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The Journal of Immunology, 2007, 179, 5768-5777
Copyright © 2007 by The American Association of Immunologists, Inc.
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Extent of Stimulation Controls the Formation of Memory CD8 T Cells1

Michael Quigley{dagger}, Xiaopei Huang* and Yiping Yang2,*,{dagger}

* Department of Medicine, Division of Medical Oncology, and {dagger} Department of Immunology, Duke University Medical Center, Durham, NC 27710


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Only a small fraction of effector CD8 T cells survives to become long-lived memory cells, whereas the majority of them die after an acute infection. What controls the formation of memory CD8 T cells remains mostly unknown. In this study, we showed CD8 T cells primed earlier during vaccinia viral infection received stronger stimulation, divided more extensively, and survived better than those primed later, leading to generation of a larger memory pool. Despite differentiation into effectors, the late-primed CD8 T cells lacked full cell division, displayed increased apoptosis, and failed to develop into memory cells, suggesting that the extent of stimulation influences the survival of effector CD8 T cells. We further demonstrated that the extent of stimulation, which included both the duration and the levels of antigenic stimulation/costimulation, during priming determined the formation of memory CD8 T cells via controlling the extent of Akt activation, and functional suppression of Akt led to defective CD8 memory formation in vivo. Collectively, our data suggest that the extent of stimulation controls CD8 memory formation via activation of Akt and may provide important insights into the design of effective vaccines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
An important goal of vaccination is to generate long-lived memory CD8 T cells to provide long-term protection against pathogenic infections and diseases. This protection is derived from the distinct capability of memory CD8 T cells to persist through self-renewal in the absence of Ag and to rapidly expand their numbers, produce effector cytokines, and destroy target cells more rapidly than naive CD8 T cells upon secondary Ag exposure (1, 2, 3). The course of memory CD8 T cell formation after acute viral infection or vaccination consists of three distinct phases, as follows: clonal expansion of Ag-specific T cells and development of effector functions, subsequent contraction of the majority (90–95%) of effector T cells via apoptosis, and generation of long-lived memory cells from the surviving cells (3, 4, 5). Studies using gene expression profiling and functional analyses have shown that transition of effector CD8 T cells into long-lived memory cells is a gradual process that may take up to several weeks after clearance of infection or Ag (2, 6). Furthermore, it has been shown that increased expression of IL-7R{alpha} on effector CD8 T cells correlates with their differentiation into long-lived memory cells (7), although recent data have suggested that IL-7R{alpha} expression might not necessarily identify memory CD8 T cell precursors (8, 9).

Despite the progress, many questions still remain and, in particular, what enables memory CD8 precursors to escape apoptosis and develop into long-term memory cells has yet to be elucidated. One model to explain the formation of memory CD8 T cells is the decreasing-potential hypothesis, which states that the main factor controlling which effector T cells die or survive to become memory cells is the duration and level of antigenic stimulation to which the T cells are exposed during priming (3, 10). This model predicts that T cells arriving late in the immune response receive only a brief and relatively weak stimulation that is sufficient to cause proliferation and differentiation into effectors, but inadequate to trigger various death pathways that are required for effector elimination, thus leading to formation of the memory cell pool. In support of this idea are the findings that overwhelming and persistent viral infections can lead to total elimination of the effector T cells (11, 12, 13), and the implication of homodimeric CD8{alpha}{alpha}, which has been shown to down-modulate TCR signals by sequestering lck away from the lipid rafts (14, 15), in promoting survival and differentiation of effectors into memory cells (16). Alternative to the decreasing-potential hypothesis is the progressive differentiation and fitness selection model, which proposes that T cells receiving strong and optimal stimulation during priming are rendered more fit and survive the contraction phase to become memory cells (17, 18). Consistent with this notion is that the initial burst size of the CD8 effector T cell response correlates the magnitude of the long-term memory pool (3, 18, 19, 20), and that the duration of stimulation influences the survival of T cells (21, 22).

In this study, we sought to better understand what promotes the differentiation of long-term memory cells using a model of influenza hemagglutinin (HA)3-specific CD8 T cell response to recombinant vaccinia virus encoding HA (rVV-HA) in vivo. We first compared the ability of HA-specific CD8 T cells that were primed early during the infection with rVV-HA with those that were primed late in the immune response to differentiate into long-term memory cells. We found that the earlier primed CD8 T cells received stronger stimulation, divided more extensively, and survived better than those primed later, leading to the development of a larger CD8 memory pool. Although the late-primed CD8 T cells that received weak stimulation could be activated to differentiate into effector T cells, they lacked full cell division, showed increased apoptosis, and failed to develop into long-lived memory cells, suggesting that the extent of stimulation during priming dictates CD8 memory formation. Using Ag-pulsed dendritic cells (DCs), we further confirmed this observation and identified that the extent of stimulation during priming controlled the activation of Akt (protein kinase B), a key downstream target of the PI3K pathway involved in regulating cell survival. Functional suppression of Akt activity led to diminished survival of activated CD8 T cells in vitro and defective CD8 memory formation in vivo. Thus, the extent of stimulation during priming determines whether effector CD8 T cells are short-lived or develop into long-term memory cells via activation of Akt.


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

B10.D2 mice were purchased from The Jackson Laboratory. The clone 4 HA-TCR transgenic mice that express a TCR recognizing a Kd-restricted HA epitope (518IYSTVASSL (526)) were provided by L. Sherman (Scripps Research Institute, La Jolla, CA) (23). These mice were backcrossed for more than nine generations into the Thy1.1, B10.D2 genetic background.

All mice used for experiments were between 8 and 10 wk of age. All experimental procedures involving the use of mice were done in accordance with protocols approved by the Animal Care and Use Committee of Duke University Medical Center.

Adoptive transfer of naive HA-specific transgenic T cells

Naive clonotypic HA-specific CD8 T cells (Thy1.1+) were prepared from clone 4 HA-TCR transgenic mice. Briefly, single-cell suspensions were prepared from spleen and lymph node of clone 4 TCR mice, and clonotypic percentage was then determined by flow cytometry analysis of CD8+Vbeta8.2+ cells, as described (23). The activation marker CD44 was also checked to ensure that these clonotypic cells were naive. For most experiments, 1 x 105 of naive clonotypic cells were transferred into recipient B10.D2 mice (Thy1.2+) through tail vein injection in 200 µl of HBSS; however, in one instance, 1 x 103 clonotypic cells were used for transfer. In some experiments, clonotypic cells were labeled with CFSE, as described (24), before injection.

Vaccinations with recombinant viruses encoding HA

rVV-HA and E1-deleted adenoviruses encoding HA (Ad-HA) were previously described (25, 26). rVV-HA was grown in TK-143B cells and purified from the cell lysate by sucrose banding. The titer of virus was determined by plaque-forming assay on TK-143B cells. For most experiments, mice were infected with 5 x 106 PFU of rVV-HA i.v.; however, in one instance, 5 x 104 PFU was used for infection.

Ad-HA was grown in 293 cells (American Type Culture Collection), purified by two rounds of CsCl density centrifugation, and desalted by gel filtration through Sephadex G-25 column (PD-10 column; Amersham Biosciences). The titer of virus was determined by plaque-forming assay on 293 cells (27). Mice were infected with 2 x 109 PFU of Ad-HA i.p.

Abs and flow cytometry

mAbs used for staining were CyChrome-conjugated anti-CD8; FITC-conjugated anti-CD44, anti-IFN-{gamma}, and anti-Thy1.1; PE-conjugated anti-Thy1.1 and annexin V; allophycocyanin-conjugated anti-IFN-{gamma}; and biotinylated anti-Thy1.1 (all from BD Pharmingen). PE-conjugated anti-Bcl-xL was purchased from Santa Cruz Biotechnology. Collection of flow cytometry data was conducted using a FACScan or FACSCanto (BD Biosciences), and events were analyzed using CellQuest software (BD Biosciences).

Intracellular cytokine staining

For intracellular staining, splenocytes were cultured in 200 µl of CTL medium (RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 50 µM 2-ME, 100 IU/ml penicillin, and 100 IU/ml streptomycin) at a concentration of 1 x 107 cells/ml in 96-well U-bottom plates for 6 h in the presence of 5 µg/ml GolgiPlug (brefeldin A; BD Pharmingen) and 2 µg/ml Kd HA518–526 peptide. Following culture, cells were surface stained with CyChrome-conjugated anti-CD8 and PE-conjugated Thy1.1 before permeabilization using the BD Pharmingen cytofix/cytoperm kit, according to the manufacturer’s instruction. Follow fixation, cells underwent intracellular staining with either allophycocyanin- or FITC-conjugated anti-IFN-{gamma}.

DC preparation

DCs were generated from bone marrow, as described (25). Briefly, femurs and tibiae of mice were harvested and bone marrow cells were flushed out with DC medium (RPMI 1640 with 5% FBS, 2 mM L-glutamine, 10 mM HEPES, 50 µM 2-ME, 100 IU/ml penicillin, and 100 IU/ml streptomycin). After lysis of RBC, the bone marrow cells were cultured in 6-well plates at a density of 3 x 106/ml in 3 ml of DC medium in the presence of mouse GM-CSF (1000 U/ml) and IL-4 (500 U/ml) (R&D Systems). GM-CSF and IL-4 were replenished on days 2 and 4. On day 5 of culture, nonadherent cells were harvested and either used for stimulation as immature DCs or placed back into culture at a concentration of 5 x 105 cells/ml in DC medium supplemented with LPS (100 ng/ml) (Sigma-Aldrich) for an additional 24 h for DC maturation. On day 6, nonadherent cells were harvested, washed twice to remove residual LPS, and used for stimulation as mature DCs. Before use in stimulation experiments, both immature and mature DCs were checked for their surface expression of CD11c, CD80, CD86, and MHC class II by FACS staining. Immature DCs were CD11c+CD80lowCD86lowclassIIlow, whereas mature DCs were CD11c+CD80highCD86highclassIIhigh. These DCs were loaded with 10 µg/ml HA peptide for 1 h at 37°C.

In vitro stimulation of T cells

Naive HA-specific CD8 T cells (Thy1.1+) were purified by positive selection using anti-CD8 microbeads (Miltenyi Biotec), according to the manufacturer’s instructions. Following selection, 2 x 106 T cells were placed into culture with 2 x 105 immature or mature DCs in a total of 2 ml of T cell medium (RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 50 µM 2-ME, 100 IU/ml penicillin,100 IU/ml streptomycin, 100 µM nonessential amino acids, 1 mM sodium pyruvate, and 100 mM HEPES) supplemented with murine IL-2 (100 U/ml) in 24-well plates. For DC titration experiments, 2 x 105, 4 x 104, or 8 x 103 DCs were cultured with 2 x 106 T cells for the 1:10, 1:50, and 1:250 (DC:T cell ratio) groups, respectively.

To remove HA-specific CD8 T cells from DC stimulation, cells were harvested from the 24-well plates and subjected to staining with a biotinylated anti-Thy1.1 Ab, followed by positive selection with streptavidin-coated microbeads (Miltenyi Biotec). Following separation, clonotypic T cells were placed back into culture in T cell medium supplemented with IL-2 (100 U/ml) or transferred into naive mice for memory formation in vivo.

Western blotting

For Western blotting, samples were separated on a 12% SDS-PAGE gel and transferred onto a nitrocellulose membrane (Bio-Rad). After overnight incubation at 4°C with anti-phospho Akt (Ser473) (Cell Signaling Technology), membranes were washed three times and probed with an AlexaFluor 680-conjugated anti-mouse Ig secondary Ab (Molecular Probes) for 1 h at room temperature. Membranes were then washed twice, and relative levels of pAkt were obtained through the use of the Odyssey infrared imaging system (LI-COR). For the loading control, membranes were stripped and reprobed with anti-total Akt (Cell Signaling Technology).

Retrovirus preparation and infection

pMSCV encoding GFP, pMSCV-dn-Akt (K179M) encoding GFP, and dominant-negative Akt retroviral vectors, gifts from Z. Songyang (Baylor College of Medicine, Houston, TX), were used to produce recombinant retroviruses through calcium chloride-mediated transfection of the Phoenix-ecotropic virus packaging cell line, as described (28). To transduce HA-specific T cells stimulated with the DCs, freshly prepared retroviral supernatants were added to the culture 24 h after DC stimulation in the presence of 8 µg/ml polybrene and IL-2 (100 U/ml). After 8-h incubation at 37°C, the retroviral supernatants were removed and replaced with T cell medium containing murine IL-2 (100 U/ml), and the cells were cultured for a total of 72 h. For the in vivo transfer of transfected cells, GFP+CD8+ T cells were sorted by FACS using a high-speed cell sorter FACSVantage (BD Biosciences) and injected i.v. into naive B10.D2 mice.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
A model of CD8 memory response to vaccinia viral infection

To study what controls the formation of long-term CD8 memory, we developed a model of HA-specific CD8 T cell response to infection with rVV-HA in vivo. A total of 1 x 105 of naive HA-specific CD8 T cells (Thy1.1+) purified from HA-TCR transgenic mice that recognize a Kd-restricted HA epitope was transferred into B10.D2 mice (Thy1.2+) that were subsequently infected with 5 x 106 PFU of rVV-HA i.v. A total of 1 x 105 of transgenic T cells was chosen for our studies to facilitate subsequent phenotypic analyses such as cell division. Consistent with previous observations in other models (3, 4, 5), clonal expansion of HA-specific T cells and effector differentiation were detected on day 7 with subsequent contraction and development into memory cell pool between days 28 and 42 (Fig. 1A). These memory cells were characterized by their ability to produce IFN-{gamma} upon short-term in vitro restimulation with HA peptide and to rapidly expand upon rechallenge with Ad-HA (Fig. 1B).


Figure 1
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  		FIGURE 1. A model of CD8 T cell memory. A, Kinetics of HA-specific CD8 T cell response to rVV-HA. A total of 1 x 105 of naive HA-specific T cells (Thy1.1+) was transferred into B10.D2 mice that were subsequently infected with rVV-HA. Splenocytes were harvested at day 0, 7, 14, 28, or 42 postinfection and stained with anti-CD8 and anti-Thy1.1. The absolute number of CD8+Thy1.1+ cells per spleen was determined via flow cytometry and plotted with SDs indicated. B, Recall response of CD8 memory. Forty-two days after infection, mice were harvested (No boost) or challenged with Ad-HA (Boost with Ad-HA) and harvested 5 days after the challenge. Splenocytes were analyzed for the expansion of HA-specific CD8 T cells (the percentages of CD8+Thy1.1+ cells among total lymphocytes are indicated) and their function by IFN-{gamma} intracellular staining (the percentages of IFN-{gamma}-producing HA-specific CD8 T cells among total CD8 T cells are indicated). Representative data of three independent experiments are shown.

 
Late-primed CD8 T cells that receive weak stimulation are able to differentiate into effectors

We next investigated the ability of HA-specific CD8 T cells that encountered the Ag early or late in the immune response to rVV-HA to differentiate into memory cells. To address this question, we first looked at clonal expansion and effector differentiation of CD8 T cells that were primed at different stages of the infection. Mice were infected with rVV-HA on day 0, and CFSE-labeled naive HA-specific CD8 T cells were transferred into mice at day 0, 3, or 7 after infection. The activation of HA-specific T cells was measured by dilution of CFSE in vivo 3 days after T cell transfer, and the clonal expansion by the percentages of HA-specific T cells and effector differentiation by the production of IFN-{gamma} were analyzed 7 days after (Fig. 2A). Extensive proliferation of T cells with at least five division cycles was detected in mice that received naive HA-specific T cells on day 0 (early primed) of the infection (Fig. 2B). This led to massive clonal expansion and effector differentiation (Fig. 2C). A significant (p < 0.001) decrease in effector size (~50% reduction) was found in mice that received T cells 3 days (intermediate-primed) after infection (Fig. 2C). When T cells were transferred 7 days (late-primed) after infection, although >95% naive T cells were recruited to divide, the cell proliferation was reduced, with the majority of T cells undergoing only four or less division cycles (Fig. 2B), leading to a greater degree of reduction in effector size (Fig. 2C). These results suggest that CD8 T cells that are primed later during infection receive weaker stimulation than those primed earlier. However, despite weak stimulation, the late-primed CD8 T cells are able to differentiate into effectors (Fig. 2C).


Figure 2
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  		FIGURE 2. Late-primed CD8 T cells receive weak stimulation, but are able to differentiate into effectors. A, A schematic view of the experimental design. B, In vivo proliferation of HA-specific CD8 T cells by CFSE. CFSE-labeled naive HA-specific CD8 T cells (1 x 105, Thy1.1+) were transferred into B10.D2 mice on day 0 (Early), 3 (Intermediate), or 7 (Late) following infection with rVV-HA or into uninfected (Naive) mice as a control. The CFSE profile of CD8+Thy1.1+ cells was analyzed 3 days following transfer. The percentages of dividing cells among total clonotypic T cells are indicated. C, Seven days after transfer, splenocytes were analyzed for clonal expansion by the percentages of CD8+Thy1.1+ population among total lymphocytes and effector function by IFN-{gamma}+ HA-specific CD8 T cells among total CD8 T cells, as indicated. Data shown are representative of three independent experiments.

 
Late-primed CD8 T cells fail to differentiate into memory cells

We next examined the ability of effector CD8 T cells primed at different stages of immune response to develop into long-term memory cells. Mice were infected with rVV-HA on day 0, followed by transfer of naive HA-specific CD8 T cells at day 0, 3, or 7 after infection. At different days after T cell transfer, the fate of effector CD8 T cells was tracked for the formation of memory T cells. Our data showed that the early-primed effector CD8 T cells efficiently developed into stable memory cells after contraction (Fig. 3, A and B). Similarly, the intermediate-primed T cells were also capable of differentiating into stable memory pool with a reduction in memory size (Fig. 3, A and B). By contrast, the late-primed effector CD8 T cells failed to develop into long-lived memory CD8 cells (Fig. 3, A and B). The lack of memory cell formation was not due to preferential sequestration of late-primed CD8 T cells in other compartments because memory cell formation from late-primed effectors in several other lymphoid and extralymphoid tissues was also severely compromised (Fig. 3C).


Figure 3
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  		FIGURE 3. Late-primed effector CD8 T cells do not develop into memory cells. A–C, B10.D2 mice were infected with 5 x 106 PFU of rVV-HA on day 0, and 1 x 105 of naive HA-specific CD8 T cells (Thy1.1+) were transferred on day 0 (Early), 3 (Intermediate), or 7 (Late) postinfection. A, Splenocytes were harvested on day 42 after transfer and stained with anti-CD8 and anti-Thy1.1, and the ex vivo percentages of the CD8+Thy1.1+ population among total lymphocytes are indicated (left panels). HA-specific CD8+ T cells were also assessed for the production of IFN-{gamma} by intracellular staining, and the percentages of IFN-{gamma}-producing HA-specific CD8 T cells among total CD8 T cells are indicated (right panels). B, Kinetics of early- vs late-primed CD8 T cell response. Splenocytes were harvested at days 7, 14, 28, and 42 after T cell transfer and stained with anti-CD8 and anti-Thy1.1 to determine the absolute number of the CD8+Thy1.1+ cells per spleen, which is shown with SDs indicated. C, Tissue distribution of early- vs late-primed memory CD8 T cells. Lymphocytes from the peripheral lymph nodes (LN), liver (Liver), and lungs (Lung) of mice were isolated and stained with anti-CD8 and anti-Thy1.1, and the ex vivo percentages of the CD8+Thy1.1+ population among total lymphocytes are indicated. D, Effect of T cell frequency on memory cell formation. Mice were infected with 5 x 106 PFU of rVV-HA on day 0, and 1 x 103 of naive HA-specific CD8 T cells (Thy1.1+) was transferred on day 0 (Early) or 7 (Late) postinfection. Splenocytes were harvested 42 days after T cell transfer, and effector function as measured by the production of IFN-{gamma} via intracellular staining was determined for both the transgenic (Thy1.1+, upper right quadrant) and endogenous (Thy1.1–, lower right quadrant) HA-specific CD8 T cell populations. E and F, Effect of infectious dose on memory cell formation. Mice were infected with 5 x 104 PFU of rVV-HA on day 0, and 1 x 105 of naive HA-specific CD8 T cells (Thy1.1+) was transferred on day 0 (Early) or 7 (Late) postinfection. E, Forty-two days later, splenocytes were harvested and stained with anti-CD8 and anti-Thy1.1, and the ex vivo percentages of the CD8+Thy1.1+ population among total lymphocytes are indicated (left panels). Clonotypic cells were also assessed for the production of IFN-{gamma} by intracellular staining, and the percentages of IFN-{gamma}-producing HA-specific CD8 T cells among total CD8 T cells are indicated (right panels). F, Absolute number of the CD8+Thy1.1+ cells per spleen from mice that were infected with 5 x 106 (5 x 10e6) or 5 x 104 (5 x 10e4) of rVV-HA is also shown with SDs indicated. Data shown are representative of three independent experiments.

 
Recent studies have shown that transfer of relatively large input numbers (~106) of transgenic T cells can suppress the endogenous T response to the same epitope, and may impact on the efficiency of memory cell formation by the transgenic T cells (29). To better model the endogenous T cell response, we titrated the input number of HA-specific T cells. We found that transfer of 103 transgenic HA-specific T cells at day 0 of infection with rVV-HA yielded a comparable degree of endogenous (0.47%) and exogenous (0.81%) HA-specific memory responses (Fig. 3D). Under this condition, we further demonstrated that transfer of transgenic T cells late after infection also led to a failure in memory generation (Fig. 3D).

It has been shown that the infection dose can affect the magnitude of CD8 T cell response by recruiting different proportions of naive T cells into the response, and those cells that are recruited undergo full activation and differentiation into memory cells independent of further Ag stimulation (30). To test whether the same was true in our system, mice were infected with 5 x 104 PFU of rVV-HA, followed by transfer of naive HA-specific CD8 T cells at day 0 or 7 after infection. Indeed, compared with mice infected with 5 x 106 PFU of rVV-HA (Fig. 3, A and B), the memory population generated from the early-primed cells was reduced, but not absent (Fig. 3, E and F). Similarly, the late-primed cells failed to form memory cells (Fig. 3, E and F). These results suggest that differential recruitment of naive T cells into the response may not explain the lack of memory formation from the late-primed cells.

Late-primed CD8 T cells display lack of full division and increased apoptosis

What then contributed to the defective memory formation from the late-primed T cells? Because the majority of the late-primed T cells underwent only four or less division cycles compared with five or more divisions with the early-primed cells at day 3 after T cell transfer (Fig. 2B), we hypothesized that the late-primed T cells might be qualitatively different from those from the early-primed T cells. To address this question, we first examined whether the late-primed T cells were able to divide fully. Analysis of CFSE dilution at day 7 after T cell transfer revealed that the late-primed T cells lacked full cell division (Fig. 4A), compared with the early-primed cells, which showed complete cell division by day 7 (Fig. 4A). We next asked whether the late-primed T cells were susceptible to increased apoptosis. We used annexin V staining to assess effector T cells undergoing early apoptosis 7 days after T cell transfer. Indeed, the late-primed T cells displayed a significant increase in annexin V+ cells compared with the early-primed T cells (Fig. 4B). This increased apoptosis may also explain why the late-primed cells showed a reduction in the percentage of dividing cells at day 7 compared with that at day 3, whereas the percentage of dividing cells among the early-primed cells remained constant (Figs. 2B and 4A).


Figure 4
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  		FIGURE 4. Late-primed effector CD8 T cells display incomplete division and reduced survival. Naive HA-specific CD8 T cells (1 x 105, Thy1.1+) were transferred into B10.D2 mice on day 0 (Early) or 7 (Late) following infection with rVV-HA or into uninfected (Naive) mice as a control. A, In vivo proliferation of HA-specific CD8 T cells by CFSE. The CFSE profile of CD8+Thy1.1+ cells was analyzed 7 days following transfer. The percentages of dividing cells among total clonotypic T cells are indicated. B, Ex vivo annexin V staining. Seven days after T cell transfer, splenocytes were analyzed for survival of effector CD8+ T cells by staining with annexin V. The percentages of annexin V+ cells among CD8+Thy1.1+ cells are indicated, as well as the mean fluorescence intensity for each in parentheses. Data shown are representative of three independent experiments.

 
Taken together, these results indicate that CD8 T cells primed earlier during the infection receive stronger stimulation, divide more extensively, and survive better to become memory cells than those primed later, suggesting the extent of stimulation during priming influences the formation of long-lived memory cells.

Extent of stimulation determines the formation of CD8 memory

To further confirm that the extent of stimulation controls the formation of long-lived memory CD8 T cells, we used an in vitro culture system in which naive HA-specific CD8 T cells are stimulated with bone marrow-derived DCs pulsed with the HA peptide. Because the weak stimulation that the late-primed T cells receive most likely reflects a short duration and low levels of antigenic stimulation/costimulation caused by elimination of Ags and changes in costimulatory ligand densities that occur over time after Ags enter the body (18), we first controlled the extent of stimulation by changing the duration of antigenic stimulation and/or the extent of costimulation. Naive HA-specific CD8 T cells were stimulated with HA-pulsed DCs, which were either immature or matured with LPS. After 4, 16, 36, or 48 h of stimulation, T cells were separated from DCs to terminate TCR triggering; cell division was measured by CFSE dilution on day 4; and the survival of activated T cells was analyzed by annexin V staining on day 7. Analysis of 4-h stimulation showed that T cell proliferation was induced by both immature and mature DCs, with the latter slightly more efficient; but the majority of T cells activated by both immature and mature DCs showed only four or less division cycles (Fig. 5A), which led to a significant portion of T cells (~50–55%) undergoing apoptosis on day 7 (Fig. 5B). More extensive T cell proliferation that correlated to the increasing duration of stimulation was noted with both immature and mature DCs compared with 4-h stimulation; however, there were more T cells that underwent five or more division cycles with mature than immature DCs, which resulted in a significant decrease in annexin V+ cells (Fig. 5, A and B), suggesting that prolonged stimulation with mature DCs was critical for acquiring resistance to apoptosis.


Figure 5
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  		FIGURE 5. Duration of stimulation influences the formation of memory CD8 T cells. Naive HA-specific CD8 T cells were stimulated in vitro with either immature (Immature DC) or LPS-matured (Mature DC) DCs or placed in culture without stimulation (Unstimulated) as a control. After 4, 16, 36, or 48 h, CD8 T cells were removed from stimulation by microbead isolation. A, Proliferation of in vitro primed HA-specific CD8 T cells. Purified, primed CD8 T cells were put back into culture in the absence of DCs for a total of 4 days, and the CFSE profile of CD8+ cells was determined by flow cytometry. B, Survival of in vitro primed HA-specific CD8 T cells. Purified, primed CD8 T cells were cultured in the absence of DCs for a total of 7 days and cells were stained with annexin V. Percentages of annexin V+ cells among CD8 T cells are indicated. C, Memory formation in vivo. Purified, in vitro primed CD8 T cells (5 x 105) were transferred into Ag-free mice; CD8 memory formation was analyzed 35 days later by IFN-{gamma} intracellular staining; and the percentages of IFN-{gamma}-producing HA-specific CD8 T cells among total CD8 T cells are indicated. Data shown are representative of four independent experiments.

 
We then assessed the ability of these differentially, in vitro primed T cells to develop into memory cells in vivo. Equal numbers of purified, in vitro primed HA-specific CD8 T cells were transferred into Ag-free hosts, and memory formation was evaluated by IFN-{gamma} intracellular staining 35 days after transfer. Very few memory cells were found in mice that received T cells stimulated for 4 h in vitro with either immature or mature DCs (Fig. 5C). By contrast, many more memory cells were detected in mice that received T cells stimulated for longer periods of time in vitro with immature DCs and mature DCs; however, the memory pool generated from T cells stimulated with mature DCs was much larger than immature DCs (Fig. 5C). These results suggest that the duration of stimulation and costimulation during priming influences T cell survival and memory formation, which is in line with a previous report in memory CD4 T cells (31).

We next looked at the impact of limiting Ag on cell survival and memory formation. We controlled the levels of antigenic stimulation by altering the numbers of DCs placed into the culture. Naive HA-specific CD8 T cells were stimulated with HA-pulsed mature DCs at different DC:T cell ratios (Fig. 6). After 48 h of stimulation, T cells were separated from DCs and T cell survival was analyzed by annexin V staining on day 7. A significant increase in annexin V+ cells that correlated with decreasing numbers of DCs was detected when compared with the control (DC:T ratio of 1:10; Fig. 6A), leading to a reduction in the formation of memory cells in vivo (Fig. 6B). These data indicate that the levels of antigenic stimulation also control T cell survival and memory formation.


Figure 6
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  		FIGURE 6. Altering DC dose influences CD8 memory T cell formation. Naive HA-specific CD8 T cells were stimulated in vitro for 48 h with LPS-matured DCs at DC to T cell ratios of 1:10, 1:50, and 1:250. After 48 h, CD8 T cells were removed from stimulation by microbead isolation. A, Survival of in vitro primed HA-specific CD8 T cells. Following withdrawal from stimulation, primed CD8 T cells were cultured in the absence of DCs for a total of 7 days and cells were stained with annexin V. Percentages of annexin V+ cells among CD8 T cells are indicated. B, Memory formation in vivo. Purified, in vitro primed CD8 T cells (5 x 105) were transferred into Ag-free mice; CD8 memory formation was analyzed 35 days later by IFN-{gamma} intracellular staining; and the percentages of IFN-{gamma}-producing HA-specific CD8 T cells among total CD8 T cells are indicated. Data shown are representative of two independent experiments.

 
Collectively, we conclude that the extent of stimulation, which includes both the duration and the levels of antigenic stimulation and costimulation, during priming controls T cell survival and formation of long-lived memory cells in vivo.

Extent of stimulation controls T cell survival via activation of Akt

We next investigated how the extent of stimulation influenced the survival of activated CD8 T cells. The serine-threonine Akt has emerged as an important regulator of cell survival in a variety of cell types, including hemopoietic cells (28, 32, 33, 34, 35). In mature T cells, Akt has been shown to be activated by TCR stimulation as well as CD28, IL-2R, and OX-40 signals (36, 37, 38, 39). We, therefore, hypothesized that the extent of stimulation regulates the extent of Akt activation in CD8 T cells during priming. To test this, we used immunoblotting to analyze lysates from HA-specific T cells that were stimulated with immature or mature HA peptide-pulsed DCs for 4, 8, 16, 24, 36, or 48 h. In T cells stimulated with mature DCs, a significant increase in phosphorylated Akt levels over the naive T cells was detected within 16 h of stimulation and a greater amount was present after 36 h of stimulation (Fig. 7A). However, in T cells stimulated with immature DCs, no significant Akt phosphorylation was observed until 48 h after initial Ag encounter (Fig. 7A). These results indicate that overall strength of stimulation during T cell priming regulates the extent of Akt activation in CD8 T cells.


Figure 7
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  		FIGURE 7. Extent of stimulation controls the survival of activated CD8 T cells through activation of Akt. A, Stimulation strength regulates the extent of Akt activation. Naive HA-specific CD8 T cells were stimulated in vitro with either immature (Immature DC) or LPS-matured (Mature DC) DCs pulsed with the HA peptide or left unstimulated (Unstimulated) as a control. After 4, 8, 16, 24, 36, and 48 h of stimulation, T cells were removed from culture and total cell lysates were collected for Western blot analysis of phosphorylated (pAKT) and total Akt (Total AKT). Data shown are a representative blot of four independent experiments. B, Akt activity controls the survival of activated CD8 T cells. Naive HA-specific CD8 T cells were stimulated in vitro with LPS-matured, HA peptide-pulsed DCs. Twenty-four hours into culture, CD8 T cells were infected with a retroviral construct encoding GFP only (Control) or GFP and dn-Akt. CD8+ cells were removed from stimulation by MACS selection after 72 h of culture, and analyzed for Bcl-xL expression by flow cytometry on day 4. Percentage of Bcl-xL+ cells among CD8+GFP+ cells is indicated. On day 7 of culture, cells were analyzed for annexin V+ cells. Percentage of annexin V+ cells within the CD8+GFP+ cell population is indicated. Data shown are representative of four independent experiments.

 
The strong correlation between Akt activation (Fig. 7A) and resistance to apoptosis (Fig. 5B) suggested that Akt activity might control the survival of activated CD8 T cells. To test this, we used a bicistronic retroviral vector encoding both GFP and dominant-negative Akt (dn-Akt) that possesses a point mutation at the ATP binding site, to transduce HA-specific T cells stimulated with HA peptide-pulsed mature DCs. Transduction was performed at 24 h after stimulation with DCs. T cells were separated from DCs to terminate TCR triggering on day 3; the expression of the antiapoptotic molecule Bcl-xL was measured by FACS on day 4; and the survival of T cells was analyzed by annexin V staining on day 7. T cells transduced with dn-Akt showed significantly reduced level of Bcl-xL expression compared with those transduced with a control retroviral vector encoding GFP only (Fig. 7B). Furthermore, the majority of dn-Akt-transduced cells (~92%) underwent apoptosis compared with only 18% in the control group (Fig. 7B). Collectively, our data indicate that extent of stimulation controls the survival of activated CD8 T cells through Akt activation.

Akt is required for CD8 memory formation in vivo

Our observations that Akt activity controls the survival of activated CD8 T cells in vitro and the extent of Akt activation (Fig. 7A) correlates well to the formation of memory CD8 T cells in vivo (Fig. 5C) suggest Akt may play a critical role in CD8 memory formation in vivo. To address this question, HA-specific CD8 T cells were primed in vitro with mature DCs pulsed with HA peptide in the presence of retroviral vector encoding GFP and dn-Akt or GFP only as a control. Three days later, GFP-positive cells were sorted by FACS and transferred into Ag-free naive mice. Forty-two days after transfer, CD8 memory formation was assayed upon recall response to Ad-HA. Splenocytes were analyzed for percentages as well as function of HA-specific CD8 T cells 5 days after challenge with Ad-HA. Our data showed that the percentage of HA-specific CD8 T cells was significantly diminished (~90% reduction) in mice transferred with dn-Akt-transduced T cells compared with those that received the control T cells (Fig. 8). These results indicate that Akt is required for CD8 memory formation in vivo.


Figure 8
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  		FIGURE 8. A critical role of Akt in CD8 memory formation in vivo. Naive HA-specific CD8 T cells were stimulated in vitro for 72 h with LPS-matured, HA peptide-pulsed DCs. Twenty-four hours into culture, CD8 T cells were infected with a retroviral construct encoding GFP only (Control) or GFP and dn-Akt. After 72 h, CD8+GFP+ cells were sorted and transferred into B10.D2 mice (2 x 104 per/mouse). Forty-two days later, mice were infected with Ad-HA. A, Five days after infection, splenocytes were analyzed for the expansion of HA-specific CD8 T cells, and the percentage of CD8+Thy1.1+ population among total lymphocytes is indicated. Cells were also subjected to IFN-{gamma} intracellular staining, and the percentage of IFN-{gamma}+ HA-specific CD8 T cells among total CD8 T cells is indicated. B, The absolute number of CD8+Thy1.1+ as well as IFN-{gamma}+CD8+Thy1.1+ cells per spleen is shown with SDs indicated. Data shown are representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Critical to the design of effective vaccine strategies is to understand what controls the formation of long-lived, memory CD8 T cells. Two models have been proposed to explain what dictates the formation of memory CD8 T cells. In the decreasing-potential hypothesis, T cells arrive that early during the immune response receive strong and prolonged stimulation leading to activation-induced cell death, whereas T cells that enter late receive short and weak stimulation and survive to become memory cells (3, 10). The progressive differentiation and fitness selection model proposes T cells that receive optimal stimulation during priming are rendered more fit and survive the contraction phase to become memory cells (17, 18).

Our study supports the progressive differentiation and fitness selection model because the early primed (i.e., day 0 of the infection) T cells received the strongest stimulation during the infection with vaccinia virus, divided more extensively, and survived better than T cells that entered the immune response later during the infection, leading to generation of the largest size of memory pool. Although the intermediate-primed (i.e., day 3 of the infection) T cells also developed into stable memory pool, the size of memory cells was reduced proportional to that of effectors. This is in line with a recent study in a model of vesicular stomatitis virus infection that the latecomer (i.e., day 4 of the infection) CD8 T cells were not preferentially recruited into the surviving pool of memory cells (40). In this study, we have further shown that despite differentiation into effector T cells, the late-primed (i.e., day 7 of the infection) T cells that received weakest stimulation showed lack of full cell division, survived poorly, and failed to develop into memory cells.

Evidence that supports the decreasing-potential hypothesis is mostly derived from models of overwhelming and persistent infection. It has been demonstrated that Ag-specific T cells are often deleted or become dysfunctional in the settings of chronic infection in mice (11, 12, 13, 41). Similar findings have also been observed in chronic HIV- and hepatitis C virus-infected humans (42, 43). Although our present study does not favor the decreasing-potential model in an acute infection, in which CD8 memory differentiation occurs in the absence of Ag, it does not argue against lack of long-lived memory formation during a chronic infection (44), in which the Ag persists. In fact, our recent study also suggests that long-lived, memory CD8 T cells cannot be generated in the presence of persistent Ag exposure (26). The implication of homodimeric CD8{alpha}{alpha} in promoting survival and differentiation of effectors into memory cells during an acute lymphocytic choriomeningitis virus infection (16) is in line with the decreasing-potential hypothesis, because CD8{alpha}{alpha} has been shown to down-modulate TCR signals by sequestering lck away from the lipid rafts (14, 15). However, as a recent study indicated (45), the role of CD8{alpha}{alpha} in promoting CD8 memory formation remains controversial because memory CD8 T cell development can occur independently of CD8{alpha}{alpha}.

Our observation that despite clonal expansion and effector differentiation, the formation of memory cells from the late-arriving T cells was severely compromised, suggests that the extent of stimulation required for T cells to commit to long-term survival is far greater than that required to initiate clonal expansion and effector differentiation. Our data suggest that the late-arriving cells probably receive insufficient stimulation in terms of a short duration and low levels of antigenic stimulation/costimulation caused by elimination of Ags and changes in costimulatory ligand densities that occur over time after Ags enter the body (18). Indeed, we further confirmed that both the duration and the levels of Ag can influence the survival of activated T cells and formation of memory cells.

How the extent of stimulation promotes T cell fitness and survival is not entirely clear. In this study, we showed that the extent of stimulation correlated well to the extent of Akt phosphorylation during priming, and that Akt activation is required for survival of the activated CD8 T cells in vitro and CD8 memory formation in vivo, suggesting the extent of stimulation promotes T cell survival through activation of Akt. Although phosphorylation of Akt can be induced by CD28, OX-40, and IL-2R signals (36, 37, 38, 39), we observed that no phosphorylation of Akt over background was induced without TCR stimulation (data not shown), suggesting TCR signaling is critical for initiation of Akt activation. It is not clear which costimulatory molecule(s) is required to induce fitness and survival of effector CD8 T cells in addition to TCR stimulation in vivo.

The mechanism underlying Akt-dependent enhancement for the survival of activated T cells remains unclear. We showed that inhibiting Akt activity in CD8 T cells could result in suppression of antiapoptotic molecule Bcl-xL. This is consistent with previous observations linking Akt to Bcl-xL and other members of the antiapoptotic Bcl-2 family (36, 39), suggesting that these molecules may be the main downstream targets of the Akt-mediated survival pathway. Akt could also inactivate its downstream substrate, FOXO, leading to the suppression of proapoptotic molecules such as Bim (46, 47). Another downstream target of Akt is GSK-3. Like FOXO, phosphorylation by Akt inactivates GSK-3, resulting in increased glycogen synthesis and cell survival (48, 49). Thus, future studies should focus on defining the downstream pathway(s) through which Akt promotes the survival of effector CD8 T cells. Delineation of such a pathway(s) will help us design effective strategies to enhance the formation of long-term, memory CD8 T cells in vivo.

In conclusion, we have presented evidence that supports the progressive differentiation and fitness selection mode of CD8 memory formation and suggests that the extent of stimulation during priming promotes the fitness and determines whether effector cells are short-lived or will develop into long-term memory cells via activation of Akt. These findings have important implications in the design of effective vaccine strategies that are targeted to boost memory CD8 T cell responses.


   Acknowledgment
 
We thank Dr. Zhou Songyang for providing the pMSCV and pMSCV-dn-Akt (K179M) retroviral constructs.


   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 work was supported by National Institutes of Health Grants CA111807 and CA047741 (to Y.Y.) and an Alliance for Cancer Gene Therapy grant (to Y.Y.). Back

2 Address correspondence and reprint requests to Dr. Yiping Yang, Departments of Medicine and Immunology, Duke University Medical Center, Box 103005, Durham, NC 27710. E-mail address: yang0029{at}mc.duke.edu Back

3 Abbreviations used in this paper: HA, hemagglutinin; Ad-HA, adenovirus encoding HA; DC, dendritic cell; dn-Akt, dominant-negative Akt; rVV-HA, recombinant vaccinia virus encoding HA. Back

Received for publication April 4, 2007. Accepted for publication August 22, 2007.


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 Introduction
 Materials and Methods
 Results
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