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Originally published online as doi:10.1189/jlb.0806535 on January 10, 2007

Published online before print January 10, 2007
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(Journal of Leukocyte Biology. 2007;81:1165-1175.)
© 2007 by Society for Leukocyte Biology

Pivotal Advance: CTLA-4+ T cells exhibit normal antiviral functions during acute viral infection

Hans-Peter Raué and Mark K. Slifka1

Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, Oregon, USA

1 Correspondence: Vaccine and Gene Therapy Institute, Oregon Health and Science University, 505 N.W. 185th Avenue, Beaverton, OR 97006, USA. E-mail: slifkam{at}ohsu.edu

ABSTRACT

Previous studies have shown that T cells, which are genetically deficient in CTLA-4/CD152 expression, will proliferate uncontrollably, resulting in lethal autoimmune disease. This and other evidence indicate that CTLA-4 plays a critical role in the negative regulation of effector T cell function. In contrast to expectations, BrdU incorporation experiments demonstrated that CTLA-4 expression was associated with normal or even enhanced in vivo proliferation of virus-specific CD4+ and CD8+ T cells following acute lymphocytic choriomeningitis virus or vaccinia virus infection. When compared with CTLA-4– T cells directly ex vivo, CTLA-4+ T cells also exhibited normal antiviral effector functions following stimulation with peptide-coated cells, virus-infected cells, plate-bound anti-CD3/anti-CTLA-4, or the cytokines IL-12 and IL-18. Together, this indicates that CTLA-4 does not directly inhibit antiviral T cell expansion or T cell effector functions, at least not under the normal physiological conditions associated with either of these two acute viral infections.

Key Words: CD8 • CD4 • LCMV • vaccinia

INTRODUCTION

Initial T cell activation begins with a primary signal through the TCR interacting with appropriate peptide/MHC molecules on the surface of an APC, in addition to a secondary signal provided by costimulation, typically via CD28. The CD28 molecule elicits proliferative and/or survival signals to the T cell after interacting with B7 proteins (B7-1/CD80 or B7-2/CD86), which are expressed mainly by professional APC [1 , 2 ]. The requirement for CD28-mediated T cell activation in vivo appears to depend on the model being studied; CD28-deficient (CD28–/–) mice mount lower T cell responses against vesicular stomatitis virus [3 4 5 ] and influenza [6 ], and normal to somewhat lower T cell responses are observed with other viruses such as lymphocytic choriomeningitis virus (LCMV) [7 8 9 ]. In contrast to the positive role of CD28 interactions during antigen-specific T cell responses, another CD28 family member, CTLA-4 (CD152), is widely recognized as a down-regulatory molecule whose interactions with B7 molecules result in sharply decreased T cell effector functions, and it is believed to play a critical role in blocking T cell proliferation. This viewpoint is based primarily on the data obtained from genetic deletion of the CTLA-4 gene, which results in lymphoproliferative disease in CTLA-4-deficient mice [10 11 12 ]. Although the current consensus is that CD28 is a strong activating molecule, and CTLA-4 acts as a counter-balancing down-regulatory molecule [1 , 2 , 13 ], this perspective is not accepted universally [14 ]. Early studies suggested that CTLA-4 might act as an activation molecule [15 ], but experiments using anti-CTLA-4 antibodies have varied, resulting in decreased or increased T cell expansion [16 ] or prevention of T cell anergy in some systems [17 ] but not others [18 , 19 ]. Moreover, lack of CTLA-4 expression by CTLA-4-deficient T cells does not absolutely result in unrestrained lymphoproliferation/generalized tissue infiltration, as CTLA-4-deficient T cells expressing transgenic TCR are often "cured" of lymphoproliferative disease [20 , 21 ], as are chimeric mice, which are reconstituted with a combination of CTLA-4-deficient and CTLA-4-competent T cells [22 23 24 25 26 ]. However, this topic remains open to debate, and CTLA-4 is often still considered to be a strictly inhibitory molecule on activated T cells. This subject remains controversial, as the proliferation of CTLA-4+ and CTLA-4– T cells has not been assessed in an unmanipulated, nontransgenic, in vivo model system. Moreover, the direct ex vivo functional attributes (e.g., cytokine kinetics, magnitude, functional avidity, responsiveness to IL-12/IL-18) of polyclonal CTLA-4+ and CTLA-4– T cells have not been compared quantitatively. To clarify the role of CTLA-4 in protection against acute viral infection under physiological conditions, we monitored antiviral T cell functions and proliferation in vivo and directly ex vivo using non-TCR-transgenic polyclonal T cell populations at different stages of T cell activation/memory.

We examined the role of CTLA-4-mediated T cell inhibition following acute infection of mice by two different viruses (LCMV and vaccinia), both of which are known to infect dendritic cells and macrophages, professional APC with the capacity to express the CTLA-4 ligands B7.1 (CD80) and B7.2 (CD86) upon activation. In these studies, we observed a paradox in which CTLA-4 expression peaked during the early stages of LCMV or vaccinia infection when antiviral T cells were undergoing their highest rates of proliferation. These results were verified by BrdU incorporation studies in vivo, demonstrating that CTLA-4+ T cells proliferated equal to or better than CTLA-4– T cells of the same antigen specificity. This was unexpected, as CTLA-4 is most noted for its inhibitory effect on T cell proliferation. Next, we analyzed several immune parameters including the kinetics and magnitude of IFN-{gamma} production following TCR-mediated T cell activation or cytokine-mediated T cell activation, as well as peptide-specific functional avidity profiles in CTLA-4+ and CTLA-4– T cell populations directly ex vivo. CTLA-4 expression was not associated with a deficit in any of these common antiviral effector functions, even when evaluated in CD28–/– mice. Moreover, if CD28–/– mice were treated with CTLA-4-Ig to block endogenous CTLA-4:B7 interactions, these mice mounted lower CD8+ T cell responses and experienced significantly higher viremia. Together, these results indicate that CTLA-4 does not directly inhibit antiviral CD4+ or CD8+ T cell expansion or effector T cell functions associated with controlling acute infection by LCMV or vaccinia virus.

MATERIALS AND METHODS

Mice and virus
BALB/c, C57BL/6, and CD28–/– mice were bred at Oregon Health and Science University (OHSU; Beaverton, OR, USA) or purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Mice (6–16 weeks old) were infected i.p. with 2 x 105 PFU LCMV-Armstrong (Arm-53b) or 2 x 106 PFU vaccinia virus (WR). For BrdU-labeling experiments, mice were injected twice daily with 0.5 mg BrdU (Sigma Chemical Co., St. Louis, MO, USA) in sterile PBS as indicated. All animal protocols were reviewed and approved by the OHSU Institutional Animal Care and Use Committee.

Cytokines, peptides, CTLA-4-Ig
IL-12 was purchased from R&D Systems (Minneapolis, MN, USA), and IL-18 was purchased from Medical and Biological Laboratories (Watertown, MA, USA). HPLC-purified (>95% pure) NP118118–126, GP3333–41, and GP6161–80 were purchased from Alpha Diagnostic International (San Antonio, TX, USA). CTLA-4-Ig (mouse IgG2a chimera) was obtained from Chimerigen Laboratories (Allston, MA, USA). On Day 0, CD28–/– mice were injected with PBS or 100 µg CTLA-4-Ig i.v. plus 100 µg CTLA-4-Ig i.p. prior to LCMV infection. PBS or CTLA-4-Ig (100 µg/dose) was i.p.-administered again on Days 2, 4, and 6 post-LCMV infection. This treatment regimen is similar to previous studies in which CD28/CTLA-4 interactions were blocked effectively in vivo [8 , 27 , 28 ]. Serum was collected to determine viral titers by plaque assay as described [29 ].

BrdU and intracellular cytokine staining (ICCS)
T cells were stimulated by adding peptide or IL-12 + IL-18 directly to the splenocyte cultures, and ICCS was performed as described [30 ]. After stimulation, cells were incubated with Fc block (1 µg/mL, Clone 2.4G2), mouse IgG (100 µg/mL, Sigma Chemical Co.), and in some cases, unconjugated hamster IgG (10 µg/mL, PharMingen, San Diego, CA, USA) for 15 min at 4°C prior to staining for surface and intracellular markers. Nonspecific IFN-{gamma} production after incubation with medium alone (typically <1–2%) was subtracted from the stimulated cultures measured at each time-point to yield the frequency of virus-specific IFN-{gamma}+ T cells. Nonspecific binding of anti-CTLA-4 antibody (UC10-4F10-11, PharMingen) was determined in parallel samples stained with a hamster IgG{kappa} isotype control (anti-TNP, PharMingen). For anti-CD3 stimulation, 96-well flat-bottom plates were coated overnight with graded amounts of anti-CD3{epsilon} (145-2C11, NA/LE, PharMingen), with or without anti-CTLA-4 (UC10-4F10-11, NA/LE, PharMingen) in sterile PBS. Plates were washed with RPMI + 5% FBS and blocked with RPMI + 5% FBS. Splenocytes (106) were incubated in the plates for 6 h at 37°C, and 2 µg/ml Brefeldin A was added for the last hour of the 6 h incubation. To identify vaccinia-specific T cells by IFN-{gamma} ICCS, 106 splenocytes were stimulated with 5 x 105 uninfected or vaccinia-infected A20 cells for 6 h in the presence of Brefeldin A as described previously [31 ]. In these experiments, A20 cells were infected with vaccinia-WR at multiplicity of infection = 1 for 16 h prior to incubation with spleen cells from vaccinia-infected mice. Nonspecific IFN-{gamma} production after incubation with uninfected A20 cells (typically <1–2%) was subtracted from the stimulated cultures measured at each time-point to yield the frequency of vaccinia-specific IFN-{gamma}+ T cells.

To measure virus-specific T cells without stimulating them with peptide antigen, T cells were measured by staining them with anti-CD8 and H-2Ld NP118–126tetramer, provided by the National Institutes of Health (NIH) Tetramer Core Facility (Atlanta, GA, USA). Samples were stained for 1 h on ice prior to being washed, fixed, and stained intracellularly for CTLA-4 as described in the ICCS protocol above. In parallel experiments, samples were stained with anti-CD8, NP118 tetramer, and anti-CTLA-4 to determine surface expression of CTLA-4. Additional samples were stained with anti-CD8, NP118 tetramer, and hamster IgGk isotype control antibody to determine the levels of nonspecific antibody binding.

To visualize BrdU incorporation, cells were surface-stained, washed, fixed with Cytofix/Cytoperm (PharMingen), washed with Permwash, permeabilized with Permwash + 10% DMSO, and washed with Permwash again. The DNA was then digested with DNAse (30 µg/100 µl, Sigma Chemical Co.) at 37°C for 1 h. Cells were washed with Permwash and incubated with anti-BrdU (3D4, Caltag, Carlsbad, CA, USA, or PharMingen), anti-IFN-{gamma} (Caltag), and anti-CTLA-4 (PharMingen). Cells were washed with Permwash and 1% FBS in PBS and resuspended in PBS containing 2% formaldehyde for acquisition on a FACSCalibur analyzed using CellQuest (Becton Dickinson, San Jose, CA, USA) or on an LSR II instrument analyzed using FlowJo software (TreeStar, Ashland, OR, USA).

Statistics
Statistical significance was tested in Microsoft Excel using a two-tailed Student’s t test with unequal variance. P values of ≤0.05 were considered statistically significant.

RESULTS

CTLA-4 expression and T cell effector function in BALB/c mice
These studies were performed in BALB/c mice, wherein the immunodominant LCMV NP118-specific CD8+ T cell response is well-characterized in terms of cytokine expression profiles and functional/structural avidity. CTLA-4 is expressed by antiviral CD8+ T cells during the acute stages of LCMV infection [32 ], and in our initial experiments, we examined the expression of CTLA-4 on virus-specific T cells directly ex vivo or after 6 h of peptide stimulation (Fig. 1 ). To identify LCMV-specific CD8+ T cells in the absence of in vitro stimulation, we used NP118 tetramers (Fig. 1a and 1c) . We found that 40–46% of NP118-specific CD8+ T cells expressed CTLA-4 at 8 days postinfection, but this was mainly intracellular storage of CTLA-4, as <1% of the NP118 tetramer+ T cells expressed detectable levels of CTLA-4 on the cell surface. After 6 h of NP118 peptide stimulation, we found that CTLA-4 was expressed by an average of 69.8% of IFN-{gamma}+ CD8+ T cells (Fig. 1b and 1c) . In contrast to unstimulated NP118 tetramer+ T cells, which do not express CTLA-4 on their surface, we found that ~44% of IFN-{gamma}+ CD8+ T cells had detectable surface CTLA-4 expression after 6 h of in vitro stimulation. These results indicate that just less than half of NP118-specific CD8+ T cells express intracellular CTLA-4 directly ex vivo and that even higher frequencies of CTLA-4+ T cells are observed shortly after peptide stimulation. Moreover, CTLA-4 is up-regulated and presented on the surface of recently activated CD8+ T cells within hours after peptide stimulation.


Figure 1
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  		Figure 1. CTLA-4 expression by virus-specific CD8+ T cells is up-regulated following peptide stimulation. BALB/c mice were infected with LCMV, and antiviral CD8+ T cell responses were examined 8 days later. To identify virus-specific CD8+ T cells directly ex vivo, samples were stained with anti-CD8 and NP118 tetramer. (a) Cells were stained with anti-CTLA-4 or hamster IgG{kappa} (i.e., isotype control) before the intracellular staining protocol to measure surface CTLA-4 or during the intracellular staining protocol to measure total (surface+intracellular) CTLA-4 expression. (b) Virus-specific CD8+ T cells were stimulated with 10–5 M NP118 peptide for 6 h with Brefeldin A added for only the last hour to allow CTLA-4 molecules to be expressed on the cell surface. After stimulation, virus-specific CD8+ T cells were identified by staining for CD8 and IFN-{gamma} in addition to CTLA-4 (or hamster IgG{kappa} isotype control), as described in (a). The dot plots are pre-gated on CD8+ T cells, and the numbers indicate the percentage of NP118 tetramer+ or IFN-{gamma}+ CD8+ T cells in each quadrant and in parentheses, the percentage of NP118 tetramer+ or IFN-{gamma}+ CD8+ T cells that express CTLA-4 or nonspecifically bind the hamster IgG{kappa} isotype control antibody. (c) The bar graph shows the percentage of CD8+ T cells that express CTLA-4 (i.e., total CTLA-4) or surface CTLA-4 directly ex vivo in the absence of stimulation (tetramer+ CD8+ T cells) or after 6 h of peptide stimulation (IFN-{gamma}+ CD8+ T cells). The dashed line indicates the percentage of virus-specific CD8+ T cells that bound hamster IgG{kappa} isotype control antibody. The data show the average ± SD and represent six mice from three independent experiments.

 
In some models, CTLA-4 is believed to have a substantial inhibitory effect on the threshold required for T cell activation [33 34 35 ]. To determine if CTLA-4 expression was associated with changes in the amount of peptide antigen required for T cell activation, we performed functional avidity curves of CTLA-4+ and CTLA-4– CD8+ T cells at 8 days after primary LCMV infection (Fig. 2 ). Intracellular staining for CTLA-4 constitutes the most sensitive method for detection of CTLA-4 protein expression and provides clear segregation between T cell subsets that actively express CTLA-4 directly ex vivo and those that do not express CTLA-4 (Figs. 1 and 2) . In these studies, when we gated on total CTLA-4-expressing CD8+ T cells (i.e., intracellular+surface) and compared them to CTLA-4– T cells (Fig. 2a) , we found no appreciable difference in the threshold of peptide-induced T cell activation. Likewise, when we stained the cells for surface-expressed CTLA-4 (Fig. 2b) , we again found no defect in the functional responsiveness of CTLA-4+ T cells exposed to graded doses of peptide antigen. Together, this indicates that the direct ex vivo functional avidity of CTLA-4+ T cells is equivalent to CTLA-4– T cells following acute LCMV infection.


Figure 2
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  		Figure 2. CTLA-4 is not associated with diminished antiviral T cell functions directly ex vivo. BALB/c mice were infected with LCMV, and antiviral T cell effector functions versus CTLA-4 expression were analyzed directly ex vivo. Functional avidity of virus-specific CD8+ T cells at 8 days after LCMV infection were (a) divided into total (intracellular+surface) CTLA-4+ and CTLA-4– cells or (b) divided into only surface CTLA-4+ and surface CTLA-4– T cell populations. IFN-{gamma} responses are expressed as a percentage of the maximum percent IFN-{gamma}+CD8+ T cell response attained with 10–5 M NP118, and the inset dot plot in each graph (gated on CD8+ T cells) is representative of the total IFN-{gamma}+ peptide-specific T cell response. (c) The kinetics of IFN-{gamma} production by CD8+ T cells were determined at 8 days postinfection following stimulation with 10–7 M NP118 peptide or (d) following stimulation with the cytokines IL-12 and IL-18 (10 ng/mL each). (e) The relative amount of IFN-{gamma} produced by CTLA-4+ and CTLA-4– T cells after stimulation with 10–7 M NP118 or (f) with IL-12 + IL-18 (10 ng/mL each) was determined by the mean fluorescence intensity (MFI) of IFN-{gamma} in CTLA-4+ and CTLA-4– CD8+ T cells at 6 h poststimulation. Brefeldin A was added for only the last hour of incubation to allow for surface CTLA-4 expression. The data show the average ± SD and represent two to six mice per group from one to four independent experiments.

 
To further characterize the functional responsiveness of virus-specific CD8+ T cells that differ in their total CTLA-4 expression, we examined the on-rate kinetics of IFN-{gamma} production following stimulation with the NP118peptide (i.e., TCR-mediated activation; Fig. 2c ) or with innate cytokines IL-12 and IL-18 (i.e., non-TCR-mediated activation; Fig. 2d ). Although peptide-induced IFN-{gamma} production is notably more rapid than cytokine-induced IFN-{gamma} production [30 , 36 ], we found no substantial difference in the kinetics of IFN-{gamma} production by CTLA-4+ or CTLA-4– T cell subsets. Moreover, there was no apparent difference in IFN-{gamma} expression in CTLA-4+ or CTLA-4– T cells following peptide (Fig. 2e) or cytokine-mediated activation (Fig. 2f) . IL-2 production was also examined; ~70% of IL-2+ CD8+ T cells examined at 8 days postinfection express CTLA-4 following direct ex vivo peptide stimulation, and these CTLA-4+ T cells expressed equivalent levels of IL-2 as their CTLA-4-negative counterparts (data not shown).

Although the spleen is a typical lymphoid organ containing physiological numbers of B7.1+ and B7.2+ APC, we were unable to identify a functional defect in CTLA-4-expressing T cells. In an attempt to cross-link the CTLA-4 molecule directly, we used an alternative approach to T cell stimulation by incubating the cells on plates coated with different doses of anti-CD3 antibody (10–0.1 µg/mL) in the absence or presence of anti-CTLA-4 antibody at a concentration shown previously to be inhibitory to naive T cells in vitro (10 µg/mL) [37 ]. Plate-bound anti-CD3 stimulation does not induce IFN-{gamma} production in naïve T cells but triggers antigen-experienced T cells to produce copious amounts of IFN-{gamma} directly ex vivo (Fig. 3 ). We found no significant difference in the percentage of IFN-{gamma}-producing T cells following anti-CD3 stimulation in the presence or absence of anti-CTLA-4 (Fig. 3a) . It is interesting that IFN-{gamma} expression was similar in surface CTLA-4+ and CTLA-4– CD4+ T cells, and IFN-{gamma} production was significantly higher (P=0.01) in CTLA-4+ CD8+ T cells compared with CTLA-4– CD8+ T cells (Fig. 3b) . This result indicates that high cytokine expression was not suppressed measurably by the presence of plate-bound anti-CTLA-4 antibody. CTLA-4 is thought to alter the threshold of T cell activation, but in these studies, we found no functional defect in CD8+ T cells (Fig. 3c) or CD4+ T cells (Fig. 3d) that express CTLA-4 on the cell surface or when total CTLA-4 protein expression (i.e., surface and intracellular) was measured.


Figure 3
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  		Figure 3. Direct ex vivo T cell responsiveness to plate-bound anti-CD3 is unaltered by anti-CTLA-4. At 8 days post-LCMV infection, IFN-{gamma} responses by CD8+ and CD4+ T cells were analyzed after stimulation with plate-bound anti-CD3 antibody in the presence or absence of plate-bound anti-CTLA-4 antibody at 10 µg/mL. (a) The percentage of IFN-{gamma}-producing CD8+ T cells and CD4+ T cells was determined after 6 h of stimulation with 10 µg/mL anti-CD3 in the presence or absence of anti-CTLA-4. (b) After direct ex vivo stimulation with anti-CD3/anti-CTLA-4 (10 µg/mL each), CD8+ and CD4+ T cells were stained for surface expression of CTLA-4, and intracellular IFN-{gamma} levels were determined by flow cytometry. Functional responsiveness to graded doses of anti-CD3 was determined in CD8+ T cells (c) and CD4+ T cells (d) in the presence or absence of anti-CTLA-4 (10 µg/mL). Brefeldin A was added for only the last hour of incubation to allow for surface CTLA-4 expression. The data show the average ± SD of three mice per group and are representative of three experiments.

 
CTLA-4 expression is not associated with diminished in vivo proliferation of LCMV-specific T cells
To examine the role of CTLA-4 expression further during in vivo T cell responses to acute viral infection, we infected C57BL/6 mice (in which virus-specific CD4+ and CD8+ T cell epitopes have been mapped) and monitored CD8+ T cell responses to the LCMV GP33 epitope and CD4+ T cell responses to the LCMV GP61 peptide epitope. CD4+ and CD8+ T cell populations proliferate vigorously during the first week of LCMV infection, and the peak in total splenic T cell numbers occurred 8–10 days postinfection (Fig. 4a ). It has been estimated previously that LCMV-specific CD4+ and CD8+ T cells proliferate at an impressive rate of one cell division every 6–10 h during the early stages of infection [38 , 39 ], and it is striking that this is when we identified the highest levels of CTLA-4 expression (Fig. 4b) . At 5 days postinfection (near the peak of in vivo proliferative responses), we found that an average of 86% of virus-specific CD8+ T cells and 98% of virus-specific CD4+ T cells expressed CTLA-4. These results show that CTLA-4 is expressed by most virus-specific T cells when cell division is at or near a physiological maximum and then declines to expression by only a subset of antiviral T cells at later stages of the immune response, when these cell populations are undergoing fewer rounds of proliferation.


Figure 4
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  		Figure 4. CTLA-4 is not associated with decreased proliferation in virus-specific CD4+ or CD8+ T cells. C57BL/6 mice were infected with LCMV, and splenic T cells were assayed for peptide-specific IFN-{gamma} production after peptide stimulation. The number of peptide-specific IFN-{gamma}+ T cells per spleen (a) and the percentage of IFN-{gamma}+ T cells expressing total CTLA-4 (b) were determined after 6 h of stimulation (Day 0; naive mice). The vertical, dashed line indicates the peak of the antiviral T cell response observed at 8 days postinfection. In vivo proliferation of total CTLA-4+ and CTLA-4– T cells in virus-specific CD8+ (c) and CD4+ T cells (e) was determined by administering BrdU to LCMV-infected mice for the last 24 or 48 h prior to analysis at Day 8 postinfection to determine BrdU incorporation rates (i.e., BrdU pulse). The 0-h time-point represents mice that did not receive BrdU injections. Virus-specific T cells were identified by IFN-{gamma} production following 6 h of peptide stimulation (10–5 M GP33 or 2x10–5 M GP61 for stimulation of CD8+ or CD4+ T cells, respectively) directly ex vivo. As an alternative approach to determining BrdU incorporation rates, we examined the rates of BrdU loss in proliferating T cells in "chase" experiments. In these studies, mice received daily BrdU injections from Days 0 to 8 (i.e., 0 h chase), Days 0 to 7 (i.e., 24 h chase), or from Days 0 to 6 postinfection (i.e., 48 h chase), followed by analysis of BrdU+ CD8+ T cells (d) or BrdU+ CD4+ T cells (f) on Day 8 postinfection. Brefeldin A was maintained throughout the in vitro culture period to allow accurate T cell quantitation, regardless of CTLA-4 function, and the data show the average ± SD and represent two to five mice per group from two or more independent experiments.

 
To verify that CTLA-4+ cells exhibit normal proliferative responses in vivo, we performed BrdU incorporation studies. Mice were infected with LCMV and BrdU was administered for 24 or 48 h prior to analysis at Day 8 postinfection. As shown in Figure 4c , GP33-specific CD8+ T cells showed substantial proliferation, and more than half of CTLA-4+ or CTLA-4– T cells incorporated BrdU into their DNA following a 24-h BrdU pulse. These T cell subsets exhibited similar BrdU incorporation rates following a longer 48-h BrdU pulse as well, indicating that CTLA-4 expression had no correlation with an inhibited ability to proliferate during the acute stages of an antiviral T cell response. An alternative explanation for these results is that the CTLA-4+ T cells may have actually been CTLA-4– during their proliferation but became CTLA-4+ (and theoretically stopped proliferating) immediately prior to direct ex vivo analysis. To test this hypothesis, we performed in vivo BrdU chase experiments in which we quantitated BrdU levels in virus-specific T cells at 8 days postinfection in mice, wherein BrdU was administered from 0 to 8 days (0 h chase), 0 to 7 days (24 h chase), or 0 to 6 days (48 h chase). If CTLA-4 is only expressed at the point in which T cells are no longer capable of proliferating, then we should observe more BrdU+ T cells in the CTLA-4+ subset, as these cells would no longer be proliferating and capable of diluting out the amount of BrdU incorporated into their DNA. Instead, CTLA-4+ T cells lost BrdU at the same rate as CTLA-4– T cells (Fig. 4d) , indicating that virus-specific CD8+ T cells that express CTLA-4 in vivo behave normally in terms of their proliferative capacity. It is believed that the inhibitory effects of CTLA-4 are more apparent with CD4+ T cells than with CD8+ T cells, so in parallel studies, we determined BrdU incorporation in pulse experiments (Fig. 4e) and BrdU loss in chase experiments (Fig. 4f) in GP61-specific CD4+ T cells. Similar to the results observed in antiviral CD8+ T cells, CTLA-4 expression did not result in any intrinsic defect in proliferation by antiviral CD4+ T cells, a result that was also confirmed at other time-points (data not shown).

Role of CTLA-4 during antiviral immune responses in the absence of CD28
One explanation for the lack of in vivo inhibition of virus-specific CTLA-4+ T cells is that coexpression of CD28 may result in a dominant phenotype in which activation through the CD28 molecule over-rides the inhibitory effects that would be triggered through the CTLA-4 pathway [2 , 34 ]. Previous studies have shown that CD28 is expressed constitutively on virus-specific CD8+ T cells following acute LCMV infection [8 , 40 ]. Therefore, to determine if CTLA-4 expression would lead to inhibition of the T cell response in the absence of CD28, we examined antiviral T cell responses in CD28-deficient (CD28–/–) mice (Fig. 5 ). At 8 days post-LCMV infection, wild-type C57BL/6 mice and CD28–/– mice showed comparable amounts of CTLA-4 expression in GP33-specific CD8+ T cells (53% CTLA-4+ vs. 40% CTLA-4+, respectively) and GP61-specific CD4+ T cell populations (77% CTLA-4+ vs. 60% CTLA-4+, respectively; data not shown). It is interesting that we observed a small but significant increase in proliferation by CTLA-4+ CD8+ T cells and CTLA-4+ CD4+ T cells compared with their CTLA-4– counterparts (Fig. 5a and 5b) . To help decipher the biological significance of CTLA-4 expression during acute LCMV infection, we determined whether loss of signaling through this pathway would have an impact on antiviral T cell responses. Administration of antibodies against CTLA-4 may lead to increased T cell responses in vivo, but it is not entirely clear if this is a result of a blocking effect or possibly through activation of CTLA-4 via cross-linking [14 ]. To circumvent this issue, we chose to block CTLA-4 ligation indirectly in vivo by administering CTLA-4-Ig. In normal mice, administration of CTLA-4-Ig results in the inhibition of CD28 as well as CTLA-4, thus making it difficult to determine the independent roles of these two closely related molecules. However in CD28–/– mice, the observed effects on antiviral T cell numbers or function would likely be a result of blockade of signaling through CTLA-4 or effects on APC functions via B7 [41 ]. Following CTLA-4-Ig injections at 0, 2, 4, and 6 days postinfection, we found that antiviral CD8+ T cell numbers in the spleen were significantly lower (Fig. 5c , P=0.002), whereas there was no effect on CD4+ T cells (Fig. 5d , P=0.2) with four of five mice demonstrating CD4+ T cell responses comparable with untreated CD28–/– controls. Although these reductions in T cell numbers were modest, administration of CTLA-4-Ig resulted in a substantial delay in viral clearance; CD28–/– mice cleared LCMV effectively from the serum by 8 days postinfection (zero of five viremic mice), whereas CD28–/– mice treated with CTLA-4-Ig still maintained infectious virus in their circulation (five of five viremic mice; Fig. 5e ). CTLA-4-Ig-treated CD28–/– mice also had higher levels of serum viremia at Day 6 postinfection (data not shown). Together, these results confirm and extend the results observed in normal mice in which CTLA-4+ T cells proliferate equal to or better than CTLA-4– T cells. Moreover, these studies rule out other confounding factors such as a required role for B7 expression on activated T cells to cause CTLA-4-mediated down-regulation [42 ] or compensatory CD28-mediated effects, which might influence the proliferative capacity of CTLA-4+ T cells in vivo.


Figure 5
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  		Figure 5. CTLA-4 plays a small but positive role in virus-specific T cell activation in CD28–/– mice, which were infected with LCMV, and antiviral T cell responses were analyzed by peptide-induced IFN-{gamma} ICCS at 8 days postinfection. Proliferation of CTLA-4+ and CTLA-4– T cells was determined by injecting mice with BrdU from Days 7 to 8 postinfection (i.e., 24 h pulse) and determining the percentage of virus-specific CD8+ T cells (a) and CD4+ T cells (b) that incorporated BrdU in vivo. To determine the role of CTLA-4 expression in T cell responses in CD28–/– mice, animals were injected with PBS (negative control) or CTLA-4-Ig to block interactions with B7 proteins in vivo. The total number of splenic, GP33-specific CD8+ T cells (c) and GP61-specific CD4+ T cells (d) at 8 days postinfection was compared among C57BL/6 (CD28+/+), CD28–/–, and CD28–/– mice given CTLA-4-Ig in vivo. (e) LCMV serum viremia in CD28–/– mice, with or without CTLA-4-Ig treatment, was determined by plaque assay (limit of detection: 50 PFU/mL) at 8 days postinfection. Brefeldin A was maintained throughout the in vitro culture period to allow accurate T cell quantitation regardless of CTLA-4 function, and the data show the average ± SD and represent four to six mice per group from two to three independent experiments.

 
In vivo CTLA-4 expression is associated with increased proliferation after vaccinia infection
To determine if the observation of normal or even enhanced T cell proliferation/effector function in CTLA-4+ T cells was unique to the LCMV model system or perhaps common to other acute viral infections as well, we examined the expression of CTLA-4 in virus-specific T cell populations induced during acute vaccinia infection (Fig. 6 ). Vaccinia-infected BALB/c mice were injected with BrdU during the last 24 h prior to analysis of antiviral T cell responses, and the rates of proliferation in CTLA-4– and CTLA-4+ T cells were determined. Vaccinia-specific T cells were identified by measuring intracellular IFN-{gamma} production following 6 h of stimulation with vaccinia-infected A20 cells as described previously [31 ]. BrdU incorporation was visualized by gating first on CD8+ T cells (Fig. 6a 6b 6c) or CD4+ T cells (Fig. 6d 6e 6f) with BrdU+ T cells colored green and BrdU– T cells colored red (Fig. 6a) . Two-color dot plots were prepared to show the relationship among IFN-{gamma} production (y-axis), CTLA-4 expression (x-axis), and recent proliferation (red vs. green events) at Day 6 (Fig. 6b and 6e) or Day 8 (Fig. 6c and 6f) after vaccinia infection. Similar to the LCMV model system, CTLA-4+ T cells expressed IFN-{gamma} as effectively as CTLA-4– T cells (Fig. 6 and data not shown), and the highest proportion of CTLA-4+ T cells was observed at the earliest time-points examined, and up to 80–90% of virus-specific T cells expressed CTLA-4 at Day 6 postinfection (Fig. 6g) . It is interesting that vaccinia-specific CTLA-4+ CD8+ T cells demonstrated significantly more proliferation than CTLA-4– CD8+ T cells at Day 8 (P=0.001) and Day 10 (P<0.001) postinfection (Fig. 6h) , whereas vaccinia-specific CTLA-4+ CD4+ T cells proliferated significantly more than their CTLA-4– counterparts at all time-points tested (P=0.01, Fig. 6i ). This shows that during vaccinia infection, CTLA-4 is expressed by the majority of T cells during the periods of highest proliferation, and as the infection is cleared, and proliferation subsides, the CTLA-4+ T cells continue to proliferate the most.


Figure 6
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  		Figure 6. Association between CTLA-4 expression and enhanced vaccinia-specific T cell proliferation. BALB/c mice were infected with vaccinia, and antiviral T cell responses were quantitated by IFN-{gamma} ICCS. Representative dot plots showing BrdU incorporation by CD8+ (a) and CD4+ T cells (d) at 6 days postinfection are colored according to whether they had incorporated BrdU in vivo during the previous 24 h (BrdU+=green; BrdU–=red). After gating on CD8+ T cells at 6 days (b) or 8 days postinfection (c) or gating on CD4+ T cells at 6 days (e) or 8 days postinfection (f), the proportion of IFN-{gamma}+ (i.e., vaccinia-specific; y-axis) T cells that express total CTLA-4 (x-axis) is shown, and the numbers indicate the percentage of T cells in each quadrant. To distinguish between IFN-{gamma}+ (vaccinia-specific) T cells, which had proliferated within the last 24 h, the events are colored green (proliferating BrdU+ T cells) or red (nonproliferating BrdU– T cells). (g) The percentage of vaccinia virus-specific (VV) CD4+ or CD8+ T cells that express total CTLA-4 was determined at 6, 8, and 10 days postinfection. The percentage of CTLA-4+ and CTLA-4– T cells that proliferated in the previous 24-h period (i.e., BrdU+) was determined for vaccinia-specific CD8+ T cells (h) and for vaccinia-specific CD4+ T cells (i) at 6, 8, and 10 days postinfection. Brefeldin A was maintained throughout the in vitro culture period to allow accurate T cell quantitation, regardless of CTLA-4 function, and the data show the average ± SD and represent four animals per group from two independent experiments.

 
DISCUSSION

In this study, we examined the association between CTLA-4 protein expression and antiviral T cell effector functions in the context of two distinct in vivo model systems: LCMV and vaccinia. To our knowledge, this is the first time that CTLA-4+ and CTLA-4– T cells from nontransgenic animals have been compared directly in terms of their responsiveness to non-TCR-mediated stimulation by IL-12 + IL-18, threshold of TCR-mediated T cell activation/functional avidity, T cell activation and in vivo proliferative responses in CD28-deficient mice, and cytokine production and proliferative potential following acute vaccinia virus infection. Although CTLA-4 is considered a potent inhibitor of T cell activation, we found that CTLA-4+ T cells induced by LCMV or vaccinia virus infection proliferated as well as CTLA-4– T cells in vivo. These results indicate that CTLA-4 may not be a universally active, down-regulatory molecule, at least in the context of either of the two acute viral infection models examined here.

Several studies have shown that CTLA-4 represses T cell responses, and the most often cited example is the dramatic lymphoproliferative disease observed in CTLA-4-deficient mice [10 11 12 ]. However, it is difficult to rule out the possibility that the lethal disease observed in these mice could be an artifact resulting from complete genetic deletion of CTLA-4 expression during all stages of T cell development and differentiation [14 ]. Although some studies find no problems with thymic selection [43 ], others have found alterations in thymic selection in the presence of anti-CTLA-4 antibodies [44 , 45 ]. However, altered negative selection may not be the only potential mechanism for CTLA-4-deficient, lymphoproliferative disease; other studies have demonstrated that adoptive transfer of CTLA-4-competent cells is able to regulate the normally uncontrollable proliferation of CTLA-4-deficient T cells in trans following adoptive transfer [23 24 25 26 ]. Although the cell type involved in this regulatory process has not been determined formally, it is thought that CTLA-4+ T cells are the most likely candidates. The use of antibodies against CTLA-4 have also added to the controversy; in some experimental models, administration of these antibodies results in decreased or increased T cell responses or have no effect [16 17 18 19 , 46 47 48 ]. It is thought that these antibodies work by blocking CTLA-4, as Fab fragments of anti-CTLA-4 antibodies are not expected to cross-link this receptor, and yet, they still augment T cell proliferation [16 ]. However, others [14 ] have pointed to other non-CTLA-4-related studies in which Fab antibodies can, in some instances, induce signaling events [49 , 50 ]. It is interesting that experiments using a bispecific single chain antibody against human CTLA-4 have shown that effective T cell activation can be triggered without cross-linking multiple CTLA-4 molecules [51 ]. In our in vivo proliferation models, CTLA-4+ and CTLA-4– T cell subsets behaved similarly under the various immunological tests used. At early stages of infection, when antiviral T cells are dividing at the high rates of once every 6–10 h [38 , 39 ], 80–90% of virus-specific CD8+ T cells and ≥95% of virus-specific CD4+ T cells are CTLA-4+ (Fig. 4) , and yet their proliferative rates within lymphoid tissue (with the highest in vivo likelihood of CD80/CD86 interactions) are at or near maximum levels. During this period, CTLA-4+ T cell proliferation appeared equal to that observed in CTLA-4– T cells (Fig. 4) . In contrast, if CTLA-4 expression was to result in intrinsic down-regulation of CTLA-4+ T cells or extrinsic regulation of neighboring CTLA-4– T cells, then these early time-points after acute viral infection should have been marked by much slower proliferation rates instead of the rapid proliferation that is common at early stages of microbial infection. Instead, CTLA-4 expression is highest during the periods with the most rapid T cell proliferation and is expressed at the lowest levels during homeostatic proliferation after the acute infection has been cleared (Figs. 4b and Fig. 6g ). It is interesting that another normally inhibitory molecule, PD-1, was shown to play a significant role in chronic LCMV Clone 13 infection, whereas it showed little or no role in regulating CD8+ T cell responses following acute LCMV Armstrong infection [19 ]. This suggests that down-regulatory molecules may play different roles depending on the virulence and pathogenesis of the virus under study. In terms of vaccinia, CTLA-4 expression was associated with increased proliferative potential in CD8+ and CD4+ T cells (Fig. 6) . It is unclear why CTLA-4+ T cells proliferate at substantially higher rates than CTLA-4– T cells after vaccinia infection, but this may be a result of differences in cell tropism of vaccinia in mice or other yet unknown characteristics of the antiviral T cell response to this virus. Further studies are necessary to determine whether this is unique to vaccinia or whether similar observations will be made in other acute infection models.

In contrast to human T cells, murine T cells constitutively express CD28 [8 , 32 ], which may interfere with the regulatory capacity of coexpressed CTLA-4. To address this issue, we examined antiviral T cell responses in CD28–/– mice (Fig. 5) . Similar to wild-type mice, we found CTLA-4+ T cells proliferated at essentially the same rates as CTLA-4– T cells following acute LCMV infection in the absence of any potential confounding regulation through the CD28 molecule. Moreover, administration of CTLA-4-Ig resulted in significantly reduced antiviral CD8+ T cell numbers (P=0.002) and increased virus titers in the serum (Fig. 5e) . One potential caveat is that CTLA-4-Ig may not only block CTLA-4 interactions by T cells but may also cause reverse signaling to trigger APC activation [41 ] and the induction of APC-derived IFN-{gamma} as well as indoleamine 2,3-dioxygenase (IDO), which catabolizes tryptophan and inhibits T cell function [41 ]. However, a recent and compelling study [25 ] used 1-methyl-DL-tryptophan at doses shown previously to effectively block IDO in vivo [41 ] and found little difference in CTLA-4+ or CTLA-4– T cell expansion following acute LCMV infection, indicating that IDO does not play a major role in modulating antiviral T cell responses in this model system. Moreover, if APC activation and innate IFN-{gamma} responses were triggered by in vivo administration of CTLA-4-Ig, then one would have expected lower levels of virus replication in CTLA-4-Ig-treated animals as a result of the antiviral effects of IFNs. Instead, we observed higher levels of virus replication after administration of CTLA-4-Ig (Fig. 5e) . CD8+ T cells play a critical role in controlling acute LCMV infection, and the decrease in antiviral CD8+ T cell numbers following CTLA-4-Ig injections likely contributed to prolonged viremia. These results, coupled with other studies demonstrating little or no role for IDO in these antiviral immune responses [25 ], suggest that CTLA-4-Ig may have decreased CD28–/– CD8+ T cell responses via direct blockade of an activation signal through CTLA-4.

The role of CD28/CTLA-4-mediated signaling events in the regulation of antigen-specific T cell responses is more complex than previously believed. It is possible that in certain model systems, CD28 plays a strictly costimulatory role, and CTLA-4 plays a strictly coinhibitory role. Alternatively, it is plausible that these molecules play similar or complementary roles in T cell activation and/or that the outcome of these signaling events is regulated by the differentiation state of the T cell being studied. Based on the results of these in vivo experiments, CTLA-4 does not appear to be associated with negative effects on T cell activation or proliferation following LCMV or vaccinia virus infections, and future studies will be needed to delineate the roles of the CD28/CTLA-4 pathways under specific immunological conditions and other infectious/autoimmune disease states.

ACKNOWLEDGEMENTS

This work was supported by NIH grants AI054458 and AI051346 and Oregon National Primate Research Center grant RR00163. The NIH Tetramer Core Facility (Atlanta, GA, USA) generously provided NP118/H-2Ld tetramer reagents.

Received August 28, 2006; revised November 29, 2006; accepted November 30, 2006.

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