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The Journal of Immunology, 2007, 179, 3524 -3534
Copyright © 2007 by The American Association of Immunologists, Inc.
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Enhanced Levels of Costimulation Lead to Reduced Effector/Memory CD8+ T Cell Functionality1

Sven Mostböck*, Silvia Vidal{dagger}, Jeffrey Schlom2,* and Helen Sabzevari*

* Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and {dagger} Department of Immunology, Hospital Sant Pau, Barcelona, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The role of different levels of costimulation in conjunction with signal 1 in the activation of memory CD8+ T cells remains elusive. In this study, we demonstrate, in a mouse model with the influenza nucleoprotein epitope NP68, that mouse early memory (effector/memory) CD8+ T cells that were generated with high levels of costimulation have reduced CTL functionality compared with those that were generated with low levels of costimulation. This reduction is associated with increased phosphorylation of the negative regulatory site 292 on Zap70 and a decrease in granzyme B levels. Furthermore, we show that enhanced costimulation reduces proliferation and cytokine production of effector/memory CD8+ T cells in response to intermediate and weak TCR stimulation, in contrast to previously described positive effects of costimulation on naive CD8+ T cells. This effect is associated with the expression of ICAM-1 on APCs. Together, our results indicate that enhanced costimulation can lead to reduced functionality in effector/memory CD8+ T cells. This compromised effector function of effector/memory CD8+ T cells in response to high levels of costimulation can have important implications for designing immunotherapeutic strategies to enhance immune responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The selectivity of T cell activation is based on the interaction of peptide-MHC complex on APCs with the TCR. APCs influence the result of T cell activation, not only via presentation of the peptide-MHC complex (1, 2, 3, 4, 5), but also via costimulatory molecules that may modulate the TCR signal (6, 7, 8, 9). Even though naive T cells require both TCR and costimulatory signals for their activation, memory T cells depend on costimulation to a much lesser degree. Therefore, the requirement for additional costimulation might depend on the functional state of T cells and the affinity of the peptide-MHC complex for a given TCR (10, 11, 12).

Several studies have demonstrated that costimulation and high-avidity ligation of the TCR can play a major role in the negative selection of T cells in the thymus and the periphery (13, 14, 15). By the use of altered peptide ligands (APLs)3 with different binding affinity to the TCR, it has been shown in CD4+ T cells that differences in TCR activation lead to uncoupling of cytokine production and cell proliferation. Moreover, activation of CD4+ T cells by low-affinity APLs can induce anergy and change cytokine secretion patterns, which bias CD4+ T cells to Th1 or Th2 subtype differentiation (16, 17, 18, 19, 20, 21, 22, 23). A few studies with naive CD8+ T cells have demonstrated the induction of cytokine production and cytotoxicity, but have rarely shown any proliferative responses to APLs (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). The modulation of memory CD8+ T responses by APLs has received even less attention. These previous studies indicated that the requirements for induction of distinct effector functions may be influenced by the peptide-MHC complex and the levels of costimulatory molecules on APCs.

Most experimental and some clinical vaccine studies have used various strategies, including the use of costimulatory molecules, to enhance T cell responses to specific tumor Ags. Among these are the use of viral or bacterial vectors for efficient delivery of costimulatory molecules and/or cytokines to improve the presentation capacity of APCs (35). The present study was designed to address the influence of optimal and suboptimal TCR binding in the context of different levels of costimulation on APCs during activation. APCs of different quality (expressing either high or low levels of costimulatory molecules) were used to assess the activation and functionality of early memory (effector/memory) CD8+ T cells in response to APLs with different TCR affinities. To this end, we have used CD8+ T cells (obtained from F5 TCR-transgenic mice) specific for the peptide NP68 (the immunodominant epitope of the influenza nucleoprotein strain A/NT/60/68) (36, 37). The MHC class I anchor sites of this peptide have been previously established and a number of APLs with similar affinities to the MHC class I molecule, but different activation of T cells, have been identified (34, 38, 39). By using exogenous APLs (that have similar affinities to the MHC class I) as Ags, we were able to obtain a similar presentation of all APLs by the APCs. This enabled us to assess the effects of various amounts of presented Ag, as well as different levels of TCR activation by Ag. In the present study, we evaluated the effects of the natural epitope NP68 and its APLs, in conjunction with APCs of different quality, on the activation of effector/memory CD8+ T cells.

Our results demonstrate for the first time that enhanced costimulation can actually restrict both the proliferative response and cytokine production of effector/memory CD8+ T cells in response to activation by low-affinity Ags. This decline in activity of effector/memory T cells is associated with high expression of ICAM-1 on APCs. Moreover, the generation of effector/memory CD8+ T cells with a high-affinity Ag in the presence of enhanced costimulation led to decreased CTL activity, without affecting the proliferative response or cytokine production of these cells.


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

F5 mice that are transgenic for an influenza NP366–374 (ASNENMDAM; NP68)-specific, H-2Db-restricted TCR (36, 37) were obtained from Taconic Farms. All the animal studies had been approved by the National Institutes of Health Animal Care and Use Committee before the experiments.

Generation of APCs

LDb is the murine fibroblast cell line L929 expressing murine B7.1 and MHC class I molecule H-2Db (34) and is a gift from J. Yewdell (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD). APClow are LDb infected as described previously (40, 41), with wild-type fowlpox virus (FP-WT; Therion Biologics) at a multiplicity of infection (MOI) of 50. These cells express similar levels of H-2Db and B7.1 as uninfected LDb, but no LFA-3 or ICAM-1. APChigh are LDb cells infected at an MOI of 50 with fowlpox carrying a triad of costimulatory molecules (ICAM-1, B7.1, and LFA-3, designated rF-TRICOM; Therion Biologics). These cells express ICAM-1 and LFA-3 as well as markedly increased levels of B7.1 compared with APClow (40). Fig. 1 demonstrates the different levels of costimulatory molecules on APClow and APChigh.


Figure 1
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  		FIGURE 1. Expression of costimulatory molecules by APChigh and APClow. A, Uninfected LDb cells. B, LDb cells were infected with 50 PFU/cell (MOI of 50) of FP-WT (APClow, black line histogram) or fowlpox carrying TRICOM (APChigh, gray histogram). The cells were stained for H-2Db MHC class I expression, B7.1, ICAM-1, or LFA-3 and analyzed by FACS. This experiment is representative of numerous FACS analyses performed on APClow and APChigh.

 
Peptides

NP68 (ASNENMDAM), NP373T (ASNENMDTM), NP4Q (ASNQNMDAM), and NP372E (ASNENMEAM) (34, 39) were synthesized by the American Peptide Company.

Naive and effector/memory CD8+ T cells

CD8+ T cells in this study were obtained from F5 mice and were transgenic for the influenza NP366–374 (NP68)-specific, H-2Db-restricted TCR. For studies of naive CD8+ T cells, CD8+ T cells were purified from freshly prepared F5 splenocytes by magnetic bead separation with CD8a (Ly-2) MicroBeads (Miltenyi Biotec) according to the manufacturer’s instructions. A surface marker staining (all Abs BD Biosciences) of the isolated CD8+ T cells confirmed their naive phenotype (CD44lowCD122–CCR7–CD62LhighLy6C–). Effector/memory CD8+ T cells were generated from untreated freshly prepared F5 splenocytes, as described below. Surface marker staining showed their memory phenotype (CD44highCD122+ CCR7+CD62LlowLy6C+).

In vitro generation of effector/memory CD8+ T cells by incubation with different peptides

Splenocytes from F5 TCR-transgenic mice were stimulated with one of the four peptides at varying peptide concentrations for 5 days. After removal of dead cells by density centrifugation (Ficoll), cells were rested for at least 2 days in the presence of 0.143 µg/ml human IL-15 (PeproTech). All resting effector cells were CD8+. Surface marker staining of the generated population demonstrated a CD44highCD122+ CCR7+CD62LlowLy6C+ memory CD8+ T cell phenotype. It has previously been shown that short-term rested effectors (which are generated by resting CD4+ T cells for 3 days after 3–4 days of activation) and long-lived memory cells (which are generated by resting effectors in vivo for 60 days or longer) have very similar phenotypes, cytokine production potential, resistance to activation-induced cell death, and gene expression. Because of the high degree of similarity between these two cell populations, the effectors that were rested for 3 days were viewed as "early memory" cells (42, 43, 44). In this study, we have termed early memory CD8+ T cells generated by a similar protocol "effector/memory CD8+ T cells."

In vitro generation of effector/memory CD8+ T cells under different levels of costimulation

Naive CD8+ T cells were purified from the splenocyte population by magnetic bead separation with CD8{alpha} (Ly-2) MicroBeads (Miltenyi Biotec). Naive CD8+ T cells were cultured with 10–4 µg/ml NP68 for 5 days with either APClow or APChigh. After Ficoll, cells were rested for 2 days in the presence of 0.143 µg/ml human IL-15. A second round of in vitro stimulation was performed in an identical manner, except for the use of 10–5 µg/ml NP68. Surface marker staining of the generated population demonstrated a CD44highCD122+CCR7+CD62LlowLy6C+ memory CD8+ T cell phenotype.

CTL assay

In vitro CTL activity of CD8+ T cells was evaluated by a standard 51Cr-release assay using LDb cells as target cells. Briefly, effector/memory CD8+ T cells were incubated with target cells at peptide concentrations ranging from 10 µg/ml to 10–8 µg/ml at an E:T ratio of 25:1 for 5–7 h. Percent-specific lysis per well was calculated as (cpmwell – cpmmin)/(cpmmax – cpmmin) x 100. For one control experiment, the target cells were pulsed with the peptide at indicated concentrations and free peptide was washed off before effector cells were added to the assay.

For the evaluation of in vivo CTL activity, splenocytes from naive untreated C57BL/6 mice were treated with ammonium chloride-potassium chloride lysing buffer (ACK; Cambrex Bio Science) to remove RBC. Cells were then incubated with either no peptide or 10 µg/ml NP68 for 2 h, washed two times with PBS, and labeled with either 0.2 or 0.04 µM CFSE, respectively, in PBS for 10 min at 37°C. After incubation, cells were washed two times in PBS, counted, and equal numbers of the two populations were combined. A total of 6.7 x 106 cells of this cell mixture were then adoptively transferred i.v. into untreated control F5 mice or vaccinated F5 mice. The next day, splenocytes from those mice were harvested, treated with ACK, and analyzed by FACS. The specific lysis was calculated as the percent reduction of the CFSE population in relation to the corresponding CFSE population in the untreated control F5 mice.

CFSE proliferation and cytokine production profile

CD8+ T cells were labeled with 1 µM CFSE. After culture with APCs and free peptides, cells were stained for CD8{alpha} (53-6.7; Abs from BD Biosciences) and analyzed by FACS. In a control experiment, APCs were pulsed with peptide and free peptide was washed off before CFSE-labeled CD8+ T cells were added. Supernatant was analyzed using the CBA Mouse Cytokine kit (BD Biosciences). For blocking experiments, APCs were preincubated with blocking Abs for B7.1 (16-10A1), ICAM-1 (3E2), LFA-3 (HM48-1), or an isotype control (A19-3) for 2 h at a concentration of 40 µg/ml. Unbound Abs were washed off before adding the CD8+ T cells. Cytokine production on the cellular level was determined by intracellular cytokine staining for IFN-{gamma} (XMG1.2) and TNF-{alpha} (MP6-XT22) using brefeldin A, following the manufacturer’s instructions (BD Biosciences).

Western blot

Either rested effector/memory CD8+ T cells or effector/memory CD8+ T cells after a 15-min restimulation with NP68-pulsed LDb cells (loaded with 10–5 µg/ml NP68) were lysed and run in a standard SDS-PAGE (10% Novex-Gel; Invitrogen Life Technologies). Protein bands were transferred to Immobilon-P polyvinylidene fluoride membranes (Millipore). Samples from rested cells were tested for perforin (1:1500; ab7203; all Abs, Abcam) and granzyme B (1:500; ab4059). Samples from stimulated cells were tested for pVAV-1 (pY160) (ab4763) or pLCK (pY505) (ab4901), pERK1 + 2 (pT185+T202) (ab4819), pZap70 (pY292) (ab12868), and pZap70 (pY315+pY319) (ab12869; all Abs 1:1000). Membranes were stripped and restained with anti-{alpha}-tubulin Ab (1:1000; Sigma T5168; Sigma-Aldrich).

Vaccination studies

F5 mice were vaccinated with 108 PFU rF-TRICOM s.c., and received either 0.5 µg of NP68 or 500 µg of NP372E in IFA (Rockland) s.c. at the same site the next day. This vaccination was repeated once a week for 3 wk. To measure the proliferative response of the CD8+ T cells in vivo, BrdU was given to the animals in the drinking water (0.8 mg/ml) for 3 days starting at day 0 and 3 days starting at day 7 following the third vaccination. Two weeks after the last vaccination, lymphocytes from the inguinal lymph nodes (LN) of two mice per group were tested for BrdU incorporation with the BrdU Flow kit (BD Biosciences), according to the manufacturer’s protocol.

For dendritic cell (DC) immunization, DC were generated from F5 mice and infected as described previously with either FP-WT (DC-WT) or rF-TRICOM (DC-TRICOM) at an MOI of 40 or 50 (45). After infection, DC-WT and DC-TRICOM were pulsed with 10 µg/ml NP68, washed, and 5 x 105 DC-WT or DC-TRICOM per mouse were adoptively transferred (i.v. injections) into F5 mice. This vaccination was repeated once a week for 3 wk. BrdU was given to the animals as an i.p. injection on the day of the third vaccination (0.8 mg of BrdU in 300 µl of PBS per mouse) and in the drinking water (0.8 mg/ml) for the first 4 days following the third vaccination. Two weeks after the last vaccination, lymphocytes from LN of one mouse per group were tested for BrdU incorporation with the BrdU Flow kit, according to the manufacturer’s protocol.

FACS analysis of splenocytes from vaccinated mice

Lymphocytes from the LN of one mouse per group were stained for the cell surface markers CD8a and CD44 with fluorochrome-labeled Abs (BD Biosciences). To identify NP68-specific cells, lymphocytes from LN were stained with ASNENDAM-pentamer (NP68-pentamer; ProImmune).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Effect of APLs on activation of CD8+ T cells in vitro

Two different APC lines were generated by transfecting the fibroblast cell line L929 expressing the MHC class I molecule H-2Db and B7.1 (LDb; Fig. 1A). APClow are LDb cells infected with FP-WT: they express H-2Db and B7.1, but no LFA-3 or ICAM-1 (Fig. 1B, black line histogram). APChigh are LDb cells infected with rF-TRICOM. These cells express ICAM-1 and LFA-3, as well as markedly increased levels of B7.1 compared with APClow (Fig. 1B, gray histogram). In this study, APClow represent APC of low costimulatory capacity (expressing just B7.1) and APChigh represent APC of high costimulatory capacity (expressing B7.1, LFA-3, and ICAM-1).

Effector/memory TCR-transgenic F5 CD8+ T cells (CD44highCD122+CCR7+CD62LlowLy6C+; generated with 0.2 µg/ml NP68 as described in In vitro generation of effector/memory CD8+ T cells by incubation with different peptides) were labeled with CFSE and cocultured with APClow in the presence of either the immunodominant epitope (NP68) or APLs (high-affinity NP373T, intermediate-affinity NP4Q, or weak-affinity NP372E) for 48 h.

As depicted in Fig. 2A, effector/memory CD8+ T cells, generated against the immunodominant epitope NP68, required a very low concentration of the immunodominant epitope (NP68) or the strong APL (NP373T) to enter the cell cycle. Effector/memory CD8+ T cells did not proliferate in response to low concentrations of intermediate or weak APLs (10–6 µg/ml, Fig. 2A); this is demonstrated by their CFSE profile, which is similar to the CFSE profile of cells cultured without any peptide (0 µg/ml, Fig. 2A). However, all effector/memory CD8+ T cells entered the cell cycle in response to an increased concentration of intermediate or weak APLs (1 µg/ml, Fig. 2A).


Figure 2
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  		FIGURE 2. Activation of effector/memory CD8+ T cells by APLs in vitro. A, Effector/memory F5 CD8+ T cells (generated to immunodominant epitope NP68) were labeled with CFSE and cultured for 48 h with APClow and either the immunodominant epitope NP68 or various APLs (NP373T, NP4Q, NP372E). Cells were cultured at three different peptide concentrations: 0 µg/ml (dotted line histogram), 10–6 µg/ml (gray histogram), and 1 µg/ml (black line histogram). Data are representative of six experiments. B, Effector/memory F5 CD8+ T cells were used in a standard 51Cr-release assay against target cells (LDb cells) loaded with the immunodominant epitope NP68 or various APLs (NP373T, NP4Q, NP372E) at an E:T ratio of 25:1. Data are representative of seven experiments. Results are reported as the mean and SD of triplicate wells.

 
In comparison, naive F5 CD8+ T cells (CD44lowCD122–CCR7–CD62LhighLy6C–) behaved similarly to memory F5 CD8+ T cells. However, the induced proliferation was generally lower than the proliferative response of effector/memory CD8+ T cells, especially for the APLs NP4Q and NP372E (data not shown). These results are in agreement with previously published findings (46).

Intermediate and weak APLs do not induce CTL killing in vitro

To study the ability of APLs to induce CTL activity in effector/memory CD8+ T cells, effector/memory CD8+ T cells were incubated with 51Cr-labeled target cells (LDb cells) with varying concentrations of several APLs for 5–7 h. As depicted in Fig. 2B, the immunodominant epitope and the strong APL (NP68 and NP373T, respectively) were able to induce the killing activity of effector/memory CD8+ T cells, while neither NP4Q nor NP372E induced any CTL activity. A similar result was obtained with peptide-pulsed target cells in the absence of free peptide. This control demonstrated that free peptide in the culture did not inhibit the CTL activity of the effector/memory cells against NP372E-loaded target cells. Enhanced expression of costimulatory molecules on the target cells (APChigh) did not trigger CTL killing by effector/memory CD8+ T cells against targets loaded with intermediate or weak APLs (data not shown).

Effector/memory CD8+ T cells generated by APLs obtain altered functionality against the immunodominant epitope in vitro

To determine the impact of different TCR affinities on the resulting effector functions of effector/memory CD8+ T cells, we cultured naive F5 CD8+ T cells with various APLs (with different TCR, but similar MHC affinities) to induce the generation of effector/memory CD8+ T cells. To ensure similar strengths of activation, we chose peptide concentrations that induced similar CFSE profiles in naive F5 CD8+ T cells (NP68 and NP373T at 10–4 µg/ml; NP4Q and NP372E at 2 µg/ml). The effector/memory CD8+ T cells generated with the immunodominant epitope or weak APL were of a CD44highCD122+CCR7+CD62LlowLy6C+ memory phenotype. To address the abilities of the differently generated effector/memory CD8+ T cells to proliferate and produce cytokines, these effector/memory CD8+ T cells were labeled with CFSE and cultured with the immunodominant epitope (NP68 at 10–6 µg/ml) and APClow for 48 h. All effector/memory CD8+ T cells generated by the various APLs demonstrated similar proliferative and cytokine responses (TNF-{alpha}, IFN-{gamma}, and IL-2) upon activation with the immunodominant epitope (Fig. 3, A and B, left panel). Intracellular studies showed that, upon reactivation with the immunodominant epitope, the percentage of IFN-{gamma}-producing effector/memory CD8+ T cells generated by either the immunodominant epitope (NP68) or the weak epitope (NP372E) was similar (Fig. 3B, right panel). The effector/memory CD8+ T cells generated with either the immunodominant epitope (NP68) or the strong APL (NP373T) were capable of killing target cells presenting the immunodominant epitope, whereas effector/memory CD8+ T cells that were generated by intermediate or weak epitopes (NP4Q, NP372E) demonstrated marginal CTL activity (Fig. 3C). Statistical analysis of the repeats of this experiment showed a significant difference in the CTL activity of NP68-generated and NP372E-generated effector/memory CD8+ T cells (paired t test, p < 0.05 for all four peptide concentrations). These results demonstrate that effector/memory CD8+ T cells that are generated by weak or intermediate APLs exhibit marginal CTL activity against the immunodominant epitope.


Figure 3
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  		FIGURE 3. Effector functions of effector/memory CD8+ T cells generated by APLs in vitro. Effector/memory F5 CD8+ T cells were generated by stimulating naive F5 CD8+ T cells in whole splenocyte culture with the immunodominant epitope NP68 or various APLs (NP373T, NP4Q, NP372E), as described in Materials and Methods. A, Effector/memory CD8+ T cells were labeled with CFSE and cultured with APClow and NP68 (10–6 µg/ml). Cells were harvested after 48 h and analyzed by FACS. Data are representative of two experiments. B, Left panel, Effector/memory CD8+ T cells were cultured with APClow and NP68 (10–6 µg/ml) and culture supernatants were harvested after 48 h and cytokines were quantitated. Data are representative of two experiments. Right panel, Effector/memory CD8+ T cells were cultured with APClow and NP68 (10–5 µg/ml) and harvested after 24 h. Cytokine-producing cells were quantitated by intracellular cytokine staining. Data are representative of two experiments. C, Effector/memory CD8+ T cells were used in a standard 51Cr-release assay against target cells loaded with the immunodominant epitope NP68 at an E:T ratio of 25:1. Data are representative of 11 repeats. Results are reported as the mean and SD of triplicate wells. D, Activated effector/memory CD8+ T cells were analyzed for the phosphorylation of Zap70 (pY292) and ERK1 + 2 (pT185+T202), as described in Materials and Methods. Rested effector/memory CD8+ T cells were analyzed for perforin and granzyme B expression.

 
Effector/memory CD8+ T cells generated by a weak APL in vitro down-regulate granzyme B expression

To study the signal pathways that may be responsible for the lack of CTL activity of effector/memory CD8+ T cells generated by a weak APL (NP372E), these cells were compared with effector/memory CD8+ T cells generated with the immunodominant epitope NP68. No differences were observed in the activation/phosphorylation of early signals such as Zap70 (pY305 and pY314), LCK, SHP-1, or VAV (data not shown), or intermediate signaling pathways ERK1 + 2 (Fig. 3D) in these cells after a 15-min restimulation with the immunodominant epitope (NP68). Effector/memory CD8+ T cells that were generated by NP68 expressed increased levels of intracellular granzyme B as compared with effector/memory CD8+ T cells that were generated with the weak APL NP372E. There was no difference in perforin levels among these two groups (Fig. 3D). These data demonstrate that weak APLs do not initiate some of the pathways (granzyme B) that are involved in CTL activity.

Reduced CTL functionality of effector/memory CD8+ T cells generated by the weak APL NP372E in vivo

To address the effects of weak APLs on the activation and CTL response of NP68-specific CD8+ T cells in vivo, F5 mice were vaccinated three times with either 500 µg of NP372 in IFA or 0.5 µg of NP68 in IFA. We administrated a 1000-fold higher amount of the weak APL NP372E to achieve a similar proliferative response of F5 CD8+ T cells in mice that were vaccinated with the weak APL NP372E compared with mice that were vaccinated with the immunodominant epitope NP68. Two weeks after the third vaccination, BrdU incorporation was measured in vaccinated mice and nonvaccinated control mice to assess the proliferative response of NP68-specific CD8+ T cells. As shown in Fig. 4A, vaccination with either NP372E or NP68 led to increased proliferation of NP68-specific CD8+ T cells in the LN compared with nonvaccinated control F5 mice (15.0 and 23.3%, respectively, compared with 6.1%). Similarly, 3 days after the third vaccination, either NP372E or NP68 led to increased numbers of CD44high NP68-specific CD8+ T cells in the LN compared with the LN from a nonvaccinated control F5 mouse (5.7 and 11.4%, respectively, compared with 1.9%). These results demonstrated that vaccination with either the weak APL NP372E or the immunodominant APL NP68 led to CD8+ T cell activation in F5 mice.


Figure 4
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  		FIGURE 4. Activation of CD8+ T cells with the weak APL NP372E in vivo leads to reduced CTL functionality. F5 mice were vaccinated three times with either the weak APL NP372E or the immunodominant epitope NP68. A, NP68-specific (NP68-pentamer stained) CD8+ T cells from the LN were analyzed for the incorporation of BrdU 2 wk after the third vaccination. Numbers are the average and SD of two mice; the marker is set according to the isotype control. B, In vivo CTL lysis activity was assayed in nonvaccinated control mice and mice vaccinated with either NP372E or NP68. Each dot represents one animal; horizontal line represents the mean. Similar results were observed in a repeat of this experiment.

 
The CTL functionality of vaccinated F5 mice was quantitated using an in vivo CTL with a NP68-loaded target population. As shown in Fig. 4B, animals that were vaccinated with the weak APL NP372E had lower lysis activities compared with animals that were vaccinated with the immunodominant epitope NP68 (32% compared with 20%).

Enhanced costimulation reduces the response of effector/memory CD8+ T cells to weak APLs in vitro

To address the effect of costimulation on effector/memory CD8+ T cells, CFSE-labeled effector/memory CD8+ T cells, which were generated against NP68, were cultured with either APClow or APChigh and various APLs for 48 h. Enhanced costimulation by APCs did not affect the proliferative response of CFSE-labeled effector/memory CD8+ T cells activated by any APL at low peptide concentration (10–6 µg/ml, data not shown). At high peptide concentration (1 µg/ml), enhanced costimulation by APCs had no effect on proliferative responses of effector/memory CD8+ T cells to the immunodominant epitope or the strong APL; however, it decreased proliferative responses to the intermediate and the weak APL (Fig. 5A). A similar result was obtained in a control experiment with peptide-pulsed APCs in the absence of free peptide in the culture. This control demonstrated that free NP372E in the culture did not lead to the reduced proliferative response of F5 effector/memory CD8+ T cells in the presence of APChigh.


Figure 5
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  		FIGURE 5. Enhanced levels of costimulation in vitro reduce response of effector/memory CD8+ T cells against weak APLs. Effector/memory F5 CD8+ T cells generated against NP68 were labeled with CFSE and cultured for 48 h with either the immunodominant epitope NP68 or various APLs (NP373T, NP4Q, NP372E) at 1 µg/ml peptide concentration. A, Cells were cultured with either APClow (black line histogram) or APChigh (gray histogram) in the presence of indicated peptides (data are representative of 10 experiments) and (B) their supernatants were analyzed at 48 h for TNF-{alpha}, IFN-{gamma}, and IL-2 (data are representative of six experiments). C, Effector/memory CD8+ T cells were cultured with the weak epitope NP372E and either APClow or APChigh. Cytokine-producing cells were quantitated after 48 h by intracellular cytokine staining for IFN-{gamma} and TNF-{alpha}. Data are representative of three experiments.

 
Activation of effector/memory CD8+ T cells with APChigh and the immunodominant epitope NP68 led to higher levels of TNF-{alpha} and IFN-{gamma} than activation with APClow. APChigh also induced stronger TNF-{alpha} production in effector/memory CD8+ T cells activated by the strong APL (NP373T) than APClow (Fig. 5B). In contrast, activation with APChigh resulted in reduction of IFN-{gamma} and TNF-{alpha} production in the presence of the intermediate APL NP4Q, and a strong reduction of IFN-{gamma} levels in the presence of the weak APL NP372E, as compared with activation with APClow (Fig. 5B). Activation of effector/memory CD8+ T cells by APChigh in the presence of the weak APL NP372E reduced the percentage of IFN-{gamma}- and TNF-{alpha}-producing cells compared with activation by APClow (Fig. 5C). Activation of effector/memory CD8+ T cells did not lead to production of IL-4, IL-5, IL-10, or IL-12 (data not shown). In contrast, the proliferation and cytokine production (IFN-{gamma}, TNF-{alpha}) of naive CD8+ T cells was greatly increased by enhanced costimulation in response to intermediate or weak APLs (NP4Q, NP372E) in a similar setting (data not shown).

Blocking of ICAM-1 on APChigh restores the response of effector/memory CD8+ T cells to weak APLs in vitro

To address the role of individual costimulatory molecules expressed on APCs in the reduction of T cell proliferation and cytokine production, CFSE-labeled effector/memory CD8+ T cells, that were generated against NP68, were cultured for 48 h with NP372E (weak APL) at a high concentration (1 µg/ml) and either APClow or APChigh, both of which had been preincubated with blocking Abs to B7.1, ICAM-1, or LFA-3. As demonstrated in Fig. 6A, the proliferative response of effector/memory CD8+ T cells to the weak APL was reduced in the presence of APChigh. Blocking of B7.1 on both APC populations (APClow and APChigh) led to decreased proliferation of effector/memory CD8+ T cells in comparison to isotype control. In contrast, blocking of ICAM-1 on APChigh led to enhanced proliferation of effector/memory CD8+ T cells, whereas blocking of LFA-3 had no effect on the proliferation of effector/memory CD8+ T cells (Fig. 6A). Consistent with the proliferation data, blocking of ICAM-1 on APChigh led to enhanced TNF-{alpha} and IFN-{gamma} production by effector/memory CD8+ T cells activated with the weak APL (Fig. 6B). We did not observe any detectable amounts of IL-2, IL-4, or IL-5 in this experiment.


Figure 6
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  		FIGURE 6. Blocking of ICAM-1 restores the response of effector/memory CD8+ T cells to the weak APL NP372E in vitro. A, APClow and APChigh were preincubated with either isotype control Ab (black line histogram) or blocking Ab to B7.1, ICAM-1, or LFA-3 (gray histogram). Effector/memory F5 CD8+ T cells generated against NP68 were labeled with CFSE and cocultured with the above-mentioned APCs in the presence of NP372E (weak APL, 1 µg/ml) for 48 h. Data are representative of three experiments. B, Effector/memory F5 CD8+ T cells were cocultured with the above-mentioned APCs in the presence of NP372E (weak APL, 1 µg/ml). After 48 h of culture, the supernatants were analyzed for TNF-{alpha} and IFN-{gamma}. Data are representative of two experiments.

 
These data collectively demonstrate that high expression of ICAM-1 in the presence of other costimulatory molecules on APC reduces the broadening T cell specificity, and that blocking of ICAM-1 on APChigh restores the proliferative and cytokine responses of effector/memory CD8+ T cells to weak APLs.

Effector/memory CD8+ T cells that are generated in vitro in the presence of high levels of costimulatory molecules in vitro have reduced CTL activity

To investigate whether CD8+ T cells activated by APCs expressing high levels of costimulation would have altered CTL activity against the natural epitope, we cultured naive F5 CD8+ T cells with APClow or APChigh and the immunodominant epitope NP68. The effector/memory CD8+ T cells generated were of a CD44highCD122+CCR7+CD62LlowLy6C+ memory phenotype. To determine the proliferative responses of these effector/memory CD8+ T cells, these cells were further CFSE labeled and cultured with the immunodominant epitope NP68 at a low peptide concentration of 10–6 µg/ml and APClow for 48 h. High levels of costimulation during the generation of effector/memory CD8+ T cells had no effect on their entry into the cell cycle in the recall response (Fig. 7A). Moreover the production of TNF-{alpha} and IFN-{gamma} by these cells was similar, regardless of how they were generated (Fig. 7B, left panel). Similar percentages of effector/memory CD8+ T cells (generated by either APClow or APChigh) that were reactivated by the immunodominant peptide produced IFN-{gamma} (Fig. 7B, right panel). However, effector/memory CD8+ T cells that were generated by APChigh showed a reduced ability to lyse target cells presenting the immunodominant epitope NP68 compared with effector/memory CD8+ T cells generated by APClow (Fig. 7C). Statistical analysis of the repeats of this experiment showed a significant difference in the CTL activity of APChigh-generated and APClow-generated memory CD8+ T cells (paired t test, p < 0.05 for peptide concentrations 10–3, 10–5, and 10–6 µg/ml). These data collectively suggest that high levels of costimulation during CD8+ T cell activation do not affect the proliferation or cytokine production of effector/memory CD8+ T cells, but clearly affect CTL function.


Figure 7
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  		FIGURE 7. Effector functions of effector/memory CD8+ T cells generated by different levels of costimulation in vitro. Effector/memory F5 CD8+ T cells were generated by stimulating naive F5 CD8+ T cells in culture with the immunodominant epitope NP68 and either APClow or APChigh. A, Effector/memory CD8+ T cells generated with APClow or APChigh were CFSE-labeled and cultured with APClow and NP68 at 10–6 µg/ml (black line histogram) or 0 µg/ml (gray histogram). After 48 h, cells were analyzed by FACS. Data are representative of three experiments. B, Left panel, Effector/memory CD8+ T cells generated with APClow or APChigh were cultured with APClow and NP68 (10–6 µg/ml). Cytokines in supernatant were quantitated after 48 h culture. Results are reported as the mean and SD of three experiments. Right panel, Effector/memory CD8+ T cells generated with APClow or APChigh were cultured with APClow and NP68 (10–5 µg/ml) for 24 h. Cytokine-producing cells were quantitated by intracellular cytokine staining. Data are representative of two experiments. C, Effector/memory CD8+ T cells generated with APClow or APChigh were used in a standard 51Cr-release assay against 51Cr-labeled target cells loaded with the immunodominant epitope NP68 at an E:T ratio of 25:1. Data are representative of 13 experiments. Results are reported as the mean and SD of triplicate wells. D, Activated effector/memory CD8+ T cells were analyzed for the phosphorylation of Zap70 (pY292) and ERK1 + 2 (pT185+T202). Blots shown are representative of two repeats. Rested effector/memory CD8+ T cells were analyzed for perforin and granzyme B expression.

 
Activation of effector/memory CD8+ T cells in the presence of APCs expressing high levels of costimulation in vitro leads to down-regulation of granzyme B expression

Effector/memory CD8+ T cells that were generated with APClow or APChigh and the immunodominant epitope NP68 were restimulated for 15 min with the natural epitope NP68. There were no differences between the activation/phosphorylation of early signals such as Zap70 (pY305, pY314), LCK, SHP-1, or VAV (data not shown) in these cells. However, we observed that effector/memory CD8+ T cells that were generated by APChigh expressed higher levels of the negative regulation site phosphorylated-Y292 on Zap70 (Fig. 7D). In addition, the effector/memory CD8+ T cells that were generated with APChigh expressed lower levels of granzyme B and a slight but reproducible decrease of the phosphorylated form of ERK1 + 2, as compared with effector/memory CD8+ T cells that were generated with APClow. There was no difference in the expression of perforin in these cells once the amount of protein was normalized using {alpha}-tubulin protein (Fig. 7D).

High levels of costimulation lead to reduced CTL functionality in vivo

To address the effects of high levels of costimulation on the activation and CTL functionality of NP68-specific effector/memory CD8+ T cells in vivo, F5 mice were vaccinated three times with either DC infected with FP-WT (DC-WT, low levels of costimulation) or DC infected with rF-TRICOM (DC-TRICOM, high levels of costimulation), both loaded with the immunodominant epitope NP68. The proliferation of NP68-specific CD8+ T cells was measured 2 wk after the last vaccination. As shown in Fig. 8A, vaccination with either DC-WT or DC-TRICOM led to increased proliferation of NP68-specific CD8+ T cells in the LN compared with a nonvaccinated control F5 mouse (16.2 and 18.7%, respectively, compared with 5.4%). The vaccinations also led to increased numbers of CD44high NP68-specific CD8+ T cells in the LN compared with a nonvaccinated control F5 mouse (15.1 and 12.0%, respectively, compared with 5.5%). These results demonstrated that the vaccinations with DC-WT or DC-TRICOM led to comparable levels of CD8+ T cell proliferation in F5 mice, similar to our in vitro data.


Figure 8
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  		FIGURE 8. High levels of costimulation lead to reduced CTL functionality in vivo. F5 mice were vaccinated three times with either DC-WT or DC-TRICOM, both loaded with peptide NP68. A, The incorporation of BrdU was analyzed in NP68-specific (NP68-pentamer stained) CD8+ T cells 2 wk after the third vaccination in the LN. The marker is set according to the isotype control. B, In vivo CTL lysis activity was assayed in nonvaccinated control mice and mice vaccinated with either DC-WT or DC-TRICOM. Each dot represents one animal; horizontal line represents the mean.

 
To determine whether high levels of costimulation lead to differences in the CTL activity of vaccinated animals, we quantitated the CTL functionality of the vaccinated F5 mice using an in vivo CTL with a NP68-loaded target population. As shown in Fig. 8B, animals that were vaccinated with DC-TRICOM had lower lysis activities compared with animals that were vaccinated with DC-WT (44% compared with 56%). These in vivo findings support our in vitro data demonstrating that repeated stimulation in the presence of high levels of costimulation can lead to reduced CTL functionality.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study addresses the impact of costimulation on the activation of early memory (effector/memory) CD8+ T cells by APLs with similar affinities for the MHC and different affinities for the TCR. We demonstrate for the first time that low-affinity TCR ligands (weak APLs) are able to induce in vitro maturation of naive CD8+ T cells into effector/memory CD8+ T cells with regular proliferative response and cytokine production, but little CTL activity against a high-affinity peptide. Our results demonstrate that this lack of CTL activity may be related to downstream signals affecting granzyme B expression. In response to weak APLs in vitro, effector/memory CD8+ T cells undergo proliferative response and cytokine production, but do not acquire any CTL activity against target cells presenting weak APLs. We also show a similar reduction of in vivo CTL activity in mice repeatedly vaccinated with the weak APL NP372E compared with mice vaccinated with the immunodominant epitope NP68.

High levels of costimulation in vitro reduce the activation of effector/memory CD8+ T cells by low-affinity APLs, leading to decreased proliferative response and cytokine production. This reduction in response is related to the high expression of ICAM-1 on APCs. Furthermore, we demonstrate that activation of effector/memory CD8+ T cells with high levels of costimulation in vitro leads to a decline of their CTL functionality, while their ability to proliferate and produce cytokines is not affected. Decreased in vivo CTL activity was seen in mice vaccinated repeatedly with DC expressing high levels of costimulation, compared with mice vaccinated with DC expressing regular levels of costimulation.

Our data demonstrate that while costimulation has beneficial effects on proliferation and cytokine production in naive CD8+ T cells, enhanced levels of costimulation can have a negative effect on the functionality of effector/memory CD8+ T cells.

It should be mentioned that using CD8+ T cells from TCR-transgenic mice allowed us to eliminate the experimental variation that might arise due to different TCR specificities in a polyclonal T cell population. Even though this system is ideal for addressing the effect of excess costimulation on the functionality of a CD8+ T cell population with defined specificity, the role of costimulation in polyclonal T cell responses may be much more complex in an in vivo setting. To this end, our laboratory is currently establishing various in vivo models that can address these questions.

It has previously been shown that distinct biochemical signals by APLs can lead to differentiation of naive CD4 T cells into either Th1 or Th2 subsets (47). In similar findings in regard to CD8+ T cells, Reis e Sousa et al. (48) have pointed out that inhibitory ligands/APLs are capable of inducing phosphorylation of signaling proteins upon TCR engagement, but fail to induce CTL effector functions. In this study, effector/memory CD8+ T cells generated with repeated stimulation by high levels of costimulation display no changes in phosphorylation of Zap70 on tyrosines 315 (LCK-binding site) and 319 (VAV-binding site) or phosphorylation/activation of LCK, but exhibit an increased phosphorylation of a negative regulatory site on Zap70 (tyrosine 292). Phosphorylation of Zap70 tyrosine 292 terminates the activation of Zap70 and attenuates lymphocyte signaling (49). The MAPK/ERK pathway, which has been shown to regulate the reorganization and release of intracellular granules containing perforin and granzymes (50, 51), constitutes a link between activation and granular release (52, 53). In our studies, lower levels of granzyme B, together with a slight but reproducible decrease in phosphorylation of ERK1 + 2, might explain the reduced CTL functionality of these cells. These results suggest that enhanced costimulation does not affect the signal pathways that are involved in proliferation or cytokine production in effector/memory cells, but influence the signal pathways that are involved in CTL function. Effector/memory CD8+ T cells generated by a low-affinity APL (delivering lower levels of TCR activation) do not demonstrate an alteration in the phosphorylation patterns of the signaling proteins we investigated. They do, however, have markedly reduced levels of granzyme B, pointing to a similar mechanism behind the reduction of CTL functionality.

One of the important issues in immunology is regulation of the number and functionality of T cells. Different mechanisms, such as T cell deletion and/or blunting of T cell functions (e.g., maintenance of T cells without any detectable CTL or cytokine function) have been shown to regulate antiviral T cell responses. Zajac et al. (54) have suggested that the degree of T cell activation is likely an important factor that determines whether CD8+ T cells are deleted or become functionally unresponsive. Our results demonstrate that regulation of functionality in effector/memory CD8+ T cells depends on both signal 1 (TCR activation) and signal 2 (level of costimulation). Enhanced costimulation might lead to the reduction of certain functional responses such as CTL activity, while not affecting other functions such as proliferation and cytokine production. Despite a number of reports demonstrating the positive role of costimulation on activation of naive CD4+ T cells (10, 55), very little attention has been paid to the modulation of memory CD8+ T cell functionality by high levels of costimulation. The adverse influence of additional costimulation on high-affinity memory CD8+ T cells might bias the immune response toward less potent CTLs. This could play a role in the containment of memory T cells in the context of strong immune reactions. Therefore, understanding the threshold for CTL functionality of memory CD8+ T cells and the role of enhanced costimulation by APCs is an important issue.

Our data also show that stimulation by peptides with low TCR affinity induces and sustains an effector/memory T cell population that is responding suboptimally to another peptide with higher TCR-binding affinity. Recognition of self-peptides by cross-reacting T cells in the periphery can play a role in T cell homeostasis (14, 56, 57, 58, 59, 60), but a possible negative impact of these low-affinity peptides on T cell populations has never been fully investigated. Based on our results, we hypothesize that interaction with weak APLs can actually reduce the functionality of memory CD8+ T cells and have a negative regulatory feedback on these cells. Previous studies have shown that the quality of APCs (related to the level of costimulatory molecule expression) is critical in enhancing the responses of naive CD4+ T cells to APLs (22, 61, 62). Our data regarding the activation of naive CD8+ T cells with APCs expressing high levels of costimulation agree with these findings. However, high levels of costimulation actually decrease the response of effector/memory CD8+ T cells to APLs. Quaratino et al. (63) demonstrated that fully competent DC may present, after natural Ag processing, a self-epitope with APL properties that can induce anergy in an autoreactive T cell clone. In conjunction with these findings, our results support the conclusion that the presentation of APLs by high-quality APCs (APCs that express high levels of costimulatory molecules) can induce and maintain an anergic state in self-reactive T cells. This might be an important regulatory mechanism for self-reactive T cells and also might be a way to ensure the high specificity of a recall response. It is important to point out that in our studies, the LDb fibroblast cell line infected with rF-TRICOM (APChigh) resembles APCs of high quality expressing high levels of MHC class I and costimulatory molecules.

For many diseases, and especially cancer, the establishment of a stable memory population may be crucial for keeping the disease in check or even curing it. Biasing the immune response toward low-affinity T cells or CD8+ T cells with reduced CTL capability might have a negative impact on the overall immune response. The tradeoff here might be the activation of high amounts of naive CD8+ cells in response to low-affinity peptides (weak APLs) in the context of high levels of costimulation that produce a strong effector function and a very good immediate response, vs reduced functionality of memory CD8+ T cells and diminished immunological memory. One of the current strategies in vaccine development is to induce high levels of costimulatory molecules on the APC, which can certainly broaden the specificity of naive T cells and augment the immunogenicity of Ags with weak peptide epitopes for naive T cells. Indeed, a recent clinical study has indicated that repeated monthly vaccination may be more beneficial than vaccinations every 3 mo in stabilizing progressive growth of solid tumors (64). Further analysis of the phenotypes involved in T cell responses following these types of vaccination protocols is thus warranted. It is important, therefore, to distinguish between the mechanisms involved in the activation of naive T cells and memory T cells.

The key question is: what is the physiological relevance of APLs and costimulatory molecules in various diseases? One can hypothesize that the immune responses of memory T cells can become compromised in the presence of high levels of costimulation, which could be a means to regulate their function and expansion, especially against cross-reacting epitopes (with weak affinity to the TCR). This may assure the high specificity of recall responses vs a broad reactivation of any cross-reacting memory T cells. In a cancer setting, high levels of costimulation could enhance the interaction of naive T cells with weak epitopes and lead to generation of large numbers of CD8+ T cells with low affinity. However, the same weak epitopes in conjunction with high costimulation could blunt the ability of high-affinity memory T cells to attack the tumor. The compromised effector function induced by APLs can favor tumor escape at the level of memory T cells. Therefore, this information is critical for designing therapeutic strategies for enhancing the immune responses in a variety of diseases, such as cancer.


   Acknowledgments
 
We thank Judith DiPietro for her technical assistance, and Debra Weingarten and Bonnie L. Casey for their editorial assistance in the preparation of this manuscript.


   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 the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. Back

2 Address correspondence and reprint requests to Dr. Jeffrey Schlom, Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Building 10 Room 8B09, 10 Center Drive, Bethesda, MD 20892. E-mail address: js141c{at}nih.gov Back

3 Abbreviations used in this paper: APL, altered peptide ligand; WT, wild type; FP-WT, WT fowlpox virus; MOI, multiplicity of infection; TRICOM, triad of costimulatory molecules (ICAM-1, B7.1, LFA-3); LN, lymph node; DC, dendritic cell. Back

Received for publication September 22, 2006. Accepted for publication July 5, 2007.


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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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