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.
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Summary and General Discussion
Chap ter 7
1. Prologue
In this thesis we explored several aspects of FcgR-mediated antigen-targeting to dendritic cells in the context of T lymphocyte-based immunotherapy. Together, these data sets highlight that FcgR-mediated antigen targeting is one of the most efficient ways of antigen loading and DC maturation we know today. Although the results of the experiments are discussed extensively in the individual chapters, some remaining issues will be discussed below. Finally, the applicability of FcgR-mediated antigen targeting to DCs for T cell-based immunotherapy and the translational challenges and opportunities of this candidate therapeutic application will be discussed.
2. Summary and discussion
Dendritic cells loaded with antigen-antibody complexes (immune complexes, IC) effectively induce antigen-specific cytotoxic T lymphocytes (CTL) that can eradicate tumor cells in vivo (chapter 2). CTL induction by IC-pre-loaded DCs is superior to in vivo DC targeting with IC. Nevertheless, cross-presentation of antigen complexed with Ig after intravenous injection is more efficient than cross-presentation of soluble non-complexed Ag (chapter 3). Cross- presentation of circulating IC in vivo is crucially dependent on complement molecule C1q. In a similar way as pre-formed IC, circulating antibodies can enhance T cell induction to vaccine antigen by in vivo formation of Ag-Ab complexes (chapter 4). Our work implicates that an ongoing B cell response can drive a T cell responses through in vivo formation of Ag-Ab complexes that are ingested and presented by DCs. This mechanism may explain some clinical observations in which antibody responses precede effective T cell responses, emphasising the clinical significance of our findings. For example, patients infected with hepatitis B virus (HBV) have a higher chance to mount a curative T lymphocyte response when they have high titers of HBV-specific antibodies compared to individuals that do not have specific antibodies (1). In addition, enhanced anti-tumor T lymphocyte responses have been found in patients that have been treated with tumor-specific monoclonal antibodies (reviewed by (2)).
In contrast, antibody-mediated T cell induction may also have a less beneficial effect in some patients. For example, antibody-mediated T cell induction may play a role in antibody-mediated allograft rejection or Ag-Ab complex-associated autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. In this view it is advisable to study the role of prolonged cross-presentation (as discussed in chapter 6) of auto-antigens that are captured by FcgR on antigen presenting cells in the damaged tissues.
To understand the mechanism of T lymphocyte induction after FcgR-mediated Ag targeting, we performed a detailed analysis of the interaction of Ag-Ab complexes with dendritic cells. RNA Microarray analysis has indicated that FcgR ligation on DCs by immune complexes induces robust DC maturation (chapter 5). FcgR-mediated DC differentiation has a unique gene expression signature compared to DC maturation via other receptors and is tightly regulated by FcγRIIb that acts as a threshold for activation. FcgR-mediated DC differentiation is characterized by many features that are associated with optimal initiation and prolongation of a T lymphocyte response (chapter 5).
Optimal CTL induction is further supported by a prolonged cross-presentation capacity of DCs that are loaded with Ag-Ab complexes compared to DCs that are loaded with minimal peptide or soluble Ag (chapter 6). CTL cross-priming requires long-lived presence of MHC class I/peptide complexes on dendritic cells for several reasons: (i) CTL priming occurs exclusively in the lymph nodes (3). Dendritic cells pick up antigen in a peripheral organ and subsequently migrate towards the lymph nodes without encountering this source of antigen again (4). This migration process takes 24-48 hours (5). (ii) For priming of naive T lymphocytes, a ten fold higher density of MHC/
peptide complexes is required compared to recognition by primed or memory T lymphocytes (6). (iii) CD8 T lymphocytes need multiple contacts with antigen-presenting DCs to develop an effector response, whereas one single contact leads to an aberrant response (7). The prolonged cross-presentation capacity observed after FcgR-mediated Ag loading is facilitated by an intracellular antigen storage compartment. This antigen source facilitates a continuous supply of exogenous-derived MHC class I ligands to replace shortlived MHC class I/peptide complexes at the cell surface. As described in chapter 6, both DCs loaded with immune complexes and TLR ligand - long peptide conjugates have a prolonged cross-presentation capacity. Prolonged cross- priming capacity in vivo is also shown for DCs transfected with lentiviral vectors containing a truncated OVA gene (8). In this model, the transfected Ag constitutes a stable source of MHC class I ligands resulting in a very efficient and prolonged CTL induction. Prolonged T cell priming capacity is also shown by studies using DNA vehicles for vaccination (9). However, since these antigen sources are formed by newly translated proteins, they are processed as endogenous antigens and are probably not stored in antigen depots as we have observed in chapter 6.
3. The role of the individual FcgRs in cross-priming of immune complexes by DCs In various chapters we have used FcgR-deficient mouse models to decipher the roles of the individual FcgRs in the interaction of DCs with IC in vitro. Uptake (chapter 6) and cross- presentation (chapter 2) of IC by DCs as well as DC maturation (chapter 5) in vitro are completely dependent on presence of the FcR g chain. More specifically, uptake (data not shown), cross-presentation (chapter 2) and DC maturation (data not shown) are dependent on the presence of either FcgRI or FcgRIII. These two activating FcgRs have a crucial but redundant role in the interaction of DCs with IC in vitro. Consequently, immunisation with IC- loaded DCs lacking both FcgRI and FcgRIII does not protect mice against a subsequent tumor challenge (chapter 2). In contrast to FcgRI and III, we did not find a significant role for FcgRIV in our models. This can be explained by a low expression level of the receptor compared to FcgRI and FcgRIII (data not shown) or the use of rabbit IgG. The affinity of rabbit IgG for mouse FcgRs has not been defined. However, the second IgG (Cg2) domain which is the most important domain for binding to FcgR and C1q is very homologues between rabbit IgG, mouse IgG2a and human IgG1 and IgG3 (10). The latter isotypes bind with high affinity to FcgRI (11) suggesting a similar preference by rabbit IgG. If rabbit IgG has indeed selective affinity for FcgRI, we might have underestimated the role of FcgRIV in our models.
FcgR-induced signalling is likely to play a role in the efficiency of the process of cross-presentation after incubation with Ag-Ab complexes; FcgR ligation induces proteasomal subunit PA28beta expression, thereby enhancing formation of PA28alpha beta complexes promoting proteasome function (12). In addition, micro-array analysis revealed upregulation of several genes associated with MHC class I presentation, such as several MHC class I loci, transporter-associated with antigen presentation 1 (TAP1) and tapasin (chapter 5). The influence of FcgR-dependent
signalling on cross-presentation was separately addressed by using BM-DCs derived from of FcR γ chain transgenic mouse that has two mutations in the ITAM that abrogate downstream signalling but has a close to normal cell surface expression of activating FcgR (13). Interestingly, uptake of IC by DCs derived from this mouse was largely abrogated indicating that uptake of IC is ITAM-dependent. In conclusion, hypothetically, it is likely that FcgR-induced signalling plays a role in enhancing the efficiency of cross-presentation; however, experimentally, the hypothesis is difficult to test.
FcgRIIb has an important role in the regulation of FcgR-mediated DC differentiation. As shown in chapter 5, several processes that are important for CTL induction such as cytokine production, upregulation of co-stimulatory molecules and molecules associated with antigen presentation in MHC class I are higher in DCs that lack FcgRIIb expression. In vitro, mouse DCs lacking FcgRIIb have a better cross-presentation capacity than WT DCs (chapter 5). FcgRIIb not only regulates the magnitude of DC activation, but also prevent spontaneous DC maturation by circulating immune complexes that are present in low numbers in serum of healthy individuals (14). The importance of the regulatory role in the immune system is highlighted by studies that show that FcgRIIb deficient mice are more susceptible to anaphylaxis (15), systemic lupus erythematosus (16) and collagen-induced arthritis (17).
The improved DC maturation and antigen presentation capacity of FcgRIIb KO DCs after FcgR ligation in vitro (described in chapter 5) suggests that FcgRIIb deficient DCs have an improved CTL priming capacity than WT DCs in vivo. However, using different read-outs such as tetramer analysis, ex vivo ELISPOT and proliferation assays, we did not find differences in T cell priming in vivo after immunisation with either WT or FcgRIIb DCs loaded with ICs. The enhanced DC maturation observed in the absence of FcgRIIb might influence especially the quality of the T cell response and the memory formation, while we focused mainly on the quantity of the T cell response in the initiation phase (data not shown). In contrast, Kalergis et al showed that absence of FcgRIIb on DCs is necessary to induce tumor protection with a vaccine composed of DCs loaded with IC (18). Two possible differences in the experimental set-up might underlie the discrepancies between the two studies. (i) We carefully optimized the Ag/Ab ratio which is different from that used by Kalergis et al. Using DCs loaded with optimally formed Ag-Ab complexes is probably sufficient to overcome the threshold for cellular activation by FcgRIIb, while suboptimal Ag-Ab complexes might not overcome the threshold. (ii) Cellular activation depends on the balance between expression of activating and inhibitory FcgRs. This balance is influenced by the cytokine milieu. For example, FcγRIIb is up-regulated after incubation with IL-4 and down-regulated after incubation with IFNγ (Table 1). The cytokine milieu in which the DCs are cultured may be different between the studies.
Together with the literature discussed, our data suggest that FcgR-mediated DC maturation is tightly regulated by FcgRIIb in both mice and humans. This mechanism prevents DC activation under steady state conditions or when suboptimal Ag-Ab complexes bind to FcgRs. During a robust immune response, optimally formed Ag-Ab complexes, comparable to the in vitro formed immune complexes, overcome the threshold for activation of DCs. Consequently, when a maximal immune response is desired but optimal Ag-Ab complexes are not formed, FcgRIIb blocking might be a way to achieve greater therapeutic efficacy (19).
Table 1: Mouse FcγR expression after O/N culture with the indicated cytokines either assessed on RNA or protein level (as indicated).
Cytokine FcγRI FcγRIIb FcγRIII FcγRIV
IL-4 (RNA) - ++ (Protein) + -
IFNγ (RNA) + - + (Protein) ++
IL-13 (Protein) nd + nd nd
IL-10 (RNA) + + (Protein) + (Protein) =
LPS (RNA) + + (Protein) + (Protein) +
Il-33 (Protein) nd + + nd
IL-6 (Protein) nd + nd nd
4. Cooperation of complement and FcγR
Immune complexes are potent initiators of the classical complement pathway through interaction with C1q. In chapter 3 we addressed the role of complement factors in the cross-presentation of immune complexes after intravenous injection. We have shown that cross-presentation of immune complexes is not dependent on complement component C3, however, is highly dependent on interaction with C1q. The dependence on C1q is mediated on the level of antigen uptake by APCs. Isolated splenic DCs from C1q deficient mice had significantly lower levels of engulfed immune complexes than WT DCs. In addition, C1q enhances uptake and cross- presentation of immune complexes by dendritic cells in vitro. Because immature DCs are an important source of C1q (20), the C1q-dependent enhancement can be mediated in the local tissues or the lymph nodes. It would be interesting to investigate the role of locally produced C1q by using a conditional KO mouse lacking C1q expression in dendritic cells
As highlighted in chapter 3, a likely explanation for our findings is functional cooperation between FcgRs and a putative C1q receptor. However, cooperation in uptake has so far only been reported for FcgR and complement receptor 3 (CR3/CD11b). CR3 facilitates binding of the particle while ingestion is mediated by FcγR (21). However, since this receptor is expressed on a limited number of DC subsets, CR3 is probably not involved in enhanced targeting of immune complexes to DCs.
Calreticulin, a candidate receptor for C1q is also of interest, since this molecule is found in our proteomics analysis of the antigen storage compartment. Further research should reveal if calreticulin is involved in enhancing the uptake of cross-presentation of immune complexes that are bound to C1q.
An alternative explanation for enhancement of uptake is a change in the composition of the immune complex when C1q is bound. C1q binds to a region on the Cg2 domain necessary for the interaction with FcgRIIb (22). By occupying this binding site it potentially prevents the interaction with FcgRIIb thus enhancing the uptake, cross-presentation or DC activation. This hypothesis could be addressed by studying DC activation in the presence of C1q or in vivo by using C1q/FcgRIIb double deficient mice.
5. Understanding antigen persistence
The prolonged cross-presentation capacity after FcgR-mediated antigen loading is facilitated by the accumulation of antigen in intracellular antigen storage compartments (chapter 6).
The antigen storage compartments have lysosome-like characteristics such as expression of LAMP. The primary function of lysosomes is degradation of antigen by proteases however in chapter 6 we observed that in our cross-presentation model Ag-Ab complexes are not rapidly degraded. This paradox raises the question how the antigen is protected from degradation. The protection can be explained by (i) a special function of the dendritic cells (ii) a characteristic of the lysosomes or (iii) the nature of the antigen.
(i) After uptake in macrophages, Ag-Ab complexes are degraded rapidly in lysosomes (23). This is in line with the classical function of lysosomes: degradation of antigen by proteases. Dendritic cells have less proteolytic activity than macrophages (24). Moreover, dendritic cells can actively regulate their lysosomal pH resulting in decreased protease activity (25). This special mechanism of dendritic cells may contribute to decreased degradation of the exogenous antigen in the endocytic route.
(ii) The Ag-Ab complexes may traffic to a different species of lysosomes with less proteolytic activity compared to classical lysosomes. Both species of lysosomes may exist in the same cell.
Retention of antigen after endocytosis and fluid-phase macropinocytosis in a lysosome-like organelle has been previously reported in immature DCs (26). However, such lysosomes have not been reported previously in mature DCs. A more detailed analysis of the protease content and the pH in the antigen storage compartments should reveal if they differ largely from classical lysosomes and if they are related to the antigen retention compartment reported previously (26).
(iii) An exclusive function of Ag-Ab complexes is binding to the neonatal Fc receptor, FcRn.
FcRn is a MHC class I-like molecule associated with b2M that has a high affinity for IgG (27).
In endothelial cells, FcRn protects IgG from catabolism in the lysosomes (28). FcRn on bone- marrow derived cells significantly contributes to serum half live of IgG (29). FcRn is expressed in mouse (chapter 5) and human dendritic cells (30). Recently, it has been shown that FcRn is involved in presentation of immune complexes to CD4 T cells (31). Therefore, it would be interesting to study the role of FcRn in cross-presentation and antigen preservation after FcgR- mediated uptake.
To understand how the nature of the antigen could further influence antigen preservation, we compared the characteristics of immune complexes with a second antigen vehicle that facilitates prolonged cross-presentation: TLR ligand-long peptide conjugates (32,33). Common characteristics of the two vaccine vehicles include:
(i) They both deliver the antigen and DC maturation stimulus in the same cargo. The so-called mechanism of toll-dependent selection has been described as a mechanism for MHC class II presentation: only vesicles that have antigen and TLR in one cargo will be a source of antigen presented in MHC class II (34). Thus, DC maturation influences antigen trafficking in the endocytic compartment (35). This could have an effect on the kinetics of cross-presentation of both antigen vehicles. In addition, DC maturation may induce mechanisms that regulate the lysosomal pH (25) or upregulate natural protease inhibitors such as cystatin F (36). These processes could either directly prevent proteolytic degradation of the antigen or prevent dissociation of the Ag-Ab complexes.
(ii) They both have structural characteristics that may prevent proteolytic cleavage of the antigen. Shielding by the Ab may protect the antigen in IC from lysosomal degradation. Western blot and electron microscopy analyses showed that the antigen in the storage compartments was at least partly present as antigen-antibody complexes. A protective effect of antibodies on degradation of antigen by proteases has been previously demonstrated in biochemical assays (37). Comparable with the shielding of the antigen by the antibody, the conjugation of the long peptide to the TLR ligand may prevent lysosomal degradation, thus increasing the half life of the antigen.
(iii) They are both efficiently presented in both MHC class I and MHC class II by travelling through the endocytic compartment. This is not a common mechanism after endocytosis. For example, soluble OVA is directed towards MHC class I and MHC class II pathways via distinct routes already determined at the receptor level (38). Since the endocytic pathway is specifically equipped for the presentation of MHC class II ligands, efficient presentation of MHC class I ligands seems puzzling. How does the DC sort peptides derived from one protein antigen either towards MHC class I or MHC class II? Our results and literature leads to the following hypothesis may be appreciated: After receptor-mediated endocytosis there is a fast redistribution of MHC class II-peptide complexes to the cell surface (35,39). Upon maturation of dendritic cells, MHC class II synthesis is subsequently downregulated (40,41). The absence of subsequent MHC class II loading may prevent total consumption of the pool of antigen in the endocytic track. The surplus of antigen stays behind in the residual lysosomes and becomes available for the slower mechanism of MHC class I. The proposed model is experimentally supported by a recent elegant study showing that decreasing the number of the MHC class II ligands by manipulating the protease activity facilitated cross-presentation in MHC class I (42).
Further research is necessary to dissect which of the proposed mechanisms is involved in the antigen persistence after loading DCs with Ag-Ab complexes or TLR ligand – long peptide conjugates. Interestingly, priming of helper T lymphocytes also requires antigen persistence (43-45). It is not clear from our experiments yet if the intracellular antigen depot contributes to the prolonged MHC class II presentation. In theory this would be not required; Prolonged MHC class II presentation is already mediated by enhanced stability of MHC class II/peptide complexes upon DC maturation (35,41).
Figure 1: Recovery of cross-presentation after peptide elution is sensitive for brefeldin A.
IgG-OVA pulse-loaded DCs were treated by mild acid elution or PBS (IgG-OVA). Samples that were treated by mild acid elution were fixed (elution) or cultured for another 6 hours in the presence (brefeldin A) or absence (recovery) of 5 ng/ml brefeldin A. All samples were fixed and analyzed for antigen presentation to OVA-specific CD8+ T cell hybridoma B3Z.
The presence of brefeldin A decreased the ability to recover antigen presentation for at least 50%. These results indicate that cross-presentation from the antigen depot is largely dependent on transport of MHC class I molecules across ER and Golgi.
6. MHC class I processing and loading from the antigen depot
The involvement of TAP and the proteasome (chapter 6) together with brefeldin A sensitivity (Figure 1) strongly suggests that MHC class I loading from the antigen depot takes place in the ER. Thus, the exogenous antigen might follow at least partly the same pathway of processing and MHC class I loading as endogenous MHC class I ligands. However, this hypothesis is challenged by the observation (chapter 6, Figure 2) that recovery after peptide elution results in enrichment of MHC class I molecules that present OVA-derived peptides, as judged by the levels of T cell recognition. How can antigen from the intracellular storage compartment compete with the endogenous ligands when they follow a shared pathway of processing and MHC class I loading? (i) The very high affinity of the Kb-binding epitope SIINFEKL could enhance formation of MHC class I/peptide complexes (46). (ii) The lower protein synthesis after DC maturation could result in less proteasomal substrates and less competition for TAP and MHC class I by endogenous ligands. (iii) Convergence of antigen-containing vesicles to the proteasome by cytoskeleton reorganisation after FcgR ligation could enhance efficiency of processing compared to endogenous ligands. A similar reorganisation polarisation of antigen- containing vesicles towards MHC class II compartments has been reported in B lymphocytes after B cell receptor signalling (47).
Alternatively, a spatial separation of processing and MHC class I loading between endogenous ligands and antigen from the antigen storage compartments might exist. One possibility is the involvement of the recently characterized insulin-related amino peptidase (IRAP) that acts as an epitope trimming peptidase for exogenous-derived antigen in the endosomes while aminopeptidases in the ER have a similar function for endogenous antigens (48). Physical association between IRAP and recycled, internalized MHC class I molecules could explain the efficiency of the IRAP-dependent cross-presentation. This pathway also fits with the observation that recovery of peptide presentation after elution was rapid, indicative for recycling of MHC class I molecules. However, so far our data do not provide evidence that cross-presentation of antigen from the antigen depot depends on IRAP. The fast recovery of MHC class I - peptide complexes after elution could also suggest that a pre-existing pool of MHC class I molecules is involved. In immature DCs, MHC class II molecules are stored in intracellular compartments.
Such a pool of pre-existing MHC class I molecules has not been described except for the newly synthesized MHC class I in the Golgi area (49). Interestingly, we have observed a significant number of intracellular MHC class I hotspots in our confocal microscopic analysis of mature DCs (chapter 6). Further research using proteomics and imaging techniques should provide us with more clues about the site of MHC class I loading from the antigen storage compartment.
7. Conclusion and translational aspects
FcgR control three important functions in DCs that facilitate superior CTL priming capacity in one natural antigen formulation: antigen uptake, DC maturation and antigen presentation to both CD4 and CD8 T lymphocytes. Moreover, DCs loaded with immune complexes have a prolonged cross-presentation capacity compared to peptide-loaded DCs. An additional advantage of FcgR- mediated antigen targeting is the possibility to target a complete tumor antigen to dendritic cells.
Whole-antigen vaccines have the benefit that they contain multiple epitopes, both inducing CTL and helper T lymphocytes and that they are not restricted to individuals with a certain HLA-type (50).
From the pre-clinical studies described in this thesis can be concluded that FcgR-mediated antigen targeting to DCs is a promising candidate strategy for immunotherapy of cancer and infectious diseases. Therefore, a further translation of this DC-based immunotherapy strategy towards clinical application should be recommended. An important translational aspect is how to target DCs. Two DC-based immune therapy methods are currently used as clinical application:
(i) adaptive transfer of ex-vivo antigen-loaded autologous DCs and (ii) targeting of antigen towards DCs in the patient. The advantages of ex vivo DC loading include the controlled loading and maturation and the high specificity and efficiency in vivo. More than 10 years of experience has revealed that DC vaccines are safe and well tolerated and induce T cell responses in a considerable number of patients (reviewed by (51)). These studies have also emphasized that the quality of DC maturation is crucial for the outcome. On the contrary, vaccines for in vivo targeting of DCs can be used off-the shelf. They target the DCs in their natural environment, however, specificity is much lower and there is no control on the DC maturation (51).
In chapter 2 we evaluated both methods with regard to FcgR targeting. Here we found that DC pre-loading requires 1000x lower antigen amounts to obtain similar results compared to intravenous injection of antigen. The relatively low efficiency of intravenously injected immune complexes to induce T cell responses could be explained by several factors; (i) Immune complexes are trapped in organs such as the lungs and liver. (ii) High affinity receptor FcgRI is constantly occupied by IgG in vivo preventing availability of this receptor for IC binding (11).
(iii) Intravenously injected immune complexes are captured by both DCs and macrophages in the spleen, however, only dendritic cells can present the complexed antigen to T lymphocytes, as has been shown specifically for CD4 T lymphocytes (52). Cross-presentation to CTL is probably also exclusively mediated by DCs. Other FcgR expressing cells such as macrophages take up more immune complexes than DCs, acting as an antigen drain (chapter 6).
A number of strategies is worthwhile considering to improve the efficiency of targeting to FcgR on DCs in vivo: (i) Depletion of macrophages to enhance the proportion of immune complexes reaching the dendritic cells (53). (ii) Addition of complement factor C1q to the IC to improve targeting to DCs (chapter 3). (iii) Specific targeting of antigen to single FcgRs. Targeting of antigen to FcgRI or FcgRIV would enhance specificity for DCs since these two receptors are less widely expressed on other celtypes than FcgRIIb and FcgRIII. FcgR selectivity would additionally reduce inhibition by FcgRIIb. FcgR selectivity could be accomplished by using IgG2a or IgG2b complexes in mouse or IgG1 or IgG3 complexes in humans. In addition, the use of engineered antibodies that have modified binding affinity for selective Fc receptors could improve targeting to activating FcgRs on DCs (54). (iv) Skewing the balance between expression levels of FcgRs.
FcgR expression is influenced by extra-cellular stimuli such as cytokines (10,22). Our work has shown that on mouse BM-DCs IL-4 up-regulates FcgRIIb while IFNg up-regulates the activating FcgRs (Table 1). Conditioning of the DCs by pre-treatment with cytokines could enhance the ratio between activating FcgR and inhibitory FcgRIIb in favor of cellular activation.
(v) Temporally elimination of FcgRIIb inhibitory function by using blocking antibodies (14).
The efficiency of targeting to dendritic cells is not the only challenge facing translation of immunization with Ag-Ab complexes to the clinic. Off-the shelf use of immune complexes will be hampered by the need for both clinical grade antibody and clinical grade antigen. In addition, our studies have shown that Ag-Ab complexes composed of polyclonal antibodies are more effective in DC maturation than monoclonal antibodies. So far, mainly monoclonal antibodies have been approved for clinical use.
To overcome this practical issue one could exploit the property of natural polyclonal Abs to drive T cell induction. More specifically, antibodies to a recall antigen such as influenza or tetanus could drive T cell responses to tumor antigens when they are conjugated to the B cell epitope of
the recall antigen. In chapter 4 we have elegantly demonstrated the proof of principle for this method by using a haptenated model antigen. We have used this model to address if circulating hapten-specific Abs can also enhance the induction of tumor-specific T cells after vaccination with haptenated protein antigen (Figure 2). Tumor outgrowth followed by death was observed in all Ab- mice compared to five out of eight of the Ab+ mice after receiving two vaccinations with haptenated antigen (Table 2). Thus, three out of eight Ab+ mice were protected against the tumor challenge and survived while none of the Ab- mice survived. Moreover, median survival after tumor challenge was 49 days in Ab+ mice, compared to 33 days in Ab- mice and 20 days in the control groups (Table 2). It is clear from the tumor outgrowth in group of control mice with circulating antibodies that the increased tumor protection in Ab+ mice is not mediated by a direct effect of the antibodies on the tumor. The increased survival in Ab+ mice is in line with the increased systemic CD8 T lymphocyte proliferation after subcutaneous injection of TNP-OVA (chapter 4). We have described that CTL are the main effector cells in this tumor model when antigen-loaded DCs are used as a vaccine (chapter 2). This data confirms that recall antigen- specific antibodies can enhance tumor-specific T cell response after vaccination with a recall B cell antigen conjugated to a tumor antigen. Clearly, the approach needs further optimization.
The capacity of such fusion antigens to induce effective T cell responses will depend on the efficiency of DC targeting and DC maturation in vivo. DC maturation by Ag-Ab complexes in vivo should be further explored, because the lack of DC maturation would induce T cell tolerance rather than immunity (55).
Figure 2: Delayed tumor outgrowth and increased survival in mice with circulating antibodies. Ab+ or Ab- animals (n=8) were sc injected twice with 2 μg TNP-OVA or PBS (control). Two weeks after the last injection, mice were challenged in the contra-lateral flank with 60.000 B16-OVA melanoma cells. Tumor outgrowth was measured twice a week. Mice were sacrificed when the tumor reached a volume of 1000 mm3.
Table 2:
Characteristics of survival per treatment group.
Treatment group Absolute Survival Median survival (days)
Control, Ab- 0/8 20
Control, Ab+ 0/8 20
TNP-OVA, Ab- 0/8 33
TNP-OVA, Ab+ 3/8a 49
aPearson Chi-square test with other groups P=0.05
Unfortunately, the induction of an effective systemic immune response by in vivo FcgR targeting to DCs can not arise without interaction with FcgRs on other cell types. We have experienced some serious adverse events after intravenous injection of immune complexes in mice including thickening of blood and death. These serious adverse events may be caused by a type I hypersensitivity reaction as a results of FcgRIII ligation on mast cells, systemic blot clotting caused by FcgR ligation on platelets (although there is no proof that platelets in mice have FcR) or anaphylactic shock. These adverse events are probable dependent on FcgRIII, since this is the only activating FcR expressed on neutrofils and mast cells and probably also platelets. Targeting of Ag-Ab complexes selectively to FcgRI and FcgRIV could potentially prevent these adverse events.
All experiments described in this thesis were performed in mouse models. The FcgR system in humans is more complex than in mouse, since humans have more FcgR genes and allelic variants than mice. In humans, IgG1 and IgG3 are the most effective IgG isotypes, comparable to IgG2a and IgG2b in mice. Although these molecular differences exist between mouse and human FcgRs, the overall concept is similar. Mouse models are and will be crucial to study the role of FcgRs in vivo. New state of the art mouse models such as cell type-specific conditional knockout mice and mice expressing human FcgRs are needed for the translation of the knowledge obtained in the currently available mouse models. Polymorphisms among individuals provide a way to predict FcgR-mediated responses in humans. A functionally relevant polymorphism is described in the second extra-cellular domain of human FcgRIIa (56,57), the functional homologue of mouse FcgRIII. Due to a single nucleotide change, the amino acid at position 131 can be either histidine or arginine. Only the histidine variant binds IgG2 (58).
In conclusion, the work presented in this thesis has shown that FcgR-mediated antigen targeting to DCs has many advantages for T lymphocyte-directed immunotherapy. Although some translational challenges exist, this thesis has highlighted several reasons why FcgR-mediated antigen targeting is worthwhile for further development. Based on the current knowledge, ex vivo loading of autologous DCs is the safest and most effective method for application of FcgR- mediated antigen targeting in humans. However, humanized FcgR mouse models will help to optimize the in vivo targeting of naturally formed Ag-Ab complexes to FcgRs on DC.
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