The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 179, 3596 -3603
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Barker, B. R.
Right arrow Articles by Letvin, N. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Barker, B. R.
Right arrow Articles by Letvin, N. L.

IL-21 Induces Apoptosis of Antigen-Specific CD8+ T Lymphocytes1

Brianne R. Barker*, Jenny G. Parvani*, Debra Meyer{dagger}, Adam S. Hey{ddagger}, Kresten Skak§ and Norman L. Letvin2,*

* Division of Viral Pathogenesis, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115; {dagger} Department of Biochemistry, University of Johannesburg, Auckland Park, South Africa; {ddagger} Antibody Pharmacology, Symphogen, Lyngby, Denmark; and § Cancer Pharmacology, Novo Nordisk, Maalov, Denmark


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IL-21, a member of the common {gamma}-chain family of cytokines, has pleiotropic effects on T, B, and NK cells. We found that IL-21 and the prototype common {gamma}-chain cytokine IL-2 can stimulate proliferation and cytokine secretion by Ag-specific rhesus monkey CD8+ T cells. However, unique among the members of this family of cytokines, we found that IL-21 drives these cells to apoptosis by down-regulation of Bcl-2. These findings suggest that IL-21 may play an important role in the contraction of CD8+ T cell responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Interleukin-21 has structural homologies and functional similarities to the five other members of the common {gamma}-chain family of cytokines (IL-2, -4, -7, -9, and -15). IL-21 has substantial sequence and structural homology to IL-15 (1). Moreover, the {alpha}-chain of the IL-21R is structurally similar to IL-2Rbeta, and also has sequence similarity to IL-2Rbeta and IL-4R{alpha} (1, 2). Like other members of the common {gamma}-chain family of cytokines, IL-21 was originally described as a growth factor for NK cells that can enhance the proliferation of TCR-stimulated T cells (1). Soon thereafter, this cytokine was shown to have diverse effects both on B and dendritic cells. IL-21R–/– mice were shown to have low concentrations of IgG1 and high concentrations of IgE in their serum (3); IL-4–/–IL-21R–/– mice were shown to have dysregulated Ab class switching, including an absence of IgE (4). In addition, other investigators have shown that IL-21 may exert suppressive effects on dendritic cells (5, 6). Interestingly, the effects of IL-21 are determined, at least in part, by the state of differentiation of its target cells.

In light of the importance of IL-2 and IL-15 in CD8+ T cell biology, the role of IL-21 in CD8+ T cell differentiation and function has been an area of particular interest. Kasaian et al. (3) demonstrated that IL-21 could enhance proliferation of T cells costimulated with anti-CD3 Abs as well as enhance T cell proliferation, cytotoxicity, and IFN-{gamma} release in response to alloantigen. IL-21 has been shown to stimulate Ag-dependent proliferation of human CMV-specific CD8+ T cells (7, 8). Recent in vivo studies indicate that IL-21 may induce tumor rejection through a CD8+ T cell-dependent mechanism (9, 10). This cytokine has also been shown to enhance CD8+ T cell functional activities such as cytokine secretion, proliferation, and cytotoxicity when administered in combination with IL-15 (3, 11, 12, 13, 14). Because many of the documented IL-21-mediated effects on CD8+ T cells overlap with the activities of IL-2 and IL-15, some investigators have suggested that IL-2 and IL-21 may have redundant roles in T cell development and function (15).

In the present study, we examined the role of IL-21 on Ag-specific CD8+ T cells. We found that IL-21, like other common {gamma}-chain cytokines, can stimulate the proliferation and function of these cells. However, we also found that IL-21 has a unique ability to drive these cells to apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Naive and SIV-vaccinated rhesus monkeys were used in this study. Two monkeys that expressed the MHC class I molecule Mamu-A*01 were vaccinated with HIV-1 89.6P env and SIVmac239 gag-pol-nef DNA vaccines on weeks 0, 4, and 8 (AW28 and AW2P), and two other Mamu-A*01+ monkeys were vaccinated with the recombinant adenoviruses AdV5-SIV gag-pol and AdV5-HIV-1 89.6P env on weeks 0 and 8 (AW13 and AV83) (16). These animals were maintained in accordance with the guidelines of the Committee on Animals for the Harvard Medical School and the Guide for the Care and Use of Laboratory Animals.

Recombinant human cytokines

Human recombinant IL-2 manufactured by Hoffmann-La Roche was obtained from the National Institutes of Health AIDS Research and Reference Reagent Program (Product No. 136; Lot No. R163990-1). Recombinant human IL-21 produced in Escherichia coli was provided by ZymoGenetics.

[3H]Thymidine incorporation assays

PBMCs were isolated from normal human volunteers or naive rhesus monkeys from EDTA-anticoagulated blood by Ficoll density gradient centrifugation. PBMCs were cultured at 37°C at 1 x 106 cells/ml in RPMI 1640 supplemented with L-glutamine (2 mM), penicillin (50 U/ml), streptomycin (40 µg/ml), and 8% FCS in triplicate wells for 3 days in 96-well plates precoated with plate-bound anti-CD3 Ab (anti-human CD3 Ab clone UCHT1; BD Pharmingen product no. 555330; anti-rhesus monkey CD3 Ab clone FN18 was generated in the laboratory). Twenty microliters of 50 µCi/ml [3H]thymidine was added to each well 18 h before terminating the culture. Cells were harvested using an automated multiwell harvester, transferred to filter paper, and the filter paper was analyzed using a scintillation counter.

Abs, tetramers, and flow cytometry

Conjugated Abs used in this study included the following (product numbers shown after clones in parentheses were obtained from BD Biosciences, unless otherwise specified): anti-CD3 Pacific Blue (clone SP34.2; product no. 558124), anti-CD3 PerCP Cy 5.5 (clone SP34.2; product no. 552852), anti-CD3 Alexa 700 (clone SP34.2; product no. 557917), anti-CD3 allophycocyanin (clone SP34.2; product no. 557597), anti-CD3 allophycocyanin Cy7 (clone SP34.2; product no. 557757), anti-CD4 Amcyan (clone L200; custom conjugate from BD Biosciences), anti-CD4 PerCP Cy5.5 (clone L200; product no. 552838), anti-CD8 Pacific Blue (clone RPA-T8; product no. 558207), anti-CD8 FITC (clone SK1; product no. 347313), anti-CD8 PerCP Cy5.5 (clone SK1; product no. 341051), anti-CD8 energy-coupled dye (clone 7PT3F9, generated in the laboratory and custom conjugated by Beckman Coulter), anti-CD8 Alexa 700 (clone RPA-T8; product no. 557945), anti-CD25 PE (clone M-A251; product no. 555432), anti-IFN-{gamma} allophycocyanin (clone B27; product no. 554702), anti-phospho-Stat5 (Y694) PE (clone 47; product no. 612567), anti-phospho-Stat3 (Y705) PE (clone 4; product no. 612569), and anti-Bcl-2 FITC (clone Bcl-2/100; product no. 65114X). Recombinant human annexin V allophycocyanin (AnnexinV05; Caltag Laboratories) was used. p11C-Mamu A*01 tetrameric complexes were generated and conjugated to PE or PE-Cy7. Purified rabbit polyclonal anti-IL-21R{alpha} was purchased from Abcam (product no. ab13268). Purified IL-21R{alpha} was conjugated to PE using the Prozyme PhycoLink R-PE Conjugation kit (product no. PJ31K). All reagents were titrated on rhesus monkey PBMCs. Samples were evaluated using a FACSCalibur (BD Biosciences) or an LSR II instrument (BD Biosciences) and analyzed using FlowJo software (Tree Star).

Intracellular phosphoepitope flow cytometry

PBMCs were isolated from normal human volunteers or naive rhesus monkeys from EDTA-anticoagulated blood by Ficoll density gradient centrifugation. PBMCs were plated at 1 x 106 cells/ml in RPMI 1640 supplemented with L-glutamine (2 mM), penicillin (50 U/ml), streptomycin (40 µg/ml), and 12% FCS at 37°C and rested overnight. Cells were then stimulated with the indicated cytokines for 15 min at 37°C. Immediately after stimulation, cells were fixed for 15 min at 37°C in tissue-culture wells by adding an equal volume of 4% paraformaldehyde. Cells were then pelleted and resuspended in 1 ml of ice-cold 90% methanol and permeabilized overnight at –20°C. Following permeabilization, cells were washed four times with 3 ml of PBS, centrifuging at 4°C. Cells were then rehydrated in 1 ml of PBS/2% FCS for 1 h on ice, resuspended in 100 µl of PBS/2% FCS and stained with anti-CD3 and anti-STAT Abs for 30 min on ice. Cells were then washed three times with PBS and analyzed on an LSR II flow cytometer (BD Biosciences). This protocol was adapted from the protocol presented in Ref. 17 .

Ag-specific T cell culture

PBMCs were cultured for 3 days at 4 x 106 cells/ml with 10 µg/ml p11C peptide (CTPYDINQM) in RPMI 1640 supplemented with L-glutamine (2 mM), penicillin (50 U/ml), streptomycin (40 µg/ml), and 12% FCS. Cytokines were added as noted and cultures were maintained with half medium changes every other day.

Ab staining

Cultured cells were washed twice in PBS/2% FCS to remove medium and then resuspended in 100 µl of PBS/2% FCS. Tetramers were incubated with the cells for 15 min, and then Abs were added and incubated for 10 additional min. Cells were then washed twice in PBS/2% FCS and then fixed with 1% formaldehyde before being analyzed by flow cytometry. When staining with annexin V in addition to the Abs, annexin binding buffer (BD Biosciences product no. 556454)/2% FCS was used in place of PBS/2% FCS, and annexin V was added to the mixture of Abs.

CFSE labeling

PBMCs were washed twice with HBSS, resuspended at 1 x 107 cells/ml HBSS, and 1 µM CFSE for 30 min at 37°C (Molecular Probes product no. C-1156), washed twice with RPMI 1640 containing 10% FCS to stop the staining reaction, and then cultured as described above.

Intracellular cytokine staining

Cultured cells were washed twice in PBS/2% FCS and resuspended in RPMI 1640/12% FCS medium containing (with BD Biosciences product numbers in parentheses): 5 µg/ml of the peptide p11C (CTPYDINQM), monensin (product no. 554724), 1 µg/ml anti-CD28 (product no. 555725), and 1 µg/ml anti-CD49d (product no. 555501), and then incubated for 6 h at 37°C. Cultured cells were then washed twice and stained with tetramer for 15 min followed by staining for cell surface molecules for 10 additional min. After fixing and permeabilization with Cytofix/Cytoperm solution (product no. 554722), cells were washed with perm/wash buffer (product no. 554723) and stained with an Ab specific for IFN-{gamma}. Cells were then washed in perm/wash buffer and fixed in 1% formaldehyde/PBS. Experimental cells were compared with cells in the absence of peptide and positive control cells incubated with 10 ng/ml PMA and 1 µg/ml ionomycin.

TUNEL staining

Cultured cells were stained with tetramer and Abs as described above and then fixed in 1% paraformaldehyde on ice for 60 min. Cells were then washed twice with PBS and permeabilized overnight in 70% ethanol. Cells were then stained with reagents from the Apo-Direct Apoptosis kit (BD Biosciences product no. 556381). Briefly, permeabilized cells were washed twice with kit wash buffer and then stained in 50 µl of staining solution containing 10 µl of reaction buffer, 0.75 µl of TdT, 5 µl of FITC-dUTP, and 34.25 µl of dH20 for 60 min at 37°C. Stained cells were then washed twice with rinse buffer and resuspended in 1% paraformaldehyde. FITC-dUTP fluorescence was compared with fluorescence seen in the positive and negative control cells included in the kit.

Intracellular Bcl-2 staining

Cultured cells were stained with tetramer and Abs as described above. Cells were then fixed and permeabilized by incubation for 30 min in Cytofix/Cytoperm (BD Biosciences product no. 554722). Cells were then washed twice in perm/wash buffer (BD product no. 554723) and stained with Abs specific for Bcl-2 for 30 min. Cells were finally washed twice with perm/wash buffer and fixed in 1% paraformaldehyde.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Recombinant human IL-21 costimulates proliferation of rhesus monkey T lymphocytes

Previous work has demonstrated that IL-21, in combination with plate-bound anti-CD3, can costimulate the proliferation of murine thymocytes, mature murine T cells, and human CD45RA+ T lymphocytes (1). To characterize further the functional activity of IL-21, we evaluated the ability of recombinant human IL-21 to stimulate rhesus monkey and human peripheral blood T lymphocyte proliferation. As previously described, recombinant human IL-21 stimulated the proliferation of anti-CD3-exposed human PBMC in a dose-dependent fashion (Fig. 1A). Moreover, no proliferation of these PBMCs was observed in response to IL-21 in the absence of anti-CD3 Ab. The efficiency with which IL-21 stimulated this proliferation was comparable to that of IL-2 stimulated proliferation, with 20 ng/ml IL-21 driving a level of proliferation comparable to 5 U/ml IL-2 and 80 ng/ml IL-21 driving a level of proliferation comparable to 20 U/ml IL-2. Like IL-2, recombinant human IL-21 also costimulated anti-CD3 Ab-exposed rhesus monkey PBMCs to proliferate in a dose-dependent fashion (Fig. 1B). This observation indicated that we could explore the functional activities of recombinant human IL-21 in rhesus monkey lymphocyte populations.


Figure 1
View larger version (30K):
[in this window]
[in a new window]

  		FIGURE 1. Recombinant human IL-21 costimulates anti-CD3- stimulated human and rhesus monkey PBMC proliferation. Human (A) or rhesus monkey 179-01 (B) PBMCs were assayed for proliferation by [3H]thymidine incorporation in response to increasing concentrations of plate-bound anti-human CD3 mAb and increasing concentrations of recombinant human IL-2 or IL-21. Values represent the mean value ± SD obtained in triplicate wells. Results shown are representative of two experiments for human and four experiments for monkey PBMCs.

 
Recombinant human IL-21 induces phosphorylation of STAT 3, but not STAT 5, in rhesus monkey T lymphocytes

Although IL-21 activates the same JAKs as other members of the common {gamma}-chain family of molecules (18, 19, 20, 21, 22), this cytokine has been reported to induce the activation of different STAT molecules. IL-2, IL-7, IL-9, and IL-15 primarily activate STAT 5A and STAT 5B (21, 23), while IL-21 has been shown to induce the activation of STAT 1 and STAT 3 and smaller amounts of STAT 5A and STAT 5B in cell lines and mouse splenocytes (2, 18, 19, 20). To determine whether this is also true in monkey PBMCs, we assessed the levels of STAT 3 and STAT 5 phosphorylation in primary human and rhesus monkey PBMCs. PBMCs were stimulated for 15 min with 20 U/ml IL-2 or 20 ng/ml IL-21, stained to detect intracellular STAT 3 and STAT 5 phosphorylation, and subjected to flow cytometric analysis (17, 24). As previously described, human T lymphocytes stimulated with recombinant human IL-2 demonstrated no up-regulation of phosphorylated STAT (pSTAT)3 3 but expressed high levels of pSTAT 5 (Fig. 2A). Similarly, rhesus monkey T lymphocytes stimulated with recombinant human IL-2 showed no up-regulation of pSTAT 3 but expressed high levels of pSTAT 5 (Fig. 2B). However, pSTAT 5 expression in IL-2-stimulated monkey T lymphocytes was lower than that seen in IL-2-stimulated human T cells. Consistent with previously published studies performed on cell lines and mouse splenocytes, primary human T lymphocytes stimulated with recombinant human IL-21 showed up-regulation of pSTAT 3 (Fig. 2C). However, in contrast to what has been described in other reports, no up-regulation of pSTAT 5 was seen in IL-21-stimulated human T lymphocytes. pSTAT 3 was also up-regulated in rhesus monkey T lymphocytes stimulated with IL-21, while no change in pSTAT 5 levels was seen (Fig. 2D). These data indicate that, although IL-2 and IL-21 share a common receptor chain and signal similarly through a JAK pathway, IL-2 and IL-21 initiate different STAT signaling. These two cytokines may therefore have different biological activities. Moreover, because recombinant human IL-21 had similar biologic activity in rhesus monkey and human cells, the monkey is a useful model for exploring IL-21 functional properties.


Figure 2
View larger version (34K):
[in this window]
[in a new window]

  		FIGURE 2. Recombinant human IL-21 induces STAT phosphorylation in human and rhesus monkey PBMCs. PBMCs from a human (A and C) or rhesus monkey AW2P (B and D) were stimulated for 15 min with 20 U/ml IL-2 (A and B) or 20 ng/ml IL-21 (C and D) at 37°C and then stained with anti-CD3 and anti-pSTAT 3 or anti-pSTAT 5 Abs. The displayed pSTAT protein staining in CD3+ lymphocytes is representative of replicate studies performed on PBMCs of two humans and four monkeys. Shaded histograms represent unstimulated cells and open histograms represent cells stimulated with the indicated cytokines.

 
The IL-21R {alpha}-chain is expressed on Ag-specific memory CD8+ T cells

Defining the expression of the IL-21R on T lymphocytes is central to understanding the effect of IL-21 on the function of these cells. Investigators have demonstrated the expression of IL-21R{alpha} on Jurkat cells but were not able to demonstrate the expression of this receptor chain on other T cell lines including EL4 and K562 cells (1). To examine whether IL-21R{alpha} is expressed on primary rhesus monkey T cells, an anti-IL-21R{alpha} Ab was conjugated to PE and used to stain rhesus monkey PBMCs in association with anti-CD3, -CD4, and -CD8 Abs. IL-21R{alpha} expression was clearly detectable on a subset of CD3+ lymphocytes (Fig. 3A). In fact, a higher percentage of CD3+ lymphocytes expressed IL-21R{alpha} than did CD3– lymphocytes. Moreover, IL-21R{alpha} expression was also detectable on a subset of CD3+CD4+ and CD3+CD8+ lymphocytes (Fig. 3, B and C). Approximately twice as many CD8+ as CD4+ T cells expressed IL-21R{alpha}. These data indicate that T cells are potentially important targets of IL-21.


Figure 3
View larger version (36K):
[in this window]
[in a new window]

  		FIGURE 3. Ag-specific memory CD8+ T cells express higher levels of IL-2R{alpha} than do unfractionated CD8+ T cells. PBMCs from rhesus monkey 179-01 were stained with an anti-human IL-21R{alpha} Ab and anti-CD3 (A), anti-CD4 (B), or anti-CD8 (C) Abs to analyze IL-21R{alpha} expression on CD3, CD4, and CD8 lymphocyte subsets. Numbers represent percent of lymphocytes in each quadrant (A) or percent of gated CD4+ or CD8+ T cells (B and C) that express IL-21R{alpha}. Shaded histograms represent total CD8+ T cells and open histograms represent p11C-specific CD8+ T cells (D and E). PBMCs from the SIV Gag-vaccinated rhesus monkey AW28 were stained with anti-CD3, anti-CD8, p11C/MamuA*01 tetramer and then either anti-CD25 (D) or anti-human IL-21R{alpha} (E). Data are representative of staining of PBMCs from three SIV Gag-vaccinated, Mamu-A*01+ rhesus monkeys.

 
We then determined whether IL-21R{alpha}, like receptors for other cytokines in the common {gamma}-chain family, is expressed on Ag-specific CD8+ T cells. PBMCs from three SIV Gag-vaccinated Mamu A*01+ rhesus monkeys were stained with Abs specific for IL-21R{alpha}, CD25, CD3, and CD8, as well as with a Mamu A*01/p11C tetramer to detect SIV Gag epitope-specific T cells. IL-21R{alpha} expression was higher on Ag-specific cells than on bulk CD8+ cells from all monkeys studied (Fig. 3E). Moreover, IL-21R{alpha} expression was comparable to the expression of CD25, the IL-2R{alpha} chain, a molecule expressed in the setting of T cell activation (Fig. 3D). These data therefore suggest that Ag-specific T cells may be important targets of IL-21.

Ag-specific CD8+ rhesus monkey T cells stimulated with IL-21 do not expand in culture

Because IL-21 drives the proliferation of Ag-stimulated CD8+ T lymphocytes, we examined the expansion of this cell subpopulation in vitro following exposure to Ag and cytokine. Although IL-2 stimulated the expansion of Ag-specific CD8+ T lymphocyte populations, IL-21 did not stimulate an increase in the number of these cells, quantitated either as percentage of total T cells (Fig. 4A) or as an absolute number of cells (Fig. 4B). These findings were paradoxical, as we expected that a cytokine that induced T cell proliferation would also induce the expansion of a T cell population.


Figure 4
View larger version (15K):
[in this window]
[in a new window]

  		FIGURE 4. Recombinant human IL-21-stimulated Ag-specific CD8+ T cells do not expand. PBMCs from the SIV Gag-vaccinated Mamu-A*01+ rhesus monkey AW28 were cultured with the dominant epitope peptide p11C for 3 days before the indicated concentrations of IL-2 or IL-21 were added. Ag-specific CD8+ cells were enumerated both as percentage (A) and absolute number (B) on days 4, 7, and 11 following the addition of cytokine.

 
Recombinant human IL-21 can induce Ag-specific rhesus monkey CD8+ T lymphocytes to proliferate and secrete IFN-{gamma}

To further examine the effect of IL-21 on the function of these Ag-specific CD8+ T cells, we assessed the proliferation of cytokine-exposed Ag-specific cells in response to peptide Ag to confirm that these cells could proliferate in response to IL-21. PBMCs from four SIV Gag-vaccinated Mamu A*01+ rhesus monkeys were evaluated. PBMCs were cultured with the dominant epitope peptide p11C for 3 days and then cytokine was added. Although epitope peptide-stimulated PBMCs did not divide in the absence of exogenous cytokine (Fig. 5A), they vigorously divided in the presence of IL-21 (Fig. 5C). Moreover, this cell division was comparable to that stimulated by peptide and IL-2 (Fig. 5B). Thus, Ag-specific CD8+ T cells proliferated following TCR triggering in the presence of IL-21.


Figure 5
View larger version (78K):
[in this window]
[in a new window]

  		FIGURE 5. Recombinant human IL-21 stimulates Ag-specific CD8+ T cell proliferation and effector function in the presence of peptide Ag. CFSE-loaded PBMCs from the SIV Gag-vaccinated Mamu-A*01+ rhesus monkey AW28 were cultured with the dominant epitope peptide p11C for 3 days before addition of medium alone, 20 U/ml IL-2, or 20 ng/ml IL-21. Four days later, CFSE dilution was assessed in the p11C/Mamu-A*01 tetramer-binding CD8+ T cells (A–C). PBMCs from the SIV Gag-vaccinated Mamu-A*01+ rhesus monkey AW13 were cultured with the dominant epitope peptide p11C for 3 days before the indicated concentrations of IL-2 (E), or IL-21 (F), or medium alone (D) were added. Four days later, IFN-{gamma} secretion was measured by flow cytometry in CD3+CD8+/Mamu-A*01 tetramer-binding lymphocytes. Data are representative of two repetitions of an experiment done on PBMCs of four monkeys.

 
To determine whether IL-21 also enhances effector function of Ag-specific CD8+ T cells, PBMCs from an SIV Gag-vaccinated (AW13) rhesus monkey were cultured with the dominant epitope peptide p11C for 3 days, IL-2 or IL-21 was then added to the cultured cells, and IFN-{gamma} secretion by CD3+, CD8+, p11C tetramer-binding lymphocytes was measured by mAb staining and flow cytometric analysis 4 days later. Intracellular cytokine staining assays performed on the lymphocytes from the vaccinated monkey indicated that IL-21 increased epitope peptide-stimulated IFN-{gamma} secretion by the Ag-specific CD8+ T cells (Fig. 5, D–F). IL-2-stimulated Ag-specific CD8+ T cells secreted more IFN-{gamma} than did IL-21-stimulated cells. This IFN-{gamma} secretion by CD3+CD8+ tetramer-negative cells from the vaccinated monkey was also increased after culture with either IL-2 or IL-21. These data therefore indicate that IL-21 can increase CTL effector function.

IL-21-stimulated Ag-specific CD8+ T cells undergo apoptotic cell death

T cells might proliferate but not expand in number in vitro following exposure to IL-21 because they might undergo apoptotic cell death. These in vitro-stimulated cells were therefore assessed by annexin V staining to determine whether IL-21-stimulated Ag-specific CD8+ T cells undergo apoptosis. On day 7, a higher fraction of tetramer+CD8+ T cells cultured in the presence of 20 ng/ml IL-21 bound annexin V than did those stimulated with no cytokine or with 20 U/ml IL-2 (Fig. 6, A and B).


Figure 6
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 6. Recombinant human IL-21 stimulates Ag-specific CD8+ T cell apoptosis in the presence of peptide Ag. PBMCs from the SIV Gag-vaccinated Mamu-A*01+ rhesus monkey AW13 were cultured with the dominant epitope peptide p11C for 3 days before addition of medium alone, 20 U/ml IL-2, or 20 ng/ml IL-21. The percent of annexin V+ Ag-specific CD8+ T lymphocytes was determined on day 7 following addition of cytokine (A and B). Percent TUNEL+ Ag-specific CD8+ T lymphocytes from rhesus monkey AW13 was also determined on day 7 following addition of cytokine (C and D). Data are representative of two repetitions of an experiment done on PBMCs of four monkeys.

 
Because rapidly dividing cells can have unstable membranes and therefore bind annexin V (25, 26, 27), we also used a TUNEL assay to evaluate IL-21-stimulated Ag-specific CD8+ T cells for apoptotic cell death. On day 7, more tetramer+CD8+ T cells cultured in the presence of IL-21 were TUNEL positive than those cells cultured in the presence of no cytokine or IL-2 (Fig. 6, C and D). These data therefore confirm that Ag-specific CD8+ T cells stimulated in the presence of IL-21 undergo apoptotic cell death.

Ag-specific CD8+ T cells stimulated with recombinant human IL-21 express lower levels of Bcl-2 than do Ag-specific CD8+ T cells stimulated with IL-2

This IL-21-induced proliferation and acquisition of effector function in association with an induction of apoptosis could be explained by various mechanisms. This cytokine may stimulate effector function but not deliver a concomitant survival signal, or it may directly stimulate apoptosis of certain cell subpopulations. To determine whether IL-21 was directly stimulating apoptosis or was leading to apoptosis because of the absence of a delivered survival signal, we examined the expression levels of the prosurvival protein Bcl-2 in these IL-21-exposed cells. PBMCs from four SIV Gag-vaccinated Mamu A*01+ rhesus monkeys were cultured for 3 days with the dominant epitope peptide p11C, cytokine was added, and 11 days later, Bcl-2 expression was measured by mAb staining and flow cytometric analysis of CD3+, CD8+, p11C tetramer-binding lymphocytes. IL-2-stimulated Ag-specific CD8+ T cells expressed higher levels of Bcl-2 than cells stimulated in the absence of cytokine (Fig. 7). In contrast, IL-21-stimulated Ag-specific CD8+ T cells expressed levels of Bcl-2 similar to or lower than those of cells cultured in the absence of cytokine (Fig. 7). Similar observations were made in studies of these stimulated cell populations by Western blot analysis (data not shown). These data suggest either that IL-21-stimulated Ag-specific CD8+ T cells lack a critical survival signal that is present in IL-2-stimulated Ag-specific CD8+ T cells or that IL-21 induces the expression of an upstream proapoptotic protein that results in the down-modulation of Bcl-2.


Figure 7
View larger version (30K):
[in this window]
[in a new window]

  		FIGURE 7. Ag-specific CD8+ T cells stimulated with human IL-21 express lower levels of Bcl-2 than those cells stimulated with IL-2. PBMCs from four SIV Gag-vaccinated Mamu-A*01+ rhesus monkey were cultured with the dominant epitope peptide p11C for 3 days before 20 U/ml IL-2 or 20 ng/ml IL-21 was added. Bcl-2 expression was measured by mAb staining and flow cytometric analysis on gated CD3+CD8+ p11C/Mamu-A*01 tetramer-binding lymphocytes 11 days after the addition of cytokine. Data are representative of four repetitions of an experiment done on PBMCs of four monkeys.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have confirmed in the present study that IL-21, like other common {gamma}-chain cytokines, can costimulate T cell proliferation and induce CD8+ T cell secretion of IFN-{gamma}. However, unlike other members of this family of cytokines, we have shown that IL-21 is unable to stimulate the growth and survival of Ag-specific CD8+ T cell populations because it stimulates low levels of the prosurvival protein Bcl-2. These data therefore indicate that IL-21 has a unique role in CD8+ T cell biology.

This unexpected finding cannot be explained by technical problems encountered in the experiments. The assessment of the function of recombinant human cytokines on rhesus monkey cells should be valid, because thymidine incorporation and STAT signaling studies indicated that rhesus monkey and human cells behave comparably in response to recombinant human IL-21. Finally, we only examined the effects of IL-21 on vaccine-induced Ag-specific CD8+ T cells. It has been shown that IL-21 has different effects on the same subpopulation of lymphocytes when the cells are in different stages of differentiation (9). Other investigators who have examined the effect of IL-21 on CD8+ T cells have seen somewhat different results. In one study, naive CD8+ T cells specific for a tumor Ag were demonstrated to expand after 7 days in culture with no difference in annexin V binding (28). In another study, it was demonstrated that IL-21 can expand tumor-specific CD8+ T cells in vivo (11). However, even if the proapoptotic effect of IL-21 is seen only in Ag-stimulated memory CD8+ T cells, this would represent a key regulatory signal on a particularly important T cell subpopulation.

The present data suggest a new T cell-specific function for the common {gamma}-chain family of cytokines. The cytokines that use the common {gamma}-chain as a component of their receptor are generally thought of as growth factors (29, 30, 31, 32). IL-2 is the only member of this cytokine family that has been previously associated with the induction of apoptosis and this was observed only in the activation-induced cell death pathway (32, 33). However, IL-21 might be expected to have different functions than other members of the common {gamma}-chain family because it signals through different STAT molecules. It is possible that all apoptotic effects of the common {gamma}-chain family are mediated as a result of STAT 1 and/or STAT 3 signaling, because IL-2, the only other member of the family associated with apoptosis, has also been associated with low levels of STAT 1 and STAT 3 signaling (21, 23). In fact, STAT 1 signaling has previously been associated with apoptosis (34, 35).

The disparate effector functions observed in the present study highlight the important unresolved question of how the same JAK and similar STAT molecules can transduce signals that lead to very different consequences. These signaling differences may be explained by different configurations of the STAT molecules or by signaling through other pathways. A recent study explored the contributions of each of the tyrosine residues of the mouse IL-21R{alpha} molecule in stimulating STAT 1, STAT 3, and STAT 5 as well as the Shc-MAPK and Akt signal transduction pathways (2). These data demonstrated that only one tyrosine (Y510) is important for optimal proliferation and stimulation of signaling pathways. It will be important to determine whether the other tyrosines are necessary for apoptosis, and which molecules transduce a signal between the tyrosine molecules and the apoptotic pathway.

Previous work has indicated that IL-21 can contribute to inducing apoptosis in B and NK cells when those cells are in certain stages of differentiation. IL-21 was shown to be proapoptotic in primary B cells and in B cells that are stimulated with LPS or anti-IgM Ab but not in B cells stimulated with anti-CD40 Ab (36, 37, 38). Similarly, NK cells stimulated with IL-21 were shown to undergo apoptosis (3, 39). IL-21 can costimulate B cell proliferation, class switching, and Ab production after CD40 and cell surface IgM are cross-linked; however, it triggers apoptosis after B cell stimulation through TLR or cell surface IgM alone (1, 37, 38). Similarly, IL-21 can induce NK cell cytotoxicity or proliferation, yet it induces apoptosis of NK cells that are in certain stages of differentiation (1, 3, 39, 40). It is of interest to determine whether the differentiation status of CD8+ T cells is important in determining whether IL-21 delivers a proapoptotic signal to CD8+ T cells.

The precise role of Bcl-2 in IL-21-stimulated B cell apoptosis has been controversial. mRNA expression data suggested that Bcl-2 levels in IL-21-stimulated primary B cells can be reduced, leading to apoptosis (36). However, this observation was not confirmed by other investigators who evaluated Bcl-2 protein levels in these stimulated cells (37). In the present experiments, Bcl-2 protein levels in IL-2-stimulated Ag-specific CD8+ T cells were substantially increased, while those in IL-21-stimulated Ag-specific CD8+ T cells were comparable to those seen in Ag-specific CD8+ T cells stimulated in the absence of cytokine. These data suggest that, while Bcl-2 levels do not differ significantly between IL-21-stimulated cells and cells stimulated in the absence of cytokine, low levels of Bcl-2 may be involved in IL-21-induced apoptosis.

We have also examined the stimulation of proapoptotic members of the Bcl-2 family of molecules in IL-21-stimulated Ag-specific CD8+ T cells. These studies suggested that IL-21-induced apoptosis of CD8+ T cells may involve the proapoptotic BH3 family member Bid. Bid activation was demonstrated by intracellular flow cytometric staining on days 7 and 11 following the addition of cytokine to cultures of PBMCs, and on days 2 and 4 following the addition of cytokine to cultures of purified CD8+ T cells. However, while findings were reproducible in experiments using cells from certain monkeys, they were not observed in cells of others of these outbred animals. The reason for the animal-to-animal variation is not clear. IL-21-induced apoptosis of CD8+ T cells also differs from IL-21-induced B cell apoptosis in the novel cytokine’s up-regulation of Bid rather than Bim (38). Bim is downstream of growth-factor withdrawal pathways, while Bid is downstream of death-receptor or caspase pathways (41, 42). Therefore, IL-21-induced CD8+ T cell apoptosis may be an active process in which IL-21 directly stimulates CD8+ T cells to die, while IL-21-stimulated B cells may undergo apoptosis after they proliferate without a concomitant survival signal.

These data raise the question of why it would be evolutionarily advantageous to have a cytokine that induces apoptosis of a differentiated, Ag-experienced CD8+ T cell population. Some have suggested that this cytokine may down-regulate the innate immune response while enhancing the adaptive immune response, facilitating the transition from an innate to a pathogen-specific adaptive immune response. Others have proposed a model in which IL-21 is proapoptotic when lymphocytes are inappropriately or incompletely activated, providing a mechanism for preventing the development of autoimmune disease (9). The data from the present study are consistent with this latter model and suggest that proper activation of primed Ag-specific CD8+ T lymphocytes may require a further signal in addition to cytokine and Ag for proper activation.

Other hypotheses might also explain the ability of IL-21 to induce CD8+ T cell apoptosis. IL-21 may be responsible for triggering the differentiation of lymphocytes to a short-lived effector population. CD8+ T cells divide rapidly upon stimulation and most effector CD8+ T cells undergo apoptosis (43). Although IL-7 and IL-15 may drive lymphocytes to become long-lived memory populations (44), IL-21 stimulation may drive them to become effector populations. Consistent with this hypothesis, the IL-7R{alpha} is preferentially expressed on Ag-specific CD8+ T cells that are committed to become memory cells (45, 46). This hypothesis is also consistent with data indicating that IL-21 production and receptor expression are dysregulated in NOD mice, potentially leading to the turnover of pathogenic T cells in these mice (10). Alternatively, IL-21 could be a critical cytokine in the contraction phase of CD8+ T cell differentiation. It is interesting to note that IL-21 is produced mainly by activated CD4+ T cells, a cell population that peaks at approximately the same time as activated CD8+ T cells. Activated CD4+ T cells may produce IL-21 and this IL-21 may signal CD8+ T cell populations to contract to avoid immunopathology.


   Acknowledgments
 
We thank Michael Gladstone for technical assistance and Michelle Lifton for flow cytometry advice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
Krester Skak is an employee of Novo Nordisk A/S.


   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 the Harvard University Center for AIDS Research, a National Institutes of Health-funded program (P30 AI060354). Back

2 Address correspondence and reprint requests to Dr. Norman L. Letvin, Division of Viral Pathogenesis, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, RE 113, P.O. Box 15732, Boston, MA 02215. E-mail address: nletvin{at}bidmc.harvard.edu Back

3 Abbreviation used in this paper: pSTAT, phosphorylated STAT. Back

Received for publication April 24, 2007. Accepted for publication July 9, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Parrish-Novak, J., S. R. Dillon, A. Nelson, A. Hammond, C. Sprecher, J. A. Gross, J. Johnston, K. Madden, W. Xu, J. West, et al 2000. Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function. Nature 408: 57-63. [Medline]
  2. Zeng, R., R. Spolski, E. Casas, W. Zhu, D. E. Levy, W. J. Leonard. 2007. The molecular basis of IL-21-mediated proliferation. Blood 109: 4135-4142. [Abstract/Free Full Text]
  3. Kasaian, M. T., M. J. Whitters, L. L. Carter, L. D. Lowe, J. M. Jussif, B. Deng, K. A. Johnson, J. S. Witek, M. Senices, R. F. Konz, et al 2002. IL-21 limits NK cell responses and promotes antigen-specific T cell activation: a mediator of the transition from innate to adaptive immunity. Immunity 16: 559-569. [Medline]
  4. Ozaki, K., R. Spolski, C. G. Feng, C. F. Qi, J. Cheng, A. Sher, H. C. Morse, 3rd, C. Liu, P. L. Schwartzberg, W. J. Leonard. 2002. A critical role for IL-21 in regulating immunoglobulin production. Science 298: 1630-1634. [Abstract/Free Full Text]
  5. Brandt, K., S. Bulfone-Paus, D. C. Foster, R. Ruckert. 2003. Interleukin-21 inhibits dendritic cell activation and maturation. Blood 102: 4090-4098. [Abstract/Free Full Text]
  6. Brandt, K., S. Bulfone-Paus, A. Jenckel, D. C. Foster, R. Paus, R. Ruckert. 2003. Interleukin-21 inhibits dendritic cell-mediated T cell activation and induction of contact hypersensitivity in vivo. J. Invest. Dermatol. 121: 1379-1382. [Medline]
  7. van Leeuwen, E. M., L. E. Gamadia, P. A. Baars, E. B. Remmerswaal, I. J. ten Berge, R. A. van Lier. 2002. Proliferation requirements of cytomegalovirus-specific, effector-type human CD8+ T cells. J. Immunol. 169: 5838-5843. [Abstract/Free Full Text]
  8. van Leeuwen, E. M., J. D. van Buul, E. B. Remmerswaal, P. L. Hordijk, I. J. ten Berge, R. A. van Lier. 2005. Functional re-expression of CCR7 on CMV-specific CD8+ T cells upon antigenic stimulation. Int. Immunol. 17: 713-719. [Abstract/Free Full Text]
  9. Leonard, W. J., R. Spolski. 2005. Interleukin-21: a modulator of lymphoid proliferation, apoptosis and differentiation. Nat. Rev. Immunol. 5: 688-698. [Medline]
  10. King, C., A. Ilic, K. Koelsch, N. Sarvetnick. 2004. Homeostatic expansion of T cells during immune insufficiency generates autoimmunity. Cell 117: 265-277. [Medline]
  11. Moroz, A., C. Eppolito, Q. Li, J. Tao, C. H. Clegg, P. A. Shrikant. 2004. IL-21 enhances and sustains CD8+ T cell responses to achieve durable tumor immunity: comparative evaluation of IL-2. IL-15, and IL-21. J. Immunol. 173: 900-909. [Abstract/Free Full Text]
  12. Zeng, R., R. Spolski, S. E. Finkelstein, S. Oh, P. E. Kovanen, C. S. Hinrichs, C. A. Pise-Masison, M. F. Radonovich, J. N. Brady, N. P. Restifo, et al 2005. Synergy of IL-21 and IL-15 in regulating CD8+ T cell expansion and function. J. Exp. Med. 201: 139-148. [Abstract/Free Full Text]
  13. Bolesta, E., A. Kowalczyk, A. Wierzbicki, C. Eppolito, Y. Kaneko, M. Takiguchi, L. Stamatatos, P. A. Shrikant, D. Kozbor. 2006. Increased level and longevity of protective immune responses induced by DNA vaccine expressing the HIV-1 Env glycoprotein when combined with IL-21 and IL-15 gene delivery. J. Immunol. 177: 177-191. [Abstract/Free Full Text]
  14. Alves, N. L., F. A. Arosa, R. A. van Lier. 2005. IL-21 sustains CD28 expression on IL-15-activated human naive CD8+ T cells. J. Immunol. 175: 755-762. [Abstract/Free Full Text]
  15. Alves, N. L., F. A. Arosa, R. A. van Lier. 2006. Common {gamma} chain cytokines: dissidence in the details. Immunol. Lett. 108: 113-120. [Medline]
  16. Santra, S., M. S. Seaman, L. Xu, D. H. Barouch, C. I. Lord, M. A. Lifton, D. A. Gorgone, K. R. Beaudry, K. Svehla, B. Welcher, et al 2005. Replication-defective adenovirus serotype 5 vectors elicit durable cellular and humoral immune responses in nonhuman primates. J. Virol. 79: 6516-6522. [Abstract/Free Full Text]
  17. Perez, O. D., D. Mitchell, R. Campos, G. Gao, L. Li, G. P. Nolan. 2005. Multiparameter analysis of intracellular phosphoepitopes in immunophenotyped cell populations by flow cytometry. J. P. Robinson, 3rd, and Z. Darzynkiewicz, 3rd, and J. Dobrucki, 3rd, and W. C. Hyun, 3rd, and A. Orfao, 3rd, and P. S. Rabinovitch, 3rd, eds. Current Protocols in Cytometry John Wiley and Sons, Inc.,
  18. Ozaki, K., K. Kikly, D. Michalovich, P. R. Young, W. J. Leonard. 2000. Cloning of a type I cytokine receptor most related to the IL-2 receptor beta chain. Proc. Natl. Acad. Sci. USA 97: 11439-11444. [Abstract/Free Full Text]
  19. Asao, H., C. Okuyama, S. Kumaki, N. Ishii, S. Tsuchiya, D. Foster, K. Sugamura. 2001. Cutting edge: the common {gamma}-chain is an indispensable subunit of the IL-21 receptor complex. J. Immunol. 167: 1-5. [Abstract/Free Full Text]
  20. Habib, T., S. Senadheera, K. Weinberg, K. Kaushansky. 2002. The common {gamma} chain ({gamma}c) is a required signaling component of the IL-21 receptor and supports IL-21-induced cell proliferation via JAK3. Biochemistry 41: 8725-8731. [Medline]
  21. Lin, J. X., T. S. Migone, M. Tsang, M. Friedmann, J. A. Weatherbee, L. Zhou, A. Yamauchi, E. T. Bloom, J. Mietz, S. John, et al 1995. The role of shared receptor motifs and common Stat proteins in the generation of cytokine pleiotropy and redundancy by IL-2, IL-4, IL-7, IL-13, and IL-15. Immunity 2: 331-339. [Medline]
  22. Johnston, J. A., C. M. Bacon, M. C. Riedy, J. J. O’Shea. 1996. Signaling by IL-2 and related cytokines: JAKs, STATs, and relationship to immunodeficiency. J. Leukocyte Biol. 60: 441-452. [Abstract]
  23. Leonard, W. J.. 2001. Cytokines and immunodeficiency diseases. Nat. Rev. Immunol. 1: 200-208. [Medline]
  24. Perez, O. D., G. P. Nolan. 2002. Simultaneous measurement of multiple active kinase states using polychromatic flow cytometry. Nat. Biotechnol. 20: 155-162. [Medline]
  25. Dillon, S. R., M. Mancini, A. Rosen, M. S. Schlissel. 2000. Annexin V binds to viable B cells and colocalizes with a marker of lipid rafts upon B cell receptor activation. J. Immunol. 164: 1322-1332. [Abstract/Free Full Text]
  26. Dillon, S. R., A. Constantinescu, M. S. Schlissel. 2001. Annexin V binds to positively selected B cells. J. Immunol. 166: 58-71. [Abstract/Free Full Text]
  27. Koopman, G., C. P. Reutelingsperger, G. A. Kuijten, R. M. Keehnen, S. T. Pals, M. H. van Oers. 1994. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84: 1415-1420. [Abstract/Free Full Text]
  28. Li, Y., M. Bleakley, C. Yee. 2005. IL-21 influences the frequency, phenotype, and affinity of the antigen-specific CD8 T cell response. J. Immunol. 175: 2261-2269. [Abstract/Free Full Text]
  29. Akashi, K., M. Kondo, U. von Freeden-Jeffry, R. Murray, I. L. Weissman. 1997. Bcl-2 rescues T lymphopoiesis in interleukin-7 receptor-deficient mice. Cell 89: 1033-1041. [Medline]
  30. Kovanen, P. E., W. J. Leonard. 2004. Cytokines and immunodeficiency diseases: critical roles of the {gamma}c-dependent cytokines interleukins 2, 4, 7, 9, 15, and 21, and their signaling pathways. Immunol. Rev. 202: 67-83. [Medline]
  31. Otani, H., M. Erdos, W. J. Leonard. 1993. Tyrosine kinase(s) regulate apoptosis and bcl-2 expression in a growth factor-dependent cell line. J. Biol. Chem. 268: 22733-22736. [Abstract/Free Full Text]
  32. Lenardo, M., K. M. Chan, F. Hornung, H. McFarland, R. Siegel, J. Wang, L. Zheng. 1999. Mature T lymphocyte apoptosis–immune regulation in a dynamic and unpredictable antigenic environment. Annu. Rev. Immunol. 17: 221-253. [Medline]
  33. Refaeli, Y., L. Van Parijs, C. A. London, J. Tschopp, A. K. Abbas. 1998. Biochemical mechanisms of IL-2-regulated Fas-mediated T cell apoptosis. Immunity 8: 615-623. [Medline]
  34. Stephanou, A., D. S. Latchman. 2003. STAT-1: a novel regulator of apoptosis. Int. J. Exp. Pathol. 84: 239-244. [Medline]
  35. Chin, Y. E., M. Kitagawa, K. Kuida, R. A. Flavell, X. Y. Fu. 1997. Activation of the STAT signaling pathway can cause expression of caspase 1 and apoptosis. Mol. Cell. Biol. 17: 5328-5337. [Abstract]
  36. Mehta, D. S., A. L. Wurster, M. J. Whitters, D. A. Young, M. Collins, M. J. Grusby. 2003. IL-21 induces the apoptosis of resting and activated primary B cells. J. Immunol. 170: 4111-4118. [Abstract/Free Full Text]
  37. Ozaki, K., R. Spolski, R. Ettinger, H. P. Kim, G. Wang, C. F. Qi, P. Hwu, D. J. Shaffer, S. Akilesh, D. C. Roopenian, et al 2004. Regulation of B cell differentiation and plasma cell generation by IL-21, a novel inducer of Blimp-1 and Bcl-6. J. Immunol. 173: 5361-5371. [Abstract/Free Full Text]
  38. Jin, H., R. Carrio, A. Yu, T. R. Malek. 2004. Distinct activation signals determine whether IL-21 induces B cell costimulation, growth arrest, or Bim-dependent apoptosis. J. Immunol. 173: 657-665. [Abstract/Free Full Text]
  39. Brady, J., Y. Hayakawa, M. J. Smyth, S. L. Nutt. 2004. IL-21 induces the functional maturation of murine NK cells. J. Immunol. 172: 2048-2058. [Abstract/Free Full Text]
  40. Toomey, J. A., F. Gays, D. Foster, C. G. Brooks. 2003. Cytokine requirements for the growth and development of mouse NK cells in vitro. J. Leukocyte Biol. 74: 233-242. [Abstract/Free Full Text]
  41. Opferman, J. T., S. J. Korsmeyer. 2003. Apoptosis in the development and maintenance of the immune system. Nat. Immunol. 4: 410-415. [Medline]
  42. Strasser, A.. 2005. The role of BH3-only proteins in the immune system. Nat. Rev. Immunol. 5: 189-200. [Medline]
  43. Seder, R. A., R. Ahmed. 2003. Similarities and differences in CD4+ and CD8+ effector and memory T cell generation. Nat. Immunol. 4: 835-842. [Medline]
  44. Schluns, K. S., L. Lefrancois. 2003. Cytokine control of memory T-cell development and survival. Nat. Rev. Immunol. 3: 269-279. [Medline]
  45. Kaech, S. M., J. T. Tan, E. J. Wherry, B. T. Konieczny, C. D. Surh, R. Ahmed. 2003. Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells. Nat. Immunol. 4: 1191-1198. [Medline]
  46. Huster, K. M., V. Busch, M. Schiemann, K. Linkemann, K. M. Kerksiek, H. Wagner, D. H. Busch. 2004. 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 101: 5610-5615. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
C. K. Holm, C. C. Petersen, M. Hvid, L. Petersen, S. R. Paludan, B. Deleuran, and M. Hokland
TLR3 Ligand Polyinosinic:Polycytidylic Acid Induces IL-17A and IL-21 Synthesis in Human Th Cells
J. Immunol., October 1, 2009; 183(7): 4422 - 4431.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
W. Hou, H. S. Kang, and B. S. Kim
Th17 cells enhance viral persistence and inhibit T cell cytotoxicity in a model of chronic virus infection
J. Exp. Med., February 16, 2009; 206(2): 313 - 328.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
M.-C. Huang, J.-J. Liao, S. Bonasera, D. L. Longo, and E. J. Goetzl
Nuclear factor-{kappa}B-dependent reversal of aging-induced alterations in T cell cytokines
FASEB J, July 1, 2008; 22(7): 2142 - 2150.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Barker, B. R.
Right arrow Articles by Letvin, N. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Barker, B. R.
Right arrow Articles by Letvin, N. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS