The TL MHC class Ib molecule has only marginal effects on the activation, survival and trafficking of mouse small intestinal intraepithelial lymphocytes

Nathalie Pardigon1,2, Sylvie Darche2, Brian Kelsall3, Jack R. Bennink1 and Jonathan W. Yewdell1

1 Laboratory of Viral Diseases, NIAID, NIH, Bethesda, MD, USA
2 Laboratoire de Biologie Moléculaire du Gene, INSERM U277, Institut Pasteur, Paris, France
3 Laboratory of Clinical Investigation, NIAID, NIH, Bethesda, MD, USA

Correspondence to: Nathalie Pardigon; E-mail: npardigon{at}niaid.nih.gov


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Thymus leukemia antigen (TL) is an MHC class Ib molecule that is highly conserved in rats and mice with no obvious human homolog. TL is expressed in mouse small intestinal epithelial cells and is known to interact with CD8{alpha}{alpha} homodimers, which are expressed by intraepithelial lymphocytes (IELs), some other T cell subsets and some non-T cells such as a subset of dendritic cells. We show here that TL is abundantly expressed on the basolateral surface of mouse small intestinal epithelial cells and that expression is abrogated in ß2m–/– mice but unaffected in TCR–/– mice or CD8{alpha} chain–/– mice. We demonstrate that the interaction between TL and CD8{alpha}{alpha} is not necessary for IEL survival in vitro or in vivo and does not modulate IEL trafficking in vivo. TL co-stimulation of {alpha}-CD3 antibody-activated IELs resulted in modestly enhanced production of IFN-{gamma} in one subset of IELs. The lack of effect on IEL survival and trafficking and the modest effect on IFN-{gamma} production suggest that the functional consequences of TL interaction with CD8{alpha}{alpha} as well as the more general biological role of TL in mucosal immunity remains to be discovered.

Keywords: intraepithelial lymphocytes, mouse small intestine, thymic leukemia ag


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mouse thymus leukemia antigen (TL) belongs to the family of MHC class Ib (‘non-classical’ class I) molecules. TL is mainly expressed by epithelial cells of the small intestine and also by immature thymocytes in TL+ strains of mice (14). The T3/T18 pseudo-alleles that encode TL have an exon–intron structure similar to MHC class Ia genes, and TL demonstrates ~70% amino acid identity with MHC class Ia molecules. Like most MHC class I molecules, TL must associate with ß2-microglobulin 2m) for stable cell surface expression. Unlike most MHC class I molecules, however, TL does not appear to act as a peptide binding/presenting protein. TL cell surface expression is TAP-independent (5), and more directly, X-ray crystallography revealed that TL lacks an obvious antigen binding groove (6). The structural changes in TL relative to MHC class Ia molecules evident in the structure were proposed to hamper peptide-specific interaction with the TCR, though there is evidence that TL can be recognized by either {gamma}{delta} or {alpha}ß TCRs (7,8).

TL is highly conserved in the subfamily Murinae, suggesting that for the past 100 million years it has played an evolutionarily significant function (9). The nature of this function is enigmatic. Potentially an important clue to TL function is the presence of substantial numbers of intraepithelial lymphocytes (IELs) residing in close proximity to TL-expressing small intestinal epithelial cells. Small intestine IELs express {alpha}ß or {gamma}{delta} TCR and most of them express a homodimeric form of the CD8{alpha} co-receptor molecule (CD8+ T cells elsewhere in the body nearly always express both CD8{alpha} and ß chains as a heterodimer CD8{alpha}ß molecule). In contrast to classical MHC class I molecules that bind CD8{alpha}ß with relatively low affinity, TL binds homodimeric CD8{alpha}{alpha} with high affinity (10). Indeed, fluorescent TL-tetramers can be used as a reagent to identify CD8{alpha}{alpha}-bearing cells (1013).

In the present study we have used a variety of approaches to characterize the interaction between TL-bearing cells and IELs and the impact of this interaction on different biological functions. Our findings indicate that neither survival, trafficking nor IFN-{gamma} production are greatly influenced by TL expression. We conclude that much remains to be learned about the biological function of TL.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
B6 and BALB/c mice were purchased from Charles River (France) and Taconic (Germantown, NY). CD3{varepsilon}–/– mice were kindly provided by Dr Anne Le Huen (Hôpital Necker, Paris, France), TCR{delta}–/– and CD8{alpha}–/– mice were bred and maintained at the Institut Pasteur animal facility, nude and TCR{alpha}–/– mice were bred and maintained in sterile isolators at the Centre des Techniques Avancées pour l'Expérimentation Animale, Orléans, France. ß2-microglobulin–/– mice were bred and maintained at the NIH animal facility.

Preparation of TL tetramers
Tetrameric TL/hß2m complexes were constructed as previously described (14). Briefly, a cDNA product of the TL molecule (T3b gene) encompassing residues 27–306 was synthesized from intestinal epithelial cells (B6 mouse strain) total RNA by PCR using the following primers: 5'-GGAATTCCATATGGGCTCACACT-3' and 5'-CGCGGATCCGGTCTGAGGAAGCTC-3'. It was then introduced into the modified pET22b(+) vector containing the BirA biotinylation substrate sequence, replacing the H-2Kd heavy chain in the construct. Complexes of TL/hß2m were formed without peptide moiety, further enzymatically biotinylated and finally mixed with PE-labeled Ultravidin (Leinco Technologies, Ballwin, MI) at a 4:1 molar ratio.

Antibodies and flow cytometry
APC-conjugated {alpha}-CD3{varepsilon} and {alpha}-TCRß, FITC-conjugated {alpha}-TCR{gamma}, {alpha}-CD8ß, {alpha}-CD4, PCP-conjugated {alpha}-CD8{alpha}, PE-conjugated {alpha}-IFN{gamma}, {alpha}-CD103 and {alpha}-Bcl-2, as well as purified {alpha}-CD3{varepsilon} mAbs were purchased from Pharmingen (San Diego, CA). {alpha}-Fc{gamma}III/II receptor CD16/CD32 mAb was produced from the supernatant of 2-4G2 hybridoma. Before all stainings, cells were treated with 2-4G2 antibody to block Fc{gamma} receptors. Cell preparations (see below) were incubated for 1 h at 4°C with PE-labeled TL tetramers, washed, and incubated with the indicated antibodies. Cryosection staining (see below) was performed using {alpha}-T3 mAb (HD168) (a kind gift from Dr Mark Solowski, Johns Hopkins School of Medicine, Baltimore, MD), followed by FITC-conjugated donkey {alpha}-rat antibodies (Jackson ImmunoResearch, Westgrove, PA) as secondary antibodies. Polyclonal rabbit {alpha}-MHC class I ({alpha}-exon 8) antiserum was prepared by immunizing rabbits against KLH-conjugated MHC class I exon 8 peptide (CKVMVHDPHSLA) and secondary antibody FITC-conjugated donkey {alpha}-rabbit was from Jackson ImmunoResearch (Westgrove, PA). For co-cultured IELs (see below), cells were surface-stained with the indicated antibodies. Intracellular staining was performed on surface-stained cells after fixation with 1% paraformaldehyde and permeabilization with 0.1% saponin. The cells were analyzed with a FACSCalibur (Becton Dickinson).

Immunochemistry
Fresh pieces of small intestine from 10–12-week-old C57BL/6 or ß2m–/– mice were snap-frozen and 8 µm sections were fixed in cold acetone, stained with {alpha}-T3 mAb (HD168) then FITC-conjugated donkey {alpha}-rat antibodies, or {alpha}-MHC class I ({alpha}-exon 8) antibodies then FITC-conjugated donkey {alpha}-rabbit antibodies, in PBS containing 5% normal donkey serum.

Cell isolation
IEL from the small bowel were isolated as previously described (15). IELs from the liver and lung were prepared as lamina propria cells (15) and peripheral lymph node lymphocytes were obtained from pooled inguinal, brachial and axillary lymph nodes.

Full length TL protein construct and cell transfection
A cDNA product of the full length TL molecule (T3b gene) encompassing residues 1–385 was synthesized from intestinal epithelial cells (B6 mouse strain) total RNA by PCR using the following primers: 5'-CCGAATTCATGAGGATGGGGACACCAGTGCCTG-3' and 5'-CTAGTCTAGATCAGGAGACCAGGTGTGGGGCAGAAGGAAGATC-3'. It was then introduced into the pCDNA3.1(+/–) vector (Invitrogen, Carlsbad, CA). Five micrograms of either full length TL protein DNA construct or a pCDNA3 DNA vector (Invitrogen, Carlsbad, CA) were electroporated in P815 using a BioRad Gene Pulser apparatus. The cells were then cultured for 24 h at 37°C in RPMI medium containing 10% FCS. In some experiments, the electroporated cells were purified on a Ficoll gradient 3 h after electroporation. Typically 20–40% P815 and 5–15% BMA cells were TL+ after electroporation.

In vitro T cell activation
96-well plates (Corning, NY) were treated overnight with {alpha}-CD3{varepsilon} mAb (1 µg/ml) in PBS. Freshly isolated IELs were co-cultured with P815 that had been transfected with TL (ratio 1:1 or 1:2) for 16 h at 37°C in RPMI medium containing 10% FCS in wells treated or not with {alpha}-CD3{varepsilon} mAb. If the IELs were to sustain intracellular staining the next day, Brefeldin A (Sigma, St Louis, MO) was added along with the transfected cells to the IELs at 10 µg/ml. In some experiments, recombinant mouse IL-15 (BioSource, Camarillo, CA) was added to the co-cultures at 15 ng/ml.

Adoptive cell transfer
IELs were isolated from 4 B6 mice. CFSE staining was performed by incubating 107 cells/ml with 5 µM CFSE (5- and 6-carboxy-fluorescein diacetate succinimidyl ester; Molecular Probes, Eugene, OR) for 10 min at 37°C in PBS. After washing, the cells were injected into either B6 or ß2m–/– mice (1.5 x 107 cells) in the tail vein. Five days later, the mice were sacrificed and their IELs were isolated; staining was performed followed by FACS analysis.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
TL-tetramers stain subpopulations of gut IELs, but not liver or lung IELs
We initially used TL-tetramers (TL-tet) to identify CD8{alpha}{alpha} bearing cells among B6 lymphocyte populations. Figure 1(A) shows the percentage of TL-tet-stained lymphocytes from various origins. Less than 3% of lymphocytes from lymph nodes, liver or the lung lamina propria were specifically stained by TL-tet. By contrast, nearly 50% of B6 IELs were TL-tet+. TL-tet were prepared using TL heavy chain encoded by the T3b gene, but they also stain gut IELs from BALB/c mice, which express the TL heavy chain encoded by the T3d gene. Although a smaller percentage of IELs are positive (30%), the positive IEL actually bind more TL-tet than B6 IEL, arguing that the lower fraction stained is not due to the decreased avidity of TL-tet for BALB/c IEL (data not shown).



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Fig. 1. Binding specificity of the TL tetramers to IELs. (A) Flow cytometry was used to determine the percentage of TL-tet+ among T lymphocytes from lymph nodes (LN), lung (LPL lung), liver (IEL liver) and small intestine of B6 (IEL gut B6) or BALB/c (IEL gut BALB/c) animals. Data represent the mean and SD of at least two mice per group. (B) Flow cytometry was used to determine TCR usage among TL-tet+ cells for small intestine IELs from B6 (open bars) or BALB/c (grey bars) animals. Data represent the mean percentages and SD of three mice per group. The mean percentage and SD of B6 and BALB/c IEL subpopulations were respectively: TCR{gamma}+: 60 ± 5 and 38 ± 6; TCRß+CD8{alpha}ß+: 14 ± 4 and 27 ± 5; TCRß+CD8{alpha}{alpha}+: 19 ± 5 and 20 ± 2.

 
Further characterization of TL-tet+ small intestinal IEL populations from B6 and BALB/c mice revealed that despite the difference in fraction of TL-tet+ cells, the IEL subpopulations present in each strain were highly similar. Approximately 50% of both B6 and BALB/c TL-tet+ IEL populations express TCR{gamma}{delta} (Fig. 1B; TCR{gamma}+) and 50% TCR{alpha}ß. Of the TCR{alpha}ß+ cells, about half express CD8{alpha}{alpha} and the other half CD8{alpha}ß (Fig. 1B; TCRß+CD8{alpha}ß+and TCRß+CD8{alpha}{alpha}+). We failed to detect significant numbers of CD8+CD4+ cells in either strain. TL-tet did not stain the fraction of CD4+ IELs and Peyer's patch and/or lamina propria-derived CD4+ cells that sometimes contaminate IEL preparations (data not shown).

Characterization of cell surface molecules required for TL-tet binding and for generation of TL-tet+-IELs in vivo
It has been reported that the interaction of TL-tet with IELs is based on the binding of TL to CD8{alpha}{alpha} (10). We confirmed this finding via several approaches (not shown). First, expression of CD8{alpha} via transient transfection is sufficient to enable TL-tet binding to CD8 cells. Second, co-expression of CD8ß with CD8{alpha} reduces TL-tet binding, probably by reducing CD8{alpha}{alpha} expression. Third, binding of TL-tet to IELs was blocked by antibodies specific for CD8{alpha} but not CD8ß.

Consistent with this conclusion, we found that although B6 mice lacking the CD8{alpha} gene possess IELs that express either TCR{alpha}ß or TCR{gamma}{delta} in proportions similar to wild-type mice, these cells fail to bind TL-tet+ above background levels (Fig. 2; CD8{alpha}–/–). We used additional mice with targeted genetic disruptions to explore the interaction of TL-tet with IELs and the requirements for the generation of TL-tet+ IELs. Mice with disruptions in either CD3{varepsilon}, TCR{alpha} or TCR{delta} genes all possess large numbers of TL-tet+ IELs, as do nude mice (Fig. 2; CD3{varepsilon}–/–, TCR{alpha}–/–, TCR{delta}–/– and nude, respectively). This indicates that TCR expression is not required for the generation of TL-tet+ IELs or for binding of TL-tet to IELs. Interestingly, TL-tet+ IEL subpopulations from CD3{varepsilon}–/–, TCR{alpha}–/– and TCR{delta}–/– animals (all of which express CD8{alpha}) demonstrate greatly reduced numbers of cells expressing CD8ß (data not shown).



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Fig. 2. TL tetramer binding to gut IELs from various knock-out mouse strains. Flow cytometry was used to determine the percentage of TL-tet+ among IELs from wild-type mice or mice with targeted disruptions in the genes indicated. Data represent the means and SD of at least two mice per group.

 
Requirement for TL expression in generation of IELs and CD8{alpha} expression in expression of TL
We next investigated the role of TL in generating IEL populations. Lacking mice with a targeted disruption in the TL gene, we turned to mice with a targeted disruption in the ß2m gene. ß2m is known to be required for TL expression in cultured cells (16). To confirm the requirement for ß2m in TL expression in vivo, we stained frozen sections of small intestine with {alpha}TL mAbs (Fig. 3). While we found intense staining of B6 enterocytes, particularly the basolateral membranes as previously described (2), ß2m–/– enterocytes failed to be stained above background levels observed when using just the secondary fluorescent antibodies (Fig. 3; {alpha}-TL). Staining of gut cryosections with a {alpha}-MHC class I mAb illustrates the difference in location between classical MHC class I and non classical MHC class I molecules. Kb molecules are expressed by all the cells in the epithelium and lamina propria while TL staining is limited to the epithelium (Fig. 3; compare {alpha}-MHC class I and {alpha}-TL).



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Fig. 3. Comparison of TL and MHC class I surface expression in B6 and ß2m–/– small intestine. TL and Kb molecules were localized in small intestine cryosections by indirect immunofluorescence using a laser scanning confocal microscope. Villi are viewed en face: their different appearance in the anti-TL and anti-Kb panels is due to the different distribution of TL and Kb molecules. Note how TL staining is localized to epithelial cells while Kb stains all cells present in the sections.

 
Despite the absence of TL in ß2m–/– mice, the fraction of TL-tet+ IELs actually increased from 50% to nearly 80% of total IEL (Fig. 2; ß2m–/–). Similar numbers of IELs were recovered from ß2m–/– and wild-type mice and could be observed in tissue sections stained with either {alpha}-CD3 or {alpha}-TCR{gamma}{delta} antibodies (data not shown). Our results are consistent with the findings of Fujiura et al. showing that 80% of gut IELs from ß2m–/– mice express CD8{alpha}{alpha} (17). We also confirmed Fujiura's finding that >90% of IELs from ß2m–/– mice express {gamma}{delta} TCR.

We next investigated the involvement of CD8{alpha}{alpha} IEL in controlling TL expression by examining TL expression in gut cryosections from CD8{alpha}–/– mice. We were unable to detect a significant difference in TL staining between wild-type and knockout mice (data not shown).

Taken together, these data suggest that expression of TL is not greatly influenced by the presence of CD8{alpha}+ IELs, and conversely, that CD8{alpha}+ IELs do not require TL expression for their generation of maintenance.

IEL survival and trafficking is independent of CD8{alpha}{alpha}–TL interaction
The seeming independence of expression of TL molecules and CD8{alpha}{alpha} co-receptor prompted us to study the possible biological function(s) of the TL–CD8{alpha}{alpha} interaction. We examined the effect of the TL–CD8{alpha}{alpha} interaction on the survival of IEL from wild-type mice and mice with various targeted disruptions in genes potentially relevant to TL function. Bcl-2 plays an important role in cell survival (18), particularly in immune cells, where loss of Bcl-2 expression contributes to apoptosis resulting from an inability to respond to external survival signals (19). We used Bcl-2 expression to monitor the effect of TL on the IEL. IELs were co-incubated for 16 h in wells coated (or not) with {alpha}-CD3 mAb with P815 cells transfected with the TL gene or an irrelevant gene. Bcl-2 levels were determined via intracellular staining and flow cytometry and are presented as a percentage of fluorescence base line for Bcl-2 expression in IELs in the absence of P815 cells (Fig. 4).The addition of P815 cells (transfected or not with TL) decreased Bcl-2 levels in IELs by 15–45% depending on the mouse strain. However, TL expression or CD3 ligation had only small and inconsistent effects on Bcl-2 expression (Fig. 4). Consistent with these findings, addition of TL-specific antibodies that block the interactions of TL-tet+ with CD8{alpha} had no significant effect on Bcl-2 expression (Fig. 4; BALB/c {alpha}-TL). We also failed to observe significant effects on IEL expression of IL-7R{alpha}, which is involved in lymphocyte survival in signaling Bcl-2 up-regulation (20) (data not shown).



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Fig. 4. Comparison of Bcl-2 expression in IELs for different mouse strains. Small intestine IELs from wild-type mice and mice with targeted disruptions in the genes indicated were incubated in {alpha}-CD3{varepsilon} mAb-coated wells in the presence of P815 cells transfected with control plasmid (P815i), or TL-expressing plasmid (P815TL). For ‘BALB/c {alpha}-TL’ {alpha}-TL antibodies were added at the start of the incubation period. For analysis, cells were stained by {alpha}-CD8{alpha} mAb, followed by intracellular staining with a {alpha}-Bcl-2 mAb. Flow cytometry was used to analyze Bcl-2 expression in CD8{alpha}+ cells, except in CD8{alpha}–/– IELs where the whole IEL population was analyzed. The level of Bcl-2 expression in the absence of P815 cells was measured in the presence or absence of {alpha}-CD3{varepsilon} mAb for each mouse strain, and the corresponding mean fluorescence value was used as a baseline. Data for the IELs co-cultured with P815 cells are expressed as the percentage of these values for each condition. Data are representative of at least two independent experiments.

 
We also examined whether the TL-CD8{alpha}{alpha} interaction could interfere with the expression of specific IEL adhesion molecule CD103 (Fig. 5). CD103 ({alpha}Eß7) is an integrin expressed on >90% of IELs that is important for IEL localization to the gut (21). CD103 is preferentially expressed on CD8-expressing cells in vivo (22). Neither TL expression nor CD3 cross-linking altered CD103 expression on the surface of BALB/c IELs co-cultured with P815 cells (Fig. 5). Similar results were obtained in the absence of {alpha}-CD3 mAb and with IELs from B6, ß2m–/–, CD8{alpha}–/– and CD8ß–/– mice (data not shown).



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Fig. 5. Comparison of integrin {alpha}Eß7 levels for gut IELs in co-cultures. Small intestine IELs from BALB/c mice incubated in {alpha}-CD3{varepsilon} mAb-coated wells in the presence of control or TL-expressing P815 cells were stained with {alpha}-CD8{alpha} and {alpha}-CD103 mAbs and analyzed by flow cytometry for CD103 expression among CD8{alpha}+ cells. Data are representative of two independent experiments.

 
These findings were made using an artificial in vitro system which may conceal the effects of TL on IEL survival or trafficking. To extend the conclusions to a more biologically relevant system we tested whether TL expression influences the survival of IELs in vivo. We isolated B6 IELs, labeled them with CFSE, transferred them into B6 or ß2m–/– mice and reisolated small intestinal IELs 5 days after transfer (Fig. 6). The absence of ß2m in recipients had only a slight effect on the number of IELs recovered (0.14% of total IELs vs 0.2% in wild-type mice). The difference was reflected in both TCRß+ cells (representing TCR{alpha}ß-expressing cells) and TCRß (representing predominantly TCR{gamma}{delta}). The intensity of CFSE staining can be used to determine the extent of cell division; with each division the intensity of cell staining diminishes as the dye is diluted. Our results show that transferred IELs divide in ß2m–/– mice, particularly among the TCR{alpha}ß+ cells (Fig. 6B, TCR{alpha}ß, dashed curve), while they tend to proliferate less when transferred into a B6 animal (Fig. 6A and B, black curves). The reason for this difference is unclear, but it may reflect negative feedback of a ß2m-dependent ligand on IEL activation and divison. Similar results were obtained when the IELs were isolated 1 or 3 days following transfer (data not shown).



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Fig. 6. Recovery of B6 IELs transferred into B6 and ß2m–/– mice. (A) B6 IELs were labeled with CFSE and introduced into B6 or ß2m–/– mice. After 5 days, IELs obtained from one mouse were stained with {alpha}-TCRß mAb and analyzed by flow cytometry. The percentage of cells in each quadrant is indicated. Similar data were obtained in two other separate experiments in which IELs were reisolated from one B6 and one ß2m–/– mouse, 1 and 3 days after transfer. (B) CFSE+ IELs recovered from recipient mice were analyzed for CFSE staining by flow cytometry according to their staining with {alpha}-TCRß mAb. Tot TCR: all CFSE cells; TCR {gamma}{delta}+: CFSE+, non-TCRß cells; TCR{alpha}ß: CFSE+, TCRß cells.

 
Altogether, these findings suggested that TL is not essential to maintain the viability of IELs in vitro or in vivo, and they demonstrate conclusively that IELs are capable of proliferating and localizing to the gut in the absence of TL in vivo.

IFN-{gamma} production by IELs is mainly independent of CD8{alpha}{alpha}/TL interaction
We next examined the effect of TL on IEL secretion of IFN-{gamma}. BALB/c IELs were co-cultured in wells coated (or not) with {alpha}-CD3 mAb, along with P815 cells transfected with the TL gene or an irrelevant gene. IFN-{gamma} secretion was measured 16 h later by ELISA (Fig. 7). CD3 cross-linking was absolutely required for INF-{gamma} secretion (Fig. 7, grey bars vs black bars). Addition of P815 cells reduced the amount of IFN-{gamma} recovered from {alpha}-CD3-activated cells (data not shown), possibly due to sequestration of IFN-{gamma} by the P815 cells. However, IFN-{gamma} production by BALB/c IELs co-cultured with TL-expressing P815 was slightly increased (Fig. 7, grey bars). Our results are in good agreement with data reported by Leishmann et al. (10), who also observed a <2-fold increase in IFN-{gamma} production in comparable experimental conditions. Due to the modesty of the increase, we repeated this experiment using either B6 or BALB/c IELs to determine its reproducibility. In respectively 4 (B6) and 13 (BALB/c) independent experiments the average increase associated with TL expression was 1.25-fold and never exceeded 2-fold (data not shown).



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Fig. 7. IFN-{gamma} production by BALB/c IELs. Small intestine IELs from BALB/c mice were cultured in wells coated (grey bars) or not (black bars) with {alpha}-CD3{varepsilon} mAb in the presence of P815 cells transfected with control plasmid (P815irr) or TL-expressing plasmid (P815TL). IFN-{gamma} production was measured by ELISA. Data are representative of two independent experiments.

 
We next used ICS to determine which BALB/c IEL subpopulations secrete INF-{gamma} (Table 1). Following incubation with {alpha}-CD3 and TL-P815 cells, nearly all of the INF-{gamma}+ cells were in the TCR{alpha}ß CD8{alpha}ß subset. A similar pattern was observed using non-TL expressing P815 cells, suggesting that the small TL-associated increase in INF-{gamma} is due to an increase in the amounts of INF-{gamma} produced and not recruitment of more INF-{gamma} secreting cells (data not shown).


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Table 1. IFN-{gamma}-producing IEL subpopulations

 
We compared BALB/c and B6 IELs for IFN-{gamma} production in co-culture conditions in the presence of {alpha}-CD3 mAb (Fig. 8). While a similar fraction of BALB/c and B6 TCR{alpha}ß+ IELs produced INF-{gamma} (Fig. 8, grey bars), we found that 6% of B6 TCR{gamma}{delta}+ IELs produce IFN-{gamma} (Fig. 8, black bars). As with BALB/c IELs, IFN-{gamma} expression occurred independently of TL expression by the co-cultured cells. In another experiment we used BMA cells (H-2b); similar results were obtained as when using P815 transfectants, so we cannot attribute the pattern of IFN-{gamma} production to allo-stimulation (data not shown). Notably, we recovered twice as many TCR{gamma}{delta}+ IELs from B6 cultures than from BALB/c-derived cultures, due to either persistence or proliferation of the cells.



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Fig. 8. Comparison between BALB/c and B6 mouse IEL subpopulations that produce IFN-{gamma}. Small intestine IELs from BALB/c and B6 mice were cultured with BFA in {alpha}-CD3{varepsilon} mAb-coated wells in the presence of TL-expressing P815 cells (BALB/c and B6). Cells were then stained with {alpha}-CD8{alpha}, {alpha}-TCRß and {alpha}-TCR{gamma}{delta} mAbs, followed by intracellular staining for IFN-{gamma}. CD8{alpha}+ gated cells were analyzed by flow cytometry. Data are expressed as the percentage of IFN-{gamma}-producing that express TCRß (grey bars) or TCR{gamma}{delta} (black bars). The means and SD of at least two mice are shown per group. Data are representative of three independent experiments.

 
To eliminate the possibility that the failure to observe stimulation of BALB/c TCR{gamma}{delta} IELs is due to contamination with other cell types or intestinal debris, we sorted the IELs by size prior to culture. Sorted cells behaved similarly to unsorted cells (data not shown).

These findings indicate that there can be non-TL related, strain-dependent differences in the properties of TCR{gamma}{delta} IELs.

Influence of IL-15 and reovirus infection on IEL IFN-{gamma} secretion
Over expression of intestinal IL-15 can induce immune pathology in the small intestine, e.g. infiltration of activated CD8{alpha}ß, TCR{alpha}ß T cells secreting Th1-type cytokines (23). It was therefore of interest to examine the effect of IL-15 on BALB/c IEL secretion of INF-{gamma} (Fig. 9A). This resulted in an ~20% increase in INF-{gamma} secretion in {alpha}-CD3-activated IELs cultured with either TL-expressing or non-expressing P815 cells (Fig. 9A, open and black bars). ICS revealed that the increase is due strictly to greater production by TCR{alpha}ß, CD8{alpha}ß IEL. We conclude from these data that IL-15 increases IEL secretion of INF-{gamma} but does so in a TL-independent manner.



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Fig. 9. Effect IL-15 or reovirus infection on IEL IFN-{gamma} production. Small intestine IELs from BALB/c mice were cultured in {alpha}-CD3{varepsilon} mAb-coated wells in the presence of P815 cells transfected with control plasmid (P815irr) or TL-expressing plasmid (P815TL). IFN-{gamma} production was measured by ELISA. Data are representative of two independent experiments. (A) Cells were cultured in the absence (open bar), or the presence (black bars) of IL-15. (B) IELs were obtained from reovirus infected (black bars) or uninfected (open bars) mice. BALB/c mice were orally infected with 107 plaque forming units of reovirus T1/L in 0.5 ml PBS. The mice were sacrificed 24 h later, at which time IELs were prepared and stained.

 
Lastly, we examined whether viral infection of the small intestine alters the ability of IELs to produce INF-{gamma} (Fig. 9B). Reovirus predominantly infects epithelial cells in the ileum and elicits virus-specific TCR{alpha}ß, CD8{alpha}ß CTLs (24,25) that interact preferentially with the basolateral membrane of intestinal epithelial cells (26). Using IELs harvested from mice infected with type 1 reovirus 1 day post-infection, we found that reovirus had no significant effect on INF-{gamma} secretion in the presence or absence of TL-transfected P815 cells (Fig. 9B, black and open bars). ICS analysis showed that IFN-{gamma} was produced mostly by TCR{alpha}ß, CD8{alpha}ß IELs from both uninfected and infected animals (data not shown).


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The best clue to the function of TL must be its highly restricted expression to small intestinal epithelial cells. Its basolateral distribution places it perfectly to interact with CD8{alpha}{alpha}+ IELs, whose origins and functions are still under debate despite their great abundance in mammals. IELs are distinct from systemic T cells, primarily in their subset composition: more than 70% of small intestinal IEL comprise CD8+ cells, mainly expressed in the absence of CD8ß, a population that is essentially absent from the circulation.

We have confirmed the observations of Leishman et al. that only ~50% of CD8{alpha}+ß IEL bind to TL-tet (10). Although in vitro tetramer binding might not entirely represent the in vivo interaction between TL and CD8{alpha}{alpha}, it is most likely that some IELs expressing CD8{alpha}{alpha} do not interact with TL, raising the question of what factors could negatively influence TL binding to CD8{alpha}{alpha}. The simplest explanation would be post-translational modification (e.g. glycosylation) of CD8{alpha} that decreases its affinity for TL. While Leishman et al. generated TL-tet from the T18d, we used the T3b gene. The two genes differ in 9% of their amino acid residues, with mainly conservative changes in exons 5, 6 and 4. The similar results obtained with the two distinct TL gene products supports the validity and generality of the binding data, as well as the other concordant observations in the two papers.

The expression of CD8{alpha}{alpha} can be induced on activated, conventional T cells (12), as well as on conventional CD4+ mature splenocytes when these cells migrate to the intestine after antigen activation (27). Furthermore, CD8ß+ IELs clearly can express CD8{alpha}{alpha} (Fig. 1) (10). Our demonstrating a TL-dependent increase of IFN-{gamma} production upon TCR triggering of TCR{alpha}ß, CD8{alpha}ß IELs suggests that CD8{alpha}{alpha} can modulate the function of these cells.

It was previously reported that TL enhances {alpha}-CD3-mediated release of INF-{gamma} by IELs (10). We confirm both this finding and the modesty of the effect. Moreover, we demonstrate that this effect is limited to one subset of IELs in BALB/c animals, namely TCR{alpha}ß, CD8{alpha}ß-bearing cells, and that in B6 mice IFN-{gamma} release by CD8{alpha}{alpha} TCR{gamma}{delta} IELs under these conditions is independent of TL. Since we used total IELs we cannot rule out that the presence of a minor IEL subset could influence which IEL subset producing IFN-{gamma} is affected by TL expression. However, it is interesting to note that no matter whether purified CD8{alpha}{alpha} IELs (10) or total IELs (this study) are used in co-cultures, the TL-dependent IFN-{gamma} increase displays the same moderate amplitude, suggesting that the presence of low affinity TL-binding CD8{alpha}ß IELs in our experimental conditions have a minimal impact on the function of CD8{alpha}{alpha} IELs.

Noticeably our study demonstrates that the presence of TL affects only the quantity of IFN-{gamma} produced, not the IEL subpopulation that produces it. Our conclusion that the small increase in IFN{gamma} production by an IEL subset is unlikely to be a significant biological response to CD8{alpha}{alpha}/TL interaction does not preclude a role for TL in modulating proliferation and cytotoxicity, as we did not study these parameters. However, any biological effect might be questionable considering the use of IELs from transgenic source and the unphysiological nature of {alpha}-CD3-mediated TCR cross-linking.

A possible role for the TL-CD8{alpha}{alpha} interaction may be to control lymphocyte division in the confined space of the intraepithelial compartment. Notably, however, the numbers of IELs are not increased in either mice lacking TL due to disruption of the ß2m gene or in mice lacking CD8{alpha}. The normal number of IELs in ß2m–/– mice also indicates that TL is not needed for IEL localization or to provide ‘viability’ signals to IELs. These conclusions are supported by our failure to observe TL-mediated alterations in Bcl-2 expression and our demonstration that IELs appear to be homed properly to their normal location in ß2m–/– mice. Another possibility is that TL serves to help retain IELs in the small intestine. {alpha}Eß7 integrin mediates T cell adhesion to epithelial cells through its binding to E-cadherin (28) and evidence has suggested that {alpha}Eß7-dependent adhesion is regulated by inside-out signals (29). However, our data demonstrate that TL does not modulate the expression of {alpha}Eß7 at the surface of CD8{alpha}{alpha} IELs.

So what is the function of TL? While we must consider it possible that TL has CD8-independent functions, the high percentage of CD8{alpha}{alpha}-expressing IELs makes it highly probable that the high affinity of TL for CD8{alpha}{alpha} is no coincidence. Clearly, it is possible that TL plays a critical role in controlling IEL activation or proliferation and that additional co-stimulatory (or inhibitory) molecules are needed to reveal its function.

On the other hand, perhaps we are looking at the interaction in a too ‘T cell-centric’ manner. As TL possesses a transmembrane domain that could potentially transmit information to epithelial cells, could TL, rather than providing signals to IELs receive them from IELs? Lacking a TL–/– mouse to examine the effect of TL on small intestinal function, the closest thing is the ß2m–/– mouse. While there are no gross alterations in gut epithelia of ß2m–/– mice, there may be subtle functional deficiencies. As a relevant example, it took years to discover that ß2m–/– mice suffer from alterations in iron metabolism due to disruption of the function of the HFE class Ib molecules (30). Deciphering the function of TL will require answering why TL is lacking in other mammals, including humans. Are there special features of murine biology that require the special functions of TL?

Several possible functions have been assigned to IELs, which unlike TL, are ubiquitous in organisms with adaptive immune systems (3133). The type of co-receptor expressed at the surface of the IELs must have an important impact on the IELs function and recognition. How and why CD8{alpha} chain forms homodimers at the surface of IELs instead of pairing with the CD8ß chain surely is bound to provide insight into understanding the relationships between the intestinal epithelium and these peculiar mucosal lymphocytes, and perhaps shed light on the true biological function of TL.


    Acknowledgements
 
We thank Drs D. Guy-Grand, D. Buzzoni-Gatel and L. Gapin for insightful discussions. Reovirus-infected animals were kindly provided by Dr M. Fleeton.


    Abbreviations
 
ß2m   ß2-microglobulin
IEL   intraepithelial lymphocyte
TL   thymic leukemia antigen
TL-tet   TL tetramer

    Notes
 
Transmitting editor: P. Ohashi

Received 25 March 2004, accepted 22 June 2004.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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