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Reduced Expression of Bcl-2 in CD8⁺ T Cells Deficient in the IL-15 Receptor -Chain¹

Tzong-Shoon Wu^2,3,*, Jan-Mou Lee^2,*,, Yein-Gei Lai^*,, Jen-Chi Hsu^*, Ching-Yen Tsai^4,*, Ying-Hue Lee^* and Nan-Shih Liao^*,5,

^* Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan; and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice that lack IL-15 or the IL-15R -chain (IL-15R) are deficient in peripheral CD8⁺, but not in CD4⁺, T cells. This CD8⁺ T cell-specific deficiency has now been investigated further by characterization of a new strain of IL-15R^-/- mice. The adult mutant mice exhibited a specific reduction in the percentage of CD8-single positive TCR^high thymocytes. The expression of Bcl-2 was reduced in both CD8⁺ thymocytes and naive T cells of the mutant animals, and the susceptibility of these cells to death was increased. Memory CD8⁺ cells were profoundly deficient in IL-15R^-/-mice, and the residual memory-like CD8⁺ cells contained a high percentage of dead cells and failed to up-regulate Bcl-2 expression compared with naive CD8⁺ cells. Moreover, exogenous IL-15 both up-regulated the level of Bcl-2 in and reduced the death rate of wild-type and mutant CD8⁺ T cells activated in vitro. These results indicate that IL-15 and IL-15R regulate the expression of Bcl-2 in CD8⁺ T cells at all developmental stages. The reduced Bcl-2 content in CD8⁺ cells might result in survival defect and contribute to the reduction of CD8⁺ cells in IL-15R^-/-mice.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Interleukin-15 is a member of the type I cytokine family and was originally identified on the basis of its ability to support T cell proliferation (1). IL-15R consists of , , and common chains (_c)⁶, with the -chain being shared by IL-2R and the _c chain being shared by receptors for IL-2, IL-4, IL-7, and IL-9 (2, 3). The - and _c chains together bind IL-15 or IL-2 with intermediate affinity and mediate signal transduction (4, 5). Whereas IL-2R serves only as a high-affinity component of the IL-2R complex (4), IL-15R binds IL-15 with high affinity in the absence of - and _c-chains (3, 6). Although IL-15R has been suggested to mediate signaling (7, 8), deletion of the cytoplasmic domain of this protein did not impair IL-15-induced proliferation of a myeloid cell line that expresses the and _c subunits (6).

IL-15 shares certain biological activities with IL-2, presumably because of the shared - and _c chains of the corresponding receptors (1, 9, 10). Functions specific to IL-15 include the ability to act as a survival factor for several T cell systems in which IL-2 promotes cell death (11). The survival of activated CD4⁺ primary T cells and CD4⁺ clones is supported to a greater extent by IL-15 than it is by IL-2 (12). Furthermore, the expression of transgenic IL-15 by CD4⁺ T cells increased their resistance to death triggered by IL-2-induced activation, and this increased resistance was abrogated by a mAb to IL-15 (13). IL-15 also promotes the survival of CD8⁺TCR and TCR intestinal intraepithelial lymphocytes (14, 15). These observations implicate IL-15 as an important determinant of T cell survival.

IL-15R and IL-15 knockout mice manifest phenotypes that differ from those of mice deficient in other type I cytokines or cytokine receptors (16, 17). Several lymphoid lineages important for innate immunity, including NK cells, NK T cells, and CD8⁺ intestinal intraepithelial lymphocytes, were shown to be markedly deficient in IL-15R^-/- and in IL-15^-/- mice. The percentage of conventional CD8⁺ cells, but not that of CD4⁺ cells, in the secondary lymphoid organs was also reduced by 50%, and the percentage of memory CD8⁺ cells was preferentially reduced by 85% in these animals. The mechanisms responsible for the deficiency of peripheral CD8⁺ cells in IL-15R^-/- and IL-15^-/- mice have not been clearly defined. Although the percentage of CD8 single-positive (SP) thymocytes was reduced by 45% in young IL-15R^-/- mice (16), the number and composition of thymocytes appeared to be normal in adult IL-15^-/- mice (17). Lymphopenia was also observed in IL-15R^-/- mice but not in IL-15^-/- mice. Lymphocyte homing and T cell proliferation were defective in IL-15R^-/- mice, and these defects were proposed to contribute to lymphopenia (16). Whether these two abnormalities are related to the specific reduction in the percentage of CD8⁺ cells in IL-15R^-/- mice remains unclear. Examination of the percentage of dead cells among lymph node (LN) cells revealed similar value in IL-15R^-/- and in wild-type mice (16), suggesting that the decrease in the percentage of CD8⁺ cells in the mutant mice was not due to defective cell survival. To investigate further the role of IL-15 and IL-15R in the development and maintenance of CD8⁺ T cells, we have now characterized thymic and peripheral T cells in IL-15R^-/- mice generated in our laboratory.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of IL-15R knockout mice

An IL-15R genomic clone was isolated from a bacterial artificial chromosome library of RW4 embryonic stem (ES) cells (Genome Systems, St. Louis, MO) derived from 129SvJ mice. A partial map of the IL-15R genomic clone was established by restriction endonuclease digestion in combination with Southern blot analysis with a murine IL-15R cDNA probe (kindly provided by Immunex, Seattle, WA). An 8.8-kb BamHI-EcoRI genomic fragment that contains exon 1 of the IL-15R gene was used to generate the targeting construct. In the targeting construct, a 1.3-kb SmaI-XbaI fragment containing exon 1 of the IL-15R gene was replaced by a 2-kb fragment containing the neomycin phosphotransferase gene (neo) under the control of the phosphoglyceraldehyde kinase gene (pgk) promoter and flanked by loxP sites. A pgk promoter-driven thymidine kinase gene (tk) cassette was also ligated to the 3' end of the short homology arm. The targeting construct was introduced into 129SvJ-derived GSI-1 ES cells (Genome Systems) by electroporation, and the cells were then subjected to selection with ganciclovir and G418. The resistant clones were screened by Southern blot hybridization with a probe that flanks the short homology arm of the targeting construct. Two ES clones were identified and used for microinjection of blastocysts. The resultant male chimeric mice were bred with C57BL/6 (B6) females, and offspring that exhibited germline transmission of the mutant allele (IL-15R^+/- mice) were bred with EIIa-cre transgenic mice (18) to delete the neo cassette. The IL-15R^+/- neo^-/- offspring were identified by Southern blot analysis and bred with B6 mice. The resultant IL-15R^+/- offspring were intercrossed to generate IL-15R^-/- and IL-15R^+/+ littermates for experiments. Mice used in this study were 6–8 wk old and had been backcrossed to B6 mice for two to five generations.

Cell preparation

Cells of the thymus, spleen, and LNs were released gently from each organ with the use of a glass tissue grinder. RBCs in the spleen cell preparation were lysed by incubation in ACK buffer (0.15 M NH₄Cl, 1 mM KHCO₃, and 0.1 mM EDTA (disodium salt), pH 7.2). Large pieces of tissue debris were removed from the single-cell suspension by gravity sedimentation. PBMCs were prepared from tail blood after lysis of RBCs with ACK buffer. For purification of CD44^lowCD8⁺ cells, LN and spleen cells were first enriched in CD8⁺ cells by negative panning on plates coated with anti-MHC class II mAb (clone BP107; prepared in our laboratory) and then on plates coated with anti-CD4 mAb (clone GK1.5; prepared in our laboratory). The nonadherent cells were collected, stained with a PE-conjugated anti-CD8 (clone 53-6.7; BD Biosciences, San Diego, CA) and an FITC-conjugated anti-CD44 (clone CT-CD4; CALTAG Laboratories, Burlingame, CA) mAb, and sorted for CD44^lowCD8⁺ cells with a FACStar^Plus (BD Biosciences).

Immunostaining and flow cytometry

Staining of cell surface molecules with mAbs was performed for 15 min at room temperature in staining buffer: Mg²⁺- and Ca²⁺-free Dulbecco’s PBS (Life Technologies, Rockville, MD) containing 1% heat-inactivated FCS and 0.1% NaN₃. For intracellular staining of Bcl-2, cells were first stained with mAbs specific for surface molecules and then fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) at 4°C for 20 min. The fixed cells were washed twice with permeabilization buffer (staining buffer plus 0.1% saponin (Sigma-Aldrich)) and then incubated for 30 min at 4°C with an FITC-conjugated mAb to Bcl-2 (clone 3F11; BD PharMingen, San Diego, CA) in permeabilization buffer; an FITC-conjugated isotype-matched mAb (clone A19-3; BD PharMingen) was used as a negative control for intracellular staining. Three-color staining was performed with mAbs conjugated with FITC, PE, or biotin, followed by incubation with streptavidin-allophycocyanin (CALTAG Laboratories), and the samples were analyzed with a FACSCalibur (BD Biosciences). Four-color staining was performed with mAbs conjugated with FITC, PE, Cy5, or biotin, followed by incubation with streptavidin-Texas Red (Zymed Laboratories, South San Francisco, CA), and the samples were analyzed with a FACStar^Plus (BD Biosciences). Data were acquired with the use of CellQuest and analyzed by FlowJo (Tree Star, San Carlos, CA) softwares. Abs used include biotin-conjugated anti-CD8 (clone CT-CD8; CALTAG Laboratories), Cy5-conjugated anti-CD8 (clone 53-6.7; BD Biosciences), biotin-conjugated anti-CD4 (clone RM4-4; BD PharMingen), biotin-conjugated anti-CD19 (clone 1D3; BD PharMingen), FITC-conjugated anti-CD19 (clone MB19-1; BD Biosciences), PE-conjugated anti-CD25 (clone PC61.5.3; CALTAG Laboratories), FITC-conjugated anti-CD44 (clone IM7; BD Biosciences), biotin-conjugated anti-CD44 (clone IM7; BD PharMingen), PE-conjugated anti-CD69 (clone H1.2F3; BD Biosciences), biotin-conjugated anti-CD69 (clone H1.2F3; BD Biosciences), biotin-conjugated anti-CD45Rb (clone 23G2; BD PharMingen), PE-conjugated anti-CD62L (clone MEL-14; CALTAG Laboratories), PE-conjugated anti-IL-2R (clone TM-1; BD PharMingen), biotin-conjugated DX5 (BD Biosciences), PE-conjugated anti-TCR (clone H57-597; BD Biosciences), and FITC-conjugated TCR (clone H57-597; prepared in our laboratory).

In vitro activation of naive CD8⁺ cells

Wells of 96-well plates were coated with anti-TCR mAb (0.5 µg/well) (clone H57-597; prepared in our laboratory) alone or in combination with anti-CD28 mAb (1 µg/well) (clone 37.51; prepared in our laboratory). Sorted CD44^lowCD8⁺ cells were activated for the indicated times in the coated wells at a density of 10⁴ cells/well in 200 µl of RPMI 10 (RPMI 1640 (Life Technologies) supplemented with 2 mM L-glutamine, 20 mM HEPES-NaOH (pH 7.2), penicillin-streptomycin (2000 U/L), 50 µM 2-ME, and 10% FCS). Recombinant mouse IL-2 (R&D Systems, Minneapolis, MN) or recombinant human IL-15 (R&D Systems) was included in the culture as indicated.

Apoptosis assay

Cells were incubated for 20 min at room temperature with FITC-conjugated annexin V (1 µl/1 x 10⁶ cells; CLONTECH Laboratories, Palo Alto, CA) and propidium iodide (1 µg/ml; Sigma-Aldrich) in annexin V binding buffer (10 mM HEPES-NaOH (pH 7.4), 140 mM NaCl, and 5 mM CaCl₂) and were then analyzed immediately with a FACSCalibur. Annexin V-positive cells were scored as dead cells. Data were acquired with the use of CellQuest and analyzed by FlowJo softwares.

Statistical analysis

Data are expressed as means or means ± SD and were compared among groups by single-classification ANOVA. A value of p < 0.05 was considered to be statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of IL-15R^-/-mice

The targeting construct for generation of IL-15R knockout mice was designed to replace a 1.3-kb region of the IL-15R gene containing exon 1 with a 2-kb loxP-flanked neo cassette by homologous recombination (Fig. 1A). Such recombination should give rise to a null allele, given that deletion of exon 1 removes both the translational start codon and the nucleotide sequence encoding the signal peptide. The neo cassette was deleted by breeding IL-15R^+/- neo^+/- mice with EIIa-cre transgenic mice. Mouse genotypes were determined by Southern blot hybridization of genomic DNA with a 0.94-kb probe that flanks the 3' end of the short homology arm of the targeting construct (Fig. 1A). The probe hybridizing with 6.5-, 7.2-, and 5.2-kb BamHI fragments corresponds to the wild-type, IL-15R^-neo⁺, and IL-15R^-neo^- alleles, respectively (Fig. 1B).

View larger version (42K): [in this window] [in a new window]   FIGURE 1. Generation of IL-15R knockout mice. A, Targeting strategy for disruption of the IL-15R gene. The targeting construct was designed to replace a 1.3-kb SmaI-XbaI fragment containing exon 1 of the IL-15R gene with a 2-kb loxP-flanked pgk-neo cassette by homologous recombination. A pgk-tk cassette was inserted at the 3' end of the short homology arm of the targeting construct. Restriction enzyme sites: B, BamHI; Sm, SmaI; Xb, XbaI; E, EcoRI. B, Analysis of mutant mouse genotype. Genomic DNA prepared from mouse tail was digested with BamHI and subjected to Southern blot analysis with a 0.94-kb probe (shown in A) that flanks the short homology arm of the targeting construct. Wild-type (WT) and knockout (KO) alleles of the IL-15R gene generate 6.5- and 7.2-kb hybridizing fragments, respectively (left panel). The neo cassette of the targeted allele was deleted by breeding the mutant mice with EIIa-cre transgenic mice. The IL-15R-neo- allele gives rise to a 5.2-kb BamHI fragment that hybridizes with the same 0.94-kb probe (right panel). Symbols above the lanes of both blots indicate the IL-15R genotype.

The lymphocyte profile of secondary lymphoid organs was analyzed to determine whether IL-15R deficiency affects lymphocyte development or maintenance. IL-15R^-/- mice exhibited similar numbers of splenic white cells but 30% fewer LN cells (p < 0.1) when compared with wild-type littermates (our unpublished data). The percentage of B cells (CD19⁺) was normal in the knockout mice, whereas the percentages of NK T cells (CD3⁺DX5⁺) and NK cells (CD3^-DX5⁺) were greatly decreased (our unpublished data). Within the T cell compartment of IL-15R^-/- mice, the percentage of CD8⁺ cells was reduced by 50% whereas that of CD4⁺ cells was increased by 20%, resulting in similar percentages of CD4⁺ plus CD8⁺ cells in wild-type and knockout animals (Fig. 2A). Among CD8⁺ T cells, a 72% decrease in the CD44^high population, which is enriched in memory cells, and a 40% decrease in the CD44^low population, which is enriched in naive cells, were detected in the knockout mice (Fig. 2B). The marked deficiency of NK, NKT, and CD44^highCD8⁺ cells apparent in our IL-15R^-/- mice is consistent with the characteristics of the IL-15R and IL-15 knockout mice described previously (16, 17). Most, if not all, memory CD8⁺ cells generated in vivo are thought to express high levels of both CD44 and IL-2R (19, 20). Moreover, the IL-15-induced proliferation of CD44^highCD8⁺ cells in vivo is largely confined to the IL-2R^high subpopulation (21). Indeed, IL-2R^high cells constituted nearly all CD44^highCD8⁺ cells in wild-type mice and were preferentially reduced in IL-15R^-/- mice (Fig. 2C). Blood lymphocyte profile in IL-15R^-/- mice was also compared with that in wild-type mice. The percentages of total and CD44^highIL-2R^highCD8⁺ T cells (Fig. 3) as well as those of NK T and NK cells (our unpublished data) were reduced in the blood to similar extents as those apparent in the spleen and LNs of the mutant mice.

View larger version (34K): [in this window] [in a new window]   FIGURE 2. Deficiency of CD8+ cells in the secondary lymphoid organs of IL-15R-/- mice. A, Percentages of CD4+, CD8+, and CD4+ plus CD8+ cells among total spleen or LN cells of IL-15R-/- mice () and wild-type littermates (•) as determined by surface staining with specific mAbs and flow cytometry. Each symbol corresponds to a different animal, and mean values are indicated by horizontal lines. *, p < 0.01; **, p < 0.001 (single-classification ANOVA). B, Flow cytometric analysis of the percentages of CD44high and CD44lowCD8+ cells among total LN cells of IL-15R knockout (-/-) and wild-type (+/+) mice. Data shown are representative of 15 independent experiments. C, Percentages of CD44highIL-2Rhigh and CD44highIL-2Rlow cells among CD8+ LN cells. Similar results were obtained in five independent experiments.

View larger version (30K): [in this window] [in a new window]   FIGURE 3. Deficiency of CD8+ cells in the blood of IL-15R-/- mice. A, Percentages of CD4+, CD8+, and CD4+ plus CD8+ cells among PBMCs of IL-15R-/- mice () and wild-type littermates (•). Mean values are indicated by horizontal lines. *, p < 0.01; **, p < 0.001 (single-classification ANOVA). B, Percentages of CD44high and CD44lowCD8+ cells among PBMCs of IL-15R knockout (-/-) and wild-type (+/+) mice. Data are representative of four independent experiments. C, Percentages of CD44highIL-2Rhigh cells among CD8+ cells. Similar results were obtained in four independent experiments.

Given that the percentage of naive CD8⁺ T cells in the periphery of IL-15R^-/- mice was reduced by 40%, we next analyzed thymocyte populations to determine whether this deficiency resulted during T cell development in the thymus. IL-15R^-/- mice exhibited normal numbers of thymocytes with a normal composition of CD4 and CD8 double-negative (DN) (6), double-positive (DP) (6), and SP populations (our unpublished data). The composition of subpopulations of DN thymocytes defined by expression of CD44 and CD25 also appeared to be normal in the IL-15R^-/- mice (our unpublished data). The TCR^high phenotype marks thymocytes that have undergone positive selection (22). Given that 35% of CD8SP thymocytes normally exhibit no or only a low level of TCR expression, we examined the percentage of CD8SP TCR^high cells among SP TCR^high thymocytes and found it reduced by 20% in IL-15R^-/- mice compared with that in wild-type mice (Fig. 4A). CD69 expression marks thymocytes that are undergoing or have just undergone positive selection (23). To examine whether the decrease in the percentage of CD8SP TCR^high thymocytes in IL-15R^-/- mice is associated with the DP to SP transition, we examined CD69 expression in TCR^med-high DP and in TCR^high CD8SP thymocytes. The percentages of CD69⁺ cells in these two thymocyte populations of IL-15R^-/- mice were similar to the corresponding values for wild-type mice (Fig. 4B). These results suggest that a normal number of DP thymocytes undergoes positive selection and progresses to the CD8SP stage in IL-15R^-/- mice, and that the decrease in the percentage of CD8SP TCR^high thymocytes in the knockout animals occurs after the DP to SP transition.

View larger version (35K): [in this window] [in a new window]   FIGURE 4. Deficiency of CD8SP TCRhigh thymocytes in IL-15R-/- mice. A, Reduction in the percentage of CD8SP TCRhigh thymocyte in IL-15R-/- mice. Total thymocytes from IL-15R-/- mice () and wild-type littermates (•) were stained with mAbs specific for CD4, CD8, and TCR. The percentages of total CD8SP TCRhigh thymocytes among total SP TCRhigh thymocytes were indicated. Mean values are provided and indicated by horizontal lines. *, p < 0.05 (single-classification ANOVA). B, Similar percentages of CD69+ cells among DP or CD8SP TCRhigh thymocytes of IL-15R-/- mice and of wild-type mice. Total thymocytes of IL-15R knockout (-/-) and wild-type (+/+) mice were stained with mAbs specific for CD4, CD8, TCR, and CD69. DP and CD8SP thymocytes were gated for analysis of CD69 and TCR expression. Data are representative of five independent experiments.

Reduced abundance of Bcl-2 in CD8⁺ T cells of IL-15R^-/- mice

The marked apoptosis and reduction in the number of thymocytes apparent in Bcl-2^-/- mice demonstrate the importance of Bcl-2 for thymocyte survival (24). The level of Bcl-2 in normal thymocytes is tightly regulated (25, 26). The abundance of this anti-apoptotic protein in DN thymocytes is greater than that in DP TCR^low thymocytes at a stage before selection. Down-regulation of Bcl-2 in DP TCR^low thymocytes is thought to facilitate positive selection by promoting the death of cells that fail this process. Thymocytes that have successfully undergone positive selection up-regulate both Bcl-2 and TCR expression and differentiate into TCR^high DP and then TCR^high SP cells (22). To determine whether the deficiency of CD8SP TCR^high thymocytes in IL-15R^-/- mice is related to defective cell survival, we examined the abundance of Bcl-2 in SP thymocytes and the preceding DP cells. The amount of Bcl-2 was normal in TCR^low and TCR^high DP thymocytes as well as in CD4SP thymocytes of IL-15R^-/- mice, but it was reduced in CD8SP TCR^high thymocytes of these animals (Fig. 5). These results demonstrate that IL-15R deficiency results in a reduced concentration of Bcl-2 specifically in CD8SP TCR^high thymocytes that are destined soon to be exported to the periphery to join the naive CD8⁺ T cell pool. The abundance of Bcl-2 in peripheral naive CD8⁺ T cells of IL-15R^-/- mice was also reduced compared with that in the corresponding wild-type cells (Fig. 6A), indicating that the Bcl-2^low phenotype is acquired during the development of CD8⁺ cells in the thymus and is conserved in the periphery. Similar to the situation with thymocytes, no difference in Bcl-2 abundance in naive CD4⁺ cells was detected between IL-15R^-/- and wild-type mice (Fig. 6A).

View larger version (25K): [in this window] [in a new window]   FIGURE 5. Reduced expression of Bcl-2 in CD8SP TCRhigh thymocytes of IL-15R-/- mice. Thymocytes were examined for surface CD4, CD8, and TCR as well as intracellular Bcl-2 expression by staining with specific mAbs and analysis by flow cytometry. Filled and open peaks demarcated by solid lines represent Bcl-2 staining of wild-type and IL-15R-/- cells, respectively. Peaks demarcated by dashed lines represent background fluorescence generated by an isotype-matched control mAb in wild-type (thick lines) or IL-15R-/- (thin lines) cells. Only one background peak is shown when wild-type and IL-15R-/- cells exhibited identical background fluorescence. Data are representative of results obtained with three different pairs of mice.

View larger version (40K): [in this window] [in a new window]   FIGURE 6. Reduced expression of Bcl-2 in peripheral CD8+ T cells of IL-15R-/- mice. Total LN and spleen cells were stained with mAbs specific for CD8, CD44, and Bcl-2 or specific for CD4, CD45Rb, and Bcl-2, and were then analyzed by flow cytometry. A, Bcl-2 expression by naive (CD44low) CD8+ cells and by naive (CD45Rbhigh) CD4+ cells. Open and filled peaks demarcated by solid lines represent IL-15R-/- and wild-type cells, respectively. Peaks demarcated by dashed lines represent the background fluorescence generated by an isotype-matched control mAb; given that IL-15R-/- and wild-type cells yielded identical background fluorescence, only one peak is shown. B, Bcl-2 expression by naive (CD44low) vs memory (CD44high) CD8+ T cells of wild-type (+/+) and IL-15R-/- mice. Open and filled peaks demarcated by solid lines represent naive and memory populations, respectively. Peaks demarcated by dashed lines represent background fluorescence generated by an isotype-matched control mAb. Data in A and B are representative of five independent experiments.

IL-15R

IL-15R

Increased sensitivity of CD8⁺ cells from IL-15R^-/- mice to death induction

Bcl-2 protects T cells from apoptosis induced by various stimuli (28, 29). Given that the abundance of Bcl-2 in CD8⁺ thymocytes and peripheral T cells of IL-15R^-/- mice is reduced compared with that in the corresponding wild-type cells, we examined the sensitivity of these cells to the induction of apoptosis. Thymocytes and resting T cells undergo apoptosis when removed from the stromal environment in vivo and cultured in vitro. This so-called "spontaneous cell death" is likely due to a lack of survival signals provided either by soluble factors present in the stromal environment or by direct contact between T cells and stromal cells or the extracellular matrix. Thymocytes are also highly sensitive to glucocorticoid-induced cell death. We therefore compared the death susceptibilities of IL-15R^-/- and wild-type thymocytes by culturing the cells in medium alone or in medium containing various concentrations of dexamethasone (Fig. 7). In the absence or presence of dexamethasone, CD8SP thymocytes from IL-15R^-/- mice exhibited a greater incidence of cell death than did their wild-type counterparts, whereas death rates were similar for either CD4SP or DP thymocytes from the knockout and wild-type mice. Thus, CD8SP, but not CD4SP or DP, thymocytes from IL-15R^-/- mice were more susceptible to both spontaneous and dexamethasone-induced death than were the corresponding wild-type cells.

View larger version (16K): [in this window] [in a new window]   FIGURE 7. Increased sensitivity of CD8SP thymocytes of IL-15R-/- mice to induction of cell death. Total thymocytes isolated from wild-type (+/+) and IL-15R knockout (-/-) mice were cultured for the indicated times in RPMI 10 (medium) in the absence or presence of 1 or 10 nM dexamethasone (dex). The percentage of apoptotic cells among CD4SP, CD8SP, and DP populations was determined by staining with mAbs to CD4 and to CD8 in combination with annexin V and propidium iodide. Annexin V-positive cells were scored as dead cells. Data are means ± SD of triplicate determinations. Similar results were obtained in three independent experiments.

IL-15R

IL-15R

IL-15R

IL-15R

IL-15R

IL-15R

IL-15R

View larger version (24K): [in this window] [in a new window]   FIGURE 8. Increased sensitivity of CD8+ LN cells of IL-15R-/- mice to death induction. A, Spontaneous cell death. Total LN cells from wild-type (+/+) and IL-15R knockout (-/-) mice were cultured in RPMI 10 for the indicated times. The percentage of dead cells in CD44low and CD44highCD8+ populations was determined by staining with mAbs to CD44 and to CD8 in combination with propidium iodide and annexin V. Data are means ± SD of duplicate determinations, and similar results were observed in two independent experiments. B, TCR-mediated death. CD44lowCD8+ cells were purified by sorting and stimulated for the indicated times with an immobilized mAb to TCR in the presence either of an immobilized mAb to CD28 or of IL-2. Dead cells were detected by staining with propidium iodide and annexin V. Data are means ± SD of triplicate determinations, and similar results were observed in four independent experiments.

The reduced abundance of Bcl-2 in CD8⁺ cells of IL-15R^-/- mice suggested that IL-15 regulates Bcl-2 expression in CD8⁺ T cells. To investigate this possibility further, we monitored both Bcl-2 concentration and cell death in naive CD8⁺ cells stimulated with a mAb to TCR in the presence of various concentrations of exogenous IL-15 (Fig. 9). IL-15 both increased the expression of Bcl-2 and reduced the incidence of cell death among activated wild-type or IL-15R CD8⁺ cells in a concentration-dependent manner. The up-regulation of Bcl-2 expression to similar levels required higher concentrations of IL-15 for IL-15R^-/-cells than for wild-type cells, consistent with that IL-15R or IL-15R_c binds IL-15 with high affinity, whereas IL-15R _c binds IL-15 with intermediate affinity (2, 3). These results thus demonstrated a direct effect of IL-15 on Bcl-2 expression in activated CD8⁺ T cells as well as a correlation between Bcl-2 expression and the survival of activated CD8⁺ T cells.

View larger version (19K): [in this window] [in a new window]   FIGURE 9. Effects of exogenous IL-15 on Bcl-2 expression in and survival of CD8+ T cells. Sorted CD44lowCD8+ LN cells from wild-type (+/+) and IL-15R knockout (-/-) mice were stimulated with immobilized anti-TCR mAb in the presence of various concentrations of IL-15 for 48 h. Bcl-2 expression was then determined by intracellular staining with a mAb to this protein; the mean fluorescence intensity (MFI) of staining shown has been corrected for the mean fluorescence intensity obtained with an isotype-matched control mAb. Dead cells were detected by staining with propidium iodide and annexin V, and the data are means of duplicates. Similar results were observed in three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

IL-15R

Specific decrease of CD8⁺ T cells was detected in IL-15R^-/- mice during T cell development in the thymus. Normal numbers of thymocytes were positively selected in the IL-15R^-/- animals, as indicated by the normal percentages of CD69⁺ cells among DP TCR^med-high and CD8SP TCR^high populations. The Bcl-2 level in postselection DP TCR^high thymocytes was also up-regulated to similar extents in IL-15R^-/- and wild-type mice. However, the subsequently derived CD8SP TCR^high cells (but not CD4SP cells) of the knockout mice contained a reduced level of Bcl-2 compared with their wild-type counterparts. Given that a Bcl-2 transgene inhibited thymocyte death induced by various stimuli, including in vitro culturing and dexamethasone treatment in vivo (28), the increased susceptibility of IL-15R CD8SP thymocytes to death induction by culturing or dexamethasone treatment in vitro is likely due to the reduced abundance of Bcl-2 in these cells. The CD8SP thymocytes of IL-15R^-/-mice might thus die readily in response to stressful conditions that arise periodically, and such cumulative death events might contribute to the observed reduction in their number.

Our IL-15R^-/- mice also manifested a specific reduction in the percentage of peripheral CD8⁺ T cells, consistent with the characteristics of IL-15R^-/- and IL-15^-/- mice described previously (16, 17). In the present study, the percentages of CD44^lowCD8⁺ cells and CD44^highCD8⁺ cells in the spleen, LNs, and blood of IL-15R^-/- mice were reduced by 40 and 70%, respectively. Given that the extent of the deficiency of these cells in the blood was similar to that in the spleen and LNs, inefficient homing of CD8⁺ cells from the blood to the periphery is unlikely the major cause for the decrease of CD8⁺ cells in the secondary lymphoid organs of IL-15R^-/- mice. The 20% reduction in CD8SP TCR^high thymocytes of IL-15R^-/- mice would be expected to compromise the supply of cells for the peripheral naive CD8⁺ T cell pool. With regard to cell maintenance, no increase in the number of dead cells was detected in freshly isolated or short-term-cultured IL-15R naive CD8⁺ cells compared with their wild-type counterparts. Therefore, the reduced abundance of Bcl-2 in naive CD8⁺ cells of IL-15R^-/- mice did not affect cell survival in the absence of specific stimulation, such as TCR cross-linking, which induced a greater extent of cell death in naive CD8⁺ cells from IL-15R^-/- mice than in the corresponding wild-type cells.

IL-15R^-/- mice are profoundly deficient in memory CD8⁺T cells. The reduced percentage of naive CD8⁺ T cells inIL-15R^-/- mice and the increased death rate of such cells after TCR stimulation likely limit the source of memory cells but would not fully account for their pronounced deficiency in IL-15R^-/- animals. The presence of low numbers of CD44^highIL-2R^highCD8⁺ cells suggests that differentiation of memory CD8⁺ cells proceeds at least to some extent in IL-15R^-/- mice. Memory CD8⁺ cells express Bcl-2 at a markedly higher level than do naive CD8⁺ cells (27). However, the residual CD44^highCD8⁺ cells of IL-15R^-/- mice did not exhibit up-regulation of Bcl-2 expression when compared with CD44^lowCD8⁺ cells. The CD44^highIL-2R^highCD8⁺ cells of IL-15R^-/- mice might therefore represent cells locked in the memory cell differentiation pathway at a stage at which they express IL-2R at a high level but are not able to up-regulate Bcl-2 expression, because they cannot use IL-15 efficiently in the absence of IL-15R. These results indicate that IL-15-IL-15R is the cytokine system that controls the up-regulation of Bcl-2 during the differentiation of memory CD8⁺ cells. Furthermore, CD44^highCD8⁺ cells of IL-15R^-/- mice were defective in survival, as demonstrated by the high incidence of apoptosis among such cells freshly isolated from LNs. These results suggest that memory CD8⁺ cells are generated but poorly maintained in IL-15R^-/- mice and that maintenance of a high Bcl-2 concentration relative to that in naive CD8⁺ cells is necessary for the survival of memory CD8⁺ T cells.

Factors that regulate Bcl-2 expression in T cells at various stages of development have not been fully defined. Mice lacking the cytokine receptor _c are markedly deficient in thymocytes and peripheral T cells (32, 33, 34) and exhibit a reduced concentration of Bcl-2 in residual SP TCR^high mature thymocytes (35). The abundance of Bcl-2 is reduced to a greater extent in CD8SP thymocytes than in CD4SP thymocytes of these mice (35). A preferential decrease in the number of CD8⁺ cells compared with that of CD4⁺ cells was also apparent in the thymus and spleen of _c knockout mice (32, 36). Given that _c is a component of IL-2, IL-4, IL-7, IL-9, and IL-15 receptors, the phenotype of _c knockout mice likely reflects the dysfunction of more than one type of cytokine receptor. IL-7-IL-7R is a nonredundant cytokine system required for expansion and survival of the earliest cells committed to the T lineage (37, 38). CD4SP and CD8SP thymocytes in IL-7R^-/- or IL-7^-/- mice are equally affected in terms of cell number and level of Bcl-2 expression (39, 40). The numbers of CD4⁺ cells and CD8⁺ cells in the peripheral blood are also reduced by similar extents in IL-7R^-/- mice (40). Characterization of IL-7R^-/- mice expressing the OT-1-transgenic TCR indicated that IL-7R is essential for the generation of Ag-specific CD8⁺ memory cells in a Bcl-2–independent manner, as up-regulation of Bcl-2 expression occurs normally in IL-7R memory CD8⁺ cells (41). Moreover, the abundance of Bcl-2 was normal in IL-7R naive CD8⁺ cells. Therefore, dysfunction of the IL-7–IL-7R system does not likely account for the preferential decrease in the number and Bcl-2 content of CD8⁺ cells in _c^-/- mice. We have now shown that the CD8⁺ T cell-specific mutant phenotypes of IL-15R^-/- mice include a reduced abundance of Bcl-2, a reduced cell number, and increased death sensitivity. These characteristics appear in TCR^high CD8SP thymocytes and persist in peripheral naive and memory CD8⁺ T cells. Therefore, among the cytokine receptors that contain _c, IL-15R-transduced signals specifically affect the Bcl-2 expression and survival of CD8⁺ T cells at all developmental stages after DP to SP transition in the thymus.

	Acknowledgments

 
We thank the transgenic core facility at the Institute of Molecular Biology, Academia Sinica, for handling ES cells, J. Kung for helpful discussion, and Y.-M. Lin for cell sorting.

	Footnotes

 
¹ This work was supported by grants from the National Science Council and Academia Sinica in Taiwan. T.-S.W. was supported by fellowships from the National Science Council and National Health Research Institute in Taiwan.

² T.-S.W. and J.-M.L. contributed equally to this study.

³ Current address: Institute of Molecular Biology, National Chung-Cheng University, Ming-Hsiung, Chia-Yi 621, Taiwan.

⁴ Current address: Level Biotechnology Inc., Shi-Jr, Taipei 221, Taiwan.

⁵ Address correspondence and reprint requests to Dr. Nan-Shih Liao, Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan. E-mail address: mbfelix{at}ccvax.sinica.edu.tw

⁶ Abbreviations used in this paper: _c, common chain; SP, single positive; DN, double negative; DP, double positive; LN, lymph node; ES, embryonic stem.

Received for publication August 30, 2001. Accepted for publication November 14, 2001.

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