implications for T lymphocyte-directed immunotherapy
Montfoort, A.G. van
Citation
Montfoort, A. G. van. (2010, November 25). Antigen targeting to Fc receptors on dendritic cells : implications for T lymphocyte-directed immunotherapy.
Retrieved from https://hdl.handle.net/1887/16177
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
Downloaded from: https://hdl.handle.net/1887/16177
Note: To cite this publication please use the final published version (if applicable).
Chap ter 1
General Introduction
1. Prologue
Immunotherapy is a medical intervention aimed to induce, enhance or suppress the immune response. The most successful immunological interventions developed so far are traditional vaccines. They are based on the empirical findings that injection of non-infectious or attenuated micro-organisms protects individuals against subsequent infection with the pathogenic micro- organism. Nowadays we know that protective immunity after prophylactic vaccination is mediated by antibodies, and that a long-term protection is guaranteed by immunologic memory, a special feature of the adaptive immune response.
Major advancements in the understanding of the immune system have provided us with the opportunity for rational design of therapeutic immunological interventions. Since T lymphocytes play an important role in the immune response to tumors and viruses, T lymphocyte-directed immunotherapy by therapeutic vaccination is a promising intervention for patients suffering from cancer and chronic infectious diseases. The requirements for induction of antigen-specific T lymphocytes have been studied extensively. The notion that dendritic cells (DC) play a crucial role in the activation of T lymphocytes has made DC biology of central importance for vaccine development (1). Accordingly, efficient delivery of antigen to DCs is one of main objectives in vaccine development.
In this thesis, antibody-mediated antigen targeting is evaluated as a potential antigen delivery strategy for therapeutic vaccination. Complexes of protein antigen and antigen-specific antibodies are natural formulations that bind to Fcg receptors. FcgR ligation on DCs leads to efficient uptake, DC maturation and presentation of the antigen to T lymphocytes (2,3). Interaction of Ag-Ab complexes with FcgRs on DCs provides a link between the humoral and cellular arms of the immune response. This thesis contains an extensive evaluation of FcgR-mediated antigen delivery to dendritic cells in the context of T lymphocyte-mediated immunotherapy. In addition, it contains a detailed analysis of FcgR function on DCs and addresses the kinetics of cross- presentation of antigen after FcgR-mediated uptake.
2. The adaptive immune response
In contrasts to the innate immune system, the adaptive immune system is specific and evolves during life. It adapts to antigenic experiences of an individual. The adaptive immune system consists of two functionally distinct arms that work together; the humoral arm and the cellular arm. They consist of B and T lymphocytes respectively. Both populations have an extensive repertoire of unique antigen receptors, selectively expressed on individual lymphocytes, that ensures specific recognition of any foreign antigen. Upon antigen recognition, antigen-specific B and T lymphocytes clonally expand, rapidly increasing their precursor frequency. Although B and T lymphocytes have distinct effector mechanisms, they interact on different levels. The role of B lymphocytes in the induction of a T cell response is discussed in chapter 4. Dendritic cells (DC) play a central role in the initiation, prolongation and coordination of both arms of the adaptive immune response. A unique function of the adaptive immune system is the establishment of immunological memory that ensures rapid activation after a second encounter with the same antigen.
2a. Humoral immune response
Antibodies (Ab), also named immunoglobulins (Ig), are the effecter molecules of the humoral immune response (Figure 1). They are antigen-specific proteins produced by B lymphocytes in response to antigens. They recognize antigenic regions in a conformation- dependent manner.
Antibody effector functions include neutralization or opsonization of antigens or micro- organisms, activation of complement and binding to Fc receptors on effecter cells.
Priming of naive lymphocytes occurs in the B cell follicles in the lymph nodes (4). Here, B lymphocytes can encounter soluble antigen infused via the lymph or antigen presented on the surface of dendritic cells (5). The B cell receptor (BCR) is a monoclonal Ig expressed on the cell surface. Upon antigen recognition, antigen-specific B lymphocytes take up the antigen and migrate to the border of the follicle and the T cell area. Here, they can receive signals from helper T lymphocytes that recognize antigen in MHC class II on the B lymphocytes initiating further proliferation and isotype switching. Some of the activated B lymphocytes become antibody-secreting plasma cells in the periphery while others become memory B cells residing in the germinal centers.
Figure 1: Antibody structure
An antibody is composed of two identical heterodimers each containing one immunoglobulin (Ig) heavy chain and one immunoglobulin light chain. The antigen binding fragment (Fab), responsible for antigen recognition, is composed of the variable regions of the heavy and light chain. The constant region of an antibody (Fc) consists of the C-terminal constant domains of the two heavy chains. The biological function of an Ab is defined by the molecules they interact with via their Fc, for example complement or Fc receptors.
2b. Cellular immune response
The cellular arm of the adaptive immune system consists of T lymphocytes. T lymphocytes recognize degraded antigenic fragments presented by MHC molecules on target cells via their T cell receptor (TCR). Two main populations of T lymphocytes are distinguished: T helper lymphocytes (Th) expressing co-receptor CD4 and cytotoxic T lymphocytes (CTL) expressing co-receptor CD8. CTL are the main effecter cells that can eradicate tumors and virus-infected cells. They continuously monitor the environment for signs of abnormality by sampling MHC/
peptide complexes. This phenomenon is referred to as immune surveillance. Th are important during the initiation, amplification and regulation of the CTL response (6). In addition, Th are important for the development of an antigen-specific B cell response. Within the population of T helper lymphocytes, several subsets with specialized functions have been identified, such as regulatory T lymphocytes, that are involved in regulation of the adaptive immune response.
CTL and Th differ in their mode of antigen recognition; CTL recognize antigenic peptides in MHC class I molecules while Th recognize antigenic peptides in MHC class II molecules on antigen-presenting cells. MHC class I molecules are expressed by all nucleated cells, while MHC class II molecules are presented exclusively on antigen-presenting cells (APC) such as B lymphocytes and DCs.
Ligands for MHC class I are generally derived from endogenous proteins after normal turnover or breakdown of misfolded proteins or defective ribosomal products (7). Viral proteins are presented by MHC class I on the infected host cell. The peptides that bind to MHC class I have a defined length of 8-10 amino acids. The main enzyme complex involved in degradation of protein antigen into peptides is the proteasome, localized in the cytosol. After transport from the cytosol to the endoplasmatic reticulum (ER) by the transporter associated with antigen processing (TAP), peptides are loaded on MHC class I molecules.
MHC class II ligands are generally derived from exogenous antigens captured by APCs. After uptake, the exogenously-derived antigen travels via the endocytic route towards the lysosomes where they are degraded into peptides. MHC class II binding peptides are longer than MHC class I binding peptides. APCs have dedicated organelles to facilitate efficient loading of antigenic peptides in MHC class II molecules (8,9). These MHC class II compartments (MIIC) are multi- vesicular endosomes that express high levels of MHC class II and are abundantly present in immature dendritic cells. Here, the optimal pH and the presence of cathepsins and HLA-DM together facilitate invariant chain degradation and exchange for antigenic peptides after fusion with the lysosomes. After encounter of maturation stimuli, MHC class II molecules are rapidly redistributed to the cell surface (8).
3. Dendritic cells as director of the T cell response
Dendritic cells (DC) are antigen presenting cells that are strategically positioned at the peripheral borders of the body. They are sentinel cells that can sense pathogens, capture antigens and transport their cargo to the lymph nodes, the exclusive site of T lymphocyte induction. Migration of DCs from tissues to the lymph node requires phenotypical changes such as upregulation of the chemokine receptor CCR7 (10). En route, the antigen is proteolytically degraded into peptides and loaded on MHC molecules that appear at the cell surface. DCs can present exogenous antigen-derived peptides both in MHC class I and MHC class II. Upon encounter of the cognate antigen in MHC, antigen-specific T lymphocytes are activated and clonally expand.
The extent of T lymphocyte activation, expansion and survival that follows antigen recognition by the T cell receptor (TCR), together called priming, is dependent on the strength of TCR stimulation, the availability of pro-survival cytokines, and the presence of co-stimulatory signals.
Dendritic cells are a unique population of cells that can provide all of these signals together (11).
Since ten years, it has been recognized that DCs are not one single cell type but actually a family of different cells with specialized functions that can be distinguished by phenotypical markers (BOX 1).
A critical requirement for effective T lymphocyte priming is DC maturation (11). During this differentiation process, DCs upregulate several molecules that are important for the interaction with T lymphocytes such as CD40 and co-stimulatory molecules of the B7 and TNF receptor families. Co-stimulatory molecules interact with counter-receptors or ligands on the cell surface of the T lymphocyte that are important for proliferation and survival. Co-stimulation lowers the threshold for antigen recognition by the TCR and qualitatively improves the effector T cell response and memory formation (22,23). Mature DCs additionally produce several soluble factors, for example cytokines such as IL-12 that are required for the generation of effector T
cell responses (24). Immature or not properly matured DCs induce anergic or apoptotic T cells (24,25).
DC maturation can be induced by various stimuli, either in a Th dependent or independent fashion (11,25). Th dependent DC maturation is mediated in the lymph node by interaction of CD40 on the DC with CD40 ligand (CD40L) on the T helper lymphocytes (25). Th independent stimuli for DC maturation are pathogen-derived signals and endogenous danger signals (24).
Several families of pathogen-recognition receptors have been described:
Toll-like receptors (TLRs) constitute a family of sensing receptors that recognize microbial structures from bacteria, viruses, fungi or parasites (26). TLRs can also sense self proteins such as interleukin-1 and heat shock proteins (24,27). TLRs 3, 7, 8 and 9 are located in the endosomes while the other TLRs are located at the cell surface. The differences in locations have functional implications for the ligands they can bind (26). In general, TLR ligation induces several signalling pathways that lead to broad DC maturation characterized by upregulation of co-stimulatory molecules, antigen presentation capacity and cytokine production (28-30).
The nucleotide oligomerization domain (NOD)-like receptors (NLRs) cover a family of receptors situated in the cytosol. Members of the NOD subfamily, composed of NOD1 and NOD2, recognize bacterial peptidoglycans and subsequently induce production of pro-inflammatory cytokines; the NALP and NAIP subfamilies recognize ligands of various nature such as bacterial peptidoglycans and uric acid crystals (26,29). Together with other proteins such as caspases they are organized in multi-subunit complexes called inflammasomes involved in induction of IL-1 family members (31). Intracellular recognition of viral DNA or RNA is mediated by cytoplasmic caspase-recruiting domain (CARD) helicases such as retinoic acid inducible gene I (RIG-I) (29,32). Pathogen-derived carbohydrate structures can be sensed by C-type lectin receptors (33,34). In addition to direct recognition, DCs can indirectly sense opsonised pathogens or antigens via Fc receptors (FcRs) (3). FcR ligation leads to DC maturation. FcR-mediated DC maturation is studied in detail in chapter 5.
4. Cross-priming
An important function of DCs is their ability to induce CTL responses to exogenous antigens, a process referred to as cross-priming (35). Cross-priming is important for the induction of immune responses against virus-infected cells (36,37) or tumors (38). These cells are incompetent to initiate CTL responses themselves because they lack the requirements to activate naive T lymphocytes. DCs obtain these requirements after maturation. Since they are not always infected by the virus or lack expression of tumor antigens, DCs have to acquire the antigens from the external milieu.
Immature DCs are very efficient in capturing exogenous antigen (39). Uptake can involve distinct processes defined by the nature of the antigen: macro-pinocytosis, phagocytosis or receptor-mediated endocytosis. Macro-pinocytosis is a process of non-specific antigen capture that is very efficient in DCs. Phagocytosis is involved in the acquisition of particulate antigen such as bacteria or apoptotic or necrotic cells (40). In addition, DCs have numerous receptors for endocytosis of exogenous soluble antigen such as C-type lectin receptors (CLR), scavenger receptors and Fc receptors. CLRs cover a diverse family of receptors that each recognize unique carbohydrate structures present on micro-organisms (34,41).
BOX 1: Mouse dendritic cell subsets
The different subtypes that together make up the heterogeneous population of dendritic cells are extensively studied in the mouse. As suggested by the name, lymph node-resident DCs can be found only in the lymph nodes. They differentiate into DCs from blood-borne precursors.
Lymph-node resident DCs can be divided in conventional DCs (cDCs) and plasmacytoid DCs (pDCs) (12). cDC can be further divided in CD8α+/CD11b- DCs and CD8α-/CD11b+
DCs. CD8α+ cDCs express many antigen receptors and pathogen recognition receptors (13) and have a superior capacity for cross-presentation of pathogens (14) and cell-bound antigen (15) compared to CD8- DCs. The capacity for CD8+ DCs to cross-present is not dictated by antigen capture, but rather by differential processing (13,16).
pDCs can be recognized by their expression of B220 and intermediate levels of CD11c.
Comparison of genome-wide expression profiles between cDC and pDCs with other leukocytes showed separate signatures for pDCs and subsets of cDC. However, in addition, a panDC signature, distinct from other families of leukocytes, was observed in both human and mouse DCs (17). pDC have been shown to present antigen to T cells, however, the significance of their role as antigen presenting cells under physiologic circumstances is not established (18). In contrast, pDCs are well-known for their ability to produce large amounts of type I interferons.
Migratory DCs represent about 50% of the DC population in the lymph nodes under steady state conditions (14). Migratory DCs include skin-derived Langerhans cells, dermal DCs or epithelial-derived DCs and can be recognized by the α-integrin CD103 or chemokine receptor CXCR3 (14,19). A subset of CD103+ cells has been described to induce regulatory T lymphocytes (19). Migratory DCs can present antigen to both Th and CTL (20), however, they mainly transfer antigen from the periphery to lymph-node resident cells (21).
BOX 2: Proposed models for cross-presentation of exogenous antigen
The intracellular mechanism of cross-presentation is intriguing because exogenous antigens have to cross at least one membrane to gain access to the MHC class I processing route.
Still exogenous antigen competes out endogenous ligands, indicating the high efficiency of the process of cross-presentation (46). The following models for cross-presentation of exogenous antigen have been reported:
1. In the endosome-to-cytosol model peptides are generated in the cytosol by proteasomal degradation (2,47) and transported into the endoplasmatic reticulum by transporter associated with antigen processing (TAP) (48) for MHC class I loading (49). In this model, the protein antigen needs to cross a membrane one time when it relocates from the endocytic track to the cytosol. Crossing the membrane may occur by simply leaking out the endocytic vesicle or by rupture, degradation or reduced stability of the vesicle membrane (50). Alternatively, a transport channel can facilitate access to the cytosol. This model is described for physiologic antigens such as cell-associated antigen (15) and Ag-Ab complexes (51).
2. In the so-called ER-phagosome model antigen-containing phagosomes are completely self- sufficient for facilitating cross-presentation through acquisition of parts of ER membrane and plasma membrane during phagocytosis (49,52). In addition, binding of the proteasome to the cytoplasmic side of the phagosome explains how the phagosome can be completely self- sufficient (53). Still, this model depends on two membrane transfers: first, the protein antigen needs to relocate from the phagosome into the cytoplasm for proteasomal degradation.
Subsequently, re-entry of the generated peptides is facilitated by TAP that is present in the phagosomal membrane (52). It is not clear how MHC class I-peptide complexes are transported to the cell surface after loading in the phagosome. The ER-phagosome model has been criticised because of the use of non-physiological antigens such as latex beads to establish this model. Moreover, a calculation of the odds predicted that the efficiency of MHC class I molecules available in the vesicle would simply be too low to effectively prime T cells (54). Nevertheless, the notion that the size of the phagocytosed body is associated with the efficiency of cross-presentation is in line with the increased need for more donor ER and plasma membrane providing the vesicle with more MHC class I molecules.
3. Alternatively, it is proposed that some soluble proteins can access the ER via retrograde transport trough the Golgi compartment (55). They can be cross-presented by hi-jacking the pathways for misfolded endogenous proteins that are pumped out of the ER for degradation.
In this model, the protein antigen needs to cross three times the ER membrane.
All three models discussed so far are dependent on translocation of the antigen from the ER or the endosome to the cytosol. The most important candidate to facilitate the translocation is the ER-resident molecule sec61 that has also been found in phagosomes (56,57).
4. In the so-called vacuolar pathway, the internalized antigen is transported, processed into peptides and loaded onto MHC class I peptides within the endosomal/lysosomal compartment.
Processing of the antigen is dependent on endosomal/lysosomal proteases. Proteases such as cathepsin S have a role in TAP/proteasome-independent cross-presentation (58). MHC class I loading in the endocytic track depends on recycling MHC class I molecules that have been internalized together with the endocytosed cargo (50). DCs constitutively endocytose 50%
of cell surface MHC class I molecules within 30 minutes (59). Reportedly, peptide exchange of recycled MHC class I molecules can occur in MHC class II compartments (60). This was associated with cross-presentation of the internalized antigen in a ammonium-chloride sensitive way, suggesting the involvement of acidic compartments (60). Although DC may be capable of generating MHC class I-peptide complexes in the endocytic route, somatic cells mainly use the proteasomal degradation pathway. Since both pathways make use of distinct enzyme systems for protein degradation, likely resulting in different peptide repertoires, this questions the relevance of the vacuolar pathway for CTL induction. On the other hand, the spatial separation of MHC class I-restricted processing and loading of internalized and endogenous antigen ensures that internalized antigen does not have to compete with the pool of endogenous antigen for loading on MHC class I molecules in the ER, which may explain the efficiency of this pathway (45).
5. An alternative way of cross-presentation without crossing a membrane is the transfer of antigenic peptides from the cytoplasm of one cell directly into to the cytoplasm of a neighbouring cell via gap junctions (61). Gap junctions are intercellular channels that facilitate communication between neighbouring cells. Although transfer of antigens via gap junctions is size restricted and the efficiency may be influenced by cytoplasmic proteases, this may be a physiological way of cross-presentation of f.e. viral antigens. In addition, direct transfer of MHC class I/peptide complexes has been reported providing an even more efficient process of cross-presentation (62).
In contrast to other cells, DCs can present antigenic fragments derived from exogenous antigen in MHC class I. Physiological forms of exogenous antigen that can be encountered by DCs in vivo include cell-bound antigen, either apoptotic cells (42), necrotic cells (43) or live cells, virus- like particles or Ag-Ab complexes. Generation of MHC class I ligands derived from exogenous antigen after ingestion is termed cross-presentation. Despite the capacity of macrophages to capture exogenous antigen, DCs have the cell biological machinery suitable for post- internalisation trafficking towards MHC class I. It was shown that DCs and macrophages have a different lysosomal protease content leading to preservation in DCs rather than destruction of the antigen in macrophages (44). Cross-presentation requires access of exogenous antigens to the MHC class I pathway. The cellular compartments involved in this process are not completely understood. BOX 2 contains an overview of various models proposed for cross-presentation.
The models are not mutually exclusive and they might operate together in one cell with different efficiencies depending on the cell type and the nature of the exogenous antigen. Recent studies suggest that the intracellular mechanism of cross-presentation is dependent on the way of antigen entry and presence of an immune stimulus (41,45). In chapter 6 we have studied the intracellular mechanisms involved in cross-presentation of Ag-Ab complexes.
5. FcgR: receptors for IgG
Fc receptors were discovered 50 years ago on macrophages as receptors for opsonized red blood cells (reviewed by (63,64)). Since their discovery, the knowledge has expanded through the successive usage of different available techniques: they were first visualized by binding of radio-labeled antibodies, then characterized by monoclonal antibodies, subsequently further characterized by biochemical approaches and finally, after cloning of the individual genes, the family of FcR was fully appreciated. FcgR KO mice have shown to be excellent tools to study the complex role of FcgRs in vivo.
The constant part (Fc) of immunoglobulins (Ig) are the natural ligands of Fc receptors. Fc receptors for all classes of Ig have been described (reviewed by (63)): Our work focuses on FcgR, a family of receptors for IgG. IgG is the most abundant Ig in blood and has the most significant effector functions.
Until recently, three different FcgR were known in mice, FcgRI, FcgRIIb and FcgRIII (64).
In 2002, a fourth type of FcgR, FcgIV was described (65) and a few years later this receptor
Figure 2: Mouse FcgRs
Activating FcgRs are multimeric receptors composed of a ligand-binding a-chain and a signal-transducing g-chain dimer. The g-chain is important for cell surface expression of the α-chain and the initiation of the signaling pathways downstream of receptor ligation (68). For this purpose it contains an immunoreceptor tyrosine based activating motif (ITAM). The inhibitory receptor is a single-chain receptor containing an immunoreceptor tyrosine based inhibitory motif (ITIM) in its cytoplasmic domain (64,67).
was functionally characterized (66). FcgRs are expressed on most hematopoietic cells (Table 1, reviewed by (63,67)). They are usually expressed at the plasma membrane but some FcgRs can be released as soluble molecules. This has been shown for FcgRIIb and FcgRIII (63).
Table 1: Characteristics of mouse Fcγ receptors
Name Function Expression pattern Isotype preference Affinity for
IgG2a
FcγRI Activation monocytes, MFs, DCs IgG2a High affinity
FcγRIIb Inhibition B cells, monocytes, MFs, DCs, neutrofils,
mast cells IgG1 > IgG2b > IgG2a Low affinity
FcγRIII Activation monocytes, Mfs, DCs, neutrofils, mast cells,
NK cells IgG1 > IgG2b > IgG2a Low affinity
FcγRIV Inhibition monocytes, MFs, DCs, neutrofils IgG2a = IgG2b Intermediate affinity
The FcgR are functionally divided in two classes – the activating receptors FcgRI, FcgRIII and FcgRIV and the inhibitory receptor FcgRIIb. These classes are also structurally different (see Figure 2). Both functional types of Fcg receptors are commonly co-expressed on the same cell. This co-expression functions as a threshold for activation. Upon ligation of both types of receptors, the concerted action of the activating and inhibitory receptor defines the outcome of the cellular response (69). The cellular outcome depends on (i) the density/class of the individual receptors on the effector cell (ii) the strength of the interaction between receptor and ligand and (iii) the level of opsonization of the Ag (70,71).
IgG has four subclasses in mice: IgG1, IgG2a, IgG2b and IgG3. The four different subclasses -also called isotypes- of IgG have different abilities to mediate effector functions. This is defined by the affinity of the ligands for the different FcgRs (72). IgG2a binds to all FcgRs, however, has a 100-1000 higher affinity for FcgRI. This has been attributed to an additional immunoglobulin- like domain in the α-chain of the receptor (73). In vivo, FcgRI is constantly saturated with monomeric IgG2a (72). Interestingly, the antigen-binding part of FcgRIIb and FcgRIII are very similar (65). This is reflected by a similar affinity for the subclasses (Table 1, reviewed by (67)):
IgG1 interacts with FcgRIIb and FcgRIII while IgG2b interacts with FcgRIIb, FcgRIII and FcgRIV. IgG3 hardly interacts with FcgRs.
Binding of FcgR by monomeric IgG molecules does not lead to cellular activation. Instead, multimeric IgGs present in Ag-Ab complexes are the physiological activators of these FcgR.
The requirement for a multimeric ligand to activate the FcgR has led to the model of receptor clustering (74); Binding of multimeric IgGs to multiple FcgRs is a physical event that aggregates the receptors (63). Presumably, each receptor is associated to a kinase that cannot phosphorylate its own receptor because of steric hindering. Aggregation of two or more receptors enables the kinases to phosphorylate each other and the immunoreceptor tyrosine-based activation motif (ITAM) of the adjacent receptor. In contrast to homo-clustering, referring to aggregation of two identical receptors, the clustering model can also include different types of receptors called hetero-clustering. An example of hetero-clustering is the aggregation of FcgR and the
complement receptor, binding to the same ligand, i.e. complement-Ag-Ab complex, leading to enhanced activation compared to ligation of FcgR alone.
The critical event after cross-linking of FcgR is phosforylation of tyrosines in ITAM by SRC family kinases LYN and HCK followed by recruitment and activation of spleen tyrosine kinase (SYK). Phosfotyrosines serve as binding sites for proteins with src homology 2 (SH2) domains, such as spleen tyrosine kinase (SYK) that is recruited. SYK binding to ITAM then leads to LYN-dependent tyrosine phosphorylation and activation of SYK (63). SYK phosphorylates downstream signal-transduction molecules finally leading to activation of transcription factors such as activator protein 1 (AP-1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) transcription factor complexes, involved in transcription of several genes such as anti-apoptotic and cytokines genes. Another important downstream effect is the generation of second messengers such as inositol (1,4,5)-triphosphate (IP3) that trigger mobilisation of intracellular calcium stores and influx of extra-cellular calcium. Calcium signaling ultimately leads to activation of nuclear factor of activated T cells (NFAT) and calcium-regulated transcription factors such as CREB (75).
Co-ligation of activating FcgRs and FcgRIIb inhibits cellular activation. The mechanism of regulating ITAM signalling is dependent on ITIM. After phosphorylation by Lyn, ITIM recruits phosphatases SHP-1, SHP-2 and SHIP that decrease the activation of molecules in the signaling cascade downstream of ITAM by de-phosphorylation.
FcgR ligation leads to ingestion of the antigen and/or activation of the effector cell. FcgRs are expressed on various cell types (Table 1). Although the intracellular signal transduction module might be similar between different cell types, the outcome of the signaling is cell-type specific (reviewed by (63)). FcgR ligation on monocytes and macrophages induces antibody-dependent cytotoxicity (ADCC), characterized by the production of superoxide and inflammatory cytokines (76). FcgR ligation on mast cells leads to fast release of inflammatory mediators, responsible for the onset of allergic reactions and anaphylaxis. In mice these responses can be induced by both IgE and IgG immune complexes, suggesting redundancy between FcgR and FceR. NK cells can kill IgG-opsonized targets by ADCC through FcgRIII. On neutrophils, FcgR ligation leads to respiratory burst.
6. FcgR function on DCs
As mentioned previously, FcgRs on DCs can facilitate uptake of antibody-bound exogenous antigen. FcgR-mediated uptake enables efficient cross-presentation of antigen to CTL (2,3).
In addition, FcgR ligation by antigen-antibody complexes leads to DC maturation. It has been demonstrated in mouse dendritic cells that Syk is non-redundant for FcgR-mediated DC maturation (77). FcgR function on DCs is investigated in detail and will be discussed in chapters 2, 5 and 6.
Expression of the individual FcgRs has been studied on different subsets of dendritic cells.
Bone-marrow derived dendritic cells express mainly FcgRI and FcgRIII and lower levels of FcgRIIb and FcgRIV (data not shown). All FcgRs are expressed on mouse Langerhans cells. On these cells, FcgRI and FcgRIIb are the most important receptors for uptake and presentation of immune complexes to T helper lymphocytes in vitro (78). Both CD8+ and CD8- resident DCs express FcgRI, FcgRIIb and FcgRIII, although CD8+ DCs have higher levels (79). Cross-
presentation of Ag-Ab complexes by CD8- splenic DCs is FcR g chain-dependent while splenic CD8+ DCs can cross-present Ag-Ab complexes in the absence of FcgR (79).
Mouse plasmacytoid dendritic cells (pDC) lack expression of activating FcgR but only express FcgRIIb. Uptake of Ag-Ab complexes by pDCs does not lead to DC maturation and antigen presentation to T lymphocytes (80). In contrast, human pDCs express at least FcgRIIA (81).
Loading of Ag-Ab complexes on pDCs via this receptor does result in cross-presentation (81).
7. Complement
The complement system is a biochemical cascade that complements the ability of antibodies to clear pathogens (82). The main physiological functions of the complement system include rapid defence against pathogens and killing of target cells. The complement system consists of more than 30 proteins found in plasma and on cell surfaces that are mainly synthesized by liver hepatocytes. They mainly circulate as pro-proteins until the amplifying cascade of proteolytic cleavages is initiated.
The complement cascade can be initiated by three different pathways: the classical pathway, the alternative pathway, and mannose-binding lectin pathway (82). Each pathway has its own activation and recognition mechanism. The three pathways converge at the central molecule C3, that is cleaved by C3-convertases into the fragments C3a and C3b. Binding of C3b enables clearance of pathogens as well as the generation of the membrane-attack complex. This pore- like complex inserts into cell membranes causing damage by lytic or nonlytic mechanisms, referred to as complement-dependent cytotoxicity. Through this mechanism complement can enhance the effector functions of monoclonal antibodies (83).
C1q is the recognition component of the classical complement pathway. The main ligands for C1q are the Fc regions of Ig molecules that are complexed with Ag (84). Binding of C1q to Ag-Ab complexes leads to activation of the complement cascade and is important for clearance of Ag-Ab molecules from the circulation (85,86). In addition, through interaction with Ag-Ab complexes C1q can co-operate with FcgR-mediated effector functions such as phagocytosis (87). Interestingly, immature dendritic cells are an important source of C1q (88). Co-operation between C1q and FcgR in targeting of Ag-Ab complexes to DCs will be discussed in chapter 3.
8. Cancer immunotherapy
Cancer cells can be immunogenic when they express abnormal proteins that serve as targets for the immune system (89,90). These targets are referred to as “ tumor antigens”. Tumor antigens can be mutated proteins, overexpressed proteins, proteins that are selectively expressed in certain tissues or virus-induced proteins (91). Spontaneous immune responses to these antigens occur when CTL notice the signs of abnormality during immune surveillance. However, in some cases this immune response is not sufficient to cause tumor regression. In addition, immune suppression by the tumor and the tumor microenvironment can dampen the immune response.
The goal of immunotherapy is to eradicate the tumor by immunological mechanisms. Optimal immunotherapy should be two-sided, on the one hand directed to overcome tolerance to the tumor by enhancing the adaptive immune response, on the other hand to deal with the mechanisms of immune suppression and immune escape (92).
Within the field of immunotherapy of cancer two main types of approaches are distinguished;
passive therapy and active therapy. Examples of passive immunotherapy include the application of tumor-specific monoclonal antibodies or adoptive T lymphocyte therapy in combination
with immune-modulating regimens. Monoclonal antibodies are the most important new drugs approved for the treatment of cancer (92). They bind either surface proteins that are highly expressed on certain tumors such as CD20, CD33, CD52, epidermal growth factor receptor (EGFR) and HER2/neu or interfere with tumor angiogenesis by binding vascular endothelial growth factor (VEGF). The antibodies have generally been designed to directly affect signaling pathways that are crucial for the malignant phenotype. Binding of the antibody to the target directly provides anti-tumor activity, however, other antibody effector functions may add to the effectiveness of this therapy as well. This will be discussed in chapter 4.
Vaccination is a main example of active immunotherapy. Therapeutic vaccination aims to induce or enhance the endogenous tumor-specific T lymphocyte response. Optimal vaccines induce both tumor-specific CTL, the cells that eventually eradicate the tumor, and helper T lymphocytes, that cooperate in the establishment of the CTL response (6,93) and may exert anti- tumor effects via activation of other immune cells. Examples of therapeutic vaccines include whole tumor cells or tumor cell lysates co-administered with e.g. GM-CSF (94) or well-defined antigen-specific vaccines (95).
Vaccination with multiple synthetic peptides is the most comprehensible vaccination strategy (95). It permits “off-the shelf ” use and can be designed in such a way that it allows immunity to subdominant epitopes. Moreover, it is an efficient way to combine epitopes of different antigens in one vaccine. Initial studies using minimal MHC class I binding epitopes showed promising results in pre-clinical models, however, therapeutic effects in patients were rare. This could be explained by the short half life of MHC-peptide complexes, the loading of the processing- independent short peptides on non-professional APCs and the lack of help by CD4 T cells (96).
Using longer processing-dependent peptides enhances immunogenicity probably because the longer peptides are dependent on cross-presentation by DCs (97,98). Multiple vaccinations with a set of overlapping synthetic long peptides (SLP) of the HPV16 onco-proteins E6 and E7 can induce effective Th and CTL responses in cervical cancer patients (99). The vaccine-induced immunological responses correlate with enhanced clinical responses in a cohort of patients with vulvar neoplasia (100). As shown in mouse models, the efficiency of a SLP vaccine can be enhanced by conjugation of the long peptide to a TLR-ligand (101).
Alternatively, whole antigens are included in therapeutic vaccines, either applied as recombinant protein, DNA or RNA. Whole antigen vaccines have the advantage that they harbor all possible CTL and helper epitopes (95). Moreover, pre-defined knowledge of the immuno-dominant epitopes is not necessary, because of natural processing and epitope selection by the host antigen presenting cells. Therefore, whole antigen vaccines can be applied to patients irrespective of HLA type.
As mentioned before, DCs are of central importance in initiation and direction of the adaptive immune response. Therefore, DCs play a central role in the design of therapeutic anticancer vaccines (102). Making use of antigen receptors present on DCs enhances the efficiency of antigen delivery to DCs (103). Receptors used for antigen targeting include integrins, sialic- acid-binding immunoglobulin-like lectin receptors (SIGLEC), C-type lectin receptors (CLR) and FcgRs (103). Many receptors used in targeting studies belong to the family of CLRs, such as CD205, DC-SIGN and the mannose receptor (MR). Two different strategies are used to target these receptors; (i) binding of their natural ligand, for example in the case of mannose or mannan as ligands of the MR (34) or (ii) targeting the receptor with antigen conjugated to receptor-specific antibodies (1,104). Antigen presentation by CLRs alone can result in tolerance, because most CLRs do not induce DC maturation. Thus, for optimal T lymphocyte induction inclusion of a separate adjuvant in the vaccine is required.
9. A short history of FcgR-mediated antigen targeting to dendritic cells
Already more than 25 years ago it was observed that antibodies specific for hepatitis B virus (HBV) envelop antigens enhance HBV-specific T cell responses (105). Since then, it has been demonstrated that targeting of Ag-Ab complexes (immune complexes, IC) to FcgR on DCs leads to profound T cell induction both in vitro (2) and in vivo (3).
In 1994, Sallusto et al demonstrated that antibodies enhance the presentation of soluble antigen by human monocyte-derived DCs to T helper lymphocytes (106). In 1999, Regnault et al showed that antibodies also enhanced cross-presentation of soluble antigen to CTL by using mouse spleen-derived and bone-marrow derived DCs (2). This study showed that cross- presentation of Ag-Ab complexes is mediated by a proteasome- and TAP-dependent pathway.
In addition, it was shown that Ag-Ab complexes induce FcgR-dependent DC maturation. In 2002, Schuurhuis et al have defined in detail the requirements of cross-presentation of Ag-Ab complexes by demonstrating that optimal cross-presentation requires an optimal ratio of Ag and Ab (3). Moreover, it was shown that in vitro pre-loading of DCs with optimal complexes efficiently induces CTL responses in vivo. The induced CTL were able to kill peptide-loaded target cells in vitro. In the same year, Rafiq et al showed that injection of DCs loaded with Ag-Ab complexes induces tumor protection (107). The ability to induce tumor protection is dependent on expression of FcR g chain, TAP, B2M and MHC class II on DCs, suggesting a role for both Th and CTLs. Moreover, it was shown in vitro that Ag-Ab complexes induce Th and CTL proliferation. The Th response was characterized by IFN-g production rather than IL-4 production.
Together, these studies suggested that FcgR-mediated antigen targeting to dendritic cells is an effective approach for T lymphocyte-based immunotherapy.
10. Outline of this thesis
In this thesis we further evaluated FcgR-mediated Ag targeting to DCs in the context of T lymphocyte-directed immunotherapy. In chapter 2, we studied induction tumor-specific T lymphocytes in mice after both ex vivo DC loading and in vivo targeting of DCs with Ag-Ab complexes. In chapter 3, we studied the role of the different FcgR and complement in the cross- presentation of immune complexes after in vivo targeting of DCs. In chapter 4, we studied the role of circulating antibodies in the induction of specific T lymphocyte responses.
The second part of this thesis zooms in on the interaction of immune complexes with FcgR on DCs to better understand the mechanisms underlying the superior T lymphocyte induction after FcgR-mediated Ag targeting. In chapter 5, we performed a detailed analysis of the differentiation in DCs after incubation with Ag-Ab complexes by micro-array. Here, we specifically focused on the role of the inhibitory Fcg receptor versus the activating Fcg receptors. In chapter 6, we studied the kinetics of cross-presentation by DCs after FcgR-mediated antigen delivery. In this study we found that intracellular antigen storage compartments facilitate prolonged cross- presentation capacity of DCs after incubation with immune complexes.
A better understanding of FcgR biology on dendritic cells provide a fundamental basis for the translation of this vaccination strategy towards clinical application. In chapter 7, the results obtained in this study will be summarized and discussed in the context of T lymphocyte-directed immunotherapy.
References
1. Steinman, R. M. 2008. Dendritic cells in vivo: a key target for a new vaccine science. Immunity. 29:319.
2. Regnault, A., D. Lankar, V. Lacabanne, A. Rodriguez, C. Thery, M. Rescigno, T. Saito, S. Verbeek, C.
Bonnerot, P. Ricciardi-Castagnoli, and S. Amigorena. 1999. Fcgamma receptor-mediated induction of dendritic cell maturation and major histocompatibility complex class I-restricted antigen presentation after immune complex internalization. J. Exp. Med. 189:371.
3. Schuurhuis, D. H., A. Ioan-Facsinay, B. Nagelkerken, J. J. van Schip, C. Sedlik, C. J. Melief, J. S. Verbeek, and F.
Ossendorp. 2002. Antigen-antibody immune complexes empower dendritic cells to efficiently prime specific CD8+ CTL responses in vivo. J. Immunol. 168:2240.
4. Pape, K. A., D. M. Catron, A. A. Itano, and M. K. Jenkins. 2007. The humoral immune response is initiated in lymph nodes by B cells that acquire soluble antigen directly in the follicles. Immunity. 26:491.
5. Wykes, M., A. Pombo, C. Jenkins, and G. G. MacPherson. 1998. Dendritic cells interact directly with naive B lymphocytes to transfer antigen and initiate class switching in a primary T-dependent response. J. Immunol.
161:1313.
6. Ossendorp, F., R. E. Toes, R. Offringa, S. H. van der Burg, and C. J. Melief. 2000. Importance of CD4(+) T helper cell responses in tumor immunity. Immunol. Lett. 74:75.
7. Yewdell, J. W. 2007. Plumbing the sources of endogenous MHC class I peptide ligands. Curr. Opin. Immunol.
19:79.
8. Kleijmeer, M., G. Ramm, D. Schuurhuis, J. Griffith, M. Rescigno, P. Ricciardi-Castagnoli, A. Y. Rudensky, F. Ossendorp, C. J. Melief, W. Stoorvogel, and H. J. Geuze. 2001. Reorganization of multivesicular bodies regulates MHC class II antigen presentation by dendritic cells. J. Cell Biol. 155:53.
9. Turley, S. J., K. Inaba, W. S. Garrett, M. Ebersold, J. Unternaehrer, R. M. Steinman, and I. Mellman. 2000.
Transport of peptide-MHC class II complexes in developing dendritic cells. Science 288:522.
10. Randolph, G. J., J. Ochando, and S. Partida-Sanchez. 2008. Migration of dendritic cell subsets and their precursors. Annu. Rev. Immunol. 26:293.
11. Steinman, R. M. 2007. Dendritic cells: understanding immunogenicity. Eur. J. Immunol. 37 Suppl 1:S53-S60.
12. Heath, W. R., G. T. Belz, G. M. Behrens, C. M. Smith, S. P. Forehan, I. A. Parish, G. M. Davey, N. S. Wilson, F. R. Carbone, and J. A. Villadangos. 2004. Cross-presentation, dendritic cell subsets, and the generation of immunity to cellular antigens. Immunol. Rev. 199:9.
13. Schnorrer, P., G. M. Behrens, N. S. Wilson, J. L. Pooley, C. M. Smith, D. El-Sukkari, G. Davey, F. Kupresanin, M. Li, E. Maraskovsky, G. T. Belz, F. R. Carbone, K. Shortman, W. R. Heath, and J. A. Villadangos. 2006. The dominant role of CD8+ dendritic cells in cross-presentation is not dictated by antigen capture. Proc. Natl.
Acad. Sci. U. S. A 103:10729.
14. Villadangos, J. A., and P. Schnorrer. 2007. Intrinsic and cooperative antigen-presenting functions of dendritic- cell subsets in vivo. Nat. Rev. Immunol. 7:543.
15. den Haan, J. M., S. M. Lehar, and M. J. Bevan. 2000. CD8(+) but not CD8(-) dendritic cells cross-prime cytotoxic T cells in vivo. J. Exp. Med. 192:1685.
16. Dudziak, D., A. O. Kamphorst, G. F. Heidkamp, V. R. Buchholz, C. Trumpfheller, S. Yamazaki, C. Cheong, K. Liu, H. W. Lee, C. G. Park, R. M. Steinman, and M. C. Nussenzweig. 2007. Differential antigen processing by dendritic cell subsets in vivo. Science 315:107.
17. Robbins, S. H., T. Walzer, D. Dembele, C. Thibault, A. Defays, G. Bessou, H. Xu, E. Vivier, M. Sellars, P.
Pierre, F. R. Sharp, S. Chan, P. Kastner, and M. Dalod. 2008. Novel insights into the relationships between dendritic cell subsets in human and mouse revealed by genome-wide expression profiling. Genome Biol. 9:R17.
18. Villadangos, J. A., and L. Young. 2008. Antigen-presentation properties of plasmacytoid dendritic cells.
Immunity. 29:352.
19. Pulendran, B., H. Tang, and T. L. Denning. 2008. Division of labor, plasticity, and crosstalk between dendritic cell subsets. Curr. Opin. Immunol. 20:61.
20. Bedoui, S., P. G. Whitney, J. Waithman, L. Eidsmo, L. Wakim, I. Caminschi, R. S. Allan, M. Wojtasiak, K.
Shortman, F. R. Carbone, A. G. Brooks, and W. R. Heath. 2009. Cross-presentation of viral and self antigens by skin-derived CD103+ dendritic cells. Nat. Immunol. 10:488.
21. Allan, R. S., J. Waithman, S. Bedoui, C. M. Jones, J. A. Villadangos, Y. Zhan, A. M. Lew, K. Shortman, W. R.
Heath, and F. R. Carbone. 2006. Migratory dendritic cells transfer antigen to a lymph node-resident dendritic cell population for efficient CTL priming. Immunity. 25:153.
22. Croft, M. 2009. The role of TNF superfamily members in T-cell function and diseases. Nat. Rev. Immunol.
9:271.
23. Peperzak, V., Y. Xiao, E. A. Veraar, and J. Borst. 2010. CD27 sustains survival of CTLs in virus-infected nonlymphoid tissue in mice by inducing autocrine IL-2 production. J. Clin. Invest 120:168.
24. Joffre, O., M. A. Nolte, R. Sporri, and Reis e Sousa. 2009. Inflammatory signals in dendritic cell activation and the induction of adaptive immunity. Immunol. Rev. 227:234.
25. Schuurhuis, D. H., S. Laban, R. E. Toes, P. Ricciardi-Castagnoli, M. J. Kleijmeer, d. van, V, D. Rea, R. Offringa, H. J. Geuze, C. J. Melief, and F. Ossendorp. 2000. Immature dendritic cells acquire CD8(+) cytotoxic T lymphocyte priming capacity upon activation by T helper cell-independent or -dependent stimuli. J. Exp.
Med. 192:145.
26. Medzhitov, R. 2007. Recognition of microorganisms and activation of the immune response. Nature 449:819.
27. Jung, S., D. Unutmaz, P. Wong, G. Sano, S. K. De los, T. Sparwasser, S. Wu, S. Vuthoori, K. Ko, F. Zavala, E. G. Pamer, D. R. Littman, and R. A. Lang. 2002. In vivo depletion of CD11c(+) dendritic cells abrogates priming of CD8(+) T cells by exogenous cell-associated antigens. Immunity. 17:211.
28. Rescigno, M., M. Martino, C. L. Sutherland, M. R. Gold, and P. Ricciardi-Castagnoli. 1998. Dendritic cell survival and maturation are regulated by different signaling pathways. J. Exp. Med. 188:2175.
29. Lee, M. S., and Y. J. Kim. 2007. Signaling pathways downstream of pattern-recognition receptors and their cross talk. Annu. Rev. Biochem. 76:447.
30. Watts, C., R. Zaru, A. R. Prescott, R. P. Wallin, and M. A. West. 2007. Proximal effects of Toll-like receptor activation in dendritic cells. Curr. Opin. Immunol. 19:73.
31. Martinon, F., A. Mayor, and J. Tschopp. 2009. The inflammasomes: guardians of the body. Annu. Rev.
Immunol. 27:229.
32. Pichlmair, A., and Reis e Sousa. 2007. Innate recognition of viruses. Immunity. 27:370.
33. Geijtenbeek, T. B., and S. I. Gringhuis. 2009. Signalling through C-type lectin receptors: shaping immune responses. Nat. Rev. Immunol. 9:465.
34. van Kooyk, Y., and G. A. Rabinovich. 2008. Protein-glycan interactions in the control of innate and adaptive immune responses. Nat. Immunol. 9:593.
35. Yewdell, J. W., C. C. Norbury, and J. R. Bennink. 1999. Mechanisms of exogenous antigen presentation by MHC class I molecules in vitro and in vivo: implications for generating CD8+ T cell responses to infectious agents, tumors, transplants, and vaccines. Adv. Immunol. 73:1.
36. Melief, C. J. 2003. Mini-review: Regulation of cytotoxic T lymphocyte responses by dendritic cells: peaceful coexistence of cross-priming and direct priming? Eur. J. Immunol. 33:2645.
37. Sigal, L. J., S. Crotty, R. Andino, and K. L. Rock. 1999. Cytotoxic T-cell immunity to virus-infected non- haematopoietic cells requires presentation of exogenous antigen. Nature 398:77.
38. Huang, A. Y., P. Golumbek, M. Ahmadzadeh, E. Jaffee, D. Pardoll, and H. Levitsky. 1994. Role of bone marrow-derived cells in presenting MHC class I-restricted tumor antigens. Science 264:961.
39. Watts, C., and S. Amigorena. 2000. Antigen traffic pathways in dendritic cells. Traffic. 1:312.
40. Albert, M. L., S. F. Pearce, L. M. Francisco, B. Sauter, P. Roy, R. L. Silverstein, and N. Bhardwaj. 1998.
Immature dendritic cells phagocytose apoptotic cells via alphavbeta5 and CD36, and cross-present antigens to cytotoxic T lymphocytes. J. Exp. Med. 188:1359.
41. Burgdorf, S., and C. Kurts. 2008. Endocytosis mechanisms and the cell biology of antigen presentation. Curr.
Opin. Immunol. 20:89.
42. Albert, M. L., B. Sauter, and N. Bhardwaj. 1998. Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature 392:86.
43. Sancho, D., O. P. Joffre, A. M. Keller, N. C. Rogers, D. Martinez, P. Hernanz-Falcon, I. Rosewell, and Reis e Sousa. 2009. Identification of a dendritic cell receptor that couples sensing of necrosis to immunity. Nature 458:899.
44. Delamarre, L., M. Pack, H. Chang, I. Mellman, and E. S. Trombetta. 2005. Differential lysosomal proteolysis in antigen-presenting cells determines antigen fate. Science 307:1630.
45. Burgdorf, S., C. Scholz, A. Kautz, R. Tampe, and C. Kurts. 2008. Spatial and mechanistic separation of cross- presentation and endogenous antigen presentation. Nat. Immunol. 9:558.
46. Yewdell, J. W. 2005. The seven dirty little secrets of major histocompatibility complex class I antigen processing. Immunol. Rev. 207:8.
47. Norbury, C. C., L. J. Hewlett, A. R. Prescott, N. Shastri, and C. Watts. 1995. Class I MHC presentation of exogenous soluble antigen via macropinocytosis in bone marrow macrophages. Immunity. 3:783.
48. Huang, A. Y., A. T. Bruce, D. M. Pardoll, and H. I. Levitsky. 1996. In vivo cross-priming of MHC class I-restricted antigens requires the TAP transporter. Immunity. 4:349.
49. Ackerman, A. L., and P. Cresswell. 2004. Cellular mechanisms governing cross-presentation of exogenous antigens. Nat. Immunol. 5:678.
50. Vyas, J. M., d. van, V, and H. L. Ploegh. 2008. The known unknowns of antigen processing and presentation.
Nat. Rev. Immunol. 8:607.
51. Rodriguez, A., A. Regnault, M. Kleijmeer, P. Ricciardi-Castagnoli, and S. Amigorena. 1999. Selective transport of internalized antigens to the cytosol for MHC class I presentation in dendritic cells. Nat. Cell Biol. 1:362.
52. Guermonprez, P., L. Saveanu, M. Kleijmeer, J. Davoust, E. P. van, and S. Amigorena. 2003. ER-phagosome fusion defines an MHC class I cross-presentation compartment in dendritic cells. Nature 425:397.
53. Houde, M., S. Bertholet, E. Gagnon, S. Brunet, G. Goyette, A. Laplante, M. F. Princiotta, P. Thibault, D.
Sacks, and M. Desjardins. 2003. Phagosomes are competent organelles for antigen cross-presentation. Nature 425:402.
54. Groothuis, T. A., and J. Neefjes. 2005. The many roads to cross-presentation. J. Exp. Med. 202:1313.
55. Ackerman, A. L., C. Kyritsis, R. Tampe, and P. Cresswell. 2005. Access of soluble antigens to the endoplasmic reticulum can explain cross-presentation by dendritic cells. Nat. Immunol. 6:107.
56. Rock, K. L. 2006. Exiting the outside world for cross-presentation. Immunity. 25:523.
57. Ackerman, A. L., A. Giodini, and P. Cresswell. 2006. A role for the endoplasmic reticulum protein retrotranslocation machinery during crosspresentation by dendritic cells. Immunity. 25:607.
58. Shen, L., L. J. Sigal, M. Boes, and K. L. Rock. 2004. Important role of cathepsin S in generating peptides for TAP-independent MHC class I crosspresentation in vivo. Immunity. 21:155.
59. Basha, G., G. Lizee, A. T. Reinicke, R. P. Seipp, K. D. Omilusik, and W. A. Jefferies. 2008. MHC class I endosomal and lysosomal trafficking coincides with exogenous antigen loading in dendritic cells. PLoS. One.
3:e3247.
60. Gromme, M., F. G. UytdeHaag, H. Janssen, J. Calafat, R. S. van Binnendijk, M. J. Kenter, A. Tulp, D.
Verwoerd, and J. Neefjes. 1999. Recycling MHC class I molecules and endosomal peptide loading. Proc. Natl.
Acad. Sci. U. S. A 96:10326.
61. Neijssen, J., B. Pang, and J. Neefjes. 2007. Gap junction-mediated intercellular communication in the immune system. Prog. Biophys. Mol. Biol. 94:207.
62. Qu, C., V. A. Nguyen, M. Merad, and G. J. Randolph. 2009. MHC class I/peptide transfer between dendritic cells overcomes poor cross-presentation by monocyte-derived APCs that engulf dying cells. J. Immunol.
182:3650.
63. Daeron M. Fc Receptors.
64. Ravetch, J. V., and S. Bolland. 2001. IgG Fc receptors. Annu. Rev. Immunol. 19:275.
65. Mechetina, L. V., A. M. Najakshin, B. Y. Alabyev, N. A. Chikaev, and A. V. Taranin. 2002. Identification of CD16-2, a novel mouse receptor homologous to CD16/Fc gamma RIII. Immunogenetics 54:463.
66. Nimmerjahn, F., P. Bruhns, K. Horiuchi, and J. V. Ravetch. 2005. FcgammaRIV: a novel FcR with distinct IgG subclass specificity. Immunity. 23:41.
67. Nimmerjahn, F., and J. V. Ravetch. 2008. Fcgamma receptors as regulators of immune responses. Nat. Rev.
Immunol. 8:34.
68. Takai, T. 1996. Multiple loss of effector cell functions in FcR gamma-deficient mice. Int. Rev. Immunol. 13:369.
69. Clynes, R., J. S. Maizes, R. Guinamard, M. Ono, T. Takai, and J. V. Ravetch. 1999. Modulation of immune complex-induced inflammation in vivo by the coordinate expression of activation and inhibitory Fc receptors. J. Exp. Med. 189:179.
70. Baudino, L., F. Nimmerjahn, S. S. zeredo da, E. Martinez-Soria, T. Saito, M. Carroll, J. V. Ravetch, J. S.
Verbeek, and S. Izui. 2008. Differential contribution of three activating IgG Fc receptors (FcgammaRI, FcgammaRIII, and FcgammaRIV) to IgG2a- and IgG2b-induced autoimmune hemolytic anemia in mice. J.
Immunol. 180:1948.
71. Fossati-Jimack, L., A. Ioan-Facsinay, L. Reininger, Y. Chicheportiche, N. Watanabe, T. Saito, F. M. Hofhuis, J. E. Gessner, C. Schiller, R. E. Schmidt, T. Honjo, J. S. Verbeek, and S. Izui. 2000. Markedly different pathogenicity of four immunoglobulin G isotype-switch variants of an antierythrocyte autoantibody is based on their capacity to interact in vivo with the low-affinity Fcgamma receptor III. J. Exp. Med. 191:1293.
72. Nimmerjahn, F., and J. V. Ravetch. 2005. Divergent immunoglobulin g subclass activity through selective Fc receptor binding. Science 310:1510.
73. Allen, J. M., and B. Seed. 1989. Isolation and expression of functional high-affinity Fc receptor complementary DNAs. Science 243:378.
74. Schamel W.W.A, and Reth M. 2008. Clustering Models. In Chapter 6; Multichain Immune Recognition Receptor Signaling: From Spatiotemporal Organization to Human Disease. Edited by Alexander B.Sigalov, ed. Landes Bioscience.
75. Ivashkiv, L. B. 2009. Cross-regulation of signaling by ITAM-associated receptors. Nat. Immunol. 10:340.
76. Fanger, M. W., L. Shen, R. F. Graziano, and P. M. Guyre. 1989. Cytotoxicity mediated by human Fc receptors for IgG. Immunol. Today 10:92.
77. Sedlik, C., D. Orbach, P. Veron, E. Schweighoffer, F. Colucci, R. Gamberale, A. Ioan-Facsinay, S. Verbeek, P. Ricciardi-Castagnoli, C. Bonnerot, V. L. Tybulewicz, S. J. Di, and S. Amigorena. 2003. A critical role for Syk protein tyrosine kinase in Fc receptor-mediated antigen presentation and induction of dendritic cell maturation. J. Immunol. 170:846.
78. Yada, A., S. Ebihara, K. Matsumura, S. Endo, T. Maeda, A. Nakamura, K. Akiyama, S. Aiba, and T. Takai.
2003. Accelerated antigen presentation and elicitation of humoral response in vivo by Fcgamma. Cell Immunol. 225:21.
79. den Haan, J. M., and M. J. Bevan. 2002. Constitutive versus activation-dependent cross-presentation of immune complexes by CD8(+) and CD8(-) dendritic cells in vivo. J. Exp. Med. 196:817.
80. Flores, M., D. D. Desai, M. Downie, B. Liang, M. P. Reilly, S. E. McKenzie, and R. Clynes. 2009. Dominant expression of the inhibitory FcgammaRIIB prevents antigen presentation by murine plasmacytoid dendritic cells. J. Immunol. 183:7129.
81. Benitez-Ribas, D., G. J. Adema, G. Winkels, I. S. Klasen, C. J. Punt, C. G. Figdor, and V. de, I. 2006.
Plasmacytoid dendritic cells of melanoma patients present exogenous proteins to CD4+ T cells after Fc gamma RII-mediated uptake. J. Exp. Med. 203:1629.
82. Walport, M. J. 2001. Complement. First of two parts. N. Engl. J. Med. 344:1058.
83. Gelderman, K. A., S. Tomlinson, G. D. Ross, and A. Gorter. 2004. Complement function in mAb-mediated cancer immunotherapy. Trends Immunol. 25:158.
84. Walport, M. J. 2001. Complement. Second of two parts. N. Engl. J. Med. 344:1140.
85. Nash, J. T., P. R. Taylor, M. Botto, P. J. Norsworthy, K. A. Davies, and M. J. Walport. 2001. Immune complex processing in C1q-deficient mice. Clin. Exp. Immunol. 123:196.
86. Sontheimer, R. D., E. Racila, and D. M. Racila. 2005. C1q: its functions within the innate and adaptive immune responses and its role in lupus autoimmunity. J. Invest Dermatol. 125:14.
87. Bobak, D. A., T. A. Gaither, M. M. Frank, and A. J. Tenner. 1987. Modulation of FcR function by complement:
subcomponent C1q enhances the phagocytosis of IgG-opsonized targets by human monocytes and culture- derived macrophages. J. Immunol. 138:1150.
88. Castellano, G., A. M. Woltman, A. J. Nauta, A. Roos, L. A. Trouw, M. A. Seelen, F. P. Schena, M. R. Daha, and C. van Kooten. 2004. Maturation of dendritic cells abrogates C1q production in vivo and in vitro. Blood 103:3813.
89. Boon, T., J. C. Cerottini, E. B. van den, B. P. van der, and P. A. Van. 1994. Tumor antigens recognized by T lymphocytes. Annu. Rev. Immunol. 12:337.
90. van der Bruggen, P., C. Traversari, P. Chomez, C. Lurquin, P. E. De, E. B. van den, A. Knuth, and T. Boon.
1991. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 254:1643.
91. Kessler, J. H., and C. J. Melief. 2007. Identification of T-cell epitopes for cancer immunotherapy. Leukemia 21:1859.
92. Dougan, M., and G. Dranoff. 2009. Immune therapy for cancer. Annu. Rev. Immunol. 27:83.
93. Bijker, M. S., C. J. Melief, R. Offringa, and S. H. van der Burg. 2007. Design and development of synthetic peptide vaccines: past, present and future. Expert. Rev. Vaccines. 6:591.
94. Soiffer, R., T. Lynch, M. Mihm, K. Jung, C. Rhuda, J. C. Schmollinger, F. S. Hodi, L. Liebster, P. Lam, S.
Mentzer, S. Singer, K. K. Tanabe, A. B. Cosimi, R. Duda, A. Sober, A. Bhan, J. Daley, D. Neuberg, G. Parry, J. Rokovich, L. Richards, J. Drayer, A. Berns, S. Clift, L. K. Cohen, R. C. Mulligan, and G. Dranoff. 1998.
Vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte-macrophage colony-stimulating factor generates potent antitumor immunity in patients with metastatic melanoma. Proc.
Natl. Acad. Sci. U. S. A 95:13141.
95. Offringa, R., S. H. van der Burg, F. Ossendorp, R. E. Toes, and C. J. Melief. 2000. Design and evaluation of antigen-specific vaccination strategies against cancer. Curr. Opin. Immunol. 12:576.
96. Bijker, M. S., S. J. van den Eeden, K. L. Franken, C. J. Melief, R. Offringa, and S. H. van der Burg. 2007. CD8+
CTL priming by exact peptide epitopes in incomplete Freund’s adjuvant induces a vanishing CTL response, whereas long peptides induce sustained CTL reactivity. J. Immunol. 179:5033.
97. Melief, C. J., and S. H. van der Burg. 2008. Immunotherapy of established (pre)malignant disease by synthetic long peptide vaccines. Nat. Rev. Cancer 8:351.
98. Bijker, M. S., S. J. van den Eeden, K. L. Franken, C. J. Melief, S. H. van der Burg, and R. Offringa. 2008.
Superior induction of anti-tumor CTL immunity by extended peptide vaccines involves prolonged, DC- focused antigen presentation. Eur. J. Immunol. 38:1033.
99. Welters, M. J., G. G. Kenter, S. J. Piersma, A. P. Vloon, M. J. Lowik, Berends-van der Meer DM, J. W.
Drijfhout, A. R. Valentijn, A. R. Wafelman, J. Oostendorp, G. J. Fleuren, R. Offringa, C. J. Melief, and S.
H. van der Burg. 2008. Induction of tumor-specific CD4+ and CD8+ T-cell immunity in cervical cancer patients by a human papillomavirus type 16 E6 and E7 long peptides vaccine. Clin. Cancer Res. 14:178.
100. Kenter, G. G., M. J. Welters, A. R. Valentijn, M. J. Lowik, Berends-van der Meer DM, A. P. Vloon, F. Essahsah, L. M. Fathers, R. Offringa, J. W. Drijfhout, A. R. Wafelman, J. Oostendorp, G. J. Fleuren, S. H. van der Burg, and C. J. Melief. 2009. Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia. N. Engl.
J. Med. 361:1838.
101. Khan, S., M. S. Bijker, J. J. Weterings, H. J. Tanke, G. J. Adema, H. T. van, J. W. Drijfhout, C. J. Melief, H. S.
Overkleeft, G. A. van der Marel, D. V. Filippov, S. H. van der Burg, and F. Ossendorp. 2007. Distinct uptake mechanisms but similar intracellular processing of two different toll-like receptor ligand-peptide conjugates in dendritic cells. J. Biol. Chem. 282:21145.
102. Melief, C. J. 2008. Cancer immunotherapy by dendritic cells. Immunity. 29:372.
103. Tacken, P. J., V. de, I, R. Torensma, and C. G. Figdor. 2007. Dendritic-cell immunotherapy: from ex vivo loading to in vivo targeting. Nat. Rev. Immunol. 7:790.
104. Mahnke, K., Y. Qian, S. Fondel, J. Brueck, C. Becker, and A. H. Enk. 2005. Targeting of antigens to activated dendritic cells in vivo cures metastatic melanoma in mice. Cancer Res. 65:7007.
105. Celis, E., V. R. Zurawski, Jr., and T. W. Chang. 1984. Regulation of T-cell function by antibodies: enhancement of the response of human T-cell clones to hepatitis B surface antigen by antigen-specific monoclonal antibodies. Proc. Natl. Acad. Sci. U. S. A 81:6846.
106. Sallusto, F., and A. Lanzavecchia. 1994. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J. Exp. Med. 179:1109.
107. Rafiq, K., A. Bergtold, and R. Clynes. 2002. Immune complex-mediated antigen presentation induces tumor immunity. J. Clin. Invest 110:71.
