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IL-15 is crucial for the development of intestinal intraepithelial lymphocytes (IEL) and delivery is mediated by a unique mechanism known as trans-presentation. Parenchymal cells have a major role in the trans-presentation of IL-15 to IELs, but the specific identity of this cell type is unknown. To investigate whether the intestinal epithelial cells (IEC) are the parenchymal cell type involved, a mouse model that expresses IL-15Rα exclusively by the IECs (Villin/IL-15Rα Tg) was generated. Exclusive expression of IL-15Rα by the IECs restored all the deficiencies in the CD8αα+TCRαβ+and CD8αα+TCRγδ+ subsets that exist in the absence of IL-15Rα. Interestingly, most of the IEL recovery was due to the preferential increase in Thy1lo IELs, which compose a majority of the IEL population. The differentiation of Thy1hiCD4−CD8− thymocytes into Thy1−CD8αα IELs was found to require IL-15Rα expression specifically by IECs and thus, provides evidence that differentiation of Thy1lo IELs is one function of trans-presentation of IL-15 in the intestines. In addition to effects in IEL differentiation, trans-presentation of IL-15 by IECs also resulted in an increase in IEL numbers that was accompanied by increases in Bcl-2, but not proliferation. Collectively, this study demonstrates that trans-presentation of IL-15 by IECs alone is completely sufficient to direct the IL-15-mediated development of CD8αα+ T cell populations within the IEL compartment, which now includes a newly identified role of IL-15 in the differentiation of Thy1lo IELs.
Intestinal intraepithelial lymphocytes (IEL)3 are a unique population of T cells residing within the gut epithelium as these cells is mostly CD8+ T cells, many of which express the CD8αα homodimer (1,2). Additionally, a large proportion of IELs express the γδ form of the T cell receptor. In general, three main populations of CD8+ T cells can be found within the IEL compartment of the small intestine. One population is comprised of conventional CD8αβ+TCRαβ+ cells that develop in the thymus and home to the gut epithelium in response to activation in the periphery (3,4); these memory CD8 T cells likely provide protection against microorganisms. The other two populations are made up of TCRαβ+ and TCRγδ+ IELs, which are considered unconventional because they express the CD8αα homodimer (5) and their origin and functions have been unclear. Previous studies have provided evidence that CD8αα IELs function in preserving the integrity of the epithelium and maintaining intestinal homeostasis (6-8). CD8αα IELs are also unique among T cells as a majority of these cells express low levels of Thy1 (9). While the relevance of low Thy1 expression is not known, it likely relates to their unconventional development and distinctive functions.
The development of unconventional CD8αα+TCRαβ+ IELs has long been controversial, with a theory that these cells are generated extrathymically; however, more recent evidence has demonstrated the existence of CD8αα+TCRαβ+ IEL precursors in the thymus (10,11). Similar to CD8αα+TCRαβ+ IELs, the development of CD8αα+TCRγδ+ IELs is not well defined and evidence exists for both thymic dependent and independent pathways (12). Despite an incomplete understanding in the development and function of the CD8αα+TCRαβ+ and CD8αα+TCRγδ+ IEL subsets, it is evident that the pleiotropic cytokine, IL-15, is crucial for their development and maintenance. IL-15 belongs to the four α-helix bundle cytokine family that utilizes three receptor subunits: IL-15Rα, IL-2Rβ, and γC. In the absence of either IL-15, IL-15Rα, or IL-2Rβ, CD8αα+TCRαβ+ and CD8αα+TCRγδ+ IEL numbers are severely reduced (13-15). In vitro, IL-15 has been shown to enhance survival and proliferation of CD8αα+TCRαβ+ and CD8αα+TCRγδ+ IELs (16,17). Additionally, it has been suggested that IL-15 can promote TCR Vγ5 rearrangement in TCRγδ+ precursors in the absence of IL-7 (18). So, whereas IL-15 can act directly on IELs to modulate survival, proliferation, and differentiation events, the main function of IL-15 in IEL development in vivo is uncertain.
Because of the unidentified roles of IL-15 in IEL development and the dual location of IEL development, the point in IEL development in which IL-15 functions remains unclear. This question may now be better addressed if one considers that IL-15 acts locally through a mechanism called trans-presentation. As first illustrated by Dubois et al., IL-15 is expressed on the cell surface bound to IL-15Rα which can stimulate neighboring cells expressing the IL-2Rβ/γC subunits (19). Evidence that trans-presentation is utilized in vivo is supported by work demonstrating that IL-15Rα expression is required by cells in the environment rather than by IELs, memory CD8 T cells, or NK cells (20-23). For development of both CD8αα+TCRαβ+ and CD8αα+TCRγδ+ IELs, parenchymal cell expression of IL-15Rα and IL-15, but not IL-2Rβ, is specifically required (21). In contrast, these IELs need to express IL-2Rβ, but not IL15Rα, for their development (21), indicating that IL-15 acts directly on the IELs. In studies addressing the possibility that thymic parenchymal cells may trans-present IL-15 to IEL precursors, Lai et al. used thymectomies along with thymic transplants to demonstrate that expression of IL-15 in the thymus was not required for development of CD8αα+ IELs (24). Altogether, these studies clearly show that parenchymal cells outside of the thymus trans-present IL-15 for IEL development, but did not precisely identify the cells providing IL-15 signals to IELs.
Since trans-presentation requires cell-cell contact and intestinal epithelial cells (IEC)3 are adjacent to IELs and express IL-15 (25), we hypothesized that IECs are the parenchymal cell type trans-presenting IL-15 to the IELs. To this end, we developed a mouse model that expresses IL-15Rα exclusively by the IECs to address the role of IL-15 trans-presented by IECs. By using this model, we demonstrate that sole expression of IL-15Rα by IECs restores the deficiencies in the CD8αα+TCRαβ+ and CD8αα+TCRγδ+ subsets present in the intestinal epithelium of IL-15Rα−/− mice. Furthermore, we identified that the main effect of transgenic IL-15Rα expression was in the appearance of Thy1loCD8α+TCR+ IELs.
C57BL/6J mice were purchased from The Jackson Laboratories (Bar Harbor, ME) and C57BL/6J Ly5.2 mice were purchased from NCI (Bethesda, MD). IL-15Rα−/− mice were generously provided by Averil Ma and backcrossed to C57BL/6 mice 15 generations (14). The full length murine IL-15Rα cDNA (from nucleotide 49−932 of precursor IL-15Rα sequence U22339) was cloned by PCR using the primers 5’-gtcactgctggggacaattg-3’ and the 5’-ggatccctaactgcccttgtatcttc-3’ from cDNA generated from C57BL/6J spleen RNA using TA Topo cloning kit (Invitrogen). A BamHI sites was added to the 3’ end of the IL-15Rα cDNA during PCR cloning. The IL-15Rα cDNA was then subcloned upstream of an IRES-EGFP reporter by ligating into the pIRES2-EGFP vector (BD/Clonetech) using EcoRI and BamHI. Cloning of the IL-15Rα-IRES-EGFP insert downstream of the Villin promoter required the introduction of a BsiWi site on the 5’ end of the IL-15Rα cDNA and a MluI site on the 3’ end of the EGFP. This was achieved by mutagenesis using QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene). Following mutagenesis, the entire IL-15Rα-IRES-EGFP region was sequenced and then subcloned into the pBS KS Villin MES SV40 poly vector (graciously provided by Sylvie Robine (26,27)) using BsiWi and MluI. The final 11.2 kb Villin-IL-15Rα-IRES-EGFP fragment was removed from the vector backbone by SalI digestion and used for microinjection into IL-15Rα+/− C57BL/6J pronuclei for generation of transgenic mice. Microinjection was performed by the Genetically-Engineered Mouse Facility at the University of Texas MD Anderson Cancer Center. Tg-positive mice were identified by PCR of tail DNA and confirmed by IL-15Rα staining and GFP expression on IECs. Tg-positive mice were bred to IL-15Rα−/− background. All mice were maintained under specific pathogen-free conditions at the University of Texas MD Anderson Cancer Center in accordance with the Institutional Animal Care and Use Committee guidelines.
IELs and IECs were isolated from mice between 6−8 weeks old as previously described (28,29). Briefly, small intestines were cut longitudinally and then into 5 mm pieces. The pieces were washed in CMF (HBSS with 1mM Hepes and 2.5 mM NaHCO3) and stirred at 37°C in CMF containing 10% FBS and 1 mM dithioerythritol (Calbiochem) for IELs or 0.5 mM dithiothreitol (Sigma) for IECs. The cells were then centrifuged on a 44−67% or 25−44% Percoll (Amersham) gradient to enrich for IELs or IECs, respectively. IELs from the colon were isolated in the same manner as described above for small intestines. For analysis of peripheral lymphocytes, single-cell suspensions were made by homogenizing spleens, peripheral and mesenteric lymph nodes, and thymuses using frosted glass slides in HBSS (containing hepes, L-glutamine, penicillin and gentamycin sulfate (HGPG)) and filtered through Nitex. Red blood cells were lysed with Tris-ammonium chloride.
Isolated lymphocytes were resuspended in PBS/0.2% BSA/0.1% NaN3 (FACS buffer) at a concentration of 1−2 × 107 cells/ml followed by incubation with 100 μl of properly diluted mAb at 4° C for 20 min. The following antibodies were used for flow cytometric analysis and were purchased from Becton Dickinson, unless otherwise noted: CD45 (30-F11)-Pacific Blue (Biolegend), CD8α (Ly-2)-PerCp-Cy5.5, γδTCR (GL3)-PE or -FITC, Thy1.2 (53−2.1)-APC or – PE-Cy7 (eBioscience), αβTCR (H57)-APC-Alexa750 (eBioscience) or -APC, CD8β (Ly3.2)-PE or –FITC, CD44 (IM7)-APC or Pacific Blue, CD4 (L3T4)-PE, and CD122 (TM-β1)-PE, IL-15Rα was detected with goat anti-IL-15Rα-biotin (R&D Systems) followed by streptavidin-APC (Jackson ImmunoResearch). Background staining was determined by staining analogous populations from IL-15Rα−/− mice and with a biotinylated Ig control (Jackson ImmunoResearch). Biotin anti-Vγ5 mAb was previously described (30) and visualized with streptavidin-APC-Alexa Fluor 750 (Caltag Laboratories). After staining, cells were washed and fixed in 3% paraformaldehyde buffer. For intracellular staining of Bcl-2, FITC conjugated Bcl-2 antibody reagent set (Becton Dickinson) was used according to manufacturer's instructions. To determine BrdU incorporation, BrdU (2mg/mouse) was injected i.p. 12 hours prior to analysis; isolated cells were stained with conjugated BrdU antibody set (Becton Dickinson) according to manufacturer's instructions and analyzed immediately by flow cytometry. All cells were acquired using a LSRII (Becton Dickinson) and analyzed using FlowJo software (Tree Star). Lymphocyte percentages and total cell numbers were calculated and evaluated by using the Students t test. Values of p<0.05 were considered statistically significant.
Small intestines were fixed in 4% paraformaldehyde and then in 30% sucrose prior to being embedded in OCT. 6μm sections of small intestine were blocked for endogenous biotin by treating with 0.001% avidin followed by 0.001% biotin in PBS. Sections were then stained with anti-mouse IL-15Rα biotin (R&D Systems) or anti-mouse goat Ig biotin (Jackson ImmunoResearch). Staining was visualized using strepavidin-Cy5 (Jackson ImmunoResearch) and analyzed using a Leica SP2 SE confocal microscope. For H&E staining, pieces from all three regions of the intestine (duodenum, jejenum, and ileum) were embedded in OCT, sectioned at 6μm, stained, and analyzed using an Olympus BX41 microscope.
Intestinal IECs were lysed in RIPA lysis buffer (Santa Cruz Biotechnology, Inc.) and protein (150,000 cells/lane) was separated on a 4−20% Tris-glycine gel (Invitrogen), transferred to a PVDF membrane (Immobilon-P; Millipore), and analyzed by western blot using anti-IL-15Rα (AZ-12; Santa Cruz Biotechnology, Inc.) as the primary antibody. The secondary antibody used was anti-rat biotin (Santa Cruz biotechnology, Inc.), followed by streptavidin-HRP (Pierce). Signals were detected using the Supersignal West Pico Chemiluminescent Substrate System (Pierce).
Thymocytes from CD45.1+ C57BL/6J mice were purified by incubating in anti-CD4 (GK1.5) and anti-CD8α (2.43) coated Dynal beads (Dynal Mouse T cell Negative Isolation Kit; Invitrogen) before separation in a magnetic field. The resulting double negative thymocytes were then incubated with CD8α (Ly-2)-PerCp-Cy5.5, Thy1.2 (53−2.1)-APC, and CD4 (L3T4)-PE (all from BD Pharmingen) and sorted for CD4−CD8α−Thy1hi thymocytes using a FACSAria (Becton Dickinson). IELs from CD45.1+C57BL/6J mice were stained with CD8α (Ly-2)-PerCp-Cy5.5, Thy1.2 (53−2.1)-APC, CD8β (Ly3.2)-PE, and CD4 (L3T4)-FITC (all from BD Pharmingen) and sorted for CD8αα+Thy1hi and CD8αα+Thy1lo IELs. The donor CD4−CD8α−Thy1hi thymocytes (approximately 750,000 cells/mouse), CD8αα+Thy1hi IELs (approximately 100,000 cells/mouse), or CD8αα+Thy1lo IELs (approximately 460,000 cells/mouse) were then injected i.v. into sub-lethally irradiated (750 cGy) Villin/IL-15Rα Tg, Wt, and IL-15Rα−/− mice (all CD45.2+). Among the CD4−CD8− Thy1hi thymocytes, approximately 25% were TCRαβ+ and 7.5% were TCRγδ+; IELs and spleens from recipient mice were analyzed 2−3 weeks later.
To develop a model where IL-15Rα is exclusively expressed by the IECs, murine IL-15Rα cDNA and an IRES-EGFP reporter sequence was cloned downstream of the villin promoter and used to generate four transgenic founder mice; these Villin/IL-15Rα-GFP transgenic mice were then backcrossed to IL-15Rα−/− mice and will be referred from here on as Villin/IL-15Rα Tg mice. Each of the four founder lines, prior to and after being backcrossed to the IL-15Rα−/− background were found to be generally healthy and did not display any defects in growth, development, or reproduction. In histological analysis, staining with IL-15Rα Ab was observed specifically in the IECs of the small intestine from Villin/IL-15Rα Tg mice, but was not observed using an Ig control (Figure 1A). Interestingly, IL-15Rα staining appeared to be more concentrated along the basolateral cell surface. Green fluorescence, due to the GFP reporter, was also observed intracellularly and appeared evenly distributed within the epithelial cells (Figure 1A). GFP could not be detected in thymus or spleen sections (data not shown), confirming previous studies demonstrating specificity of the villin promoter to the intestinal epithelium (31-35). Expression of both GFP and IL-15Rα was also detected in CD45-negative cells in Villin/IL-15Rα Tg mice by flow cytometric analysis (Figure 1B). In side-by-side comparisons, the four founder lines expressed similar levels of IL-15Rα and GFP by IECs (data not shown). Detection of IL-15Rα by flow cytometry was insufficient in discerning differences in IL-15Rα expression in Wt and IL-15Rα−/− mice, which was likely due to the high level of background fluorescence of IECs. As such, IL-15Rα expression of IECs was examined by western blot analysis and was indeed expressed by IECs in Wt and Tg mice but not IL-15Rα−/− mice (Figure 1C). Overall, Villin/IL-15Rα Tg mice specifically express IL-15Rα by IECs, which correlates with GFP expression.
To provide evidence that functional activities of IL-15 are not present in the periphery of Villin/IL-15Rα Tg mice, lymphocytes were analyzed for deficiencies in CD8 T cells, memory-phenotype CD8 T cells (CD44hi) and NK cells, which are characteristic of an IL-15Rα deficiency (14). Lymphocytes isolated from spleen and peripheral and mesenteric lymph nodes of Villin/IL-15Rα Tg mice had a similar phenotype to those of IL-15Rα−/− mice, where total CD8 T cells and NK cells were decreased and displayed a deficiency in memory phenotype (CD44hi) CD8 T cells (Figure 1D, E). The composition of lymphocytes in the spleens of Villin/IL-15Rα Tg mice crossed to the Wt background (Villin/IL-15Rα Tg (Wt)) did not appear different from normal Wt mice (supplemental data). The phenotype of the lymphocytes in the secondary lymphoid tissues of Villin/IL-15Rα Tg mice is biological evidence that IL-15Rα is not expressed outside the intestine.
Upon isolation of cells from the IEL compartment, the total number of CD45+ and CD45-negative cells isolated from the intestinal epithelium was compared among the different groups of mice. Among multiple experiments, the total number of CD45+ cells was increased 3.3 fold in the Villin/IL-15Rα Tg mice compared to the Wt mice (Figure 2A). Conversely, there was only a slight increase in the number of CD45-negative cells cell in the Villin/IL-15Rα Tg mice compared to Wt (Figure 2A). This was reflected by an increased proportion of CD45+ cells from 28% ± 2.4 present in Wt mice to 48% ± 0.8 in Villin/IL-15Rα Tg mice (Figure 2B). As increased cell numbers could be due to an increase in surface area, the general structure of the intestinal villi and crypts was examined in all three regions of the small intestines (duodenum, jejenum, and ileum). Between the three groups of mice, the length of villi and depths of crypts were similar (Figure 2C). Therefore, IL-15Rα expression by IECs enhances the overall number of lymphocytes in the intestinal epithelium.
To determine whether exclusive IL-15Rα expression by IECs affects the composition of IEL populations, IELs were isolated from Villin/IL-15Rα Tg mice and compared to that of Wt and IL-15Rα−/− mice. Among CD45+ IELs, the frequency of CD8α+TCRγδ+ IELs was reduced in IL-15Rα−/− mice compared to Wt mice by approximately 75% (Figure 3A) as previously described (13). In contrast, the IELs from Villin/IL-15Rα Tg mice contained a similar or higher proportion of CD8α+TCRγδ+ cells than Wt mice (Figure 3A). As CD8α+TCRγδ+ IELs contain subsets with different Vγ usage and varying levels of Thy1 expression, the proportion of these subsets were assessed in the different groups of mice. Whereas CD8α+TCRγδ+ IELs in Wt mice predominately use Vγ5 and have a high frequency of Thy1lo subset, the Vγ5+ and Thy1lo TCRγδ+ IELs were virtually absent in IL-15Rα−/− mice (Figure 3A). In the Villin/IL-15Rα Tg mice, the proportion of Vγ5+ and Thy1lo TCRγδ+ IELs was restored to normal (Figure 3A). The differences in frequency of TCRγδ+ IEL subsets correlated to changes in absolute numbers (Figure 3B). Although not previously described, the total number of both Vγ5+ and Vγ5−Thy1loCD8α+TCRγδ+ IELs was severely reduced by approximately 95% in IL-15Rα−/− mice compared to Wt mice (p<0.01 and p<0.05 respectively, Figure 3B). In contrast, the number of Thy1hi CD8α+TCRγδ+ IEL subsets was not significantly different between Wt and IL-15Rα−/− mice and was independent of Vγ5 usage (Figure 3B). In the Villin/IL-15Rα Tg mice, the total numbers of all CD8α+TCRγδ+ IEL subsets were dramatically increased (p<0.05, Figure 3B) which was largely due to the increased number of total lymphocytes that exist in the intestinal epithelium of Tg mice. Overall, exclusive expression of IL-15Rα by IECs restored the deficiency in Thy1loCD8α+TCRγδ+ IELs that occurs in the absence of IL-15Rα expression as well as enhanced the numbers of all CD8α+TCRγδ+subsets.
In the analysis of TCRαβ IELs, similar percentages of CD8α+TCRαβ+ IELs were present in each group of mice (i.e. Villin/IL-15Rα Tg mice, Wt, and IL-15Rα−/− mice) (Figure 3C); however, within this population, the CD8αα+TCRαβ+ IELs are the most heavily dependent on IL-15 (13,14). Therefore, since CD8α+TCRαβ+ IELs contain CD8αβ+ as well as Thy1hi and Thy1loCD8αα+ cells, the effect of IL-15Rα on each of these CD8α+TCRαβ+ IELs subsets were examined (Figure 3C). In Wt mice, the CD8β− IELs (CD8αα) make up a majority of the CD8α+TCRαβ+ IELs, with most of those cells expressing low levels of Thy1 (Figure 3C). The frequency of CD8αα+TCRαβ+IELs was affected in IL-15Rα−/− mice with a preferential deficiency in the Thy1lo IELs and an enhancement in the CD8αβ+ IELs (Figure 3C). Most significantly, the Villin/IL-15Rα Tg mice had normal percentages of all CD8α+TCRαβ+ IEL subsets (Figure 3C). Comparison of absolute numbers indicated that the Thy1lo subset of CD8αα+ IELs was dramatically lost in the IL-15Rα−/− mice (97% decrease, p<0.001), but the Thy1hi CD8αα+ IELs were not deficient, similar to that observed for the CD8+TCRγδ+ IELs (Figure 3D). In Villin/IL-15Rα Tg mice, cell numbers of both Thy1lo and Thy1hi CD8αα+TCRαβ+ IELs were restored to levels beyond that found in Wt mice due to the increase in total lymphocyte numbers (p<0.05 for Thy1lo, Figure 3D). Surprisingly, the proportion and total numbers of CD8αβ+Thy1hi cells was higher in IL-15Rα−/− and Villin/IL-15Rα Tg mice compared to Wt mice (p<0.01, Figure 3C and D). Since an increase in CD8αβ+TCRαβ+ IELs in IL-15Rα−/− mice has not been previously described and cannot be presently explained, further investigation is required. Regardless, these findings show that IL-15Rα expression by IECs similarly recovers the defective development of Thy1loCD8αα+TCRαβ+ IELs while enhancing the Thy1hiCD8α+TCRαβ+ IELs.
The composition of lymphocytes in the IEL compartment was also examined in Villin/IL-15Rα Tg mice crossed to the Wt background. Overall, the proportion of CD8αα+TCRγδ+ subsets based on Vγ5 and Thy1 expression did not differ between Wt mice and Villin/IL-15Rα Tg (Wt) mice (supplemental data). In addition, no differences in the proportions of CD8αα+TCRαβ+ IELs were observed among the various groups of mice (supplemental data). Furthermore, in Villin/IL-15Rα Tg (Wt) mice, the lymphocytes in the spleen displayed a normal phenotype with no evidence that IELs migrated out of the intestines (supplemental data). This analysis demonstrates that transgenic expression of IL-15Rα by the IECs, in combination with normal endogenous expression of IL-15Rα, does not have an additional impact on IEL development.
Villin promoter expression, in addition to being active in the small intestine, is also active in the large intestine. As such, phenotypic analysis of IELs from the large intestine was also examined in this study. Similar to that observed in the small intestine, TCRγδ+ IELs were deficient in IL-15Rα−/− mice but were restored in theVillin/IL-15Rα Tg mice beyond that of Wt levels (Figure 3E,F). For the TCRαβ+ IELs, a deficiency was also present in the IL-15Rα−/− mice but to a greater degree in the colon than in the small intestine. In contrast to the TCRγδ+ IELs, transgenic expression of IL-15Rα was less effective in restoring the defect in CD8αα+TCRαβ+ IELs (Figure 3E,G). Upon further analysis of the TCRαβ+ and TCRγδ+ IELs, Thy1lo subsets were preferentially affected by IL-15Rα expression as the Thy1lo cells were absent in IL-15Rα−/− mice and regained when IL-15Rα was expressed by IECs. Therefore, IL-15Rα expression by IECs in the colon has similar effects on IEL development as in the small intestine by preferentially affecting Thy1lo CD8αα+TCRαβ+ and TCRγδ+ IELs.
In an attempt to identify a mechanism by which IL-15Rα increases overall IEL numbers and preferentially restores Thy1lo IELs, cell survival and proliferation were examined among IEL subsets. Since IL-15 mediates survival by regulating Bcl-2(36), Bcl-2 expression in IELs was analyzed in each group of mice. Bcl-2 expression in Wt mice was higher than that of IL-15Rα−/− mice in all IEL subsets indicating that IL-15 normally regulates Bcl-2 levels in IELs (Figure 4A). In Villin/IL-15Rα Tg mice, expression of Bcl-2 was comparable to that of Wt mice in all IEL subsets, except the CD8αα+Thy1hi subset, which had lower Bcl-2 levels than Wt mice but still higher than that observed in IL-15Rα−/− mice. The restored deficiencies in Bcl-2 expression in most IEL subsets suggest that trans-presentation of IL-15 by IECs helps maintain overall IEL survival. This upregulation of Bcl-2 may result in a decrease in IEL turnover, thus causing the overall increase in IEL cell numbers observed in the Villin/IL-15α Tg mice.
To determine whether effects of IEC expression of IL-15Rα were due to IL-15-mediated cell expansion, BrdU incorporation of IELs was measured after 12 hours of BrdU treatment. In general, each of the CD8αα+TCRγδ+ and the CD8αα+TCRαβ+ IEL subsets from Villin/IL-15Rα Tg mice had a similar level of BrdU incorporation as Wt mice, with the exception of CD8αβ+TCRαβ+ cells which actually had less BrdU incorporation than the Wt mice (Figure 4B,C). Surprisingly, proliferation was increased among Thy1lo subsets regardless of TCR expression in IL-15Rα−/− mice compared to Wt mice (Figure 4B,C). Increased cell division in IL-15Rα−/− mice could be due to a compensatory mechanism that occurs when lymphocyte numbers are deficient or a result of defective development. In general, these findings suggest that trans-presentation of IL-15 does not specifically enhance the basal rate of IEL proliferation and thus, does not provide a mechanism for the increased number of lymphocytes observed in Villin/IL-15Rα Tg mice. In contrast to the predicted role of IL-15 on IEL proliferation, our data provides evidence that the absence of IL-15Rα results in a dysregulation of proliferation in Thy1lo IELs.
Previous reports have demonstrated that expression of the IL-2/15Rβ chain (CD122) correlates with the level of IL-15 responsiveness (37). As such, we determined whether the expression of IL-15Rα affects the expression level of CD122 among CD8α+ IELs subsets. In Wt mice, expression of CD122 was higher in Thy1lo IELs than Thy1hi IEL for both CD8αα+TCRαβ+ and TCRγδ+ subsets, suggesting that Thy1lo IELs are more sensitive to IL-15 than Thy1hi IELs (Figure 4D). Among CD8αβ+TCRαβ+ IELs, the CD122 expression was very low (data not shown) as previously described (38). In comparing the different groups of mice, CD122 expression was similar within the TCRγδ+ (regardless of Thy1 expression levels) and Thy1hiCD8αα+ TCRαβ+ IELs (Figure 4D). Conversely, CD122 expression in Thy1loCD8αα+TCRαβ+ IELs was decreased in the absence of IL-15Rα−/− but restored to normal levels in Villin/IL-15Rα Tg mice (Figure 4D). This finding suggests that Thy1loCD8αα+TCRαβ+ IELs in IL-15Rα−/− mice failed to become adequately responsive to IL-15, which might explain the deficiency in this population.
Previous studies identified Thy1hiCD4−CD8−TCRαβ+ and CD4−CD8−TCRγδ+ thymocytes as precursors for CD8αα+TCRαβ+ and CD8αα+TCRγδ+ IELs, respectively(11,39,40). To determine whether trans-presentation of IL-15 by IECs drives the differentiation of Thy1hi CD4−CD8− thymic IEL precursors into Thy1loCD8αα+IELs, CD4−CD8− Thy1hi thymocytes were sorted from congenic Wt mice to a purity of >99% and injected into irradiated Villin/IL-15Rα Tg, Wt, and IL-15Rα−/− mice. Two to three weeks after transfer, the presence of donor cells in the IEL and spleen were compared among the three different recipients. In Wt hosts, donor-derived cells in the IEL were mostly TCRγδ+ (~90−95%) suggesting that this thymocyte population has a greater potential for development into TCRγδ+ IELs (Figure 5A). This developmental potential or expansion of thymocytes into TCRγδ+ IELs was not observed in IL-15Rα−/− mice but was restored in Villin/IL-15Rα Tg mice (Figure 5A). Upon gating on the donor-derived CD8α+TCRγδ+ IELs (>95%), almost all the cells expressed low levels of Thy1 in Wt and Villin/IL-15Rα Tg mice while this population was predominately Thy1hi in IL-15Rα−/− mice (Figure 5A). Among donor-derived CD8α+TCRαβ+ IELs, Thy1 expression was low in Wt mice and high in IL-15Rα−/− similar to that observed for TCRγδ+ IELs. In contrast, only a small portion of CD8α+TCRαβ+ IELs were Thy1lo in the Villin/IL-15Rα Tg mice suggesting the TCRαβ+ thymic progenitors may not receive an adequate IL-15 signal from IECs. Regardless of the host, a small percent of donor-derived cells were detected in the spleen, which were a mixture of TCRαβ+ and TCRγδ+ cells and were all Thy1hi (Figure 5B). In addition, all the donor-derived CD8+TCRαβ+ splenocytes expressed the CD8αβ heterodimer (Figure 5B). These data demonstrate that Thy1hi CD4−CD8− thymocytes, upon migration to the intestinal epithelium, preferentially differentiate into Thy1loCD8+TCRγδ+ IELs through the signals delivered by intestinal epithelium expressing IL-15Rα. This does not exclude the possibility that intermediary stages of development may occur in the thymus prior these cells migrating into the intestinal epithelium. In contrast, while Thy1hi thymocytes can differentiate into Thy1loTCRαβ+ IELs in an IL-15Rα-dependent fashion, this differentiation is only minimally influenced by trans-presentation of IL-15Rα by IECs.
To examine differentiation of CD8αα+ IELs at a later stage of development, Thy1hi CD8αα+ IELs were sorted from congenic mice, transferred into irradiated Villin/IL-15Rα Tg, Wt, and IL-15Rα−/− mice, and examined 2 weeks later. In Villin/IL-15Rα Tg mice, donor-derived TCRαβ+ and TCRγδ+ IELs were both mostly Thy1lo (Figure 5C). Down-regulation of Thy1 also occurred in TCRαβ+ IELs from Wt recipients, but was less dramatic in TCRγδ+ IELs (Figure 5C). In IL-15Rα−/− mice, both donor-derived TCRαβ+ and TCRγδ+ IELs remained Thy1hi (Figure 5C). When Thy1loCD8αα+ IELs were transferred, the donor-derived TCRαβ+ and TCRγδ+ IELs remained Thy1lo in both Villin/IL-15Rα Tg and Wt recipients (Figure 5D). Interestingly, the transfer of Thy1lo CD8αα+ IELs into IL-15Rα−/− recipients resulted in the upregulation of Thy1 expression in these mice (Figure 5D). After all IEL transfers, no donor-derived cells were detected in the spleen (data not shown). These experiments suggest that IL-15 trans-presentation by IECs induces the down regulation of Thy1 and maintains this phenotype in established CD8αα+ IELs.
Trans-presentation of IL-15 by parenchymal cells has been shown to drive IL-15-mediated development of IELs (21); however, the identity of the parenchymal cell type involved had yet to be determined. Since IECs have long been recognized as a source of IL-15 in the intestines and are adjacent to IELs, IECs were speculated to be a likely cell type trans-presenting IL-15 for IEL development. As studies have demonstrated that some IEL precursors are thymic-derived, have increased levels of CD122 expression, and are responsive to IL-15 (11), it is possible that this early stage of development may require IL-15 regulation by thymic parenchymal cells. In this current study, we demonstrate that expression of IL-15Rα solely by IECs was completely sufficient to restore the deficiencies in both the CD8αα+TCRαβ+ and CD8αα+TCRγδ+ subsets observed in the absence of IL-15Rα. These findings indicate that the development of IELs does not require the expression of IL-15Rα by the parenchymal cells of the thymus or in any other site outside of the intestine and are in agreement with the study conducted by Lai et al. (24). Whereas Lai et al. demonstrated that regulation of CD8αα+ IELs does not require IL-15 in the thymus (24), this study did not precisely identify the cells providing IL-15 signals to IELs. Up until now, the point at which IL-15 becomes important for IEL development was not known, but our study provides evidence that IL-15 is not essential until the IEL precursors arrive in the intestinal epithelium.
In addition to demonstrating that IECs trans-present IL-15 for IEL development, our study also elucidates a novel role of IL-15 in IEL development. Previous studies have reported that IL-15 or IL-15Rα deficiencies preferentially affect the Vγ5 subset (21) and this correlated to the finding that rearrangements of Vγ5 were deficient in the absence of IL-15 (18). Vγ5 IELs are unusual T cells in that they are predominantly Thy1lo, whereas the other Vγ1 and Vγ2 IELs are predominantly Thy1hi. Upon a more thorough analysis, we found that Thy1hiVγ5+ cells are present at normal levels in IL-15Rα−/− mice while the Thy1loVγ5+ cells are clearly deficient. The same is true for Vγ5− (Vγ1+ and Vγ2+) IELs, where the small portion of Thy1loVγ5− IELs are deficient in the absence of IL-15 signals but the Thy1hiVγ5− IELs are not. Since this deficiency was also observed in the Thy1loCD8αα+ subpopulation of the TCRαβ+CD8α+ IELs in IL-15Rα−/− mice, it suggests that the major effect of IL-15 on development is in the differentiation and expansion of Thy1lo IELs and is independent of TCR rearrangement.
While a number of models for IEL development have been proposed, most do not include alterations in Thy1 expression. As such, the relationship between the Thy1hi and Thy1lo cells is not clear. Two possibilities exist, where these cells could be two independently-derived subsets or could be developmental intermediates of each other. Our results showing that Thy1hiCD4−CD8− thymocytes develop into Thy1loTCR+CD8αα+IELs, indicates that the transition from Thy1hi to Thy1lo is along the same pathway of development. This hypothesis was further strengthened upon demonstrating that resident Thy1hiCD8αα+ IELs also converted to Thy1loCD8αα+IELs. Most importantly, these conversion events were dependent on IEC expression of IL-15Rα, which provide evidence that IL-15 trans-presentation mediates this differentiation. Interestingly, the transition from Thy1hiCD4−CD8− thymocytes to Thy1loTCRαβ+CD8αα+ IELs was only partially driven by IEC expression of IL-15Rα and could be an indication that additional IL-15 signals outside the intestinal epithelial compartment are required for this particular event. In contrast, once TCRαβ+CD8αα+ IELs are present in the IEL compartment, IL-15 trans-presentation was efficient in down regulating Thy1 expression. Lastly, while we initially suspected that conversion of Thy1hi to Thy1lo IELs may be unidirectional, Thy1lo IELs re-expressed Thy1 in the absence of IL-15Rα. Therefore, IL-15 signals delivered by IECs appear to down regulate Thy1 expression and maintain this expression. One model of IEL differentiation did include the variable expression of Thy1; however, Thy1 was suggested to be down regulated before TCR expression (12). Altogether, our findings support a model of IEL development in which Thy1hi IEL precursors migrate to the intestinal epithelium, acquire expression of CD8αα, and is followed by the IL-15-mediated down regulation of Thy1 expression.
Our model of restricted IL-15 trans-presentation also supports past findings that IEC-derived IL-15 is important for IEL survival by regulating Bcl-2 levels in vivo. In IL-15Rα−/− mice, all CD8α+ IEL subsets had lower levels of Bcl-2 expression than those found in Wt mice, indicating that IL-15Rα is important for maintaining Bcl-2 levels. Concurrently, Bcl-2 levels in IELs from Villin/IL-15Rα Tg mice were comparable to those found in Wt mice, demonstrating that IL-15 trans-presentation specifically by IECs was delivering a signal that regulated Bcl-2 levels. Unfortunately, we were unable to correlate the levels of Bcl-2 with indicators of IEL apoptosis, such as Annexin V staining. This inability to detect apoptotic cells may be due to the close proximity to the gut lumen, as apoptotic cells could be easily expulsed. Our results follow those observed by Nakazato et al., which demonstrated that the transgenic expression of Bcl-2 in IL-15Rα−/− mice could restore the numbers of TCRγδ+ IELs (36); however, that study did not examine the contribution of transgenic Bcl-2 expression on Thy1lo and Thy1hi TCRγδ+ or TCRαβ+ IELs. While our data demonstrates that IL-15 regulates Bcl-2 levels in all IEL subsets, it is possible that Thy1lo IELs may be more sensitive to effects of IL-15-mediated survival than Thy1hi IELs thus contributing to the increase in the Thy1lo population. This idea is supported by our observation that Thy1lo IELs express higher levels of CD122.
In addition to its effects on IEL survival, IL-15 is also known for the ability to enhance proliferation of isolated IELs in vitro (16). Therefore, it was surprising that BrdU incorporation of IELs was not defective in IL-15Rα−/− mice. Moreover, the absence of IL-15Rα expression actually enhanced BrdU incorporation in the Thy1lo IELs. Presently, this cannot be explained but could be a result of the severely reduced cell numbers and an over-compensation mediated by an alternative mechanism, such as IL-7. Indeed, TCRγδ IELs in IL-7−/− mice also have enhanced BrdU incorporation compared to Wt mice(41). Regardless of the high proliferation rate in the absence of IL-15Rα, these IELs are present at very low levels.
In summary, we have generated a novel model to study the specific effects of IL-15 trans-presented by the intestinal epithelium for IEL development. Using this model, we have provided further evidence that IL-15 trans-presentation is the main mechanism by which IL-15 is delivered to IELs. We have also given conclusive evidence that cells of the intestinal epithelium are the only cell type that needs to trans-present IL-15 to IELs. In addition to identifying the cell source and localization of IL-15, this study identifies a new function of IL-15 in the preferential differentiation of Thy1loCD8αα+ IELs. Since the specific effect on Thy1lo IELs applied to both TCRαβ+ and TCRγδ+ IELs, a common link likely exists in the differentiation between these two separate lineages.
Characterization of Villin/IL-15Rα Tg mice on a Wt background (IL-15Rα+).
(A) Flow cytometric analysis of TCRγδ+ IEL subsets from Wt, Villin/IL-15Rα Tg mice on a Wt background, and Villin/IL-15Rα Tg on an IL-15Rα−/− background. Top panel shows proportion of CD8α+TCRγδ+ cells among CD45+ IELs. Bottom panel shows staining for Thy1 and Vγ5 expression after gating on CD8α+TCRγδ+ cells, which is indicated on the left and depicted by the circle in the above row. (B) Flow cytometric analysis of TCRαβ+ IEL subsets. Top panel shows proportion of CD8α+TCRαβ+ cells among CD45+ IELs. Bottom panel shows staining for Thy1 and CD8β expression after gating on CD8α+TCRαβ+ cells, which is indicated on the left and depicted by the circle in the above row. (C) Phenotype of lymphocytes isolated from spleens of the respective groups of mice. Top rows shows flow cytometric staining of TCRαβ+ and TCRγδ+ cells after gating on lymphocytes. The remaining rows show lymphocyte analysis after gating on the population denoted on the left.
We would like to thank Eliseo Castillo for irradiating mice, Bhavin Shah for technical assistance, Pam Grant for tissue sectioning, Anna Zal for assisting with confocal microscopy, the Genetically-Engineered Mouse Facility at the University of Texas MD Anderson Cancer Center in part supported by CCSG grant NCI# CA016672, and Sylvie Robine for generously providing the Villin promoter construct.
1Research is supported by NIH grant AI070910 and the MD Anderson Trust Fellowship (to K.S.).
The authors declare no competing financial interests.
IEL, intraepithelial lymphocyte; IEC, intestinal epithelial cells