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. 2009 Sep;128(1 Pt 2):e471–e478. doi: 10.1111/j.1365-2567.2008.03001.x

Cytotoxic T-lymphocyte antigen 4 blockade augments the T-cell response primed by attenuated Listeria monocytogenes resulting in more rapid clearance of virulent bacterial challenge

Jared H Rowe 1, Tanner M Johanns 1, James M Ertelt 1, Joseph C Lai 1, Sing Sing Way 1
PMCID: PMC2753913  PMID: 19191906

Abstract

Cytotoxic T-lymphocyte antigen 4 (CTLA-4) uniformly suppresses antigen-specific T cells during chronic infection with bacterial, parasitic or viral pathogens. However, the importance of CTLA-4 in controlling the T-cell response during acute infection or after priming with live attenuated vaccine vectors has not been well characterized. Since strategies aimed at blocking CTLA-4 are being actively developed to therapeutically augment T-cell-mediated immunity, the effects of CTLA-4 blockade on T-cell activation during these conditions need to be more clearly defined. We have examined the role of CTLA-4 in a prime-challenge model of acute bacterial infection using both attenuated and virulent strains of the intracellular bacterium Listeria monocytogenes. Although Foxp3+ CD4+ T cells are the predominant CTLA-4-expressing cell type in naïve mice, antigen-specific Foxp3− CD4+ cells upregulate CTLA-4 expression after primary L. monocytogenes infection. Blockade of CTLA-4 results in increased numbers of L. monocytogenes-specific CD4 and CD8 T cells after primary infection with attenuated L. monocytogenes, and confers more rapid bacterial clearance after secondary challenge with virulent L. monocytogenes. Accordingly, CTLA-4 plays an important suppressive role in T-cell priming and protective immunity in a prime-challenge model of acute bacterial infection.

Keywords: bacteria, costimulation, infection, T cell, vaccine

Introduction

The mammalian immune system contains multiple intricately regulated networks of opposing stimulatory and inhibitory signals that allow protective antigen-specific responses to be rapidly generated and mobilized during infection, and quickly silenced once infection resolves to minimize immune-mediated injury to the host. For example T-cell activation is controlled in part by a pair of homologous receptors, CD28 and cytotoxic T-lymphocyte antigen 4 (CTLA-4; CD152), that play stimulatory and suppressive roles, respectively.1 The CTLA-4-mediated suppressive effects on pathogen-specific T-cell activation have been experimentally demonstrated primarily with in vivo models of chronic bacterial, viral and parasitic infection using monoclonal antibodies that specifically block CTLA-4.2–10 CTLA-4 blockade in each of these experimental models of chronic infection uniformly augments the pathogen-specific T-cell response magnitude, and in most cases is associated with reductions in pathogen burden. Accordingly, in vivo CTLA-4 blockade is actively being evaluated as a therapy for chronic infection and in other settings where increased antigen-specific T-cell immunity is desired.11–13 Especially intriguing is the direct correlation between increased CTLA-4 expression on virus-specific T cells and disease progression during ongoing human immunodeficiency virus (HIV) infection.14 These results suggest that CTLA-4 blockade may therapeutically bolster T-cell immunity against HIV. Paradoxically, however, after simian immunodeficiency virus infection in non-human primates, CTLA-4 blockade caused increases in both T-cell activation and viral replication.15 Similarly, in murine models of Plasmodium infection, the augmented T-cell response attributable to CTLA-4 blockade leads to increased immune-mediated pathology.4,5 Therefore the effects of CTLA-4 blockade on infection outcome are context dependent and vary with each specific infection condition. Interestingly, and in striking contrast to the uniformly described CTLA-4-mediated suppressive effects on T-cell activation, a recent study revealed that CTLA-4 blockade during acute infection with lymphocytic choriomeningitis virus (LCMV) in some settings can dampen the virus-specific T-cell response, resulting in increased circulating levels of virus within the first 8 days after infection.16 Whether these apparent T-cell stimulation roles for CTLA-4 are specific to the experimental conditions of acute LCMV infection or more broadly reflect a T-cell stimulatory role for CTLA-4 during infection with pathogens that primarily cause acute infection is unclear.

Listeria monocytogenes is an intracellular Gram-positive bacterium that primarily causes acute localized infections in the gastrointestinal tract in immune-competent individuals, and more severe systemic infections in immune-compromised individuals. Infection with L. monocytogenes primes a robust antigen-specific CD8+ and CD4+ T-cell response, and represents a widely used infection model whereby antigen-specific T-cell priming, and the protective effects of these T cells, can be evaluated.17 Moreover, the existence of highly attenuated yet immunogenic mutant L. monocytogenes make recombinant strains expressing heterologous antigens a promising class of live attenuated vaccine vectors.18–21 Accordingly, in this study, the role of CTLA-4 in T-cell priming and immunity was investigated using a prime-challenge model of L. monocytogenes infection. We first characterized the kinetics whereby CTLA-4 is expressed on T cells after primary infection with the attenuated L. monocytogenesΔactA mutant that triggers a robust L. monocytogenes-specific T-cell response, but does not cause a productive infection because of defects in both intracellular and intercellular spread.22 This mutant is rapidly cleared after infection even in neonatal mice or adult mice with targeted defects in critical components of innate host defence.23–25 We then determined the effects of CTLA-4 blockade on antigen-specific T-cell response magnitude after primary L. monocytogenesΔactA infection, and the kinetics whereby virulent L. monocytogenes is cleared after secondary challenge.

Materials and methods

Mice

C57BL/6 (H-2b) female mice were purchased from the National Cancer Institute and used at 6–8 weeks of age. OT-II T-cell receptor transgenic mice were intercrossed with CD90.1 mice and maintained on a RAG-1-deficient background.26 All experiments were performed under University of Minnesota IACUC approved protocols.

Bacterial infections

Listeria monocytogenes ovalbumin (Lm-OVA), and Lm-OVA ΔactA derived from Lm-OVA through targeted disruption of the actA gene were used allowing for the immune response to the surrogate L. monocytogenes-specific antigens H-2Kb OVA257–264 and I-Ab OVA323–339 to be more precisely characterized.24,26 For infections, L. monocytogenes was grown to early log phase (optical density at 600 nm 0·1) in brain–heart infusion medium at 37°, washed, and diluted with saline to 200 μl and injected intravenously. At the indicated time-points after infection, the numbers of recoverable L. monocytogenes colony-forming units (CFUs) in the spleens of infected mice were quantified by homogenization in saline containing Triton-X (0·05%), and plating serial dilutions of the organ homogenate on brain–heart infusion agar plates.24,27

Reagents, in vitro cultures, cell staining and adoptive transfer

For in vivo CTLA-4 blockade, purified hamster anti-mouse CD152 (CTLA-4; clone UC10-4F10) immunoglobulin G (IgG) or hamster IgG control antibodies were purchased from BioXCell (West Lebanon, NH), and injected intraperitoneally in the following manner: 1 day before primary infection (500 μg per mouse), days 4 and 8 after primary infection and day 1 before rechallenge (250 μg per mouse). For in vivo T-cell depletion, 500 μg anti-mouse CD8 (clone 2.43) was inoculated intraperitoneally 1 day before virulent L. monocytogenes challenge.28 Antibodies and other reagents for cell surface and intracellular staining were purchased from BD Biosciences (San Jose, CA) or eBioscience (San Diego, CA). For in vitro culture, splenocytes were plated in 96-well round-bottom plates (5 × 106 cells/ml), and stimulated with the indicated peptides (10−6 m) with Brefeldin-A (BD GolgiPlug reagent) for 5 hr as described previously.24,27 For adoptive transfer, 105 CD4+ T cells from OT-II (CD90.1+) were transferred intravenously into recipient (CD90.2+) mice 1 day before Lm-OVA infection.

Statistics

The differences in number and percentage T cells, and geometric mean CFUs between groups of mice were evaluated using the Student’s t-test with P < 0·05 taken as statistically significant (graphpad, prism software, La Jolla, CA).

Results

CTLA-4 expression by CD4+ T cells after L. monocytogenesΔactA infection

Our initial studies examined the level and kinetics whereby CTLA-4 is expressed after primary L. monocytogenesΔactA infection. For both CD4+ and CD8+ T cells, increased levels of CTLA-4 were detected by staining cells before and after permeabilization compared with cell surface staining alone, and are in agreement with other studies reporting maximal CTLA-4 detection after intracellular staining.16,29 Using this technique, we found dramatically lower levels of CTLA-4 expression by CD8+ compared with CD4+ T cells before and at each time-point after infection (Fig. 1a,b). In naïve mice, CTLA-4 expression by ∼ 7% of all CD4 T cells was consistent with the expected percentage (5–10%) of Foxp3+ regulatory T cells known to constitutively express CTLA-4.29–31 After L. monocytogenesΔactA infection, an increased percentage of CD4+ T cells expressed CTLA-4, reaching maximal levels on day 5 when an approximately twofold increased percentage of CTLA-4+ cells among all CD4+ T cells was recorded (16% compared with 7%) (Fig. 1b). This increased percentage of CTLA-4+ cells among CD4+ T cells also reflected an increase in absolute numbers of CTLA-4+ CD4+ cells among splenocytes reaching maximal levels on day 5 after infection (Fig. 1c). Comparatively, neither the percentage nor the total number of CTLA-4+ CD8+ T cells changed significantly from baseline levels during the same time period after infection. These results indicate that within the first week after L. monocytogenesΔactA infection, CTLA-4 expression by CD4+ T cells was dynamically regulated.

Figure 1.

Figure 1

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Cytotoxic T-lymphocyte antigen 4 (CTLA-4) expression among CD8+ T cells (a) and CD4+ T cells (b) at the indicated time-points after infection with 106Listeria monocytogenes ovalbumin (Lm-OVA) ΔactA. CTLA-4 expression was measured by staining with anti-CTLA-4 antibody (open histograms) or isotype control antibody (shaded histograms) either before cell permeabilization (surface), or staining before and after permeabilization (surface + intracellular). The numbers indicate the percentage of CTLA-4+ cells compared with isotype control antibody. (c) Absolute number of CTLA-4+ CD8 and CD4 T cells among splenocytes at the indicated time-points after Lm-OVA ΔactA infection. These data are representative of four to six mice per time-point from two independent experiments with similar results. Bar indicates standard error.

To determine if increased CTLA-4 expression among CD4+ cells after L. monocytogenesΔactA infection reflects the expansion of Foxp3+ CTLA-4+ cells or induced CTLA-4 expression by Foxp3− CD4+ cells, we examined CTLA-4 expression among Foxp3+ and Foxp3− cells (Fig. 2a). These studies revealed that most CTLA-4+ CD4 cells were Foxp3+ before infection, and the percentage of this cell population did not change significantly after infection. In contrast, CTLA-4 expression among Foxp3− CD4+ T cells compared with naïve mice was increased at each time-point after infection, reaching maximal levels at day 5 (Fig. 2a). Consequently, while Foxp3+ CD25+ CD4+ cells comprise the majority of CTLA-4+ cells before infection, Foxp3− CD25− CD4+ T cells upregulate CTLA-4 expression in response to L. monocytogenesΔactA infection.

Figure 2.

Figure 2

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(a) Cytotoxic T-lymphocyte antigen 4 (CTLA-4) expression determined by both cell surface and intracellular staining compared with Foxp3 expression among CD4+ T cells at the indicated time-points after infection with 106Listeria monocytogenes ovalbumin (Lm-OVA) ΔactA. The numbers indicate the percentage of CD4+ cells in each gate and are representative of four mice per time-point from two independent experiments. (b) Histogram plots indicating CTLA-4 expression among OT-II CD90.1+ OVA323–339-specific CD4+ T cells at the indicated time-points after infection. CTLA-4 expression was measured by staining with anti-CTLA-4 antibody (open histograms) or isotype control antibody (shaded histograms) either before cell permeabilization (surface), or staining before and after permeabilization (surface + intracellular). The numbers indicate the percentage CTLA-4+ cells compared with isotype control antibody, and is representative of four to six mice per time-point from two independent experiments with similar results.

To more precisely characterize the level and kinetics whereby CTLA-4 is expressed by CD4+ T cells after infection, we examined CTLA-4 expression among congenically marked (CD90.1+) antigen-specific CD4+ T cells from T-cell receptor transgenic (OT-II) mice after adoptive transfer and at various time-points after Lm-OVA ΔactA infection. CD4 T cells from OT-II mice have specificity to the OVA323–339 class II peptide contained within Lm-OVA ΔactA used in these experiments. One day before infection, 105 purified CD4+ T cells from OT-II mice (CD90.1+) were transferred intravenously into naïve recipient B6 (CD90.2+) mice, and tracked ex vivo by staining for CD90.1+ CD4+ cells. After Lm-OVA ΔactA infection, CTLA-4 levels increased significantly on these adoptively transferred OVA323–339-specific CD4+ T cells, and with similar kinetics to those by which L. monocytogenes infection induces CTLA-4 expression on all CD4 T cells, reaching maximal levels on day 5 after infection (Fig. 2b). These results demonstrate that CTLA-4 expression is dynamically regulated by antigen-specific CD4+ T cells during the first week after primary L. monocytogenes infection. Moreover by eliminating the potentially self-reactive components of adaptive immunity through intercrossing these OT-II mice with RAG1−/− mice, > 99% of these OVA323–339-specific (CD90.1+) CD4+ T cells were Foxp3−, both directly ex vivo before adoptive transfer and at each time-point within the first week after infection. Together these results demonstrate both constitutive CTLA-4 expression by Foxp3+ CD4 cells and induced CTLA-4 expression among antigen-specific Foxp3− CD4 cells after primary L. monocytogenes infection.

CTLA-4 blockade increases the antigen-specific T-cell response magnitude

CTLA-4 in experimental models of chronic bacterial, parasitic and viral infection suppresses the antigen-specific T-cell response because CTLA-4 blockade uniformly augments the antigen-specific T-cell response magnitude.2–10 In contrast, CTLA-4 blockade during defined conditions after acute LCMV infection inhibits virus-specific T-cell expansion, resulting in increased levels of circulating virus.16 To evaluate whether these discordant roles for CTLA-4 are caused by inherent differences in the costimulation signals required for optimal T-cell activation after acute and chronic infection, we examined the effects of CTLA-4 blockade on the antigen-specific T-cell response after primary L. monocytogenesΔactA infection. For these experiments, mice were treated with either anti-mouse CTLA-4 monoclonal antibody (UC10-4F10) used previously by other investigators for in vivo CTLA-4 blockade or hamster IgG isotype control antibody beginning 1 day before infection and throughout the experiment.2–10 Eight days after primary L. monocytogenesΔactA infection, mice treated with CTLA-4 blocking antibody compared with control antibody had a ∼ 2·5-fold increased percentage (P < 0·05) and total numbers of both antigen-specific CD8+ and CD4+ T cells enumerated by intracellular cytokine staining after stimulation with the Lm-OVA-specific major histocompatibility complex class I OVA257–264 or class II listeriolysin-O (LLO) 189–201 peptides, respectively (Fig. 3a). Similarly, by day 30 after infection, and despite a significantly reduced magnitude response compared with day 8, increased percentage and total numbers of antigen-specific T cells were maintained for mice treated with CTLA-4 blocking antibody compared with control antibody-treated mice (Fig. 3b). These results demonstrate that CTLA-4 blockade increases the antigen-specific T-cell response magnitude at both early (day 8) and memory (day 30) time-points after primary L. monocytogenesΔactA infection.

Figure 3.

Figure 3

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(a) Percentage and total number of interferon-γ (IFN-γ) producing CD8+ (top) and CD4+ (bottom) T cells among splenocytes from mice treated with hamster isotype control immunoglobulin G (h-IgG) or cytotoxic T-lymphocyte antigen 4 (CTLA-4) blocking antibodies on day 8 after infection with 106Listeria monocytogenes ovalbumin (Lm-OVA) ΔactA and stimulation with OVA257–264 peptide [major histocompatibility complex (MHC) class I], lipid-linked oligosaccharide 189–201 peptide (LLO189–201 MHC class II), or no stimulation. (b) Percentage and total number of IFN-γ-producing CD8+ (top) and CD4+ (bottom) T cells among splenocytes from hamster isotype control (h-IgG) or CTLA-4 blocking antibody-treated mice on day 30 after infection with 106 Lm-OVA ΔactA and stimulation with OVA257–264 peptide (MHC class I), LLO189–201 peptide (MHC class II), or no stimulation. These data are representative of six mice per experimental group from two independent experiments with similar results. Bar represents standard error. *P < 0·05.

Since L. monocytogenes antigen load, as measured by the number of recoverable CFUs 24 hr after infection, directly correlates with the L. monocytogenes-specific T-cell response magnitude,32 we evaluated the possibility that the increased magnitude of the antigen-specific T-cell response attributable to CTLA-4 blockade could result from differences in L. monocytogenesΔactA antigen load at early time-points after infection between mice treated with CTLA-4 blocking antibody and control antibody-treated mice. Consistent with our previous results characterizing the kinetics of L. monocytogenesΔactA clearance in other mice,24,27 only ∼ 10% of the initial L. monocytogenesΔactA inoculum was recovered after 24 hr, and the infection was completely cleared by 72 hr for most mice treated with either CTLA-4 blocking antibody or control antibody (Fig. 4). Importantly no significant differences in bacterial counts between mice treated with CTLA-4 blocking antibody and those given isotype control antibody were detected after L. monocytogenesΔactA infection (Fig. 4). These results indicate that differences in L.monocytogenes antigen load at early time-points after infection do not account for the increased magnitude of the antigen-specific T-cell response in mice treated with CTLA-4 blocking antibody.

Figure 4.

Figure 4

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Number of recoverable Listeria monocytogenes colony-forming units (CFUs; log 10) per spleen for cytotoxic T-lymphocyte antigen 4 (CTLA-4) blocking antibody (•, grey line) or hamster isotype control (h-IgG, ▮, black line) antibody-treated mice after infection with 106Listeria monocytogenes ovalbumin (Lm-OVA) ΔactA. These data are representative of four mice per experimental group. Bar, standard error.

CTLA-4 blockade increases the kinetics of bacterial clearance after virulent L. monocytogenes challenge

Lastly, we evaluated how the increased magnitude antigen-specific T-cell response conferred by CTLA-4 blockade after priming with L. monocytogenesΔactA would alter the kinetics of bacterial clearance after challenge with virulent L. monocytogenes. In our recent studies, suppressing the antigen-specific T-cell response magnitude with anti-PDL-1 blocking antibody delays the kinetics whereby virulent L. monocytogenes is cleared.27 Accordingly we hypothesized that CTLA-4 blockade resulting in increased antigen-specific response magnitude would increase the clearance kinetics of virulent L. monocytogenes after secondary challenge. To test this hypothesis, groups of naïve mice or mice initially primed with L. monocytogenesΔactA each treated with either CTLA-4 blocking or control antibody were challenged with an inoculum of virulent L. monocytogenes that was normally lethal for naïve mice (1 × 50% lethal dose). Within the first 72 hr after virulent L. monocytogenes challenge, both CTLA-4 blocking antibody-treated and control antibody-treated naïve mice had progressively increasing numbers of L. monocytogenes CFUs, became moribund and were killed (Fig. 5). Importantly there were no significant differences in numbers of recoverable L. monocytogenes CFUs between naïve mice treated with CTLA-4 blocking antibody or control antibody, indicating that CTLA-4 does not play an important role in innate host defence against virulent L. monocytogenes. Consistent with the highly efficient nature whereby antigen-specific T cells primed with L. monocytogenesΔactA confer protection against virulent L. monocytogenes challenge, greater than 100-fold decreased L. monocytogenes CFUs were present for control antibody-treated, L. monocytogenesΔactA-primed mice compared with naïve mice beginning 24 hr after challenge (Fig. 5). Remarkably, the rate of bacterial clearance was further enhanced for CTLA-4 blocking antibody-treated mice because an additional ∼ 10-fold reduction (P < 0·05) in recoverable L. monocytogenes CFUs was present for L. monocytogenesΔactA-primed, CTLA-4 blocking antibody-treated mice compared with control antibody-treated mice 24 hr after challenge with L. monocytogenes (Fig. 5). By 72 hr post-challenge, virulent L. monocytogenes was cleared for the majority of L. monocytogenesΔactA-primed mice regardless of CTLA-4 blockade whereas increased numbers of L. monocytogenes CFUs were found for both groups of naïve control mice (Fig. 5). The CTLA-4 blockade increases the kinetics whereby virulent L. monocytogenes challenge is eradicated in L. monocytogenesΔactA-primed mice.

Figure 5.

Figure 5

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Number of recoverable Listeria monocytogenes CFUs (log 10) per spleen at the indicated time-point after infection with 105Listeria monocytogenes ovalbumin (Lm-OVA) for naïve mice (left-sided panel), mice primed 30 days previously with 106 Lm-OVA ΔactA (middle panel), and Lm-OVA ΔactA-primed mice depleted of CD8 T cells one day before challenge (right-sided panel) each treated with cytotoxic T-lymphocyte antigen 4 (CTLA-4) blocking (•, grey line) or hamster isotype control (h-IgG, ▮, black line) antibody. These data are representative of six to eight mice per experimental group from two independent experiments with similar results. Bar, standard error. *P < 0·05.

Since CD8 T cells are the adaptive immune effectors that mediate protective immunity to L. monocytogenes infection as demonstrated in various mice with unique targeted immune defects,23,25,28 we sought to examine if CD8 T cells were responsible for the increased kinetics of virulent L. monocytogenes clearance attributable to CTLA-4 blockade in L. monocytogenesΔactA-primed mice. Accordingly, we quantified the effects of CD8 T-cell depletion before challenge with virulent L. monocytogenes in groups of L. monocytogenesΔactA-primed mice treated with either anti-CTLA-4 antibody or control antibody. Beginning at day 1 post-challenge, dramatically more L. monocytogenes CFUs were recovered from both anti-CTLA-4 antibody-treated and control antibody-treated mice depleted of CD8 T cells (Fig. 5). Importantly, CD8 T-cell depletion eliminated the difference in kinetics of virulent L. monocytogenes clearance between mice treated with anti-CTLA-4 antibody and mice given control antibody (Fig. 5). Consequently, after CTLA-4 blockade, CD8 T cells are the primary mediators of protective immunity to virulent L. monocytogenes infection. Taken together, these results demonstrate CTLA-4 suppresses the L. monocytogenes-specific CD8 T-cell response during primary L. monocytogenesΔactA infection, resulting in delayed protective immunity after secondary virulent L. monocytogenes challenge.

Discussion

In this report we demonstrate, using a prime-challenge model of attenuated followed by virulent L. monocytogenes infection, that CTLA-4 plays a suppressive role in antigen-specific T-cell activation. These results are consistent with other studies that have characterized the role of CTLA-4 in antigen-specific T-cell activation after infection that together uniformly indicate a T-cell suppressive role for CTLA-4.1–11 In each of these studies and the results presented here, CTLA-4 blockade augments the T-cell response magnitude. Although antigen-specific T cells play important roles in host protection during infection, the increased magnitude T-cell activation conferred by CTLA-4 blockade can either improve or worsen infection outcome. For example, CTLA-4 blockade in some models of chronic bacterial and parasitic infection results in more rapid pathogen clearance and protective immunity,2,6–10 whereas in other infections the benefits of more rapid pathogen clearance are outweighed by the immune-mediated host tissue injury caused by CTLA-4 blockade.4,5 Interestingly for viruses such as simian immunodeficiency virus that are capable of establishing latent infection within T cells, CTLA-4 blockade causing non-specific T-cell activation increases viral replication levels.15 These results may be directly related to increased transcription of latent viral genes after the non-specific immune activation triggered by Mycobacterium tuberculosis or Mycobacterium avium infection in transgenic mice containing integrated copies of the HIV genome.33,34 Nevertheless, these studies together with the data we present here clearly indicate that CTLA-4 confers important suppressive effects on T-cell activation and expansion during infection.

It remains unclear why CTLA-4 does not play a suppressive role in T-cell activation following acute LCMV or vaccinia virus infection, and in the setting of CD28 deficiency after acute LCMV infection appears to play an important stimulatory role in priming virus-specific CD8+ T cells. Although protective immunity to both LCMV and L. monocytogenes infection is mediated primarily by pathogen-specific CD8+ T cells, a striking difference between our results and the data reported for acute LCMV infection is the dramatically increased CTLA-4 expression by CD8+ T cells after LCMV infection that does not occur after primary L. monocytogenesΔactA infection. Whether differences in CTLA-4 expression by CD8+ T cells triggered by LCMV compared with L. monocytogenes alone account for the discordant roles for CTLA-4 in T-cell activation and expansion between these infection models is unclear. Although CTLA-4 was not upregulated to any appreciable extent on CD8 T cells after L. monocytogenes infection, our results indicate that in addition to constitutive expression on Foxp3+ regulatory T cells, CTLA-4 is also transiently upregulated on newly activated antigen-specific CD4 T cells. However, using our current experimental conditions, we cannot distinguish whether the augmented L. monocytogenes-specific CD8 and CD4 T-cell response after anti-CTLA-4 antibody treatment is attributable to CTLA-4 blockade on Foxp3+ and/or Foxp3− CD4 T cells. Since others have demonstrated that CTLA-4 blockade also blocks the suppressive function of regulatory T cells (Tregs) that results in autoimmunity, the augmented L. monocytogenes-specific CD8 and CD4 T-cell response after CTLA-4 blockade could be consistent with overall blockade of Tregs.29–31 However, since these studies did not specially prime conditions where CTLA-4 is upregulated on non-Treg CD4 cells, the potential role for CTLA-4 expression on these cells cannot be excluded. Our ongoing studies are designed to more specifically characterize how CTLA-4 and CD4 T cells control protective immunity during L. monocytogenes and other infections.

Acknowledgments

This work was supported through funding from the following sources: NICHD/NIH-K08HD51584, Infectious Disease Society of America, March of Dimes Foundation, Vikings Children’s Fund and the Minnesota Medical Foundation.

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