Farmington, CT 06030; and Department of Dermatology, Yale University School of Medicine, New Haven, CT 06520

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
TCR-transgenic IL-7^-/- mice were generated to determine whether T cells containing productively rearranged TCR genes have additional requirements for IL-7 within the thymus or peripheral lymphoid tissues. Differences in developmental requirements for IL-7 by TCR cells were noted and were linked to derivation from fetal- vs adult-type precursors in the thymus. Although TCR cells are absent from IL-7^-/- mice, TCR cells were restored to the thymus and periphery by expression of TCR transgenes. Endogenous TCR chains were expressed by IL-7^+/- but not IL-7^-/- TCR-transgenic mice, providing direct support for findings that IL-7 is necessary for rearrangement and expression of TCR genes. The number of TCR thymocytes was 10-fold reduced in TCR-transgenic IL-7^-/- embryos; however, adult TCR-transgenic IL-7^-/- or IL-7^+/- mice had similar numbers of fetal thymus-derived TCR cells in their skin. Thus, fetal TCR cells required IL-7 for TCR rearrangement, but not for proliferation or survival in the periphery. In contrast, the numbers of TCR cells in other tissues of TCR-transgenic IL-7^-/- mice were not completely restored. Moreover, coincident with the transition from the first to second wave of T cell precursors maturing in neonatal thymus, thymus cellularity of TCR-transgenic IL-7^-/- mice dropped significantly. These data indicated that in addition to TCRV gene rearrangement, TCR cells differentiating from late fetal liver or adult bone marrow precursors have additional requirements for IL-7. BrdU incorporation studies indicated that although IL-7 was not required for TCR cell proliferation, it was required to prolong the life span of mature TCR cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Interleukin-7 is required for development of mature TCR cells. TCR cells are absent from thymus, spleen, skin (dendritic epidermal T cells (DETCs)⁴) and intestinal epithelium (intraepithelial lymphocytes (IEL)) of mice deficient for IL-7, either component of the IL-7R (IL-7R or common chain, _c), or the _c signaling molecule Jak3 (1, 2, 3, 4, 5, 6, 7, 8). It has been reported that IL-7^-/- and _c^-/- fetal thymus contain a few immature TCR^low heat stable Ag (HSA)^high thymocyte precursors to DETCs (3, 4, 6). However, neither thymus has mature TCR^highHSA^low cells, and no DETCs are detectable in the epidermis of IL-7^-/- or _c^-/- mice (3, 9, 10). Thus, generation of mature TCR cells absolutely requires IL-7/IL-7R interactions.

IL-7 is synthesized by thymus stromal cells (10, 11, 12, 13, 14, 15). We have shown that development of thymus-derived TCR cells absolutely requires intrathymic IL-7 but does not require additional IL-7 in peripheral tissues (16). Thus, IL-7 must act on TCR precursors within the thymus, although the molecular events downstream of an IL-7 signal and whether there are multiple stages affected by IL-7 that ultimately result in thymic production and maintenance of mature TCR cells in the periphery have not been completely defined.

In vitro data demonstrate that IL-7 stimulates rearrangement of murine TCR genes. Addition of IL-7 to cultures of E14 fetal liver cells, adult BM T cell precursors, or mature TCR HSA^lowCD4⁺ thymocytes yields in-frame, junctionally diverse TCRV1.2, TCRV2, or TCRV4 transcripts (8, 17, 18, 19, 20). In all instances, TCR mRNAs are not found in cells cultured without IL-7. Analyses of TCR rearrangements in thymocytes from gene-deleted mice (IL-7R^-/- (two independently derived lines), _c^-/-, or Jak3^-/- mice) also demonstrated that IL-7R signaling facilitates TCRV gene rearrangement (2, 3, 7, 8, 21, 22, 23, 24, 25). IL-7R signaling regulates germline transcription and the accessibility of recombinase machinery to the TCRV locus (26, 27, 28, 29). These data suggest that IL-7R signaling is required for the initiation of TCR gene rearrangement.

In contrast, other groups have reported that PCR analyses of IL-7R^-/-, _c^-/-, or Jak3^-/- thymocytes reveal that TCRV-J gene rearrangements are present but severely reduced in comparison with wild-type thymocytes (6, 30, 31). In the case of IL-7^-/- fetal thymus, it was reported that in addition to being severely reduced in quantity, TCRV3 rearrangements are developmentally delayed (30). These data suggest that although _c cytokines greatly facilitate TCRV rearrangements, they are not absolutely required. If that is true, then _c cytokines must also be critically important for later stages of TCR thymocyte development because IL-7^-/-, IL-7R^-/-, _c^-/-, and Jak3^-/- mice all lack mature TCR cells.

Both chains of the heterodimeric IL-7R are integral components of other cytokine receptors. The -chain of the IL-7 receptor pairs with a novel chain to make up the thymic stromal lymphopoietin receptor (32, 33). Numerous cytokine receptors that influence T cell development/survival, i.e., IL-2, -4, -7, -9, -15, and -21 use _c and Jak3 (34). Therefore, it cannot be assumed that all of the defects observed in IL-7R^-/- mice are due solely to the absence of an IL-7-mediated signal. To definitively determine whether IL-7 is required for TCR gene rearrangement and to elucidate its contribution to later steps in TCR cell maturation, we generated IL-7^-/- mice (1) expressing rearranged G8 TCR transgenes (35, 36), thus bypassing any need for IL-7 during TCR gene rearrangement. Mature TCR^high cells were present in thymus and peripheral tissues of G8 IL-7^-/- mice. Moreover, endogenous TCRV chains were expressed on the cell surface of T cells isolated from G8 IL-7^+/- mice but not by T cells isolated from G8 IL-7^-/- mice. These data indicated that IL-7 was absolutely required for protein expression of endogenously rearranged TCRV genes. The number of TCR cells isolated from G8 IL-7^-/- animals was drastically reduced when compared with control G8 IL-7^+/- littermates. Decreased production of TCR cells by the thymus only partially accounted for this reduction. Pulse-chase experiments with BrdU revealed that the turnover rate of peripheral TCR cells was higher in G8 IL-7^-/- animals than in G8 IL-7^+/- littermate control animals, suggesting that IL-7 prolonged the life span of mature TCR cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

IL-7^-/--Ly5.1 mice were originally obtained from DNAX Research Institute of Molecular and Cellular Biology (Palo Alto, CA) and were maintained on a C57BL/6 x 129/Ola hybrid background, as previously described (1). A single line of G8 TCR-transgenic mice (35) was originally obtained from Steve Hedrick (University of California, San Diego, CA), and was maintained in our animal facility. IL-7^-/- females and G8 males were crossed to obtain H-2^b/d IL-7^+/- G8^+/- F₁ progeny. Male and female F₁ mice were intercrossed to obtain F₂ progeny. G8 TCRV2⁺ cells are deleted by H-2^b; therefore, peripheral blood leukocytes from F₂ animals were screened, and H-2^b/b and H-2^b/d animals were removed from the breeding colony. H-2^d/d animals were screened for IL-7 by Southern blot. F₂ H-2^d/d G8^+/+ IL-7^+/- were bred with H-2^d/d G8^-/- IL-7^-/- mice, and the resultant F₃ H-2^d/d G8^+/- IL-7^+/- or IL-7^-/- animals were analyzed. All G8^+/- mice used in this study were derived from a single line of G8 mice; therefore, G8 transgene copy number and integration site were held constant. All mice were fed sterile food and water and were housed in microisolators under specific pathogen-free conditions. Their welfare was in accordance with institutional and Office of Laboratory Animal Welfare guidelines.

Southern blotting

Approximately 10 µg of genomic DNA (prepared from 0.25 inch of tail) were digested with XbaI (Life Technologies, Gaithersburg, MD) and BamHI (New England Biolabs, Beverly, MA) and electrophoresed in a 0.8% Seakem Gold agarose gel (FMC Bioproducts, Rockland, ME). The DNA was transferred by capillary action to a Nytran membrane (0.45 µm, net neutral charge; Schleicher and Schuell, Keene, NH) in 10x standard saline citrate phosphate/EDTA. DNA was UV cross-linked to the membrane (2400 J) and hybridized overnight at 55°C in hybridization buffer (0.1 M Tris-HCl, 5 mM EDTA, 5 mg/ml heparin, 0.1% sodium pyrophosphate, 0.5% Sarkosyl, 10% dextran sulfate, 1 M NaCl, 30% formamide, 0.1 mg/ml sheared salmon sperm, pH 7.5) with a probe containing 3100 bp of intronic IL-7 gene sequence 3' of exon 5. DNA (25 ng) was labeled with [-³²P]dATP (Amersham Life Sciences, Cleveland, OH) using a Random Primers DNA Labeling System (Life Technologies). Excess, unincorporated [-³²P]dATP was removed by filtration through a Sephadex G-50 Quick Spin column (Boehringer Mannheim, Indianapolis, IN). IL-7^-/- mice have a neomycin cassette in place of IL-7 exon 4, which results in loss of an XbaI site present in the wild-type gene (1). A 6-kb band represents the wild-type gene, whereas a 14-kb band represents the exon 4-deleted IL-7 gene.

All blots were washed in 2x SSC for 15 min at room temperature, followed by one 30-min and then one 20-min wash in 0.1x SSC plus 0.2% SDS at 65°C. Bands were visualized by exposing membranes to Biomax MS film (Kodak, Rochester, NY) for >5 h with a BioMax TranScreen-HE intensifying screen (Kodak).

Lymphocyte isolation

Lymphocytes were isolated from thymus, spleen, and lymph nodes using a glass homogenizer and then passed through 100-µm pore size Nitex nylon mesh (Tetko, Kansas City, MO) to remove connective tissue. Splenic RBCs were lysed via two sequential incubations in Tris-ammonium chloride (13 mM Tris, 135 mM NH₄Cl, pH 7.2) for 4 min at 37°C. Before staining for flow cytometric analyses, splenocyte FcR were preblocked with affinity purified mouse IgG (200 µg/ml; Jackson ImmunoResearch Laboratories, West Grove, PA).

Isolation of intestinal IEL

Small intestine (gastroduodenal junction to the ileocecal junction) was cut longitudinally, and then into 5-mm pieces, and washed twice with Ca²⁺, Mg²⁺-free HBSS containing 1 mM HEPES and 2.5 mM NaHCO₃ (pH 7.3), and 2% FCS. Washed intestinal pieces were combined, and stirred at 37°C for 20 min in Ca²⁺, Mg²⁺-free HBSS containing 1 mM HEPES and 2.5 mM NaHCO₃ (pH 7.3), with 10% FCS and 1 mM dithioerythritol (Calbiochem, La Jolla, CA). This step was repeated and the cells in the supernatants from both treatments were combined and rapidly filtered through scrubbed nylon wool (NEN, Boston, MA). Cells were then centrifuged in a 44%/67.5% Percoll (Pharmacia, Piscataway, NJ) gradient. Viable cells at the interface were collected and prepared for flow cytometric analysis.

Isolation of DETC

To prepare DETC suspensions for fluorescence flow cytometric analysis abdominal and back skin was shaven with a straight razor, excess fat and blood vessels were removed, and the skin was cut into 1-cm-wide strips. Pieces were incubated epidermal side up in 0.3% trypsin (type XI; Sigma-Aldrich, St. Louis, MO) in 0.17% glucose, 0.88% NaCl, and 0.04% KCl (pH 7.6) at 37°C for 1.5 h. Epidermal sheets were separated from the underlying dermis by scraping and placed in fresh 0.3% trypsin with 0.01% DNase (ICN Nutritional Biochemicals, Cleveland, OH) at 37°C for 10 min with shaking. An equal volume of cold MEM with 10% FCS, 0.01% DNase, 100 U/ml penicillin, and 100 mg/ml streptomycin was added to inactivate the trypsin. Clumps of stratum corneum were removed by filtering the suspension through Nitex. The filtrate was then centrifuged at 1000 rpm for 10 min at 4°C in a Sorvall RTH-750. Pellets were resuspended in 4 ml of MEM with FCS, penicillin, and streptomycin and then underlaid with an equal volume of Histopaque 1083 (Gallard Schlesinger Chemical Manufacturing, Carle Place, NY). Gradients were centrifuged at 1200 rpm for 20 min at room temperature. Epidermal cells harvested at the interface (IEC) were washed once with MEM plus FCS, penicillin, and streptomycin and then counted. Viability was assessed via trypan blue exclusion. Before staining, IEC were cultured overnight in RPMI 1640 supplemented with 10% FBS, 25 mM HEPES, 20 mM L-glutamine, 10 mM sodium pyruvate, 30 mM 2-ME, nonessential amino acids, and penicillin-streptomycin, to allow reexpression of trypsin-sensitive epitopes. IEC suspensions were stained as described below.

In vivo proliferation experiments

Mice were provided with water ad libitum supplemented with 0.8 mg/ml BrdU (Sigma) for 7 days as described by Tough and Sprent (37). Where indicated, following the 1-wk BrdU labeling period, mice were returned to normal, unsupplemented water for an additional 14 days and then analyzed.

Monoclonal Abs

The following mAbs were used: anti-Thy-1.2-FITC or -PE (53-2.1); anti-CD3-FITC (145-2C11) (38) or anti-CD3-biotin (500A2); anti-TCR-PE or -biotin (GL3) (39); anti-TCRV5-FITC or -biotin (GL1) (39); anti-V2-FITC or -biotin (UC3-10A6); anti-TCRV1 (2.11) was a generous gift of P. Pereira (40); anti-TCRV3-FITC or -biotin (F536) (41); anti-TCRV4-FITC (GL2) (39); anti-TCR-FITC, -Cy-Chrome, or -PE (H57.597); anti-CD8-FITC (3.168) (42) or anti-CD8-PE (53-6.7); anti-CD8-FITC or -biotin (H35-17-2); anti-CD4-FITC (RM4–4) or anti-CD4-PE (GK1.5) (Becton Dickinson Collaborative Technologies, Bedford, MA) or anti-CD4-TriColor (CT-CD4) (Caltag Laboratories, South San Francisco, CA); anti-CD44-PE (IM7); anti-CD25-FITC (7D4); anti-HSA (CD24)-PE (M1/69); anti-CD62L-PE (Mel-14); anti-H-2K^b-PE (AF6-88.5); anti-H-2K^d-FITC (SF1-1.1); anti-BrdU-FITC (BD Biosciences, San Jose, CA). All mAbs were obtained from BD PharMingen (San Diego, CA) unless otherwise noted. Biotin-conjugated Abs were visualized with Streptavidin Red 670 (Life Technologies), streptavidin-PE, or streptavidin-Cy5 (Jackson ImmunoResearch Laboratories). Relative fluorescence intensities were measured with a FACScan or FACSCalibur (BD Biosciences).

Immunofluorescence analysis

A single-cell suspension of lymphocytes in PBS containing 0.2% BSA and 0.1% NaN₃ (PBS-BSA-NaN₃) was incubated with properly diluted mAb at 4°C for 20 min. After staining, cells were washed twice with PBS-BSA-NaN₃, and relative fluorescence intensities were measured by fluorescence flow cytometry. Fluorescence intensity is presented on a 4-decade log scale. A minimum of 10,000 cells within the forward scatter vs side scatter lymphocyte gate were analyzed in each sample.

Anti-BrdU staining was done as described by Tough and Sprent (37). Briefly, cells were stained with mAb conjugated to fluorochromes detected in FL2 (PE), and either FL3 (Red-670, Tricolor, or Cy-Chrome) or FL4 (APC or Cy5) as above, washed in PBS, fixed in 70% ethanol for 30 min, and then permeabilized overnight in 1% paraformaldehyde, 0.01% Tween at 4°C. Following two washes in PBS, samples were incubated with 50 U/ml DNase (DNase I from bovine pancreas; Roche Molecular Biochemicals, Mannheim, Germany) in 0.9% NaCl (pH 5.0), for 10 min at 37°C, washed once with PBS-BSA-NaN₃ and once with PBS, and then stained with FITC-conjugated anti-BrdU mAb (1/10) for 30 min at room temperature. Following washing with PBS/BSA/NaN₃, samples were resuspended in PBS and analyzed immediately by fluorescence flow cytometry.

Statistical analyses

All two-tailed Student t tests were conducted using InStat Instant Biostatistics (GraphPad Software, San Diego, CA). Error bars represent the SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
TCR transgenes restored TCR cells in IL-7^-/- mice

IL-7^-/- mice lack mature TCR cells (4). To determine whether fully rearranged TCR transgenes were able to bypass the need for IL-7 during TCR cell development, we looked for TCR lymphocytes in the peripheral lymphoid tissues of G8 TCR-transgenic IL-7^-/- (G8 IL-7^-/-) mice. As expected, TCR cells were absent from nontransgenic IL-7^-/- animals and present in lymph node, spleen, and thymus of G8 TCR-transgenic IL-7^+/- (G8 IL-7^+/-) mice. TCR cells were also present in lymph node, spleen, small intestinal IEL, and peripheral blood of G8 IL-7^-/- mice (Fig. 1 and data not shown). This demonstrated that expression of TCR transgenes overcame the absence of IL-7 in generation of TCR lineage cells. Moreover, because the G8 TCRV2 transgene was under control of its endogenous regulatory regions, IL-7 was not absolutely required for either transcription or cell surface expression of the productively rearranged TCR gene.

View larger version (40K): [in this window] [in a new window]   FIGURE 1. TCR transgenes restored TCR cells in peripheral tissues of IL-7-/- mice. Lymphocytes were isolated from IL-7-/-, G8+/-IL-7-/-, or G8+/-IL-7+/- mice and stained with mAb against CD3, TCR, and TCRV2. Total CD3+ cells were positively gated and then analyzed for expression of TCR and the transgenic TCRV2. Numbers in the upper right quadrant represent the percentage of T cells expressing the transgenic TCR among total T cells. LN, Lymph nodes.

View larger version (28K): [in this window] [in a new window]   FIGURE 2. Some G8 transgenic T cells coexpressed TCR and TCR. SI-IEL were isolated from a 10-wk-old G8+/-IL-7-/- mouse and stained with mAb specific for CD3, TCR, and TCR or for TCRV2, CD8, and CD4. CD3+ cells were positively gated and then analyzed for expression of TCR or TCR (left). Total IEL were analyzed for expression of V2 along with CD8 or CD4 (middle or right). Similar results were obtained with G8+/-IL-7-/- lymph nodes and spleen cells and lymphocytes isolated from G8+/-IL-7+/- mice.

IL-7 was required for surface expression of endogenous TCR chains

The ability of TCR transgenes to preempt the requirement for IL-7 in the thymus suggested that IL-7 was critical for a stage of T cell development that preceded expression of a TCR. This was consistent with either IL-7 directly stimulating TCR gene rearrangement in normal mice or an indirect effect, i.e., premature expression of TCR providing a survival signal to TCR precursors. To begin to dissect this, the expression of endogenous TCR genes was examined. TCR genes are not strictly allelically excluded at the level of gene rearrangement (44). Therefore, we reasoned that if IL-7 merely provided a survival signal to TCR precursors, then both G8 IL-7^+/- and G8 IL-7^-/- mice would have some TCR cells that expressed endogenous TCR chains on their cell surface. However, if IL-7 was required to initiate TCR rearrangements, then endogenous TCR could be present only on the surface of T cells from G8 IL-7^+/- mice and not on T cells from G8 IL-7^-/- mice.

TCRV usage varies with anatomic location. Most peripheral blood and splenic TCR cells express TCRV2, the same V region encoded by the transgene. Therefore, we analyzed IEL from the small intestine or skin of G8 IL-7^-/- mice or G8 IL-7^+/- littermates for surface expression of their characteristic TCRV chains. TCRV5 is the predominant TCRV region used by small intestinal IEL (40, 45, 46), and TCRV3 is used exclusively by DETC in murine skin (47, 48). Although the vast majority of TCR IEL in the small intestine or skin of G8 mice were TCRV2⁺, it was not the only population of TCR cells in G8 IL-7^+/- mice (Fig. 3A). A small population of small intestinal IEL (2%) isolated from G8 IL-7^+/- mice expressed other TCRV, either alone (TCRV5 or TCRV1), or dual TCRV2/TCRV5 or TCRV2/TCRV1 (Fig. 3A). A larger percentage of DETC (30–40%) isolated from G8 IL-7^+/- mice expressed other TCRV, either exclusively TCRV3⁺ or dual TCRV2/TCRV3 (Fig. 3B, top and middle). This result was consistent with another TCRV2-transgenic mouse line, KN6, that has TCR⁺ DETC that do not express the transgenic TCRV2/V5 (49). T cells expressing endogenous tissue-characteristic TCRV were absent in IEL and DETC of IL-7^-/- mice (Fig. 3). Even after 3 wk of expansion in vitro with Con A plus IL-2, 10% of G8 IL-7^+/- DETC expressed TCRV3, whereas G8 IL-7^-/- DETC expressed only TCRV2⁺ (Fig. 3B, bottom). Thus, IL-7 had a direct effect on TCRV gene expression within TCR cells that ultimately resided in intestinal or skin epithelium.

View larger version (33K): [in this window] [in a new window]   FIGURE 3. IL-7 was required for expression of endogenous TCR chains. A, Small intestinal IEL were isolated from adult G8+/-IL-7-/- or G8+/-IL-7+/- mice and stained with mAb specific for TCR, TCRV2, and TCRV5 or for TCR, TCRV2, and TCRV1. TCR+ cells were positively gated and then analyzed for TCRV usage. Numbers indicate the percentage of cells expressing the indicated TCRV region among total TCR IEL. B, Skin epidermal cells (DETC) were isolated from 8.5-wk-old mice and then stained with mAb against TCR and TCRV2 or against TCRV2 and TCRV3 immediately (top and middle) or after 21 days of stimulation in vitro with IL-2 plus Con A (bottom). Numbers indicate the percentage of cells within the lymphocyte gate expressing the indicated TCRV region.

Fewer transgenic TCR cells developed in the thymus of IL-7^-/- mice

The presence of TCR cells in peripheral lymphoid tissues of G8 IL-7^-/- mice demonstrated that by directing surface expression of TCR, TCR transgenes bypassed at least the earliest requirement for IL-7 during TCR cell development. In vitro, IL-7 enhances proliferation and survival of TCR cells (50, 51, 52). Therefore, we analyzed the number of TCR cells in G8 IL-7^-/- mice. The density of DETC in the skin of 8.5-wk-old mice was not significantly different between G8 IL-7^-/- and G8 IL-7^+/- littermates (Fig. 4). However, the absolute number of TCRV2⁺ cells in all other peripheral lymphoid tissues of adult G8 IL-7^-/- mice was significantly reduced when compared with age-matched G8 IL-7^+/- control mice (Fig. 4). Such decreases could have resulted from reduced production, proliferation, or life span of TCR cells. The contribution of these three nonmutually exclusive possibilities was evaluated.

View larger version (25K): [in this window] [in a new window]   FIGURE 4. G8+/-IL-7-/- mice had reduced numbers of peripheral TCR cells. Lymphocytes were isolated from 5- to 35-wk-old G8+/-IL-7-/- or G8+/-IL-7+/- mice, stained with mAb against CD3, TCR, and TCRV2. The absolute number of TCRV2+TCR- cells in lymph node (LN), spleen, and IEL was calculated by multiplying the total number of cells isolated by the percentage of CD3+TCRV2+TCR- cells determined by fluorescence flow cytometry. The density of DETC in epidermal sheets from individual 8.5-wk-old mice was determined by counting the number of CD3+ cells per mm2 in four to six 1.5-cm2 pieces of skin prepared from each mouse. Values of p were calculated using unpaired Student t tests. Error bars, SEM.

View larger version (18K): [in this window] [in a new window]   FIGURE 5. IL-7 dependence of TCR thymocytes increased with age. Thymocytes isolated from G8+/-IL-7-/- or G8+/-IL-7+/- mice of increasing age were counted and stained with CD3, TCRV2, and TCR. The number of TCRV2+TCR- cells was calculated by multiplying the total number of cells isolated by the percentage of CD3+TCRV2+TCR- cells determined by fluorescence flow cytometry. Each data point represents the mean of 2–11 mice analyzed individually. Note the 10-fold difference in scale for IL-7-/- and IL-7+/- mice.

TCR cells proliferated more but had a decreased life span in IL-7^-/- mice

The phenotype of splenic and lymph node TCRV2 cells was assessed in G8 IL-7^-/- mice and their G8 IL-7^+/- littermates. The percentages of TCRV2 cells expressing Thy-1, CD62 ligand, CD45RB, or HSA were not significantly different (p 0.3) between G8 IL-7^-/- and G8 IL-7^+/- mice (Fig. 6). Although some difference in expression of CD44 was observed (p = 0.067), the biological significance of changes in expression of this particular phenotypic marker when accompanied by negligible changes in the others is uncertain. In addition, the paradigm of assignment of activation status as applied to TCR cells may not be as relevant for TCR cells (53). Thus, our conclusion was that although drastically reduced numbers of TCR cells were present in the periphery of G8 IL-7^-/- mice, few, if any, significant phenotypic differences were detected. This suggested that similar populations of TCR cells developed in both the presence and the absence of IL-7.

View larger version (36K): [in this window] [in a new window]   FIGURE 6. Similar populations of TCR cells were present in lymph nodes of G8+/-IL-7-/- or G8+/-IL-7+/- mice. Lymph node cells from several G8+/-IL-7-/- or G8+/-IL-7+/- mice were isolated, pooled, and stained with V2, TCR, and a panel of mAb that typify distribution of T cell phenotypes. TCRV2+TCR- cells were positively gated and analyzed for the markers indicated. Data are the means of two determinations, each of which analyzed cells isolated from groups of three to five mice.

View larger version (19K): [in this window] [in a new window]   FIGURE 7. G8 TCR cell proliferation in the absence of IL-7. A, Lymphocytes were isolated from 6.5-wk-old G8+/-IL-7-/- or G8+/-IL-7+/- mice that had consumed BrdU-supplemented water for 7 days. Cells were stained with mAb against TCRV2, TCR, and BrdU. TCRV2+TCR- cells were analyzed for incorporated BrdU. The IEL and lymph node data were obtained from the same G8+/-IL-7-/- or G8+/-IL-7+/- mouse. Data shown were obtained from analyses of G8+/-IL-7-/- and G8+/-IL-7+/- littermates on the same day, using the same anti-BrdU Ab preparation. The percentages indicate the fraction of total BrdU+ cells among TCRV2+TCR- cells. The MFI represents is that of total TCRV2+TCR- BrdU+ cells. The differences in MFI are representative of six mice analyzed individually.

View larger version (62K): [in this window] [in a new window]   FIGURE 8. IL-7 prolonged the life span of mature G8 TCR cells. A, Lymphocytes were isolated from 8.5-wk-old G8+/-IL-7-/- or G8+/-IL-7+/- mice that had consumed BrdU-supplemented water for 7 days (on 7), and then were returned to normal water for an additional 14 days (off 14). Cells were stained with mAb against TCRV2, TCR, and BrdU. TCR+ cells were negatively gated, and the remaining cells were analyzed for expression of TCRV2 and the presence of incorporated BrdU. Numbers in the upper right quadrant indicate the percentage of BrdUhigh cells among total TCRV2+- cells. B, Means of data obtained from analysis of two to three mice. Error bars, SEM.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Because productively rearranged TCR genes were sufficient to bypass the developmental blockade, IL-7 must have been required for rearrangement of TCRV genes, either directly by influencing rearrangement itself or indirectly by stimulating survival of precursors to a developmental stage at which they could attempt TCR rearrangement. The latter was less likely because TCR and TCR loci undergo rearrangement concurrently beginning at the pro-T cell stage (CD44⁺CD25⁺) (54, 55) and TCR rearrangements are normal in IL-7R^-/- mice (6, 8, 21, 22, 23). Moreover, although the thymus cellularity of E14 IL-7R^-/-, IL7^-/-, or Jak3^-/- embryos is drastically reduced, the relative percentages of CD44/CD25 triple-negative subsets are normal, indicating that immature thymocytes differentiate normally (56).

We showed that T cells expressing nontransgenic or dual transgenic/endogenous TCR proteins on the cell surface were present in G8 IL-7^+/- mice but absent in G8 IL-7^-/- mice. These data strongly suggested that IL-7 stimulated rearrangement of endogenous TCR genes and, in the case of IEL, were consistent with the absence of TCRV5 rearrangements in IL-7R^-/-, _c^-/-, or Jak3^-/- thymocytes (6, 7, 8, 21, 22, 23, 31). Moreover, they are the first cell surface protein expression data showing that murine TCR loci are not strictly allelically excluded. Because TCR genes are also allelically included (44), any given TCR cell has the potential to express four different TCR on its surface simultaneously. Perhaps the expression of TCRV genes in ordered waves (40, 54, 57, 58) is a means of limiting the number of different TCR expressed on individual cells.

IL-7R signaling has been shown to directly influence TCR rearrangement. IL-7 affects accessibility of the TCRV locus to the recombinase machinery. IL-7-mediated activation of STAT5 (28, 33, 59) up-regulates the sterile transcripts that precede the appearance of TCR gene rearrangements. Both TCR enhancers and 5'-HsA, a newly described regulatory region upstream of TCRV2 that is required for consistent rearrangement of TCRV2 transgenes, have STAT5 binding motifs (60, 61, 62). STAT5 activated in response to IL-7 stimulation leads to sterile TCR transcripts in IL-7-dependent pre-B cell lines, and a constitutively active form of STAT5A restores TCRV-J rearrangements in IL-7R^-/- thymocytes (7). Also, it has been shown that IL-7 renders TCR loci accessible to the recombinase machinery by modifying histone acetylation (26, 28, 29). Like many silenced genes, TCRV loci are highly methylated in IL-7R^-/- thymocytes, and pretreatment of IL-7R^-/- precursors with a histone acetylase (TSA) restores TCRV-J rearrangements in FTOC (23).

IL-7 was not absolutely required for either terminal differentiation steps within the thymus or survival of mature TCR cells. TCR⁺TCR^- cells were present in the thymus and peripheral tissues of G8 IL-7^-/- mice. DETC develop exclusively in fetal thymus, from fetal precursors; thus, DETC in the skin of adult animals are the progeny of cells that arose during the fetal period (47, 48). The presence of TCR DETC in adult G8 IL-7^-/- mice indicated that once a TCR was expressed, TCR cells developed normally in fetal thymus, and then survived >8 wk in the absence of IL-7. This is in agreement with the pattern of expression of IL-7R in that precursors to DETC in the fetal thymus express IL-7R, but mature TCR cells in the skin do not (L. Puddington, J. M. Lewis, and R. E. Tigelaar, unpublished observations). The ability of TCR cells to survive in the periphery without IL-7 was consistent with the presence of splenic TCR cells in TCRV2-transgenic IL-7R^-/- mice (8) and the results of our earlier thymus grafting experiments, in which IL-7⁺ thymus graft-derived TCR cells were found in spleen and IEL of IL-7^-/- hosts up to 10 wk postgrafting (16).

The developmental requirements of fetal vs adult thymocytes differed in their dependence upon IL-7. Fetal thymus-derived DETC (47, 48) are absent from the skin of non-TCR-transgenic IL-7^-/- mice (10). A TCR transgene restored a normal density of TCRV2⁺ DETC to the skin of G8 IL-7^-/- mice. Similar results were obtained in the skin of TCRV3/V1 (the canonical fetal DETC-type TCR)-transgenic IL-7R^-/- mice (63). This indicated that in addition to a role in the maintenance of T cell progenitors (56), fetal TCR thymocytes were dependent on IL-7 for rearrangement of TCR. In contrast, beginning at 4 wk of age, the number of TCRV2 thymocytes sharply declined, until very few TCRV thymocytes matured in the thymus of adult G8 IL-7^-/- animals. This correlated with the transition from the first to the second wave of lymphoid precursor cells that seed fetal thymus (64), suggesting that TCR thymocytes developing later in ontogeny also required IL-7 for survival and/or proliferation. These results were consistent with the paucity of TCR cells found in adult TCRV3/V1-transgenic IL-7R^-/- mice, TCRV1-transgenic _c^-/- mice, or G8 TCR-transgenic Jak3^-/- mice (6, 31, 63). Moreover, a similar conclusion was reached studying TCR development in IL-7R^-/- mice, i.e., survival of adult, but not fetal, CD25⁺ double-negative thymocytes is IL-7R dependent (65).

The concept that fetal and adult lymphocyte progenitor cells are inherently different was first suggested by the differential usage and junctional diversity of TCRV gene segments in fetal vs adult thymocytes (46, 47, 48). It has now become a common finding in developmental immunology and is exemplified by mice deficient in cytokines or chemokines, i.e., stem cell factor, IL-7, stromal cell-derived factor-1 (65, 66, 67, 68, 69, 70), transcription factors, i.e., Ikaros, T cell factor-1 (71, 72), adhesion molecules, i.e., ₄ integrins (73), or receptors for hormones, i.e., estrogen (74). In light of this, it is important to reevaluate the conclusions reached in previous studies where the effects of age were not considered and to better control for age-related parameters in future studies.

A similar decline in thymocyte number was not reported in 2- to 5-wk-old TCRV2-transgenic IL-7R^-/- mice (8). In that study, it is not clear that dual TCR/ cells (see Fig. 2) were gated out during FACS analysis of TCRV2-transgenic IL-7R^-/- cells. Inclusion of dual TCR/TCRV2 cells in the absolute cell numbers could have masked the onset of TCRV2⁺ thymocyte decline that would have only just begun in 4- to 5-wk-old IL-7R^-/- mice. Another possibility is the TCRV2-transgenic IL-7R^-/- mice were on a pure B6 background, whereas the data presented here were obtained from the study of mice on a mixed background (B6 x 129/Ola x BALB/c). Background genes can modulate the IL-7 dependence of developing thymocytes, as exemplified by exon 3 IL-7R^-/- mice. On a mixed B6 x 129/J background, 70% of mice have thymocytes that are completely arrested at the double-negative stage (24, 65), whereas on a pure B6 background, IL-7R^-/- TCR thymocyte development progresses through the single positive stage in all mice (30). Similarly, IL-7^-/- mice on a FVB/N background have a more severe phenotype than IL-7^-/- mice on a B6 x 129/Ola or a 129/Ola background (K. Laky, U. von Freeden Jeffry, B. E. Rich, R. Murray, and L. Puddington, unpublished observations).

In lymph node or small intestinal IEL, similar or greater percentages of TCR cells were proliferating in IL-7^-/- mice than IL-7^+/- mice. Presumably, the same was also true for TCR cells in the skin because TCRV2⁺ cells were 10-fold reduced in E15-E18 fetal thymus, but not in the skin of G8 IL-7^-/- mice. The stimulus for that proliferation was unclear, but it was not IL-7, and presumably it was not Ag driven, because all G8 IL-7^-/- T cells express a single, clonotypic TCR. It is possible that IL-2 or IL-15 cytokine signaling via IL-2R could have provided this survival signal. Whereas expression of V3/V1 transgenes rescues development of fetal thymocytes and DETC in IL-7R^-/- mice, it is not able to rescue DETC in IL-2R^-/- mice (63). In G8 IL-7^-/- mice of all ages, the number of peripheral TCR cells was reduced 100-fold. It is possible that cells were merely undergoing homeostatic proliferation in an attempt to fill the available space in IL-7^-/- mice. Such a space-filling model was first suggested by experiments in which mature thoracic duct lymphocytes were adoptively transferred to lymphocyte-deficient scid or nude mice and observed to expand in a manner inversely proportional to the size of the inoculum, before being maintained at a steady state number (75, 76, 77, 78). If this were the case, it would represent a difference between TCR and TCR cells because IL-7 has been found to regulate of homeostatic proliferation of TCR cells (79).

Similar percentages of lymph node T cells proliferated in the presence or absence of IL-7. In contrast, the percentage of IEL dividing in G8 IL-7^-/- mice was greater than in G8 IL-7^+/- mice. This reemphasizes the differential regulation of TCR cell homeostasis in the small intestinal IEL compartment vs peripheral tissues. We have previously shown that T cell requirements for stem cell factor/c-Kit interaction differ at these two anatomic locations. In W/W^v and Sl/Sl^d mice, both TCR and TCR IEL homeostasis is disrupted in the absence of stem cell factor-c-Kit interactions, whereas peripheral T cell populations are unaffected (66, 67).

In the absence of IL-7 the life span of TCR cells was shortened. The pulse chase experiments described here did not allow discrimination between cell death and dilution of label due to division. However, we favor an explanation that includes at least a component of the latter. During the 7-day pulse period, BrdU⁺ cells in G8 IL-7^-/- animals had higher MFIs, results that could suggest that cells were proliferating at a higher rate in the absence of IL-7. It is possible that they continued to do so during the following 14-day chase period. Moreover, we have previously shown that IL-7 is not required for survival of mature TCR cells in the periphery (Ref.16 and see above).

In summary, we have used a TCR-transgenic IL-7^-/- model system to evaluate the requirements of TCR cells for IL-7 in the thymus and peripheral lymphoid tissues of mice ranging from E15 through 35 wk of age. With regard to thymocytes, IL-7 was required for endogenous TCR gene rearrangements in both fetal and adult thymus, and adult thymocytes also required IL-7 for survival. With regard to peripheral TCR cells, IL-7 was not required for the survival or proliferation of TCR cells, although it prolonged the life span of individual TCR cells.

	Acknowledgments

 
We acknowledge DNAX Research Institute of Molecular and Cellular Biology for their gift of IL-7^-/- mice; Pablo Pereira for mAb against TCRV1; Elizabeth G. Lingenheld for technical assistance; and Leo Lefrançois, David Tough, and B. J. Fowlkes for helpful discussions of the data.

	Footnotes

 
¹ This work was supported by United States Public Health Service Grants DK51505 (to L.P.), AI35917 (to L.L. and L.P.), AI27404 and AI27855 (to R.T.) and by National Institutes of Health Predoctoral Training Grant AI07080 (to K.L.).

² Current address: National Institutes of Health/National Institute of Allergy and Infectious Diseases/Laboratory of Cellular and Molecular Immunology, 4 Center Drive, Building 4, Room 407, Bethesda, MD 20892-0420.

³ Address correspondence and reprint requests to Dr. Lynn Puddington, Department of Medicine, MC-1319, University of Connecticut Health Center; 263 Farmington Avenue, Farmington, CT 06030-1319. E-mail address: lpudding{at}neuron.uchc.edu

⁴ Abbreviations used in this paper: DETC, dendritic epidermal T cells; HSA, heat stable Ag; IEC, epidermal cells harvested from the interface; IEL, intraepithelial lymphocytes; MFI, mean fluorescence intensity.

Received for publication August 13, 2002. Accepted for publication February 5, 2003.

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