Elsevier

Vaccine

Volume 24, Issue 24, 12 June 2006, Pages 5119-5132
Vaccine

The adjuvant effects of the toll-like receptor 3 ligand polyinosinic-cytidylic acid poly (I:C) on antigen-specific CD8+ T cell responses are partially dependent on NK cells with the induction of a beneficial cytokine milieu

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Abstract

Poly (I:C), a TLR3 ligand, has shown promise as a vaccine adjuvant to CD8+ T cell responses. The underlying mechanisms involved in creating this adjuvant response in vivo, however, have not been well defined. In this study, we explored the contribution of NK cells and inflammatory cytokines in mediation the poly (I:C) adjuvant effects. Enhanced antigen-specific CD8+ T cell responses were observed only when poly (I:C) was administered within 4 h of peptide vaccination. Poly (I:C) treatment was associated with a rapid induction of inflammatory cytokines in the serum, including IL-6, IL-10, MCP-1, TNF-α, IFN-α, and IFN-γ, and selective increases in the numbers of NK (NK1.1+CD11b+) cells and Mϕ (NK1.1CD11b+), but not NK T (CD3+NK1.1+) cells. NK cells were required for the adjuvant effects of poly (I:C). Poly (I:C) treatment in TNF-α, type I IFNR, IFN-γ, IL-6, IL-12Rβ2, or IL-15 gene-deficient mice revealed a reciprocal interaction and interdependence in the induction of these cytokines, where the absence of one cytokine impacted on the production of others. Further, the adjuvant effects of poly (I:C) were dependent on the endogenous levels of type I IFNs, TNF-α, IFN-γ, IL-12, and IL-15. IFN-α and IFN-β, but not TNF-α or IL-6, were able to mimic the adjuvant effects of poly (I:C). We conclude that the adjuvant effects of poly (I:C) on antigen-specific CD8+ T cells appeared to be exquisitely dependent on the rapid induction of certain beneficial cytokines produced in part by NK cells.

Introduction

Conserved microbial structures, termed pathogen-associated molecular patterns (PAMPs), are recognized by receptors of the innate immune system such as Toll-like receptors (TLRs) [1], [2]. Ligation of TLRs by their specific PAMPs results in the induction of pro-inflammatory mediators, including NF-κB, cytokines, and costimulatory molecules by cells of the innate immune system such as NK cells, macrophages (Mϕ), and dendritic cells (DCs). So far, 13 TLRs (1–13) have been found to be expressed on the cell surface of sentinel cells of the innate immune system [3]. Double-stranded RNA (dsRNA), a natural product of viral replication, has recently been identified as a specific ligand for TLR3 [4]. TLR3 is expressed on NK cells, Mϕ, DCs [5], and CD4 T cells [6]. Polyriboinosinic polyribocytidylic acid (poly (I:C)), a synthetic dsRNA mimic copolymer, is also a specific TLR3 ligand [7], [8].

Recent in vitro studies have shown that poly (I:C) can efficiently mature both murine and human DCs, and induce cross-presentation of different antigens by these cells [9], [10], [11], [12]. Furthermore, stimulation of TLR3 alone or in combination with CD40 triggering during active vaccination appears to enhance CD8+ T cell responses [13], [14], [15]. We have found that treatment of poly (I:C) can enhance antigen-specific CD8+ T cell responses to both self and non-self peptides, and is associated with effective antitumor immunity [16]. However, the mechanisms underlying the adjuvant effects of poly (I:C) on CD8+ T cells have not been fully explored in vivo. Several components of the innate immune system including NK cells, Mϕ, and DCs are considered to be the initial targets of PAMPs [3]. After ligation of TLRs, these cells produce an array of inflammatory cytokines and chemokines, leading to autocrine and paracrine stimulation of both the innate and adaptive immune systems [17]. Previous studies have shown that ligation of TLR3 by poly (I:C) results in the activation of DCs, as well as the production of inflammatory cytokines by NK cells, and DCs [4], [18], [19], [20], [21]. However, it remains to be determined which of the pleiomorphic effects of poly (I:C) is primarily responsible for mediating the adjuvant effects of this agent on adaptive CD8+ T cell responses in vivo.

Specific goals of the present study include defining the following parameters associated with poly (I:C) treatment: (1) the optimal timing of treatment, (2) the early activation kinetics of the innate immune system following treatment, (3) the nature, kinetics, source, reciprocal interaction, and adjuvant effects of cytokines induced by poly (I:C), and (4) the role of NK cells in mediating the adjuvant effects of poly (I:C). Ultimately, defining the mechanisms underlying the adjuvant effects of poly (I:C) on antigen-specific CD8+ T cells will improve our ability to rationally and predictably apply this promising TLR3 agonist as a cancer vaccine adjuvant.

Section snippets

Mice

B6.SJL (Ly5.1), C57BL/6 (Ly5.2), BALB/c, TNF-α, IL-6, IL-12Rβ2, and IFN-γ deficient mice on the C57BL/6 background, and CD1 deficient mice on the BALB/c background were purchased from Jackson Laboratory (Bar Harbor, ME). IFN-α/βR KO mice are a gift from Dr. Jonathan Sprent (The Scripps Research Institute, La Jolla, CA). IL-15 deficient mice were obtained from Taconic (Germantown, NY). OT-1 TCR transgenic (Vα2/Vβ5) mice (Jackson Lab.) were bred with B6.SJL mice to generate Ly5.1/Ly5.1 mice

The adjuvant effects of poly (I:C) are exquisitely dependent on the timing of its treatment relative to peptide vaccination

We have previously shown that treatment of poly (I:C) at the time of peptide vaccination results in a dramatic enhancement of the antigen-specific CD8+ T cell response, with decreased CD8+ T cell apoptosis, and enhanced CD8+ T cell proliferation and function [16]. To initially explore the mechanisms underlying the adjuvant effects of poly (I:C) treatment, the response to peptide vaccination and poly (I:C) treatment was determined where poly (I:C) was administered immediately before, at the time

Discussion

Primary CD8+ T cell responses are characterized by a dramatic expansion phase followed by an equally dramatic contraction phase [29]. It is becoming increasingly apparent that cytokines may be involved in modulating this process [30], [31], and that the timing of cytokine production and/or treatment may significantly impact on the survival of CD8+ T cells [22], [32], [33], [34], [35], [36], [37], [38]. We have recently demonstrated that poly (I:C) treatment at the time of peptide vaccination

Acknowledgement

This work was supported by the National Institutes of Health Grant 1 R01 CA94856-01.

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