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IL-15 Does Not Affect IEL Development in the Thymus but Regulates Homeostasis of Putative Precursors and Mature CD8⁺ IELs in the Intestine¹

Yein-Gei Lai^*,, Mau-Sheng Hou, Yaw-Wen Hsu, Chin-Ling Chang^*,, Yae-Huei Liou, Ming-Han Tsai, Fan Lee and Nan-Shih Liao^2,*,

^* Graduate Institute of Life Sciences, National Defense Medical Center, Institute of Molecular Biology, Academia Sinica, Taipei, and Division of Nephrology, Chang Gung Memorial Hospital, Taoyuan, Taiwan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Mice devoid of the IL-15 system lose over 90% of CD8⁺ TCRβ and TCR intestinal intraepithelial lymphocytes (iIELs). Previous work revealed that IL-15R and IL-15 expressed by parenchymal cells, but not by bone marrow-derived cells, are required for normal CD8⁺ iIEL homeostasis. However, it remains unclear when and how the IL-15 system affects CD8⁺ iIELs through their development. This study found that IL-15R is dispensable for the thymic stage of CD8⁺ TCRβ and TCR iIEL development but is required for the maintenance and/or differentiation of the putative lineage marker negative precursors in the intestinal epithelium, especially for the most mature CD8 single positive subset. Moreover, the IL-15 system directly supports the survival of mature CD8⁺ iIEL in vivo. Taken together, this study suggests that regulation of CD8⁺ iIEL homeostasis by the IL-15 system does not occur in the thymus but involves mature cells and putative precursors in the intestine.

	Introduction

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Interleukin-15 (IL-15) is a pleiotropic cytokine expressed in many cell and tissue types (1). The IL-15R consists of -, β-, and -chains. The IL-15R -chain confers specificity for IL-15, for which it possesses high affinity, and is expressed widely as its ligand (2). The β-chains are common to IL-15R and IL-2R, bind both soluble IL-2 and IL-15 with intermediate affinity, and are expressed mainly by hematopoietic cells (2). Lymphocytes signal with IL-15 through two modes of high-affinity receptor-ligand interaction: one uses soluble IL-15 via the β receptor (2), whereas the other uses IL-15 bound to IL-15R on neighboring cells via the β receptor (3). The latter mode has been termed "in trans presentation" and appears to be the major mode used in homeostatic control of several lymphoid lineages in vivo (4).

Intraepithelial lymphocytes (IELs)³ in the small intestine reside between the columnar epithelial cells and are composed of multiple T cell populations. In C57BL/6J (B6) mice, 50% of intestinal IELs (iIELs) are CD8 single positive (SP), in which TCRβ⁺ (β) and TCR⁺ () cells are found at a ratio of 1:4. Mice lacking the IL-15 system, including Il15^–/– (5), Il15ra^–/– (6), and Il15rb^–/– (7) mice, show a severe reduction in CD8⁺ β and iIEL. This deficiency may result from either a developmental or a maintenance defect. Bone marrow (BM) chimera experiments demonstrated that IL-15 and IL-15R expressed by non-BM-derived parenchymal cells are essential for normal CD8⁺ β and iIEL homeostasis, whereas IL-15 and IL-15R expressed by BM-derived cells are dispensable (8). However, just when and how the IL-15 system affects CD8⁺ iIEL homeostasis in vivo remains elusive.

Normal CD8⁺ iIEL homeostasis requires the presence of a thymus in neonatal life, as nude mice and neonatal thymectomized mice harbor few CD8⁺ β iIELs and 10 times less CD8⁺ iIELs than normal mice (9, 10), and neonatal thymus transplants restore both iIEL subsets in athymic mice (11). Recent studies identified CD8⁺ iIEL precursors in the thymus and have led to two different models for CD8⁺ iIEL differentiation involving thymic precursors. One model proposes that CD8⁺ β iIELs complete TCR selection in the thymus, based on the presence of thymic CD4⁺CD8β⁺CD8⁺TCR^– preselection and CD4^–CD8^–TCRβ⁺ post-selection precursors in B6 mice (12). A role of IL-15 in the development of these thymic iIEL precursors was suggested by the fact that both pre- and post-selection precursors gave rise to cells with the mature CD4^–CD8⁺ TCRβ⁺ phenotype when cultured in the presence of IL-15, despite the differentiation of the preselection precursors requiring thymic epithelial cells and cognate antigenic peptides (12). This model is consistent with findings that suggest positive selection of CD8⁺ β iIEL repertoire by agonist self-Ags at the CD4⁺CD8⁺ thymocyte stage (13, 14). Another model proposes that CD4^–CD8^–TCR^– (triple negative, TN) thymocytes at the TN2 and TN3 stages, as defined by CD44⁺CD25⁺ and CD44^–CD25⁺, respectively, contain CD8⁺ iIEL precursors (15). These early precursors migrate to the intestine, where they colonize and give rise to mature CD8⁺ β and iIELs, as shown by the generation of CD8⁺ β and iIELs in Rag2^–/–_c^–/– mice transferred with TN2-TN3 thymocytes (15). Although the two models disagree on the differentiation steps taken by CD8⁺ iIEL precursors in the thymus, they agree on the presence of a thymus stage during CD8⁺ iIEL development. Given that a neonatal thymus and the IL-15 system are required for normal CD8⁺ iIEL homeostasis, that the thymic epithelium expresses IL-15 (16) and IL-15R (unpublished data), and that cultured thymic CD8⁺ β iIEL precursors survive and differentiate in the presence of IL-15, it is logical to examine whether the IL-15 system participates in CD8⁺ iIEL development in the thymus.

In addition to the recently identified thymic precursors described above, TCR^– precursors for CD8⁺ iIEL have been known to exist in the cryptopatches (CP) and in the iIEL compartment of the intestine (17, 18, 19). Despite the fact that the CP precursors give rise to the iIEL precursors (19), mice lacking CP structures possess a normal CD8⁺ iIEL compartment (20). Both extrathymic and intrathymic differentiation pathways contribute to the TCR^– iIEL precursor pool, as shown by the presence of CD3^– iIEL in nude and in day 3-thymectomized mice (10, 21) and by generation of lineage marker negative (lin^–) CP precursors and mature CD8⁺ iIELs from TN2-TN3 thymocytes transferred into thymectomized Rag2^–/–Il2rg^–/– mice (15), respectively. One study reported three lin^– iIEL subsets with precursor-product relationships determined by their generation kinetics from BM precursors and TCR rearrangements (19). These lin^– iIEL subsets can be distinguished by differential expression of Thy1 and CD8 molecules: the Thy1 SP cells give rise to Thy1 CD8 double positive (DP) and then to CD8 SP cells. Whether the IL-15 system affects these putative iIEL precursors resided in the intestine has not been determined.

For mature iIELs, IL-15 has been shown to increase Bcl-2 expression and support the survival of primary iIELs and CD8⁺ β iIELs in vitro in the absence of TCR stimulation (22, 23). However, over-expression of Bcl-2 only partially restored CD8⁺ iIELs in Il15^–/– mice (24) and did not rescue CD8⁺ β iIELs in Il15ra^–/– mice (unpublished data). Therefore, the role of the IL-15 system in the survival of mature CD8⁺ iIELs in vivo remains to be determined. In this study, we examined the role of the IL-15 system during CD8⁺ iIEL development in the thymus and in the intestine.

	Materials and Methods

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Mice

B6 and B6-nude (Foxn1^nu) mice were purchased from The Jackson Laboratory and bred in our animal facility. Il15ra^–/– mice were generated in this lab (25) and backcrossed to B6 for at least 17 generations. Ubiquitin promoter-driven enhanced GFP (eGFP) transgenic mice on a B6 background were kindly provided by Dr. C.-Q. Wang (Institute of Molecular Biology, Academia Sinica, Taiwan) and bred in our animal facility. All mice used were 12–18 wk of age. All experiments using mice were approved by the Institutional Animal Care and Use Committee at Academia Sinica and conformed to the relevant regulations.

Total iIEL preparation and immunofluorescence

In brief, after removal of Peyer’s patches (PP), the small intestine was cut into 5 mm pieces and stirred in Ca²⁺-Mg²⁺-free Dulbecco’s PBS (Invitrogen Life Technologies) containing 1 mM DTT (Merck), 1 mM EDTA (Merck), and 2% FBS at 37°C for 35 min. Supernatant was passed through nylon wool (Cellular Products) in a column, and IELs were enriched by a discontinuous 43–67% Percoll (Pharmacia) gradient. Cells were stained in staining buffer (PBS-2%FBS-0.02%NaN₃) containing mAbs, following standard protocols, and analyzed on FACScalibur or LSRII (BD Biosciences). The following mAbs conjugated with fluorochrome or biotin (prepared in our lab or purchased from eBioscience or Biolegend) were used: CD3 (2C11), CD4 (GK1.5), CD8 (53.6.7), CD8β (53.5.8), Thy1.2 (30-H12), TCRβ (H57.597), TCR (GL3), V2 (UC3–10A6), and V5 (GL1 was kindly provided by Dr. L. Lefrancois, University of Connecticut, Farmington, CT). Samples were stained with biotin-conjugated Ab followed with streptavidin (SA)-allophycocyanin or SA-allophycocyanin-Cy7 (eBioscience or Biolegend).

Adult mice thymectomized and bone marrow re-constituted (ATXBM) chimera preparation

B6 and Il15ra^–/– mice at 6–8 wk of age were thymectomized as described (26), rested for 2 wk, and engrafted with B6 or Il15ra^–/– neonatal thymus. Two weeks later, the mice were irradiated (1000 rads, ¹³⁷Cs source) and injected i.v. with wild-type (WT) eGFP⁺ T cell-depleted BM cells (4 x 10⁶/mouse) prepared by treating femur and tibia BM cells with mAbs specific for Thy1 (J1J), CD4 (RL172.4), and CD8 (3.155) plus guinea pig complement at 37°C for 20 min.

Thymus engraftment

ATXBM chimera or nude mice were anesthetized with tribromoethanol (27). An incision was made through the skin and the muscle covering the left kidney and then through the kidney capsule. A whole thymus, taken from B6 or Il15ra^–/– mice within 24 h after birth, was placed under the kidney capsule. The muscle incision was sutured, and the skin incision was closed with a wound clip. Ten to 12 wk later, iIELs were isolated, counted, and analyzed for cell composition by staining with propidium iodide (PI) and mAbs specific for TCRβ, TCR, CD4, CD8, and CD8β.

CD8⁺ iIEL preparation

Total iIEL were prepared as described above up to the nylon wool filtration step. Cells were re-suspended in 33% Percoll and centrifuged to remove mucus. Intestinal epithelial cells and CD4⁺ cells were removed by complement-mediated lysis with mAbs specific for MHC class II (BP107.2, 28-16-8s, 25-5-16s) and CD4 (RL172.4). Live IEL were recovered by 43–67% discontinuous Percoll gradient centrifugation and stained with anti-CD4-PE, anti-CD8β-PE, and anti-CD8-biotin mAb. CD8⁺ cells were isolated by depletion of CD4⁺ and CD8β⁺ cells with anti-PE mAb-conjugated beads (Miltenyi Biotec) and then by positive selection with SA MicroBeads (Miltenyi Biotec) using autoMACS (Miltenyi Biotec). The resultant preparation contained 95–98% CD8⁺ cells, which consisted of 25.0 ± 0.8% β cells, 72.3 ± 0.8% cells, and 1.6 ± 0.1% TCR^– cells.

IEL precursor analysis

Total iIEL were isolated as described (19). Cells were incubated with anti-CD16/32 (2.4G2; prepared in this lab) to block FcR and then stained with biotinylated lineage marker mAbs (CD3, CD19, erythroid cells (TER119), Gr-1 (8C5)), PE-conjugated mAbs against TCRβ, TCR, goat anti-mouse IgM (Southern Biotech Associates), and CD11b, Pacific blue (PB)-anti-CD8, PE-Cy7-anti-Thy1.2 (53.2.1), FITC-anti-CD44 (1M7), and allophycocyanin-anti-B220 (RA3–6B2). For IL-15R analysis, cells were stained with PE-conjugated lineage marker mAbs (CD3, CD19, erythroid cells, Gr-1, TCRβ, TCR, and IgM), PB-anti-CD8, PE-Cy7-anti-Thy1.2, FITC-anti-CD44, and biotinylated goat anti-mIL15R (R&D Systems), IL-2Rβ (5H4), or anti-_c (4G3) followed with SA-allophycocyanin. Cells were analyzed on LSRII for lin^– iIEL precursor subsets and surface marker expression. All mAbs were purchased from Pharmingen, eBioscience, or Biolegend.

In vivo cell survival assay

CD8⁺ iIELs were prepared from B6 or egfp transgenic mice. The former were labeled with CFSE (5 µM) using Vybrant CFDA SE CellTracer kit (Molecular Probes) following the manufacturer’s instructions. CD8⁺ iIELs were injected into recipient mice (5 7.5 x 10⁶ cells/mouse) via the tail vein. Total iIEL, spleen cells and PP cells were isolated from the recipient mice at indicated time points, suspended in 1 ml of staining buffer, and analyzed for CFSE⁺ or eGFP⁺ cells after collecting 10⁷ cells by LSRII. The volume of the remaining cell suspension was measured and used to deduce the total number of CFSE⁺ or eGFP⁺ cells isolated from each lymphoid tissue.

In vitro cell survival assay

CD8⁺ iIELs were cultured in 96-well plates (1 x 10⁵ cells/200 µl/well) with indicated amounts of IL-2 (R&D Systems) and IL-15 (eBioscience) or in RPMI 1640 (Invitrogen Life Technologies)-10% FBS only. Anti-IL-2Rβ mAb (TM-β1; eBioscience) was added to some cultures to block IL-2/15Rβ. All cultures were conducted in triplicate. Cells were collected at indicated times, stained with PI (0.25 µg/ml in PBS containing 2% FBS and 0.02% NaN₃), and analyzed by FACScalibur. PI⁺ and PI^– represent dead and live cells, respectively.

Statistical analysis

As resultant data was generally normally distributed, Student’s t test was applied to all data analyses with GraphPad Prism (GraphPad).

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
IL-15R expressed by the thymic epithelium plays little role in CD8⁺ iIEL development

As the Il15ra^–/– mouse line used in this study was generated with a different construct to the other Il15ra^–/– mice line reported previously (6, 25), we first confirmed the CD8⁺ iIEL deficiency phenotype in our mouse line. Il15ra^–/– mice harbored 15 and 6 times less CD8⁺ β and iIELs, respectively, than WT mice at the age of 7–12 wk (Fig. 1). The number of CD8⁺ iIELs increased with age in WT mice but remained unchanged in KO mice, which led to a 39- and 15-fold reduction of CD8⁺ β and iIELs, respectively, in KO mice compared with WT mice at the age of 17–20 wk (Fig. 1).

View larger version (27K): [in this window] [in a new window]   FIGURE 1. CD8+ iIEL phenotype in Il15ra–/– mice. Total iIELs were isolated from WT and Il15ra–/– mice of indicated age, and the number of live cells was counted by trypan blue exclusion under the light microscope. The percentage of CD8+ β iIEL was determined by staining total iIELs with biotinylated anti-TCR, CD4 and CD8β mAb, and anti-CD8-PE. The percentage of CD8+ iIELs was determined by staining total iIELs with anti-TCR-biotin and anti-CD8-PE mAb. Stained cells were analyzed by flow cytometry, and cell numbers (mean ± SEM) were deduced from total cell counts and percentages of each subset. Each dot represents one mouse.

View larger version (53K): [in this window] [in a new window]   FIGURE 2. Thymic epithelium IL-15R plays little role in CD8+ iIEL development. A, Analysis of WT donor BM-derived CD8+ iIEL in ATXBM chimera engrafted with WT or Il15ra–/– neonatal thymus. The number of donor BM-derived (eGFP+) CD8+ β and iIELs were determined by cell counting and flow cytometry. Each dot represents one mouse. B, Analysis of iIELs in WT nude mice engrafted with WT or Il15ra–/– neonatal thymus. Total iIELs were isolated from sham-operated and from WT or KO thymus-engrafted nude mice and analyzed for TCR type (left) and for the percentage of CD8+ population in (central) and in β (right) cells by cell counting and flow cytometry. C, Analysis of TN2 and TN3 thymocytes in WT and Il15ra–/– mice. Total thymocytes were stained with anti-CD4-PE, anti-CD8-PE, anti-CD3-PE, anti-CD44-FITC, and anti-CD25-allophycocyanin mAbs. CD4–CD8–CD3– cells were gated and analyzed for TN subpopulations. Data are representative of six pairs of mice. D, Analysis of thymic post-selection iIEL precursors in WT and Il15ra–/– mice. Total thymocytes were stained with anti-CD4-Alexa 405, anti-CD8-Alexa 405, anti-B220-PB, anti-TCRβ-FITC, anti-NK1.1-PE-Cy7, and anti-CD5-PE mAbs. CD4–CD8–B220– cells were analyzed for NK1.1 and TCRβ expression. The NK1.1–TCRβ+ subset, which represents the post-selection iIEL precursor, was examined for CD5 expression. Data are representative of six pairs of mice.

View this table: [in this window] [in a new window]   Table II. Analysis of V5+ CD8+ iIELa

Lin^– iIEL precursors in the intestinal epithelium of Il15ra^–/– mice are reduced in number and display altered IL-15R and surface marker patterns

We next examined whether the IL-15 system affects the three putative lin^– iIEL precursor populations in the intestinal epithelium (19). WT and Il15ra^–/– mice were examined at 5–6 wk and at 7–9 wk of age. In WT mice, similar numbers of Thy1 SP cells existed in both age groups, whereas the number of DP and CD8 SP cells increased 2 and 1.5 times, respectively, in the older mice (Fig. 3A). Compared with WT mice, KO mice harbored 2, 3.5, and 6 times less Thy1 SP, DP, and CD8 SP cells, respectively, in both age groups (Fig. 3A). All three precursor subsets expressed surface IL-15R in WT mice but not in KO mice (Fig. 3B). The patterns of IL-15Rβ expression by Thy1 SP and by DP cells were similar between WT and KO mice, whereas the level of IL-15Rβ was considerably elevated on CD8 SP cells in WT mice but not on their KO counterparts (Fig. 3B). The expression of _c by all three precursor subsets was similar in KO and WT mice (Fig. 3B). We further examined expression of CD44 and B220 by the lin^– iIEL precursor subsets, as CD44 is down-regulated while B220 is up-regulated along their differentiation in normal mice (19, 29). The expression patterns in WT and KO cells were relatively similar for the Thy1 SP and DP subsets but distinct for the CD8 SP subset, in which higher levels of CD44 and B220 were detected on the KO cells (Fig. 3C). Taken together, the reduction in cell number and the alteration in surface marker expression imply a role for the IL-15 system in both differentiation and maintenance of the putative lin^– iIEL precursors in the intestinal epithelium, especially for the most mature CD8 SP subset.

View larger version (31K): [in this window] [in a new window]   FIGURE 3. Lin– iIEL precursors in the intestinal epithelium of Il15ra–/– mice are reduced in number and displayed altered IL-15R and surface marker patterns. A, Analysis of lin– iIEL precursors in the intestine epithelium. The numbers of Thy1 SP, Thy1+CD8+, and CD8 SP lin– iIEL precursors in WT (closed symbols) and Il15ra–/– (open symbols) mice at indicated ages were determined by cell counting and flow cytometry. Each dot represents one mouse. B, Analysis of IL-15R, IL-15Rβ, and c expression by Thy1 SP, Thy1+CD8+, and CD8 SP lin– iIEL in WT and Il15ra–/– mice. The number displayed in each panel indicates the percentage of positive cells. Negative controls, comprising cells stained with all mAb and SA-conjugated fluorochrome except the specified anti-IL-15R chain mAb, contained <0.001% of positive cells. Data are representative of four pairs of mice. C, Analysis of CD44 and B220 expression by Thy1 SP, Thy1+CD8+, and CD8 SP lin– iIELs in WT and Il15ra–/– mice. Data are representative of four pairs of mice.

Maintenance of mature CD8⁺ iIEL requires IL-15R expression by peripheral parenchymal cells

We then examined the role of IL-15R in the maintenance of mature CD8⁺ iIEL in vivo by transferring WT eGFP⁺ CD8⁺ iIELs into un-manipulated WT and Il15ra^–/– mice and quantifying them in the spleen, Peyer’s patches (PP), and iIEL compartment 4, 12, and 20 days later. The number of eGFP⁺ cells decreased in the spleen of WT and KO recipients over time (Fig. 4A). This decrease likely reflected migration of the i.v.-delivered donor cells from the blood system to the lymphoid tissues, as suggested by the concomitant increase of eGFP⁺ cells in PP cells and in iIEL of WT recipients (Fig. 4A). The day 20 eGFP⁺ iIELs in WT recipient contained 31% β cells and 69% cells, a composition similar to that before transfer. However, donor cells in iIEL and PP failed to accumulate in KO recipients on day 20 (Fig. 4A). The numbers of eGFP⁺ β and iIEL between WT and KO recipients were similar on days 4 and 12 but reduced by 80% and 60%, respectively, in KO recipients compared with those in WT recipients on day 20 (Fig. 4B). Several factors may affect the number of donor cells in the iIEL compartment, including homing efficiency, cell division, and cell survival. The homing efficiency of donor cells appears similar in WT and KO recipients, as similar numbers of eGFP⁺ cells were recovered from the iIEL compartment of both recipients on days 4 and 12 (Fig. 4A). To examine the involvement of cell division, we transferred CFSE-labeled CD8⁺ iIELs. The CFSE level of donor cells in WT and KO iIELs appeared similar and showed no obvious dilution (Fig. 4C), implying that cell division had little effect on donor cell number in WT and KO recipients. Taken together, the reduction of donor CD8⁺ iIELs in the iIEL compartment of Il15ra^–/– recipients on day 20 likely reflects the defective maintenance of mature CD8⁺ iIELs in an Il15ra^–/– environment.

View larger version (24K): [in this window] [in a new window]   FIGURE 4. Maintenance of mature CD8+ iIELs requires IL-15R expression by peripheral parenchymal cells. A, Quantification of donor CD8+ iIELs in the lymphoid tissues of WT and Il15ra–/– recipient mice. WT eGFP+ CD8+ iIELs were transferred i.v., and the number of eGFP+ cells in total splenocytes, iIEL, and PP cells were determined by cell counting and flow cytometry at the indicated days after transfer. Data are representative of three experiments. B, Quantification of β and cells in eGFP+ iIELs isolated from WT and Il15ra–/– recipient mice. Data are representative of three experiments. C, Analysis of CFSE level in donor CD8+ iIEL. WT CD8+ iIEL were labeled with CFSE and transferred i.v. into WT and Il15ra–/– mice. The CFSE level of donor and host iIELs isolated from recipient mice were determined by flow cytometry 12 and 20 days after transfer. Data are representative of three experiments.

IL-15 exerts a direct prosurvival effect on CD8⁺ iIEL in vitro

To determine whether IL-15 affected CD8⁺ iIEL maintenance directly, cells were isolated and treated with IL-15 in vitro. IL-15 supported the survival of CD8⁺ iIELs in a dose-dependent manner (Fig. 5A, left). More live cells existed in the IL-15 culture than in the IL-2 or medium alone culture at various time points (Fig. 5A, middle). The ratio of β and cells in CD8⁺ iIELs before and after culturing in IL-15 remained constant (Fig. 5A, right), which indicates that IL-15 protected the two iIEL subsets with similar efficiency. IL-15 has been shown to induce signal transduction in T cells via the β receptor (30). Previous BM chimera experiments also showed that IL-15Rβ expressed by BM-derived cells, in addition to IL-15R expressed by parenchymal cells, is necessary for CD8⁺ iIEL homeostasis (8). We thus examined whether IL-15 functions via IL-15R on CD8⁺ iIELs and found that IL-15Rβ-blocking mAb completely inhibited IL-15’s prosurvival activity (Fig. 5B). Together, these results demonstrate that IL-15 exerts a direct prosurvival effect on CD8⁺ iIELs in an IL-15Rβ-dependent manner.

View larger version (19K): [in this window] [in a new window]   FIGURE 5. IL-15 supports CD8+ iIEL survival in vitro in an IL-15Rβ-dependent manner. A, IL-15 supports survival of CD8+ iIELs in vitro. Cells were cultured with various amounts of IL-15 or IL-2 for 40 h (left), or with 50 ng/ml IL-15 or IL-2 for indicated time periods (middle). Cell survival was determined by PI exclusion. The proportion of β and cells was determined by staining cells with TCRβ- and TCR-specific mAbs (right). Data are representative of three experiments. B, CD8+ iIEL were preincubated with indicated concentrations of anti-IL-2Rβ or isotype control mAb for 1 h, cultured with IL-15 (50 ng/ml) for 40 h, and analyzed for survival by PI exclusion. Data are representative of two experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

The dispensability of thymic epithelium IL-15R in CD8⁺ iIEL development is demonstrated by the similar capacity of WT and Il15ra^–/– neonatal thymus grafts to support the generation of CD8⁺ β and iIEL in WT ATXBM chimera and in WT nude mice. One recent study identified TCR^– preselection and TCR⁺ post-selection precursors for CD8⁺ β iIEL in the thymus (12). The two precursor populations were found to differentiate and survive in culture containing IL-15 (12), suggesting a role of IL-15 in their development in the thymus. For the CD4^–CD8^–TCRβ⁺ post-selection precursors, IL-15 induced CD8 and down-regulated CD5 expression, so the cells acquired a phenotype resembling mature CD8⁺ β iIELs. The authors speculated that this step might take place in the intestine where IL-15 expression is abundant, as the post-selection precursors readily generated CD8⁺ CD5^low β iIELs when transferred into Rag2^–/– mice. For the CD4⁺CD8β⁺CD8⁺TCR^– preselection precursors, thymic epithelial cells and cognate peptides were essential to drive their differentiation into TCRβ⁺ cells in vitro, in which about half of them acquire the mature CD8⁺ phenotype with the rest being CD4⁺CD8⁺ or CD4^–CD8^–. The role of IL-15 in this preselection precursor culture was not specified. Based on their results from the post-selection precursor culture experiment, we would speculate that IL-15 might act on cells that had differentiated in response to stimulation by thymic epithelial cells and Ags and drive them to the mature CD8⁺ phenotype. Consistently, we found that Il15ra^–/– mice harbor a normal number of thymic post-selection iIEL precursors, which is in agreement with a minimal role of IL-15 in the development of thymic iIEL precursors. Interestingly, despite the presence of normal numbers of post-selection iIEL precursors in the thymus, CD8⁺ β iIELs are still much reduced in the intestine of Il15ra^–/– mice. It is thus possible that these precursors are IL-15-independent in the thymus and acquire IL-15 dependency for further differentiation in the intestine. Another possibility is that the mature CD8⁺ β iIELs generated from this differentiation pathway stay IL-15-independent and constitute a small fraction of the mature iIEL pool, which would account for the low number of CD8⁺ β iIELs in Il15ra^–/– mice.

Generation of CD8⁺ iIELs involves both thymus-dependent and thymus-independent pathways. The thymus-dependent pathway appears to be dominant in normal mice, because CD8⁺ iIELs are reduced by 90% in nude and in neonatally thymectomized mice. In this study, ATXBM chimera engrafted with either WT or Il15ra^–/– thymus generated similar results to an earlier study by Schluns et al. (8) using BM chimera without thymus transplantation. Both studies revealed a 10–20-fold reduction of CD8⁺ iIEL in Il15ra^–/– recipients compared with WT recipients. The study by Schluns et al. (8) also found that V5⁺ cells were predominant in CD8⁺ iIEL of WT recipients but were preferentially reduced in Il15ra^–/– recipients. This observation is consistent with a later study reporting that IL-15 differentially promotes rearrangement of the V5 locus (31). In this study, we found that Il15ra^–/– thymus is able to restore Thy1^– V5⁺ CD8⁺ iIEL to WT level in nude mice, implying that the IL-15-promoted V5 rearrangement in iIEL precursors occurs outside of the thymus and thus favors the model that CD8⁺ iIEL precursors exit the thymus before TCR rearrangement and colonize the intestine to generate CD8⁺ iIEL (15). This supposition is consistent with the observation that sterile transcripts of un-rearranged V5 gene were more abundant than that of un-rearranged V2 gene in Rag1^–/– iIELs, whereas the reverse was found in Rag1^–/– thymocytes (31).

This study found that the IL-15 system affects the putative lin^– iIEL precursors in the intestinal epithelium. All three lin^– iIEL subsets express similar levels of IL-15R and _c, whereas the IL-15Rβ level is sharply up-regulated in the most mature CD8 SP subset. This result implies that the CD8 SP subset is more responsive to and dependent on IL-15 than the Thy1 SP and Thy1⁺CD8⁺ subsets, an implication consistent with the fact that the CD8 SP subset displays the most affected phenotypes in Il15ra^–/– mice, including a 6–7-fold reduction in cell number and an alteration in CD44 and B220 expression. The reduction of CD8 SP precursors in KO mice may result from either survival or differentiation defects. A possible scenario explaining this result is that the up-regulated IL-15Rβ expression makes WT CD8 SP precursors accessible to the prosurvival effect of IL-15, as for the mature CD8⁺ iIEL, whereas the lack of IL-15Rβ up-regulation on KO CD8 SP precursors renders them incapable of using IL-15 for survival. In contrast, the possibility of impaired differentiation is suggested by the high B220 expression on lin^– CD8 SP iIELs in Il15ra^–/– mice, as this pattern of B220 expression resembles that on CD3^–CD8⁺ iIELs in Cd3e^–/–, Rag^–/–, and Lck^–/–Fyn^–/– mice, which show a development block in CD8⁺ iIELs (32, 33). Whether TCR gene rearrangement is affected in the lin^– CD8 SP iIEL of Il15ra^–/– mice as in the CD3^–CD8⁺ iIEL of Cd3e^–/– and Rag^–/– mice remains to be determined. Regardless of the mechanism, one way the IL-15 system may regulate CD8⁺ iIEL homeostasis is by affecting their precursors in the intestine.

For mature CD8⁺ iIELs, a prosurvival function of the IL-15 system was confirmed in vitro and in vivo. Both the mature cell transfer and the thymus grafting ATXBM chimera experiments indicate the involvement of IL-15R expressed by peripheral parenchymal cells, presumably the intestinal epithelial cells, in CD8⁺ iIEL homeostasis. It is, therefore, possible that IL-15 affected CD8⁺ iIEL homeostasis indirectly by induction of secondary effector(s) via interaction with IL-15R on parenchymal cells. However, we believe a direct effect of IL-15 on CD8⁺ iIELs is more likely, based on the following observations. First, exogenous IL-15 supported the survival of CD8⁺ iIELs in vitro in the absence of parenchymal cells and in an IL-15Rβ-dependent manner. Second, IL-15Rβ⁺ BM is necessary for the generation of CD8⁺ iIELs in WT recipients (8). The requirement of IL-15Rβ both in vitro and in vivo implies a direct use of IL-15 by CD8⁺ iIELs involving the receptor β-chain.

What is the mode of IL-15 usage by CD8⁺ iIELs in vivo? Results from the BM chimera experiment by Schluns et al. (8) and from the thymus grafting and the mature CD8⁺ iIEL transfer experiments of this study all imply the in trans mode of IL-15 usage. Similar implications are made by experiments examining the requirement of the IL-15 system for homeostasis of memory CD8⁺ T cells and NK cells in vivo (8, 34, 35). This probable preferred mode of IL-15 usage by lymphocytes in vivo may result from situations that limit the availability of soluble IL-15. One possible situation is that IL-15 exclusively complexes with IL-15R intracellularly, as suggested by two previous studies (36, 37). Another possible situation is that the amounts of IL-15R expressed on the cell surface are abundant enough to retain soluble IL-15 as a cell-bound form. In fact, the in trans mode of IL-15 usage was discovered by adding soluble IL-15 to IL-15R-expressing cells in vitro (3). Moreover, injection of soluble IL-15 restored NK and memory CD8⁺ T cells in Il15^–/– mice (5), demonstrating that exogenous soluble IL-15 can carry out similar functions as endogenous IL-15. These studies indicate that soluble IL-15 and cell-bound IL-15 function similarly and suggest that soluble IL-15 may be used in a cell-bound manner due to the high affinity interaction between soluble IL-15 and cell surface IL-15R. We think a similar scenario might occur for the CD8⁺ iIELs cultured in IL-15 in vitro. In the absence of parenchymal cells, the adjacent IL-15R-expressing CD8⁺ iIEL cells in the well might have presented IL-15 in trans to one another. Thus, the direct prosurvival effect of IL-15 on pure CD8⁺ iIELs in vitro likely reflects the function of IL-15 in vivo, which is supported by the CD8⁺ iIEL transfer experiment.

In summary, this study excludes a role for the IL-15 system in CD8⁺ iIEL development in the thymus and suggests that the IL-15 system is necessary for the maintenance or/and differentiation of the putative lin^– precursors and the maintenance of mature CD8⁺ iIEL in the intestine epithelium.

	Acknowledgments

 
We thank Yu-Chia Su (Academia Sinica) for guidance in thymectomy and thymus engraftment.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
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 Taiwanese National Science Council Grant 95-2320-B-001-033 (to Y.-G.L. and Y.-H.L.). M.-S.H. and Y.-W.H. were supported by Academia Sinica.

² Address correspondence and reprint requests to Dr. Nan-Shih Liao, Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan. E-mail address: mbfelix{at}imb.sinica.edu.tw

³ Abbreviations used in this paper: IEL, intraepithelial lymphocyte; iIEL, intestinal IEL; BM, bone marrow; ATXBM, adult mice thymectomized and bone marrow re-constituted; PP, Peyer’s patches; PI, propidium iodide; lin ^–, lineage marker negative; CP, cryptopatch; SP, single positive; DP, double positive; TN, triple negative; SA, streptavidin; PB, Pacific blue; B6, C57BL/6J; β, TCRβ⁺; , TCR⁺; eGFP, enriched GFP; WT, wild type.

Received for publication September 14, 2007. Accepted for publication January 11, 2008.

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