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Neonatal and Adult CD4⁺CD3^– Cells Share Similar Gene Expression Profile, and Neonatal Cells Up-Regulate OX40 Ligand in Response to TL1A (TNFSF15)¹

Medical Research Council Centre for Immune Regulation, Institute for Biomedical Research, Birmingham Medical School, Birmingham, United Kingdom

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
We report here the quantitative expression of a set of immunity-related genes, including TNF family members, chemokine receptors, and transcription factors, in a CD4⁺CD3^– accessory cell. By correlating gene expression between cell-sorted populations of defined phenotype, we show that the genetic fingerprint of these CD4⁺CD3^– cells is distinct from dendritic cells, plasmacytoid dendritic cells, T cells, B cells, and NK cells. In contrast, it is highly similar to CD4⁺CD3^– cells isolated from embryonic and neonatal tissues, with the exception that only adult populations express OX40L and CD30L. We have previously reported that IL-7 signals regulate CD30L expression. In the present study, we show that both neonatal and adult CD4⁺CD3^– cells express the TNF family member, death receptor 3 (TNFRSF25), and that addition of TL1A (TNFSF15), the ligand for death receptor 3, up-regulates OX40L on neonatal CD4⁺CD3^– cells. Finally, we demonstrate that this differentiation occurs in vivo: neonatal CD4⁺CD3^– cells up-regulate both CD30L and OX40L after adoptive transfer into an adult recipient.

	Introduction

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We have previously reported that CD4⁺CD3^–CD11c^–B220^– cells (CD4⁺CD3^– cells) provide survival signals to activated CD4 T cells via their constitutive expression of OX40L (CD252 and TNFSF4) and CD30L (CD153 and TNFSF8) in adult mouse (1, 2). These cells are located in B follicles but also T cell areas, especially the outer T zone. In B follicles they are attached to the T cells that select germinal center (GC)³ B cells, and in the absence of OX40 and CD30 signals, GC T cells fail to survive, with consequent failure of affinity maturation of the Ab responses (2). Even more significantly, T cell memory for Ab responses is abrogated in OX40 and CD30 double-deficient mice (2), and we have speculated that memory T cells are normally maintained by their associations with these cells in the outer T zone.

When we investigated the presence of these cells in neonatal tissues, we found a similar population: however, OX40L and CD30L, the T cell survival molecules, were lacking (3). These studies demonstrated that the expression of OX40L and CD30L was regulated independently: IL-7 signals were important for CD30L but not OX40L expression (3). Our failure to induce OX40L on neonatal CD4⁺CD3^– cells raised the possibility that they were different cells from those that we found in adult mice. In the present study, we report three independent pieces of evidence that further support a relationship. We show that cells of related lineage show strong correlations in the quantitative mRNA expression of a 96-gene set of immunity related genes: for example, subsets of dendritic cells (DCs), T and B cells, are closely correlated. This relationship also holds for neonatal and adult populations of CD4⁺CD3^– cells. Of particular interest was the shared high levels of mRNA for the TNF ligands, lymphotoxin (LT) , LT, TNF-, and TNF-related activation-induced cytokine (TRANCE) (TNFSF11). Like OX40L and CD30L on adult CD4⁺CD3^– cells, these cells appear to express high levels of these ligands constitutively. They also express receptor activator of NF-B (RANK) (TNFRSF11A), death receptor 3 (DR3) (TNFRSF25), IL-2R (CD25), IL-7R (CD127), common cytokine receptor -chain (_c) (CD132), CCR7, and CXCR5.

Because we found that both neonatal and adult CD4⁺CD3^– cells expressed high levels of DR3, we added recombinant TL1A (TNFSF15) to both neonatal and adult populations. This signal induced high levels of OX40L expression on embryonic/neonatal populations, and the expression of OX40L was further augmented on adult cells. Finally, we show that embryonic CD4⁺CD3^– cells following transfer into an adult recipient up-regulate OX40L and CD30L expression to comparable levels to the adult host CD4⁺CD3^– cells in vivo.

	Materials and Methods

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Mice

All experiments were performed in accordance with the U.K. laws and with the approval of the local ethics committee. Normal, RAG1^–/–, and T cell-deficient mice were bred and maintained in our animal facility. Neonatal lymph node or spleen CD4⁺CD3^– cells were isolated from 1- to 2-day-old normal BALB/c litters or RAG1^–/– mice. Spleens from normal C57BL/6 or BALB/c E15 embryos were used to isolate E15 CD4⁺CD3^– cells.

Preparation of CD4⁺CD3^– cells, plasmacytoid DCs (pDCs), DCs, and other cells

Cell suspensions for isolation of CD4⁺CD3^– cells, DCs, and pDCs were made from the spleens of adult RAG^–/– mice as described previously (1, 3). Neonatal CD4⁺CD3^– cells were isolated from either BALB/c or C57BL/6 mice that were 1 or 2 days old. Briefly, CD11c⁺ cells were positively enriched by using CD11c-coated magnetic beads (Miltenyi Biotec) and then FACS sorted into CD8⁺ and CD8^– populations. CD4⁺ cells were enriched from CD11c⁺-depleted populations using CD4-coated magnetic beads, and the resulting CD4⁺-enriched populations sorted into CD4⁺CD3^–B220^–CD11c^– (CD4⁺CD3^–) and CD4⁺CD3^–B220⁺CD11c^low (pDC) populations. CD45^– stromal cells were FACS sorted from BALB/c mice.

For the preparation of E15 CD4⁺CD3^– cells, embryos from normal pregnant mice of gestation day 15 were obtained and the spleens removed. The spleens were placed in culture medium with 100 ng/ml IL-7 (PeproTech) on a 0.8-µm sterile Nuclepore filter (Millipore) on a sterile arti wrap sponge. The petri dish was then cultured in a contained humid environment in a 10% CO₂ incubator for 5 days. On day 5, cultured spleens were teased apart with fine forceps and CD4 cells enriched as above.

Follicular B (CD21^lowCD23⁺IgM^int) cells, marginal zone B (CD21^highCD23^–IgM⁺) cells, and NK cells from normal mice were sorted to make cDNA. Th1 and Th2 cells were prepared under Th1 conditions (10 ng/ml IL-12 and 10 µg/ml anti-IL-4) and Th2 conditions (10 ng/ml IL-4 and 10 µg/ml anti-IL-12) for 6 days in vitro culture.

Stimulation of E15 or neonatal or adult CD4⁺CD3^– cells

Prepared cells were cultured with a wide range (0.1–100 ng/ml) of recombinant mouse TL1A (R&D Systems) for 2 or 6 days of culture and then stained for flow cytometry analysis or for MoFlo cell sorting.

FACS staining

CD4⁺CD3^– cells were stained with anti-CD4 PE, anti-CD3 FITC, anti-CD11c FITC, and anti-B220 FITC mAbs or anti-B220 allophycocyanin mAbs (BD Biosciences) and then stained with biotinylated mAbs against OX40L, CD30L, and CXCR4 (BD Biosciences) or TRANCE (R&D systems) in conjunction with streptavidin CyChrome (BD Biosciences) as the second-step staining reagent.

TaqMan low-density array analysis

TaqMan primer sets are designed to work with an efficiency approaching 100%, enabling the quantitative comparison of mRNA expression for different genes not only within a cell type, but also between cells of different lineages. Housekeeping genes (-actin, ₂-microglobulin (₂m), or 18S rRNA) were used to correct for total mRNA.

TaqMan low-density real-time PCR arrays (Applied Biosystems) were designed with a 96-gene format. A list of all of the genes measured is as follows: chemokines (CCL19, CXCL12, and CXCL13), chemokine receptors (CCR7, CXCR3, and CXCR5), cytokines (IL-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p35, IL-12p40, IL-13, IL-15, IL-18, TSLP, IFN-1, IFN-, IFN-, and TGF-1), cytokine receptors (IL-2R, IL-2R, IL-2R, IL-4R, IL-7R, IL-10R, IL-10R, IL-12R1, IL-12R2, IFN-R1, and IFN-R2), costimulatory molecules (CD80, CD86, CTLA4, ICOS, and ICOSL), DC marker (DC-SIGN, cathepsin S, and integrin _x), housekeeping (CD4, -actin, 18S RNA, and ₂m), MHC class II (CD74), TLR (MyD-88, TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9), TNF family (LT, TNF-, LT, OX40L, CD40L, FASL, CD70, CD30L, 4-1BBL, TRANCE, TWEAK, APRIL, BAFF, LT-related inducible ligand that competes for glycoprotein D binding to HVEM on T cell (LIGHT), and TL1), TNFR family (TNFR1, TNFR2, LTR, OX40, CD40, FAS, CD30, 4-1BB, RANK, TWEAKR, BAFFR, HVEM, GITR, and DR3), transcription factors (Bcl-2, Bcl-6, Bcl-x_L, ROR, GATA3, foxP3, and T-bet), and others (perforin and granzyme B).

cDNA was mixed with TaqMan Universal PCR Master Mix (Applied Biosystems). This was added to the TaqMan Low-Density Array, and PCR was performed in a 7900HT Fast Real-Time PCR System (Applied Biosystems) according to manufacturer’s recommendations.

The relative signal per cell was quantified by setting a threshold within the logarithmic phase of the PCR and determining the cycle number at which the fluorescence signal reached the threshold (C_t). The C_t for the target gene was subtracted from the C_t for -actin. The relative amount was calculated as 2^–Ct x 10².

Conventional PCR analysis

Signals for RORt, LT, TL1A, and -actin were analyzed by conventional PCR. PCR products were analyzed by ethidium bromide gel electrophoresis and identified by fragment size using Syngene Gel Documentation Gene Tools software.

The primer sequences were as follows: -actin, forward (5'-ATC TAC GAG GGC TAT GCT CTC C-3') and reverse (5'-CTT TGA TGT CAC GCA CGA TTT CC-3'); RORt, forward (5'-ACC TCC ACT GCC AGC TGT GTG CTG TC-3') and reverse (5'-CAA GTT CAG GAT GCC TGG TTT CCT C-3'); LT, forward (5'-CTC CAT CCT GAC CGT TGT TT-3') and reverse (5'-TAG ACC CAC AAA AAC CCT GC-3'); TL1A, forward (5'-AGTCCCAGTGGAAGTGCTG-3') and reverse (5'-GTGCTAAGTCCTGCGAGGAT-3').

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Embryonic/neonatal and adult CD4⁺CD3^– cells display similar genetic fingerprints that readily distinguish them from other cell types

Embryonic/neonatal CD4⁺CD3^– cells share a common surface phenotype with the cells that we have described in adult mice: CD4⁺CD3^–CD11c^–B220^–IL-7R⁺, cytokine _c⁺, CD45⁺, Thy1⁺, TRANCE⁺, RANK⁺, and MHC class II^low (4). Although relatively few genes are cell specific, we reasoned that if we compared the levels of mRNA expression for a more comprehensive set of immunity-related genes, cells of related lineage would share a genetic fingerprint, particularly if we looked at genes linked with immune function and migration. Therefore, we designed TaqMan arrays for a panel of immunity-related genes (see Materials and Methods). Comparison of genes expressed within the two major subsets of DCs in mice, CD8⁺CD11c⁺ and CD8^–CD11c⁺ DCs (correlation coefficient (CC) = 0.94), Th1- and Th2-differentiated T cells (CC = 0.86), and marginal zone and follicular B cells (CC = 0.95), revealed a strong CC for the related cells (Fig. 1A, top panel), but there was little correlation between cells of different types, lending validity to the use of the fingerprinting method. Comparison of gene expression between adult CD4⁺CD3^– cells and either NK (CC = 0.66), pDCs (CC = 0.59), CD8⁺ DCs (CC = 0.68), follicular B (CC = 0.70), marginal zone B cells (CC = 0.65), Th2 (CC = 0.63), or Th1 (CC = 0.76) showed much weaker CCs. In contrast, after OX40L and CD30L gene expression was excluded from the analysis, the gene expression in adult CD4⁺CD3^– cells was strongly correlated with embryonic (E15) spleen CD4⁺CD3^– cells (CC = 0.86) and with neonatal (D2) spleen CD4⁺CD3^– cells (CC = 0.90); neonatal lymph node and neonatal spleen CD4⁺CD3^– cells were also strongly correlated (CC = 0.88) (Fig. 1, A and B). The values of individual gene expression are shown in supplemental Fig. 1 data.⁴

View larger version (29K): [in this window] [in a new window]   FIGURE 1. Correlation between the gene expression patterns of CD4+CD3– cells and a range of other cell populations. Adult cell populations of CD4+CD3– cells, pDCs, and DCs were isolated from RAG–/– spleens. E15 embryonic, day 2 neonatal CD4+CD3– cells, follicular B (fol B), marginal zone (MZ) B, and NK cells were from normal mice. Th1 and Th2 cells were differentiated in vitro for 6 days. A, Correlation of gene expressions between two cell types. Over 0.01% expression of individual mRNAs of -actin signals were plotted. Axes show levels of mRNA expression relative to the -actin signal (-actin signal = 100%, log scale). Each plot compares two cell types, one on each axis (as labeled). B, CCs between cell populations. These results are representative of on average five separate experiments. C, Gene expression profile normalized to -actin signals (a), to 2m (b), and to 18 rRNA (c). The left panel shows correlation of gene expressions between adult and D2 CD4+CD3– cells. The middle panel shows correlation of gene expressions between adult CD4+CD3– cells and CD8+ DCs. The right panel shows correlation of gene expressions between adult CD4+CD3– cells isolated from RAG-deficient and T cell-deficient mice.

RAG^–/– or T cell-deficient mice were used as a source of adult CD4⁺CD3^– cells because of the technical difficulties in purifying CD4⁺CD3^– cells from mice with an intact repertoire of CD4 T cells, which adhere to and contaminate the CD4⁺CD3^– population (1). We have previously reported that 10⁵ CD4⁺CD3^– cells can be isolated from an individual RAG^–/– spleen and more from T cell-deficient mice (3). We think it likely, however, that this underestimates their number as they attach to VCAM-1⁺ stromal cell populations in both B and T cell areas, making their isolation difficult (our unpublished observations). By confocal microscopy, we think that there are as many CD4⁺CD3^– cells as DCs, but the latter are much more readily isolated from tissues (1).

Cross-correlation of gene expression between CD4⁺CD3^– cells from adult RAG^–/– and T cell-deficient mice that have normal numbers of B cells (CC = 0.92) (Fig. 1C) indicates that the pattern of gene expression does not depend on B cells. Furthermore, the slope of the correlated genes is 1, indicating that levels of genes expressed are not influenced significantly by B cells.

Expression of TNF/TNFR family members in CD4⁺CD3^– cells

There are currently 17 identified TNF and 30 TNFR family members (www.gene.ucl.ac.uk/nomenclature/genefamily/tnftop.html). Our gene array focused on TNF/TNFR family members linked with survival. The details of the distinctive gene profile of TNF and TNFR (TNF/TNFR) family members established for the CD4⁺CD3^– cell type are tabulated (Tables I and II). Expression of mRNA in embryonic E15 CD4⁺CD3^– cells (Tables I and II) was similar to D2 neonatal CD4⁺CD3^– cells (data not shown). Our analysis focused on genes expressed at high levels (mRNA expressed at >0.2% of the -actin signal). To simplify analysis, gene expression has been categorized into four groups relative to expression of -actin: 1) +++ >10%; 2) ++ 1–10%; 3) + 0.2, 1%; and 4) – <0.2%.

Neonatal and adult CD4⁺CD3^– cells coordinately expressed 7 of the 14 TNFR family members, 5 of them strongly (++). The TNFR family members can also be grouped into those involved in lymph node development and B:T segregation (5, 7) (LTR, TNFR1, and RANK) and those linked with T cell activation (HVEM, TNFR2, 4-1BB, and DR3) (8). These four T cell-associated TNFR family members come from a gene cluster of seven TNFR family members on human chromosome 1 and mouse chromosome 4. Neither neonatal nor adult CD4⁺CD3^– cells express CD30 or OX40.

Expression of non-TNF/TNFR family genes

Fig. 2 summarizes the expression of non-TNF/TNFR family genes expressed on CD4⁺CD3^– cell populations normalized to -actin (Fig. 2A) or ₂m (Fig. 2B). All of them were expressed at comparable levels in adult and embryonic/neonatal populations independently on housekeeping genes. There are three gene groups of particular interest: the chemokine receptors, the survival genes, and the GC-specific genes. CD4⁺CD3^– cells express both of the chemokine receptors, CXCR5 and CCR7, but not the pDC-related receptor, CXCR3 (9). We looked for but did not find mRNA for the ligands of CXCR5 (CXCL13) and CCR7 (CCL21 and CCL19), which occur in stromal populations (10, 11). Because TaqMan primer for CXCR4 was not available, we stained with mAbs and identified expression of CXCR4 on both neonatal and adult CD4⁺CD3^– cells (Fig. 2C).

View larger version (27K): [in this window] [in a new window]   FIGURE 2. Expression of Bcl family members and chemokines/chemokine receptors. A, Relative mRNA expression of Bcl-2, Bcl-xL, CCR7 and its ligand (CCL19), CXCR5 and its ligand (CXCL13), and CXCR3 normalized to -actin signals. This result is representative of three separate experiments. B, Relative mRNA expression normalized to 2m signals. C, Surface CXCR4 expression on neonatal D2 and adult CD4+CD3– cells. Shaded histograms show control staining with biotinylated rat Abs.

RORt mRNA is expressed in adult CD4⁺CD3^– cells, and DR3 signals up-regulate OX40L expression on embryonic/neonatal CD4⁺CD3^– cells

Because of the similar genetic fingerprint of embryonic/neonatal and adult CD4⁺CD3^– cells, we looked for mRNA expression of the splice variant of the retinoic acid orphan receptor, RORt (12), a gene essential for the function of CD4⁺CD3^– cells in lymph node development. Both adult CD4⁺CD3^– cells and embryonic/neonatal CD4⁺CD3^– cells expressed mRNA for RORt, but levels were 4-fold higher in embryonic CD4⁺CD3^– cells than in adult CD4⁺CD3^– cells (Fig. 3A) and both expressed both TNFR1 ligands and LTR ligands (Fig. 3A and Tables I and II).

View larger version (23K): [in this window] [in a new window]   FIGURE 3. Effects of TL1A signals on CD4+CD3– cells. A, Genes differentially expressed in embryonic (E15) and adult CD4+CD3– cells. B, Adult CD4+CD3– cells cultured with or without 100 ng/ml TL1A for 2 days. C, E15 CD4+CD3– cells cultured with or without 100 ng/ml TL1A for 2 days. D, Correlation of gene expression in OX40L+ vs OX40L– FACS-sorted E15 CD4+CD3– cells after treatment with TL1A for 2 days. Axes show levels of mRNA expression relative to the -actin signal (-actin signal = 100%, log scale). These results are representative of at least two separate experiments.

Addition of TL1A to embryonic (E15) CD4⁺CD3^– cells also down-regulated RORt expression with up-regulation of both TRANCE and particularly OX40L (Fig. 3C). The effects on mRNA were reflected by changes in protein expression at the cell surface on adult splenic CD4⁺CD3^– cells (Fig. 4A), adult lymph node CD4⁺CD3^– cells (Fig. 4B), neonatal (D2) splenic CD4⁺CD3^– cells (Fig. 4C), and E15 splenic CD4⁺CD3^– cells (Fig. 4D). In all three groups, 48 h of DR3 signals up-regulated OX40L and TRANCE expression but had little effect on CD30L expression. Costimulation of neonatal CD4⁺CD3^– cells with TL1A for 6 days up-regulated OX40L, TRANCE, and also CD30L, whereas IL-7 alone up-regulated CD30L and TRANCE but not OX40L (Fig. 4E). Together, IL-7 and TL1A showed additive effects. However, the fact that mice deficient in _c or IL-7 signals have normal levels of OX40L but not CD30L and TRANCE shows an important role for IL-7 signals in CD30L expression (3), and therefore, the effects of TL1A in the 6 day experiments may be indirectly mediated through IL-7.

View larger version (49K): [in this window] [in a new window]   FIGURE 4. Effect of TL1A on TNF ligand protein expression on CD4+CD3– cells in vitro. A, Adult splenic CD4+CD3– cells cultured with/without 100 ng/ml TL1A for 2 days. B, Adult lymph node CD4+CD3– cells cultured with/without 100 ng/ml TL1A for 2 days. C, Neonatal splenic day 2 CD4+CD3– cells cultured with/without 100 ng/ml TL1A for 2 days. D, Embryonic splenic E15 CD4+CD3– cells cultured with/without 100 ng/ml TL1A for 2 days. E, Neonatal splenic day 2 CD4+CD3– cells cultured with/without 100 ng/ml TL1A and/or 100 ng/ml IL-7 for 6 days. Shaded histograms show control staining with biotinylated rat Abs. This result is representative of four separate experiments.

Fetal CD4⁺CD3^– cells up-regulate expression of both OX40L and CD30L after transfer into adult recipients

To test directly whether embryonic CD4⁺CD3^– cells were capable of up-regulating OX40L and CD30L in vivo, CD4⁺CD3^– cells were prepared from CD45.2 embryonic spleens and transferred into an adult CD45.1 recipient that lacked T cells (13) (isolation of CD4⁺CD3^– populations from T cell-sufficient mice is technically difficult (3)). Five days later, CD4⁺ cells were enriched from the spleen and stained with the allotype marker to identify transferred embryonic CD4⁺CD3^– cells and OX40L and CD30L (Fig. 5). Transferred CD4⁺CD3^– cells were clearly identifiable in adoptive recipients, and while they were negative for CD30L and OX40L before cell transfer, they showed expression levels of OX40L and CD30L comparable to host adult CD4⁺CD3^– cells, indicating that fetal CD4⁺CD3^– cells acquire hallmarks of adult CD4⁺CD3^– cells in vivo.

View larger version (37K): [in this window] [in a new window]   FIGURE 5. In vivo up-regulation of OX40L and CD30L on embryonic CD4+CD3– cells (CD45.2) after transfer into an adult mouse (CD45.1). Five days after transfer of embryonic (E15) CD4+CD3– cells into an adult mouse, the host spleen was taken, and the CD11c-depleted CD4-enriched population was overnight cultured and immunostained the following day. Shaded histograms show control staining with biotinylated rat Abs. This result is representative of two separate experiments.

Because TL1A protein induced OX40L expression in embryonic CD4⁺CD3^– cells, we hypothesized that TL1A expression would be minimal in E15 spleen. Due to lack of reagents to detect TL1A protein, we tested for mRNA expression from total mRNA isolated from E15, D1, and adult spleen (Fig. 6). TL1A mRNA was clearly expressed in E15 tissues but not E15 CD4⁺CD3^– cells. We do not know, however, whether TL1A is expressed at the protein level, but it is clearly not able to signal through DR3 expressed on CD4⁺CD3^– cells.

View larger version (41K): [in this window] [in a new window]   FIGURE 6. TL1A mRNA expression in embryonic, neonatal, and adult spleens. cDNA was prepared from embryonic E15, neonatal D1, and adult wild-type mice and E15 CD4+CD3– cells. Expression of TL1A and -actin was assessed by PCR. Legend to supplemental data Fig. 1.4 The values of individual mRNA expression normalized to the 2m signal (2m signal = 100). Each plate contains four samples derived from FACS-sorted cell populations. Analysis from four chips is shown in this figure. PCR was done for 40 cycles. The signal for each gene is the cycle number at which the fluorescence signal reached the threshold (Ct). Ct was subtracted from the signal for housekeeping gene, in this case 2m (Ct–Ct2m). The difference between each gene and the housekeeping gene was calculated as 2–(Ct–Ct2m) x 102. This value gives individual mRNA expression normalized to the 2m signal. The 2m signal is 100.

	Discussion

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Although embryonic/neonatal and adult CD4⁺CD3^– cells share a similar genotype compared with other cell types and also share expression of similar set of protein markers at the cell surface, they clearly differ in their expression of the T cell survival proteins, CD30L and OX40L, which may help explain why T cell priming in the neonate results in tolerance rather than autoimmunity (3). We have previously reported that CD30L expression can be induced on neonatal CD4⁺CD3^– cells in vitro with IL-7, and the expression of CD30L in vivo also appears to be IL-7 dependent (3). In the present study, we have shown that a signal from another TNF family member, TL1A, through DR3 expressed on both embryonic/neonatal and adult CD4⁺CD3^– cells rapidly up-regulates the expression of the T cell survival ligand, OX40L, on embryonic/neonatal CD4⁺CD3^– cells and induces further up-regulation of OX40L on adult CD4⁺CD3^– cells.

TL1A has been reported to be inducible by TNF- and IL-1 (14) and thought to circulate as a homotrimeric soluble form (15). TL1A is produced by cerebral endothelial cells in mouse (16) and umbilical vein endothelial cells in human (17) and inhibits angiogenesis (18). In addition, membrane TL1A is expressed by human CCR9⁺ mucosal and gut-homing peripheral blood T cells (19). DR3 is the receptor for TL1A containing a death domain with the highest homology to TNFR1 (20, 21) and, in addition to being expressed on CD4⁺CD3^– cells, is expressed by activated T cells (15). Although DR3 contains a death domain, DR3/TL1A engagement also leads to activation of NF-B and survival rather than apoptosis (14, 22). Our data demonstrating normal expression of OX40L on CD4⁺CD3^– cells in T cell-deficient mice (3) suggest that T cell-derived TL1A is not essential for expression of OX40L on CD4⁺CD3^– cells.

However, neonatal CD4⁺CD3^– cells are found clustered close to blood vessels in spleen (our unpublished data), which could therefore potentially provide these DR3 signals (14). TNF- expressed by neonatal CD4⁺CD3^– cells has the potential to induce TL1A (14) and also IL-7 (23) expression on endothelium, providing a signaling loop leading to their own maturation to OX40L and CD30L expressing adult phenotype CD4⁺CD3^– cells. This conclusion is further supported by our observations that neonatal CD4⁺CD3^– cells up-regulate expression of both OX40L and CD30L to levels comparable to that found on adult cells following adoptive transfer of embryonic CD4⁺CD3^– cells into adult mice in vivo. Because TL1A mRNA is detected in E15 spleen, lack of TL1A mRNA could not explain the failure of embryonic CD4⁺CD3^– cells to express OX40L. One possibility is that TL1A protein is not present in E15 tissues; alternatively, it may be sequestered inside cells and only available in response to some as yet unidentified signal present in postnatal life. This is currently under investigation.

Both embryonic/neonatal and adult CD4⁺CD3^– cells express a second set of TNF family members, including LT, LT, TNF-, and LIGHT, that are linked with the organized B:T segregation observed in lymphoid tissues (5). These CD4⁺CD3^– cells have a similar phenotype to lymphoid tissue inducer cells, which, via their expression of LT, LT, TNF-, and LIGHT, elicit the development of lymph nodes and gut-associated lymphoid tissue (7) and have been associated with the initial B:T segregation that occurs in the neonatal lymph node (24). Although definitive proof that lymphoid tissue inducer cells give rise to adult CD4⁺CD3^– cells awaits cell fate mapping experiments (25), we think the data provided here are suggestive of this sequence of events.

In summary, in this report, we provide evidence that the precursors of adult CD4⁺CD3^– cells are present in embryonic tissues but lack expression of the TNF ligands that we have linked with T cell memory, OX40L and CD30L. We demonstrate, however, that the embryonic population differentiates into adult phenotype cells following adoptive transfer in vivo in adult hosts. Furthermore, we demonstrate that TL1A, the ligand for the TNFR, DR3, expressed on both embryonic/neonatal and adult populations, rapidly up-regulates OX40L expression on the populations.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by a Wellcome Programme Grant (to P.J.L.L.).

² Address correspondence and reprint requests to Dr. Peter J. L. Lane, Medical Research Council Centre for Immune Regulation, Institute for Biomedical Research, Birmingham Medical School, Birmingham B15 2TT, U.K. E-mail address: p.j.l.lane{at}bham.ac.uk

³ Abbreviations used in this paper: GC, germinal center; ₂m, ₂-microglobulin; CC, correlation coefficient; DC, dendritic cell; DR3, death receptor 3; _c, common -chain; HVEM, herpes virus entry mediator; LT, lymphotoxin; LIGHT, LT-related inducible ligand that competes for glycoprotein D binding to HVEM on T cell; pDC, plasmacytoid DC; TRANCE, TNF-related activation-induced cytokine; RANK, receptor activator of NF-B.

⁴ The online version of this article contains supplemental material.

Received for publication April 4, 2006. Accepted for publication June 16, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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