Intrinsic requirement for the vitamin D receptor in the development of CD8αα expressing T cells

Abstract

Vitamin D and vitamin D receptor (VDR) deficiency results in severe symptoms of experimental inflammatory bowel disease in several different models. The intraepithelial lymphocytes of the small intestine contain large numbers of CD8αα⁺ T cells that have been shown to suppress the immune response to antigens found there. Here we determined the role of the VDR in the development of CD8αα⁺ T cells. There are fewer total numbers of TCRαβ⁺ T cells in the gut of VDR knockout (KO) mice and that reduction was largely in the CD8αα⁺ TCRαβ⁺ cells. Conversely TCRγδ⁺ T cells were normal in the VDR KO mice. The thymic precursors of CD8αα⁺ TCRαβ⁺ cells (triple positive for CD4, CD8αα, and CD8αβ) were reduced and less mature in VDR KO mice. In addition, VDR KO mice had a higher frequency of the CD8αα⁺ TCRαβ⁺ precursors (double negative (DN) TCRαβ⁺ T cells) in the gut. The proliferation rates of the DN TCRαβ⁺ gut T cells were less in the VDR KO compared to WT. Low proliferation of DN TCRαβ⁺ T cells was a result of the very low expression of the IL-15R in this population of cells in the absence of the VDR. Bone marrow transplantation showed that the defect in VDR KO CD8αα⁺ TCRαβ⁺ cells was cell intrinsic. Decreased maturation and proliferation of CD8αα⁺ TCRαβ⁺ cells in VDR KO mice, results in fewer functional CD8αα⁺ TCRαβ⁺ T cells that likely explain the increased inflammation in the gastrointestinal tract of VDR KO and vitamin D deficient mice.

The human body is comprised of approximately 100 trillion cells and 10 times that many bacteria reside in the lumen of the intestine (1). The intestinal epithelial layer not only forms a physical barrier to protect from invading pathogens but also contains a highly specialized immune system. The gut associated lymphoid tissue has evolved to have effector responses to invading pathogens while maintaining tolerance to harmless commensal flora (2). When the balance between effector and tolerogenic response is lost intestinal inflammation can occur like that seen in inflammatory bowel disease (IBD) (2). The intestinal epithelial layer contains intraepithelial lymphocytes (IEL) that are responsible for maintaining intestinal health.

The IEL contains several unique cell types including CD8αα⁺ T cells. Unlike the TCR co-receptor CD8αβ, CD8αα does not act as a co-receptor and T cells that express CD8αα are not MHC I class restricted (3, 4). CD8αα has been shown to bind to the non-classical MHC molecule Thymic Leukemia antigen with a higher affinity than MHC class I (5). CD8αα⁺ TCRαβ⁺ IEL are self-reactive but not self-destructive and are believed to be regulatory T cells that help to maintain tolerance in the gut (6). In addition, CD8αα⁺ TCRαβ⁺ IEL have been shown to suppress intestinal inflammation in the T cell transfer model of IBD (7). The homodimeric form of CD8 can be expressed on both αβ and γδ T cells in the gut and expression of CD8αα is IL-15 dependent (8, 9). In addition, IL-15 has been shown to induce maturation, enhance survival and proliferation of both CD8αα⁺ TCRαβ⁺ and CD8αα⁺ TCRγδ⁺ IEL (9).

The intestine can support lymphopoiesis as is evident by the presence of CD8αα⁺ IEL in athymic nude mice and in irradiated neonatal thymectomized mice reconstituted with bone marrow (BM) (4). However, the CD8αα⁺ IEL in athymic mice are largely of the TCRγδ variety (4, 10). More recent data suggests that the thymus is required for the CD8αα⁺ TCRαβ⁺ IEL (8). TCRγδ⁺ cells diverge from the TCRαβ⁺ cells at an early double negative stage in the thymus. Like conventional TCRαβ⁺ T cells, CD8αα⁺ TCRαβ⁺ IEL progenitors develop from double positive (DP) thymocytes (8). The DP thymocytes that become CD8αα⁺ TCRαβ⁺ IEL precursors become triple positive (TP) expressing CD4, CD8αβ and CD8αα (8). The development of these self-reactive T cells requires exposure to self-agonist peptides for selection in the thymus like other regulatory T cell populations (4). After surviving agonist selection, CD8αα⁺ TCRαβ⁺ IEL precursors down regulate expression of CD4 and CD8 to become double negative (DN) TCRαβ⁺ thymocytes that express CD5 (8). Unlike conventional T cells, DN TCRαβ⁺ thymocytes egress the thymus and migrate directly to the intestine (11). Upon entering the IL-15 rich environment of the intestine DN TCRαβ⁺ cells down regulate CD5 and become mature CD8αα⁺ TCRαβ⁺ IEL (8). Even though the gut contains both CD8αα⁺ TCRαβ⁺ and TCRγδ⁺ T cells and there may be some overlap in function; the two cell types are developmentally distinct.

The vitamin D receptor (VDR) is a member of the steroid hormone family of nuclear receptors (12). The VDR contains a DNA-binding domain that is accountable for the high affinity binding of the active form of vitamin D (1,25 dihydroxyvitamin D3), for dimerization with retinoid X receptor (RXR) and for binding other transcription factors (12). The hetrodimeric complex of VDR and RXR binds to vitamin D response elements and regulates transcription of the target genes (12).

Vitamin D is an important modulator of the immune system. Signaling through the VDR has been shown to suppress multiple models of Th1 and Th17 driven autoimmune diseases including IBD (13). Vitamin D can affect T cell function as well as the development of specific T cell populations. In vitro, supplementation with 1,25D3 limits secretion of IFN-γ by CD4 T cells and promotes IL-5 and IL-10 which favors Th2 responses over Th1 (14, 15). In addition, VDR knockout (KO) TCRαβ⁺ cells show an impaired ability to migrate to the intestine when adoptively transferred to Rag KO mice (16). VDR deficient mice have normal numbers of conventional CD4 and CD8 T cells in the peripheral lymphoid organs. VDR KO mice have increased proportions of Th1 cells, reduced Th2 responses and fewer iNKT cells and CD8αα⁺ TCRαβ⁺ T cells than wildtype (WT) (16–18). CD4 T cells from VDR KO mice overproduce IFN-γ and proliferate twice as much in mixed lymphocyte reactions (13). VDR KO and vitamin D deficient WT mice have a significant reduction in the number of CD4⁺ IEL that co-express CD8αα (16).

We show here that intrinsic defects occur during the development of VDR KO CD8αα⁺ TCRαβ⁺ T cells that results in the impaired development of these cells in the IEL. VDR KO mice have normal numbers of CD8αα⁺ TCRγδ⁺ IEL. There is a significant difference in the percentages and total numbers of T cells expressing TCRαβ in the VDR KO IEL compared to WT. WT bone marrow can reconstitute VDR KO IEL to normal levels but VDR KO CD8αα⁺ TCRαβ⁺ IEL fail to develop normally in a WT host. The number of TP thymocytes and the frequency of maturing TCRβ⁺ TP thymocytes are significantly reduced in neonatal VDR KO mice. The less mature VDR KO DN TCRαβ⁺ IEL are more prevalent in the IEL, fail to become IL-15R⁺ and do not mature and proliferate in response to IL-15. Our results suggest that the VDR is an important factor in the development and maturation of CD8αα⁺ TCRαβ⁺ IEL.

Materials and Methods

Mice

Age- and sex-matched VDRKO and WT C57BL/6 mice were produced at the Pennsylvania State University (University Park, PA). VDR KO and WT mice were housed in the same room and under the exact same housing conditions. For embryonic thymocytes, timed breedings were performed. Breeding pairs were caged together in the evening and the following morning females were inspected for seminal plugs. Females with plugs were separated to establish embryonic day 0 and monitored for weight gain as an indicator of pregnancy. Experimental procedures received approval from the Office of Research Protection Institutional Animal Care and Use Committee at the Pennsylvania State University.

T cell isolation and cell culture

For IEL, the small intestine was removed and flushed with HBSS (Sigma-Aldrich, St. Louis, MO) containing 5% FBS and the Peyer's patches were removed. The intestine was cut into 0.5-cm pieces. The pieces were incubated twice in media containing 0.15 μg/ml DTT (Sigma-Aldrich) and stirred at 37 °C for 20 min. Supernatants were collected and the IEL were collected at the interface of 40/80% Percoll gradients (Sigma-Aldrich). Thymocytes were prepared with 70 μm nylon strainer and suspended in RPMI 1640 supplemented with 10% FCS (Thermo-Fisher Scientific, Rockford, IL). For in vitro stimulation thymocytes were suspended at 1.0 × 10⁶ cells/ml in RPMI 1640 supplemented with 10% FCS (Thermo-Fisher Scientific, Rockford, IL), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin, and 5 mM 2-mercaptoethanol (Invitrogen, Carlsbad, CA) and stimulated with 0.5 ng/ml anti-CD3 antibody (BD Bioscience, San Jose, CA) and 100 ng/ml recombinant IL-15 (R&D Systems, Minneapolis, MN).

Immunofluorescence

Cells were stained according to standard procedures and analyzed on a FC500 bench top cytometer (Beckman Coulter, Brea, CA). The following antibodies were used: ECD anti-CD4 (Southern Biotech, Birmingham, AL), PE anti-CD8β, FITC anti-CD122, PE anti-CD25, FITC anti-TCRγδ, PE-Cy5 anti-TCRβ, FITC anti-NK1.1, PE anti-CD5, FITC CD45.1 or CD45.2 and PE-Cy7 anti-CD8α and appropriate isotype controls including: FITC IgG1,λ (A110-1), PE-Cy7 IgG2a, κ (R35–95), PE IgG2b, κ (A95-1), FITC IgG2a, κ (G155–178) or PE-Cy5 IgG2, λ (Ha 4/8) (BD Bioscience). Isotype controls were used to set appropriate gating. CD8αα was detected with PE labeled TL-tetramer (T3b) (8). The tetramers were a gift from Dr, Hilde Cheroutre (La Jolla Institute for Allergy and Immunology, La Jolla, CA) and the National Institute of Health Tetramer Core Facility (Atlanta, GA). Data were evaluated with WinMDI 2.9 software (Scripps Institute, La Jolla, CA).

Bone marrow transplantation

Donor bone marrow cells were isolated and transferred into sub-lethally irradiated CD45 allele mismatched recipients. Mice were allowed 6 weeks to recover and reconstitution of BM was evaluated in the blood by flow cytometry and staining for donor CD45 expression.

BrdU incorporation assay

2 week old mice were injected i.p. with 50μl of 25mg/ml BrdU dissolved in PBS at day 0, 2 and 4 of the experiment. The mice were sacrificed on day 7 and the IEL were analyzed for BrdU incorporation, using a BrdU flow kit (BD Pharmingen) according to the manufacturer's instructions. PE-IgG1,κ (MOPC-)21 was used as isotype control (BD Pharmingen).

Statistics

Bar graphs are represented as mean ± SEM. Data was analyzed by two- tailed unpaired t test using Prism 5.0 statistical software (GraphPad Software, La Jolla, CA). P values of 0.05 or less were considered statistically significant.

RESULTS

Gastrointestinal TCRαβ T cells are reduced in the absence of vitamin D signaling

Equal total numbers of cells were isolated from WT and VDR KO IEL (Fig. S1A). The frequency of innate immune cells including dendritic cells (WT: 42 ± 4%, VDR KO: 30 ± 4%), macrophage (WT: 6 ± 2%, VDR KO: 11 ± 2%), and NK cells (WT: 2.3± 0.3%, VDR KO: 3.2 ± 0.6%) were the same in the IEL of WT and VDR KO mice. The frequency of both CD4⁺ and CD8αβ⁺ T cells were not different in VDR KO and WT mice (16). Staining for the TCRγ chain showed that WT and VDR KO mice had similar frequencies of γδ T cells in the IEL (Fig 1A). Staining for TCRβ showed significantly fewer TCRαβ⁺ T cells in VDR KO mice (Fig. 1A). Over 40% of the IEL in WT mice expressed the TCRαβ compared to only 25% of the VDR KO IEL (Fig. 1A). CD8αα can be expressed on many different cell types but primarily on γδ and αβ T cells (6). The majority of the CD8αα⁺ IEL are γδ T cells in WT mice (Fig. 1B). In the VDR KO IEL the frequency of CD8αα⁺ TCRγδ⁺ (VDR KO: 67%, WT: 54%) and CD8αα⁺ non-T cells in the IEL (VDR KO: 12 %, WT: 5%) were higher than WT (Fig. 1B). However, the total numbers of CD8αα⁺ TCRγδ⁺ and CD8αα⁺ non-T cells in WT and VDR KO IEL were the same (Fig. S1B, and D). Conversely, the frequency and total cell number of CD8αα⁺ TCRβ⁺ T cells in WT IEL were higher than the VDR KO values (WT: 39%, VDR KO: 17%; Fig. 1B and S1C). In the absence of the VDR fewer TCRαβ⁺ and fewer CD8αα expressing TCRαβ⁺ T cells were present in the gut.

Fig. 1.

Intrinsic defect in VDR KO mice. The T cell subsets in the IEL of WT and VDR KO mice were characterized by flow cytometry. A) The percentage of γδ and αβ TCR⁺ IEL in WT and VDR KO mice, n=3 per group and 1 representative experiment of 2. B) The percentage of the CD8αα IEL from WT and VDR KO mice that are TCRγδ, TCRαβ positive and TCR negative, n=3 per group and 1 representative experiment of 2. C) The percentage of donor derived cells in the blood of recipient mice (Donor→Recipient), n=5 per group. D) Percent of donor derived CD8αα⁺ TCRαβ⁺ IEL in the transplant recipients, n=5 per group. Values are mean ± SEM of 3–5 mice per group. Data represent 1 of 2 experiments. E) Gating strategy to identify donor specific CD45 and TCRβ⁺ expressing cells (top left). Dot plot of one recipient of each of the three transplants shown in C and D.

Intrinsic defect in VDR KO cell CD8αα⁺ TCRαβ⁺ T cells

Reciprocal BM chimeras were produced using VDR KO and WT mice. Reconstitution of WT-WT (donor –recipient), VDR KO-WT, and WT-VDR KO mice was over 80% complete in the blood of all mice tested (Fig. 1C). Importantly, VDR KO mice were reconstituted with the same efficiency as WT mice (Fig. 1C). The IEL of WT mice reconstituted with WT BM (WT-WT) had 43% of the CD8αα⁺ TCRαβ⁺ T cells of donor origin (Fig. 1D and E). VDR KO recipients of WT BM (VDR KO-WT) had slightly lower reconstitution with WT donor cells but the values were not significantly different than WT-WT (Fig.1D and E). Fewer CD8αα⁺ TCRαβ⁺ T cells of VDR KO origin were recovered from WT recipients (VDR KO-WT) and the difference was significantly different than both the WT-WT and WT-VDR KO chimeras (Fig. 1D and E). The data suggest that VDR KO BM has a cell intrinsic defect in the generation of CD8αα⁺ TCRαβ⁺ T cells that reside in the IEL.

Early CD8αα⁺ TCRαβ⁺ thymic precursors in the VDR KO mice

CD8αα⁺ T cell precursors are first detectable in the thymus at embryonic (e) day 16 of fetal development (8). Expression of CD8αα along with CD8αβ and CD4 result in a TP cell type (8). The TP thymocytes in WT mice make up over 40% of the thymus at e16, peak at e17 (58%) and are only 10% by birth (d1, Fig. 2A). Similar frequencies of TP thymocytes with the same kinetics were found in the VDR KO thymus (Fig. 2A). Frequencies of double positive (DP) thymocytes were also not different in the fetal thymus of VDR KO and WT mice.

Fig. 2.

The development of CD8αα⁺ TCRαβ⁺ IEL and thymic precursors in fetal and neonatal VDR KO mice. The percentages of CD8αα⁺ TCRαβ⁺ thymic precursors and CD8αα⁺ TCRαβ⁺ IEL were evaluated in fetal and neonatal WT and VDR KO mice. A) The percentage of the thymus that is TP (CD4⁺/CD8αβ⁺/CD8αα⁺) in fetal (e16, e17, and e18) and newborn (d1) WT and VDR KO mice, n=4 per group. B) The percentages of TP thymocytes in 1, 2, 3, and 6 wk old WT and VDR KO mice, n=3 per group. C) The frequency of DP thymocytes of WT and VDR KO neonatal mice, n=3 per group. D) The percentage of maturing (TCRβ⁺) TP cells in WT and VDR KO mice, n=3 per group. E) The percentage of the total IEL of WT and VDR KO mice that express TCRαβ at 1, 2, 3, and 6 wks of age, n=3 per group. F) The percentage of WT and VDR KO CD8αα⁺ TCRαβ⁺ IEL in neonatal mice, n=3 per group. These data represent 1 of 2 independent experiments. Values are mean ± SEM. * VDR KO values are significantly different from WT, p<0.05.

From birth to 3 wks of age WT mice maintained the percentage of TP thymocytes (~10%) while the percentage of VDR KO TP thymocytes significantly decreased (Fig. 2B). The frequency of TP cells in the thymus then declined to those found in the adult thymus by 6 wks of about 5% and at 6 wks there were equal numbers of TP cells in WT and VDR KO thymuses (Fig. 2B). The frequencies of DP thymocytes are not different in WT and VDR KO mice regardless of age (Fig. 2C). Expression of the TCR is a step in the maturation of the CD8αα⁺ T cells precursors (8).

There were more WT TP cells that express the TCRβ receptor than VDR KO TP cells at 2 and 3 wks of age. The kinetics for the appearance of TCRβ⁺ TP thymocytes was the same in VDR KO and WT mice but the WT mice had a higher frequency of TCRβ⁺ CD8αα precursors (Fig. 2C). By 6 wks when the frequency of TP cells was low and not different between WT and VDR KO mice the expression of TCRβ on TP cells was also not different (Fig. 2D).

The first 3 weeks of life are a critical time for the appearance of CD8αα⁺ TCRαβ⁺ T cells in the IEL (4). WT IEL contain 20% TCRαβ⁺ T cells by 1 wk of age and that percentage gradually increased as the mouse matures to the adult levels of 30% (Fig. 2E). CD8αα⁺ TCRαβ⁺ IEL can be found in low frequencies at 1 and 2 wks in WT mice and a significant increase occurs between 2 and 3 wks of age to when over 40% of the TCRαβ⁺ T cells express CD8αα (Fig. 2F). VDR KO TCRαβ⁺ IEL fail to appear in the intestine at the same rate as WT T cells and this defect can be seen as early as the first week of life (Fig. 2E). The frequency of VDR KO CD8αα⁺ TCRαβ⁺ T cells in the gut are the same as WT at 1 and 2 wk of age (Fig. 2F). By 3 wks there are lower frequencies of VDR KO CD8αα⁺ TCRαβ⁺ IEL than WT and the numbers of the CD8αα⁺ TCRαβ⁺ fail to increase further as the mice age (Fig. 2F).

Reduced proliferation of TCRαβ⁺ IEL in the absence of the VDR

At the time of CD8αα⁺ TCRαβ⁺ appearance in the IEL (between 2 and 3 wks of age in WT mice, Fig. 2F), mice were treated with BrdU to measure the in vivo proliferation of the cells. Total BrdU incorporation of the IEL of WT mice showed that 34% of the cells had proliferated (Fig. 3A). Only 22% of the VDR KO IEL incorporated BrdU over the same time period, which was significantly less than WT (Fig. 3A). In order to identify what population of IEL were proliferating at a slower rate in the VDR KO mice; the cells were stained for TCRβ, CD8αα and BrdU. Incorporation of BrdU by the TCRβ⁻ IEL (γδ T cells, NK cells, etc.) was similar in the VDR KO and WT mice (Fig. 3B). Incorporation of BrdU in the CD8αα⁺ TCRβ⁻ IEL (largely the CD8αα⁺ TCRγδ⁺ cells Fig. 1B) was higher but not significantly higher in VDR KO (32%) than WT (24%) mice (Fig. 3C). The frequency of proliferating TCRαβ⁺ T cells in WT IEL was twice as high as VDR KO IEL (Fig. 3D). Different TCRβ⁺ subsets proliferated at different rates. In WT mice, the frequency of proliferating DN TCRβ⁺ T cells was significantly greater compared to CD8αα⁺ TCRαβ⁺ T cells (Fig. 3D). The frequency of proliferating DN TCRαβ⁺ from VDR KO mice was not different than CD8αα⁺ TCRαβ⁺ T cells (Fig. 3D). There was a significant decrease in the frequency of proliferating DN TCRαβ⁺ and CD8αα⁺ TCRαβ⁺ IEL in VDR KO mice compared to WT (Fig. 3D). Only the VDR KO CD8αα⁺ TCRβ⁺ IEL and their precursors had impaired homeostatic proliferation.

Fig. 3.

Reduced proliferation of VDR KO CD8αα⁺ TCRαβ⁺ IEL precursors. Proliferation was evaluated by BrdU incorporation of the IEL subsets of WT and VDR KO mice between 2–3 wks of age. A) BrdU incorporation of the total IEL from WT and VDR KO mice at 3 wks of age, n=3 per group (p<0.01). B) Total BrdU incorporation of TCRαβ negative IEL from 3 wk old WT and VDR KO mice, n=3 per group. C) BrdU incorporation in the CD8αα⁺ and TCRβ⁻ cells, n=3 per group. D) The frequency of proliferating TCRβ⁺, DN TCRαβ⁺ and CD8αα⁺ TCRαβ⁺ IEL in 3wk old WT and VDR KO mice (each gated on TCRβ⁺ cells first), n=3 per group. One representative histogram or dot plot (panels A–D) is shown from 2 independent experiments. The shaded histograms are isotype staining, whereas open histograms are BrdU staining. Values are mean ± SEM of the MFI or percentages. * VDR KO values are significantly different from WT, p<0.05.

Increased frequency but decreased numbers of CD8αα⁺ TCRαβ⁺ IEL precursors in VDR KO mice

CD8αα⁺ TCRαβ⁺ IEL mature from DN TCRαβ⁺ IEL (8). In the WT IEL there was a minor population of less mature DN TCRαβ⁺ T cells that had not upregulated CD8αα⁺ in the IEL (Fig. 4A). VDR KO mice had a higher frequency of DN TCRαβ⁺ IEL than WT IEL (Fig. 4A). Because VDR KO mice have fewer TCRαβ⁺ cells the total number of immature T cells was less in the VDR KO IEL than the WT IEL (Fig. 4B and 4C). The increased frequency of DN TCRαβ⁺ IEL in the VDR KO IEL was not due to an increase in NKT cells since the percentage of TCRαβ⁺/NK1.1⁺ expressing cells was also less in the VDR KO than WT mice (Fig. 4D).

Fig. 4.

Immature CD8αα⁺ TCRαβ⁺ precursors in the VDR KO IEL. CD8αα⁺ TCRαβ⁺ IEL precursors in the intestine of WT and VDR KO mice were evaluated by flow cytometry. A) The frequency of DN (CD4⁻/CD8⁻) TCRαβ⁺ IEL in WT and VDR KO mice, n=4 per group. B) The total number of TCRαβ⁺ IEL isolated from WT and VDR KO mice, n=4 per group. C) The total number of DN TCRαβ⁺ IEL in WT and VDR KO mice, n=4 per group. D) The percentage of NKT cells or DN TCRαβ⁺ and NK1.1⁺ cells in the IEL, n=4 per group. The E) frequency and F) total numbers of CD5⁺ DN TCRαβ⁺ cells in the IEL of WT and VDR KO mice, n=4 per group. These represent 1 of 2 experiments. Values are mean ± SEM.

Immature DN TCRαβ⁺ IEL first express CD5 that is later down regulated as the cell matures and expresses CD8αα (8). Of the DN cells in the IEL of WT mice 25% express CD5 (Fig. 4E). Over 42% of the DN IEL in the VDR KO expressed CD5, which was significantly more than the WT DN cells (Fig. 4E). As a result of the reduced numbers of TCRαβ⁺ cells the total number of VDR KO CD5⁺ DN TCRαβ⁺ IEL was significantly less than the WT mice (Fig. 4F). VDR KO mice had a higher frequency of immature DN TCRαβ⁺ IEL than WT mice.

IL-15 unresponsiveness of VDR KO CD8αα precursors in the VDR KO mice

IL-15 is required to induce the maturation of CD8αα⁺ TCRαβ⁺ IEL (8, 19). In addition, IL-15 is required for CD8αα⁺TCRγδ⁺ IEL, CD44^highCD8⁺ memory T cells and NK cells (20). CD8αα⁺TCRγδ⁺ IEL numbers were not different in WT and VDR KO mice (Fig. 1 and Fig. S1B). Of the CD8+ T cells in the spleen 49 ± 3% WT and 45 ± 6% VDR KO were of the memory phenotype or CD44^high. Similarly the frequencies of NK cells (NK1.1⁺ and CD3⁻) were similar in the spleen of VDR KO and WT mice (4–5%). Of the 4 cell types that are IL-15 dependent only the CD8αα⁺ TCRαβ⁺ IEL is affected by VDR deficiency.

Expression of the IL-15R was measured on T cells in the thymus and IEL by measuring CD122 on the CD25⁻ T cells. The mean fluorescence intensity (MFI) of the IL-15R on DN T cells in the thymus of WT mice was low (Fig. 5A). The level of IL15R expressed on WT and VDR KO DN T cells in the thymus was similar (Fig. 5A). Expression of the IL-15R in all IEL of WT and VDR KO mice was also low and similar (Fig. 5B). The MFI of the IL-15R was slightly higher on VDR KO (MFI 6.9) CD8αα⁺ TCRαβ⁺ IEL than WT (MFI 5.9) but the difference did not reach significance (Fig. 5C, P=0.07). The MFI of the IL-15R in the CD8αα⁺ TCRβ⁻ cells (largely γδ T cells, Fig. 1B) was about 4.0 in both the WT and VDR KO IEL (Fig. 5D). The level of expression of IL-15R on WT DN TCRαβ⁺ IEL was significantly higher (MFI, 7.0) than VDR KO DN TCRαβ⁺ IEL (MFI 4.1, P=0.01). The frequency of IL-15R expression was low on all CD8αα⁺ cells in the IEL of either VDR KO or WT mice: CD8αα⁺ TCRαβ⁺ (2–3%, Fig. 5E), CD8αα⁺TCRβ⁻ (1.8–2.3%) and CD8αα⁺ TCR γδ (0.9–1.2%). Only 2% of WT and 3% of VDR KO CD8αα⁺ TCRαβ⁺ IEL express high levels of the IL-15R (Fig. 5E). Conversely, 37% of DN TCRβ⁺ cells expressed the IL-15R in the WT IEL (Fig. 5F). VDR KO DN TCRβ⁺ IEL failed to express the IL-15R (0.6%) (Fig. 5F). Exogenously delivered IL-15 induces the expression of CD8αα in activated thymocytes (8). Twenty percent of the cells expressed CD8αα when WT thymocytes were activated and cultured with IL-15 (Fig. 5G). Ten percent or half as many of the similarly treated VDR KO thymocytes upregulated CD8αα in response to IL-15 (Fig. 5G). The data point to defects in DN cell expression of the IL-15R and intrinsic defects in the response to exogenous IL-15 in the absence of the VDR.

Fig. 5.

Immature CD8αα precursors express low levels of the IL-15R and respond poorly to IL-15 in the absence of the VDR. Cell surface expression of the IL-15R (CD122⁺ CD25⁻) on A) DN TCRαβ⁺ thymocytes, B) TCRβ⁺ IEL, C) CD8αα⁺ TCRαβ⁺ IEL and D) CD8αα⁺ TCRβ⁻ IEL, n=3 per group. Values are mean MFI ± SEM. *Values for VDR KO are significantly different from the WT values, p<0.05. The frequency of E) CD8αα⁺ TCRαβ⁺ IEL and F) DN TCRαβ⁺ IEL that express the IL-15R, n=3 per group. These data are 1 representative dot plot from 2 experiments. Values are mean ± SEM. There is a significant difference between WT and VDR KO DN TCRαβ⁺ IEL, p<0.001. IL-15 induces CD8αα expression on WT and VDR KO thymocytes in vitro. G) The percentage of WT and VDR KO T cells that are CD8α and TL tetramer⁺ after IL-15 treatment, p<0.05. One representative dot plot is shown and gates were set from negative stain and isotype control. Cultures were performed in triplicate. Data represent 1 of 3 experiments. Values represent mean ± SEM percentage for VDR KO and WT cultures.

DISCUSSION

We show here that there is a T cell intrinsic defect in VDR KO CD8αα T cell development that is TCRαβ T cell specific. In VDR KO mice, TP thymocyte development was normal during fetal development and in the adult thymus but there are fewer TP thymocytes during the first 3 wks of life. VDR KO DN precursors failed to mature and proliferate under the influence of IL-15 due to the low expression of the IL-15R. These results identify the VDR as an important regulator of homeostasis, development, maintenance and proliferation of CD8αα⁺ TCRαβ⁺ cells in the IEL. The GALT can support extrathymic development of CD8αα⁺ T cells as evident by the discovery that T cells can be found in the IEL compartment of athymic nude mice (10). The majority of the T cells found in athymic mice are CD8αα⁺ TCRγδ⁺ T cells but the numbers were 4 fold less than normal euthymic mice and very few CD8αα⁺ TCRαβ⁺ IEL existed in athymic mice (10). These discoveries indicated that the thymus is more important for the development of CD8αα⁺ TCRαβ⁺ than CD8αα⁺ TCRγδ⁺ IEL. Under normal physiological conditions TCRγδ⁺ T cell development occurs both thymically and extrathymically during fetal development and continues through adulthood (21). The discovery that TP cells are thymic precursors of CD8αα⁺ TCRαβ⁺ IEL supports a thymic requirement for these cells (8). The ability of CD8αα⁺ TCRγδ⁺ T cells to develop normally in VDR KO mice suggests that signals specific for CD8αα⁺ TCRαβ⁺ T cell development such as thymic selection may be VDR dependent.

The development of both CD8αα⁺ TCRαβ⁺ and CD8αα⁺ TCRγδ⁺ IEL is severely impaired in the absence of IL-15, IL-15Rα or IL-15/IL-2Rβ (9). In addition, NK cells and memory CD8⁺ T cells fail to develop in the absence of IL-15 and/or the IL-15 receptors (9). IL-15 is produced and trans-presented via the IL-15Rα by the epithelial cells of the intestine (9). Because VDR KO mice had normal numbers of CD8αα⁺ TCRγδ⁺ IEL and WT CD8αα⁺ TCRαβ⁺ IEL developed normally in a VDR KO host, expression and presentation of IL-15 by intestinal epithelial cells must be adequate for induction of these IL-15 dependent cell types. Furthermore, the selective defect in only the CD8αα⁺ TCRαβ⁺ IEL suggest that there are not global VDR mediated effects on the IL-15 pathway. We found that VDR KO DN TCRαβ⁺ T cells failed to become IL-15R positive and proliferated less than WT DN T cells in the IEL. The severe reduction in the level of proliferation in VDR KO DN TCRαβ⁺ reflects the loss of IL-15 signaling in only this cell type. In addition, thymocytes from VDR KO mice expressed normal amounts of the IL-15R but failed to respond to exogenous IL-15 as well as the WT thymocytes to upregulate CD8αα expression. This data indicates that in the VDR KO mice, CD8αα precursors also have a defect in IL-15R signaling. The maturation of CD5⁺ DN TCRαβ⁺ T cells requires IL-15 and VDR KO IEL contain a higher frequency of CD5⁺ DN TCRαβ⁺ T cells (8). The increased percentage of VDR KO DN TCRαβ⁺ T cells that remain in an immature CD5⁺ state also reflects a lack of IL-15 signaling and likely contributed to the overall reduction of the mature CD8αα⁺ TCRαβ⁺ T cells in the IEL of VDR KO mice. The data suggests that expression of the VDR and other transcription factors found only in the precursor of the CD8αα⁺ TCRαβ⁺ are required for IL-15R signaling and to upregulate IL15R expression that results in survival, maturation and CD8αα⁺ expression in the IEL. Other IL-15 dependent cells do not use vitamin D to regulate IL15R expression.

iNKT cell development is also impaired in VDR KO mice (18). CD8αα⁺ TCRαβ⁺ T cells have been called “gut NKT cells” because of their expression of NK receptors (6). Like NKT cells, CD8αα⁺ TCRαβ⁺ T cells express NK receptors including members of the Ly49 family; CD94 and NKR-PI and use the invariant signaling component FcεRIγ as a part of their CD3 complex (6, 22). Both cells have a memory phenotype and rapidly produce cytokines upon stimulation (6, 22). In addition, both cells can develop in the absence of MHC class I or class II but do require β2 microglobulin (6, 11, 22). An interesting similarity between CD8αα⁺ TCRαβ⁺ T cells and NKT cells is their developmental dependence on expression of the NF-κB family transcription factor RelB (6). RelB deficiency is associated with autoimmunity that is believed to be linked to dysfunctional T cell selection in the thymus which allows the survival of conventional T cells that express high affinity receptors that would normally be deleted during the selection process (23, 24). The reduced numbers of CD8αα⁺ TCRαβ⁺ and NKT cells and the survival of conventional T cells with self-reactive TCR suggests that RelB plays a role in thymic function during agonist selection (6). Interestingly, RelB is transcriptionally regulated by vitamin D and has been shown to have VDR response elements in its promoter in both mice and humans (25). It would be interesting to determine what the role of RelB is in the VDR mediated development of CD8αα⁺ TCRαβ⁺ cells.

The process of agonist selection allows T cells carrying “forbidden repertoires” to survive the selection process but reprograms the cell to be regulatory in nature, carrying a self-reactive TCR while maintaining a non-destructive function (6, 10). Both CD8αα⁺ TCRαβ⁺ and NKT cells undergo agonist selection in the thymus as DP cells (6, 22). Each cell type then diverges along a separate developmental pathway. NKT cells that survive selection downregulate CD8 or CD4 and CD8, rapidly expand and upregulate expression of CD44 and NK1.1 (22). At the DP stage CD8αα⁺ TCRαβ⁺ precursors transition to a TP stage in a preTCR signal dependent manner (8). The TP thymocytes complete TCR rearrangement and undergo agonist selection (8). After surviving selection, TP cells downregulate all CD4 and CD8 becoming DN TCRαβ⁺ T cells that egress the thymus and continue their maturation in the gut under the influence of IL-15 (8). The similarities in the selective requirement for the VDR in CD8αα⁺ TCRαβ⁺ and NKT cells suggests a common mechanism for regulation. Further investigation is needed to determine the role of vitamin D signaling in the thymus during agonist selection.

Vitamin D and signaling through the VDR are important for regulating intestinal health. VDR deficiency increases the severity of inflammation in several mouse models of intestinal disease (13). The prevalence of Crohn's disease and ulcerative colitis is higher in northern versus southern climates and urban versus rural areas (26, 27). These are also factors that correlate with vitamin D deficiency. Several studies have reported deficiencies in adult and pediatric patients with IBD and vitamin D deficiency is common even when the patient is in remission (28, 29). VDR KO and vitamin D deficient mice are highly susceptible to multiple different models of experimental IBD (13, 30, 31). VDR KO mice do not develop overt inflammation but there is microscopic evidence of increased TNF-α, and IFN-γ in the gastrointestinal tract of the VDR KO mouse and as it ages the amounts and variety of cytokines increased (IL-12 was detected,(32)). The data suggests that inflammatory bowel disease is not a vitamin D deficiency disease but rather vitamin D may be an environmental factor that regulates inflammation in the gut. Genome wide association studies have shown that polymorphisms within the VDR gene increase susceptibility to IBD (33, 34). It seems likely that reduced signaling through the VDR would limit the development of CD8αα⁺ TCRαβ⁺ T cells in the human gut.

We have shown that there is an intrinsic and specific defect in the development of VDR KO CD8αα⁺ TCRαβ⁺ T cells. TP precursors in the thymus are reduced in neonatal VDR KO mice. Immature DN TCRαβ⁺ T cells fail to upregulate the IL-15R, proliferate less and maintain a more immature phenotype. CD8αα⁺ TCRαβ⁺ T cells are important regulators of intestinal immune responses and their failure to mature in VDR KO mice likely contributes to the increased susceptibility of these mice to intestinal inflammation.

Nonstandard abbreviations

DN

double negative

DP

double positive

KO

knockout

IBD

inflammatory bowel disease

IEL

intraepithelial lymphocyte

TP

triple positive

VDR

vitamin D receptor

WT

wildtype