Combined TLR and CD40 triggering induces potent CD8+ T cell expansion with variable dependence on type I IFN

Abstract

Toll-like receptors are important in the activation of innate immunity, and CD40 is a molecule critical for many T and B cell responses. Whereas agonists for either pathway have been used as vaccine adjuvants, we show that a combination of Toll-like receptor (TLR)7 and CD40 agonists synergize to stimulate CD8+ T cell responses 10-20-fold greater than the use of either agonist alone. Antigen-specific CD8+ T cells elicited from combination CD40/TLR7 treatment demonstrated both lytic activities and interferon (IFN)gamma production and an enhanced secondary response to antigenic challenge. Agonists for TLRs 2/6, 3, 4, and 9 also synergized with CD40 stimulation, demonstrating that synergy with the CD40 pathway is a property of TLR-derived stimuli in general. The CD8+ T cell expansion induced by CD40/TLR7 triggering was independent of CD4+ T cells, IFNgamma, and IL-12 but dependent on B7-mediated costimulation and surprisingly on type I IFN. These studies provide the rational basis for the use of TLR and CD40 agonists together as essential adjuvants to optimize vaccines designed to elicit protective or therapeutic immunity.

Figure 1.

Anti-CD40 and the TLR7 agonist 27609 synergize to induce enhanced CD8⁺ T cell expansion. (A) C57BL/6 mice were immunized i.p. with 500 μg of whole ovalbumin protein, 50 μg of the anti-CD40 antibody FGK45, and 100 μg of 27609 in the combinations indicated above. Mice were killed 6 d after immunization, and spleen cells were isolated and stained with tetramer as described in Materials and Methods to identify ovalbumin-specific T cells. The data shown has been gated on all CD8⁺, B220⁻ events. The percentages given in the top right quadrant are the percentage of tetramer staining cells out of total CD8⁺ T cells. (B and C) Mice were immunized as in A with increasing amounts of peptide i.v. (B) or whole ovalbumin (C) and anti-CD40 ± 27609 i.p. 6 d after priming, the spleen cells were stained and analyzed as in A for ovalbumin-specific T cells. Percentages given are tetramer staining cells out of total CD8⁺ T cells. Error bars represent SD of three mice per group. (D) Mice were immunized as in A with whole ovalbumin, anti-CD40, and 27609, and at the times indicated after priming, the spleen cells were removed and analyzed as in A. The data shown was gated on all live, CD8⁺, B220⁻ events. The percentages given in the top right quadrant are the percentage of tetramer staining cells out of total CD8⁺ T cells.

Figure 2.

CD40/TLR7 triggering induces functional CTL. (A) Mice primed with 500 μg whole ovalbumin ± CD40 and/or S-27609 were assessed by using an in vivo cytotoxicity assay as described in Materials and Methods. The number in the top left of the histograms indicates the ratio of nonantigen pulsed, low CFSE-labeled spleen cells to antigen pulsed, high CFSE-labeled spleen cells. (B) As in A, mice were immunized and analyzed by tetramer staining and in vivo cytotoxic activity at the time points indicated. (C) Cells from mice treated as in A (day 7 after priming) were incubated in the presence of brefeldin A with or without SIINFEKL peptide for 6 h at 37°. The cells were then stained for CD8, fixed, permeabilized, and stained for intracellular IFNγ as described in Materials and Methods. The data shown is gated on all CD8⁺ events. Numbers in the top right quadrant indicate the percentage of IFNγ⁺ cells out of the total CD8⁺ cells.

Figure 3.

TLR/CD40 triggering produces long-term T cell immunity. (A) Mice were immunized i.p. with ovalbumin, anti-CD40, and S-27609 as in Fig. 1. 30 d later, the mice were rechallenged i.p. with either 100 μg SIINFEKL peptide or 500 μg ovalbumin protein ± 27609/CD40 as indicated. At days 3 and 5 after rechallenge, spleen cells were isolated and stained with tetramer as described in Materials and Methods. Peak responses for peptide or protein responses are shown (day 3 for peptide rechallenge and day 5 for protein rechallenge). The data shown was gated and analyzed as in Fig. 1 A. The data is representative of three experiments performed.

Figure 4.

Synergy with CD40 in the induction of CD8⁺ T cell proliferation and differentiation is a property of multiple TLR agonists. (A) Mice were challenged i.p. with ovalbumin and the indicated TLR agonists (30 μg LPS as a TLR4 agonist, 100 μg CpG 1826 as a TLR9 agonist, 25 μg Malp-2 as a TLR2/6 agonist, 50 μg poly IC as a TLR3 agonist, and 100 μg 27609 as the TLR7 agonist), with or without anti-CD40. 6 d after challenge, spleens cells were isolated and stained with tetramer as described in Fig. 1. The data shown was gated and analyzed as in Fig. 1 A. (B and C) The average percentage of tetramer staining T cells and their SDs from three mice per treatment primed with whole protein (B) or peptide (C) were analyzed and calculated. The data shown is representative of three to eight experiments performed, depending on the TLR agonist.

Figure 5.

TLR/CD40 triggering of CD8⁺ T cell expansion and effector function is largely dependent on costimulation via CD80/86 but independent of CD4 cells, IFNγ, IL-12, or IL-23. The indicated genetically deficient mice were immunized with 500 μg ovalbumin plus 50 μg anti-CD40 and 200 μg 27609 by i.p. injection. On day 6 postimmunization, in vivo lytic activity was measured as in Fig. 2 A, and splenocytes were isolated and analyzed as in Fig. 1 but using anti-CD8α PE and APC-labeled SIINFEKL/H-2K^b tetramers. Data is representative of at least three mice per group. Numbers in the top right quadrant of the dotplots indicates the percentage of tetramer⁺ CD8 T cells out of total CD8 T cells. The number in the top left of the histograms indicates the ratio of nonantigen pulsed, low CFSE-labeled spleen cells to antigen pulsed, high CFSE-labeled spleen cells.

Figure 6.

CD40 synergy with IFNα/β-inducing TLR agonists is critically dependent on type I IFN. Either wt control B6/129 F1 or IFNαβR KO mice were immunized with 100 μg of SIINFEKL peptide (A and B) or 500 μg ovalbumin protein (C), 50 μg anti CD40, and the indicated TLR agonist (30 μg LPS as a TLR4 agonist, 100 μg CpG 1826 as a TLR9 agonist, 25 μg Malp-2 as a TLR2/6 agonist, 50 μg poly IC as a TLR3 agonist, and 100 μg 27609 as the TLR7 agonist) as in Fig. 3. On day 6, spleen cells were stained with tetramer and analyzed as in Fig. 1 A. The legend above the figure indicates the TLR agonist used. (B and C) The percentage of tetramer staining cells in the mice from A after either peptide (B) or whole protein (C) challenge were calculated as the percentage of tetramer staining cells out of total CD8⁺ T cells. This percentage was then divided by the percentage of tetramer staining cells from wild-type mice challenged with the same TLR agonist. The data is expressed as the percentage of maximum synergy seen in the IFNαβR KO mice compared with the wild type for the given TLR agonist. The data shown is an average from three mice per treatment group, and error bars indicate the calculated SD. The data represents three experiments performed.