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

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.

Figure 1

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.

Figure 2

(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⁺ OVA_323–339-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.

Figure 3

(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 OVA_257–264 peptide [major histocompatibility complex (MHC) class I], lipid-linked oligosaccharide 189–201 peptide (LLO_189–201 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 OVA_257–264 peptide (MHC class I), LLO_189–201 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.

Figure 4

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.

Figure 5

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.