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The Journal of Immunology, 2004, 173: 3131-3139.
Copyright © 2004 by The American Association of Immunologists

Proliferation and Differentiation of CD8+ T Cells in the Absence of IL-2/15 Receptor {beta}-Chain Expression or STAT5 Activation

Ryan M. Teague1,*,{dagger},¶, Richard M. Tempero1,*,§, Sunil Thomas{dagger},{ddagger}, Kaja Murali-Krishna{dagger},{ddagger} and Brad H. Nelson2,*,{dagger},¶

* Benaroya Research Institute, Virginia Mason, Seattle, WA; {dagger} Department of Immunology, {ddagger} Washington National Primate Center, and § Department of Otolaryngology-Head and Neck Surgery, University of Washington School of Medicine, Seattle, WA; and Fred Hutchinson Cancer Research Center, Seattle, WA


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Major gains in the efficacy of T cell-based therapies for cancer and infectious diseases could be realized through improved understanding of the signals that control expansion and differentiation of CD8+ cytolytic T cells. IL-2, IL-15, and the downstream transcription factor STAT5 have all been implicated as important regulators of these processes, yet there are conflicting data regarding their contribution to in vivo T cell responses. We used a murine adoptive T cell transfer model to examine the contribution of IL-2 and IL-15 signaling to the proliferation and differentiation of naive, CD8+ T cells bearing an OVA-specific TCR transgene (OT-I). OT-I T cells failed to express the high affinity IL-2R (CD25) while proliferating in vivo, irrespective of the mode of Ag delivery. Moreover, OT-I T cells rendered genetically deficient in the shared IL-2/IL-15R{beta} subunit (IL-2R{beta}) demonstrated normal Ag-induced proliferation and cytolytic activity in vivo. Accordingly, activation of STAT5 was not detected in proliferating IL-2R{beta}-deficient OT-I T cells, thus implicating a STAT5-independent cytokine or costimulatory pathway in this process. Even though IL-2 and IL-15 were dispensable for CD8+ T cell proliferation, systemic infusion of IL-2 nevertheless promoted the expansion of OT-I T cells in vivo. Thus, IL-2 and IL-15 signals are not essential for CD8+ T cell proliferation or differentiation, but IL-2 can promote supraphysiological expansion when supplied exogenously. These findings challenge current models that place CD8+ T cell proliferation under the control of STAT5-dependent cytokines and suggest new approaches to the therapeutic manipulation of T cell numbers in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Despite the clinical use of IL-2 to promote immune responses in patients with renal cell carcinoma, melanoma, lymphoma, and AIDS (1, 2, 3, 4, 5, 6, 7), the role of IL-2 in T cell proliferation and differentiation in vivo remains poorly understood and highly controversial. IL-2 is unquestionably a potent growth factor for T cells in vitro; however, IL-2-deficient mice are not lymphopenic or immunocompromised, but rather develop a fatal, T cell-mediated, multiorgan autoimmune syndrome (8). This surprising phenotype led to the notion of IL-2 being a negative regulator of T cell activity in vivo. In this regard, IL-2 was subsequently implicated as essential for activation-induced cell death, a mechanism by which effector T cells are eliminated upon completion of immune responses (9, 10, 11). More recently, IL-2 has been shown to be essential for the development and expansion of regulatory T cells, which are thought to suppress autoreactive and possibly other T cell responses in vivo (12, 13, 14). Adding to the complexity, others have demonstrated a positive role for IL-2 in promoting T cell expansion in both mouse models and human patients (4, 15, 16, 17). It appears, therefore, that IL-2, like many cytokines, can play different roles depending on the developmental stage, responding cell population, and physiological context. Clearly, we need to better understand these divergent roles if we are to fully realize the clinical potential of this important cytokine.

To understand the physiological function of IL-2, one must also consider the related cytokine IL-15, which is a potent T cell growth factor (1, 18, 19). IL-2 and IL-15 use similar heterotrimeric receptors comprised of the IL-2R {beta}-chain (IL-2R{beta}), the common cytokine receptor {gamma}-chain, and private {alpha}-chains that allow discriminatory binding of IL-2 or IL-15 (18, 20, 21). The shared {beta}- and {gamma}-chains are responsible for receptor signal transduction, and hence, IL-2 and IL-15 appear to induce identical intracellular signaling events, including tyrosine phosphorylation of the Janus kinases JAK1 and JAK3, the adaptor protein Shc, and the transcription factor STAT5 (22, 23). The STAT5 pathway in particular is thought to be critical for T cell proliferation as STAT5-deficient T cells fail to proliferate in vitro in response to TCR or IL-2 stimulation (24, 25, 26).

Despite generating similar intracellular signals, IL-2 and IL-15 demonstrate widely divergent physiological properties in vivo. As mentioned above, evidence suggests that IL-2 plays a negative regulatory role for T cells in vivo as IL-2-, IL-2R{alpha}-, and IL-2R{beta}-deficient mice develop profound lymphoid hyperplasia and systemic autoimmunity (8, 27, 28). Moreover, IL-2 has been shown to negatively regulate T cell homeostasis and memory (19, 29, 30). In contrast, IL-15- and IL-15R{alpha}-deficient mice display a lymphopenic phenotype (31, 32), and infusion of IL-15 into wild-type (WT)3 mice promotes T cell expansion, especially of the memory subset (33, 34). Thus, IL-15 is now considered one of the more important cytokines for promoting T cell homeostasis and expansion in vivo (19, 29). This has led to the current view that IL-2 and IL-15 play opposing roles in the regulation of T cell proliferation in vivo, with the relative balance of these two cytokines dictating the magnitude of T cell responses (19).

In this study, we examine the effects of IL-2 and IL-15 on the proliferation and differentiation of CD8+ T cells in vivo using a murine adoptive transfer model involving OT-I TCR transgenic (Tg) CD8+ T cells. Our results lead to the unexpected conclusion that neither IL-2 nor IL-15 signals are required for the primary expansion or cytolytic differentiation of CD8+ T cells in vivo. Moreover, we observed strong CD8+ T cell proliferation in the absence of STAT5 activation, which excludes additional cytokines such as IL-7 and IL-9 as essential mediators of this process. Therefore, we conclude that the proliferation of naive CD8+ T cells is mediated by a STAT5-independent cytokine or costimulatory pathway. These results prompt a new model for the regulation of T cell proliferation and suggest new possibilities for the therapeutic manipulation of T cell numbers in patients with viral or malignant diseases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and genotypic screening

OT-I Tg C57BL/6 mice expressing a H-2kb-restricted TCR specific for the OVA epitope (SIINFEKL) (35) were a gift from M. Bevan (University of Washington). OT-I mice were bred with IL-2R{beta}–/– mice carrying a thymus-specific IL-2R{beta} transgene (36), a gift from T. Malek (University of Miami, Miami, FL). Mice were genotyped using PCR and the following primer sets: IL-2R{beta} WT F-TTGGTATCATACCGATTGGT and WT R-AGAGGCTAAGCAAACAGCCT, IL-2R{beta} mutant F-TGGCCTTGTCCGAAAGGTCA and mutant R-CTTGACGAGTTCTTCTGAGG, and Tg F-AGATCCTCCCCATGTCATGG and Tg R-CAAGGCATTGGGCAGATGGA. PCR conditions were 95°C for 60 s, 60°C for 30 s, and 72°C for 60 s, followed by 10-min extension at 72°C. Products were visualized by ethidium bromide on an agarose gel. Before use, mice were also screened for expression of the V{alpha}2 and V{beta}5 subunits of the OT-I Tg TCR by flow cytometric analysis of PBL. Briefly, PBL were obtained by saphenous venipuncture. A small volume of blood was collected in a heparin tube, and RBCs were lysed with ammonium chloride, potassium carbonate, Tris-EDTA (AKT) buffer (150 mM NH4Cl, 0.1 mM EDTA, 1 mM KHCO3, pH 7.2). Samples were washed in flow buffer (PBS with 2% FBS) and incubated at 25°C with anti-CD8 CyChrome, anti-V{alpha}2 FITC, and anti-V{beta}5 PE. C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Animal studies were approved by and complied with the institutional Animal Care and Use Committee.

CFSE labeling, in vitro stimulation, and T cell adoptive transfer

Lymph node (LN) cells (axillary, brachial, superficial inguinal, cervical) and splenocytes were harvested and processed to a single cell suspension. Red cells were lysed with AKT buffer, and remaining cells were washed twice in PBS. Cells were labeled with 2 µM CFSE (Molecular Probes, Eugene, OR) in PBS for 15 min at 37°C and then washed three times with PBS. For in vitro experiments, 5 x 106/ml CFSE+ total OT-I LN and spleen cells were stimulated with 5 µg/ml anti-CD3 Ab (BD Pharmingen, San Diego, CA) or 1 mg/ml OVA (Sigma-Aldrich, St. Louis, MO) in 96-well plates. For in vivo experiments, 10 x 106 CFSE+ total OT-I LN and spleen cells were administered by tail vein injection into sex-matched nonirradiated C57BL/6 recipients. A total of 1 mg OVA protein (Sigma-Aldrich) or 5 x 106 EL4/EG.7 tumor cells (a gift from M. Bevan) was administered s.c. between the scapulae. Alternatively, 1 x 106 PFU vesicular stomatitis virus (a gift from L. Lefrancois, University of Connecticut, Farmington, CT) was administered by tail vein injection. All viral work was performed at the University of Washington biosafety level 3 facility.

Flow cytometry

For adoptive transfer studies or in vivo cytotoxicity experiments, recipient C57BL/6 mice were sacrificed, and draining LNs or spleens were collected. RBC were lysed from splenocytes in AKT buffer. Single cell suspensions were prepared from splenocytes and LN cells and washed in flow buffer. Cell preparations were labeled at room temperature with anti-CD8 CyChrome (BD Pharmingen) and then split into aliquots for staining with anti-CD25 PE, anti-CD44 PE, anti-CD69 PE, anti-CD62L PE, and the appropriate isotype controls conjugated with PE (BD Pharmingen). Multiple-color flow analyses were performed using a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA).

Western blotting

T cell lysates were prepared, as described previously (37). Total protein was quantified in each sample, and equal amounts of protein were separated by SDS-PAGE (Invitrogen Life Technologies, Carlsbad, CA). Proteins were transferred to nitrocellulose, blotted with anti-STAT5, anti-phospho-STAT5, anti-STAT1, or anti-phospho-STAT1 Abs (Cell Signaling Technology, Beverly, MA), followed by a secondary goat anti-rabbit Ab conjugated to HRP (Caltag Laboratories, Burlingame, CA), and detected by ECL (Cell Signaling Technology).

In vivo cytolytic assay

Nonlabeled OT-I T cells were adoptively transferred and stimulated in vivo, as described above. Five days posttransfer, 2.5 x 106 irradiated C57BL/6 splenocytes pulsed with 1 µg of SIINFEKL peptide and labeled CFSElow (0.2 µM) were transferred into these mice by tail vein injection, along with 2.5 x 106 splenocytes pulsed with 1 µg of HSV-2 peptide and labeled CFSEhigh (2.0 µM). Eighteen hours posttarget cell transfer, spleens were removed, and the presence of CFSE high and low cells was analyzed by flow cytometry.

IL-2 administration

OT-I T cells were adoptively transferred into normal C57BL/6 recipients, which were then immunized with 1 mg of OVA. On day 5 following adoptive transfer and immunization, mice were injected s.c. with 30,000 IU of human IL-2 (Chiron, Emeryville, CA) or 200 µl of PBS daily for 7 days. In some experiments, mice were sacrificed and LN were analyzed at the indicated time points. In others, serial blood draws were taken over a 2-wk period by saphenous venipuncture. A small volume of blood was collected in heparin tube, and RBCs were lysed with AKT buffer. LN or PBL samples were washed in flow buffer and incubated at 25°C with anti-CD8 CyChrome, anti-V{alpha}2 FITC, and anti-V{beta}5 PE. After a final wash, the frequency of adoptively transferred OT-I T cells was determined by flow cytometry using a gating strategy to identify the percentage of CD8+ V{alpha}2+ V{beta}5+ lymphocytes vs all other CD8+ cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-2R{alpha} is expressed on proliferating CD8+ T cells stimulated in vitro, but not in vivo

To analyze the role of IL-2 and IL-15 signaling in CD8+ T cell proliferation and differentiation, we used naive CD8+ T cells expressing the well-characterized OT-I TCR transgene, which confers recognition of the SIINFEKL epitope from chicken OVA (35). As expected, OT-I T cells stimulated with soluble OVA in vitro underwent vigorous proliferation and displayed an activated T cell phenotype, including increased expression of CD25 (IL-2R{alpha}) and CD44 and decreased expression of CD62L (L-selectin) (Fig. 1). When CFSE-labeled OT-I cells were adoptively transferred into WT syngeneic hosts and stimulated with soluble OVA, they again proliferated vigorously, but, unexpectedly, showed no detectable expression of CD25 (Fig. 2A). Time course experiments revealed that proliferation began in LN 1–3 days after OVA immunization and persisted until at least day 7; however, CD25 expression was not detected at any time point in vivo (including days 1, 2, 3, 5, and 7; Fig. 2 and data not shown).



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  		FIGURE 1. Proliferating CD8+ T cells stimulated in vitro express IL-2R{alpha}. CFSE+ OT-I LN and spleen cells were left nontreated or stimulated in vitro with 1 mg/ml OVA and allowed to proliferate for 72 h. Cells were then removed from culture and stained with anti-CD25 PE or an isotype control PE Ab and anti-CD8 CyChrome. CFSE+ CD8+ OT-I cells were identified (inset), and a gating strategy was used to show expression of CD25 (solid line) vs an isotype control stain (shaded area) in the histograms. The same cells were also analyzed for the expression of CD44 (solid line) and CD62L (dashed line). These data are representative of 11 separate experiments.

 


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  		FIGURE 2. Proliferating CD8+ T cells stimulated in vivo do not express detectable IL-2R{alpha}. CFSE+ OT-I LN and spleen cells were adoptively transferred into syngeneic hosts, which were then immunized with A, 1 mg of soluble OVA, or B, infected with 1 x 106 PFU VSV or VSV-OVA for 72 h. Cells were then removed from host LN and stained with anti-CD25 PE or an isotype control PE Ab and anti-CD8 CyChrome. CFSE+ CD8+ OT-I cells were identified (inset), and a gating strategy was used to show expression of CD25 (solid line) vs an isotype control stain (shaded area). These data are representative of eight separate soluble OVA and three separate VSV experiments.

 
The above results indicate that the physiological context of Ag stimulation (i.e., in vitro vs in vivo) can dictate whether or not proliferating T cells express CD25. Therefore, we evaluated whether other forms of Ag delivery might induce CD25 expression in vivo. The addition of CFA to soluble OVA Ag induced OT-I T cell proliferation as before, but failed to trigger CD25 expression (data not shown). Similarly, a syngeneic lymphoma cell line engineered to express OVA (EG.7) induced OT-I proliferation, but again without detectable CD25 expression (data not shown). Finally, to create an inflammatory context, we examined OT-I proliferation and CD25 expression during viral challenge. In this study, T cells were transferred into syngeneic recipients, which were then challenged with a derivative of vesicular stomatitis virus (VSV) engineered to express OVA (VSV-OVA). Following 24, 48, and 72 h in vivo stimulation with virus, OT-I T cells had proliferated and demonstrated several classic signs of T cell activation, including increased cell size, increased CD44 expression, and decreased CD62L (Fig. 2B and data not shown). However, despite inducing CD8+ T cell proliferation, VSV-OVA failed to induce detectable expression of CD25 in vivo at any of the time points tested. As expression of CD25 is thought to be essential for T cells to respond to physiological levels of IL-2 (27), the foregoing results suggested that the primary expansion of OT-I T cells was not dependent on IL-2 signaling.

Signals from IL-2 and IL-15 are not required for naive CD8+ T cell proliferation

To definitively test whether primary expansion of CD8+ T cells was IL-2 independent, we generated IL-2R{beta}-deficient OT-I mice. The IL-2R{beta} chain is an essential component of both the IL-2R and IL-15R complexes; therefore, deletion of IL-2R{beta} renders T cells unresponsive to IL-2 and IL-15 (14, 18, 28, 36, 38, 39). IL-2R{beta}-deficient mice become fatally ill at an early age (4–6 wk) due to the spontaneous development of a severe T cell-dependent autoimmune syndrome (28). Expression of the OT-I Tg TCR suppressed autoimmunity in these mice by several weeks, although mice still demonstrated signs of lymphadenopathy and splenomegaly (Fig. 3). Moreover, unstimulated OT-I IL-2R{beta}–/– T cells expressed high levels of CD44 and reduced levels of CD62L (Fig. 4B), indicating they had become spontaneously activated in vivo. Nevertheless, upon OVA stimulation, OT-I IL-2R{beta}–/– T cells demonstrated a robust proliferative response comparable to WT OT-I T cells both in vitro and in vivo (Fig. 4B). Flow cytometry confirmed that OT-I IL-2R{beta}–/– T cells did not express the IL-2R{beta} subunit and had little or no expression of CD25 (data not shown). These results suggest that CD8+ T cells require neither IL-2 nor IL-15 signaling for primary expansion in vivo, although this conclusion was confounded by the fact that the OT-I IL-2R{beta}–/– T cells already showed signs of activation at the start of the experiment.



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  		FIGURE 3. Genotype and phenotype of OT-I mice with differential IL-2R{beta} expression. LN and spleens were removed from OT-I/IL-2R{beta}+/–, OT-I/IL-2R{beta}–/– Tg+, and OT-I/IL-2R{beta}–/– Tg– mice at 8 wk of age and compared by size and mass. A, The combined mass of the indicated LN and spleen for each mouse is expressed in parentheses. B, Specific primers for the IL-2R{beta} mutant genotype (Mu), the IL-2R{beta} WT genotype (Wt), and the IL-2R{beta} transgene (Tg) were used to amplify genomic DNA. These data are representative of at least 10 mice from each genotype.

 


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  		FIGURE 4. In vivo activation and proliferation of CD8+ OT-I cells in the absence of IL-2R{beta} expression. CFSE+ LN and spleen cells from WT OT-I (A), OT-I IL-2R{beta}–/– (B), or OT-I IL-2R{beta}–/– Tg+ (C) mice were transferred i.v. into C57BL/6 recipients and were stimulated with an s.c. injection of PBS or 1 mg of OVA for 72 h. The upper row of dot plots displays CFSE content (y-axis) vs CD8 expression (x-axis). The cells within the gated region (inset) were analyzed for expression of CD44 (solid line) and CD62L expression (dashed line) and compared with an isotype control stain (shaded area), shown in the lower row of histograms. These results are representative of three separate experiments.

 
This latter caveat was addressed using OT-I IL-2R{beta}–/– T cells engineered to express an IL-2R{beta} transgene during thymic development, but not in the periphery (OT-I IL-2R{beta}–/– Tg+) (36, 38). Thymic expression of IL-2R{beta} has previously been shown to suppress the autoimmune syndrome that otherwise befalls IL-2R{beta}–/– mice (28, 36, 38). Consistent with this, OT-I IL-2R{beta}–/– Tg+ mice were long-lived and showed little evidence of lymphadenopathy or splenomegaly by 8 wk (Fig. 3). Furthermore, nonstimulated OT-I IL-2R{beta}–/– Tg+ T cells displayed a naive phenotype based on CD44 and CD62L expression (Fig. 4C). Following adoptive transfer and immunization with OVA, OT-I IL-2R{beta}–/– Tg+ T cells proliferated vigorously and showed an activated phenotype with respect to CD44 and CD62L (Fig. 4C). Flow cytometry revealed no detectable IL-2R{beta} and little or no expression of CD25 on these activated cells (data not shown). Thus, these experiments provide definitive genetic proof that IL-2 and IL-15 signals are dispensable for the primary expansion of CD8+ T cells.

In addition to IL-2 and IL-15, other T cell growth factors include IL-7, IL-9, and, under some circumstances, IL-21. Common to all of these cytokines is the activation of STAT5, which is reported to be essential for T cell proliferation (24, 25, 26). To investigate whether any STAT5-dependent cytokine signals are active in proliferating CD8+ T cells, immunoblotting was performed using phospho-STAT5-specific Abs. Polyclonal T cells from C57BL/6 mice treated with anti-CD3 or IL-2 demonstrated strong phosphorylation of STAT5 (Fig. 5A), as did OT-I T cells stimulated with OVA (Fig. 5B). In both cases, phosphorylation of STAT5 was readily detected by 8 h, peaked by 24 h, and persisted for at least 48 h. By contrast, OT-I T cells deficient in IL-2R{beta} demonstrated no detectable phospho-STAT5 in response to OVA or IL-2 treatment (Fig. 5B), despite undergoing a proliferative response equal in magnitude to WT OT-I cells (Fig. 4). Both WT OT-I and IL-2R{beta}–/– OT-I T cells demonstrated phosphorylation of STAT1 (Fig. 5B), which most likely reflects autocrine IFN-{gamma} signaling (40). Thus, IL-2R{beta} deficiency caused selective impairment of the STAT5 pathway while sparing other signaling events. Collectively, these results indicate that: 1) STAT5 phosphorylation is normally associated with CD8+ T cell proliferation in vitro; 2) IL-2 and/or IL-15 are the most likely source of this signal (because STAT5 is not phosphorylated in the absence of IL-2R{beta}); and 3) in contrast to prior reports (24, 25, 26), STAT5 activation is not essential for T cell proliferation.



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  		FIGURE 5. Proliferation of OT-I IL-2R{beta}–/– T cells in the absence of detectable STAT5 phosphorylation. LN and speen cells from (A) C57BL/6, WT OT-I, and (B) OT-I IL-2R{beta}–/– Tg+ mice were isolated and stimulated with 5 µg/ml anti-CD3, 1 mg/ml OVA, or 100 U/ml IL-2 in culture. Cells were harvested at the indicated time points (hours), and cytoplasmic fractions were removed. Western blotting was performed, as described in Materials and Methods. Briefly, protein lysates from cells treated in culture for the indicated time points (hours) were separated by SDS-PAGE, transferred to nitrocellulose, and blotted with anti-phospho-STAT5, anti-STAT5, anti-phospho-STAT1, or anti-STAT1. These data are representative of three separate anti-CD3 and five separate soluble OVA experiments.

 
In vivo CD8+ T cell differentiation and cytolytic activity in the absence of IL-2R{beta}

Having excluded an essential role for IL-2 and IL-15 in CD8+ T cell proliferation, we performed in vivo cytotoxicity experiments to test whether either of these cytokines is required for CTL differentiation and target cell lysis. In this study, IL-2R{beta}+/– or IL-2R{beta}–/– Tg– OT-I cells were adoptively transferred into host mice, which then received PBS or immunization with OVA s.c. Five days later, peptide-pulsed syngeneic splenocytes were infused i.v. to serve as cytolytic target cells. One-half of the splenocytes were pulsed with the SIINFEKL peptide from OVA and labeled with a low concentration of CFSE (CFSElow). The remaining splenocytes were pulsed with a negative control peptide from HSV-2 and labeled with a high concentration of CFSE (CFSEhigh). Eighteen hours later, spleens were harvested from all mice, and the relative abundance of specific target cells (CFSElow) compared with control cells (CFSEhigh) was evaluated by flow cytometry. Significant epitope-specific lytic activity was detected in both IL-2R{beta}+/– and IL-2R{beta}–/– Tg– OT-I recipient mice provided mice were primed with OVA Ag (Fig. 6A). Because IL-2R{beta}–/– Tg– OT-I T cells demonstrate a preactivated phenotype (Fig. 4B), which may conceivably increase cytolytic activity of these cells in the absence of IL-2, we also tested the lytic capability of IL-2R{beta}–/– Tg+ T cells. Virtually identical results were observed in mice receiving WT, IL-2R{beta}–/– Tg–, or IL-2R{beta}–/– Tg+ OT-I T cells when activated with soluble OVA protein (Fig. 6B). Therefore, in contrast to previous reports (39), IL-2 and IL-15 signals are not required for the differentiation of CD8+ T cells into functional cytotoxic effectors capable of specific target cell lysis in vivo.



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  		FIGURE 6. IL-2 and IL-15 are not required for CTL differentiation and cytolytic activity in vivo. In vivo cytotoxicity assays were performed by transferring naive T cells from the indicated mice i.v. into syngeneic recipients, followed by an s.c. injection of PBS or OVA. On day 5 after immunization, SIINFEKL and HSV peptide-pulsed C57BL/6 syngenic target splenocytes, stained with 0.2 µM or 2 µM CFSE, respectively, were transferred i.v. into the same recipient mice. On day 6, recipient spleens were removed and analyzed for the presence or absence of CFSE-labeled target cell populations. Histograms display fluorescence intensity of CFSE (x-axis) vs cell number (y-axis). The results in A are representative of two separate experiments, and the results in B are representative of three separate experiments from each genotype.

 
Exogenous IL-2 directly promotes the expansion of WT OT-I, but not IL-2R{beta}–/– OT-I T cells in vivo

Our data suggest that, in vivo, CD8+ T cells do not require IL-2 or IL-15 signaling to proliferate or differentiate into effector CTL. This conclusion seems at odds with multiple studies showing that systemic administration of IL-2 or IL-15 can enhance T cell numbers and Ag-specific T cell responses (3, 4, 15, 16, 17, 41, 42). We addressed this apparent contradiction by analyzing the number of OT-I T cells over the course of a primary immune response with or without systemic infusion of IL-2. As before, WT OT-I T cells were transferred into recipient mice, followed by immunization with OVA. On days 5–12, mice received either 30,000 U of IL-2 or 200 µl of PBS by s.c. injection at the site of immunization. Serial blood draws were taken, and OT-I T cells were enumerated in peripheral blood based on their expression of CD8, V{alpha}2, and V{beta}5. In control mice that received PBS instead of IL-2, OT-I expansion peaked at day 4 and contracted to baseline by day 8 (Fig. 7A). In striking contrast, infusion of IL-2 greatly increased the frequency of OT-I T cells such that they came to constitute 40% of the total PBL CD8+ population (Fig. 7A). Similar, although less dramatic increases were observed in draining LN, indicating that these increases did not simply reflect mobilization of OT-I T cells from LN to PBL (data not shown). Thus, this model recapitulates the results of previous studies indicating that systemic IL-2 infusion can enhance the size of the responding CD8+ T cell population (15, 43).



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  		FIGURE 7. IL-2 administration prevents the contraction of WT OT-I T cells, but not OT-I T cells deficient in IL-2R{beta}. C57BL/6 (A, solid lines) or IL-2R{beta}-deficient (B, dashed line) recipient mice received lymphocyte and splenocyte donor cells harvested from either WT OT-I or OT-I/IL-2R{beta}–/– Tg+ mice. Recipient mice also received an s.c. injection of 1 mg of soluble OVA Ag. At days 5–12 (shaded area), PBS or 30,000 U/day IL-2 was infused s.c. Peripheral blood was drawn at the indicated time points (days), and the frequency of OT-I T cells was assessed by expression of CD8, V{alpha}2, and V{beta}5. Data are representative of three separate experiments.

 
When given systemically, IL-2 can potentially act on a number of lymphocyte subsets. For example, IL-2 is critical for the development and peripheral expansion of regulatory T cells, which in turn can control the proliferation of effector T cells (12, 13, 14). We therefore sought to determine whether systemic IL-2 infusion promotes CD8+ T cell expansion by a direct or indirect mechanism. To this end, we first examined whether OT-I T cells need to express a functional IL-2R to benefit from the effects of systemic IL-2 infusion. The above OVA immunization and IL-2/PBS infusion studies were repeated using IL-2R{beta}-deficient OT-I T cells in place of WT OT-I T cells. Although OT-I IL-2R{beta}–/– T cells expanded well in response to OVA immunization, they did not show increased expansion in the presence of IL-2 (Fig. 7B). The converse experiment was also performed, in which WT OT-I T cells were adoptively transferred into IL-2R{beta}–/– host mice, followed by OVA immunization and IL-2 infusion. In this case, IL-2 infusion led to a clear increase in the number of OT-I T cells despite the lack of functional IL-2Rs on any endogenous cells (Fig. 7B). Thus, systemic IL-2 therapy acts directly on the responding CD8+ T cell population (in this case, OT-I T cells), with little or no contribution from endogenous cell populations. Collectively, our results demonstrate that endogenous IL-2 is not critical for the proliferation or differentiation of CD8+ T cells, but that exogenously supplied IL-2 can nonetheless act directly on these cells to enhance their in vivo expansion.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The work presented in this study provides new insight into the role of IL-2R and IL-15R signaling during the expansion and differentiation of CD8+ T cells. Our in vitro experiments demonstrated that proliferating CD8+ OT-I T cells express the high affinity IL-2R and show robust activation of the transcription factor STAT5, which is consistent with the conventional view that CD8+ T cell proliferation is driven, in part, by IL-2 or a related cytokine such as IL-15. Unexpectedly, however, experiments with IL-2R{beta}-deficient OT-I T cells revealed that neither IL-2 nor IL-15 signaling is essential for CD8+ T cell proliferation in vitro or in vivo. Rather, IL-2R{beta}-deficient T cells were equivalent to WT T cells with respect to the magnitude and duration of T cell proliferation, and they retained the ability to differentiate into CTL effectors in vivo. Moreover, IL-2R{beta}-deficient T cells underwent strong proliferation in the absence of STAT5 phosphorylation, which indicates that a STAT5-independent cytokine or costimulatory pathway must be responsible for the proliferative signal. Because these findings seemed at odds with numerous reports that IL-2 and IL-15 can promote CD8+ T cell proliferation in vivo, we assessed whether systemic infusion of IL-2 could promote T cell expansion in our model. Consistent with prior reports, systemic IL-2 infusion led to a profound increase in the number of OT-I T cells in peripheral blood after immunization. Using combinations of WT and IL-2R{beta}–/– T cells, we showed that the infused IL-2 acts directly on the OT-I T cells rather than through an endogenous regulatory T cell subset. Our findings reveal a striking difference between the physiological effects of endogenous vs exogenous IL-2. Whereas endogenous IL-2 is dispensable for the primary expansion of CD8+ T cells, exogenous IL-2 can nevertheless enhance the overall magnitude of expansion. Thus, IL-2 is sufficient, but not necessary for CD8+ T cell proliferation. These results challenge current views about the regulation of T cell proliferation by IL-2 and other STAT5-dependent cytokines, and suggest the existence of other regulatory pathways that might serve as appropriate targets for the therapeutic manipulation of T cell numbers and activity.

Several previous studies have addressed the role of IL-2 and IL-15 in CD8+ T cell proliferation. Because T cells invariably express CD25 and the other IL-2R subunits when activated in vitro and proliferate vigorously in response to IL-2, it was assumed for many years that IL-2 would also represent the physiological stimulus for T cell proliferation in vivo. However, as shown in this work, proliferating CD8+ T cells do not necessarily express CD25 in vivo (Fig. 2), which precludes responses through the high affinity IL-2R. Moreover, in other in vivo models, CD25 expression on proliferating CD8+ T cells is negligible or transient and does not necessarily coincide with the peak proliferative response (15, 19, 34, 41, 44). Indeed, studies using IL-2- or IL-2R-deficient mice have led to the general consensus that IL-2 signaling is partly or entirely dispensable for the primary expansion of CD4+ (45) and CD8+ T cells (43) in peripheral lymphoid tissues, but may play a more prominent role in nonlymphoid tissues such as gut (41). CD4+CD25+ T regulatory cells represent an exception to this rule, as they appear to be highly dependent on IL-2 not only during thymic development, but for expansion in the periphery as well (14). Marrack and colleagues (29) infused neutralizing Abs to IL-2 and IL-2R{alpha} into WT mice and showed enhanced proliferation of CD8+ memory T cells, which they attributed to the inhibition of IL-2-mediated activation-induced cell death. By contrast, infusion of Abs to IL-2R{beta} (which blocks both IL-2 and IL-15 signaling) strongly inhibited the expansion of CD8+ memory T cells, suggesting this process is dependent on IL-15. Accordingly, exogenous IL-15 causes expansion of CD8+ memory T cells in vivo (33, 34, 46), and IL-15-deficient mice have diminished CD8+ memory T cell numbers and activity (31, 42). This latter phenotype reflects defects in both the primary expansion of CD8+ T cells and the maintenance of the memory subset (42). Collectively, the above studies have led to a widely accepted model in which CD8+ T cell expansion is promoted by IL-15 and opposed by IL-2.

The present results appear to contradict this model, as we found that IL-2R{beta}-deficient CD8+ T cells undergo normal Ag-induced expansion and cytolytic differentiation in vivo despite lacking functional IL-2R or IL-15R. This apparent discrepancy may be attributable to several factors. First, IL-2/IL-15 signals may be dispensable for the initial proliferation of naive CD8+ T cells, as measured in this study, but then become increasingly important for subsequent cell divisions, especially those underlying the homeostatic proliferation of memory T cells (38, 43). Second, IL-2/IL-15 signaling may be more important for T cell proliferation in nonlymphoid tissues (43), whereas the present studies analyzed responses in peripheral blood and LN. Third, it was recently reported that memory CD8+ T cell proliferation does not require IL-15R{alpha} expression on the T cells themselves, but rather on one or more other cell types, yet to be defined, that promote the proliferation of memory T cells by an indirect mechanism (47). Thus, our results support an emerging body of evidence that CD8+ T cell proliferation is promoted by IL-2- and IL-15-independent signals in specific physiological circumstances.

In light of the above conclusion, it may seem somewhat contradictory that systemic administration of IL-2 can enhance OT-I T cell expansion in vivo (Fig. 7), especially because these cells fail to initially express CD25 and do not require IL-2R{beta} to proliferate (Figs. 2 and 4). We considered the possibility that systemic IL-2 may enhance OT-I T cell proliferation by an indirect mechanism, for example by acting on a CD25+ regulatory T cell subset. However, use of IL-2R{beta}-deficient OT-I T cells, or infusion of WT OT-I cells into IL-2R{beta}-deficient hosts clearly revealed that systemic IL-2 acts directly on the responding OT-I T cells (Fig. 7). Blattman et al. (15) also examined the effect of systemic IL-2 infusion on the expansion of CD8+ T cells in vivo and found that proliferating T cells, although initially positive for CD25, unexpectedly showed maximum response to IL-2 when CD25 was low or not detectable. Presumably, a sufficiently high concentration of exogenous IL-2 can trigger the intermediate affinity IL-2R comprised of IL-2R{beta} and common {gamma}-chain. This in turn would induce expression of CD25, which is a target gene of the IL-2 pathway (48), thereby further increasing the sensitivity of T cells to IL-2. Thus, therapeutic administration of IL-2 can potentially convert T cell responses from an IL-2-independent state to one that is driven by IL-2. It will be interesting in the future to examine the consequences of such a signaling conversion on the ultimate magnitude and character of the T cell response.

In addition to proliferation, IL-2 and IL-15 have been reported to promote the differentiation of CD8+ T cells to cytolytic effector cells (CTL) (49, 50, 51, 52). Accordingly, IL-2R{beta}-deficient mice demonstrate reduced numbers of IFN-{gamma}-secreting CD8+ T cells following vaccinia virus challenge, and T cells from these mice have decreased expression of perforin and granzyme B (36, 38, 39). Despite these deficiencies, IL-2R{beta}-deficient mice have been shown to reject primary and secondary allogeneic skin grafts and mount effective secondary responses against vaccinia virus (39). Moreover, as shown in this study, IL-2R{beta}-deficient OT-I T cells efficiently killed peptide-pulsed syngeneic splenocytes in vivo, demonstrating that they had undergone productive CTL differentiation. Thus, as with proliferation, CD8+ T cell differentiation can be driven by IL-2- and IL-15-independent signals.

Perhaps the most unexpected finding from the present study was that IL-2R{beta}-deficient CD8+ T cells undergo vigorous proliferation without any detectable tyrosine phosphorylation of STAT5. From studies of mice, it has been argued unequivocally that STAT5 is essential for T cell proliferation (24, 25, 26). Specifically, T cells from STAT5-deficient mice fail to up-regulate cell cycle genes (cyclin D2, cyclin D3, and cdk6) or undergo cell proliferation in response to anti-CD3 or IL-2 stimulation (24, 25). Reintroduction of STAT5 by retroviral transduction restores the proliferative response of these T cells, suggesting that their otherwise nonresponsive state does not reflect an irreversible developmental defect (24), an important concern given the prominent role of STAT5 in IL-7R signaling. Mutation of a critical tyrosine residue in STAT5 (Y693) abrogates its ability to rescue T cell proliferation (24), indicating that tyrosine phosphorylation of STAT5 is essential to its role in promoting T cell proliferation. In the present study, tyrosine phosphorylation of STAT5 was not detected in proliferating IL-2R{beta}-deficient CD8+ T cells, indicating that STAT5 was inactive by this definition. It is formally possible that our Western blot assay failed to detect a very low level of STAT5 phosphorylation that is nevertheless functionally relevant. Unfortunately, we were unable to test this by genetic means, as we were unsuccessful at generating STAT5-deficient OT-I mice, owing to a high rate of postpartum mortality. However, we note that IL-2R{beta}-deficient OT-I T cells fail to up-regulate CD25, which is a well-characterized STAT5 target gene (53, 54), indicating that there was little, if any, activated STAT5 in these cells. Furthermore, we could easily detect inducible phosphorylation of STAT1 in the same cells, which verifies the reliability of our detection methods. Finally, tyrosine phosphorylation of STAT5 was readily detected in WT OT-I T cells under the same conditions (Fig. 5). Thus, we propose that the tyrosine phosphorylation of STAT5, and all subsequent events, including dimerization and nuclear translocation, are not required for CD8+ T cell proliferation, and that some other signaling pathway may normally drive this process. We suggest that the general state of nonresponsiveness of STAT5-deficient T cells may reflect developmental or secondary effects that are evidently reversible upon reintroduction of STAT5. Of note, IL-2R{beta}-deficient T cells were originally reported to be nonresponsive to numerous mitogenic stimuli (28), but later work showed the proliferative capacity of these T cells could be restored either by thymic expression of Tg IL-2R{beta} or by i.v. transfer of WT regulatory T cells into neonatal IL-2R{beta}-deficient mice (14, 36, 38). In this regard, STAT5-deficient mice are now appreciated to have multiple, serious hemopoietic defects, including a loss of regulatory T cells (55, 56, 57), which opens the possibility for secondary effects on T cell proliferation.

A major outstanding question arising from this study concerns the nature of the signal that drives T cell proliferation in the absence of IL-2R{beta} or STAT5 activity. One can speculate that it might involve a STAT5-independent cytokine such as IL-6, which can provide a differentiation signal and serve as a potent costimulatory growth factor for naive T cells (58, 59, 60, 61), including CD8+ OT-I T cells (R. Teague, unpublished observations). However, although some defects in B cell and CD4+ T cell responses have been observed in IL-6-deficient animals, defects in CD8+ T cell activity have not been reported (62, 63). The signal could also arise from a cytokine-independent pathway, such as CD28, OX40, or 4-1BB, all of which have been shown to enhance CD8+ T cell proliferation in cooperation with an Ag signal (58, 64, 65, 66, 67, 68). Finally, initial rounds of CD8+ T cell proliferation could reflect a programmed, intrinsic response to TCR stimulation alone (69, 70, 71, 72), without the need for other signals.

The nature of the proliferative signal is not only of academic interest, but could also represent an important target for the therapeutic manipulation of CD8+ T cell numbers in vivo. Many groups have shown that systemic infusion of IL-2 or IL-15 can enhance CD8+ T cell responses to vaccination or tumors (1, 2, 3, 4, 15, 34, 73, 74), and we have shown in this study that this reflects a direct effect on the responding T cell population. However, IL-2 therapy has yielded less than ideal clinical results, owing not only to the serious nonspecific toxicity of this cytokine, but also to the often transient nature of its effects. Our demonstration in this study that the primary expansion of CD8+ T cells may be driven by signals other than IL-2 and IL-15 brings into question whether IL-2 or IL-15 represents the optimal signal to deliver to CD8+ T cells for therapeutic purposes. If the in vivo expansion of CD8+ T cells initially occurs by an IL-2/IL-15-independent signaling pathway, as our data suggest, then infusion of IL-2 could potentially interfere with this process by diverting T cells down a differentiation or possibly even apoptotic pathway. Perhaps delivery of a more physiologically relevant proliferative signal would keep CD8+ T cells in a primary expansion mode for a longer period of time, resulting in more profound and durable increases in the number of effector T cells.


   Acknowledgments
 
We thank Dr. Thomas Malek for IL-2R{beta}–/– Tg+ mice; Julie Stewart, Tim Martyak, and Meghan Crawford for technical assistance; and Dr. James Moon and Bryan Carson for helpful discussions.


   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 R.M.T. and R.M.T. contributed equally to this work. Back

2 Address correspondence and reprint requests to Dr. Brad H. Nelson, British Columbia Cancer Agency, 2410 Lee Avenue, Victoria, British Columbia, V8R 6V5 Canada. E-mail address: bnelson{at}bccancer.bc.ca Back

3 Abbreviations used in this paper: WT, wild type; AKT, ammonium chloride, potassium carbonate, Tris-EDTA; LN, lymph node; Tg, transgenic; VSV, vesicular stomatitis virus. Back

Received for publication March 17, 2004. Accepted for publication June 23, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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