Published online before print June 6, 2007
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,1
* Maisonneuve-Rosemont Hospital Research Center, Departments of
Medicine and
Microbiology and Immunology, University of Montreal, Montreal, Quebec, Canada
1 Correspondence: Research Center, Maisonneuve-Rosemont Hospital, 5415 boul. de l’Assomption, Montreal, Qc, Canada HIT 2M4. E-mail: nathalie.labrecque{at}umontreal.ca
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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chain (c) in its receptor. As members of the c cytokine/cytokine receptors family play crucial role in the differentiation, activation, and survival of lymphocytes, we have investigated if IL-21 could promote T cell survival and thus, contribute to T cell homeostasis and expansion. Unlike most c cytokine receptors, we report that IL-21R is constitutively expressed by all mature T lymphocytes and that stromal cells of lymphoid organs are a constitutive source of IL-21. These observations are reminiscent of what is observed for IL-7/IL-7R, which control T cell survival and homeostasis and suggest a role for IL-21 in T cell homeostasis. Indeed, our results show that IL-21 is a survival factor for resting and activated T cells. Moreover, the ability of IL-21 to costimulate T cell proliferation is mediated by enhancing T cell viability. Further investigation of how IL-21R signaling induces T cell survival shows for the first time that IL-21 binding to its receptor activates the PI-3K signaling pathway and induces Bcl-2 expression. Moreover, the activation of the PI-3K signaling pathway is essential for IL-21-mediated T cell survival. Our data provide a new role for IL-21 in the immune system, which might be used to improve T cell homeostasis in immunocompromised patients.
Key Words: cytokine • cytokine receptor • homeostasis • Bcl-2
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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chain (c), including IL-2, IL-4, IL-7, IL-9, IL-15, and the recently identified IL-21 (c cytokines). Each of these cytokines binds to a receptor composed of one or two specific chain(s) associated with c [1
]. It is important that most of these cytokines and receptors are not expressed constitutively on T lymphocytes but are rather induced following activation.
The development and life of lymphocytes are regulated by discrete c cytokines, which orchestrate homeostasis of the immune system. The use of mice deficient in cytokines and receptors of the c family has revealed their importance for lymphoid development, survival, and activation. In particular, c cytokines play pivotal roles in T cell development and in regulating mature T cell homeostasis. For example, IL-7 is essential for T cell development in the thymus [2
3
4
5
6
]. Moreover, the maintenance of the naïve T cell pool is regulated by IL-7, which promotes naïve T cell survival [7
8
9
] and allows homeostatic expansion of naïve T cells in a lymphopenic situation [8
9
10
]. It is interesting that the survival and maintenance of CD8+ memory T lymphocytes require another c cytokine member, IL-15 [10
11
12
13
14
15
], and IL-7 promotes CD4+ memory T cell survival [16
, 17
]. Distinct c cytokines are involved in the regulation of T cell proliferation and differentiation following Ag encounter. Although IL-2 and IL-15 are potent inducers of T cell proliferation during in vitro stimulation, they are dispensable in vivo [13
, 14
, 18
19
20
21
22
23
24
25
]. It is still unclear which cytokines drive in vivo expansion of Ag-specific T cells. However, IL-2 is critical in the re-establishment of T cell homeostasis after Ag clearance [22
, 23
, 26
]. Concomitant with T cell death occurring at the end of the T cell response to Ag, some T lymphocytes differentiate further into memory T cells, a process dependent on c cytokines. IL-7 promotes CD4+ and CD8+ memory T cell generation [9
, 16
, 27
]. We postulated that the identification of a new c cytokine, IL-21, may bring new insights into the regulation of T cell survival, proliferation, and differentiation.
IL-21 and its receptor (IL-21R
) are the most recent c cytokine/receptor pair identified [28
29
30
]. Activated CD4+ T lymphocytes and NKT cells are the only identified source of IL-21 in the immune system [28
, 30
31
32
]. Conversely, IL-21R is widely expressed in the immune system, including T cells, B cells, NK cells, NKT cells, and some cells of myeloid origin [28
29
30
, 33
34
35
]. This wide distribution of IL-21R expression in the immune system reflects the pleiotropic action of IL-21 on immune cells (for review, see ref. [30
]). In T lymphocytes, IL-21 augments TCR-induced proliferation in addition to increasing IFN- production and effector functions in CTLs [28
, 36
37
38
39
40
]. Furthermore, IL-21 enhances anti-tumor activity of CD8+ T cells [37
, 38
, 41
42
43
44
45
] and NK cells [37
, 46
, 47
]. IL-21 was also found to synergize with IL-15 and to a lesser extent, with IL-7, in regulating the function and expansion of naive and memory-phenotype CD8+ T lymphocytes [41
]. Moreover, Ag-driven CD8+ T cell expansion and function are diminished in IL-21R-deficient mice [41
]. In addition to its effect on T cells, IL-21 influences B cell responses (for review, see ref. [30
]). In summary, IL-21 plays a critical role in the regulation of B cell function, as IL-21R–/– mice have higher production levels of IgE but lower IgG1 than wild-type animals following immunization [48
]. IL-21 was also shown to promote memory B lymphocyte and plasma cell differentiation [49
, 50
]. However, in the absence of CD4+ T cell help, IL-21 induces B cell apoptosis [33
, 51
].
In this study, we have investigated if IL-21 could promote T cell survival and thus contribute to T cell homeostasis and expansion. It is interesting that we have identified a new, constitutive source of IL-21 in lymphoid organs. We have also demonstrated that IL-21 promotes naive T cell survival and that IL-21 enhances T cell proliferation by promoting survival. We have investigated further how IL-21R signaling induces T cell survival. We show for the first time that IL-21 binding to its receptor activates the PI-3K signaling pathway and induces Bcl-2 expression. Moreover, the activation of the PI-3K signaling pathway is essential for IL-21-mediated T cell survival.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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chain [52
] and C57BL/6 (B6) mice were bred under specific, pathogen-free (SPF) conditions.
Production of IL-21Fc
Murine IL-21 cDNA was amplified by RT-PCR on mRNA extracted from splenocytes, which were stimulated with anti-CD3 antibodies (1 µg/ml) coated on plates for 16 h. PCR was performed on cDNA using mouse IL-21 primers: 5' primer, 5'-AAC TGC AGG GTG GCA TGG AGA GGA CCC TT-3' (PstI and nucleotides 48–68 of IL-21); 3' primer, 5'-CGG GGT ACC GGA GAG ATG CTG ATG AAT CAT-3' (nucleotides 471–491 of IL-21 and KpnI). The PCR product was digested with PstI and KpnI and ligated into the PstI–KpnI-digested CosFcLink vector. IL-21Fc fusion protein was produced by transient transfection of Cos-7 cells. After 96 h, the supernatant containing IL-21Fc was recovered and quantified by dot blot.
Antibodies and flow cytometry
Anti-CD4 (L3/T4), anti-CD25 (2541), and goat F(ab')2 anti-human IgG (Fc-specific) antibodies were purchased from Cedarlane Laboratories (Hornby, Ontario, Canada). Anti-CD4 (CT-CD4), anti-CD8 (CT-CD8a), anti-B220 (RA3-6B2), anti-mouse IgM, anti-CD25 (PC61 5.3), anti-CD44 (1M7.8.1), and anti-CD62L (Mel-14) antibodies were from Caltag (Burlingame, CA, USA). Anti-CD8 (53.6.7), anti-CD3 (145-2C11), anti-TCR 
(GL3), anti-CD45RB (16A), anti-IA (AF16-120.1), and anti-BrdU antibodies were purchased from BD Biosciences (San Jose, CA, USA). Anti-IL-7R (A7R34) antibody was from eBioscience (San Diego, CA, USA). Fluorescently labeled streptavidins were purchased from BD Biosciences.
Cell stainings were performed on ice in FACSwash (DMEM without phenol red, 3% heat-inactivated horse serum, 0.03 M Hepes, and 0.1% NaN3). Samples were analyzed on a FACSCalibur or a FACScan using CellQuest software (Becton Dickinson, Mountain View, CA, USA).
Cell size was evaluated by flow cytometry by determining the forward light-scatter (FSC).
Detection of IL-21R expression
Cells (106) were incubated with 50 ng IL-21Fc or human IgG for 30 min on ice. Binding of IL-21Fc to IL-21R was detected with biotin goat F(ab')2 anti-human IgG (Fc-specific) followed by streptavidin-APC or -PerCP.
Intracellular Bcl-2 staining
Lymphocytes were permeabilized in 0.1% saponin/FACSwash (Sigma-Aldrich, Oakville, ON, Canada) and stained with anti-Bcl-2 antibodies (3F11, BD Biosciences) or isotype control (A19-3) diluted in 0.1% saponin/FACSwash for 30 min at room temperature. Cells were washed once in 0.1% saponin/FACSwash and twice in FACSwash before surface staining.
RT-PCR
Lymphocyte subpopulations (20,000) were sorted directly in 1 ml Trizol (Invitrogen, Burlington, Ontario, Canada). Stromal cells were enriched from C–/– lymph nodes (LN) as follows. LN were mechanically disrupted, and the nonsuspendable fraction was used to extract stromal cell RNA [53
], which was also extracted from various organs using Trizol, according to the manufacturer’s instructions. cDNA was synthesized using Superscript RT and synthetic oligo(dT) (Invitrogen). PCR was performed with mouse IL-21 primers: forward, 5'-AAC TGC AGG GTG GCA TGG AGA GGA CCC TT-3', and reverse, 5'-CGG GGT ACC GGA GCG ATG CTG ATG AAT CAT-3'. The primers for the internal control hypoxanthine guanine phosphoribosyl transferase (HPRT) were: forward, 5'-GTA ATG ATC AGT CAA CGG GGG AC-3', and reverse, 5'-CCA GCA AGC TTG CAA CCT TAA CCA-3'. The stromal cell-specific primers from the gp38 gene [53
, 54
] were: forward, 5'-CTCTGGTACCAACGCAGAGA-3', and reverse, 5'-TTAGGGCGAGAACCTTCCA-3'.
T cell stimulation
LN cells from B6 mice were resuspended at 107 cells/ml in PBS and incubated with 0.5 mM CFSE (Molecular Probes, Eugene, OR, USA) at 37°C for 10 min and then washed in FBS [55
]. CFSE-labeled cells (2x106) were stimulated in 24-well plates coated with different concentrations (0.3, 0.6, 0.9 µg/ml) of anti-CD3 antibodies in the presence or absence of IL-21Fc (50 ng/ml). After 72 h, cells were stained with anti-CD4 and anti-CD8 antibodies. Cell viability was determined using the death exclusion dye Trypan blue (Invitrogen).
In vitro T cell survival assay
LN cells (2x106) or purified T lymphocytes were cultured in complete RPMI (Invitrogen) without stimulation for 24, 48, and 72 h at 37°C in the presence or absence of 25 ng/ml mouse recombinant (mr)IL-21 (R&D Systems, Minneapolis, MN, USA) or 1 ng/ml mrIL-7 (Medicorp, Montreal, QC, Canada) in 24-well plates. At each time-point, viability was evaluated by staining cells with anti-CD4 antibodies, anti-CD8 antibodies, and 7-amino-actinomycin (7-AAD; BD Biosciences). In some experiments, T cells were enriched by depletion of B220+ and MHC Class II+ cells; 108 cells were incubated with antibodies against B220 and Class II for 30 min on ice, washed, and incubated with magnetic beads coated with anti-rat antibodies (Dynal, Lake Success, NY, USA) for 30 min. Cells coated with beads were removed magnetically. After depletion, T cell purity was at least 95%. CD4+ and CD8+ T lymphocytes were also sorted based on CD44 expression (CD44low, CD44high) using a FACSVantage SE (Becton Dickinson). In some experiments, 10 µM of the PI-3K inhibitor LY294002 (Calbiochem, La Jolla, CA, USA) was added to the culture [56
].
Western blotting
Lymphocytes were cultured for 2, 4, 8, 24, and 48 h in the presence or absence of rIL-21 25 ng/ml or rIL-7 1 ng/ml or were freshly isolated. Cells were lysed in 0.5% Nonidet P-40, 100 mM NaCl, 2 mM DTT, 1 mM EDTA, 20 mM Hepes, 1 mM PMSF, and 1 µg/ml aprotinin [57
]. Lysates containing 3 x 106 cells were run on 15% SDS-PAGE, and proteins were transferred to a nitrocellulose membrane (Amersham Biosciences Inc., Baie d’Urfé, Quebec, Canada). Blots were probed with polyclonal anti-Bcl-xL (BD Biosciences), anti-Akt, antiphospho-Akt (Ser473), antiphospho-BAD (Ser112, Cell Signaling Technology, Pickering, Ontario, Canada), and anti-GAPDH antibodies (Chemicon International Inc., Mississauga, ON, Canada) and subsequently, with anti-rabbit or mouse HRP-conjugated antibodies (Cell Signaling Technology or Sigma-Aldrich). All blots were developed using ECL Western blotting reagents and exposed to film (Amersham Biosciences Inc.).
Statistics
The two-tailed unpaired Student’s t-test was used for statistical analysis.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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When we started this study, the expression pattern of IL-21R by immune cells was not well characterized as a result of the lack of a mAb specific for this receptor. To identify which immune cells express IL-21R, we used our IL-21Fc protein, as the binding of this protein to its receptor can be measured by FACS using antibodies against the Fc portion of the fusion protein. First, we tested whether our IL-21Fc fusion protein can be used to detect IL-21R on B lymphocytes, a cell type known to express this receptor [28
, 29
]. As shown in Figure 1A
, our IL-21Fc reagent could efficiently detect IL-21R on B cells. We then asked whether mature T cells from LN expressed IL-21R. We easily detected IL-21R expression by all T cells, including CD4+, CD8+, and + T lymphocytes (Fig. 1A)
. This staining is not an artifact of our IL-21Fc fusion protein, as the binding of IL-21Fc to T cells was totally inhibited by the addition of rIL-21 (Supplemental Fig. 2). Moreover, we have observed transcription of the IL-21R gene in purified CD4+ and CD8+ T cells (not shown). A previous report was unable to detect IL-21R expression by resting human T cells using a biotinylated form of IL-21 [28
]. However, IL-21R expression by murine T cells was detected using a novel mAb [33
], confirming our observation. Knowing that mature T lymphocytes expressed IL-21R, we then sought to determine at which step of their differentiation IL-21R expression originated on T cells. As shown in Figure 1B
, IL-21R expression was low on CD4+CD8+ (DP) thymocytes and was up-regulated in thymocytes at the SP stage of T cell maturation. It is interesting that the expression of IL-21R on T cells is reminiscent of what is observed for IL-7R. The comparison of IL-7R and IL-21R expression on T cell subsets (Fig. 1A
and 1B)
confirmed the parallel expression of IL-21R and IL-7R during T cell differentiation. As the IL-7R expression is also regulated tightly during the maturation of DN, immature thymocytes, we further investigated if IL-21R expression were also occurring in immature thymocytes. Figure 1C
shows that the early, immature DN1 (CD44+CD25–) subset of thymocytes expressed IL-21R at a low level, and expression is shut-off at the DN2 stage. The low level of expression of IL-21R by DN1 and DP thymocytes was confirmed by RT-PCR analysis (data not shown). The observation that IL-21R expression is regulated like IL-7R during T cell maturation prompted us to determine if a similar phenomenon was occurring during B cell development. IL-21R is present at every stage of B cell development, from early, pro-B cells to mature B cells (Fig. 1D)
. In contrast to IL-7R expression, IL-21R expression is not down-regulated on mature, bone marrow B lymphocytes (Fig. 1D)
.
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Figure 1. IL-21R expression on lymphocyte subsets. (A) IL-21R and IL-7R expression on CD4+, CD8+, +, and B cells. LN or spleen cells were stained with IL-21Fc fusion protein or with anti-IL-7R antibody in combination with anti-CD4, anti-CD8, anti-TCR, anti-CD3, anti-B220, and anti-IgM antibodies. IL-21R and IL-7R surface expression (thick lines) is shown for the different subsets relative to isotype control staining (thin lines, human IgG for IL-21R and rat IgG2a for IL-7R). (B) IL-21R and IL-7R expression on double-negative (DN), double-positive (DP), CD4+ single-positive (CD4SP), and CD8+ SP (CD8SP) thymocytes, which were stained for surface IL-21R or IL-7R in combination with anti-CD4 and anti-CD8 mAb. (C) IL-21R and IL-7R expression on DN thymocyte subpopulations. DN thymocyte subsets were analyzed by excluding lineage-positive cells and stained with anti-CD44 and anti-CD25 antibodies. IL-21R and IL-7R expression is shown on DN1 (CD44+CD25–), DN2 (CD44+CD25+), DN3 (CD44–CD25+), and DN4 (CD44–CD25–). (D) IL-21R and IL-7R expression on bone marrow, small, pre-, immature, and mature B cells. Bone marrow cells were stained for surface IL-21R or IL-7R in combination with anti-B220 and anti-IgM antibodies.
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Figure 2. IL-21 expression in lymphoid cells and organs. (A) IL-21 mRNA expression by CD4+, CD8+, and B cells. IL-21 and HPRT-specific RT-PCRs were performed on RNA extracted from sorted CD4+, CD8+, or B220+ LN cells. (B) Expression of IL-21 by activated/memory CD4+ T cells. IL-21 and HPRT-specific PCRs were performed on RNA extracted from CD4+CD25–CD44–CD62L+ (naive), CD4+CD44+ (activated/memory), CD4+CD25–CD44+ (activated/memory), and CD4+CD25+CD44+ (activated)-sorted cells. (C) IL-21 mRNA expression by different subsets of CD4+ T lymphocytes. IL-21 and HPRT-specific PCRs were performed on RNA extracted from 10,000 CD4+CD25–CD45RBlow (memory) and CD4+CD25+CD45RBlow (regulatory)-sorted cells. (D) Constitutive expression of IL-21 by nonlymphoid cells. RNA was extracted from LN, spleen (S), thymus (T), and kidney (K) of B6 and C–/– (C°) mice using Trizol. (E) Lack of expression of IL-21 by dendritic cells (DC), monocytes, , and NK cells. RNA was extracted from 20,000 sorted DC (CD11c+Class II+), monocytes (CD11b+Class II+), T cells, and NK cells (NK+CD3–). RT-PCRs were performed using specific IL-21 and HPRT primers. (F) IL-21 is produced by nonlymphoid stromal cells in C–/– LN. RNA was extracted from LN cell suspension obtained after mechanical disruption (LN cells) and from the remaining nonsuspendable fraction (LN stroma). Semiquantitative PCR was performed on serial dilutions (1:5) of cDNA using specific IL-21 primers. RT-PCR, using gp38-specific primers, was performed to monitor for stromal cell enrichment. Data are representative of at least three independent experiments.
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ß T cells (C–/–) and thus lacking CD4+ and CD8+ T lymphocytes. As shown in Figure 2D
, IL-21 mRNA was detected in LN of C–/– mice. We could also detect IL-21 mRNA in LN derived from RAG-deficient animals (lacking ß+, +, and B cells; not shown). These results suggest the existence of another cell type transcribing the IL-21 gene in lymphoid organs, as the IL-21 mRNA was undetectable in B cells (Fig. 2A)
, T cells, monocytes, NK cells, and DC (Fig. 2E)
. This leads us to test if stromal cells from lymphoid organs transcribe the IL-21 gene. To do so, we have enriched stromal cells from the LN of C–/– mice and analyzed IL-21 expression by RT-PCR. It was important to perform this analysis with the stromal cell fraction isolated from C-deficient mice to avoid any possible contamination from CD4+ T cells and NKT cells, which are the main source of IL-21 mRNA [28
, 30
31
32
]. As shown in Figure 2F
, IL-21 mRNA was enriched in the stromal cell fraction isolated from C–/– LN. We were also able to detect IL-21 mRNA in the spleen and thymus, but not kidneys, from C–/– mice (Fig. 2D)
, suggesting that stromal cells from all lymphoid organs can transcribe the IL-21 gene. Further studies are needed to confirm that IL-21 protein is made by stromal cells, but this requires the development of a better reagent to detect the IL-21 protein.
IL-21 enhances T cell stimulation via survival
Previous reports have shown that IL-21 costimulates T cell proliferation to anti-CD3 antibodies [28
, 36
], but it is still unknown if T cell proliferation of CD4+ and CD8+ T cells is enhanced by IL-21. Until now, IL-21 has only been shown to increase the proliferation of antigen-specific CD8+ T cells [30
, 40
41
42
]. Therefore, we evaluated if IL-21 could costimulate the proliferation of CD4+ and CD8+ T cells using CFSE-labeled T cells. As expected, we detected enhanced thymidine incorporation when IL-21 was added to LN cells stimulated with anti-CD3 antibodies (Supplemental Fig. 1). It is interesting that this was not associated with an increase in the number of cell divisions made by CD4+ and CD8+ T cells when this was monitored using CFSE-labeled cells (Fig. 3A
). Moreover, IL-21 did not increase the percentage of T cells, which have divided (Fig. 3A)
, nor modify the frequency of T cells, which have reached a certain number of divisions (Fig. 3A)
. Furthermore, IL-21 did not accelerate T cell division, as no difference in CFSE dilution was observed after 48 h of anti-CD3 stimulation in the presence or absence of IL-21 (data not shown). This apparent discrepancy between the thymidine incorporation and CFSE results was reconciled when we estimated the total T cell number, the number of cells present at each division, and the viability of cells at the end of the culture (Fig. 3B
3C
3D)
. As shown in Figure 3B
, there were more T cells, which were recovered at each division when IL-21 was added to the culture. This was statistically significant (P<0.05) for divisions 1–3 in CD4+ cells and for divisions 1 and 2 in CD8+ lymphocytes. Moreover, the number of recovered cells and the viability at the end of the culture were higher in the presence of IL-21 when a suboptimal dose of anti-CD3 antibodies was used (Fig. 3C
and 3D)
. The results presented in Figure 3
were seen consistently in eight independent experiments. These results suggest that the principal effect of IL-21 on T cells stimulated with anti-CD3 antibodies consists of enhancing T cell survival but not proliferation.
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Figure 3. Effect of IL-21 on T cell proliferation is mediated by enhanced cell survival. (A) Proliferation of CD4+ and CD8+ T cells stimulated with anti-CD3 antibodies in the presence or absence of IL-21Fc. CFSE-labeled LN cells were in vitro-stimulated with anti-CD3 antibodies (0.6 µg/ml) for 72 h in the presence or absence of IL-21Fc (50 ng/ml). CFSE profiles gated on CD4+ or CD8+ T cells are shown for cells stimulated in the presence of IL-21Fc (bold lines) or absence (thin lines). (B) Number of cells present at each division in the absence or presence of IL-21Fc (50 ng/ml). The number of cells in each division was obtained by multiplying the total number of cells by the percentage of cells in each division. (C) Total cell number recovery at the end of 72 h of anti-CD3 stimulation in the presence or not of IL-21Fc. (D) Cell viability following anti-CD3 stimulation in the presence or absence of IL-21. The percentage of viability in the presence or absence of IL-21Fc was obtained with Trypan blue staining. The data are representative of eight independent experiments; each dot represents one mouse. Statistical differences were determined with a Student’s t-test (*, P<0.05, and **, P<0.01).
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Figure 4. IL-21 promotes T cell survival. (A) Viability of CD4+ and CD8+ T cells in the presence or absence of rIL-21 or rIL-7. Purified T cells from B6 mice were cultured in vitro for 72 h without stimulation in the presence or absence of rIL-21 (25 ng/ml) or rIL-7 (1 ng/ml). The percentage of viability was obtained by staining with 7-AAD in combination with anti-CD4 and anti-CD8 antibodies at different time-points. (B) Dose-dependent effect of IL-21 on the survival of CD4+ and CD8+ T cells. Lymphocytes from B6 mice were cultured in vitro for 72 h without stimulation in the presence of 1, 5, 10, or 25 ng/mL rIL-21. The percentage of viability was obtained by staining with 7-AAD in combination with anti-CD4 and anti-CD8 antibodies at different time-points. Statistical differences were evaluated with a Student’s t-test (**, P<0.01). (C) Lack of proliferation of CD4+ and CD8+ T cells in the presence of rIL-21. Lymphocytes from B6 mice were cultured in vitro for 72 h in the presence or absence of rIL-21 (25 ng/mL), rIL-7 (1 ng/mL), or anti-CD3 antibodies (1 µg/mL). BrdU (10 µM) was added at the start of cell culture, and BrdU incorporation was measured in CD4+ and CD8+ T cells by flow cytometry. Each experiment was done in triplicate, and the data are representative of at least three independent experiments.
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Figure 5. IL-21 mediates naïve CD4+ and CD8+ as well as activated/memory CD8+ T lymphocyte survival. (A) Viability of CD4+ and CD8+ T cells based on their CD44 expression in the presence or absence of rIL-21 or rIL-7. T lymphocytes from B6 mice were cultured in vitro for 72 h without stimulation in the presence or absence of rIL-21 (25 ng/mL) or rIL-7 (10 ng/mL). The percentage of viability was obtained by staining cells with 7-AAD in combination with antibodies against CD4, CD8, and CD44 after 72 h of culture. (B) Viability of sorted CD4+ and CD8+ T cells based on their CD44 expression in the presence or absence of rIL-21 or rIL-7. LN T lymphocytes were sorted as CD4+CD44low, CD4+CD44high, CD8+CD44low, or CD8+CD44high and cultured in vitro for 72 h at 106/mL in medium containing rIL-21 (25 ng/mL) or rIL-7 (10 ng/mL). The percentage of viability was obtained by staining cells with 7-AAD after 72 h of culture. (C) IL-21 does not induce differentiation of naïve T lymphocytes. The CD44 expression level before cell sorting (thin lines), after sorting (dotted lines), and after a 72-h culture with rIL-21 (bold lines) for CD4+ and CD8+ T cells is shown. Data are representative of three independent experiments. Statistics were done using a bilateral Student’s t-test (*, P<0.05; **, P<0.01; ****, P<0.0005; and *****, P<0.00005). ns, Not significant.
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Figure 6. IL-21 promotes T cell survival and cell size maintenance by activating the PI-3K pathway. (A) Viability of CD4+ and CD8+ T cells in the presence of the PI-3K pathway inhibitor, LY294002 (LY). Total LN cells were cultured in vitro for 72 h without stimulation in the presence or absence of rIL-21 (25 ng/ml) or rIL-7 (1 ng/ml) and in the presence or absence of LY294002 (10 µM). The percentage of viability was obtained by staining with 7-AAD in combination with anti-CD4 and anti-CD8 antibodies at different time-points. Each experiment was done in triplicate, and the data are representative of at least three independent experiments. Statistical differences were evaluated with a Student’s t-test (*, P<0.05). (B) IL-21 maintains T cell size. Total LN cells were cultured in vitro for 72 h without stimulation in the presence or absence of rIL-21 (25 ng/ml) or rIL-7 (1 ng/ml) and in the presence or absence of LY294002 (10 µM). The size of T cells was determined by FACS analysis of the mean FSC. This experiment was done in duplicate and is representative of at least three independent experiments. Statistical differences were evaluated with a Student’s t-test (**, P<0.01).
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Figure 7. IL-21 biochemical pathway promoting T cell survival. (A) Bcl-2 expression by CD4+ and CD8+ T cells in the presence or absence of rIL-21 or rIL-7. Total LN cells were cultured in vitro for 72 h without stimulation in the presence or absence of rIL-21 (25 ng/ml) or rIL-7 (1 ng/ml). Bcl-2 expression was determined by intracellular staining with anti-Bcl-2 FITC or isotype control FITC antibodies, followed by surface staining with anti-CD4 and anti-CD8 antibodies. (B) Bcl-xL expression by lymphocytes in the presence or absence of rIL-21 or rIL-7. (C) Phosphorylation of Ser473 of Akt (P-Akt Ser473) in the presence or absence of rIL-21 or rIL-7. (D) Phosphorylation of Bad (Ser112; P-Bad Ser112) in the presence or absence of rIL-21 or rIL-7. Levels of Bcl-xL, phospho-Akt, and phospho-Bad were determined by Western blot on cell lysates, which were made from total LN cells cultured in vitro for the indicated time in the presence of absence of rIL-21 (25 ng/ml) or rIL-7 (1 ng/ml) or from freshly isolated lymphocytes. Membranes were then stripped and rehybridized with antibodies against GAPDH or Akt to control for loading.
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c can also lead to an increase in the Bcl-XL level, but in our hands, neither IL-7 nor IL-21 led to up-regulation of this antiapoptotic protein in resting T cells (Fig. 7B)
. We have also confirmed that the PI-3K pathway is induced in T cells cultured in the presence of IL-21 by looking at the levels of Akt and Bad phosphorylation, which are downstream targets of PI-3K. As shown in Figure 7C
and 7D
, the levels of Akt and Bad phosphorylation were increased in the presence of IL-21 and IL-7. It is interesting that IL-21-induced phosphorylation of Akt is faster and stronger than for IL-7 (Fig. 7C)
. Moreover, Bad phosphorylation is more sustained with IL-21 than IL-7 (Fig. 7D)
. The induction of the PI-3K signaling pathway by IL-7 in T cells is well documented [58
59
60
61
62
63
] and is not essential for IL-7-mediated T cell survival [58
]. However, the induction of the PI-3K signaling pathway by IL-7 is critical for the maintenance of the metabolic state of the cell and thus, of its size (known as the trophic effect) [58
]. As IL-21 activates the PI-3K signaling pathway, we have also determined if IL-21 can maintain the size of T cells and if this were dependent on the activation of PI-3K. As shown in Figure 6B
, IL-21 is able to maintain cellular size, and this effect was lost when LY294002 was added. |
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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mAb [33
], except that we only detect low levels of IL-21Fc binding to DP thymocytes. This could be a result of different sensitivities of the assay or to the absence of a functional IL-21R on DP thymocytes, despite detection of IL-21R expression by the mAb.
The constitutive expression of IL-21R by CD4+ and CD8+ T cells is intriguing, as most Type I cytokine receptors are induced following T cell activation. IL-21R expression is similar to another Type I cytokine receptor, IL-7R, which plays a critical role in T cell survival and in the induction of homeostatic proliferation in lymphopenic mice [7
8
9
10
]. The expression of IL-21R by T cells is probably important for their survival and/or to allow them to be responsive to IL-21 as soon as an immune response is elicited. Our identification of a possible constitutive production of IL-21 by stromal cells of lymphoid organs is also in favor of a role for IL-21 in regulating T cell survival in physiological conditions. Further experiments are needed to address this issue. Moreover, we have shown that IL-21 can prolong T cell life in vitro. No defect in the number of T cells in lymphoid organs has been reported in IL-21R–/– mice, but T cell survival was never assessed directly (i.e., determination of the half-life of peripheral T cells in the absence of thymic output). It is interesting that a recent report showed that the treatment of mice with an IL-21R-Fc fusion protein leads to a reduction in the number of CD4+ and CD8+ T cells in the spleen [64
]. These observations and our results open the possibility that IL-21 contributes to T cell survival and homeostasis in vivo. Furthermore, our data are in agreement with the report of Zeng et al. [41
], who have observed enhanced in vitro survival of resting CD8+ T cells in the presence of IL-21.
Our study also highlights the mechanism by which IL-21 costimulates T cell proliferation. Unlike other Type I cytokines, IL-21 does not increase the number of divisions but increases the clonal burst size by enhancing cell viability. This is in contrast with other studies, which have reported that IL-21 increases antigen-induced CD8+ T cell proliferation in vivo [41 , 42 ]. Increased proliferation is probably observed in vivo as a result of the presence of IL-15, which is able to synergize with IL-21 in enhancing CD8+ T cell proliferation [41 , 65 ]. Although IL-21 does not increase the proliferation rate of T cells, the ability of IL-21 to promote T cell survival might be beneficial toward increasing T cell expansion and differentiation of effector cells into memory cells during an immune response.
We have also determined the intracellular signaling pathway involved in the promotion of T cell survival by IL-21. We show for the first time that IL-21 binding to its receptor on T cells leads to the activation of the PI-3K signaling pathway. The presence of the consensus site for the docking of the p85 regulatory subunit of PI-3K, YXXM [66
], in the cytoplasmic tail of the mouse IL-21R chain (Tyrosine-397) suggests direct recruitment of PI-3K to the IL-21R. However, we cannot exclude the possibility that PI-3K is activated without a direct recruitment to the IL-21R, as reported with other cytokine/cytokine receptor systems [67
68
69
70
71
]. Further studies are needed to determine if PI-3K is recruited directly to the IL-21R or if PI-3K is activated indirectly by other signaling molecules such as insulin receptor substrate [67
68
69
]. In agreement with our results, a recent report by Zeng et al. [72
] described a weak induction of Akt phosphorylation in CD8+ T cells by IL-21. It is interesting that IL-21-mediated survival of T cells is dependent on a different biochemical pathway than IL-7. The inhibition of the PI-3K pathway, using the specific inhibitor LY294002, abrogates the survival effect of IL-21 on T lymphocytes, and its does not interfere with T cell survival mediated by IL-7. As IL-21 and IL-7 induce the phosphorylation of Akt and Bad, which are downstream targets of PI-3K, it indicates that the activation of PI-3K is not necessary for the induction of T cell survival by IL-7, and it is absolutely necessary for IL-21. IL-7 is probably able to induce T cell survival without the induction of the PI-3K pathway, as a result of its ability to increase the Bcl-2 level in T cells, and IL-21 will only allow for normal levels of Bcl-2 to be maintained. In the absence of PI-3K signaling, Bad is not phosphorylated and translocates to the mitochondria, where it inhibits the antiapoptotic function of Bcl-2 [73
74
75
76
]. Therefore, it is possible that the inhibition of Bad via its phosphorylation is not necessary for IL-7-mediated T cell survival as a result of the high level of Bcl-2 expression in T cells treated with IL-7. The lower level of Bcl-2 induction in IL-21-treated cells might require Bad inactivation and therefore PI-3K activation to promote survival. Moreover, like IL-7, IL-21 was able to prevent cellular atrophy, suggesting that IL-21 not only promotes survival but also metabolic activity of T cells. The concomitant survival and trophic effects of IL-21 on T lymphocytes are probably crucial for the maintenance of a pool of metabolically active T cells, which are ready to respond rapidly to antigenic challenge. These properties render IL-21 attractive to use as a therapeutical agent to help maintain the T cell pool in patients with thymic atrophy.
Our results about the ability of IL-21 to promote naïve T cell survival contrast with the described effect of IL-21 on naïve B lymphocytes [33 , 49 , 51 ]. IL-21 has proapoptotic effects on naive B cells and on B cells, which are stimulated without T cell help or without signals mimicking T cell help [33 , 51 ]. The apoptosis of B cells in the presence of IL-21 correlates with a decrease in the level of the antiapoptotic molecule Bcl-XL and an increase in the level of the proapoptotic molecule Bim [33 , 51 ]. The differential effect of IL-21 on naïve T cells versus naïve B cells could also be a result of the use of different doses of IL-21 in the assay. For T cells, we used from 5 to 20 ng/ml IL-21, and 200 ng/ml was used for B cells [51 ]. It is thus possible that IL-21 would induce T cell death at higher doses, but we never saw induction of death, even when we used IL-21 at a high concentration (200 ng/ml; data not shown). The ability of IL-21 to induce naïve T cell survival or naïve B cell death probably results from the differential coupling of the IL-21R to the signaling machinery in B cells versus T lymphocytes. Moreover, IL-21 promotes proliferation and differentiation of B lymphocytes when they are activated in the presence of CD4+ T cell help [28 , 33 , 49 ]. Furthermore, another Type I cytokine, IL-2, can induce T cell growth or promote death of activated T cells [77 78 79 ], suggesting that signaling via the cytokine receptor will differ between cell types and activation status.
The ability of IL-21 to promote T cell survival should be helpful to maintain the T cell pool in immunodeficient patients. IL-21 might be a better therapeutic agent than IL-7, as it was shown that treatment of naive T cells with IL-7 render them permissive to HIV infection [80 , 81 ]. Moreover, the use of IL-7 in therapy might be hampered by its ability to increase Bcl-2 expression in T cells, which might be detrimental to T cell activation by inhibiting cell cycle progression and NFAT activation [82 , 83 ].
Received August 3, 2006; revised May 14, 2007; accepted May 16, 2007.
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  J. Leignadier, M.-P. Hardy, M. Cloutier, J. Rooney, and N. Labrecque Memory T-lymphocyte survival does not require T-cell receptor expression PNAS, December 23, 2008; 105(51): 20440 - 20445. [Abstract] [Full Text] [PDF]
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