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The Journal of Immunology, 2007, 179, 2074-2081
Copyright © 2007 by The American Association of Immunologists, Inc.
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Generation of CD8 T Cell Memory Is Regulated by IL-121

Erika L. Pearce2 and Hao Shen3

Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Various signals during infection influence CD8 T cell memory generation, but these factors have yet to be fully defined. IL-12 is a proinflammatory cytokine that has been shown to enhance IFN-{gamma}-producing T cell responses and has been widely tested as a vaccine adjuvant. In this study, we show that IL-12-deficient mice generate a weaker primary CD8 T cell response and are more susceptible to Listeria monocytogenes infection, but have substantially more memory CD8 T cells and greater protective immunity against reinfection. Kinetic analyses show that in the absence of IL-12 there is a reduced contraction of Ag-specific CD8 T cells and a gradual increase in memory CD8 T cells as a result of increased homeostatic renewal. By signaling directly through its receptor on CD8 T cells, IL-12 influences their differentiation to favor the generation of fully activated effectors, but hinders the formation of CD8 T cell memory precursors and differentiation of long-term CD8 T cell memory. These results have implications for understanding memory T cell development and enhancing vaccine efficacy, and offer new insight into the role of IL-12 in coordinating the innate and adaptive immune response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The CD8 T cell responses to infection are characterized by distinct phases. Naive CD8 T cells rapidly develop into effectors upon encounter with Ag. As infection subsides, effector cell populations contract, leaving a stable pool of memory cells that mediate protective immunity against reinfection (1). Unlike naive T cells, memory T (TM)4 cells respond quickly to Ag stimulation and possess the stem cell-like quality of self-renewal, which allows them to be maintained for long periods of time in the absence of Ag (2). Thus, Ag-independent renewal, along with faster expansion and greater protection from reinfection, are the hallmark characteristics of TM. More recently, TM populations have been characterized and divided into categories based on homing capabilities and effector functions (3, 4). It has also been shown that Ag-specific T cells during the early stage of infection are not a homogeneous population, but consist of numerous effector T (TE) cells and a small subset of TM precursors (5, 6, 7, 8). Expression of IL-7R{alpha} during the effector stage marks CD8 TM precursors that are destined to become TM (5). Differentiation of TM precursors into TM cells and their long-term maintenance depend on IL-7R{alpha} expression and correlate with the ability of these cells to produce IL-2 (4, 5, 9). However, the factors regulating the expression of these traits that are important for the formation of TM precursors and differentiation of long-term TM remain to be elucidated.

IL-12 is a proinflammatory cytokine consisting of 35-kDa (p35) and 40-kDa (p40) subunits (10). It is produced by phagocytes in response to microbial stimulation and is an important early mediator in host defense (11, 12). The IL-12R is composed of two subunits, IL-12Rbeta1 and IL-12Rbeta2, and is expressed mainly by activated T cells and NK cells (13). Most studies to date have focused on the ability of IL-12 to form a link between innate and adaptive immunity by inducing IFN-{gamma} production and polarizing naive CD4 T cells to become Th1 cells (12, 14, 15). In addition to these well-known properties, IL-12 has been shown to enhance CD8 T cell homeostatic proliferation and provide a third signal that promotes full activation and survival of activated CD8 T cells (16, 17, 18, 19). Collectively, many studies have shown that IL-12 is a potent inducer of TE cells, and this has led to its testing as a vaccine adjuvant (12, 20, 21, 22, 23). However, relatively little is known about how IL-12 affects CD8 TM generation.

In this study, we show that IL-12-deficient (p35–/–) mice have substantially more CD8 TM, despite a weaker TE response following immunization with recombinant Listeria monocytogenes expressing OVA (rLmOVA) (24). As a result of more CD8 TM and stronger recall response, p35–/– mice become more resistant to reinfection although they are more susceptible to a primary infection. In the absence of IL-12, more TM precursors are formed that express IL-7R{alpha} and IL-2, while fewer fully activated TE are generated. Addition of IL-12 enhances effector functions such as IFN-{gamma} and radical oxygen species (ROS) while suppressing IL-7R{alpha} and IL-2 expression. The ability of IL-12 to regulate these traits and CD8 TM development is dependent on direct signaling via the IL-12R on CD8 T cells. Thus, IL-12 regulates gene expression in CD8 T cells in a way that favors the generation of fully activated TE cells, but hinders the formation of CD8 TM precursors and differentiation of long-term CD8 TM. These results have implications for understanding TM differentiation and enhancing vaccine efficacy while offering new insight into the role IL-12 plays in coordinating innate and adaptive immune responses.


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

C57BL/6 (WT) and B6.Ly5.1/Cr (Ly5.1+ WT) mice were purchased from the National Cancer Institute (Frederick, MD). C57BL/6 (WT), C57BL/ 6-Il12atm1Jm (p35–/–), C57BL/6-Il12btm1Jm (p40–/–), B6.129S1-Il12rb2tm1Jm/J (IL-12Rbeta2–/–), and OT-I mice were purchased from The Jackson Laboratory. All animals were cared for according to the Animal Care Guidelines of the University of Pennsylvania.

Immunizations

Age-matched mice were immunized i.v. with a sublethal dose of 3 x 104 CFU of rLmOVA or 1 x 106 CFU of rLmOVA deleted for actA ({Delta}actA rLmOVA). For secondary immunizations, mice were challenged i.v. with 1 x 106 CFU of rLmOVA or with 1 x 107 CFU {Delta}actA rLmOVA. Acute infections were resolved and bacteria were cleared within 7–14 days in WT and p35–/– mice. CFUs per spleen and liver were determined 3 days after infection as described previously (25).

BrdU incorporation

Mice were injected with 1 mg of BrdU i.p. at day 45 postinfection and were then fed BrdU in their drinking water for 14 days at a concentration of 0.8 mg/ml until sacrificed.

Ampicillin treatment

Mice were fed ampicillin at a concentration of 2 mg/ml in their drinking water starting on day 14 postinfection until sacrificed.

Flow cytometry and intracellular cytokine staining

All fluorochrome-conjugated mAbs were purchased from BD Pharmingen with the exception of anti-IL-7R{alpha} (anti-CD127), which was purchased from eBioscience. Surface staining and intracellular cytokine staining were performed as previously described (26). For ex vivo intracellular cytokine staining, splenocytes were cultured at 37°C for 5 h in complete medium supplemented with 50 U/ml recombinant human IL-2/1.0 µl/ml GolgiStop in either the presence or absence of OVA257–264 peptide at 1.0 µg/ml. OVA-specific CD8+ T cells were also quantified by direct staining with H2-Kb/OVA257–264 (Kb/OVA) MHC/peptide tetramers. All cells from in vitro cultures were restimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) 4 h before analysis. GolgiStop was only added for intracellular cytokine staining at 1.0 µl/ml 2 h before analysis. For intracellular BrdU staining, the protocol outlined in the BD Pharmingen BrdU Flow Kit was followed (catalog no. 559619). Intracellular levels of ROS were analyzed by FACS using dichlorofluorescein diacetate (DCFDA; Molecular Probes) as a fluorescent probe (27). Cells were loaded with 5 µM DCFDA for 30 min at 37°C and incubated with DCFDA continuously throughout the experiment. Cells were stained for surface molecules before FACS analysis (27). All FACS plots are from a representative mouse from each group and data in all graphs are the average of each group. All error bars indicate SD unless percentages have been calculated, where bars represent SE. Values of p were calculated using Student’s t test. All experiments were performed at least twice with three to five mice per group.

In vitro cell stimulation

Splenocytes from C57BL/6 (WT), B6.Ly5.1/Cr (Ly5.1+ WT), C57BL/6-Il12atm1Jm (p35–/–), C57BL/6-Il12btm1Jm (p40–/–), and B6.129S1-Il12rb2tm1Jm/J (IL-12Rbeta2–/–) mice were stimulated in vitro using soluble anti-CD3 and anti-CD28 mAbs (both at 0.5 µg/ml; BD Pharmingen) in the presence of human rIL-2 (100 U/ml; BD Pharmingen). OT-I splenocytes were stimulated with 1.0 µg/ml OVA257–264 peptide. Recombinant murine IL-12 (5 ng/ml; PeproTech) and the neutralizing mAb anti-IFN-{gamma} (hybridoma clone XMG-6; 40 µg/ml) were added to the indicated wells.

Bone marrow chimeras

B6.Ly5.1/Cr mice were sublethally irradiated and partially reconstituted with bone marrow from IL-12Rbeta2–/– mice. After 60 days, the animals were bled and T cells were analyzed by surface staining and flow cytometry to confirm chimerism. Mice were then immunized with rLmOVA and analyzed 7 days or 60 days after immunization.

Adoptive transfer

Splenocytes from WT and p35–/– mice were stained with Kb/OVA tetramer to determine numbers of OVA-specific cells, and splenocytes containing 1 x 104 of OVA-specific cells were transferred i.v. to recipient mice followed by challenge infection as indicated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Greater CD8 TM in p35–/– mice after rLmOVA immunization

During the course of our studies to investigate the ability of Th1 cells to assist CD8 TM development, we unexpectedly discovered that IL-12-deficient mice had substantially more CD8 TM cells despite a weaker TE response after immunization with rLmOVA. The numbers of OVA-specific CD8 TE cells in p35–/– mice 7 days after rLmOVA immunization were roughly one-half of those in WT mice, while 60 days postimmunization there were 3-fold or 1.4 x 105 more OVA-specific CD8 TM cells in p35–/– mice compared with WT (Fig. 1A). This increase in OVA-specific CD8 TM cells was consistently observed when calculated as percentages of CD8 T cells or as absolute numbers per spleen and when measured by tetramers or intracellular IFN-{gamma} staining. The difference in CD8 TM is particularly striking in light of the weaker primary response in p35–/– animals. Although the number of CD8 TM cells was 3% of OVA-specific CD8 TE cells in WT mice, the number of CD8 TM cells was >30% of OVA-specific CD8 TE cells in p35–/– mice (Fig. 1B). The increase in CD8 TM formation was also observed in the lymph nodes, liver, and bone marrow (Fig. 1C) and in IL-12p40–/– mice (data not shown). Taken together, our results clearly show that although IL-12 promotes the CD8 TE cell response, there is enhanced generation of CD8 TM in the absence of IL-12.


Figure 1
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  		FIGURE 1. More memory CD8 T cells in p35–/– mice after L. monocytogenes infection. WT and p35–/– mice were immunized with rLmOVA and OVA-specific cells were analyzed 7 days or 60 days postinfection by intracellular IFN-{gamma} and Kb/OVA tetramer staining (A and B). A, Dot plots show IFN-{gamma}-producing splenic CD8 T cells (numbers indicate percentage of CD8 T cells that produce IFN-{gamma}). Bar graphs represent the total number of Kb/OVA tetramer+ CD8 T cells per spleen (means ± SD). B, Bar graph shows the percentage of TM over TE cells (number of TM cells on day 60 divided by the number of TE cells on day 7). C, Dot plots show OVA-specific memory CD8 T cells in the liver, spleen, lymph node, and bone marrow 60 days postinfection. D, WT and p35–/– mice were immunized with 5 x 105 CFU of rLm for naive mice (left panel, primary) and 5 x 106 CFU of rLm for immune mice (right panel, challenge). Bar graphs show CFU per g of liver 2 days postimmunization (means ± SD). E, WT and p35–/– mice were immunized with rLmOVA and challenged 60 days later with rLmOVA. OVA-specific cells in the spleens of challenged mice 8 days after secondary immunization (2°TE) and unchallenged mice (TM) were detected by intracellular cytokine staining. Numbers represent the percentage of CD8 T cells that were IFN-{gamma}+. The bar graphs show the numbers of OVA-specific CD8 T cells per spleen (means ± SD). F and G, Equal numbers of OVA-specific CD8 T cells from WT and p35–/– mice (Thy1.2+) immunized with rLmOVA were injected into WT recipients (Thy1.1+) that were then challenged with rLmOVA. Splenocytes were restimulated with the OVA peptide, and intracellular IFN-{gamma} was measured 3 days postchallenge (F) and bacterial clearance was measured in the spleen and liver (G). Bar graphs represent the number of Kb/OVA+ CD8 T cells (F) or CFU per organ (G) (means ± SD).

 
IL-12-deficient mice are more susceptible to primary infection, but more resistant to reinfection

Because p35–/– mice have greater CD8 TM, we investigated whether they were more protected from reinfection. We measured bacterial numbers in naive and immune WT and p35–/– mice 2 days after infection with rLmOVA. As expected, naive p35–/– mice were more susceptible and had 1 log more bacteria than WT mice after primary infection (Fig. 1D, left panel). However, immune p35–/– mice had 3 logs fewer bacteria compared with immune WT after challenge infection (Fig. 1D, right panel). Thus, although IL-12-deficient mice were more susceptible to primary infection, they became more resistant to reinfection. Increased resistance of IL-12-deficient mice to reinfection correlated with a robust recall response in p35–/– mice; 8 days postchallenge, ~19% of CD8 T cells in p35–/– mice were OVA specific compared with only 8% in WT mice (Fig. 1E). These results show that a stronger secondary TE response is generated as a result of more TM cells in p35–/– mice that confer greater protective immunity to reinfection.

To compare the functionality of CD8 TM on a per cell basis, we adoptively transferred equal numbers of OVA-specific TM cells from immune WT and p35–/– mice into naive congenic WT recipients. By day 3 postchallenge with rLmOVA, there were more OVA-specific secondary TE derived from p35–/– donor TM cells than from WT donor TM cells (Fig. 1F). Mice that received p35–/– donor TM had 2 logs fewer bacteria compared with the mice that received WT donor TM (Fig. 1G). These results demonstrate that CD8 TM cells from IL-12-deficient mice are fully capable of mounting enhanced recall responses that mediate greater protective immunity to reinfection.

More CD8 TM is not due to exaggerated infection in IL-12-deficient mice

IL-12-deficient mice are more susceptible to rLm infection although they are capable of clearing a sublethal infection (28). Although p35–/– had higher bacterial loads at day 3 (Fig. 1D, left panels), both WT and p35–/– mice cleared infection by 7 days after rLmOVA immunization (data not shown). To examine whether enhanced CD8 TM in p35–/– mice is due to differences in bacterial load and Ag stimulation, we immunized WT and p35–/– mice with a highly attenuated strain of rLmOVA ({Delta}actA rLmOVA) that is unable to spread from cell to cell and is quickly cleared in both WT and p35–/– mice. On day 2 postinfection, there were comparable levels of bacteria in the spleens and livers between WT and p35–/– mice (Fig. 2A). By day 4 postinfection, both WT and p35–/– mice cleared the infection in the spleen and liver and no bacteria were detected in other organs at any time points (Fig. 2A). To further eliminate any possible persisting bacteria, we treated {Delta}actA rLmOVA-immunized WT and p35–/– mice with antibiotics and measured OVA-specific CD8 TM 60 days later. More CD8 TM cells were still present in p35–/– mice than in WT (Fig. 2B). Together, these results show that greater CD8 TM in p35–/– mice is not due to differences in the level or duration of Ag stimulation during infection.


Figure 2
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  		FIGURE 2. More memory OVA-specific CD8 T cells in p35–/– mice after immunization with attenuated L. monocytogenes and antibiotic treatment. A, WT and p35–/– mice were immunized with a highly attenuated strain of rLmOVA ({Delta}actA rLmOVA), which renders the bacteria unable to spread from cell to cell in the host. Bacterial clearance was measured in the spleen 2 and 4 days postimmunization. Bar graphs show the CFU per spleen (means ± SD). B, WT and p35–/– mice were immunized with {Delta}actA rLmOVA treated with 2 mg/ml ampicillin in their drinking water starting 14 days postinfection until sacrificed 70 days postinfection. Splenocytes were stained with Kb/OVA tetramer or restimulated with OVA peptide and intracellular IFN-{gamma} was measured (CD8+ T cell gated). Numbers indicate the percentage of CD8 T cells. Bar graphs represent the number of IFN-{gamma}+ CD8 T cells per spleen (means ± SD).

 
More CD8 TM in IL-12-deficient mice is not simply due to reduced contraction

A recent study by Badovinac et al. (6) has shown that without inflammation there is reduced contraction of CD8 T cell populations following infection. Another study by Van Faasen et al. (29) demonstrates the role of T cell stimulation, governed by inflammation, in negatively influencing commitment to TM cells. Because IL-12 is a proinflammatory cytokine, we considered the possibility of reduced contraction in p35–/– mice leading to more CD8 TM. Because the majority of TE cell death occurs between 2 and 3 wk after infection, we performed kinetic analyses of OVA-specific CD8 T cells in WT and p35–/– mice from days 7 to 21 following {Delta}actA rLmOVA immunization to more carefully study the amount of actual TE cell contraction that occurs in p35–/– mice.

At day 7 following immunization, the magnitude of CD8 TE cell expansion in p35–/– mice was lower than in WT mice (Fig. 3, A–C), similar to the results we observed following rLMOVA infection (Fig. 1A). This is consistent with published findings showing that IL-12 is important for CD8 TE cell responses (17, 18, 19). Like in IFN-{gamma}–/– mice (30), there is a reduced contraction of Ag-specific CD8 T cells in p35–/– mice. The fold contraction in WT animals is ~9- to 10-fold, whereas it is ~3- to 4-fold in p35–/– mice, as determined by comparing the numbers of OVA-specific CD8 T cells at days 7 and 21. Although the fold contraction was less in p35–/– mice, the numbers of OVA-specific cells in WT and p35–/– mice were similar at day 21 (1.2% and 1.2% of CD8 T cells or 1.4 x 105 and 1.5 x 105 cells/spleen, respectively; Fig. 3, A–C). Thus, at the end of the contraction phase, WT and p35–/– mice had similar numbers of total OVA-specific CD8 T cells, and the greater number of long-term TM cells in p35–/– mice is not simply due to reduced contraction.


Figure 3
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  		FIGURE 3. More CD8 TM precursors are generated in p35–/– mice. WT and p35–/– mice were immunized with {Delta}actA rLmOVA and OVA-specific cells analyzed in the spleen on days 7, 14, and 21 after infection (A–G). A, Dot plots show splenocytes stained with Kb/OVA tetramer (Kb/OVA+ gate). Numbers indicate the percentage of CD8 T cells that are Kb/OVA+. Line graphs represent the (B) percentage of CD8 T cells that were Kb/OVA+ (means ± SE) or (C) the total number of Kb/OVA+ cells per spleen (means ± SD). D, Histograms show surface expression of IL-7R{alpha} (Kb/OVA+ gated for days 7–21 and isotype control; total CD8+ gated for naive mouse; WT, no fill; p35–/–, shaded). The ability of OVA-specific CD8 T cells to produce IL-2 was determined by intracellular cytokine staining (E–G). E, Dots plots show CD8+ gated cells; numbers indicate the percentage of IFN-{gamma}+ cells that produced IL-2. Line graphs represent the percentage (means ± SD) (F) or the number (means ± SE) (G) of IFN-{gamma}+ cells that produced IL-2. H, WT and p35–/– mice were administered BrdU for a period of 14 days (days 41–55 postimmunization with {Delta}actA rLmOVA). Dot plots (CD8+ gated) show BrdU incorporation by OVA-specific CD8 T cells and total CD8 T cells, as determined by intracellular IFN-{gamma} and BrdU staining. The percentages of IFN-{gamma}+ cells and total CD8 T cells that have incorporated BrdU are indicated by the numbers in the upper and lower right quadrants, respectively, and are shown in bar graphs (means ± SE; *, statistical significance, p = 0.02).

 
More CD8 TM precursors in p35–/– mice

Ag-specific CD8 T cells responding to infection are not a homogeneous population of effectors, but consist of a small number of memory precursors, which express IL-7R{alpha} and IL-2 and gradually differentiate into long-term TM (5). To examine whether more TM precursors were generated in the absence of IL-12, we measured expression of IL-7R{alpha} and IL-2 in OVA-specific cells following {Delta}actA rLmOVA immunization. Greater percentages of OVA-specific CD8 T cells in p35–/– mice expressed high levels of IL-7R{alpha} and IL-2 compared with WT mice (Fig. 3, D–F). These differences were observed as early as day 7 at the peak of the primary response and continued through the contraction phase. Although the overall number of OVA-specific cells was less in p35–/– mice at day 7 (Figs. 1A and 3C), there were greater numbers of IL-2-producing OVA-specific TM precursors in p35–/– mice compared with WT mice (Fig. 3G). As the total number of OVA-specific cells contracted to a similar level by day 21, the number of IL-2-producing OVA-specific TM precursors remained higher in p35–/– than in WT mice. These data suggest that during the primary response more OVA-specific TM precursors are generated in IL-12-deficient animals, thus leading to greater OVA-specific CD8 TM.

IL-7R{alpha} and IL-2 expression by Ag-specific CD8 T cells correlate with the ability of these cells to undergo proliferative renewal. This proliferative process is critical for maturation of TM precursors into fully differentiated TM cells and their long-term maintenance (2, 4, 5, 9, 31). We examined the proliferation of OVA-specific CD8 TM cells in rLmOVA-immunized p35–/– and WT animals by administering BrdU. We found that 39% of OVA-specific CD8 T cells in p35–/– mice had incorporated BrdU compared to only 14% in WT mice. This enhanced proliferation in p35–/– mice was only observed for Ag-specific CD8 TM cells, while the total CD8 T cells incorporated BrdU at the same rate between the groups (Fig. 3H). Thus, there is either enhanced proliferation of Ag-specific CD8 cells or more of them undergoing proliferative renewal in p35–/– mice than in WT mice. These results further support our finding that more long-term CD8 TM cells are generated and maintained in p35–/– mice.

IL-12 regulation of CD8 TM development is dependent on signaling via the IL-12R on CD8 T cells

IL-12 could suppress CD8 TM either by signaling through its receptor on CD8 T cells or by signaling through its receptor on an intermediate cell. For example, IL-12 can activate NK cells to produce large amounts of IFN-{gamma} (32) that might influence CD8 TM development. To distinguish these two possibilities, we generated IL-12Rbeta2 bone marrow chimeras, which have one population of T cells expressing the IL-12R (Ly5.1+) and another population lacking a functional IL-12R (Ly5.2+), thus allowing a direct comparison of these two populations within the same animal. On day 7 after rLmOVA immunization, OVA-specific CD8 T cells were comprised equally of IL-12Rbeta2+/+ and IL-12Rbeta2–/– populations (Fig. 4, A and B). By day 60 >80% of the OVA-specific TM cells were now of IL-12Rbeta2–/– origin (Fig. 4, A and B). Furthermore, greater numbers of OVA-specific IL-12Rbeta2–/– cells expressed high levels of IL-7R{alpha} and produced IL-2 compared with IL-12RB2+/+ cells (Fig. 4, C and D). These results show that IL-12 signaling via its cognate receptor on CD8 T cells regulates the generation of CD8 TM precursors and their differentiation into long-lasting TM.


Figure 4
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  		FIGURE 4. IL-12 signaling via the IL-12R on CD8 T cells inhibits CD8 TM generation. OVA-specific CD8 cells from IL-12Rbeta2+/+ (Ly5.1+)/IL-12Rbeta2–/– (Ly5.2+) bone marrow chimera mice were analyzed 7 and 60 days after immunization with rLmOVA. A, OVA-specific CD8 T cells were detected by Kb/OVA tetramer (left panels, numbers indicate the percentage of CD8 T cells that are Kb/OVA+) and the composition of this population was assessed for IL-12Rbeta2+/+ (Ly5.1+) or IL-12Rbeta2–/– (Ly5.2+) origin (right panels, Kb/OVA+ gated). B, OVA-specific CD8 T cells were detected by intracellular IFN-{gamma} staining and the composition of this population was assessed for IL-12Rbeta2+/+ (Ly5.1+) or IL-12Rbeta2–/– (Ly5.2+) origin (gated on IFN-{gamma}+ cells). C, IL-7R{alpha} expression on OVA-specific IL-12Rbeta2+/+ cells (no fill, Kb/OVA+ Ly5.1+ gated) vs IL-12Rbeta2–/– cells (shaded, Kb/OVA+ Ly5.2+ gated). Numbers indicate the percentage of Kb/OVA+ cells that express high levels of IL-7R{alpha}. D, The ability of OVA-specific CD8 T cells to produce IFN-{gamma} and IL-2 were measured by intracellular cytokine staining (CD8+ gated; numbers indicate percentage of IFN-{gamma}+ cells that are IL-2+).

 
IL-12 enhances activation of CD8 T cells and effector functions while suppressing IL-2 and IL-7R{alpha} expression

Our in vivo results suggest that IL-12 enhances the generation of fully activated CD8 TE cells while inhibiting the formation of CD8 TM precursors (expressing IL-7R{alpha} and IL-2) during a primary response, leading to reduced long-term CD8 TM. To examine the mechanism by which IL-12 regulates CD8 TM development, we took a reductionist approach and stimulated OT-I cells in vitro in the presence or absence of exogenous IL-12. OT-I cells cultured with IL-12 produced more IFN-{gamma} (compare mean fluorescence intensity of 131 and 98 with and without IL-12, respectively). However, fewer OT-I cells produced IL-2 or expressed IL-7R{alpha} when cultured with exogenous IL-12 (Fig. 5A). These data recapitulate our in vivo results and clearly show that IL-12 enhances the effector function of IFN-{gamma} production, while suppressing the expression of IL-2 and IL-7R{alpha} that are conducive for TM development.


Figure 5
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  		FIGURE 5. IL-12 suppresses IL-2 and IL-7R{alpha} expression while enhancing ROS production in CD8 T cells. A and B, OT-I splenocytes were stimulated with OVA peptide in the presence or absence of exogenous IL-12 for 3 days. A, Dot plots show IFN-{gamma}-producing CD8 T cells. The numbers indicate the mean fluorescent intensity of IFN-{gamma} for IFN-{gamma}-producing cells (top panels), percentage of IFN-{gamma}-producing cells that coproduce IL-2 (middle panels), and the percentage of IL-2-producing cells that express high levels of IL-7R{alpha} (IFN-{gamma}+ gated; bottom panels). B, Histograph shows ROS production and numbers indicate the percentage of CD8 T cells that produce high levels of ROS (CD8+ gated; +IL-12, shaded; no IL-12, unshaded). C, WT and p35–/– mice were immunized with rLm and ROS was measured in splenocytes directly ex vivo 2 days later. Histograms show ROS production by CD8 T cells from WT (solid line), p35–/– (dashed line), and the negative control (shaded area). D and E, Equal numbers of IL-12Rbeta2+/+ and IL-12Rbeta2–/– splenocytes were mixed and stimulated with anti-CD3/CD28 with or without exogenous IL-12. D, Histograms show IL-2 production by activated CD8 T cells (IL-12Rbeta2+/+, no fill; IL-12Rbeta2–/–, shaded). The numbers represent the percentage of activated CD8 T cells that produce IL-2. E, ROS production was measured in CD8 T cells cultured in the presence of IL-12 and/or anti-IFN-{gamma}. Numbers within the histographs represent the percentage of CD8 T cells that produce high levels of ROS.

 
Activation increases the amount of ROS in T cells and these molecules have been shown to modulate the apoptosis/survival of activated T cells (33, 34, 35, 36). We tested whether IL-12-driven TE produced more ROS because IL-12 enhances T cell activation and the expression of effector functions. OT-I cells stimulated in vitro with OVA peptide and exogenous IL-12 produced substantially more ROS (43% compared 18% without IL-12; Fig. 5B). Consistent with this in vitro finding, more CD8 T cells from L. monocytogenes-infected WT mice expressed high levels of ROS compared to CD8 T cells from infected p35–/– mice (Fig. 5C). These data indicate that IL-12 drives greater ROS production in CD8 TE and suggest that IL-12-driven CD8 TE are more likely to undergo apoptosis and thus less likely to differentiate into TM cells.

To ascertain a direct role of IL-12, we examined whether signaling directly through its cognate receptor on CD8 T cells was required or whether it involved intermediates such as IFN-{gamma}, which is highly induced by IL-12. Splenocytes from IL-12Rbeta2+/+ and IL-12Rbeta2–/– mice were cocultured in the same well and stimulated with anti-CD3/CD28 mAbs in the presence or absence of IL-12. Addition of IL-12 reduced the number of IL-2-producing IL-12Rbeta2+/+ cells (28% without IL-12 and 15% with IL-12), but had no effect on IL-2 expression by IL-12Rbeta2–/– cells (39 and 41% without and with IL-12, respectively; Fig. 5D). In the absence of IL-12, a similar percentage of IL-12Rbeta2+/+ and IL-12Rbeta2–/– cells produced ROS (27 and 28%, respectively; Fig. 5E). However, addition of IL-12 resulted in an increase in the percentage of IL-12Rbeta2+/+ (40%) but not IL-12Rbeta–/– cells (21%) that produced high levels of ROS (Fig. 5E). Furthermore, neutralization of IFN-{gamma} had no effect on the ability of IL-12 to suppress IL-2 production (data not shown) or to enhance ROS expression (Fig. 5E). These data demonstrate a direct role for IL-12 in augmenting ROS production and in suppressing IL-2 and IL-7R{alpha} expression. Together, our results suggest a mechanism by which IL-12 influences CD8 TM cell development. By signaling directly through its receptor on CD8 T cells, IL-12 regulates gene expression in CD8 T cells to favor the generation of fully activated TE cells, while hindering the formation of CD8 TM precursors and differentiation of long-term CD8 TM.

Prime boost immunization in the absence of IL-12 induces more TM precursors and long-lasting TM

Because prime boost immunization is a common vaccination protocol to induce long-lived CD8 TM, we examined the effects of IL-12 on the efficacy of prime boost immunization. WT and p35–/– mice were immunized and then 60 days later reimmunized with rLmOVA. On day 7 following the boost, 70% of OVA-specific secondary TE in p35–/– mice expressed high levels of IL-7R{alpha}, as compared with only 30% in WT mice (Fig. 6A). Thus, the difference in IL-7R{alpha} expression between p35–/– and WT mice seen in primary TE became strikingly more apparent in secondary TE. These data further support the conclusion that IL-12 regulates the generation of CD8 TM precursors by modulating IL-7R{alpha} expression, and suggest that prime boost immunization in the absence of IL-12 would induce more long-lasting CD8 TM cells. Indeed, when we measured OVA-specific CD8 TM cells 60 days after boost, the p35–/– mice maintained a larger population that expressed high levels of IL-7R{alpha} compared to WT mice (Fig. 6B). These results show that in the absence of IL-12, substantially more CD8 TM precursors are generated among secondary TE following prime boost immunization, leading to greater numbers of long-lived CD8 TM cells.


Figure 6
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  		FIGURE 6. Generation of more CD8 TM precursors and long-lasting TM cells following prime boost immunization in the absence of IL-12. A, WT (no fill) and p35–/– (shaded) mice were primed with rLmOVA and one group was boosted with rLmOVA 60 days after the first immunization. Histograms (Kb/OVA+ gated) show surface expression of CD62L and IL-7R{alpha} on OVA-specific TM (60 days after prime) and secondary TE (8 days after boost). B, WT and p35–/– mice were primed and then boosted 60 days later with rLmOVA. Two months after boost, OVA-specific CD8 TM cells were detected by Kb/OVA tetramer (top panels, CD8+ gated) and their expression of CD62L and IL-7R{alpha} was examined (bottom panels; Kb/OVA+ gated).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Most studies have focused on the roles of Ag dose and persistence in regulating CD8 T cell expansion and TM differentiation (37, 38). In this study, we describe the novel finding that the cytokine IL-12 has the opposing properties of promoting the TE cell response while inhibiting the development of CD8 TM. We show that IL-12-deficient mice have a reduced CD8 TE cell expansion and contraction following L. monocytogenes immunization, but a gradual increase in the numbers of CD8 TM cells formed due to enhanced homeostatic renewal.

To examine the mechanism by which IL-12 regulates CD8 TM, we first considered whether increased CD8 TM in p35–/– mice resulted from altered infection. Our results with the attenuated mutant {Delta}actA rLmOVA, which is cleared at similar rates in WT and p35–/– mice, as well as those with the antibiotic treatment indicate that differences in the level or duration of Ag stimulation following infection do not account for greater CD8 TM in the absence of IL-12. Furthermore, our studies of IL-12R bone marrow chimeras conclusively rule out this possibility because experiments were performed within the same chimeric mouse, where the rate of bacterial clearance is not an issue, and still more IL-12Rbeta2–/– than IL-12Rbeta2+/+ cells developed into CD8 TM cells. These results also demonstrate that IL-12 regulation of CD8 TM depends on direct signaling via its receptor on CD8 T cells, rather than through intermediate factors such as IFN-{gamma}. This is further supported by our in vitro studies using cocultures of IL-12Rbeta2–/– and IL-12Rbeta2+/+ cells with IFN-{gamma} neutralization (Fig. 5).

Previous studies have shown reduced CD8 T cell contraction in the absence of inflammation (6). We examined whether this was the mechanism underlying increased CD8 TM in p35–/– mice. Our results show that the total number of Ag-specific CD8 T cells contract to a similar level in p35–/– and WT mice. However, the quality of the Ag-specific CD8 T cell populations in WT and p35–/– mice begins to diverge early in the response in terms of the percentage of cells that express TE or TM phenotypes (Fig. 3, B–E). This divergence in quality resolves the conundrum of why the overall numbers of Ag-specific cells during the contraction phase in the WT and p35–/– mice are similar (Fig. 3A), while the latter animals go on to have greater numbers of TM cells.

Why are more TM precursors generated in p35–/– mice? In accordance with the signal strength model (39), CD8 T cells are driven less vigorously to become TE in the absence of IL-12, and thus are more likely to become TM precursors that develop into long-term TM. Our in vitro studies further show that IL-12 acts on CD8 T cells to enhance effector characteristics such as IFN-{gamma} and ROS production, while inhibiting their expression of IL-2 and IL-7R{alpha}. In addition to inducing IFN-{gamma} and ROS production, it is likely that IL-12 up-regulates a panel of genes that are key for inducing TE, just as it down-regulates another set of genes in addition to IL-2 and IL-7R{alpha}, which are critical for TM generation. Together, these results provide a mechanistic explanation for how IL-12 has the opposite effect of promoting a primary T cell response while inhibiting TM development. By signaling directly through its receptor on CD8 T cells, IL-12 regulates gene expression and influences CD8 T cell differentiation in a way that favors the generation of fully activated TE, while hindering the formation of CD8 TM precursors and differentiation of long-term CD8 TM. A recent study (40) has shown that IL-12 represses the expression of the transcription factor Eomesodermin in Ag-specific CD8+ T cells during infection eomesodermin has previously been shown to play an important role in controlling CD8 T cell function and fate (41, 42).

Our findings suggest a paradigm in which the early innate response not only promotes the adaptive TE response, but also regulates TM differentiation. This makes biological sense in the context of an infection. When dendritic cells produce IL-12 in response to microbial stimulation, they ensure maximal IFN-{gamma} production and TE cell activation, which are important for pathogen clearance. The adverse effect of IL-12 on TM development might ensure that the diversion of Ag-specific cells away from the TE pool into the TM pool occurs only after the infection is controlled and inflammation has subsided, indicating "no more danger" (43). Consistent with this model, a recent study by Williams and Bevan (44) has suggested that the stimuli received during the early stages of infection promote the generation of TE, whereas cues at the later stages of infection influence TM differentiation. Our data indicate that IL-12 provides a key signal through which the innate response orchestrates the subsequent adaptive response to ensure the induction of potent TE that first clear the pathogen, followed by the generation of long-lasting TM that mediates protective immunity against reinfection.

One of the most surprising aspects of this study was the observation that p35–/– mice are more resistant to reinfection while they are more susceptible to primary infection. The fact that the p35–/– mice have a smaller primary TE response, but more TM clearly underlies this difference in susceptibility and illustrates the important point that a large primary response does not de facto dictate a large population of long-lived TM cells. This finding is of significance for prophylactic vaccine development, because it illustrates that: 1) a priming immunization need not induce a large TE response and 2) IL-12 and other proinflammatory adjuvants may not be the most favorable choices for inducing long-lived CD8 TM. Although IL-12 helps therapeutic vaccines that aim to drive large TE responses (23), the use of IL-12 as an adjuvant in prophylactic vaccines may not achieve its intended goal of inducing a large population of long-lived TM cells. Instead, our results suggest that new strategies exploiting the opposite roles of IL-12 on the primary response and memory formation will achieve an optimal effect for enhancing vaccine efficacy.


   Acknowledgments
 
We thank Kathy Foulds, Jiu Jiang, Amy Troy, John Northrop, Connie Krawczyk, Rusty Jones, Jie Sun, and Edward Pearce for discussion, advice, and assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This research was supported by the National Institutes of Health Grant AI45025 (to H.S.). Back

2 Current address: Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 421 Curie Boulevard, Philadelphia, PA 19104 Back

3 Address correspondence and reprint requests to Dr. Hao Shen, Department of Microbiology, University of Pennsylvania School of Medicine, 3610 Hamilton Walk, Philadelphia, PA 19104. E-mail address: hshen{at}mail.med.upenn.edu Back

4 Abbreviations used in this paper: TM, memory T; TE, effector T; rLmOVA, recombinant Listeria monocytogenes expressing OVA; ROS, reactive oxygen species; WT, wild type; DCFDA, dichlorofluorescein diacetate; rLm, recombinant L. monocytogenes. Back

Received for publication July 12, 2006. Accepted for publication June 5, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Serbina, N., E. G. Pamer. 2003. Quantitative studies of CD8+ T-cell responses during microbial infection. Curr. Opin. Immunol. 15: 436-442. [Medline]
  2. 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: 1377-1381. [Abstract/Free Full Text]
  3. 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: 708-712. [Medline]
  4. Wherry, E. J., V. Teichgraber, T. C. Becker, D. Masopust, S. M. Kaech, R. Antia, U. H. von Andrian, R. Ahmed. 2003. Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat. Immunol. 4: 225-234. [Medline]
  5. Kaech, S. M., J. T. Tan, E. J. Wherry, B. T. Konieczny, C. D. Surh, R. Ahmed. 2003. Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells. Nat. Immunol. 4: 1191-1198. [Medline]
  6. Badovinac, V. P., B. B. Porter, J. T. Harty. 2004. CD8+ T cell contraction is controlled by early inflammation. Nat. Immunol. 5: 809-817. [Medline]
  7. Badovinac, V. P., K. A. Messingham, A. Jabbari, J. S. Haring, J. T. Harty. 2005. Accelerated CD8+ T-cell memory and prime-boost response after dendritic-cell vaccination. Nat. Med. 11: 748-756. [Medline]
  8. Marzo, A. L., K. D. Klonowski, A. Le Bon, P. Borrow, D. F. Tough, L. Lefrancois. 2005. Initial T cell frequency dictates memory CD8+ T cell lineage commitment. Nat. Immunol. 6: 793-799. [Medline]
  9. Schluns, K. S., W. C. Kieper, S. C. Jameson, L. Lefrancois. 2000. Interleukin-7 mediates the homeostasis of naive and memory CD8 T cells in vivo. Nat. Immunol. 1: 426-432. [Medline]
  10. Trinchieri, G., S. Pflanz, R. A. Kastelein. 2003. The IL-12 family of heterodimeric cytokines: new players in the regulation of T cell responses. Immunity 19: 641-644. [Medline]
  11. Kobayashi, M., L. Fitz, M. Ryan, R. M. Hewick, S. C. Clark, S. Chan, R. Loudon, F. Sherman, B. Perussia, G. Trinchieri. 1989. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J. Exp. Med. 170: 827-845. [Abstract/Free Full Text]
  12. Trinchieri, G.. 2003. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat. Rev. Immunol. 3: 133-146. [Medline]
  13. Presky, D. H., H. Yang, L. J. Minetti, A. O. Chua, N. Nabavi, C. Y. Wu, M. K. Gately, U. Gubler. 1996. A functional interleukin 12 receptor complex is composed of two beta-type cytokine receptor subunits. Proc. Natl. Acad. Sci. USA 93: 14002-14007. [Abstract/Free Full Text]
  14. Manetti, R., P. Parronchi, M. G. Giudizi, M. P. Piccinni, E. Maggi, G. Trinchieri, S. Romagnani. 1993. Natural killer cell stimulatory factor (interleukin 12 [IL-12]) induces T helper type 1 (Th1)-specific immune responses and inhibits the development of IL-4-producing Th cells. J. Exp. Med. 177: 1199-1204. [Abstract/Free Full Text]
  15. Hsieh, C. S., S. E. Macatonia, C. S. Tripp, S. F. Wolf, A. O’Garra, K. M. Murphy. 1993. Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science 260: 547-549. [Abstract/Free Full Text]
  16. Kieper, W. C., M. Prlic, C. S. Schmidt, M. F. Mescher, S. C. Jameson. 2001. Il-12 enhances CD8 T cell homeostatic expansion. J. Immunol. 166: 5515-5521. [Abstract/Free Full Text]
  17. Valenzuela, J., C. Schmidt, M. Mescher. 2002. The roles of IL-12 in providing a third signal for clonal expansion of naive CD8 T cells. J. Immunol. 169: 6842-6849. [Abstract/Free Full Text]
  18. Curtsinger, J. M., C. M. Johnson, M. F. Mescher. 2003. CD8 T cell clonal expansion and development of effector function require prolonged exposure to antigen, costimulation, and signal 3 cytokine. J. Immunol. 171: 5165-5171. [Abstract/Free Full Text]
  19. Orange, J. S., S. F. Wolf, C. A. Biron. 1994. Effects of IL-12 on the response and susceptibility to experimental viral infections. J. Immunol. 152: 1253-1264. [Abstract]
  20. Afonso, L. C., T. M. Scharton, L. Q. Vieira, M. Wysocka, G. Trinchieri, P. Scott. 1994. The adjuvant effect of interleukin-12 in a vaccine against Leishmania major. Science 263: 235-237. [Abstract/Free Full Text]
  21. Gurunathan, S., C. Prussin, D. L. Sacks, R. A. Seder. 1998. Vaccine requirements for sustained cellular immunity to an intracellular parasitic infection. Nat. Med. 4: 1409-1415. [Medline]
  22. Calarota, S. A., D. B. Weiner. 2004. Enhancement of human immunodeficiency virus type 1-DNA vaccine potency through incorporation of T-helper 1 molecular adjuvants. Immunol. Rev. 199: 84-99. [Medline]
  23. Colombo, M. P., G. Trinchieri. 2002. Interleukin-12 in anti-tumor immunity and immunotherapy. Cytokine Growth Factor Rev. 13: 155-168. [Medline]
  24. Foulds, K. E., L. A. Zenewicz, D. J. Shedlock, J. Jiang, A. E. Troy, H. Shen. 2002. Cutting edge: CD4 and CD8 T cells are intrinsically different in their proliferative responses. J. Immunol. 168: 1528-1532. [Abstract/Free Full Text]
  25. Shen, H., J. F. Miller, X. Fan, D. Kolwyck, R. Ahmed, J. T. Harty. 1998. Compartmentalization of bacterial antigens: differential effects on priming of CD8 T cells and protective immunity. Cell 92: 535-545. [Medline]
  26. San Mateo, L. R., M. M. Chua, S. R. Weiss, H. Shen. 2002. Perforin-mediated CTL cytolysis counteracts direct cell-cell spread of Listeria monocytogenes. J. Immunol. 169: 5202-5208. [Abstract/Free Full Text]
  27. Lee, J. R., G. A. Koretzky. 1998. Production of reactive oxygen intermediates following CD40 ligation correlates with c-Jun N-terminal kinase activation and IL-6 secretion in murine B lymphocytes. Eur. J. Immunol. 28: 4188-4197. [Medline]
  28. Oxenius, A., U. Karrer, R. M. Zinkernagel, H. Hengartner. 1999. IL-12 is not required for induction of type 1 cytokine responses in viral infections. J. Immunol. 162: 965-973. [Abstract/Free Full Text]
  29. van Faassen, H., M. Saldanha, D. Gilbertson, R. Dudani, L. Krishnan, S. Sad. 2005. Reducing the stimulation of CD8+ T cells during infection with intracellular bacteria promotes differentiation primarily into a central (CD62LhighCD44high) subset. J. Immunol. 174: 5341-5350. [Abstract/Free Full Text]
  30. Badovinac, V. P., A. R. Tvinnereim, J. T. Harty. 2000. Regulation of antigen-specific CD8+ T cell homeostasis by perforin and interferon-{gamma}. Science 290: 1354-1358. [Abstract/Free Full Text]
  31. Osborne, L. C., S. Dhanji, J. W. Snow, J. J. Priatel, M. C. Ma, M. J. Miners, H. S. Teh, M. A. Goldsmith, N. Abraham. 2007. Impaired CD8 T cell memory and CD4 T cell primary responses in IL-7R {alpha} mutant mice. J. Exp. Med. 204: 619-631. [Abstract/Free Full Text]
  32. Chan, S. H., B. Perussia, J. W. Gupta, M. Kobayashi, M. Pospisil, H. A. Young, S. F. Wolf, D. Young, S. C. Clark, G. Trinchieri. 1991. Induction of interferon {gamma} production by natural killer cell stimulatory factor: characterization of the responder cells and synergy with other inducers. J. Exp. Med. 173: 869-879. [Abstract/Free Full Text]
  33. Goldstone, S. D., N. H. Hunt. 1997. Redox regulation of the mitogen-activated protein kinase pathway during lymphocyte activation. Biochim. Biophys. Acta 1355: 353-360. [Medline]
  34. Devadas, S., L. Zaritskaya, S. G. Rhee, L. Oberley, M. S. Williams. 2002. Discrete generation of superoxide and hydrogen peroxide by T cell receptor stimulation: selective regulation of mitogen-activated protein kinase activation and Fas ligand expression. J. Exp. Med. 195: 59-70. [Abstract/Free Full Text]
  35. Hildeman, D. A., T. Mitchell, T. K. Teague, P. Henson, B. J. Day, J. Kappler, P. C. Marrack. 1999. Reactive oxygen species regulate activation-induced T cell apoptosis. Immunity 10: 735-744. [Medline]
  36. Hildeman, D. A., T. Mitchell, J. Kappler, P. Marrack. 2003. T cell apoptosis and reactive oxygen species. J. Clin. Invest. 111: 575-581. [Medline]
  37. Zinkernagel, R. M.. 2002. On differences between immunity and immunological memory. Curr. Opin. Immunol. 14: 523-536. [Medline]
  38. Wherry, E. J., R. Ahmed. 2004. Memory CD8 T-cell differentiation during viral infection. J. Virol. 78: 5535-5545. [Free Full Text]
  39. Gourley, T. S., E. J. Wherry, D. Masopust, R. Ahmed. 2004. Generation and maintenance of immunological memory. Semin. Immunol. 16: 323-333. [Medline]
  40. Takemoto, N., A. M. Intlekofer, J. T. Northrup, E. J. Wherry, S. L. Reiner. 2006. Cutting edge: IL-12 inversely regulates T-bet and eomesodermin expression during pathogen-induced CD8+ T cell differentiation. J. Immunol. 177: 7515-7519. [Abstract/Free Full Text]
  41. Pearce, E. L., A. C. Mullen, G. A. Martins, C. M. Krawczyk, A. S. Hutchins, V. P. Zediak, M. Banica, C. B. DiCioccio, D. A. Gross, C. A. Mao, et al 2003. Control of effector CD8+ T cell function by the transcription factor eomesodermin. Science 302: 1041-1043. [Abstract/Free Full Text]
  42. Intlekofer, A. M., N. Takemoto, E. J. Wherry, S. A. Longworth, J. T. Northrup, V. R. Palanivel, A. C. Mullen, C. R. Gasink, S. M. Kaech, J. D. Miller, et al 2005. Effector and memory CD8+ T cell fate coupled by T-bet and eomesodermin. Nat. Immunol. 6: 1236-1244. [Medline]
  43. Matzinger, P.. 2002. The danger model: a renewed sense of self. Science 296: 301-305. [Abstract/Free Full Text]
  44. Williams, M. A., M. J. Bevan. 2004. Shortening the infectious period does not alter expansion of CD8 T cells but diminishes their capacity to differentiate into memory cells. J. Immunol. 173: 6694-6702. [Abstract/Free Full Text]



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