The high affinity IgG receptor, FcγRI (CD64), is found exclusively on cells of the myeloid lineage. This receptor comprises an IgG binding α-chain complexed with an ITAM-containing γ-chain dimer. The ITAM within the γ-chain is critical to an array of FcγRI-mediated effector functions including phagocytosis, cytokine production and antibody-dependent cell-mediated cytotoxicity. In addition to these functions, FcγRI also facilitates antigen presentation. However, while integrity of the γ-chain ITAM appears to be important for antigen presentation mediated by other Fc activating receptors, this may not be the case for FcγRI. Specifically, FcγRI has been reported to promote antigen presentation independently of a functional γ-chain ITAM [29
]. On the other hand, the α-chain appears to be critical not only for antigen uptake, but also for antigen presentation through targeting of receptor-ligand complexes to the appropriate antigen processing compartments within the APC.
In the early 1990’s, the effects of targeting antigen to FcγRI were studied by conjugating antigens to the anti-human FcγRI mAb 22.2. This mAb had been shown to bind to FcγRI via its Fab region outside the Fc binding domain making it feasible to target antigens to the receptor despite occupancy by its normal ligand, IgG. Targeting antigens to human FcγRI using mAb 22 was shown to enhance T cell responses in vitro
in human-based systems and augment humoral responses in vivo
in a transgenic mouse model [30
]. Collectively, these findings pointed to enhanced immunogenicity mediated by FcγRI targeting using mAb 22.
A humanized version of anti-CD64 mAb 22 (H22) was developed which was shown to stimulate receptor internalization, an important first step in antigen processing ()[32
]. This trivalent antibody cross-links FcγRI through binding by its Fc end as well as its Fab ends [33
]. Interestingly, targeting antigenic peptides using just the monovalent form of H22 (Fab) enhanced both antigen-specific CD4+ T cell proliferation and cytokine production in vitro
]. Using a similar approach to target prostate specific antigen to FcγRI in the human myeloid cell line THP-1 led to enhanced killing of those cells by antigen-specific cytotoxic T lymphocytes [35
]. Thus, in addition to facilitating presentation of antigen on MHC class II molecules to CD4+ T cells, exogenous antigens targeted to FcγRI using H22 can also be presented to CD8+ T cells in the context of MHC class I. Moreover, a monovalent form of H22 is sufficient to mediate these effects.
The Immunomodulatory Capacity of the Novel Allergen Variant, H22-Fel d 1
In 2001, Platts-Mills and colleagues reported a novel type of immune response (modified Th2
response) in children living with a cat who were exposed to high levels of the major cat allergen, Fel d 1 [36
]. This response, which was characterized by the presence of anti-Fel d 1 IgG antibodies in the serum without IgE and without allergic symptoms, was proposed to represent a form of high dose respiratory tolerance. Subsequent T cell studies performed in modified
responders suggested a role for enhanced recognition of an immunodominant region within polypeptide chain 2 of Fel d 1 which contained IL-10- and IFN-γ-inducing T cell epitopes [37
]. Marked increases in cytokine production to these chain 2 epitopes were also observed in cultures from cat-allergic patients receiving conventional immunotherapy using cat extract. Collectively, these findings supported a role for IL-10 and IFN-γ in protective responses to Fel d 1.
In 2002, Vailes and colleagues developed a recombinant fusion protein which linked Fel d 1 to the single-chain antibody fragment variable regions (sFv) of H22 (H22-Fel d 1)()[38**
]. The sFv version of H22 (sFv22) binds monovalently and thus, does not cross-link FcγRI, but nevertheless leads to receptor internalization. Interestingly, this effect requires IgG suggesting that occupancy of the ligand binding domain of FcγRI is necessary for receptor uptake mediated by sFv22 [39
]. Subsequently, H22-Fel d 1 was shown to bind to FcγRI on monocytes. Moreover, H22-Fel d 1 retained the ability to bind Fel d 1-specific IgE ab, suggesting that fusion of Fel d 1 to sFv22 did not influence allergen folding [38**
We speculated that targeting Fel d 1 to FcγRI may enhance presentation of major T cell epitopes, including those which induce IL-10 or IFN-γ. Thus, the immunomodulatory properties of H22-Fel d 1 were investigated. In initial studies using PBMC cultures, H22-Fel d 1 stimulated increased T cell proliferation as compared with non-receptor-targeted allergen (rFel d 1)[40**
]. This phenomenon was examined further at both the APC and the T cell level. High level expression of CD64 was observed on monocyte-derived dendritic cells (MDCs) confirming that this was an appropriate APC type for analysis. Interestingly, MDCs pulsed with H22-Fel d 1 showed a semi-mature phenotype as judged by production of inflammatory cytokines with no change in surface markers of maturation (HLA-DR, CD40, CD80, CD86). Specifically, H22-Fel d 1 induced increased secretion of Th1-promoting and inflammatory cytokines (IL-1β, IL-12, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, RANTES) as compared with rFel d 1. Moreover, levels for selected inflammatory cytokines (MCP-1, MIP-1β) were as high as those induced by the TLR4 ligand, LPS. H22-Fel d 1 also induced increased IL-10, whereas no changes in Th2-attracting chemokines were observed [40**
Next, we examined the T cell repertoire induced using CD4+ T cells cultured with H22-Fel d 1-pulsed MDCs. Given that semi-mature DCs were previously reported to tolerize T cells, we theorized that changes at the DC level could translate to induction of IL-10-producing T cells [41
]. Flow cytometry analysis showed that H22-Fel d 1 amplified both IL-5+ and IL-10+ CD4+
T cells as compared with rFel d 1. This effect was observed for T cells isolated from cat-allergic subjects, but not those from modified
responders or serum antibody-negative controls. Since many cytokine-positive T cells induced by H22-Fel d 1 expressed multiple cytokines, we further assessed the nature of these cells by analyzing the following six subtypes: (1) IFN-γ+ only; (2) IL-5+ only; (3) IL-10+ only; (4) IFN-γ+IL-5+; (5) IFN-γ+IL-10+; (6) IL-10+IL-5+. Interestingly, H22-Fel d 1 amplified diverse T cell subtypes which were characteristic of Th0 cells (IFN-γ+IL-5+) and different types of regulatory T cells including T regulatory type 1 (IL-10+ only), regulatory Th1 (IL-10+IFN-γ+) and regulatory Th2 (IL-10+IL-5+) cells. Importantly, the T cell repertoire induced by H22-Fel d 1 did not resemble that induced by rFel d 1 in subjects with a modified Th2
response, showing that it constituted a distinct response. Given that IL-5+ T cells were amplified in response to H22-Fel d 1, we compared the T cell repertoire with that induced by the prototypic dust mite allergen, Der p 1. Notably, despite its Th2 elements, the repertoire induced by H22-Fel d 1 was markedly more diverse as judged by increases in all IL-10-expressing subtypes, as well as Th0 cells [40**
]. Thus, targeting Fel d 1 to FcγRI induced a novel variation of the Th2 response in subjects with cat allergy which was characterized by increased T cell diversity ().