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IL-15R Is a Negative Regulator of TCR-Activated Proliferation in CD4⁺ T Cells¹

Jan-Mou Lee^*,, Chen-Yen Chung, Wei-Wei Chiang, Yae-Huei Liou, Chian-Feng Chen^*, and Nan-Shih Liao^2,*,

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

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although IL-15 is known to be a T cell growth factor, the function in T cells of IL-15R, its high affinity receptor, remains unclear. We found that murine IL-15R^–/– CD4⁺ T cells hyperproliferated in response to TCR stimulation, in vitro and in vivo, and displayed a lower TCR activation threshold than wild-type CD4⁺ T cells. TCR-induced activation of Zap70 and of the phospholipase C-1-NFATp, Ras-ERK-c-Fos, and Rac-JNK-c-Jun pathways was all augmented in IL-15R^–/– CD4⁺ T cells. This in turn led to earlier IL-2R induction and higher IL-2 production, which most likely contribute to the hyperproliferation of IL-15R^–/– CD4⁺ T cells. Exogenous IL-15 reduced levels of TCR-activated signals, transcription factors, IL-2, and IL-2R, and division in wild-type CD4⁺ T cells. These results reveal IL-15R to be a negative regulator for CD4⁺ T cell activation and demonstrate a novel layer of regulation of TCR signaling by a cytokine system.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
T cell proliferation is growth factor dependent; one such T cell growth factor is IL-2 (1, 2, 3). Resting T cells express the intermediate affinity IL-2R, but not the high affinity receptor component IL-2R or IL-2 itself. Upon stimulation of TCR, T cells express both IL-2R and IL-2 (4). IL-15 also uses IL-2R as its intermediate affinity receptor (5), which reflects the commonality of many of the functions of IL-15 and IL-2, including supporting lymphocyte growth (6). In contrast, each cytokine receptor has a private -chain, which appears to be responsible for the unique functions of each respective cytokine. Indeed, IL-15R and IL-15 knockout (ko)³ mice exhibit a phenotype distinct from IL-2R and IL-2 ko mice. IL-15R^–/– and IL-15^–/– mice are both deficient in CD8⁺ T cells, CD8⁺ intestinal intraepithelial lymphocytes, and NK cells (7, 8, 9), but do not develop the lymphoproliferative disorder that characterizes IL-2R^–/– and IL-2^–/– mice (10, 11). The unique phenotype of IL-15R^–/– and IL-15^–/– mice is indicative of the unique function of the IL-15 cytokine system and the critical role played by IL-15R in that system.

Although IL-15 is a known T cell growth factor, the role played by IL-15R in T cell proliferation is not yet fully understood. IL-15R, alone or together with the -chains, binds IL-15 with high affinity (12, 13). Moreover, it was recently shown that IL-15R expressed on the surface of APCs bind IL-15 and present it to neighboring T cells (14), i.e., by concentrating IL-15 on their surface in this way, APC make the cytokine available to intermediate affinity receptors expressed on neighboring lymphocytes. But while these findings provide important information about the function of IL-15R expressed on environmental cells in contact with T cells, they shed little light on function of IL-15R expressed on T cells.

It is known, however, that IL-15 induces Jak1 phosphorylation in a Jurkat T cell subline expressing receptor but not the -chain, and induces proliferation in a myeloid cell line that expresses receptor and cytoplasmic tail-truncated IL-15R (13). This suggests IL-15R is dispensable for IL-15-induced cell proliferation. In contrast to that interpretation, Raji B-lymphoblastoid cells, which express IL-15R, but not -chain, proliferate moderately in response to IL-15 (15). Moreover, IL-15 induces association of IL-15R with cytosolic signaling molecules, such as Syk kinase in primary B cells (15) and TNFR-associated factor 2 in a fibroblast line (16), indicating IL-15R to have a broader function, beyond high affinity binding of IL-15. It is against this background that we conducted the present study with the aim of clarifying the function of IL-15R expressed on T cells. To address that question, we compared IL-15R^–/– and wild type (wt) T cells on TCR-activated proliferation and signal transduction, and on TCR activation threshold. We also examined the effects of IL-15 on the activation of wt and ko CD4⁺ T cells. Taken together, our findings indicate that IL-15R is a negative regulator of CD4⁺ T cell activation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

IL-15R^–/– mice were generated, as described (9), and backcrossed to C57BL/6 (B6) background for at least 10 generations. IL-15R^+/– and IL-15R^–/– mice expressing transgenic (tg) AND TCR that recognizes a pigeon cytochrome c (PCC) peptide presented by E^k were generated by crossing IL-15R^–/– mice with AND TCR tg mice. Mice were housed under specific pathogen-free conditions at the animal facility of Institute of Molecular Biology, Academia Sinica, and used between 6 and 10 wk old, unless indicated otherwise.

Flow cytometry

For surface molecules, cells were stained with specific Abs conjugated with FITC, PE, or biotin, followed by allophycocyanin-conjugated streptavidin (SAV) in staining buffer (Mg²⁺/Ca²⁺-free PBS (Invitrogen Life Technologies, Carlsbad, CA), 1% FCS, 0.1% Na₃N). For intracellular IL-2, cells were stained for surface molecules, fixed by 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) in PBS, washed with permeabilization buffer (staining buffer, 0.1% saponin (Sigma-Aldrich)), and then stained with PE-conjugated anti-IL-2 (JES6-5H4) or isotype control (keyhole limpet hemocyanin (KLH)/G2b-1-2) mAb in permeabilization buffer. Data were acquired on FACSCalibur (BD Biosciences, San Jose, CA) and analyzed by FlowJo (Tree Star, Ashland, OR). Used were biotin-conjugated Abs specific for CD8 (53-6.7), CD4 (GK1.5), CD19 (nb19-1), TCR (GL3), DX-5 (DX5), CD11b (M1/70), CD25 (PC61.5), CD44 (IM7), and IL-15R (R&D Systems, Minneapolis, MN); PE-conjugated mAbs specific for CD4 (GK1.5), CD8 (53-6.7), and TCRV8 (F23.1; BD Pharmingen, San Diego, CA); and allophycocyanin-conjugated anti-CD4 (GK1.5). All Abs were purchased from eBioscience, unless indicated otherwise.

Isolation and activation of CD4⁺ T cells in vitro

CD25^–CD4⁺ T cells were isolated by depleting CD8⁺, CD19⁺, TCR⁺, DX5⁺, CD11b⁺, and CD25⁺ cells from lymph node (LN) cells using biotinylated Abs, SAV beads (Miltenyi Biotec, Auburn, CA), and MACS (Miltenyi Biotec). This yielded CD4⁺ T cells that were >93% pure. CD44^lowCD25^–CD4⁺ T cells were further isolated from CD25^–CD4⁺ T cells by depleting CD44^high cells using anti-CD44 mAb and MACS.

CD4⁺ T cells were activated for indicated times in wells precoated with the indicated amounts of anti-TCR (H57.597; prepared in our laboratory) and anti-CD28 (37.51; prepared in our laboratory) mAb in RPMI 1640 (Invitrogen Life Technologies) supplemented with 2 mM L-glutamine, 20 mM HEPES-NaOH (pH 7.2), penicillin-streptomycin (2000 U/L), 50 mM 2-ME, and 10% FCS (RPMI 10). Addition of IL-2R-blocking mAb (3C7; BD Pharmingen) or mouse rIL-15 (eBioscience, San Diego, CA) was as indicated. AND TCR⁺ CD25^–CD4⁺ T cells (10⁵/well) were activated with equal numbers of -irradiated (750 rad), T-depleted B10.A splenocytes in the presence of antigenic peptides. T-depleted splenocytes were prepared by complement-mediated lysis of T cells in the presence of T cell-specific mAbs. PCC₈₈₁₀₄ (KAERADLIAYLKQATAK) and K99Q mutant (KAERADLIAYLQQATAK) peptides were purchased from SynPep (Dublin, CA); moth cytochrome c (MCC₈₈₁₀₃-T102S) mutant peptide (ANERADLIAYLKQASK) was from Biosynth International (Lewisville, TX).

Proliferation assays

For [³H]TdR incorporation assays, cells were pulsed for 12 h with [³H]TdR (1 µCi/well) (Amersham Biosciences, Cardiff, U.K.) and then harvested. [³H]TdR incorporation was determined using a LS 6500 scintillation counter (Beckman Coulter, Fullerton, CA). For cell division assays, T cells were labeled with 5 mM CFSE (Molecular Probes, Eugene, OR) and then activated, as described in the figure legend or in the text. Activated cells were stained with 2 µg/ml propidium iodide, after which CFSE levels in live (propidium iodide^–) cells were determined using FACSCalibur.

Cell extracts and Western blotting

Cell lysate was prepared by lysing cells in lysis buffer (50 mM Tris-HCl, pH 7.4, 0.5% Nonidet P-40, 0.15 M NaCl, 2 mM EDTA, 10 mM NaF, 100 µg/ml PMSF, and 1 µg/ml aprotinin and leupeptin). Nuclear and cytosolic extracts were prepared, as described (17). Boiled lysates were separated by 10% SDS-PAGE, after which the resolved proteins were transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA) for Ab detection. The immunoblots were developed using ECL (Amersham Biosciences), following the manufacturer’s instructions. The primary Abs used were specific for p27^kip (57; BD Transduction Laboratories, Lexington, KY), cyclin D3 (1; BD Transduction Laboratories), cyclin A (E23; Zymed Laboratories, San Francisco, CA), cyclin E (M-20; Santa Cruz Biotechnology, Santa Cruz, CA), -actin (AC-15; Sigma-Aldrich), NFATc (7A6; Santa Cruz Biotechnology), NFATp (4G6-G5; Santa Cruz Biotechnology), c-Fos (4; Santa Cruz Biotechnology), p-c-Jun (KM-1; Santa Cruz Biotechnology), p65 (N-20; Santa Cruz Biotechnology), Zap70 (29; BD Transduction Laboratories), p-Zap70 (Cell Signaling Technology, Beverly, MA), phospholipase C (PLC)-1 (E-12; Santa Cruz Biotechnology), Vav (B-2; Santa Cruz Biotechnology), phosphotyrosine (4G10; Upstate Biotechnology, Lake Placid, NY), Erk1,2/p-Erk1,2 (Cell Signaling Technology), and JNK/p-JNK (Cell Signaling Technology). The secondary Abs used were HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and goat anti-rabbit IgG (Caltag, Burlingame, CA).

Quantitative real-time PCR

RNA was extracted using TRIzol (Invitrogen Life Technologies), after which first strand cDNAs were synthesized using Superscript II RNase H^– reverse transcriptase (Invitrogen Life Technologies). Quantitative real-time PCR was conducted in a LightCycler (Roche, Basel, Switzerland) using the following primers and TagMan probes (TIB Molbiol, Berlin, DE). Murine IL-2: sense, 5'-CCTGAGCAGGATGGAGAATTACA; antisense, 5'-TCCAGAACATGCCGCAGAG; probe, 5'-6FAM-TTACTTGCCCAAGCAGGCCACAGAA-TAMRA. Murine IL-2Rµ: sense, 5'-CCCCATAACCACCACAGACTT; antisense, 5'-GAAGAGGCAGCTGGCCACT; probe, 5'-6FAMACCCACAGAAACAACTGCAATGACGGA-TAMRA. -actin: sense, 5'-CACACTGTGCCCATCTACGA; antisense, 5'-CTTGCGCTCAGGAGGAGC; probe, 5'-6FAM-CATCCTGCGTCTGGACCTGGC-TAMRA.

Recall response of in vivo KLH-primed CD4⁺ T cells

Mice were i.p. injected with PBS or KLH (50 µg in PBS; Sigma-Aldrich) emulsified with 50 µl of CFA (Sigma-Aldrich). Thirteen days later, CD25^–CD4⁺ T cells were isolated from the spleens by MACS, and restimulated at 10⁴/well for 4 days with equal numbers of irradiated (750 rad) T-depleted B6 splenocytes in the presence of KLH. Proliferation was determined by [³H]TdR incorporation.

Stimulation of adoptively transferred CD4⁺ T cells by staphylococcal enterotoxin B (SEB) in vivo

CFSE-labeled wt or IL-15R^–/– CD44^lowCD25^–CD4⁺ T cells were injected into wt or IL-15R^–/– mice (5 x 10⁶/recipient) via the tail vein. Twelve to 14 h after cell transfering, each mouse received PBS or SEB (80 µg; Sigma-Aldrich) i.p. Three days after the injection, the CFSE levels of donor cells (CD4⁺TCRV8⁺ cells) from the spleen were examined. Twelve hours after SEB injection, expression of IL-2R and intracellular IL-2 by donor cells (CFSE⁺V8⁺ cells) in the spleen was analyzed by Ab staining and flow cytometry.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hyperproliferation of IL-15R^–/– CD4⁺ T cells in response to TCR stimulation in vitro

Freshly isolated CD4⁺ and CD8⁺ T cells both expressed IL-15R at levels that increased with cell activation (Fig. 1A). Examination of the pan-T cell response elicited by stimulating total LN cells with anti-CD3 mAb revealed that more viable cells were present in cultures of IL-15R^–/– cells than wt cells (Fig. 1B). Analysis of coreceptor expression revealed significantly higher numbers of CD4⁺ cells in the mutant cell cultures, although the numbers of CD8⁺ T cells were similar in both cultures (Fig. 1B).

View larger version (30K): [in this window] [in a new window]   FIGURE 1. IL-15R–/– CD4+ T cells were hyperactive. A, Expression of IL-15R. The wt LN cells were activated with soluble anti-CD3 mAb (1 µg/106/ml) for the indicated times and stained for expression of CD4, CD8, and IL-15R. Cells stained with allophycocyanin-SAV alone served as negative controls (Neg.). B, Viable cell counts. The wt and IL-15R–/– LN cells were stimulated with soluble anti-CD3 mAb (1 µg/106/ml) for the indicated times. Live cell counts were determined by trypan blue exclusion. Percentages of CD4+ and CD8+ cells were determined by immunostaining and flow cytometry. C, [3H]TdR incorporation. The wt or IL-15R–/– CD25–CD4+ and CD8+ T cells (104/well) were activated for 2 days using the indicated amounts of immobilized anti-TCR and anti-CD28 (1 µg/well) mAb. All data presented were from one representative experiment of three.

The enhanced incorporation of [³H]TdR by activated IL-15R^–/– CD4⁺ T cells might reflect enhanced proliferation and/or better survival. IL-15R^–/– CD4⁺ T cells divided more vigorously than wt cells in response to low, optimal, and high doses of anti-TCR mAb stimulation (Fig. 2A). The percentages of surviving cells were similar in wt and ko CD4⁺ T cells stimulated with an optimal dose of anti-TCR mAb, but were higher in ko than wt cells stimulated with low or high doses of anti-TCR mAb (Fig. 2A). This means that increased proliferation contributed to the hyperresponse of IL-15R^–/– CD4⁺ T cells to optimal TCR stimulation, while increases in both proliferation and survival contributed to the hyperresponse of IL-15R^–/– CD4⁺ T cells to high or low TCR stimulation.

View larger version (40K): [in this window] [in a new window]   FIGURE 2. Elevated proliferation of IL-15R–/– CD4+ T cells in response to TCR stimulation in vitro. A, Cell division. CFSE-labeled wt and IL-15R–/– CD25–CD4+ T cells (104/well) were activated for 3 days using the indicated amounts of immobilized anti-TCR and anti-CD28 (1 µg/well) mAb. The percentage of surviving cells is indicated in the upper left corner of each histogram. B, Expression of p27kip and cyclins. The wt and IL-15R–/– CD25–CD4+ T cells (106/well on 6-well plate) were activated by optimal dose of plate-bound anti-TCR (1.65 µg/well) and anti-CD28 (30 µg/well) mAb for the indicated times. Cell lysates were analyzed by Western blotting. The number below each band indicates its OD. The OD of -actin in all samples varied from 125 to 130 AU/mm2. All data presented were from one representative experiment of three.

Reduction of the TCR activation threshold in IL-15R^–/– CD4⁺ T cells

The hyperproliferation of IL-15R^–/– CD4⁺ T cells induced by low dose anti-TCR mAb stimulation (Figs. 1C and 2A) suggested that the activation threshold is lowered in these cells. To test this possibility further, we stimulated IL-15R^+/– and IL-15R^–/– CD4⁺ AND TCR⁺ cells with an agonist, PCC peptide (PCC_88–104) (Fig. 3A); a weak agonist, MCC_88–103-T102S (Fig. 3B); or a weak antagonist, altered PCC peptide (PCC_88–104-K99Q) (Fig. 3C). All three of these peptides stimulated more vigorous division in ko than IL-15R^+/– cells, and the difference between the responses of the two cell types increased with decreases in the stimulating capacity of the antigenic peptides. In the presence of exogenous CD28 stimulation, the weak antagonist barely stimulated the IL-15R^+/– CD4⁺ T cells, but induced significant division in IL-15R^–/– CD4⁺ T cells, which is indicative of the reduction in the TCR activation threshold.

View larger version (29K): [in this window] [in a new window]   FIGURE 3. Reduction of TCR activation threshold in IL-15R–/– CD4+ T cells. IL-15R+/– and IL-15R–/– AND TCR+ CD25–CD4+ cells were labeled with CFSE and activated for 3 days with B10.A APC and indicated concentrations of PCC88104 (A), MCC8810 (T102S) (B), or PCC (K99Q) (C) peptides. Anti-CD28 mAb (5 µg/ml) was added into all cultures in the experiments of group C, but not of groups A and B. All data presented were from one representative experiment of three.

Augmentation of TCR-activated signals in IL-15R^–/– CD4⁺ T cells

To further understand the hyperactivity of IL-15R^–/– CD4⁺ T cells in response to TCR stimulation, we compared the TCR-activated signals in wt and ko CD4⁺ T cells. TCR stimulation results in the activation of three intracellular signaling pathways: the PLC-1-initiated inositol 1,4,5-triphosphate-Ca²⁺-calcineurin pathway, which leads to nuclear translocation of NFAT; the Ras-ERK pathway, which leads to expression of c-Fos; and the Rac/vav-JNK pathway, which leads to phosphorylation of c-Jun (18, 19). The wt and IL-15R^–/– CD4⁺ T cells were stimulated for 4 min via TCR and CD4 in the absence or presence of IL-15, and then measured levels of a key proximal molecule, Zap70, as well as representative molecules from the three signaling pathways mentioned above. We found that levels of p-Zap70, pPLC-1, pERK1/2, pVav, and pJNK were all higher in stimulated ko than wt cells (Fig. 4A), and were all reduced in wt, but not in ko, cells by exogenous IL-15 (Fig. 4A). We also examined the level of TCR-induced transcription factors in the nuclei of wt and ko CD4⁺ T cells (Fig. 4B). After 1.5 h of activation, levels of NFATc and p65 in the nuclei of wt and ko cells were similar, but there was 1.5-fold more NFATp and c-Fos and 1.9-fold more p-c-Jun in the nuclei of ko cells. Treatment with exogenous IL-15 caused a 50% reduction of NFATp and c-Fos and an 80% reduction of p-c-Jun in wt cells, but did not affect levels of these transcription factors in ko cells. Taken together, these results indicate that TCR-mediated signaling is enhanced in IL-15R^–/– CD4⁺ T cells, and that IL-15 negatively regulates TCR-activated signals.

View larger version (55K): [in this window] [in a new window]   FIGURE 4. Augmentation of TCR-activated signals in IL-15R–/– CD4+ T cells. A, The wt and IL-15R–/– CD44lowCD25– CD4+ T cells were stimulated by cross-linking CD3, CD28, and CD4 with specific mAbs and SAV for 4 min in the absence or presence of exogenous IL-15 (200 ng/ml). Cell lysates were analyzed by Western blotting. The number below each band indicates its OD normalized to the OD of the same molecule in wt cells stimulated by cross-linking CD3, CD28, and CD4 with specific mAb and SAV alone. B, The wt and IL-15R–/– CD44lowCD25–CD4+ T cells (106/well) were activated using immobilized anti-TCR (1.65 µg/well) and anti-CD28 (30 µg/well) mAbs in the absence or presence of 200 ng/ml IL-15 for 1.5 h. Nuclear and cytosolic extracts were prepared and analyzed by Western blotting. The number below each band indicates its OD normalized to the OD of the same molecule in wt cells without IL-15. All data presented were from one representative experiment of three.

Elevated expression of IL-2 and IL-2R by IL-15R^–/– CD4⁺ T cells

Previous studies have shown that during T cell activation, expression of IL-2 and IL-2R is induced at the level of transcription (20, 21, 22). As NF-ATp, c-Fos, and c-Jun are critical transcription factors for the expression of IL-2 and IL-2R in T cells, the increased level of these transcription factors in IL-15R^–/– CD4⁺ T cells suggests an increase in the expression of IL-2 and IL-2R. Consistently, our quantitative PCR analysis showed that after 2 h of activation, IL-15R^–/– CD4⁺ T cells expressed more IL-2 and IL-2R mRNA than did wt cells, while little IL-2 and IL-2R mRNA was detected in wt and ko cells before stimulation or after 2 h of culturing without stimulation (Fig. 5A).

View larger version (29K): [in this window] [in a new window]   FIGURE 5. Elevated expression of IL-2 and IL-2R by IL-15R–/– CD4+ T cells. A, Expression of IL-2R and IL-2 mRNA by wt and IL-15R–/– CD44lowCD25–CD4+ T cells (106/sample) were analyzed by quantitative real-time PCR before stimulation (0 h) and after culturing for 2 h with or without stimulation by immobilized anti-TCR (1.65 µg/well) and anti-CD28 (30 µg/well) mAb. IL-2R and IL-2 mRNA levels were first normalized to -actin mRNA level of the same cells. The values obtained for all samples were normalized to that of wt cells before stimulation. The values shown were the average of four independent experiments. Paired Student’s t test was used to compare the wt and ko groups (*, p < 0.05; **, p < 0.001). B, The wt and ko cells (104/well) were activated using immobilized anti-TCR (0.055 µg/well) and anti-CD28 (1 µg/well) or cultured without activation for indicated times and analyzed for expression of surface IL-2R. The mean fluorescence indexes (MFI) of IL-2R on activated wt and ko cells (wt/ko) are indicated. C, The wt and ko cells were activated, as described in B, or cultured without activation for the indicated times. The expression of IL-2 was determined by intracellular staining, and the data are displayed as single-parameter dot plot. Left panel, Cells were activated for 10 h. Right panel, Cells were activated for 3 days and then restimulated with PMA and ionophore for 5 h. The percentages of IL-2-positive cells are indicated. The isotype control Ab stained <1% of cells. D, The wt and ko cells were labeled with CFSE and activated, as described in B, for 2 days in the presence of 3.7 µg/ml anti-IL-2R or isotype control mAb. All data presented in B, C, and D were from one representative experiment of three.

Intracellular IL-2 was not detected in either wt or ko cells before stimulation (Fig. 5C). After 10 h of activation, the percentage of IL-2-producing cells was higher among ko than wt cells, while no IL-2-producing cell was detected in either wt or ko cells cultured for 10 h without stimulation (Fig. 5C). By the end of 3 days of stimulation, the percentage of cells that had produced IL-2 as revealed by PMA and ionophore restimulation was 1.4-fold higher among ko cells than in wt cells (Fig. 5C). Up to 90% of wt and ko cells died after culturing for 3 days without stimulation (data not shown). The above results demonstrate that TCR stimulation triggered faster IL-2R induction and more IL-2 expression in IL-15R^–/– CD4⁺ T cells than in their wt counterparts. The observed hyperactivity of IL-15R^–/– CD4⁺ T cells did not occur spontaneously, as IL-2R and IL-2 were not induced in freshly purified cells or in unstimulated cultured cells.

We then used an IL-2R blocking mAb to evaluate the relationship between the elevated expression of IL-2/IL-2R and hyperproliferation, and found that blocking IL-2R reduced the level of ko cell division to that seen in wt cells (Fig. 5D), which is consistent with the idea that the elevated expression of IL-2 and IL-2R contributes to the hyperproliferation of IL-15R^–/– CD4⁺ T cells.

IL-15R deficiency directly resulted in hyperproliferation of CD4⁺ T cells

To determine whether the hyperproliferation is a direct result of the IL-15R deficiency, wt and ko CD4⁺ T cells were activated in the presence of exogenous IL-15, which dose dependently inhibited induction of IL-2R and IL-2 and cell division in wt cells, but had no effect in ko cells (Fig. 6). Neither induction of IL-2R or IL-2 nor cell division occurred in wt and ko cells in the absence of TCR and CD28 stimulation (Fig. 6). This result is consistent with the inhibitory effect of IL-15 on TCR-activated signals in wt CD4⁺ T cells (Fig. 4), and implies that hyperproliferation of IL-15R^–/– CD4⁺ T cells is a direct result of the absence of IL-15R.

View larger version (32K): [in this window] [in a new window]   FIGURE 6. IL-15 inhibited the activation of CD4+ T cells. The wt and IL-15R–/– CD44lowCD25–CD4+ T cells were activated, as described in Fig. 5B, or cultured without activation in the presence of indicated amounts of IL-15. Surface IL-2R, intracellular IL-2, and cell division were determined after 6, 12, and 60 h of activation, respectively. The thin-dashed lines in the top panel represent cells stained with allophycocyanin-SAV alone. The IL-2R MFI and the percentage of IL-2-positive cells are indicated. For intracellular IL-2 staining, the isotype control Ab stained <1% of cells. All data presented were from one representative experiment of three.

Hyperproliferation of IL-15R^–/– CD4⁺ T cells in response to TCR stimulation in vivo

We next evaluated the in vivo responses of IL-15R^–/– CD4⁺ T cells to TCR stimulation in two types of experiment. First, the recall responses of CD4⁺ T cells isolated from in vivo KLH-primed wt and IL-15R^–/– mice were determined. We found that in response to in vitro KLH restimulation ko CD4⁺ T cells showed higher [³H]TdR incorporation (Fig. 7A) and greater cell division (data not shown) than did wt CD4⁺ cells. CD4⁺ cells isolated from PBS-primed wt or ko mice proliferated minimally to KLH restimulation (Fig. 7A). Second, the response of wt and IL-15R^–/– CD4⁺ T cells to SEB was determined in an adoptive transfer system that enabled us to distinguish the function of IL-15R in donor CD4⁺ T cells from that in the recipient environmental cells. CD44^lowCD25^–CD4⁺ cells isolated from wt and ko mice were labeled with CFSE and then transferred into wt and ko mice, after which the responses of TCRV8⁺ donor cells to SEB were examined. The ko donors divided more vigorously than the wt donors in either wt (kowt > wtwt) or ko (koko > wtko) recipients 3 days after SEB injection, while donor cells did not divide in recipients that received PBS only (Fig. 7B). The ko donor cells also showed higher IL-2R expression and included more IL-2-producing cells than did the wt donor cells 12 h after SEB injection, whereas no difference in the levels of IL-2R - and -chains was found between wt and ko donor cells (Fig. 7C). These results demonstrated that IL-15R^–/– CD4⁺ T cells are hyperresponsive to TCR stimulation in vivo as well as in vitro.

View larger version (23K): [in this window] [in a new window]   FIGURE 7. IL-15R–/– CD4+ T cells hyperproliferated in response to TCR stimulation in vivo. A, Recall response to KLH. Splenic CD25–CD4+ T cells isolated from wt or IL-15R–/– mice primed with KLH or PBS for 13 days before were restimulated for 4 days with B6 APC and indicated amounts of KLH. Proliferation was determined by [3H]TdR incorporation. B, Division of adoptively transferred wt or IL-15R–/– CD44lowCD25–CD4+ T cells in response to SEB stimulation in wt or in ko recipients. CFSE levels were analyzed 3 days after SEB or PBS injection. C, Expression of IL-2R and intracellular IL-2 by donor T cells in the spleen was analyzed 12 h after SEB injection. The dashed lines in the upper three panels represent cells stained with allophycocyanin-SAV alone. The MFI of IL-2R staining and the percentages of IL-2-positive cells are indicated. For intracellular IL-2 staining, the isotype control Ab stained <1% of cells. All data presented were from one representative experiment of three.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

IL-15R exerts its negative regulatory effect, at least in part, by inhibiting TCR-mediated signal transduction, thereby reducing IL-2R and IL-2 expression during the early phase of CD4⁺ T cell activation. Activation of Zap-70 is a key proximal event in such TCR signaling. Zap-70 kinases provide the physical link between the activated TCR complex and downstream signaling events by binding to p-ITAM motifs on the TCR complex, where they are activated, and then phosphorylate PLC-1 and the scaffold proteins, linker for activation of T cells (LAT) and SH2 domain-containing leukocyte protein of 76 kDa (SLP-76) (25, 26, 27). Phosphorylated LAT serve as docking sites for various adaptor molecules and enzymes downstream of TCR stimulation, includingPLC-1 (28). By associating with p-LAT, Grb2-son of sevenless is brought to the site of Ras, activating a Ras-mediated MAPK pathway. Gads specifically interacts with SLP-76, bringing it to the LAT complex. Phosphorylated SLP-76 in turn recruits vav, which activates the Rac-MAPK pathway. Bearing these various relationships in mind, it was not surprising that the higher level of p-Zap-70 seen in activated IL-15R^–/– CD4⁺ T cells was associated with increased activity in three downstream signaling pathways: pPLC-1-Ca²⁺-NF-ATp, pERK1/2-c-fos (Ras-MAPK pathway), and vav-pJNK-p-c-Jun (Rac-MAPK pathway). Moreover, all three affected transcription factors are critical for the induction of IL-2R and IL-2, which is consistent with our finding that IL-2 and IL-2R mRNA and proteins were more abundant in ko cells during the early phase of activation, contributing at least in part to the hyperproliferation of IL-15R^–/– CD4⁺ T cells. Its effect on the level of p-Zap-70 indicates that IL-15R acts very early during TCR-mediated signal transduction, perhaps mediating dephosphorylation of Zap-70 or event(s) upstream of Zap-70 activation, e.g., recruitment of Zap-70 or phosphorylation of the TCR signaling modules. Because the intracytoplasmic domain of IL-15R does not possess enzymatic activity, IL-15R most likely exerts its negative regulatory function in two general ways: by associating with certain negative regulatory molecule(s) or by affecting the distribution of signaling molecules, e.g., by affecting raft movement. We are currently examining these possibilities.

An earlier study by Kumaki et al. (29) reported that IL-15 up-regulated IL-2R in human peripheral blood T cells that were activated by PHA for 2 days. The inhibitory effect of IL-15R on IL-2R expression reported in this study occurred at early phase during T cell activation (within 18 h of activation; Fig. 5B) and is restricted to CD4⁺ T cells. Because IL-15R^–/– CD8⁺ T cells proliferated much worse than wt CD8⁺ T cells (Fig. 1C), IL-15 most likely helps the proliferation of CD8⁺ T cells, which may also involve the regulation of IL-2R expression. If that is the case for human T cells, the level of IL-2R on activated human peripheral blood T cells is contributed by the level of IL-2R on CD4⁺ and CD8⁺ T cells, which most likely differ in response to IL-15 treatment. Moreover, unlike the TCR-specific stimulation by anti-TCR mAb or antigenic peptide/MHC, PHA most likely stimulates other surface glycoprotein(s) than TCR, which may or may not affect the IL-15/IL-15R-regulated expression of IL-2R. Therefore, the difference in cell population, in stimulatory agents, and in stimulation time might have caused the appeared discrepancy between Kumaki’s and our observations on the role of IL-15/IL-15R in expression of IL-2R.

According to this study, a model illustrating the way in which IL-15R regulates CD4⁺ T cell proliferation is depicted in Fig. 8. First, IL-15R expressed on CD4⁺ T cells negatively regulates proliferation by reducing TCR-induced expression of IL-2 and IL-2R. Second, the in trans presented IL-15 by IL-15R on the environmental cells (APC) delivers a positive proliferation signal to CD4⁺ T cells via receptors, as suggested by the finding that wtwt cells proliferated more vigorously than wtko cells in the SEB experiment. Third, IL-15R^–/– CD4⁺ T cells activated in an IL-15R^–/– environment mount a strong proliferation response, as koko donor cells in the SEB experiment showed the greatest proliferation among the four donor/recipient combinations (koko > kowt > wtwt > wtko). Therefore, based on the following observations, we suggest that the strong proliferation reflects, at least in part, greater interaction between IL-2 and IL-2R on ko than wt cells (1). The koko cells presumably produced more IL-2 than donor cells in other donor/recipient combinations, as koko cells contained the highest percentage of IL-2-producing cells after SEB stimulation (2). The ko donor cells proliferated more vigorously in the absence of the in trans presented IL-15 (koko > kowt). Given that activated koko and kowt cells expressed similar levels of surface IL-2/15R - and -chains (Fig. 7C), we suggest that the - and -chains on koko donor cells were used solely to form IL-2R, but were shared by IL-2R and IL-15R on kowt cells. Consequently, the number of IL-2R complexes on koko cells was higher than on kowt cells, which led to stronger proliferation of the former. The number of IL-2R on CD4⁺ T cells in wt mice might be even lower than on the kowt cells, because the - and -chains on CD4⁺ T cells are most likely shared among IL-2R, IL-15R, and IL-15R (Fig. 8, left panel). This model focuses on the inhibition of IL-2/IL-2R expression by IL-15R as a mechanism by which it negatively regulates CD4⁺ T cells. The presence of other pro-proliferation receptor/ligand pairs or signals that are negatively regulated by IL-15R is not excluded. Our results and the proposed model are consistent with the previous report that IL-2 production by activated IL-15 tg T cells is lower than in non-tg T cells, as CD4⁺ T cells are most likely the predominant IL-2 producer (30). The inhibitory effect of IL-15 on TCR-activated signals, IL-2/IL-2R expression, and division of primary CD4⁺ T cells is also consistent with the ability of IL-15 to maintain a quiescent state of a CD4⁺ T cell clone (31, 32).

View larger version (15K): [in this window] [in a new window]   FIGURE 8. Schematic diagrams illustrating the role of IL-15R in CD4+ T cell activation. Stimulation of TCR and CD28 on T cells by peptide/MHC and B7 molecules on APC triggers signal transduction from the TCR complex that leads to the expression of IL-2 and IL-2R [1]. IL-2 is secreted and acts as an autocrine factor, binding to IL-2R on the activated T cell and thereby driving T cell proliferation [2]. In wt mice (left panel), IL-15R expressed by T cells bind IL-15, and attenuate TCR signaling and downstream IL-2 and IL-2R expression. The in trans presented IL-15 by IL-15R on APCs also delivers a proliferation signal via the receptors on T cells. In ko mice (right panel), the IL-15R deficiency eliminates the negative regulation on TCR-activated IL-2 and IL-2R expression, and the competition for IL-2/15R results in T cell hyperproliferation. IL-15R (), IL-2R (), IL-15 (circles), and IL-2 (triangles).

In summary, we have shown for the first time that IL-15R negatively regulates TCR-activated signals in naive CD4⁺ T cells, which most likely contribute to the hyperproliferation of IL-15R^–/– CD4⁺ T cells. Although further study will be required before a complete picture of the ways in which IL-15R affects TCR signaling emerges, the present findings raise concerns about the manipulation of the IL-15 cytokine system for purposes of suppressing immune responses.

	Acknowledgments

 
We acknowledge the advice of Dr. Fang Liao.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Health Research Institute, Academia Sinica, and National Science Council of Taiwan.

² Address correspondence and reprint requests to Dr. Nan-Shih Liao, Institute of Molecular Biology, Academia Sinica, No. 128, Academia Road, Section 2, Taipei 11529, Taiwan. E-mail address: mbfelix{at}ccvax.sinica.edu.tw

³ Abbreviations used in this paper: ko, knockout; KLH, keyhole limpet hemocyanin; LAT, linker for activation of T cells; LN, lymph node; MCC, moth cytochrome c; MFI, mean fluorescence index; PCC, pigeon cytochrome c; PLC, phospholipase C; SAV, streptavidin; SEB, staphylococcal enterotoxin B; SLP, SH2 domain-containing leukocyte protein; tg, transgenic; wt, wild type.

Received for publication March 29, 2004. Accepted for publication June 25, 2004.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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