1.1. DCs Are Crucial for Effective Helper and Cytotoxic T-cell Activation

As suggested before, the effective activation of a T cell fully depends on its interaction with APCs ^[1][16]. They require an Ag to be presented in a rather short peptide sequence in a peptide:protein complex by the APC. The full activation of T cells requires the interplay of three different signals. Signal 1 is the recognition of the specific peptide presented to the T-cell receptor (TCR) by the major histocompatibility complex (MHC) molecules (either MHC-I for cytotoxic CD8⁺ T cells or MHC-II for CD4⁺ T cells) presented on APCs ^[2][3][17,18]. MHC-I-restricted peptides are mostly of cytoplasmic origin, while MHC-II-restricted peptides are of extracellular origin ^[3][18]. Through a process termed MHC-II cross presentation, extracellularly derived antigen peptides can be presented to CD8 T cells. This process itself is not sufficient to trigger the effective activation of Ag-specific T cells. In addition, they require Signal 2, characterized by the interaction between costimulatory molecules on T cells (e.g., CD28) and their counterparts on the APCs, such as CD80 and CD86 (also termed B7.1 and B7.2). Finally, to define the type of response, Signal 3 is required in the form of cytokines. Together, these three signals induce Ag-specific CD4⁺ or CD8⁺ T-cell responses ^[4][19]. Since DCs have the special ability to ingest virus-infected cells or tumor cells, they are able to present Ags derived from these to specific CD8⁺ T cells. The DCs activate them through a process termed cross-presentation via a separate MHC-I pathway ^[5][6][7][9,20,21].

DCs themselves become activated upon contact with foreign Ags ^[8][22]. DC activation can occur upon the engagement of conserved bacterial or viral Ags, so-called pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs). In resting conditions, immature DCs (imDCs) are equipped with several types of PRRs, including Toll-like receptors (TLRs), membrane-associated C-type lectin receptors (CLRs) ^[9][23], and mannose receptors ^{[10][11][12][13][14][15]}[24,25,26,27,28,29]. Following the recognition of pathogens, imDCs can remain in a tolerogenic state ^[16][30] or undergo a maturation process where they lose their endocytic ability while increasing the Ag processing and presentation capacity ^[17][18][31,32]. PRR engagement activates mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) signaling ^[19][33], which, in mature DCs (maDCs), induces the expression of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12), and IL-6 ^[20][34]. This is particularly important for the activation and clonal expansion of proinflammatory Th1-type CD4⁺ T cells ^[21][35]. MaDCs also upregulate chemokine receptors like CCR7 that drive their homing to lymph nodes (LN) ^[22][23][36,37]. The secretion of cytokines is reflected in a profound transcriptional change in DC gene expression that also results in the upregulation of Signal-2 markers, such as MHC-II, CD80, CD86, and CD40 ^[24][25][38,39]. Importantly, DC activation may alternatively trigger anti-inflammatory Th2 CD4⁺ T-cell activation or invigorate other specialized T-helper subsets, such as Th17, Th22, or regulatory T cells (Treg), depending on the context. We illustrate an overview of proinflammatory DC-mediated T-cell activation ^[26][40] in Figure 1.

Figure 1. DC response to the antigen challenge. DCs can process either host-derived (self) proteins (blue, left-hand side) or foreign antigens (red, right-hand side). The latter could be from an exogeneous source (e.g., bacteria as illustrated) or cancer cell-derived neo-Ags. Self-protein processing and the presentation to T-cell receptors (signal 1) via peptide–MHC complexes (pMHC) leads to tolerance. In addition to signal 1, foreign antigens can lead to a strong DC activation, for instance, through the co-stimulation of TLRs or other receptors (not shown), which entails the upregulation of co-stimulatory molecules such as CD80 or CD86 at the DC surface (signal 2). These prolong and intensify the TCR-driven activation of antigen-specific T cells. Finally, cytokines such as IL-1β, IL-12, IL-6, IFN-γ, and TNF-α are released (signal 3) by both the DC and the T cell, which further shape the antigen-induced T-cell response. TCR: T cell receptor; pMHC-II: Peptide-MHC-II receptor; imDC: Immature DC; maDC: Mature DC.

PRRs are also relevant with regards to vaccine development, where the effective activation of T cells is critical. Although from a mechanistic point of view, targeting DCs seems like a promising avenue for vaccine development; it has been demonstrated that many DC vaccines alone do not achieve full T-cell activation ^[27][41]. In an effort to overcome this limitation, adjuvants can be used as key molecules aimed to promote stronger T-cell responses by inducing DC maturation and prolonging their exposure to antigens ^[27][41]. The efforts to create effective adjuvants have focused on the use of microbial compounds and selective TLR ligands ^[28][29][30][42,43,44]. However, PRRs are not only expressed on APCs but on a wide variety of myeloid cells, including neutrophils ^[31][45], monocytes ^[32][33][46,47], and macrophages ^[32][46], as well as nonimmune cells such as endothelial cells ^[34][48]. Consequently, the use of microbial compounds seemed to elicit a very broad inflammatory immune cell activation caused by non-DC PRR activation. Currently, even though adjuvants like TLR ligands ^[12][35][36][26,49,50], aluminum, or saponin-based particles are being studied to maximize the immunogenicity of vaccines ^{[37][38][39][40]}[51,52,53,54], this strategy may still entail the risk of inducing general inflammation.

1.2. FcγR Crosslinking on DCs Leads to Effective T-Cell Activation and Proliferation

In addition to PRRs, DCs express Fc-gamma receptors (FcγR) that can lead to a highly effective internalization of Ag and subsequent DC activation ^[41][55]. FcγRs, when crosslinked through IgG antibody-complexed (“opsonized”) soluble Ag, allow for rapid internalization and cellular activation ^[42][43][56,57]. This immune complex (IC) will then be shuttled to endolysosomal DC compartments, where the Ag will subsequently be degraded. This facilitates the subsequent MHC:peptide generation and Ag presentation on the DC ^[42][44][56,58]. In humans, three groups of FcγRs have been described across a variety of cell types: FcγRI (CD64), FcγRIIA/B (CD32A/B), and IIIA/B (CD16A/B) ^[45][59]. Concerning DC activation, all FcγRs except FcγRIIB are considered activators; FcγRIIB acts as an inhibitory receptor. FcγRs bind—albeit, with different affinities ^[46][60]—to the Fc (fragment, crystallizable) portion of IgG antibodies ^[44][58]. On DCs, the expression of FcγRs depends on the cell subtype. FcγRI is expressed on monocyte-derived DCs (moDCs) ^[47][61]. FcγRIIA has been described on conventional DCs, which were also shown to express FcγRIIB ^[48][62]. In addition, human, as well as murine, pDCs were described to express FcγRII ^{[49][50][51][52]}[63,64,65,66]. Overall, human and mouse DCs express largely overlapping FcγR subsets ^[53][54][67,68]. However, the balance between activating and inhibitory receptors on APCs critically depends on the tissue of origin, and the numbers of cell surface receptors can be different between the species ^[55][69].

Several lines of evidence, both in humans and mice, have convincingly demonstrated that IgG:Ag ICs induce a superior DC activation compared to the delivery of uncoated, “naked” Ag ^[42][43][56,57]. For example, pulsing in vitro human moDCs with polyclonal tetanus IgG ICs led to a stronger increase in DC activation, including the release of proinflammatory cytokines compared to “naked” tetanus toxoid Ag ^[43][56][57,70]. Similarly, in a pivotal mouse study, ovalbumin (OVA) preincubated with anti-OVA IgG was taken up much more efficiently by splenic mouse DCs ex vivo than “naked” OVA. Importantly, in mice transplanted with OVA-specific CD8⁺ or CD4⁺ T cells, OVA:IgG ICs induced both CD4⁺ and CD8⁺ T-cell proliferation more efficiently than “naked” OVA ^[57][71]. Similar experiments were repeated with henn egg lysozyme IgG ICs and with mice selectively lacking activating, as well as inactivating, FcγRs ^[41][57][58][55,71,72]. In addition, mechanistic confirmation was achieved using mouse models where signal transduction downstream of FcγRI and FcγRIIIA was impaired ^[41][59][55,73]. Another study showed that IC-mediated FcγR crosslinking in mouse DCs was required to induce long-lasting transcriptional changes reflected in the induction of T cell-polarizing genes, such as IL2, IL6, IL10, IL15, IL23a, IL27, and Ifnb1 ^[60][74]. These experiments provide the mechanistic basis to target FcγRs via IgG ICs, and this holds promise for DC-specific vaccination strategies ^[61][75].