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International Immunology Advance Access originally published online on September 6, 2007
International Immunology 2007 19(9):1039-1048; doi:10.1093/intimm/dxm072
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© The Author 2007. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved.
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Phenotypic changes induced by IL-12 priming regulate effector and memory CD8 T cell differentiation

Jee-Boong Lee1,2, Kyoo-A Lee2 and Jun Chang1,2

1 College of Pharmacy, Ewha Womans University, 11-1 Dae-Hyun Dong, Seo-Dae-Mun Gu, Seoul 120-750
2 Rheumatism Research Center, Catholic University of Korea, Seoul 137-701, Republic of Korea

Correspondence to: J. Chang; E-mail: tcell{at}ewha.ac.kr


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 Funding
 References
 
In addition to TCR and co-stimulatory signals, inflammatory cytokines such as IL-12 provide important signals for differentiation and survival of activated CD8 T cells. In the present study, to investigate the mechanisms by which IL-12 priming contributes to activation and enhanced survival of CD8 T cells, we searched the differentially regulated genes and markers by IL-12 during antigenic stimulation. Here, we show that IL-12 priming results in the increased subpopulation of CD127hi cells, which differentiates into long-lived memory cells. We also found that IL-12 priming induces IL-10 expression from activated CD8 T cells, which is distinct from CD127 up-regulation. Direct IL-10 priming of CD8 T cells results in the significant increase of effector and memory CD8 T cell population after adoptive transfer, and this priming effect is closely associated with less susceptibility to apoptosis. Although IL-10 is known as a cytokine with anti-inflammatory and immunosuppressive properties, our results have shown that IL-10 has a direct and positive effect on the survival of CD8 T cells. Together, we suggest that IL-10-dependent and independent effects of IL-12 play important roles in regulating differentiation and survival of activated CD8 T cells into effector and memory cells.

Keywords: CD8 T cells, CD127, IL-10, IL-12, survival


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary data
 Funding
 References
 
Antigen-specific naive CD8+ T cells exist at very low frequencies. After antigenic encounter, these cells undergo massive clonal proliferation followed by the contraction phase in which most expanding T cells undergo apoptosis. The remaining small populations of T cells (~5%) become memory cells, which have the capability for a faster and stronger recall response against secondary exposure of antigens. By virtue of memory T cells, prolonged, sometimes life-long, protective immunity could be provided against repetitive lethal infections. Thus, many efforts have been made to elucidate how memory T cells are generated and how to efficiently elicit higher and longer memory T cell responses.

Activation of naive CD8+ T cells to proliferate and generate effector and memory cells requires TCR stimulation via MHC/peptide and B7-1/2 recognition by CD28. Besides TCR and co-stimulatory signals, it has been suggested that additional cytokine signals from the immunological environment are required for the generation and maintenance of memory T cells. For example, cytokines such as IL-4, IL-7 and IL-15 are reported to be essential for induction, survival or turnover of memory T cells, respectively (1–3). Also, IL-12 was shown to provide an additional signal for optimal activation and enhanced survival of CD8+ T cells (4, 5). Recently, it has been proposed that CD127(IL-7R{alpha}) expression level of CD8+ T cells in the effector phase might be a critical marker to determine the precursors which have the potential to develop into long-lived memory cells (6). However, the mechanisms which mediate the selective survival of memory precursors and regulatory signals that are involved in this developmental process still remain elusive.

IL-12, an inflammatory cytokine, is well-known for regulating Th1/Th2 differentiation (7) and increasing survival of CD4+ T cells by preventing apoptosis (8). For CD8+ T cells, IL-12 has been suggested as a critical signal for the generation of effector and memory cells. For example, in vivo stimulation with antigen in the absence of IL-12 results in induction of tolerance (9–11), suggesting the role of IL-12 in the differentiation of effector and memory CD8+ T cells. Recently, we have shown that IL-12 priming during the primary stimulation dramatically increased the population of memory CD8+ T cells (5), proposing that development of memory cells could be regulated by early IL-12 signaling. While IL-12 signaling was shown to prevent activation-induced cell death by increasing anti-apoptotic molecules such as Bcl-3 and FLIP (4, 12), the detailed mechanisms through which IL-12 promotes the generation of memory CD8+ T cells are not determined yet. In this regard, our IL-12 priming method (5) might be a relevant experimental model to identify the early regulatory events and genes controlling the fate of memory CD8+ T cells.

In this study, we sought to identify critical markers regulated by IL-12 priming, which functionally mediate the generation of high frequencies of memory CD8+ T cells. We used T cell-specific oligonucleotide arrays to examine genome-wide changes in gene expression by IL-12. Our results demonstrate that the expression of CD127 and IL-10 is significantly up-regulated by IL-12 priming and IL-10 functionally mediates the memory-generating effect of IL-12 by enhancing the survival of activated CD8+ T cells.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary data
 Funding
 References
 
Mice
Female C57BL/6 mice (Charles River Laboratories Inc., Yokohama, Japan) and OT-I TCR-transgenic mice (The Jackson Laboratory, Bar Harbor, ME) were housed under specific pathogen-free conditions and were used between 6 and 12 weeks of age following Institutional Animal Care and Use Committee protocols.

Antibodies and reagents
All antibodies were purchased from BD Biosciences Pharmingen (San Diego, CA) unless specified otherwise. Recombinant human IL-2 and murine IL-12 were purchased from R&D Systems (Minneapolis, MN), and recombinant murine IL-10 from the eBioscience (San Diego, CA). CD8a+ T cell isolation kits were obtained from Miltenyi Biotec (Auburn, CA)

In vitro T cell activation and adoptive transfer
Spleen cells from OT-I TCR-transgenic mice were stimulated with 100 nM ovalbumin peptide (OVA)257–264 (SIINFEKL; referred to as OVA) for 72 h in complete IMDM supplemented with 2 mM L-glutamine, 50 µM 2-ME and 10 U/ml human rIL-2 in the presence of rIL-12 (5 ng/ml) or rIL-10 (20 ng/ml). rIL-10 was added 24 h after stimulation. For blocking the activity of endogenous IL-10, cells were treated daily with 10 µg/ml anti-IL-10 neutralizing mAb (clone JES5-16E3). After in vitro stimulation, the CD8+ T cells were purified by negative selection using magnetic bead separation (MACS) according to the manufacturer's instruction (Miltenyi Biotec), and then 2 x 106 CD8+ T cells in 200 µl of PBS were transferred into naive C57BL/6 mice via tail vein injection. For antibody stimulation, MACS-purified CD8+ cells were stimulated for 3 days with plate-bound anti-CD3 (clone 145-2C11, 10 µg/ml) and anti-CD28 (clone 37.51, 1 µg/ml) mAbs.

Oligonucleotide array
Splenocytes from OT-1 TCR-transgenic mice were stimulated with OVA for 72 h in the presence or absence of rIL-12 (5 ng/ml). CD8+ T cells were purified by MACS and then mRNA was isolated with Dynabeads mRNA DIRECT kit (Invitrogen, Carlsbad, CA). For biotinylated cRNA sample preparation, 100 ng of mRNA was used as starting material. The sample preparation was performed using Truelabeling-AMPTM 2.0 kit (SuperArray Bioscience Corporations, Frederick, MD) according to the instructions provided by the manufacturer. Twelve micrograms of each cRNA sample were hybridized to 3 sets of Oligo GEArray® immunology-focused microarrays (Th1-Th2-Th3, T cell and B cell activation and inflammatory cytokine and receptor microarrays from the SuperArray Bioscience Corporations) containing 282 different genes that are involved in lymphocyte activation and function. After overnight hybridization, array images were detected using chemiluminescent detection kit (SuperArray Bioscience Corporations) and captured with Kodak image station. The data were analyzed with GEArray Expression Analysis Suite software (SuperArray Bioscience Corporations) and normalized to several housekeeping genes (ribosomal protein S27a, ß2 microglobulin, heat shock protein 1, and peptidylpropyl isomerase A, etc.).

Surface staining, intracellular staining and flow cytometric analysis
For counting a total number of donor T cells, recipient mice were sacrificed and cells from spleens and mediastinal lymph nodes were isolated. The lungs were perfused with 5 ml of PBS containing 10 U/ml heparin (Sigma–Aldrich, St Louis, MO) through the right ventricle using a syringe fitted with 25-gauge needle. The tissues were then processed through a steel screen to obtain single-cell suspension and particulate matter was removed by passing through 70-µm Falcon cell strainer (BD Labware, Franklin Lakes, NJ). Freshly explanted cells were purified by density gradient centrifugation and re-suspended in FACS buffer (1% fetal bovine serum, 0.03% sodium azide in PBS) at a concentration of 1 x 107 cells per ml. A total of 100 µl of these cells (1 x 106 cells) was stained for CD8 (clone 53-6.7), CD44 (IM7), CD62L (MEL-14), CD127 (A7R34), CD210 (1B1.3a), TCR V{alpha}2 (B20.1) or Vß5 (MR9-4), and samples were acquired on FACSCaliburTM (BD Biosciences, San Jose, CA). PE- or APC-conjugated OVA-specific MHC I tetramer, Kb/OVA-Tet, was produced as described elsewhere (5), and optimal concentration was determined by titration. Cells were stained for 40 min at 4°C using fluorochrome conjugate antibodies and Kb/OVA-Tet, washed, and fixed in PBS containing 2% formaldehyde before analysis by flow cytometry. For intracellular staining, cells were first stained for surface markers, washed, fixed and permeabilized with FACS buffer containing 0.5% saponin (Sigma–Aldrich, Seoul, Korea). Then, cells were stained with anti-IFN-{gamma} (XMG1.2), IL-10 (JES5-16E3) or Bcl-2 (3F11). Gates were set on lymphocytes by forward and side scatter profiles, and the data were analyzed using CellQuestTM Pro (BD Biosciences), FlowJoTM software (Windows version 5.7.2, TreeStar, San Carlos, CA) and WinMDI version 2.8 software (The Scripps Research Institute, La Jolla, CA).

Cytokine assays
The OT-I or purified CD8 T cells were stimulated in the presence or absence of cytokines for 72 h, and the culture supernatants were harvested at the indicated time points. The levels of specific cytokines were quantitated by ELISA kits for IL-10 and IFN-{gamma} (eBioscience) according to the manufacturer's protocol. For detection of osteopontin (OPN) by sandwich ELISA, Maxisorb plastic wells (Nalge Nunc International, Roskilde, Denmark) were coated with purified anti-OPN antibody (AF808 antibody, R&D Systems) in coating buffer (0.1 M sodium carbonate, pH 9.5). Detection was performed with biotinylated anti-OPN antibody (BAF808 antibody, R&D Systems) followed by avidin–HRP conjugate. Recombinant IL-10 or OPN protein was used as standards for calculating cytokine concentrations in the culture supernatants. The assays were carried out in triplicate wells.

Detection of apoptotic death
Apoptosis of in vitro activated T cells was determined by Annexin V and 7-amino-actinomycin D (7-AAD) staining, as recommended by the manufacturer (BD Biosciences). In brief, purified CD8 T cells were seeded in 24-well flat bottom plate (1 x 106 cells per well) without any stimulants in 2 ml of complete IMDM. At indicated time points, these cells were washed, re-suspended in Annexin V binding buffer at a concentration of 1 x 106 cells per ml, incubated with Annexin V–FITC and 7-AAD and analyzed by flow cytometry.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary data
 Funding
 References
 
Identification of genes and markers regulated by initial IL-12 priming during primary CD8+ T cell stimulation
Previously, we have shown that IL-12 priming during initial antigenic stimulation resulted in the significantly enhanced survival of activated CD8 T cells and increased memory population after adoptive transfer (5). Recent studies have suggested that CD127(IL-7R{alpha})hi subpopulation of effector cells preferentially survive and differentiate into long-lived memory cells (6, 13). Therefore, to define the basis of the IL-12 priming effect, we first examined expression of CD127 on OT-I cells stimulated with cognate OVA peptide (SIINFEKL) in the presence or absence of the IL-12. The level of CD127 on naive cells was relatively high (mean fluorescence intensity, ~100), but immediately after antigenic stimulation most OT-I started to express much less CD127 (6–48 h; Fig. 1A). There was no significant difference in the CD127 expression between non-primed and IL-12-primed cells at this period. However, from beginning at 72 h, IL-12-primed cells showed a relatively higher proportion of CD127 positive cells than non-primed cells, and this selective up-regulation of IL-7R by a subpopulation of effector cells was observed more clearly at day 4 after transfer of stimulated cells (21.5% versus 7.5% for IL-12-primed or non-treated cells, respectively; Fig. 1A and B). At this point, the group of mice that received IL-12-primed cells already showed a difference in the number of surviving OT-I cells compared with the non-primed groups (Fig. 1B). At memory phase (at day 40 after transfer), almost all the donor OT-I cells remained CD127hi regardless of IL-12 priming, even though the number of donor OT-I cells in non-primed group was too low to compare the relative expression levels of CD127 between two groups (Fig. 1B). Consistent with our previous finding (5), the absolute numbers of IL-12-primed OT-I cells survived in the recipient mice were at least 10-fold higher than those of non-primed cells at later time points (data not shown). These results indicate that IL-12 priming during in vitro antigenic stimulation selectively enriches a subpopulation of CD127hi effector cells which might eventually develop into long-lived memory cells.


Figure 1
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  		Fig. 1. IL-7R expression is up-regulated by IL-12 priming during in vitro stimulation. (A) OT-I TCR-transgenic cells were stimulated with OVA in the presence or absence of IL-12 (5 ng/ml), and at each time point, cells were harvested, stained with anti-CD8{alpha}, Kb/OVA-Tet and CD127 and analyzed by flow cytometry. The levels of anti-CD127 antibody staining (filled area) were determined among CD8+Tet+ gated populations compared with isotype antibody staining (open histogram) and the percentages of CD127hi cells are indicated in each histogram. The results are representative of three independent experiments with similar results. (B) For adoptive transfer experiments, CD8+ cells were MACS purified 72 h after stimulation, and 2 x 106 cells were intravenously injected into normal C57BL/6 mice. After 4 days, donor OT-I cells in the spleens of recipient mice were identified by CD8{alpha} and Kb/OVA-Tet staining and the levels of CD127 were determined among CD8+Tet+ gated populations. The percentages of CD127hiV{alpha}2+ subpopulation among CD8+Tet+ gated populations are indicated for each group. At day 40, the frequency of donor OT-I cells in spleens of the recipient mice were measured and the levels of CD127 were determined among CD8+Tet+ gated populations. The results shown are derived from pooled spleens of 5 mice for each group and are representative of three independent experiments with similar results.

 
To further identify differentially regulated genes by IL-12 during antigenic stimulation, we compared gene expression profiles of CD8 T cells stimulated in the presence or absence of IL-12 using three pathway-focused DNA microarrays, as described in the Materials and Methods. These arrays contain oligonucleotides representing genes related to T cell differentiation and activation including various transcription factors, signaling molecules, chemokines, cytokines and their receptors. These analyses show that IL-12-primed cells express a distinct molecular signature, that is, consistent and statistically significant changes in RNA levels of several genes (Table 1). Seven out of 282 genes including IL-10 exhibited reproducibly increased expression levels (≥2-fold) while 10 genes including OPN and Tim-3 exhibited decreased expression levels (≤2-fold). Among these genes, IL-10 and OPN were identified in common by all three microarrays and they were repeatedly shown to be increased or decreased in similar levels in each of the separate experiments, respectively.


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  		Table 1. DNA array analysis of gene expression in IL-12-primed CD8 T cells

 
Expression of IL-10 by initial IL-12 priming during primary CD8+ T cell stimulation
We next checked whether increased mRNA level of IL-10 by initial IL-12 priming leads to increased level of IL-10 protein expression. The splenocytes from OT-1 mice were stimulated with OVA in the presence or absence of IL-12, and supernatants were collected at 24, 48 and 72 h to measure the level of IL-10 in the culture. As expected, dramatically increased level of IL-10 was detected in the culture supernatant of IL-12-primed cells compared with non-primed cells from 48 h (Fig. 2A), indicating that IL-12 priming up-regulates the expression of IL-10. To ensure that IL-10 induced by IL-12 priming is derived from CD8 T cells, we also examined the levels of IL-10 induced by anti-CD3/CD28 stimulation of purified CD8 T cells from normal C57BL/6 mice. Again, we observed increased IL-10 production from stimulated CD8 T cells with anti-CD3/CD28 and IL-12 (Fig. 2B), indicating the induction of IL-10 by IL-12 treatment also occurs in normal purified CD8 T cells. These results suggest that IL-10 is produced directly by activated CD8 T cells themselves upon IL-12 treatment. These results are consistent with the previous report that human CD8+ T cell clones stimulated with anti-CD3 antibodies in the presence of IL-12 showed up-regulation of IL-10 (14). We further confirmed the production of IL-10 from CD8 T cells by intracellular staining. As expected, ~7% of CD8+ T cells stimulated with OVA/IL-12 produced IL-10 upon stimulation when compared with OVA-stimulated cells (Fig. 2C). Interestingly, we also detected IFN-{gamma} production from the same culture supernatant (5) (data not shown). This result is in agreement with the recent work suggesting that IL-10 is actually not a Th1- or Th2-specific cytokine, and IL-10 is co-expressed with other Th1 or Th2 cytokines by a subset of T cells (15).


Figure 2
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  		Fig. 2. Induction of IL-10 mediated by initial IL-12 priming during primary CD8+ T cell stimulation. (A) The splenocytes from OT-I TCR-transgenic mice were stimulated with the peptide in the presence or absence of IL-12 (5 ng/ml) for 72 h. At indicated time points, the levels of IL-10 cytokine in the culture supernatant were determined by ELISA. (B) Purified untouched CD8+ T cells from C57BL/6 mice were stimulated with anti-CD3/CD28 antibodies in the presence or absence of IL-12 and the levels of IL-10 were determined at indicated time points. (C) Activated CD8+ T cells at 72 h after stimulation were intracellular stained for TCR V{alpha}2 and IL-10. CD8+ gated cells were shown and the percentages of V{alpha}2+IL-10+ are shown in the upper right quadrant. Quadrant markers were set for each plot based on isotype antibody staining. All data are representative of three independent experiments.

 
According to our data from DNA microarray experiments, OPN expression appears to be significantly reduced by IL-12 priming (Table 1). In mice deficient in OPN gene expression, IL-12 production is diminished and IL-10 production is increased (16), and myelin-reactive T cells in OPN–/– mice produce more IL-10 than in wild-type mice (17). Thus, it is quite likely that there is a regulatory network between OPN and Th1/Th2 signature cytokines such as IL-12 and IL-10. When we measured the level of secreted OPN in the culture supernatant of activated CD8 T cells by sandwich ELISA, it was indeed reduced approximately by half at 72 h in the presence of IL-12, but IL-10 production from activated CD8 T cells was not altered by the addition of anti-OPN neutralizing antibody (data not shown).

The effect of IL-12 on the generation of effector and memory CD8+ T cells is partly mediated by IL-10
Since IL-10 has been shown to inhibit the activation and function of T cells (18–20), dramatic induction of IL-10 by IL-12 priming was an unexpected finding. Although the effects of IL-10 on Th cells have been well-characterized, less is known about the role of IL-10 signaling in the generation of effector and memory CD8 T cells. Thus, it was of interest to examine whether IL-10 induced by IL-12 priming plays an important role in positively regulating the generation of effector and memory cells. To this end, OT-I splenocytes were stimulated with OVA for 72 h in the presence of IL-10, IL-12 or IL-12 plus neutralizing anti-mouse IL-10 antibody and the expression levels of CD127 were examined first. However, we failed to observe any significant difference in the level of CD127 on IL-10-treated cells compared with non-treated cells (Supplementary Data 1 are available at International Immunology Online). Also, addition of anti-IL-10 antibody to IL-12-primed culture had no inhibitory effect on the up-regulation of CD127. These results indicate that IL-12 effect on CD127 expression is not mediated by induction of IL-10. To further examine the role of IL-10 in the generation of effector and memory cells, purified activated OT-I cells were adoptively transferred into normal C57BL/6 mice and the survival of donor OT-I cells was monitored. At day 6 after adoptive transfer for primary effector phase and at day 40 for memory phase, the frequency and absolute numbers of OT-I cells in the spleens of recipient mice were analyzed by Kb-OVA tetramer staining and flow cytometry. As shown in Fig. 3A and B, direct treatment of IL-10 resulted in a statistically significant increase of primary and memory OT-I cells compared with non-primed control groups. During the transition of effector to memory cells (day 10 and 30 after transfer), differences in the percentages of surviving donor OT-I cells in the peripheral blood were maintained between two groups (Supplementary Data 2 are available at International Immunology Online). As a negative control, naive OT-I cells treated with the cytokine alone did not show any sign of primary expansion and memory cell generation (data not shown). When endogenously produced IL-10 was neutralized by the addition of anti-IL-10 antibody (clone JES5-16E3) during IL-12 priming, the number of transferred OT-I cells was, in part, decreased at effector phase (Fig. 3A). These results demonstrate that endogenous IL-10 up-regulation by IL-12 priming at the time of antigenic stimulation functionally contributes to the increased survival of effector and memory CD8+ T cells and that exogenous IL-10 treatment instead of IL-12 is also effective for the enhanced survival of effector and memory CD8 T cells. Interestingly, the effect of IL-12 priming was not completely blocked by the addition of neutralizing anti-IL-10 antibody in these adoptive transfer experiments, suggesting that mechanisms other than IL-10 up-regulation such as induction of CD127 expression also mediate the IL-12 priming effect independently.


Figure 3
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  		Fig. 3. The effect of IL-10 on the generation of effector and memory CD8+ T cells. (A and B) OT-I splenocytes was stimulated with OVA for 72 h in the presence of IL-10 (20 ng/ml), IL-12 (5 ng/ml) or IL-12 plus neutralizing antibody to IL-10 ({alpha}IL-10, 10 µg/ml). After 3 days culture, 2 x 106 MACS-purified CD8+ T cells were adoptively transferred into five C57BL/6 mice for each group. Donor cell recovery was determined at day 6 (A) and day 40 (B) after transfer from the spleens of recipient mice by the tetramer staining. Values represent the absolute numbers (A) or both the percentages and absolute numbers (B) of CD8+Tet+CD44+ cells among lymphocyte-gated cells in one experiment and are shown as the mean ± SD of five mice. Similar results were obtained using CD8, V{alpha}2 and Vß5 triple staining. All data are representative of two independent experiments with similar results. (C) Activated OT-I cells were transferred into normal host, and 48 days after transfer, recipient mice were infected with the recombinant adenovirus expressing OVA intranasally. At day 5 after challenge, lung tissues, mediastinal lymph nodes (MLN) and spleens were isolated, and the frequency of donor OT-I cells was measured by the tetramer staining as described above. (D) Surface expression of IL-10R (CD210) on activated OT-I cells were determined 48 h after peptide stimulation by staining with anti-CD8, TCR V{alpha}2 and IL-10R antibodies. Live CD8+V{alpha}2+ cells were gated and mean fluorescence intensities for IL-10R staining of each gated population are shown in parentheses on the histogram. Filled area, OVA/IL-12 treated; solid line, OVA treated; dashed line, isotype control staining of OVA/IL-12-treated cells.

 
Previously, it has been shown that CD8 T cells previously exposed to IL-10 did not expand to the same level as the untreated control upon re-stimulation (21). To examine whether proliferative capacity of IL-10-primed cells is impaired on secondary response, we challenged the recipient mice 48 days after adoptive transfer with recombinant adenovirus expressing OVA intranasally and determined both the absolute numbers and the percentages of OT-I cells recruited to the lung tissue at the peak (day 5 after infection). Most of the CD8 T cells detected in the lung were donor OT-I cells, and the percentages of Tet+CD8+CD44+ cells among lymphocyte-gated mononuclear cells were 2-fold higher in IL-10-primed group compared with the untreated group (Fig. 3C). We also examined the numbers and the percentages of donor cells in the draining lymph nodes and spleens of challenged mice at the same time point, and found that the frequencies of Tet+CD8+CD44+ cells were linearly correlated with those in the lung tissues (Fig. 3C). These results indicate that the differences in the number of donor cells between two groups are not due to a different migration property. Thus, it is unlikely that IL-10 treatment during primary stimulation affects proliferative potential of memory cells and subsequently diminishes secondary responses.

We have obtained similar results in the adoptive transfer experiments with purified CD8 T cells from Thy1.1 mice (data not shown), suggesting that IL-10 might have a direct effect on CD8 T cells. Thus, surface expression of IL-10R on CD8 T cells was examined after peptide stimulation of OT-I cells (Fig. 3D). Nearly 70% of activated cells expressed IL-10R at 48 h after stimulation, while we found no significant difference in mean fluorescence intensity of IL-10R staining between non-treated and IL-12-treated cell populations. Thus, it is likely that CD8 T cells rapidly up-regulate IL-10R after antigenic stimulation, supporting the possibility that IL-10 acts directly on CD8 T cells to increase the number of responding cells (21).

The increase of CD8+ T cell population by IL-10 is caused by decreased apoptosis and higher Bcl-2 expression
Since IL-12-primed cells have differential susceptibility to cell death throughout their life cycle (5, 12), we sought to determine if IL-10 treatment of CD8 T cells also affects the susceptibility of activated cells to apoptosis. To determine whether initial IL-10 priming reduces cell death during primary expansion, 72 h-activated OT-I cells were rested without stimulation and monitored for apoptotic cell death at the various time points by 7-AAD and Annexin V staining. IL-10-treated cells showed similar death rate to untreated cells by 12 h during the resting period, but exhibited a significant reduction of apoptotic death by 48 h (Fig. 4A). As expected, the level of protection from apoptosis was highest in OVA/IL-12-stimulated cells. Interestingly, neutralization of IL-10 by the addition of anti-IL-10 antibody to the IL-12-primed culture diminished anti-apoptotic effect of IL-12. These results clearly demonstrated that the reduction of apoptotic CD8 T cell death by IL-12 priming is partially mediated by induction of IL-10. To examine involvement of intracellular anti-apoptotic molecule, the level of intracellular Bcl-2 was analyzed after 48 h of resting culture period. As expected, statistically significant differences in the frequency of Bcl-2-positive cells were observed between OVA/IL-10-stimulated cell population as well as in OVA/IL-12-treated cells and OVA-stimulated cells (Fig. 4B; P < 0.05). In addition, the level of Bcl-2 expression was decreased by the addition of anti-IL-10 neutralizing antibody to OVA/IL-12-stimulated culture (Fig. 4B; P ≤ 0.01). Similar results were obtained with purified normal CD8 T cells stimulated with anti-CD3/anti-CD28 antibodies (Supplementary Data 3 are available at International Immunology Online), suggesting that the effects are not due to endogenous IL-10 and/or other cytokines produced by splenocytes. These results are consistent with other reports that IL-10-mediated inhibition of cell death have been noted for T cells (22–24), and IL-10 has been associated with increase of Bcl-2 expression in B and T cells (25, 26). Taken together, our results indicate that initial IL-10 priming increases the survival of effector and memory CD8 T cells by reducing apoptotic cell death.


Figure 4
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  		Fig. 4. The increase of CD8 T cell population by IL-10 is due to their reduced death and higher Bcl-2 expression. (A) Spleen cell suspensions from naive OT-I mice were cultured for 3 days with 100 nM OVA in the presence or absence of IL-12 (5 ng/ml), IL-10 (10 ng/ml) or IL-12 plus anti-IL-10 neutralizing antibody (10 µg/ml). Then, activated OT-I cells were MACS purified and rested for additional 2 days without any stimulant. At indicated time points, cells were harvested and apoptotic cell death was determined by Annexin V and 7-AAD double staining. The samples were assayed in triplicate and the error bars represent the standard deviation values of the mean. The results shown are representative of three separate experiments. (B) The levels of Bcl-2 in each cell population were determined at 48 h after resting by intracellular staining. The percentages of cells within the marker are indicated for each population. The results shown are representative of two independent experiments with similar results. Filled histogram, Bcl-2 staining; open histogram, isotype control staining.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary data
 Funding
 References
 
For many years, researchers have tried to elucidate the mechanisms of memory cell development in T cell immunity, yet the factors that determine this process remain largely unknown. Our previous findings suggest that initial IL-12 signaling has a strong influence on the programming of memory T cell development (5). To study regulatory mechanisms exerted by IL-12 priming in the generation of memory T cell population, we tried to identify differentially expressed markers by IL-12 during in vitro antigenic stimulation. We found that IL-12 priming of CD8 T cells resulted in the increase of CD127(IL-7R{alpha})hi subpopulation during initial antigenic stimulation. CD127 expression was previously shown to be a marker that distinguishes effector cells that will develop into functional memory cells from those that are short-lived (6, 13). In addition, several studies have reported that survival of naive and memory CD8 T cells is dependent on IL-7 signal and CD127(IL-7R{alpha}) expression is also necessary for the development of memory CD8 T cells during the interval of effector-to-memory transition (6, 27–30). However, regulatory mechanisms controlling the expression of CD127 on activated CD8 T cells are not clearly defined yet. One possible regulatory mechanism has been recently proposed that IL-7R expression is reduced on resting T cells in response to IL-7 and other prosurvival cytokines, and this down-regulation is mediated by suppressing IL-7R{alpha} transcription (31). Our results that up-regulating CD127 expression on a subpopulation of activated CD8 T cells mediates survival advantage provoked by early IL-12 signaling suggest another mechanism for the regulation of CD127 expression on effector CD8 T cells. To our knowledge, this is the first report showing that cytokine signaling positively regulates CD127 expression on a subset of activated CD8 T cells. It will be of interest to identify and characterize novel regulatory mechanisms and signaling molecules involving IL-12-mediated IL-7R up-regulation on effector CD8 T cells in future studies.

Another phenotypic change induced by IL-12 priming was the high-level induction of IL-10 from activated CD8 T cells. It is likely that in vitro antigenic stimulation induces high affinity IL-12R on activated CD8 T cells (32) and IL-12 signaling through this receptor subsequently activates STAT3 and induces transcription of IL-10 gene (33). The effects of IL-10 are mostly inhibitory on T cells, monocytes and/or other immune cells (34). For example, reduced IFN-{gamma} expression and cytotoxicity in mouse CD8+ T cell clone by IL-10 was reported, suggesting IL-10 is also inhibitory for the function of CD8+ T cells (35). On the other hand, IL-10 has been shown to increase the frequency of cytotoxic CD8+ T cells through Con A and IL-2 stimulation (36), and IL-10 is required for the optimal generation of effector CTL (36, 37). Furthermore, it has been reported that IL-10 is involved in the generation of effector and memory T cells (37, 38) and more recently that IL-10 is required for optimal CD8 T cell memory following bacterial infection (39). Thus, the exact role of IL-10 on the developmental phases of effector and memory CD8 T cells still remains controversial. Our in vitro priming and adoptive transfer experiments clearly showed that the positive effect of IL-12 is in part mediated by the induction of IL-10, which is independent of IL-12 effect on CD127 up-regulation. This beneficial effect of IL-10 was of interest, since the regulatory role of IL-10 signaling on activated CD8 T cells has been largely unknown. The positive effect of IL-10 on CD8 T cell may occur by direct and/or indirect mechanisms. Supporting a direct role, IL-10 has been shown to enhance the proliferation of CD8 T cells both in vitro (21) and in vivo (39), and IL-10R has been shown to be up-regulated on activated CD8 T cells compared with resting T cells in vivo (39). Our results showing that IL-10R is up-regulated on activated CD8 T cells also support the hypothesis that IL-10 acts directly on CD8 T cells to alter susceptibility to apoptosis and differentiation into memory cells. In addition, it has been shown that the increased accumulation of IL-10-treated CD8 T cells during antigen-driven stimulation was due to an increase in cell viability (21). This is consistent with our results that augmented survival of IL-10-primed CD8 T cells both in vitro and in vivo resulted from decreased apoptosis of activated cells and increased frequency of Bcl-2-expressing cells. Taken together, previous reports and our results both support the idea that IL-10 is an important cytokine positively regulating the survival of effector and memory CD8 T cell populations.

In conclusion, the present study shows that the effect of IL-12 priming on the prolonged survival of activated CD8 T cells is mediated by two distinct mechanisms: up-regulation of CD127 expression and induction of IL-10 production. Exogenous IL-10 treatment enhances the survival, suggesting that IL-10 has the potential to directly regulate the generation and survival of effector/memory CD8 T cell in a stimulatory fashion. Inclusion of IL-10 as an adjuvant might be beneficial for enhanced T cell memory following vaccination since it could prolong the duration of antigen presentation by diminishing innate immunity and/or act directly on CD8 T cells to enhance their survival. Although further studies may be needed to characterize the exact mechanisms of IL-10 for the regulation of CD8 T cell responses both in vitro and in vivo, our findings propose that IL-10 priming may be useful in developing strategies for in vitro generation of antigen-specific CTLs for adoptive immunotherapy and/or for improving the efficacy of vaccines.


   Supplementary data
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
Funding
 References
 
Supplementary data 1, 2 and 3 are available at International Immunology Online.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 Funding
References
 
Korea Science and Engineering Foundation (R11-2002-098-05005-0); NCRC program of the Ministry of Science and Technology and the Korea Science and Engineering Foundation (R15-2006-020).


   Acknowledgements
 
We thank So-Young Choi and Sang-Chun Lee for dedicated technical support. We also thank P. Kilgore for critically reading the manuscript.

Funding to pay the Open Access publication charges for this article was provided by NCRC program of the Ministry of Science and Technology and the Korea Science and Engineering Foundation (R15-2006-020).


   Abbreviations
 
7-AAD, 7-amino-actinomycin D
OPN, osteopontin
OVA, ovalbumin peptide

   Notes
 
Transmitting editor: J. Borst

Received 2 November 2006, accepted 1 June 2007.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 Funding
 References
 

  1. Schluns KS, Kieper WC, Jameson SC, Lefrancois L. Interleukin-7 mediates the homeostasis of naive and memory CD8 T cells in vivo. Nat. Immunol. (2000) 1:426.[CrossRef][Web of Science][Medline]
  2. Schluns KS, Williams K, Ma A, Zheng XX, Lefrancois L. Cutting edge: requirement for IL-15 in the generation of primary and memory antigen-specific CD8 T cells. J. Immunol. (2002) 168:4827.[Abstract/Free Full Text]
  3. Huang LR, Chen FL, Chen YT, Lin YM, Kung JT. Potent induction of long-term CD8+ T cell memory by short-term IL-4 exposure during T cell receptor stimulation. Proc. Natl Acad. Sci. USA (2000) 97:3406.[Abstract/Free Full Text]
  4. Valenzuela JO, Hammerbeck CD, Mescher MF. Cutting edge: Bcl-3 up-regulation by signal 3 cytokine (IL-12) prolongs survival of antigen-activated CD8 T cells. J. Immunol. (2005) 174:600.[Abstract/Free Full Text]
  5. Chang J, Cho JH, Lee SW, Choi SY, Ha SJ, Sung YC. IL-12 priming during in vitro antigenic stimulation changes properties of CD8 T cells and increases generation of effector and memory cells. J. Immunol. (2004) 172:2818.[Abstract/Free Full Text]
  6. Kaech SM, Tan JT, Wherry EJ, Konieczny BT, Surh CD, Ahmed R. Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells. Nat. Immunol. (2003) 4:1191.[CrossRef][Web of Science][Medline]
  7. Trinchieri G. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity. Annu. Rev. Immunol. (1995) 13:251.[Web of Science][Medline]
  8. Yoo JK, Cho JH, Lee SW, Sung YC. IL-12 provides proliferation and survival signals to murine CD4+ T cells through phosphatidylinositol 3-kinase/Akt signaling pathway. J. Immunol. (2002) 169:3637.[Abstract/Free Full Text]
  9. Marth T, Zeitz M, Ludviksson BR, Strober W, Kelsall BL. Extinction of IL-12 signaling promotes Fas-mediated apoptosis of antigen-specific T cells. J. Immunol. (1999) 162:7233.[Abstract/Free Full Text]
  10. Curtsinger JM, Lins DC, Mescher MF. Signal 3 determines tolerance versus full activation of naive CD8 T cells: dissociating proliferation and development of effector function. J. Exp. Med. (2003) 197:1141.[Abstract/Free Full Text]
  11. Schmidt CS, Mescher MF. Adjuvant effect of IL-12: conversion of peptide antigen administration from tolerizing to immunizing for CD8+ T cells in vivo. J. Immunol. (1999) 163:2561.[Abstract/Free Full Text]
  12. Lee SW, Park Y, Yoo JK, Choi SY, Sung YC. Inhibition of TCR-induced CD8 T cell death by IL-12: regulation of Fas ligand and cellular FLIP expression and caspase activation by IL-12. J. Immunol. (2003) 170:2456.[Abstract/Free Full Text]
  13. Huster KM, Busch V, Schiemann M, et al. Selective expression of IL-7 receptor on memory T cells identifies early CD40L-dependent generation of distinct CD8+ memory T cell subsets. Proc. Natl Acad. Sci. USA (2004) 101:5610.[Abstract/Free Full Text]
  14. Gerosa F, Paganin C, Peritt D, et al. Interleukin-12 primes human CD4 and CD8 T cell clones for high production of both interferon-gamma and interleukin-10. J. Exp. Med. (1996) 183:2559.[Abstract/Free Full Text]
  15. Cousins DJ, Lee TH, Staynov DZ. Cytokine coexpression during human Th1/Th2 cell differentiation: direct evidence for coordinated expression of Th2 cytokines. J. Immunol. (2002) 169:2498.[Abstract/Free Full Text]
  16. Ashkar S, Weber GF, Panoutsakopoulou V, et al. Eta-1 (osteopontin): an early component of type-1 (cell-mediated) immunity. Science (2000) 287:860.[Abstract/Free Full Text]
  17. Chabas D, Baranzini SE, Mitchell D, et al. The influence of the proinflammatory cytokine, osteopontin, on autoimmune demyelinating disease. Science (2001) 294:1731.[Abstract/Free Full Text]
  18. de Waal Malefyt R, Haanen J, Spits H, et al. Interleukin 10 (IL-10) and viral IL-10 strongly reduce antigen-specific human T cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class II major histocompatibility complex expression. J. Exp. Med. (1991) 174:915.[Abstract/Free Full Text]
  19. Fiorentino DF, Zlotnik A, Vieira P, et al. IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells. J. Immunol. (1991) 146:3444.[Abstract]
  20. Ding L, Shevach EM. IL-10 inhibits mitogen-induced T cell proliferation by selectively inhibiting macrophage costimulatory function. J. Immunol. (1992) 148:3133.[Abstract]
  21. Kang SS, Allen PM. Priming in the presence of IL-10 results in direct enhancement of CD8+ T cell primary responses and inhibition of secondary responses. J. Immunol. (2005) 174:5382.[Abstract/Free Full Text]
  22. Taga K, Chretien J, Cherney B, Diaz L, Brown M, Tosato G. Interleukin-10 inhibits apoptotic cell death in infectious mononucleosis T cells. J. Clin. Invest. (1994) 94:251.[Web of Science][Medline]
  23. Sieg S, King C, Huang Y, Kaplan D. The role of interleukin-10 in the inhibition of T-cell proliferation and apoptosis mediated by parainfluenza virus type 3. J. Virol. (1996) 70:4845.[Abstract]
  24. Taga K, Cherney B, Tosato G. IL-10 inhibits apoptotic cell death in human T cells starved of IL-2. Int. Immunol. (1993) 5:1599.[Abstract/Free Full Text]
  25. Itoh K, Hirohata S. The role of IL-10 in human B cell activation, proliferation, and differentiation. J. Immunol. (1995) 154:4341.[Abstract]
  26. Cohen SB, Crawley JB, Kahan MC, Feldmann M, Foxwell BM. Interleukin-10 rescues T cells from apoptotic cell death: association with an upregulation of Bcl-2. Immunology. (1997) 92:1.[CrossRef][Web of Science][Medline]
  27. Goldrath AW, Sivakumar PV, Glaccum M, et al. Cytokine requirements for acute and basal homeostatic proliferation of naive and memory CD8+ T cells. J. Exp. Med. (2002) 195:1515.[Abstract/Free Full Text]
  28. Maraskovsky E, Teepe M, Morrissey PJ, et al. Impaired survival and proliferation in IL-7 receptor-deficient peripheral T cells. J. Immunol. (1996) 157:5315.[Abstract]
  29. Sudo T, Nishikawa S, Ohno N, Akiyama N, Tamakoshi M, Yoshida H. Expression and function of the interleukin 7 receptor in murine lymphocytes. Proc. Natl Acad. Sci. USA (1993) 90:9125.[Abstract/Free Full Text]
  30. Tan JT, Dudl E, LeRoy E, et al. IL-7 is critical for homeostatic proliferation and survival of naive T cells. Proc. Natl Acad. Sci. USA (2001) 98:8732.[Abstract/Free Full Text]
  31. Park JH, Yu Q, Erman B, et al. Suppression of IL7Ralpha transcription by IL-7 and other prosurvival cytokines: a novel mechanism for maximizing IL-7-dependent T cell survival. Immunity (2004) 21:289.[CrossRef][Web of Science][Medline]
  32. Desai BB, Quinn PM, Wolitzky AG, Mongini PK, Chizzonite R, Gately MK. IL-12 receptor. II. Distribution and regulation of receptor expression. J. Immunol. (1992) 148:3125.[Abstract]
  33. Jacobson NG, Szabo SJ, Weber-Nordt RM, et al. Interleukin 12 signaling in T helper type 1 (Th1) cells involves tyrosine phosphorylation of signal transducer and activator of transcription (Stat)3 and Stat4. J. Exp. Med. (1995) 181:1755.[Abstract/Free Full Text]
  34. Moore KW, de Waal Malefyt R, Coffman RL, O'Garra A. Interleukin-10 and the interleukin-10 receptor. Annu. Rev. Immunol. (2001) 19:683.[CrossRef][Web of Science][Medline]
  35. Rohrer JW, Coggin JH Jr. CD8 T cell clones inhibit antitumor T cell function by secreting IL-10. J. Immunol. (1995) 155:5719.[Abstract]
  36. Chen WF, Zlotnik A. IL-10: a novel cytotoxic T cell differentiation factor. J. Immunol. (1991) 147:528.[Abstract]
  37. Yang G, Hellstrom KE, Mizuno MT, Chen L. In vitro priming of tumor-reactive cytolytic T lymphocytes by combining IL-10 with B7-CD28 costimulation. J. Immunol. (1995) 155:3897.[Abstract]
  38. Groux H, Bigler M, de Vries JE, Roncarolo MG. Inhibitory and stimulatory effects of IL-10 on human CD8+ T cells. J. Immunol. (1998) 160:3188.[Abstract/Free Full Text]
  39. Foulds KE, Rotte MJ, Seder RA. IL-10 is required for optimal CD8 T cell memory following Listeria monocytogenes infection. J. Immunol. (2006) 177:2565.[Abstract/Free Full Text]

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