Multiple innate signaling pathways cooperate with CD40 to induce potent, CD70-dependent cellular immunity

Abstract

We have previously shown that Toll-like receptor (TLR) agonists cooperate with CD40 to generate CD8 T cell responses exponentially larger than the responses generated with traditional vaccine formulations. We have also shown that combined TLR agonist/anti-CD40 immunization uniquely induces the upregulation of CD70 on antigen bearing dendritic cells (DCs). In contrast, immunization with either a TLR agonist or a CD40 stimulus alone does not significantly increase CD70 expression on DCs. Furthermore, the CD8⁺ T cell response generated by combined TLR agonist/anti-CD40 immunization is dependent on the expression of CD70 by DCs, as CD70 blockade following immunization dramatically decreases the CD8 T cell response. Here we show that other innate pathways, independent of the TLRs, can also cooperate with CD40 to induce potent, CD70 dependent, CD8 T cell responses. These innate stimuli include Type I IFN (IFN) and α-galactosylceramide (αGalCer) or aC-GalCer, glycolipids that are presented by a nonclassical class I MHC molecule, CD1d, and are able to activate NKT cells. Furthermore, this combined IFN/anti-CD40 immunization generates protective memory against bacterial challenge with Listeria monocytogenes. Together these data indicate the importance of assessing CD70 expression on DCs as a marker for the capacity of a given vaccine formulation to potently activate cellular immunity. Our data indicate that optimal induction of CD70 expression requires a coordinated stimulation of both innate (TLR, IFN, αGalCer) and adaptive (CD40) signaling pathways.

Introduction

Toll-like receptor (TLR) stimulation elicits a pro-inflammatory response involving the induction of the transcription factor NFκB and MAP kinases, leading to the production of pro-inflammatory cytokines such as TNFα, IL-12, IL-6, and IL-1 by dendritic cells (DCs) and macrophages [1], [2]. Triggering of TLRs 3, 4, 7, and 9 also leads to production of Type I Interferons (IFN), cytokines which have been shown to be important in the generation of adaptive immunity [3], [4], [5], [6], [7], [8], [9], [10]. Given the ability of TLRs to induce the production of these pro-inflammatory responses, TLR agonists are generally thought to be useful vaccine adjuvants [11]. Indeed, TLR agonists are relatively successful at augmenting humoral immune responses [12]. However, when used to enhance the generation of cellular immune responses, TLR agonists by themselves have largely been a disappointment [13], [14], [15], [16], [17], [18], [19], [20], [21], [22]. Indeed, when used alone as a vaccine adjuvant, we previously showed that they are unable to generate cellular immune responses capable of protecting against viruses or other intracellular infections [23].

The paucity with which TLR agonists initiate cellular immunity reveals the fact that the precise quantity and/or quality of DC maturation that promotes the highest magnitude T cell response is still poorly understood. In general there is a prevailing view that “more is better”. Beyond that however, a precise characterization of how much of which costimulatory molecules are required for optimal T cell expansion is as of yet unknown, a somewhat surprising fact given the degree of effort that is put into investigating both DC and T cell activation. CD70 is a costimulatory marker expressed primarily by antigen presenting cells (APCs)[24]. It is a member of the TNF ligand superfamily and binds to its receptor CD27, which is expressed mainly on T cells but is also expressed on B cells and NK cells. CD70 has been shown to be important for the priming of CD8 T cell responses [23], [25], [26], [27], [28], [29], [30], [31], [32] and blockade of CD70/CD27 interactions often dramatically reduces the generated CD8 T cell response [23], [25], [26], [28], [29], [30]. Importantly, CD70 has also been shown to be upregulated on APCs during some natural infections, and is known in this setting to be important for CD8 T cell priming [30]. Additionally, the constitutive expression of CD70 on immature DCs is enough to overcome tolerance and prime CD8⁺ T cells that can infiltrate into tumor sites and induce tumor regression [27]. Given the potency of CD70 for inducing cellular immunity, an established method for inducing its expression on DCs would be of great benefit for the purposes of vaccine development.

Toward this end, we previously demonstrated that immunization with both a TLR agonist and an agonistic anti-CD40 antibody induces a degree of CD8⁺ T cell expansion that is 10–50-fold greater than that observed in response to immunization in the presence of either agonist alone [14], [23]. The magnitude of these T cell responses often matches or even exceeds that seen in response to infectious agents such as LCMV or Listeria monocytogenes [18], [19], [20], [21]. When comparing the phenotype of dendritic cells stimulated with a TLR agonist, anti-CD40, or both, we observed that upregulation of the TNF ligand superfamily member CD70 on both CD8 and CD11b DC subsets was unique to only the combined TLR agonist/anti-CD40 stimulus [23]. The CD8 T cell response generated by the combined TLR agonist/anti-CD40 stimulus was dependent on this DC CD70 expression, since blocking CD70 from its receptor CD27, dramatically reduced the CD8 T cell response. Thus, CD70 expression in vivo is uniquely regulated by the combined stimulation of a TLR and CD40.

Here we show that the innate signaling pathways able to elicit the generation of potent CD70-dependent CD8 T cell responses in combination with CD40 are not limited to the TLRs. We demonstrate that Type I IFN (IFN) or NKT ligands (αGalCer or αC-GalCer) similarly induce the upregulation of CD70 on DCs when used in combination with anti-CD40, leading to the exponential expansion of antigen-specific T cells. While αGalCer alone can induce an increase in CD70 expression on DCs in vivo [29], maximal CD70 expression, leading to maximal CD8⁺ T cell expansion, is induced only when used in combination with anti-CD40 antibody. In contrast to αGalCer but similar to the TLR agonists [23], IFN alone induces no CD70 expression at all, but synergizes effectively with anti-CD40 to induce CD70 upregulation and the subsequent induction of CD8⁺ T cell memory that is protective against infectious challenge. Therefore, multiple innate pathways (TLRs, Type I IFN, NKT agonists) are able to work in synergy with CD40 to generate large CD8 T cell responses through a CD70-dependent mechanism, demonstrating the importance of CD70 as a marker in identifying vaccine strategies with efficacy in generating cellular immunity.