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Human T Cells Constitutively Express IL-15 That Promotes Ex Vivo T Cell Homeostatic Proliferation through Autocrine/Juxtacrine Loops¹

Department of Rheumatology, Hospital Universitario La Paz, Madrid, Spain

	Abstract

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Homeostatic proliferation of T cells in vivo is responsible for the maintainance of the T cell pool, and IL-15 is a pivotal cytokine implicated in this process. Known cell sources providing physiologically active IL-15 are monocytes/macrophages, dendritic cells, and stromal cells. T lymphocyte expression of functionally active IL-15 and its possible role in T cell biology have not been investigated. In this study, we demonstrate that human T cells constitutively express IL-15 that acts through autocrine/juxtacrine loops to promote ex vivo homeostatic T cell proliferation.

	Introduction

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The T lymphocyte pool is maintained constant in adult animals: as a population, mature T cells have an almost indefinite life span (1). Long-term persistence of T cells is the result of the balance between cell longevity and cell proliferation (1). Kinetic studies in mice and healthy humans have shown that T lymphocytes proliferate in vivo, and memory T (T_M)³ cells divide at higher rates than naive T (T_N) cells (2, 3). Although contact with Ag may help preserve T_M cell numbers (4), it has been shown that Ag is not essential for this function (5, 6, 7), and T cell memory is sustained by cytokines that promote cell survival and slow homeostatic proliferation (8). IL-7 and IL-15 control mouse CD8 T_M cell survival and division in the absence of Ag (9, 10); in contrast, mouse naive and CD4 memory cells require IL-7 and TCR ligands (9, 11, 12), but do not respond to IL-15 (10). Importantly, human CD4 T_M cells have been described to proliferate in response to IL-15 in a TCR-independent fashion and with slow kinetics (13, 14), suggesting different roles for IL-15 in mouse and human CD4 T_M cell homeostasis (15).

IL-15 acts through a heterotrimeric receptor consisting of a specific high affinity binding -chain (IL-15R), plus the IL-2R - and common -chain that mediate signaling (16, 17), and is able to activate T lymphocytes in the absence of Ag (18). The high affinity of IL-15R conditions an extremely rapid uptake of secreted IL-15, preventing detection of IL-15 in culture supernatants (19). Most of the IL-15 detected on cell surfaces is bound to IL-15R (19) and can stimulate in trans both - and IL-15R--bearing cells (19). In addition, IL-15 may interact with the plasma membrane independent of its receptor (20, 21).

Known cell sources inducing T cell proliferation in vivo through IL-15 are dendritic cells, monocytes/macrophages, bone marrow stromal cells, and fibroblasts (19, 20, 21). Although not detected initially (16, 22), T cells were later shown by more sensitive techniques to express IL-15 mRNA (23). In addition, Neely et al. (24) described IL-15 protein expression in normal human T cells, and Thurkow et al. (25) described IL-15 protein expression in synovial tissue T cells of rheumatoid arthritis patients. However, despite later evidence on the presence of IL-15 in T cells both at the mRNA and protein level (23, 24, 25), numerous reports assume that normal human T lymphocytes do not express IL-15 (18, 19, 20), and the possible role of T cell IL-15 expression on T cell biology has not been investigated.

Ex vivo, isolated human T lymphocytes are able to survive for long periods of time (26), and longevity depends on cell density (26). However, it has not been investigated whether isolated T cells are able to proliferate ex vivo in the absence of exogenous stimuli or contact with other cell lineages. Our objective was to determine the ex vivo homeostatic proliferation rate of isolated human T cells and study the factors implicated in this process. We observed that homotypic cell contact is crucial for isolated T cell division, and that IL-15 is a key player in this phenomenon.

	Materials and Methods

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Isolation and culture of human T lymphocytes

Blood was drawn from 20 healthy controls, 13 female, 7 male, with a mean age of 25 years, median 26, range 23–42. The study was approved by the Hospital Ethics Committee. PBMCs were separated by Ficoll-Hypaque (Amersham) gradient. CD3⁺ T cells were prepared from PBMCs by immunomagnetic negative selection in an Automacs (Miltenyi Biotec) with a negative isolation kit (Miltenyi Biotec). Purity of T lymphocytes was >99% CD3⁺ (Fig. 1). Functional purity was assessed by the lack of proliferative responses to superantigens, lectins, and/or soluble anti-CD3. T_N and T_M cells were isolated from CD3⁺ cells by negative selection using anti-CD45RO or anti-CD45RA microbeads (Miltenyi Biotec). Central memory T (T_CM) and effector memory T (T_EM) cells were isolated from T_M cells by positive or negative selection, respectively, with an anti-CCR7 Ab (BD Pharmingen), followed by anti-mouse microbeads (Miltenyi Biotec). Purity of the T_N, T_CM, and T_EM subpopulations was >97% (Fig. 1). T cells were cultured in RPMI 1640 (Invitrogen Life Technologies) with 10% autologous serum, glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 mg/ml). To track cell division by flow cytometry, before initiating culture cells were labeled with CFSE (Molecular Probes) at a final concentration of 8 mM. For functional inhibition experiments, neutralizing mAbs to IL-15, IL-2/IL-15R (CD122) (R&D Systems), HLA class I (Sigma-Aldrich), or isotype control (R&D) were added to the culture at 10 µg/ml. A neutralizing polyclonal goat IgG anti-IL-15R Ab or control goat IgG (R&D Systems) was used at 1 mg/ml. A human rIL-15R/Fc chimera (R&D Systems) or control human IgG was added at 100 ng/ml. Transwell inserts (0.4 µm) (Corning Glass) were used in some experiments. This setup allows division of the T cell culture system into two compartments sharing soluble factors exchanged through the Transwell pores, while preventing direct contact between cells in the upper and lower compartments.

View larger version (31K): [in this window] [in a new window]   FIGURE 1. Flow cytometry analysis of cell purity after magnetic isolation of CD3+ T cells, and of TN, TM, TCM, and TEM subsets. A, CD3+ T cells were prepared from PBMCs by immunomagnetic negative selection. Purity of T lymphocytes was >99% CD3+. B, TN and TM cells were isolated from CD45RO+ CD3+ cells by negative selection using anti-CD45RO or anti-CD45RA microbeads. TCM and TEM cells were isolated from TM cells by positive or negative selection, respectively, with an anti-CCR7 Ab, followed by anti-mouse microbeads. Purity of the TN, TCM, and TEM subpopulations was >97%.

Eighteen hours before the termination of the cultures, the plates were pulsed with 0.5 µCi/well [³H]thymidine (Amersham). The cells were harvested on paper filters and [³H]thymidine uptake was measured in a liquid scintillation counter and expressed as inhibition indices, calculated by dividing the counts in conditions with neutralizing Ab by the counts in conditions without neutralizing Ab.

Intracellular cytokine staining, surface staining, and flow cytometry

For intracellular IL-15 staining, T cells were washed with PBS/2% FCS/0.01% NaN₃, permeabilized for 10 min with FACS permeabilizing solution 2 (BD Pharmingen), washed again, and incubated on ice for 1 h with an anti-IL-15 mAb or an irrelevant IgG1 isotype control mAb (R&D Systems). Cells were then washed and incubated on ice for 30 min with a FITC-conjugated goat anti-mouse Ab (Jackson ImmunoResearch Laboratories). After washing once with PBS/2% FCS/0.01% NaN₃ and once with PBS, cells were resuspended in 1% paraformaldehyde and analyzed in a FACSCalibur flow cytometer using CellQuest software (BD Biosciences). No brefeldin A was needed for intracellular IL-15 detection. For surface staining, the permeabilization step was omitted. Surface IL-15R components were detected with an anti-IL-2/IL-15R mAb, anti-common -chain mAb, or an anti-IL-15R mAb (R&D Systems), followed by a FITC-conjugated goat anti-mouse Ab (Jackson ImmunoResearch Laboratories). Fluorochrome-conjugated mAbs from BD Pharmingen were used to examine the expression of phenotypic markers that define T_N cells (CD45RA⁺, CD27⁺, CD28⁺, CD62 ligand⁺ (CD62L⁺), CCR7⁺, CD11a^low, CD95^–), T_CM cells (CD45RO⁺, CD27⁺, CD28⁺, CCR7⁺, CD62L⁺, CD11a^high, CD95⁺), and T_EM cells (CD45RO⁺, CD27^–, CD28^–, CD62L^–, CCR7^–, CD11a^high, CD95⁺) (27). Flow cytometric quantification of variable element (BV) usage of T lymphocytes was performed using the mAbs anti-TCRBV1S1/2, anti-TCRBV2, anti-TCRBV3, anti-TCRBV5S2, anti-TCRBV7, anti-TCRBV8S1/2, anti-TCRBV12S1, anti-TCRBV14S1, and anti-TCRBV17S1 (all Beckman Coulter). Mean fluorescence intensity (MFI) is given as the difference between the MFI of tested cells and the MFI of background staining.

ELISAs

ELISAs for IL-15, IL-2, and IL-7 were performed in cell-free supernatants using DuoSet kits (R&D Systems).

RT-PCR and quantitative RT-PCR

Total RNA was isolated using the RNeasy mini kit (Qiagen) with DNase treatment. A total of 1 µg of RNA was subjected to reverse transcription using Advantage RT for PCR kit (BD Clontech). Aliquots (1 µl) of the reverse-transcription products were subjected to PCR in a PerkinElmer 9600 thermal cycler (Applied Biosystems), with Taq polymerase from Qiagen. Primers used to detect IL-15 mRNA recognize two distinct IL-15 isoforms corresponding to the cytokine with the short (21-aa) and long (48-aa) signal peptides, respectively (28): IL-15 sense, 5'-GGA TTT ACC GTG GCT TTG AGT AAT GAG- 3'; IL-15 antisense, 5'-CAA TCA ATT GCA ATC AAG AAG TG-3' (product size, 643/524). IL-15R primers are located in exon 2 (sense) and exon 5 (antisense) to amplify mRNA encoding all IL-15R variants that contain exon 2, and thus, bind IL-15: IL-15R sense, 5'-GGA ATT CAT CAC GTG CCC TCC CCC CAT G-3'; IL-15R antisense, 5'-CGG GAT CCT CAA GTG GTG TCG CTG TGG CCC TG-3' (product size, 543/444). IL-2/IL-15R sense, 5'-ACC TCT TGG GCA TCT GCA GC-3'; IL-2/IL-15R antisense, 5'-CGT CTC CAG GCA GAT CCA TT-3' (product size, 531); common -chain sense, 5'-CCA GAA GTG CAG CCA CTA TC-3'; common -chain antisense, 5'-TCA CTC CAA TGC TGA GCA CT-3' (product size, 420). Quantitative PCR was performed in triplicate in the LightCycler (Roche Molecular Biochemicals) using the FastStart DNA Master SYBR Green I kit (Roche Molecular Biochemicals), as previously described (29). The transcript of human -actin served as an external standard; primers were: -actin sense, 5'-GAG CGG GAA ATC GTG CGT GAC ATT-3'; -actin antisense, 5'-GAA GGT AGT TTC GTG GAT GCC-3' (product size, 225). Quantities of specific mRNA in the sample were measured according to the corresponding gene-specific standard curve. The results are expressed as fold of induction: (cDNA sample cultured cells/-actin-cultured cells)/(cDNA sample freshly isolated cells/-actin freshly isolated cells).

Statistical analysis

Comparison between groups was by Mann-Whitney U test. Paired samples were compared using a Wilcoxon matched pairs signed rank sum test.

	Results

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Isolated resting human T cells divide ex vivo in the absence of exogenous stimuli; division rate is dependent on cell density

Flow cytometry of CFSE-labeled isolated human T lymphocytes demonstrated that T cells divide ex vivo in the absence of exogenous growth factors (Fig. 2). Division rate was dependent on cell density (Fig. 2). Specifically, when cultured in 24-well plates, the percentage of divided cells after 12 days was minimal when density was below 1 x 10⁶ cells/well (0.5, 0.15, 0.07 x 10⁶/well) (Fig. 2A). In contrast, at densities of 1, 2, or 4 x 10⁶ cells/well, increasing dividing rates were observed (Fig. 2, A and B). At 1 x 10⁶ cells/well (24-well plate), the first division occurred after a lag period of 144 h (6 days) and cells divided approximately every 40 h. At 2 x 10⁶/well, the first division was observed at 120 h and cells divided every 35 h. At 4 x 10⁶/well, the first division was seen at 96 h and cells divided every 30 h.

View larger version (28K): [in this window] [in a new window]   FIGURE 2. Isolated human T cells divide ex vivo. A, CFSE-labeled human T cells were cultured for 12 days in 24-well plates at increasing cell densities, without () or with () Transwell inserts separating 50% T cells in the top and 50% T cells in the lower compartment. Shown is percentage of divided cells. Each bar represents the mean ± SD of 20 subjects per group. *, p < 0.01 vs T cells cultured at the immediately lower density; , p < 0.01 vs T cells cultured at the same density, without Transwell. B, Representative CFSE dilution histograms of T cells cultured for 12 days without Transwell. C, A total of 2 x 106 T cells was cultured for 12 days in 2 ml of medium in 24-well (area per well, 1.9 cm2), 12-well (well area, 3.8 cm2), or 6-well plates (well area, 9.5 cm2). Shown is percentage of divided T cells. (Bars: mean ± SD of 20 subjects.) *, p < 0.01 vs T cells cultured at the immediately lower area. D, A total of 2 x 105 T cells was cultured for 12 days in 200 µl of medium, in flat-bottom or round-bottom 96-well plates. Shown is percentage of divided cells. (Bars: mean ± SD of 20 subjects.) *, p < 0.01 vs T cells cultured in flat-bottom wells.

Subset analysis by flow cytometry of CFSE-labeled T cells demonstrated that divided cells bear the CD45RO⁺ phenotype, while no division was observed among CD45RA⁺ cells (Fig. 3A). In addition, when cells were gated on CD45RO expression, it was evident that the majority of divided T_M cells express CCR7 and CD62L (Fig. 3B), markers of nonpolarized T_CM cells (27). Cell division was observed in both CD4 and CD8 T cell subsets (Fig. 3C). A higher percentage of dividing CD4 T lymphocytes only reflected the higher proportion of CD4 T cells in human peripheral blood together with a higher frequency of CD45RO phenotype among CD4 T lymphocytes (30). Flow cytometry of cells cultured for 12 days revealed that purity of T cells was still 99% CD3⁺, thereby ruling out expansion of the 1% non-T cell population that was present immediately after isolation.

View larger version (69K): [in this window] [in a new window]   FIGURE 3. Subset analysis of cultured CFSE-labeled T cells. Dot plots show CFSE-labeled human T cells cultured in 24-well plates at 4 x 106 cells/well for 12 days (representative of 20 experiments). A, CD45RO and CD45RA expression on CD3+ cells. B, CD62L and CCR7 expression on CD45RO+CD3+ cells. C, CD4 and CD8 expression on CD3+ cells.

T cells were cultured for 12 days in round-bottom 96-well plates at 4 x 10⁵ cells/well in triplicates. Despite the absence of soluble IL-15 in culture supernatants, a neutralizing anti-IL-15 mAb, a goat polyclonal anti-IL-15R Ab, a soluble IL-15RFc chimera, and an anti-IL-2/IL-15R mAb significantly inhibited T cell division as determined by CFSE dilution and [³H]thymidine incorporation (Fig. 4). Subset analysis by flow cytometry of CFSE-labeled cells confirmed that inhibition was observed on both CD4 and CD8 T cells (Fig. 4A). An isotype control mAb, normal goat IgG, normal human IgG, and an anti-MHC class I mAb had no effect (Fig. 4). This suggests that T cell IL-15 induces T cell proliferation through autocrine/paracrine loops. Therefore, we next examined T cells for IL-15 and IL-15R expression.

View larger version (29K): [in this window] [in a new window]   FIGURE 4. Effect of neutralizing Abs on ex vivo homeostatic T cell division. CFSE-labeled T cells were cultured for 12 days in round-bottom 96-well plates at 4 x 105 cells/well in triplicates. A, T cell division is markedly decreased by an anti-IL-15 mAb, a goat polyclonal anti-IL-15R, a chimeric IL-15R-Fc, and an anti-IL-2R mAb, but not by an isotype control mAb, goat IgG, human IgG, or an anti-MHC class I mAb. Subset analysis by flow cytometry confirmed that this effect was observed on both CD4 and CD8 T cells. (Bars: mean ± SD of 20 subjects.) *, p < 0.01 vs same subset cultured in medium alone. B, Representative CFSE dilution histograms of CD3 cells cultured for 12 days in the presence or absence of neutralizing Abs. C, [3H]Thymidine incorporation of CD3+ cells at 12 days culture in the presence or absence of neutralizing mAbs. The inhibition index is calculated by dividing the counts in conditions with neutralizing Ab by the counts in conditions without neutralizing Ab. (Bars: mean ± SD of 20 subjects.) *, p < 0.01 vs cells cultured in medium alone.

RT-PCR of freshly isolated human T cells showed mRNA expression for IL-15, IL-15R, IL-2/IL-15R, and common -chain (Fig. 5A). Because RT-PCR is a very sensitive technique, it was important to exclude that the 1% contaminating cells could account for the detected IL-15 mRNA expression. Purity of our T cell preparations was >99% CD3⁺. Contaminating cells were <0.85% CD3^–/CD45⁺/CD19⁺, <0.15% CD3^–/CD45⁺/CD14⁺ (Fig. 1A). IL-15 mRNA was quantified by real-time RT-PCR in magnetically sorted CD3⁺, CD14⁺, and CD19⁺ cells. An analysis of (IL-15 mRNA/-actin mRNA) ratios in magnetically sorted CD3⁺ cells, magnetically sorted CD19⁺ cells, and magnetically sorted CD14⁺ cells demonstrated that at least 99% of the IL-15 mRNA detected in sorted CD3⁺ cells is derived from CD3⁺ lymphocytes and not from the contaminating cells. Specifically, the ratio (IL-15mRNA (CD14⁺ cells)/-actin mRNA (CD14⁺ cells))/(IL-15mRNA (CD3⁺ cells)/-actin mRNA (CD3⁺ cells)) was 13.0 ± 2.1, whereas the ratio (IL-15 mRNA (CD19⁺ cells)/-actin mRNA (CD19⁺ cells))/(IL-15mRNA (CD3⁺ cells)/-actin mRNA (CD3⁺ cells)) was 1.1 ± 0.2 (mean ± SD of six different healthy human subjects).

View larger version (36K): [in this window] [in a new window]   FIGURE 5. Expression of IL-15 on human T cells. A, RT-PCR demonstrates expression of mRNA for IL-15, IL-15R, IL-15/IL-2R, and common -chain. The IL-15 pair of primers recognizes sequences encoding both IL-15 isoforms: a 524-bp-long secretory/membrane-bound and a 643-bp-long cytosolic protein. IL-15R primers recognize two alternative splicing isoforms containing (543 bp) or lacking exon 3 (444 bp). B, Flow cytometry shows IL-15 intracellular stores in permeabilized T cells. The histograms on the left demonstrate staining with an anti-IL-15 mAb (solid line) and with an isotype control Ab (dotted line). On the right, shown is inhibition of IL-15 staining in the presence of increasing doses of rhIL-15 (0, 0.5, 1, 2, and 5 µg/ml, represented by histograms numbers 1, 2, 3, 4, and 5, respectively). C, Intracellular IL-15 expression in T cell subsets. Freshly isolated T cells were permeabilized, and indirectly labeled with an anti-IL-15 mAb (upper dot plots) or an isotype control mAb (lower dot plots), followed by staining for subset markers CD4, CD8, CD45RO, CD45RA, and CD62L. Dot plots are gated on forward/side light scatter and CD3 expression (black dots) or on forward/side light scatter, CD3, and CD45RO expression (gray dots). D, Flow cytometry of nonpermeabilized T cells demonstrates that surface IL-15 is not detected on freshly isolated T cells. After 3 and 5 days in culture, surface IL-15 expression is seen. E, Flow cytometry analysis of surface IL-15 expression on T cell subsets at baseline and after 3 and 5 days in culture. (Bars: mean ± SD of 20 subjects.) *, p < 0.05 vs TN cells; , p < 0.05 vs TCM cells. F, Real-time quantitative analysis of IL-15 mRNA expression in T cells cultured for 48 h with medium alone (nonstimulated cells, NS), with an anti-IL-15 mAb, or with human rIL-15, 10 ng/ml (rhIL-15). Shown is fold induction referred to expression in freshly isolated T cells. (Bars: mean ± SD of 10 subjects.) *, p < 0.05 vs T cells cultured in medium.

In parallel, quantitative RT-PCR showed an up-regulation of IL-15 mRNA expression in T lymphocytes cultured for 48 h in medium alone, which was abrogated by a neutralizing anti-IL-15 mAb and significantly increased by rIL-15 (10 ng/ml) (Fig. 5F). Up-regulation of IL-15 mRNA expression observed in culture is attributable to an increased IL-15 mRNA expression in T cells and not in contaminating cells, as demonstrated by quantitative RT-PCR analysis of magnetically sorted CD3⁺ cells, magnetically sorted CD19⁺ cells, and magnetically sorted CD14⁺ cells, resting and stimulated with human rIL-15. Specifically, when stimulated with rhIL-15 (10 ng/ml), the observed up-regulation of IL-15 mRNA expression was (mean ± SD of six independent experiments done in duplicate with six different healthy human subjects) 6.8 ± 1.3-fold for isolated CD3⁺ cells, 7.7 ± 1.5-fold for isolated CD14⁺ cells, and 7.1 ± 1.2-fold for isolated CD19⁺ cells.

All components of the IL-15R complex were detected by flow cytometry in freshly isolated human T cells (Fig. 6). Expression of the three receptor chains was higher on T_M (T_CM and T_EM) when compared with T_N cells (Fig. 6B). CD4 T lymphocytes expressed higher levels of all three receptor chains when compared with CD8 T lymphocytes, and this is attributable to the greater proportion of CD45RO cells within the former subset (30). Among T_M cells, T_CM expressed higher levels of IL-15R when compared with T_EM. In contrast, T_EM expressed higher levels of IL-2/IL-15R and common -chain than T_CM cells, although this difference was not statistically significant (Fig. 6B).

View larger version (24K): [in this window] [in a new window]   FIGURE 6. Expression of IL-15R chains on human T cells. A, Representative histograms show expression of IL-15R, IL-2/IL-15R, and common -chain on freshly isolated human T cells. B, Flow cytometry subset analysis of IL-15R expression on freshly isolated human T cells. (Bars: mean ± SD of 20 subjects.) *, p < 0.05 vs TN cells; , p < 0.05 vs TCM cells.

Magnetically sorted T_N, T_CM, and T_EM cells were labeled with CFSE and cultured in 96-well round-bottom plates in triplicates at 2 x 10⁵ cells/well for up to 12 days. Division was first observed at 3 days in the T_CM subset, at 6 days in the total CD3 population, at 7 days in the T_EM subset, and at 10 days in the T_N subset (Fig. 7A). The percentage of divided cells at 12 days was significantly higher in the T_CM subset when compared with total CD3, T_EM, and T_N cells (Fig. 7). During the culture period, no phenotypic changes were observed in the isolated subpopulations, i.e., T_CM cells did not lose CD62L or CCR7 expression, T_EM cells did not acquire CD62L or CCR7 expression, and T_N cells maintained CD45RA and did not acquire CD45RO expression (Fig. 7B). Analysis by flow cytometry of CFSE-labeled cells demonstrated that division was observed on both CD4 and CD8 T cells (Fig. 7C). The percentage of divided cells at 12 days was significantly decreased in all subsets by a neutralizing anti-IL-15 mAb and an anti-IL-2/IL-15R mAb, but not by an isotype control mAb (Fig. 7C). Analysis by flow cytometry of CFSE-labeled cells confirmed that inhibition was observed on both CD4 and CD8 T cells (Fig. 7C).

View larger version (27K): [in this window] [in a new window]   FIGURE 7. Ex vivo homeostatic division of isolated TCM, TEM, and TN cells. Magnetically sorted TCM, TEM, and TN cells were labeled with CFSE and cultured in 96-well round-bottom plates in triplicates at 2 x 105 cells/well. A, Percentage of divided cells over 12 days of culture as determined by CFSE dilution. (Bars: mean ± SD of 10 subjects.) B, Representative dot plots of CFSE-labeled TCM, TEM, and TN cells cultured for 12 days. Divided TCM cells maintained CD62L, divided TEM cells did not aquire CD62L, and divided TN cells maintained CD45RA and did not acquire CD45RO expression (C). Effect of an anti-IL-15 and an anti-IL-2/IL-15R mAb on ex vivo homeostatic division of isolated T cell subsets. Flow cytometry demonstrated that division was observed on CD4 and CD8 cells, and inhibition was effective on CD4 and CD8 cells. (Bars: mean ± SD of 10 subjects.) *, p < 0.05 vs same T cell subset cultured in medium; , p < 0.05 vs TCM cultured in medium.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

Pilling et al. (26) have demonstrated that resting T lymphocytes cultured at high cell density (4 x 10⁶/ml) are able to survive for long periods in the absence of survival factors. Our results confirm and extend this observation, showing that high T cell density favors ex vivo homeostatic proliferation of isolated T lymphocytes. Experiments in which the same total number of cells were cultured in the same volume of medium, but on increasing culture areas, and experiments with Transwell inserts indicate that the observed homeostatic proliferation is not attributable to soluble factors or to exogenous contaminants. In addition, the cytokines IL-2, IL-7, and IL-15 were below 10 pg/ml in culture supernatants.

Experiments with neutralizing Abs directed to IL-15 and to the IL-15R complex unequivocally suggest that, despite the absence of IL-15 in culture supernatants, this cytokine plays an important role in ex vivo homeostatic T cell division. This is in agreement with reports indicating that although IL-15 is constitutively expressed by several cell lineages, secretion to the extracellular space is hardly detected (31). In contrast, IL-7, a cytokine that plays an important role in in vivo T cell homeostasis, is readily detected by ELISA after secretion. IL-7 is essentially a tissue-derived cytokine (32), with the primary sources being stromal and epithelial cells in various locations (32), whereas bone marrow-derived dendritic cells appear to be relatively minor sources of IL-7 (32) and IL-7 expression has not been detected in normal lymphocytes (32).

The secretion of IL-15 is exquisitely regulated and not yet well understood (31). IL-15R has a very high affinity for IL-15 (K_d = 10 pM) compared with the low affinity of IL-2R for IL-2 (K_d = 10 nM) (31). It has been proposed that the high affinity of IL-15R results in an extremely rapid uptake of secreted IL-15 by producer cells and also by neighboring cells, thereby preventing detection of IL-15 in culture supernatants (19, 33). This mechanism implies autocrine as well as paracrine activation and has been termed intercellular recirculation of IL-15 (33). Furthermore, Dubois et al. (19) demonstrated that most of the IL-15 detected on cell surfaces is bound to IL-15R, and that the complex IL-15/IL-15R is biologically active on neighboring cells through direct cell contact (19): in fact, IL-15R is able to present IL-15 in trans to cells expressing the IL-15R and IL-15R chains (19). Surface, IL-15R-bound IL-15 has a wider biological activity than soluble IL-15 because the signal induced in trans by the IL-15/IL-15R complex can stimulate efficiently at picomolar concentrations the proliferation of both - and IL-15R--bearing cells in a similar manner (19). IL-15R-bound IL-15 on the cell surface is internalized and recycled (19), and the level of surface IL-15 expression results from an equilibrium between IL-15 secretion and internalization of the IL-15/IL-15R complex. We have observed in this study that the basal surface IL-15 expression in total resting circulating peripheral blood T cells is below the level of detection by flow cytometry, whereas a very low surface IL-15 expression was observed on freshly isolated T_CM. Surface IL-15 expression was up-regulated in cultured T cells, consistent with an autocrine loop whereby T cell IL-15 stimulates its own production and modifies the equilibrium between IL-15 secretion and internalization.

Flow cytometric analysis demonstrated that ex vivo homeostatic division occurs in CD4 and CD8 T cells, and that Abs directed to IL-15 are effective at decreasing proliferation in both subsets. This is consistent with previous evidence suggesting that IL-15 plays different roles in mouse and human CD4 T_M cell homeostasis (15) given that human as opposed to murine CD4 T cells proliferate in response to IL-15 in a TCR-independent fashion (13, 14). Several differences have been described between human and mouse T cells (34). Interestingly, in humans, common -chain deficiency results in X-linked SCID, a disease in which CD4 and CD8 T cells are absent (35). In contrast, unlike X-linked SCID patients, a few T cells do develop in -chain-deficient mice, as young adult mice possess 1–3% of the normal number of thymocytes (36). More remarkably, the spleens of older mice contain a near normal number of CD4⁺, but not CD8⁺, T cells (37). Thus, as opposed to humans, there is not an absolute requirement for -chain in the production of murine T cells, and another cytokine receptor can deliver signals that partially compensate for -chain deficiency (36). One candidate is c-Kit, because mice doubly deficient in -chain and c-Kit, the receptor for stem cell factor, lack any T cells as assayed phenotypically or by the detection of TCR rearrangements using a sensitive PCR-based approach (38).

T_N and T_M cells are present in human peripheral blood CD4 and CD8 T lymphocytes. T_M cells comprise at least two functionally distinct subsets (27): 1) nonpolarized T_CM cells lack effector function, express CD62L and the chemokine receptor CCR7, and home to the T cell areas of secondary lymphoid organs; 2) polarized T_EM cells do not express CCR7 or CD62L, and have effector funcion together with the capacity to migrate to nonlymphoid tissues (27). In our system, phenotypic analysis by flow cytometry demonstrated that most divided T cells express T_CM surface markers. This is in accordance with the observation that T lymphocytes can be maintained in a nonpolarized memory state by culture in IL-15 (39).

Whereas intracellular IL-15 expression was comparable among subsets, freshly isolated T_CM lymphocytes displayed the highest IL-15R and surface IL-15 levels. A highest IL-15R expression would allow T_CM cells to compete for IL-15 with other subsets, given the high affinity of IL-15R and the low physiological production of T cell IL-15. And this competition would account for the observed predominance of divided T_CM cells in total CD3⁺ T cell cultures. Competition is possible because human IL-15 has at least two binding sites for IL-15R (40), allowing the IL-15R-IL-15 complex on one T cell to stimulate IL-15R on another, neighboring T cell (20, 40). In addition, membrane-bound IL-15 participates in reverse signaling (20, 21). Therefore, when IL-15 is captured by two IL-15R molecules on the surface of two distinct T lymphocytes, it can activate both cells simultaneously (20). This mechanism would amplify the T cell responsiveness to T cell IL-15, not only favoring competition of T_CM with other subpopulations, but also contributing to the highest division rate observed in isolated T_CM cultures.

Magnetically sorted T_CM, T_EM, and T_N cells were established in culture to determine whether, in the absence of competing T_CM cells, endogenous IL-15 is functionally active in T_EM and T_N cells. Isolated T_CM showed a higher division rate when compared with unsorted total T cells, isolated T_EM, and isolated T_N cells. As opposed to cultures of total T cells, in which T_EM division was minimal and T_N cells did not divide, division was observed among isolated T_EM and isolated T_N cells. However, the lag period until the first division was considerably longer in cultures of isolated T_EM and isolated T_N than observed in isolated T_CM and total T cell cultures. The lag period preceding each T cell division has been shown to be related to the nature and strength of the stimulus (14). Geginat et al. (14) described that stimulation of T_N cells with dendritic cells pulsed with superantigen induced a rapid growth in which the first division occurred after a lag period of 40 h and cells subsequently divided rapidly, approximately every 10 h. In contrast, when exposed to a combination of cytokines, T cells divided with slower kinetics: the first division occurred after 72 h and the division time was 30 h (14). In our system, cells bearing lower levels of IL-15R might need a longer period of time to allow the IL-15 autocrine loop increase IL-15 production up to the threshold level needed to initiate cell division. Importantly, during the culture period, no phenotypic changes were observed in the isolated subpopulations, i.e., T_CM cells did not lose CD62L⁺ or CCR7 expression, T_EM cells did not acquire CD62L or CCR7 expression, and T_N cells maintained CD45RA and did not acquire CD45RO expression. These results suggest that the predominant CD45RO⁺/CD62L⁺ phenotype in cultures of total CD3 T cells is not acquired while entering cell division.

In summary, T cell IL-15 may contribute to the maintenance of the T cell pool in areas of T cell aggregation. Specifically, it would promote the persistence of T_CM cells in the absence of prolonged exposure to Ag (41), when T cells in the lymph node paracortex are in closer contact facilitated by a decreased number of APCs (42). In addition, T cell IL-15 may play an important role in the interaction of T lymphocytes with other cell lineages (29).

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

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

¹ This work is supported by Ministerio de Ciencia y Tecnología Programa Ramón y Cajal (to M.-E.M.-C.), Ministerio de Ciencia y Tecnología Grant SAF 2003-01670 (to M.-E.M.-C. and M.B.-M.), and Hospital La Paz Research Grant (to M.A.L.).

² Address correspondence and reprint requests to Dr. María-Eugenia Miranda-Carús, Department of Rheumatology, Hospital La Paz, Paseo de la Castellana, 261, 28046 Madrid, Spain. E-mail address: eugeniamiranda{at}telefonica.net

³ Abbreviations used in this paper: T_M, memory T; MFI, mean fluorescence intensity; rhIL, human rIL; T_CM, central memory T; T_EM, effector memory T; T_N, naive T.

Received for publication April 19, 2005. Accepted for publication July 5, 2005.

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