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The Journal of Immunology, 2002, 169: 4990-4997.
Copyright © 2002 by The American Association of Immunologists

Selective Delivery of Augmented IL-2 Receptor Signals to Responding CD8+ T Cells Increases the Size of the Acute Antiviral Response and of the Resulting Memory T Cell Pool1

Laurence E. Cheng*,{ddagger} and Philip D. Greenberg2,*,{dagger},{ddagger}

Departments of * Immunology and {dagger} Oncology, University of Washington, Seattle, WA 98195; and {ddagger} Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD8+ T cells respond to IL-2 produced both endogenously and by CD4+ Th during an antiviral response. However, IL-2R signals can potentially promote CD8+ T cell death as well as proliferation, making it unclear whether IL-2R signals provide a predominantly positive or negative effect upon CD8+ T cell responses to viral infection. To more precisely define the direct role of IL-2R signaling on CD8+ T cells during the response to a virus, we examined the effect of delivering augmented IL-2R signals selectively to CD8+ T cells responding to lymphocytic choriomeningitis virus infection. Although naive CD8+ T cells are competent to produce IL-2, CD8+ T cells lose this capacity upon differentiation into effector CD8+ T cells. However, effector CD8+ T cells do retain the capacity to produce GM-CSF upon Ag stimulation. Thus, to deliver enhanced autocrine IL-2R signals to CD8+ T cells, we established a transgenic mouse strain expressing a chimeric GM-CSF/IL-2R (GMIL2R). As GM-CSF production is Ag dependent, the GMIL2R delivers an augmented IL-2R signal exclusively to CD8+ T cells responding to Ag. Following lymphocytic choriomeningitis virus infection, GMIL2R transgenic mice exhibited an increase in both the peak CD8+ T cell response achieved and the size of the resulting memory pool established. Upon secondary viral challenge, the GMIL2R also enhanced the proliferative response of memory CD8+ T cells. Thus, our findings indicate that IL-2 delivery to responding CD8+ T cells is a limiting factor in both the acute and memory antiviral responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In response to viral infection, Ag-specific CD8+ T cells undergo extensive proliferation to form a large pool of cytolytic effector cells to eliminate the pathogen (1). Following this peak of expansion and associated clearance of virus, memory CD8+ T cells are formed and provide the host with a pool of virus-specific CD8+ T cells capable of mounting an accelerated T cell response and protective immunity in the event of reinfection with the virus. In mice previously infected with lymphocytic choriomeningitis virus (LCMV),3 the size of the memory CD8+ T cell pool established has been shown to correlate with the size of the peak response, and determines both the magnitude and effectiveness of the response to rechallenge with the virus (2, 3). Similarly, in humans, the presence of a memory CD8+ T cell response to persistent viruses such as CMV and EBV has been associated with resistance to disease following reactivation of the virus (4, 5, 6, 7).

Establishment of the memory CD8+ T cell pool following clearance of a viral infection is in part determined by the presence of a concurrent CD4+ Th response during the response to acute infection (2). Although in some settings CD4+ Th cells are absolutely required for induction of a CD8+ T cell response, based on the necessity to activate and license APCs (8, 9, 10), many viruses including LCMV can activate APC independent of CD4+ Th cells and induce an antiviral CD8+ T cell response (11, 12). However, even with such viruses, CD4+ T cells provide an additional helper function that promotes formation of a memory CD8+ T cell response. IL-2 production is an effector function of CD4+ Th cells not alternatively provided by the virus. IL-2 delivers signals through the heterotrimeric IL-2R, comprised of a unique {alpha}-chain (CD25) required for high affinity binding of IL-2, as well as the signaling {beta}- and {gamma}-chains shared with the IL-15R (13). Although both naive CD8+ T cells and CD4+ Th cells can produce IL-2 following initial activation, only CD4+ T cells retain this capacity after differentiation into effector T cells (14, 15).

Defining the in vivo role of IL-2 in the regulation of CD8+ T cells responding to a virus has been complicated by the myriad effects of IL-2 in the immune system. In vitro studies have demonstrated that signals through the IL-2R can lead to enhanced CD8+ T cell proliferation and survival, or alternatively cell death following T cell activation (16, 17, 18), and data from some in vivo models have suggested that IL-2 may have a predominantly negative regulatory role on T cell responses by promoting activation-induced cell death (AICD). IL-2-/- mice display only a slight decrease in the magnitude of the peak CD8+ T cell response to viral infection (19, 20, 21), and suffer from a T cell-mediated lymphoproliferative autoimmune syndrome (22). Ab blockade of IL-2R signaling in vivo leads to a selective increase in the total population of CD8+ T cells with a memory phenotype, suggesting IL-2 may naturally function to induce cell death in memory CD8+ T cells (23). However, these global modulations of IL-2 biology may not reflect the direct role of IL-2R signaling in CD8+ T cells responding to a pathogen, but rather, the roles of IL-2R signals in T cell development and/or maintenance of regulatory CD25+ CD4+ T cells (24, 25, 26, 27).

To assess the direct role of IL-2R signals in the generation of antiviral CD8+ T cell responses and the establishment of memory CD8+ T cells, we designed a strategy to deliver augmented IL-2R signals only to responding CD8+ T cells during the period of the active response to Ag. Therefore, transgenic (Tg) mice were generated with a murine chimeric GM-CSF/IL-2R (GMIL2R) under the control of a CD8{alpha} enhancer/promoter, which results in transgene expression exclusively in mature CD8+ T cells, thereby obviating any impact on T cell development (28, 29). The GMIL2R is a two-chain receptor consisting of the extracellular ligand-binding domains of the GM-CSFR {alpha}- and {beta}-chains fused to the intracellular signaling domains of the IL-2R {gamma}- and {beta}-chains, respectively. The GM-CSFR ectodomains were selected for the chimeric receptor because the receptor is not normally expressed on resting or activated murine CD8+ T cells (30), and the GM-CSFR shares nearly identical on/off rates and a dissociation constant for ligand binding as the IL-2R (31, 32). Binding of GM-CSF dimerizes the chimeric GMIL2R chains and delivers a signal to T cells indistinguishable, either kinetically or biochemically, from the native IL-2R (33, 34, 35). Moreover, GM-CSF, unlike IL-2, continues to be produced following target recognition by CD8+ T cells that have differentiated from naive to effector T cells (14, 36). Thus, following Ag stimulation, GMIL2R+ effector CD8+ T cells retain via GM-CSF production an autocrine mechanism to sustain proliferation without a requirement for exogenous sources of IL-2 (29).

Enhanced growth of CD8+ T cells expressing the GMIL2R in response to immunization with irradiated tumor cells was previously demonstrated both in vitro and in vivo (29). Although these data indicated a positive regulatory role for IL-2R signals in the generation of CD8+ T cell responses to immunization, as has been suggested by vaccine studies that incorporate IL-2 during immunization (37), the availability of Ag and subsequent TCR signaling are short-lived in these settings due to the inability of the immunogen to replicate. Because promotion of AICD by IL-2 occurs primarily following repetitive stimulation of effector CD8+ T cells in the presence of high Ag loads (18, 38, 39), the negative regulation of CD8+ T cell responses by IL-2R-mediated AICD may be more relevant following recognition of replicating or abundant Ags (40, 41), such as might occur in humans following EBV or HIV infections. Therefore, we examined infection of GMIL2R Tg mice with LCMV, a well-described murine virus that generates high viral burdens, and for which strategies have been developed to precisely evaluate the magnitude of the CD8+ T cell response and the contributions of CD4+ Th function. Our data demonstrate that following LCMV infection, the physiologically provided IL-2R signals limit the magnitude of acute CD8+ T cell expansion and the resultant pool of memory CD8+ T cells formed during the primary antiviral response. Additionally, although a concurrent CD4+ Th response to the virus increases the size of the memory CD8+ T cell pool established, these contributions by CD4+ Th can be supplanted by provision of increased IL-2R signals to responding CD8+ T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and viral infection

All mice were handled according to University of Washington and American Association for the Accreditation of Laboratory Animal Care guidelines. B6 and Thy-1.1 congenic mice were purchased from The Jackson Laboratory (Bar Harbor, ME). GMIL2R Tg mice were generated on a pure B6 background, and expression of the GMIL2R in CD8+ T cells did not lead to alterations in T cell development, the size of lymphoid T cell populations, nor expression of activation/memory markers on CD8+ T cells in the absence of Ag stimulation (29). For assessing primary infections, mice received 2.5 x 105 PFU of the Armstrong strain of LCMV i.p. For assessing recall responses, 5 x 106 PFU of the more virulent LCMV clone 13 was administered i.v.

In vivo CD4+ T cell depletion

Culture supernatants from the anti-CD4-producing GK1.5 hybridoma were isolated, and Ab precipitated with 50% saturated ammonium sulfate solution (42). Precipitates were resuspended and dialyzed 50-fold against 1x PBS before use. Mice were injected i.p. with 0.75 ml of purified GK1.5 Ab, which led to deletion of >99% of CD4+ T cells within 2 days of Ab depletion (42). The CD4-deficient state was maintained by weekly Ab administration (42).

Intracellular cytokine staining and flow cytometry

Spleens were harvested, homogenized, and passed through nylon filters to create single cell suspensions, and RBC lysed via resuspension in ACK (150 mM NH4Cl, 10 mM KHCO3) lysis buffer. A total of 106 splenocytes was then cultured in 96-well flat-bottom plates with 10 U/ml IL-2, and 0.1 µg/ml of either NP396–404 or gp33–41 peptide in RPMI supplemented with 10% FBS (10% RPMI). One hour later, 10 µg/ml of brefeldin A (Sigma-Aldrich, St. Louis, MO) was added, and 4 h later cells were harvested, stained with anti-CD8 FITC mAb, fixed, permeabilized with cytofix/cytoperm solution, and stained with anti-IFN-{gamma} PE mAb (BD PharMingen, La Jolla, CA). Cells were analyzed on a FACSCalibur, and data analysis was performed using CellQuest software (BD Biosciences, San Jose, CA).

Isolation of lymph node, lung, and liver T cells

Mice were perfused with HBSS supplemented with 10 U/ml heparin. Inguinal and mesenteric lymph nodes were isolated and homogenized to form single cell suspensions. Lung and liver tissue were resected as intact exsanguinated organs and homogenized, and lymphocytes were isolated similar to published protocols (43).

Adoptive transfer for examination of memory T cell turnover

Single Tg mice expressing TCRs specific for the gp33 peptide of LCMV (TCR) and double Tg TCR x GMIL2R mice were infected with 2.5 x 105 PFU of LCMV i.p. 30 days after infection, and splenocytes were harvested and labeled with CFSE for 1 h at 37°C in 10% RPMI. A total of 3 x 106 T cells was then transferred i.v. into LCMV-immune Thy-1.1 congenic mice also infected 30 days prior with LCMV. Ten days after transfer, spleens from congenic hosts were isolated, and the transferred T cells were examined via flow cytometry.

Plaque assay

Plaque assays were performed, as previously described (44). Briefly, Vero cell monolayers were infected with serial dilutions of splenic homogenate or serum from LCMV-infected mice at 37°C for 1 h. Infected monolayers were then overlayed with 5% media 199 in 0.5% agarose. Plaques were counterstained with neutral red 4 days later and enumerated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Delivery of augmented autocrine IL-2R signals to CD8+ T cells leads to increased splenomegaly after LCMV infection

Following acute LCMV infection, mice develop splenomegaly due in part to an increase in splenic CD8+ T cells (45). Therefore, CD4-intact B6 and GMIL2R Tg mice were infected with 2.5 x 105 PFU of the Armstrong strain of LCMV i.p., and spleens were removed at the peak of T cell expansion on day 8 (45). Infected B6 mice developed enlarged spleens (Fig. 1Go), which included an increase in the number of splenic CD8+ T cells from 6.5 x 106 in uninfected control mice (n = 5) to 2.9 x 107 cells. Uninfected B6 and GMIL2R Tg mice had equivalent numbers of splenic CD8+ T cells (both 6.5 x 106 CD8+ T cells), but following LCMV infection, GMIL2R Tg mice (n = 6) consistently showed more marked splenomegaly, which contained 6.9 x 107 CD8+ T cells/spleen compared with 2.9 x 107 in infected B6 mice. Similar expansion of the CD8+ population at the peak on day 8 was observed in CD4-deficient Tg and B6 LCMV-infected mice, with CD4-deficient GMIL2R Tg mice containing 7.8 x 107 splenic CD8+ T cells compared with 2.8 x 107 in B6 mice. Mice from all groups displayed nearly identical serum viral titers of LCMV by plaque assay on day 3 (1.3–1.7 x 103 PFU/ml) and completely cleared LCMV infection by day 9 postinfection.



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  		FIGURE 1. Spleens from GMIL2R Tg mice display increased splenomegaly after LCMV infection. B6 or GMIL2R Tg mice were either left uninfected or infected with 2.5 x 105 PFU of LCMV i.p., and spleens were harvested 8 days later. Spleens depicted are representative of four separate experiments.

 
GMIL2R Tg mice generate an increased number of LCMV-specific T cells at the peak of expansion, but exhibit a similar rate of contraction

To determine whether the increased number of CD8+ T cells in spleens of infected GMIL2R Tg mice reflected a larger LCMV-specific CD8+ T cell response, LCMV-specific T cells reactive to the two codominant immunogenic epitopes of LCMV, NP396–404 (NP396) and gp33–41 (gp33), were quantitated by intracellular staining for IFN-{gamma} production (45). On day 8 following infection of B6 mice (n = 5), 9.2% of all splenic CD8+ T cells produced IFN-{gamma} in response to NP396, representing 2.6 x 106 NP396-specific splenic CD8+ T cells (Fig. 2Go, A and B). B6 mice lacking CD4+ T cells (n = 6) had similar frequencies (9.5%) and numbers (2.4 x 106) of NP396-specific T cells compared with the CD4-intact B6 mice (Fig. 2GoB), consistent with reports that CD4+ T cells do not significantly enhance the peak effector response to LCMV (42). Analysis of splenic gp33-specific CD8+ T cells revealed similar results (Fig. 2Go, A and C). In CD4-intact B6 mice, 9.6% of CD8+ T cells were gp33 specific, representing 2.8 x 106 gp33-specific T cells, whereas in CD4-deficient mice, 7.8% of CD8+ T cells were gp33-specific, representing 2.3 x 106 T cells/spleen.



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  		FIGURE 2. GMIL2R Tg mice display an increased number of antiviral CD8+ T cells at the peak of expansion and at the memory phase. A, Splenocytes from a CD4-intact B6 mouse infected with 2.5 x 105 PFU of LCMV i.p. 8 days prior were assessed for IFN-{gamma} production by intracellular staining. The peptide stimulus for in vitro culture, along with the percentage of CD8+ T cells specific for each peptide, are indicated. B and C, Cohorts of CD4-intact B6 ({blacksquare}) and GMIL2R Tg mice (•), and CD4-deficient B6 ({square}) and GMIL2R Tg ({circ}) mice were infected with 2.5 x 105 PFU of LCMV i.p., and spleens were harvested on days 8, 14, 25, and 50 postinfection. At each time point, the number and frequency of CD8+ T cells specific for NP396 (B) or gp33 (C) were quantitated by intracellular staining for IFN-{gamma}. Results are compiled from four separate experiments, with SEs indicated for each time point.

 
At the peak of expansion following infection, both CD4-intact and CD4-deficient GMIL2R Tg mice displayed antiviral CD8+ T cell responses that were greater than those generated in B6 mice. On day 8, CD4-intact GMIL2R Tg mice contained 7.9 x 106 NP396-specific and 9 x 106 gp33-specific splenic CD8+ T cells (n = 6) at frequencies of 11.6 and 13% (Fig. 2Go, B and C). These responses were >3-fold higher than the response observed in CD4-intact B6 mice (p < 0.001). Similarly, spleens from CD4-deficient GMIL2R Tg mice (n = 6) contained 9.5 x 106 NP396-specific and 7.3 x 106 gp33-specific CD8+ T cells/spleen at frequencies of 11.6 and 9.5% (Fig. 2Go, B and C), which were >3-fold greater than responses in CD4-deficient B6 mice (p < 0.001).

By day 14 after infection in both CD4-intact and deficient B6 mice, the NP396-specific and gp33-specific CD8+ T cell pools had contracted >80% (Fig. 2Go, B and C), most likely due to apoptosis following viral clearance and removal of the antigenic stimulus (1, 39, 46). The NP396- and gp33-specific CD8+ T cell pools of CD4-intact and deficient GMIL2R Tg mice similarly contracted >80% from the peak, suggesting that enhanced signaling through the chimeric IL-2R during the response to the virus did not lead to either improved survival or proportionately greater cell death from cytokine withdrawal following viral clearance.

GMIL2R Tg mice generate an increased number of LCMV-specific memory T cells

By day 25 postinfection, spleens of infected mice return nearly to normal size, and a stable long-lived memory CD8+ T cell pool to LCMV is established (45). At this time, 1.8 x 105 NP396-specific and 2.2 x 105 gp33-specific CD8+ T cells were present in spleens of CD4-intact B6 mice (n = 10), representing 2.0 and 2.7% of CD8+ T cells (Fig. 2Go, B and C). Although the absence of CD4+ T cells did not impact the magnitude of the peak antiviral CD8+ T cell response achieved in response to acute infection, CD4-deficient mice exhibited a significant decrease in the size of the memory pool established, consistent with previous reports (2). In CD4-deficient mice (n = 9), the NP396-specific CD8+ T cell memory pool of 0.83 x 105 cells and frequency of 1.7% were >50% smaller (p < 0.025) than the pool in CD4-intact mice (Fig. 2GoB). Similarly, there was a nearly 60% decline (p < 0.025) in formation of the gp33-specific memory CD8+ T cell pool of 0.93 x 105 cells at a frequency of 1.9% of CD8+ T cells (Fig. 2GoC).

At day 25 postinfection, spleen sizes in GMIL2R Tg mice were only 1.3- to 1.4-fold larger than B6 mice, as compared with the >2-fold increases on day 8 postinfection. This in part reflected the significantly increased memory CD8+ T cell pools in infected GMIL2R Tg animals compared with infected B6 mice. On day 25 postinfection, spleens from CD4-intact GMIL2R Tg mice (n = 11) infected with LCMV contained 4.7 x 105 NP396-specific and 4.9 x 105 gp33-specific CD8+ T cells at frequencies of 3.5 and 3.9% of CD8+ T cells (Fig. 2Go, B and C), which were 2.6- and 2.2-fold greater than infected CD4-intact B6 mice (p < 0.001). In CD4-deficient GMIL2R Tg mice (n = 8), the NP396-specific and gp33-specific CD8+ T cell splenic pools of 3.6 x 105 and 3.0 x 105 at frequencies of 3.6 and 3.2% were 4.3- and 3.2-fold larger (p < 0.001) than the pools in infected CD4-deficient B6 mice (Fig. 2Go, B and C). As with B6 mice, there were fewer total memory NP396- and gp33-specific CD8+ T cells in CD4-deficient GMIL2R Tg mice compared with CD4-intact GMIL2R Tg mice, but the magnitude of these differences was smaller. The NP396-specific and gp33-specific memory CD8+ T cell pools in CD4-deficient GMIL2R Tg mice were only 24 and 38% less than the pools in CD4-intact GMIL2R Tg mice, as contrasted to the 50 and 60% decreases in these memory pools in CD4-deficient B6 mice compared with intact B6 mice. Indeed, the sizes of the memory populations established in CD4-deficient GMIL2R Tg mice exceeded the sizes achieved in B6 mice with an intact CD4+ Th response. The increase in the size of the memory CD8+ T cell pools in GMIL2R Tg mice was stable and maintained long-term, with no change from days 25 to 50 in both B6 and GMIL2R Tg mice (Fig. 2Go, B and C).

The increased peak responses and memory pool size detected in GMIL2R Tg mice presumably reflected the autocrine proliferative response to GM-CSF produced during the period CD8+ T cells recognized LCMV-infected targets. CD8+ T cells isolated from mice either during the period of acute viral infection or following viral clearance only produced GM-CSF if specifically stimulated with Ag, and no GM-CSF from exogenous sources could be detected either spontaneously from spleen cells or in the serum of mice that had cleared infection (data not shown).

GMIL2R+ CD8+ memory T cells exhibit similar rates of turnover compared with wild-type memory CD8+ T cells

Despite constitutive expression of the GMIL2R on CD8+ T cells, GM-CSF does not cause naive GMIL2R+ T cells to proliferate in the absence of Ag in vitro (29). GM-CSF is not measurable in the serum of mice (47), but, because it is potentially produced by a variety of cell types (48, 49), such local production of GM-CSF might lead to activation and proliferation of resting memory GMIL2R+ CD8+ T cells. Resting memory CD8+ T cells do normally exhibit a steady proliferative rate, with ~20–30% of memory CD8+ T cells dividing during a 7-day period (50), and the observed stability of the size of the LCMV-specific memory CD8+ T cell pools in GMIL2R Tg mice between days 25 and 50 suggested GMIL2R+ CD8+ T cells did not have a higher turnover rate. However, we directly examined the turnover of GMIL2R+ and GMIL2R- memory CD8+ T cells in an adoptive transfer model (50). To generate a large population of LCMV-specific memory T cells, Tg mice expressing a TCR specific for gp33 (TCR) were crossed to GMIL2R Tg mice (TCR x GMIL2R) and infected with 2.5 x 105 PFU of LCMV (51). Before infection, CD8+ T cells from TCR and TCR x GMIL2R mice had a uniformly naive phenotype, with fewer than 10% of CD8+ T cells expressing CD44. However, 30 days following infection, >85% of CD8+ T cells displayed a CD44high memory phenotype (data not shown). CD8+ T cells from TCR and TCR x GMIL2R Tg mice were harvested and loaded with CFSE to monitor T cell proliferation and turnover. To closely mimic the environment that the transferred memory CD8+ T cell would normally be exposed, the CFSE-labeled cells were transferred into nonirradiated LCMV-immune Thy-1.1 congenic hosts that had also been infected 30 days prior. Ten days after transfer, spleens were harvested and CFSE content was examined in Thy-1.2+ CD8+ T cells (Fig. 3Go). At day 10 posttransfer, ~31% of TCR T cells had divided at least once, consistent with published results (50). TCR x GMIL2R T cells exhibited a similar turnover rate, with 30% of TCR x GMIL2R T cells having undergone at least one cell division. Similar numbers of TCR and TCR x GMIL2R were recovered from recipient mice (data not shown).



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  		FIGURE 3. Memory GMIL2R+ CD8+ T cell turnover is similar to wild-type memory CD8+ T cells. GMIL2R- and GMIL2R+ memory CD8+ T cells were generated from TCR and TCR x GMIL2R Tg mice infected with LCMV 30 days prior. Memory CD8+ T cells were then isolated from spleens of TCR and TCR x GMIL2R Tg mice, labeled with CFSE, and transferred into LCMV-immune congenic Thy-1.1 hosts. CFSE content of the Thy-1.2+-transferred cells was examined 10 days after transfer. The percentage of cells in a particular cell division is indicated.

 
The increased memory T cell pools in GMIL2R Tg mice are observed in both lymphoid and nonlymphoid memory CD8+ T cell populations

In mice, memory T cells have been divided into two subpopulations based in part on distinct migratory patterns to either lymphoid or nonlymphoid tissues (43, 52, 53). Lymphoid and nonlymphoid memory T cells also display unique phenotypic and functional characteristics, such as differences in cytokine expression or effector function upon target recognition (43, 53). Because GMIL2R Tg mice exhibited an increased CD8+ T cell response in the spleen, we next examined whether this reflected altered differentiation impacting on the CD8+ T cell pools established in nonlymphoid organs. CD4-intact B6 and GMIL2R Tg mice were infected with 2.5 x 105 PFU of LCMV i.p., and 30 days later spleen, lymph nodes, liver, and lung were removed and perfused to eliminate blood lymphocytes. Liver and lung lymphocytes were isolated as previously described (43). CD4-intact GMIL2R Tg mice contained 2.5-fold more NP396- and gp33-specific CD8+ T cells (p < 0.005) in spleen and lymph nodes compared with CD4-intact B6 mice (Fig. 4GoA). The virus-specific CD8+ T cell populations of CD4-intact GMIL2R Tg mice residing in liver and lung were also expanded relative to CD4-intact B6 mice, with 2.2- and 1.8-fold increases observed in liver and lung, respectively (p < 0.05). In CD4-deficient GMIL2R Tg mice, the virus-specific lymphoid CD8+ T cell pool was 3.3-fold greater (p < 0.001) than in CD4-deficient B6 mice and 1.7-fold greater (p < 0.02) in both lung and liver (Fig. 4GoB). Thus, the enhanced memory CD8+ T cell pools in GMIL2R Tg mice included both lymphoid and nonlymphoid compartments.



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  		FIGURE 4. GMIL2R Tg mice contain increased memory CD8+ T cell pools in nonlymphoid organs. A, CD4-intact B6 (filled bars) and GMIL2R Tg (gray bars) mice, and B, CD4-deficient B6 (filled bars) and GMIL2R Tg (gray bars) mice were infected with 2.5 x 105 PFU of LCMV i.p. 30 days after infection; mice were perfused; and spleens, lymph nodes, liver, and lung were isolated. The total number of NP396- and gp33-specific CD8+ T cells in each organ was determined by intracellular staining for IFN-{gamma}. Data are compiled from three experiments with four to six mice per group.

 
GMIL2R Tg memory T cells expand following viral rechallenge

A functional memory response must be capable of responding to rechallenge with the Ag. Because excessive proliferation of CD8+ T cells has been associated with exhaustion of the responding cells (41), we examined whether the increased T cell proliferative response of GMIL2R Tg mice during primary LCMV infection affected the capacity to mount a recall response to a secondary LCMV infection. To eliminate the potentially confounding effects of anti-LCMV-neutralizing Abs on the viral load achieved during secondary infection that could alter the magnitude of the CD8+ T cell proliferative response (54), we examined the recall response in B6 and GMIL2R Tg mice lacking CD4+ Th function, because such mice are unable to generate neutralizing Abs following LCMV infection (44). Thus, cohorts of CD4-deficient B6 and GMIL2R Tg mice initially infected with 2.5 x 105 PFU of LCMV were rechallenged 30 days later, after the memory responses had been established, with 5 x 106 PFU of a more virulent LCMV strain, clone 13, i.v. (45). Following rechallenge, mice rapidly clear even the more virulent virus and display a peak recall response at 5 days after rechallenge (45, 55). Both groups of mice had no detectable virus in spleens by plaque-forming assay at day 5 postrechallenge. The number of virus-specific CD8+ T cells in CD4-deficient B6 mice increased 5.6-fold during these 5 days (Fig. 5Go). CD4-deficient GMIL2R Tg mice increased the anti-LCMV CD8+ T cell pool 13.7-fold at 5 days following reinfection compared with preinfection levels. Thus, the GMIL2R+ memory CD8+ T cells were not only capable of mounting a functional response to viral rechallenge, but the size of the recall response was proportionally larger than the response of wild-type memory CD8+ T cells.



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  		FIGURE 5. GMIL2R Tg memory CD8+ T cells expand following viral reinfection. Cohorts of CD4-deficient B6 (filled bars) and GMIL2R Tg (gray bars) mice were infected with 2.5 x 105 PFU of LCMV i.p., and 30 days later rechallenged with 5 x 106 PFU of clone 13 i.v. Five days after reinfection with clone 13, spleens were harvested, and the total number of NP396- and gp33-specific CD8+ T cells was quantitated by intracellular staining for IFN-{gamma}. Data are compiled from three experiments with four to six mice at each time point.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IL-2 can potentially mediate multiple effects during an antiviral CD8+ T cell response. Our data demonstrate that provision of augmented IL-2R signals to CD8+ T cells during the period of target recognition increases the magnitude of the CD8+ T cell response to LCMV, increases the size of the resulting long-lived memory CD8+ T cell pool, and compensates for the absence of CD4+ Th function in generating the antiviral memory CD8+ T cell response. These results suggest that even under high Ag loads with potentially repetitive TCR stimulation, which could predispose CD8+ T cells to AICD, the primary role of IL-2R signals in responding CD8+ T cells is to promote continued proliferation and expansion rather than cell death. Thus, the conclusions regarding the negative effects of IL-2R signaling on CD8+ T cell responses drawn from IL-2-/- mice or Ab-mediated blockade of IL-2 activity appear to reflect global changes in T cell development and/or function, rather than direct effects on responding CD8+ T cells.

Following acute LCMV infection, CD4+ Th cells have little impact on the magnitude of the peak CD8+ T cell response achieved. The lack of a requirement for CD4+ Th to license APC for effective stimulation of CD8+ T cells is most likely attributable to the direct maturation/activation of APC by LCMV infection (11). However, the basis for the apparent lack of a contribution by IL-2 produced by responding CD4+ Th cells to the peak of CD8+ T cell expansion is less clear, because our data demonstrate that the availability of IL-2 limits CD8+ T cell expansion. Several reasons may account for this. First, the peak magnitude of the CD4+ Th response to LCMV infection is >20-fold lower than the CD8+ T cell response (56). Thus, CD4+ Th cells may only be able to provide paracrine IL-2 to a minor population of responding CD8+ T cells. Second, because viral infection with LCMV directly infects and matures APC, CD8+ T cells can produce IL-2 and proliferate at sites in which CD4+ Th function might be scarce or absent, particularly because the responding CD8+ T cells can lyse the APC. Finally, the outcome of IL-2R signaling in CD8+ T cells may be tied to the timing of IL-2R signals (18). Because CD4+ Th production of IL-2 is not synchronized to activation of individual responding CD8+ T cells, exposure to paracrine IL-2 could cause both enhanced CD8+ T cell proliferation and cell death, leading to no net effect on the T cell response.

Although CD4+ Th function does not appear to contribute to the magnitude of the acute phase of CD8+ T cell expansion in B6 mice, CD4+ Th cells do play a role in determining the size of the memory CD8+ T cell pool formed following viral clearance (2). The contributions of CD4+ Th function to memory CD8+ T cell formation were much less evident in GMIL2R Tg because the number of memory CD8+ T cells formed after viral infection in CD4-deficient GMIL2R Tg mice was only slightly smaller than in intact GMIL2R Tg mice, whereas in wild-type mice, the presence of CD4+ T cells more than doubled the number of memory CD8+ T cells formed. Augmented IL-2R signals delivered via the chimeric GMIL2R could have several effects on CD8+ T cells, which might directly enhance the generation of memory CD8+ T cells and account for the diminished CD4 dependence in GMIL2R Tg mice. If memory CD8+ T cells are directly derived from the predominant population represented by the peak of expansion, enhanced signaling through an IL-2R during the expansion phase could result in increased expression of antiapoptotic factors such as bcl-2 (57), making the cells better able to survive the contraction phase. Alternatively, if memory CD8+ T cells are derived from a distinct subset of cells responding to the Ag, delivery of signals through an IL-2R might directly promote their proliferation and survival, and/or facilitate differentiation into memory cells.

Increased CD8+ T cell proliferation in the context of a persistent viral infection has been associated with exhaustion and deletion of Ag-specific CD8+ T cell responses (41). This exhaustion of CD8+ T cells has been thought to at least in part be attributable to excessive proliferation of Ag-specific CD8+ T cells Although GMIL2R Tg mice mounted an enhanced acute phase CD8+ T cell response to primary LCMV infection, the increased proliferative response did not limit the capacity of the memory CD8+ T cell pool in GMIL2R Tg mice to mount a secondary response to reinfection. Indeed, rather than exhibiting diminished responses, CD8+ T cells from GMIL2R Tg mice demonstrated stronger responses to rechallenge than wild-type CD8+ T cells, consistent with a positive effect from augmentation of IL-2R signals in secondary responses of CD8+ T cells, as was observed in primary responses. Thus, our data suggest that increased proliferation of CD8+ T cells does not predispose these cells to exhaustion, and are consistent with preliminary observations that exogenous IL-2 can promote the survival and effector function of CD8+ T cells that might normally be deleted in the face of persistent viral infection (41).

Our data suggest different conclusions than those drawn from recent work examining the effects of the loss of IL-2 function on CD8+ T cells during an antiviral CD8+ T cell response (58). Following adoptive transfer of TCR Tg IL-2-/- CD8+ T cells into wild-type or IL-2-/- mice and infection of the recipient mice with a recombinant vesicular stomatitis virus, IL-2 availability did not appear to be a limiting factor for the splenic CD8+ T cell response. By contrast, our data, which demonstrate significantly enhanced anti-LCMV responses in mice with augmented autocrine IL-2R signals, suggest IL-2 availability is limiting. This disparity may be attributable to differences in the immunobiology of LCMV and vesicular stomatitis virus infection, but alternatively the lack of requirement for autocrine IL-2 observed by D’Souza et al. could be due to either altered development of IL-2-/- donor T cells (20), paracrine IL-2 production by CD4+ T cells in wild-type recipient mice, or the loss of contributions by IL-2 during T cell development in the recipient IL-2-/- mice, leading to modified peripheral T cell function, such as from failure to develop CD4+ regulatory T cells (24, 59).

The GMIL2R transduces a signal that is biochemically indistinguishable from the native IL-2R (33). However, the IL-2R{beta}- and common {gamma}-chains are activated by binding of IL-15 as well as IL-2, with both cytokines capable of enhancing CD8+ T cell proliferation in vitro, making it possible the GMIL2R is mimicking the effects of either or both IL-2 and IL-15. Although the two cytokines share these two signaling receptor chains, IL-2 and IL-15 receptors have distinct {alpha}-chains required for high affinity ligand binding, and the biologic effects of the cytokines are not entirely overlapping. For example, T cells become susceptible to AICD following repetitive TCR stimulation in vitro. Although AICD can be enhanced in vitro by the presence of pharmacologic doses of IL-2 during TCR activation, exposure of activated T cells to pharmacologic doses of IL-15 does not appear to increase apoptosis (60), and may actually enhance T cell survival (61). Given the common signaling pathways engaged by IL-2 and IL-15 and the fact that the cytokines were added at the initiation of culture, such findings are difficult to explain. The disparate effects might reflect distinct kinetics of signaling, such as could occur if IL-15R{alpha} expression is delayed compared with IL-2R{alpha}, making the IL-15 signal more prominent when T cells have completed an initial proliferative burst and have become less susceptible to AICD (62). Alternatively, the distinct effects of IL-15 might be mediated by a unique signaling capability of the IL-15R{alpha} chain, as compared with the nonsignaling IL-2R{alpha} chain (63, 64, 65). Such unique signaling events would make the chimeric GMIL2R more like the IL-2R, because it lacks a signaling {alpha}-chain. The patterns of cytokine expression also differ between IL-2 and IL-15, and again might make the signals delivered through the GMIL2R more similar to IL-2R signals. IL-2, similar to GM-CSF, is produced by activated T cells and is rapidly extinguished following Ag withdrawal. IL-15, in contrast, is produced primarily by stromal cells and APC, but not T cells (66), and may not be abundantly available to rapidly proliferating CD8+ T cells, particularly in the context of viral infection of APC (67). However, it should be noted that IL-15R signaling may contribute to the acute phase of the antiviral CD8+ T cell response (68), although it remains unclear whether this reflects effects of IL-15 on T cells or APC (69). Thus, we cannot exclude the possibility that some of the effects of the GMIL2R may reflect IL-15R-like rather than IL-2R-like signals.

Provision of augmented IL-2R signals to CD8+ T cells enhanced T cell responses both during the acute and recall responses, which could potentially be useful clinically. For example, administration of IL-2 to HIV patients during bursts of viral replication such as following structured treatment interruption might be an effective means of enhancing CD8+ T cell immunity to HIV (70, 71, 72). However, regimens for delivering supplemental IL-2 will require adequate penetration of secondary lymphoid organs and other sites at which targets are recognized. Administration of high doses of IL-2 may be able to accomplish this, but can be associated with toxicity that could limit clinical utility (73, 74). For therapeutic settings in which adoptive T cell transfer with Ag-specific CD8+ T cells is an option, it might be possible to overcome these obstacles to effective IL-2 therapy by genetically modifying memory/effector CD8+ T cells with the chimeric GMIL2R before transfer. Such an approach might also obviate the requirement for long-term IL-2 administration to sustain specific T cell responses, and could permit establishment of long-term functional immunity in patients in which a concurrent CD4+ T cell response against either viruses or tumors is chronically deficient or absent.


   Acknowledgments
 
We thank R. Ahmed for kindly providing LCMV-Armstrong and clone 13 virus stocks. We also thank S. Riddell, K. Murali-Krishna, and J. N. Blattman for experimental advice and thoughtful discussions, as well as A. Shur and X. Tan for expert technical assistance.


   Footnotes
 
1 This work was supported by a Poncin Foundation award to L.E.C. and grants from the National Institutes of Health (CA33084, CA18029, and AI43650) to P.D.G. Back

2 Address correspondence and reprint requests to Dr. Philip D. Greenberg. University of Washington, Box 356527, Seattle, WA 98195. E-mail address: pgreen{at}u.washington.edu Back

3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; AICD, activation-induced cell death; Tg, transgenic; GMIL2R, GM-CSF/IL-2R. Back

Received for publication July 8, 2002. Accepted for publication August 30, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ahmed, R., D. Gray. 1996. Immunological memory and protective immunity: understanding their relation. Science 272:54.[Abstract]
  2. Von Herrath, M. G., M. Yokoyama, J. Dockter, M. B. Oldstone, J. L. Whitton. 1996. CD4-deficient mice have reduced levels of memory cytotoxic T lymphocytes after immunization and show diminished resistance to subsequent virus challenge. J. Virol. 70:1072.[Abstract]
  3. Ochsenbein, A. F., U. Karrer, P. Klenerman, A. Althage, A. Ciurea, H. Shen, J. F. Miller, J. L. Whitton, H. Hengartner, R. M. Zinkernagel. 1999. A comparison of T cell memory against the same antigen induced by virus versus intracellular bacteria. Proc. Natl. Acad. Sci. USA 96:9293.[Abstract/Free Full Text]
  4. Reusser, P., S. R. Riddell, J. D. Meyers, P. D. Greenberg. 1991. Cytotoxic T-lymphocyte response to cytomegalovirus after human allogeneic bone marrow transplantation: pattern of recovery and correlation with cytomegalovirus infection and disease. Blood 78:1373.[Abstract/Free Full Text]
  5. Li, C. R., P. D. Greenberg, M. J. Gilbert, J. M. Goodrich, S. R. Riddell. 1994. Recovery of HLA-restricted cytomegalovirus (CMV)-specific T-cell responses after allogeneic bone marrow transplant: correlation with CMV disease and effect of ganciclovir prophylaxis. Blood 83:1971.[Abstract/Free Full Text]
  6. Walter, E. A., P. D. Greenberg, M. J. Gilbert, R. J. Finch, K. S. Watanabe, E. D. Thomas, S. R. Riddell. 1995. Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor. N. Engl. J. Med. 333:1038.[Abstract/Free Full Text]
  7. Rickinson, A. B., D. J. Moss. 1997. Human cytotoxic T lymphocyte responses to Epstein-Barr virus infection. Annu. Rev. Immunol. 15:405.[Medline]
  8. Ridge, J. P., F. Di Rosa, P. Matzinger. 1998. A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell. Nature 393:474.[Medline]
  9. Bennett, S. R., F. R. Carbone, F. Karamalis, R. A. Flavell, J. F. Miller, W. R. Heath. 1998. Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 393:478.[Medline]
  10. Schoenberger, S. P., R. E. Toes, E. I. van der Voort, R. Offringa, C. J. Melief. 1998. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 393:480.[Medline]
  11. Ruedl, C., M. Kopf, M. F. Bachmann. 1999. CD8+ T cells mediate CD40-independent maturation of dendritic cells in vivo. J. Exp. Med. 189:1875.[Abstract/Free Full Text]
  12. Borrow, P., A. Tishon, S. Lee, J. Xu, I. S. Grewal, M. B. Oldstone, R. A. Flavell. 1996. CD40L-deficient mice show deficits in antiviral immunity and have an impaired memory CD8+ CTL response. J. Exp. Med. 183:2129.[Abstract/Free Full Text]
  13. Nelson, B. H., D. M. Willerford. 1998. Biology of the interleukin-2 receptor. Adv. Immunol. 70:1.[Medline]
  14. Aruga, A., E. Aruga, M. J. Cameron, A. E. Chang. 1997. Different cytokine profiles released by CD4+ and CD8+ tumor-draining lymph node cells involved in mediating tumor regression. J. Leukocyte Biol. 61:507.[Abstract]
  15. Tham, E. L., P. Shrikant, M. F. Mescher. 2002. Activation-induced nonresponsiveness: a Th-dependent regulatory checkpoint in the CTL response. J. Immunol. 168:1190.[Abstract/Free Full Text]
  16. Gillis, S., K. A. Smith. 1977. Long term culture of tumor-specific cytotoxic T cells. Nature 268:154.[Medline]
  17. Maraskovsky, E., W. F. Chen, K. Shortman. 1989. IL-2 and IFN-{gamma} are two necessary lymphokines in the development of cytolytic T cells. J. Immunol. 143:1210.[Abstract]
  18. 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.[Medline]
  19. Schorle, H., T. Holtschke, T. Hunig, A. Schimpl, I. Horak. 1991. Development and function of T cells in mice rendered interleukin-2 deficient by gene targeting. Nature 352:621.[Medline]
  20. Bachmann, M. F., H. Schorle, R. Kuhn, W. Muller, H. Hengartner, R. M. Zinkernagel, I. Horak. 1995. Antiviral immune responses in mice deficient for both interleukin-2 and interleukin-4. J. Virol. 69:4842.[Abstract]
  21. Cousens, L. P., J. S. Orange, C. A. Biron. 1995. Endogenous IL-2 contributes to T cell expansion and IFN-{gamma} production during lymphocytic choriomeningitis virus infection. J. Immunol. 155:5690.[Abstract]
  22. Sadlack, B., H. Merz, H. Schorle, A. Schimpl, A. C. Feller, I. Horak. 1993. Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell 75:253.[Medline]
  23. Ku, C. C., M. Murakami, A. Sakamoto, J. Kappler, P. Marrack. 2000. Control of homeostasis of CD8+ memory T cells by opposing cytokines. Science 288:675.[Abstract/Free Full Text]
  24. Bassiri, H., S. R. Carding. 2001. A requirement for IL-2/IL-2 receptor signaling in intrathymic negative selection. J. Immunol. 166:5945.[Abstract/Free Full Text]
  25. Papiernik, M., M. L. de Moraes, C. Pontoux, F. Vasseur, C. Penit. 1998. Regulatory CD4 T cells: expression of IL-2R{alpha} chain, resistance to clonal deletion and IL-2 dependency. Int. Immunol. 10:371.[Abstract/Free Full Text]
  26. Piccirillo, C. A., E. M. Shevach. 2001. Cutting edge: control of CD8+ T cell activation by CD4+CD25+ immunoregulatory cells. J. Immunol. 167:1137.[Abstract/Free Full Text]
  27. Murakami, M., A. Sakamoto, J. Bender, J. Kappler, P. Marrack. 2002. CD25+CD4+ T cells contribute to the control of memory CD8+ T cells. Proc. Natl. Acad. Sci. USA 99:8832.[Abstract/Free Full Text]
  28. Ellmeier, W., M. J. Sunshine, K. Losos, F. Hatam, D. R. Littman. 1997. An enhancer that directs lineage-specific expression of CD8 in positively selected thymocytes and mature T cells. Immunity 7:537.[Medline]
  29. Cheng, L. E., C. Ohlen, B. H. Nelson, P. D. Greenberg. 2002. Enhanced signaling through the IL-2 receptor in CD8+ T cells regulated by antigen recognition results in preferential proliferation and expansion of responding CD8+ T cells rather than promotion of cell death. Proc. Natl. Acad. Sci. USA 99:3001.[Abstract/Free Full Text]
  30. Park, L. S., U. Martin, R. Sorensen, S. Luhr, P. J. Morrissey, D. Cosman, A. Larsen. 1992. Cloning of the low-affinity murine granulocyte-macrophage colony-stimulating factor receptor and reconstitution of a high-affinity receptor complex. Proc. Natl. Acad. Sci. USA 89:4295.[Abstract/Free Full Text]
  31. Matsuoka, M., T. Takeshita, N. Ishii, M. Nakamura, T. Ohkubo, K. Sugamura. 1993. Kinetic study of interleukin-2 binding on the reconstituted interleukin-2 receptor complexes including the human {gamma} chain. Eur. J. Immunol. 23:2472.[Medline]
  32. Niu, L., D. W. Golde, J. C. Vera, M. L. Heaney. 1999. Kinetic resolution of two mechanisms for high-affinity granulocyte-macrophage colony-stimulating factor binding to its receptor. Blood 94:3748.[Abstract/Free Full Text]
  33. Nelson, B. H., J. D. Lord, P. D. Greenberg. 1994. Cytoplasmic domains of the interleukin-2 receptor {beta} and {gamma} chains mediate the signal for T-cell proliferation. Nature 369:333.[Medline]
  34. Nelson, B. H., J. D. Lord, P. D. Greenberg. 1996. A membrane-proximal region of the interleukin-2 receptor {gamma}c chain sufficient for Jak kinase activation and induction of proliferation in T cells. Mol. Cell. Biol. 16:309.[Abstract]
  35. Nelson, B. H., B. C. McIntosh, L. L. Rosencrans, P. D. Greenberg. 1997. Requirement for an initial signal from the membrane-proximal region of the interleukin 2 receptor {gamma}c chain for Janus kinase activation leading to T cell proliferation. Proc. Natl. Acad. Sci. USA 94:1878.[Abstract/Free Full Text]
  36. Troutt, A. B., E. Maraskovsky, L. A. Rogers, M. H. Pech, A. Kelso. 1992. Quantitative analysis of lymphokine expression in vivo and in vitro. Immunol. Cell Biol. 70:51.
  37. Barouch, D. H., S. Santra, J. E. Schmitz, M. J. Kuroda, T. M. Fu, W. Wagner, M. Bilska, A. Craiu, X. X. Zheng, G. R. Krivulka, et al 2000. Control of viremia and prevention of clinical AIDS in rhesus monkeys by cytokine-augmented DNA vaccination. Science 290:486.[Abstract/Free Full Text]
  38. Li, X. C., G. Demirci, S. Ferrari-Lacraz, C. Groves, A. Coyle, T. R. Malek, T. B. Strom. 2001. IL-15 and IL-2: a matter of life and death for T cells in vivo. Nat. Med. 7:114.[Medline]
  39. Van Parijs, L., A. K. Abbas. 1998. Homeostasis and self-tolerance in the immune system: turning lymphocytes off. Science 280:243.[Abstract/Free Full Text]
  40. Razvi, E. S., R. M. Welsh. 1993. Programmed cell death of T lymphocytes during acute viral infection: a mechanism for virus-induced immune deficiency. J. Virol. 67:5754.[Abstract/Free Full Text]
  41. Moskophidis, D., F. Lechner, H. Pircher, R. M. Zinkernagel. 1993. Virus persistence in acutely infected immunocompetent mice by exhaustion of antiviral cytotoxic effector T cells. Nature 362:758.[Medline]
  42. Matloubian, M., R. J. Concepcion, R. Ahmed. 1994. CD4+ T cells are required to sustain CD8+ cytotoxic T-cell responses during chronic viral infection. J. Virol. 68:8056.[Abstract/Free Full Text]
  43. Masopust, D., V. Vezys, A. L. Marzo, L. Lefrancois. 2001. Preferential localization of effector memory cells in nonlymphoid tissue. Science 291:2413.[Abstract/Free Full Text]
  44. Ahmed, R., L. D. Butler, L. Bhatti. 1988. T4+ T helper cell function in vivo: differential requirement for induction of antiviral cytotoxic T-cell and antibody responses. J. Virol. 62:2102.[Abstract/Free Full Text]
  45. Murali-Krishna, K., J. D. Altman, M. Suresh, D. J. Sourdive, A. J. Zajac, J. D. Miller, J. Slansky, R. Ahmed. 1998. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8:177.[Medline]
  46. Arch, R. H., C. B. Thompson. 1999. Lymphocyte survival: the struggle against death. Annu. Rev. Cell. Dev. Biol. 15:113.[Medline]
  47. Metcalf, D., C. G. Begley, D. J. Williamson, E. C. Nice, J. De Lamarter, J. J. Mermod, D. Thatcher, A. Schmidt. 1987. Hemopoietic responses in mice injected with purified recombinant murine GM-CSF. Exp. Hematol. 15:1.[Medline]
  48. Gasson, J. C.. 1991. Molecular physiology of granulocyte-macrophage colony-stimulating factor. Blood 77:1131.[Free Full Text]
  49. Paine, R., III, A. M. Preston, S. Wilcoxen, H. Jin, B. B. Siu, S. B. Morris, J. A. Reed, G. Ross, J. A. Whitsett, J. M. Beck. 2000. Granulocyte-macrophage colony-stimulating factor in the innate immune response to Pneumocystis carinii pneumonia in mice. J. Immunol. 164:2602.[Abstract/Free Full Text]
  50. Murali-Krishna, K., L. L. Lau, S. Sambhara, F. Lemonnier, J. Altman, R. Ahmed. 1999. Persistence of memory CD8 T cells in MHC class I-deficient mice. Science 286:1377.[Abstract/Free Full Text]
  51. Pircher, H., K. Burki, R. Lang, H. Hengartner, R. M. Zinkernagel. 1989. Tolerance induction in double specific T-cell receptor transgenic mice varies with antigen. Nature 342:559.[Medline]
  52. Sallusto, F., D. Lenig, R. Forster, M. Lipp, A. Lanzavecchia. 1999. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401:708.[Medline]
  53. Reinhardt, R. L., A. Khoruts, R. Merica, T. Zell, M. K. Jenkins. 2001. Visualizing the generation of memory CD4 T cells in the whole body. Nature 410:101.[Medline]
  54. Planz, O., S. Ehl, E. Furrer, E. Horvath, M. A. Brundler, H. Hengartner, R. M. Zinkernagel. 1997. A critical role for neutralizing-antibody-producing B cells, CD4+ T cells, and interferons in persistent and acute infections of mice with lymphocytic choriomeningitis virus: implications for adoptive immunotherapy of virus carriers. Proc. Natl. Acad. Sci. USA 94:6874.[Abstract/Free Full Text]
  55. Suresh, M., J. K. Whitmire, L. E. Harrington, C. P. Larsen, T. C. Pearson, J. D. Altman, R. Ahmed. 2001. Role of CD28–B7 interactions in generation and maintenance of CD8 T cell memory. J. Immunol. 167:5565.[Abstract/Free Full Text]
  56. Homann, D., L. Teyton, M. B. Oldstone. 2001. Differential regulation of antiviral T-cell immunity results in stable CD8+ but declining CD4+ T-cell memory. Nat. Med. 7:913.[Medline]
  57. Lord, J. D., B. C. McIntosh, P. D. Greenberg, B. H. Nelson. 2000. The IL-2 receptor promotes lymphocyte proliferation and induction of the c-myc, bcl-2, and bcl-x genes through the trans-activation domain of Stat5. J. Immunol. 164:2533.[Abstract/Free Full Text]
  58. D’Souza, W. N., K. S. Schluns, D. Masopust, L. Lefrancois. 2002. Essential role for IL-2 in the regulation of antiviral extralymphoid CD8 T cell responses. J. Immunol. 168:5566.[Abstract/Free Full Text]
  59. Sadlack, B., J. Lohler, H. Schorle, G. Klebb, H. Haber, E. Sickel, R. J. Noelle, I. Horak. 1995. Generalized autoimmune disease in interleukin-2-deficient mice is triggered by an uncontrolled activation and proliferation of CD4+ T cells. Eur. J. Immunol. 25:3053.[Medline]
  60. Dai, Z., A. Arakelov, M. Wagener, B. T. Konieczny, F. G. Lakkis. 1999. The role of the common cytokine receptor {gamma}-chain in regulating IL-2-dependent, activation-induced CD8+ T cell death. J. Immunol. 163:3131.[Abstract/Free Full Text]
  61. Marks-Konczalik, J., S. Dubois, J. M. Losi, H. Sabzevari, N. Yamada, L. Feigenbaum, T. A. Waldmann, Y. Tagaya. 2000. IL-2-induced activation-induced cell death is inhibited in IL-15 transgenic mice. Proc. Natl. Acad. Sci. USA 97:11445.[Abstract/Free Full Text]
  62. Boehme, S. A., M. J. Lenardo. 1993. Propriocidal apoptosis of mature T lymphocytes occurs at S phase of the cell cycle. Eur. J. Immunol. 23:1552.[Medline]
  63. Bulfone-Pau, S. S., E. Bulanova, T. Pohl, V. Budagian, H. Durkop, R. Ruckert, U. Kunzendorf, R. Paus, H. Krause. 1999. Death deflected: IL-15 inhibits TNF-{alpha}-mediated apoptosis in fibroblasts by TRAF2 recruitment to the IL-15R{alpha} chain. FASEB J. 13:1575.[Abstract/Free Full Text]
  64. Bulanova, E., V. Budagian, T. Pohl, H. Krause, H. Durkop, R. Paus, S. Bulfone-Paus. 2001. The IL-15R{alpha} chain signals through association with Syk in human B cells. J. Immunol. 167:6292.[Abstract/Free Full Text]
  65. Pereno, R., J. Giron-Michel, A. Gaggero, E. Cazes, R. Meazza, M. Monetti, E. Monaco, Z. Mishal, C. Jasmin, F. Indiveri, et al 2000. IL-15/IL-15R{alpha} intracellular trafficking in human melanoma cells and signal transduction through the IL-15R{alpha}. Oncogene 19:5153.[Medline]
  66. Ma, A., D. L. Boone, J. P. Lodolce. 2000. The pleiotropic functions of interleukin 15: not so interleukin 2-like after all. J. Exp. Med. 191:753.[Free Full Text]
  67. Werling, D., R. A. Collins, G. Taylor, C. J. Howard. 2002. Cytokine responses of bovine dendritic cells and T cells following exposure to live or inactivated bovine respiratory syncytial virus. J. Leukocyte Biol. 72:297.[Abstract/Free Full Text]
  68. Schluns, K. S., K. Williams, A. Ma, X. X. Zheng, L. Lefrancois. 2002. Cutting edge: requirement for IL-15 in the generation of primary and memory antigen-specific CD8 T cells. J. Immunol. 168:4827.[Abstract/Free Full Text]
  69. Ohteki, T., K. Suzue, C. Maki, T. Ota, S. Koyasu. 2001. Critical role of IL-15-IL-15R for antigen-presenting cell functions in the innate immune response. Nat. Immun. 2:1138.
  70. Lisziewicz, J., E. Rosenberg, J. Lieberman, H. Jessen, L. Lopalco, R. Siliciano, B. Walker, F. Lori. 1999. Control of HIV despite the discontinuation of antiretroviral therapy. N. Engl. J. Med. 340:1683.[Free Full Text]
  71. Ortiz, G. M., D. F. Nixon, A. Trkola, J. Binley, X. Jin, S. Bonhoeffer, P. J. Kuebler, S. M. Donahoe, M.-A. Demoitie, W. M. Kakimoto, et al 1999. HIV-1-specific immune responses in subjects who temporarily contain virus replication after discontinuation of highly active antiretroviral therapy. J. Clin. Invest. 104:R13.
  72. Altfeld, M., B. D. Walker. 2001. Less is more? STI in acute and chronic HIV-1 infection. Nat. Med. 7:881.[Medline]
  73. Edwards, R. P., W. Gooding, B. C. Lembersky, K. Colonello, R. Hammond, C. Paradise, C. D. Kowal, A. J. Kunschner, M. Baldisseri, J. M. Kirkwood, R. B. Herberman. 1997. Comparison of toxicity and survival following intraperitoneal recombinant interleukin-2 for persistent ovarian cancer after platinum: twenty-four-hour versus 7-day infusion. J. Clin. Oncol. 15:3399.[Abstract/Free Full Text]
  74. Baluna, R., J. Rizo, B. E. Gordon, V. Ghetie, E. S. Vitetta. 1999. Evidence for a structural motif in toxins and interleukin-2 that may be responsible for binding to endothelial cells and initiating vascular leak syndrome. Proc. Natl. Acad. Sci. USA 96:3957.[Abstract/Free Full Text]



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S. Mostbock, M. E. C. Lutsiak, D. E. Milenic, K. Baidoo, J. Schlom, and H. Sabzevari
IL-2/Anti-IL-2 Antibody Complex Enhances Vaccine-Mediated Antigen-Specific CD8+ T Cell Responses and Increases the Ratio of Effector/Memory CD8+ T Cells to Regulatory T Cells
J. Immunol., April 1, 2008; 180(7): 5118 - 5129.
[Abstract] [Full Text] [PDF]

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H. Jin, D. Gong, D. Adeegbe, A. L. Bayer, C. Rolle, A. Yu, and T. R. Malek
Quantitative assessment concerning the contribution of IL-2R{beta} for superantigen-mediated T cell responses in vivo
Int. Immunol., April 1, 2006; 18(4): 565 - 572.
[Abstract] [Full Text] [PDF]

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A. C. Moore, A. Gallimore, S. J. Draper, K. R. Watkins, S. C. Gilbert, and A. V. S. Hill
Anti-CD25 Antibody Enhancement of Vaccine-Induced Immunogenicity: Increased Durable Cellular Immunity with Reduced Immunodominance
J. Immunol., December 1, 2005; 175(11): 7264 - 7273.
[Abstract] [Full Text] [PDF]

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R. M. Teague, R. M. Tempero, S. Thomas, K. Murali-Krishna, and B. H. Nelson