FcγR expressed on T-cell hybrids: Specificity,behavior and relationship with Ia antigens

The fine specificity of receptor for the Fc portion of IgG (FcγR) expressed on T-cell hybrids secreting soluble FcγR (sFcγR) which suppresses antibody production, was investigated. FcγR was found to bind IgG from mouse, human, and rabbit species. It reacted with mouse IgG1 and IgG2a but not IgG2b, and human IgG1 and IgG3 but not IgG4. Mouse IgG and their subclasses bound more avidly to FcγR than human and rabbit IgG. FcγR of T-cell hybrids was sensitive to pronase and resistant to trypsin. In kinetics experiments, the behavior of FcγR on the membrane of T-cell hybrids was analyzed and compared to that of I-region-coded antigens expressed on these hybrids. Upon incubation at 37 °C in balanced salt solution (BSS), T-cell hybrids released FcγR into the medium. The reexpression of FcγR, after pronase cleavage or shedding, was complete within 3 hr of incubation in culture medium and required protein synthesis. I-A-coded antigens, present on these hybrids, disappeared simultaneously with FcγR upon incubation of cells at 37 °C in BSS. Within 3 hr of incubation in culture medium, although the reexpression of Fc°R was complete, no Ia antigens could be detected. They were reexpressed later, as tested after 19 hr of culture. During a single growth cycle, the expression of FcγR was maximal during log phase.     

Suppression of the late stages of mitogen-induced human B cell differentiation by FC gamma receptors (FC gamma R) released from polymorphonuclear neutrophils

During short incubation in serum-free medium, polymorphonuclear neutrophils (PMN) release soluble material that can be characterized as receptors for Fc IgG (Fc gamma R) on the following evidence: it agglutinates erythrocyte-IgG antibody (EAG) complexes, it prevents the binding of EAG to EAG-rosette-forming cells, and it binds to EAG-rosette-forming cells after modulation of their Fc gamma R, allowing the formation of 'passive' rosettes. These Fc gamma R were isolated by affinity chromatography on sepharose 4B-IgG. This material was shown to interfere with the differentiation of peripheral blood B cells into Ig-secreting cells in cultures stimulated by pokeweed (PWM) or Nocardia opaca (NOC) extracts. The number of Ig-secreting cells determined by a reverse hemolytic plaque assay using protein A-coated sheep erythrocytes was decreased by addition of Fc gamma R over a wide range of dilutions. The number of Ig-containing cells was diminished in PWM-stimulated cultures, but not in cultures stimulated with NOC. Fc gamma R did not affect cell viability, nor did they interfere with plaque-forming cell assay. Fc gamma R was not suppressive when added before the 3rd day or after the 6th day of culture. The suppressor factor was shown to remain associated with Fc gamma R after elution at acidic pH; it was removed by absorption on Sepharose 4B-IgG, but not on pepsin-digested F(ab')2 fragments. The suppressive factor as well as the capacity to restore EAG rosette formation by modulated lymphocytes were destroyed by heating (56 degrees C, 30 min) or by freezing and thawing. Properties of Fc gamma R released from PMN in this system are similar to those of Fc gamma R released from unstimulated human peripheral blood lymphocytes (PBL) and to those of mouse Ig-binding factor produced by alloactivated T cells or T cell lines.     

Isotype Specificity and Heterogeneity of Fcγ-Receptors on Guinea Pig Splenic B and T Lymphocytes

The isotype specificity of Fc-receptors for IgG (Fcγ-Rs) on normal guinea pig splenic B and T cells was determined by a rosette assay using sheep erythrocytes sensitized with either homologous IgG1 or IgG2 anti-sheep erythrocyte antibody [EA(IgG1) or EA(IgG2)]. Approximately 70% of the lymphocytes in the highly purified B-cell fraction could form rosettes with EA(IgG2), and 55% of the cells with EA(IgG1). Inhibition experiments with soluble complexes of IgG1 or IgG2 antibody with ovalbumin demonstrated that approximately 20% of the EA(IgG2) rosette-forming B cells bore the Fcγ-R monospecific for IgG2, whereas 80% of the cells had two distinct Fcγ-Rs simultaneously; one monospecific for IgG2 and the other bispecific for IgG1 and IgG2. The existence of a B cell bearing the Fcγ-R monospecific for IgG1 was not definitively demonstrated in the B-cell fraction. In the T cell-enriched fraction, approximately 40% of the cells could form rosettes with EA(IgG2). The EA(IgG1)rosette-forming cells, however, comprised only 6% of the total cells, indicating that most of the EA(IgG2) rosette-forming T cells bear essentially the Fcγ-R monspecific for IgG2 alone. The results obtained revealed that guinea pig splenic lymphocytes bear two distinct Fcγ-Rs, which are not equally distributed on the B- and T-cell populations and also on their respective subsets.     

Fcγ receptors inhibit mouse and human basophil activation

Besides high-affinity IgE receptors (FcεRI), human basophils express activating (FcγRIIA) and inhibitory (FcγRIIB) low-affinity IgG receptors. IgG receptors (FcγR) were also found on mouse basophils, but not identified. We investigated in this study FcγR and the biological consequences of their engagement in basophils of the two species. We found the following: 1) that mouse basophils also express activating (FcγRIIIA) and inhibitory (FcγRIIB) low-affinity FcγR; 2) that activating FcγR can activate both human and mouse basophils, albeit with different efficacies; 3) that negative signals triggered by inhibitory FcγR are dominant over positive signals triggered by activating FcγR, thus preventing both human and mouse basophils from being activated by IgG immune complexes; 4) that the coengagement of FcεRI with inhibitory and activating FcγR results in a FcγRIIB-dependent inhibition of IgE-induced responses of both human and mouse basophils; 5) that FcγRIIB has a similar dominant inhibitory effect in basophils from virtually all normal donors; and 6) that IL-3 upregulates the expression of both activating and inhibitory FcγR on human basophils from normal donors, but further enhances FcγRIIB-dependent inhibition. FcγR therefore function as a regulatory module, made of two subunits with antagonistic properties, that prevents IgG-induced and controls IgE-induced basophil activation in both mice and humans.     

FcγRΙΙB controls the potency of agonistic anti-TNFR mAbs

Isotype plays a crucial role in therapeutic monoclonal antibody (mAb) function, mediated in large part through differences in Fcγ receptor (FcγR) interaction. Monoclonal Abs such as rituximab and alemtuzumab, which bind target cells directly, are designed for efficient recruitment of immune effector cells through their activatory FcγR engagement to mediate maximal target cell killing. In this setting, binding to inhibitory FcγRIIB is thought to inhibit function, making mAbs with high activatory/inhibitory (A/I) FcγR binding ratios, such as mouse IgG2a and human IgG1, the first choice for this role. In contrast, exciting new data show that agonistic mAbs directed against the tumour necrosis factor receptor superfamily member CD40 require interaction with FcγRIIB for in vivo function. Such ligation activates antigen-presenting cells, promotes myeloid and CTL responses and potentially stimulates effective anti-cancer immunity. It appears that the role of FcγRIIB is to mediate mAb hyper-crosslinking to allow CD40 downstream intracellular signalling. Previous work has shown that mAbs directed against other TNFR family members, Fas and death receptor 5 and probably death receptor 4, also require FcγRIIB hyper-crosslinking to promote target cell apoptosis, suggesting a common mechanism of action. In mouse models, IgG1 is optimal for these agents as it binds to FcγRIIB with tenfold higher affinity than IgG2a and hence has a relatively low A:I FcγR binding ratio. In contrast, human IgG isotypes have a universally low affinity for FcγRIIB, but in the case of human IgG1, engineering the Fc to increase its affinity for FcγRIIB can potentially overcome this problem. Thus, modifying the A/I binding ratio of human IgG Fc can be used to optimise different types of therapeutic activity by enhancing cytotoxic or hyper-crosslinking function.     

The herpes simplex virus 1 IgG fc receptor blocks antibody-mediated complement activation and antibody-dependent cellular cytotoxicity in vivo

Herpes simplex virus 1 (HSV-1) glycoprotein E (gE) mediates cell-to-cell spread and functions as an IgG Fc receptor (FcγR) that blocks the Fc domain of antibody targeting the virus or infected cell. Efforts to assess the functions of the HSV-1 FcγR in vivo have been hampered by difficulties in preparing an FcγR-negative strain that is relatively intact for spread. Here we report the FcγR and spread phenotypes of NS-gE264, which is a mutant strain that has four amino acids inserted after gE residue 264. The virus is defective in IgG Fc binding yet causes zosteriform disease in the mouse flank model that is only minimally reduced compared with wild-type and the rescue strains. The presence of zosteriform disease suggests that NS-gE264 spread functions are well maintained. The HSV-1 FcγR binds the Fc domain of human, but not murine IgG; therefore, to assess FcγR functions in vivo, mice were passively immunized with human IgG antibody to HSV. When antibody was inoculated intraperitoneally 20 h prior to infection or shortly after virus reached the dorsal root ganglia, disease severity was significantly reduced in mice infected with NS-gE264, but not in mice infected with wild-type or rescue virus. Studies of C3 knockout mice and natural killer cell-depleted mice demonstrated that the HSV-1 FcγR blocked both IgG Fc-mediated complement activation and antibody-dependent cellular cytotoxicity. Therefore, the HSV-1 FcγR promotes immune evasion from IgG Fc-mediated activities and likely contributes to virulence at times when antibody is present, such as during recurrent infections.     

Biological Activities of Murine Low-Affinity Fc Receptors for IgG

Murine low-affinity Fc receptors for IgG (FcγRIIbl, FcγRIIb2, and FcγRIII) bind the same IgG subclasses and are not distinguished by available anti-FcγRII/III mAbs (2.4G2). They trigger various biological activities, among which are the internalization of soluble and particulate immune complexes, cell activation, and its regulation. To determine the biological properties of the three murine receptors, each was expressed by stable transfection of corresponding cDNAs in two model cells: the murine lymphoma B cell IIA1.6 and the rat basophilic leukemia cell RBL-2H3. Biological activities of recombinant receptors were triggered with soluble immune complexes or 2.4G2 IgG in IIA1.6 cells, which express no FcγR, and with 2.4G2 Fab or F(ab′)₂, cross-linked with mouse anti-rat F(ab′)₂ in RBL, which express rat FcγR. Conditions for studying cell activation and endocytosis in both cell models are described, as are conditions for studying phagocytosis in RBL cells and antigen presentation or regulation of cell activation in IIA1.6 cells. Internalization of immune complexes was triggered by FcγRIIb2 and FcγRIII, but not by FcγRIIb1. Intracytoplasmic sequences required for phagocytosis and endocytosis could be distinguished in FcγRIIb2, but not in FcγRIII. Cell activation was restricted to FcγRIII. FcγRIII-mediated endocytosis, phagocytosis, and cell activation involved the consensus tyrosine-containing activation motif found in the intracytoplasmic domain of the γ subunit. Regulation of cell activation was induced by both FcγRII isoforms and depended on the same sequence as endocytosis. As a consequence, a single motif can determine more than one biological response of the cell, and a given response may be triggered by several motifs, borne by different FcγR.     

IgG Fc variant cross-reactivity between human and rhesus macaque FcγRs

Non-human primate (NHP) studies are often an essential component of antibody development efforts before human trials. Because the efficacy or toxicity of candidate antibodies may depend on their interactions with Fcγ receptors (FcγR) and their resulting ability to induce FcγR-mediated effector functions such as antibody-dependent cell-meditated cytotoxicity and phagocytosis (ADCP), the evaluation of human IgG variants with modulated affinity toward human FcγR is becoming more prevalent in both infectious disease and oncology studies in NHP. Reliable translation of these results necessitates analysis of the cross-reactivity of these human Fc variants with NHP FcγR. We report evaluation of the binding affinities of a panel of human IgG subclasses, Fc amino acid point mutants and Fc glycosylation variants against the common allotypes of human and rhesus macaque FcγR by applying a high-throughput array-based surface plasmon resonance platform. The resulting data indicate that amino acid variation present in rhesus FcγRs can result in disrupted, matched, or even increased affinity of IgG Fc variants compared with human FcγR orthologs. These observations emphasize the importance of evaluating species cross-reactivity and developing an understanding of the potential limitations or suitability of representative in vitro and in vivo models before human clinical studies when either efficacy or toxicity may be associated with FcγR engagement.     

Natural and Recombinant Soluble Low-Affinity FcγR: Detection,Purification, and Functional Activities

Studies on the identification, cloning, and biochemical characterization of natural and recombinant human and mouse low-affinity soluble FcγR (sFcγR) have been developed using various methods. RT-PCR and/or biochemical analyses have demonstrated that low-affinity sFcγR (i) are generated by enzymatic cleavage of membrane-associated receptors or by an alternative splicing of the transmembrane region encoding exon and (ii) comprise only the extracellular domains or these domains plus the intracellular region of the membrane-associated molecules, respectively. Functional studies indicated that recombinant sFcγR bind mouse and human IgG subclasses with a binding profile identical to that of their membrane counterparts and inhibit FcγR-mediated functions such as immune complex binding or ADCC. In addition, it has been demonstrated that a mouse recombinant truncated sFcγRII inhibits antibody responses to T-dependent antigens as well as B-cell proliferation and that a human recombinant truncated sFcγRIIIB blocks the Ig production by activated human peripheral blood mononuclear cells. Finally, different immunoassays devised to detect and quantitate circulating sFcγR showed that sFcγR serum levels vary in circumstances such as injections of protein antigens, in parasitic infections, in tumor-bearing mice, in patients with multiple myeloma (MM), or upon infusions of IgG or Fcγ fragments in MM or immune thrombocytopenic purpura patients. The use of recombinant sFcγR, as well as the availability of monoclonal and polyclonal antibodies directed against different regions of these molecules, makes it possible to characterize further the biological effects of sFcγR and their biochemical and immunochemical characteristics, as well as to define their putative ligands on cell membranes.     

Efficient expression and purification of human aglycosylated Fcγ receptors in Escherichia coli

Effector Fc gamma receptors (FcγRs) are expressed on the surface of a variety of cells of hematopoietic lineage and serve as a bridge between adaptive and innate immune responses. The interaction between immune complexes, formed by IgG class antibodies that are crosslinked with antigen, and FcγRs triggers signaling cascades that result in numerous cellular responses including the activation or donwregulation of cytotoxic responses, cytokine release, and antibody synthesis. Here, the extracellular domains of the human type I transmembrane FcγRs were expressed in Escherichia coli and their interactions to subclass IgGs (IgG1, IgG2, IgG3, and IgG4) antibodies were analyzed. Expression using fully synthetic E. coli codon optimized FcγR genes and optimization of sequences for N‐terminal translation initiation region through mRNA secondary structure prediction enabled us to achieve high yield of purified, bacterially expressed receptors, including FcγRI and FcγRIIIa which have not been successfully expressed in bacteria until now. The aglycosylated FcγRs showed similar IgG subclass binding selectivity compared to the respective glycosylated FcγRs expressed in mammalian cells. Biotechnol. Bioeng. 2010;107: 21–30. © 2010 Wiley Periodicals, Inc.     

IgG1 is pathogenic in Leishmania mexicana infection

There are >2 million new cases of leishmaniasis annually, and no effective vaccine has been developed to prevent infection. In murine infection, Leishmania mexicana, which lives intracellularly in host macrophages, has developed pathways to hijack host IgG to induce a suppressive IL-10 response through FcγRs, the cell-surface receptors for IgG. To guide vaccine development away from detrimental Ab responses, which can accompany attempts to induce cell-mediated immunity, it is crucial to know which isotypes of IgG are pathogenic in this infection. We found that IgG1 and IgG2a/c induce IL-10 from macrophages in vitro equally well but through different FcγR subtypes: IgG1 through FcγRIII and IgG2a/c through FcγRI primarily, but also through FcγRIII. In sharp contrast, mice lacking IgG1 develop earlier and stronger IgG2a/c, IgG3, and IgM responses to L. mexicana infection and yet are more resistant to the infection. Thus, IgG1, but not IgG2a/c or IgG3, is pathogenic in vivo, in agreement with prior studies indicating that FcγRIII is required for chronic disease. This calls into question the assumption that macrophages, which should secrete IL-10 in response to IgG1 and IgG2a/c immune complexes, are the most important source of IL-10 generated by IgG-FcγR engagement in L. mexicana infection. Further investigations are required to better determine the cell type responsible for this immunosuppressive FcγRIII-induced IL-10 pathway and whether IgG2a/c is protective.     

Affinity of human IgG subclasses to mouse Fc gamma receptors

Human IgG is the main antibody class used in antibody therapies because of its efficacy and longer half-life, which are completely or partly due to FcγR-mediated functions of the molecules. Preclinical testing in mouse models are frequently performed using human IgG, but no detailed information on binding of human IgG to mouse FcγRs is available. The orthologous mouse and human FcγRs share roughly 60–70% identity, suggesting some incompatibility. Here, we report binding affinities of all mouse and human IgG subclasses to mouse FcγR. Human IgGs bound to mouse FcγR with remarkably similar binding strengths as we know from binding to human ortholog receptors, with relative affinities IgG3>IgG1>IgG4>IgG2 and FcγRI>>FcγRIV>FcγRIII>FcγRIIb. This suggests human IgG subclasses to have similar relative FcγR-mediated biological activities in mice.     

Fc receptor heterogeneity: immunofluorescent studies of B, T, and "third population" lymphocytes in human blood with rabbit IgG b4/anti-b4 complexes.

IgG Fc receptors on human peripheral blood lymphocytes (PBL) were characterized by immunofluorescence studies with defined rabbit IgG b4 allotype/anti-allotype complexes. Three discrete types of Fc receptor-bearing cells, totaling approximately 33% of PBL, were identified. Fc receptors of the three types differed in their sensitivity to trypsin and in either absolute or localized density (topography) as determined by variable requirements for anti-IgC cross-linking in order to visualize bound complexes microscopically. The question of additional heterogeneity related to differences in individual Fc receptor affinity for complexed IgG was not approached in this study. Ten to 15% of PBL had pronase-sensitive, trypsin-resistant Fc receptors readily detected by direct immunofluorescence by using large fluorescein-conjugated complexes prepared near equivalence. Double label and lymphocyte fractionation experiments established this population to be largely distinct from suface IgM+ B cells and T cells, and identical to EA Ripley rosette-forming cells. Approximately 50% of surface IgM+ B cells and approximately 10% of T cells had lower density Fc receptors identified by indirect immunofluorescence with small complexes prepared in antigen excess or by cross-linking fluorescein-conjugated complexes with anti-rabbit IgG anti-serum. An additional approximately 15% peripheral T and B cells had very low density Fc receptors detectable by complexing the IgG on the cell surface by sequential incubations of cells with b4 IgG and anti-b4. Fc receptors on B and T cells were sensitive to both pronase and trypsin digestion. The heterogeneity of IgG Fc receptors on different lymphocyte subpopulations as defined by these these experiments may be of relevance for further analysis of normal and abnormal immune function.     

Interaction with FcγRIIB is critical for the agonistic activity of anti-CD40 monoclonal antibody

A high activatory/inhibitory FcγR binding ratio is critical for the activity of mAb such as rituximab and alemtuzumab that attack cancer cells directly and eliminate them by recruiting immune effectors. Optimal FcγR binding profiles of other anti-cancer mAb, such as immunostimulatory mAb that stimulate or block immune receptors, are less clear. In this study, we analyzed the importance of isotype and FcγR interactions in controlling the agonistic activity of the anti-mouse CD40 mAb 3/23. Mouse IgG1 (m1) and IgG2a (m2a) variants of the parental 3/23 (rat IgG2a) were engineered and used to promote humoral and cellular responses against OVA. The mouse IgG1 3/23 was highly agonistic and outperformed the parental Ab when promoting Ab (10-100-fold) and T cell (OTI and OTII) responses (2- to >10-fold). In contrast, m2a was almost completely inactive. Studies in FcγR knockout mice demonstrated a critical role for the inhibitory FcγRIIB in 3/23 activity, whereas activatory FcγR (FcγRI, -III, and -IV) was dispensable. In vitro experiments established that the stimulatory effect of FcγRIIB was mediated through Ab cross-linking delivered in trans between neighboring cells and did not require intracellular signaling. Intriguingly, activatory FcγR provided effective cross-linking of 3/23 m2a in vitro, suggesting the critical role of FcγRIIB in vivo reflects its cellular distribution and bioavailability as much as its affinity for a particular Ab isotype. In conclusion, we demonstrate an essential cross-linking role for the inhibitory FcγRIIB in anti-CD40 immunostimulatory activity and suggest that isotype will be an important issue when optimizing reagents for clinical use.     

Impact of N-glycosylation on Fcγ receptor / IgG interactions: unravelling differences with an enhanced surface plasmon resonance biosensor assay based on coiled-coil interactions

The N-glycosylation profile of immunoglobulin G (IgG) is considered a critical quality attribute due to its impact on IgG-Fc gamma receptor (FcγR) interactions, which subsequently affect antibody-dependent cell-based immune responses. In this study, we investigated the impact of the FcγR capture method, as well as FcγR N-glycosylation, on the kinetics of interaction with various glycoforms of trastuzumab (TZM) in a surface plasmon resonance (SPR) biosensor assay. More specifically, we developed a novel strategy based on coiled-coil interactions for the stable and oriented capture of coil-tagged FcγRs at the biosensor surface. Coil-tagged FcγR capture outperformed all other capture strategies applied to the SPR study of IgG-FcγR interactions, as the robustness and reproducibility of the assay and the shelf life of the biosensor chip were excellent (> 1,000 IgG injections with the same biosensor surface). Coil-tagged FcγRs displaying different N-glycosylation profiles were generated either by different expression systems, in vitro glycoengineering or by size-exclusion chromatography, and roughly characterized by lectin blotting. Of salient interest, the overlay of their kinetics of interaction with several TZM glycoforms revealed key differences on both association and dissociation kinetics, confirming a complex influence of the FcγR N-glycosylation and its inherent heterogeneity upon receptor interaction with mAbs. This work is thus an important step towards better understanding of the impact of glycosylation upon binding of IgGs, either natural or engineered, to their receptors.     

Murine IgA binding factors (IgA-BF) suppressing IgA production: characterization and target specificity of IgA-BF

Chemical and functional properties of IgA binding factor(s) (IgA-BF) from both murine Con A-activated spleen cells and Fc gamma R+, Fc alpha R+ T hybridoma cells (T2D4) were studied. IgA-BF produced from the cells after preculture with IgA were purified with IgA-Sepharose. Purified IgA-BF inhibited the binding of IgA to Fc alpha R+ L5178Y T lymphoma cells, and class-specifically suppressed in vitro IgA synthesis of the pokeweed mitogen (PWM)-stimulated murine spleen cells. Both IgA-specific suppressive activity and IgA binding activity of the factor(s) were co-fractionated between BSA and OVA in gel filtration analysis. SDS-PAGE analysis of IgA-BF biosynthetically labeled with [35S]methionine showed a specific band on 56,000. Suppressive activity of IgA-BF was absorbed with lentil-lectin-Sepharose and was eluted with 0.2 M alpha-methyl-D-mannoside. The suppressive activity obtained from T2D4 cells (H-2k) and BALB/c Con A blasts (H-2d) was absorbed with the corresponding anti-H-2 and anti-I-A column and recovered in the acid-eluate. The activity was not absorbed with the unrelated anti-H-2 column. Despite the presence of MHC products, IgA-BF from both cell sources equally suppressed IgA-specific responses of BALB/c (H-2d), C3H/He (H-2k), and C57BL/10 (H-2b) spleen cells. They also suppressed IgA production as well as IgA synthesis of PWM-stimulated culture of human peripheral blood lymphocytes without affecting IgM and IgG responses. Suppression of murine and human IgA responses both in mouse and human were mediated by the molecules having the same Ia products, suggesting that there is no MHC, as well as species restriction, for the interaction between IgA-BF and their target cells. IgA-specific suppressive activity was absorbed with human B blastoid cells bearing surface IgA (Dakiki) but not with those bearing surface IgG (CESS) or murine and human T cell line cells (BW5147, L5178Y, HPB-ALL, and MOLT4), indicating that IgA-BF interact with B cells bearing IgA to suppress their differentiation.     

A nonfucosylated variant of the anti-HIV-1 monoclonal antibody b12 has enhanced FcγRIIIa-mediated antiviral activity in vitro but does not improve protection against mucosal SHIV challenge in macaques

Eliciting neutralizing antibodies is thought to be a key activity of a vaccine against human immunodeficiency virus (HIV). However, a number of studies have suggested that in addition to neutralization, interaction of IgG with Fc gamma receptors (FcγR) may play an important role in antibody-mediated protection. We have previously obtained evidence that the protective activity of the broadly neutralizing human IgG1 anti-HIV monoclonal antibody (MAb) b12 in macaques is diminished in the absence of FcγR binding capacity. To investigate antibody-dependent cellular cytotoxicity (ADCC) as a contributor to FcγR-associated protection, we developed a nonfucosylated variant of b12 (NFb12). We showed that, compared to fully fucosylated (referred to as wild-type in the text) b12, NFb12 had higher affinity for human and rhesus macaque FcγRIIIa and was more efficient in inhibiting viral replication and more effective in killing HIV-infected cells in an ADCC assay. Despite these more potent in vitro antiviral activities, NFb12 did not enhance protection in vivo against repeated low-dose vaginal challenge in the simian-human immunodeficiency virus (SHIV)/macaque model compared to wild-type b12. No difference in protection, viral load, or infection susceptibility was observed between animals given NFb12 and those given fully fucosylated b12, indicating that FcγR-mediated activities distinct from FcγRIIIa-mediated ADCC may be important in the observed protection against SHIV challenge.     

Murine IgA binding factors produced by Fc alpha R(+) T cells: role of Fc gamma R(+) cells for the induction of Fc alpha R and formation of IgA-binding factor in Con A-activated cells

The relationship between the production of a T cell factor having affinity for IgA (IgA-binding factor(s); IgA BF) and the expression of Fc receptors specific for IgA (Fc alpha R) was studied by using murine spleen cells activated with concanavalin A (Con A blasts). Fc alpha R was detected by the cytophilic binding of anti-TNP murine IgA myeloma protein (MOPC 315 IgA) to Con A blasts as determined by an indirect rosette method with trinitrophenylated sheep red blood cells (TNP-SRBC). After 18 hr preculture with IgA, Fc alpha R was expressed on 15 to 20% of Con A blasts, which released IgA BF suppressing the in vitro IgA synthesis of the spleen cells stimulated with pokeweed mitogen (PWM). Without preculture with IgA, there was neither induction of Fc alpha R nor the production of IgA BF from Con A blasts. Fc alpha R was not induced on Con A blasts by IgA if Fc gamma R(+) cells were depleted from the blasts by rosetting with SRBC sensitized with rabbit IgG antibody (EA gamma). Even after preculture with IgA, the suppressive IgA BF was undetectable in the culture supernatant of Con A blasts depleted of the Fc gamma R(+) cell population. By using a double rosette method with EA gamma and trinitrophenylated quail red blood cells, Fc alpha R proved to be co-expressed on Fc gamma R(+) precursor T cells in the Con A blasts. The results suggested that both Fc gamma R and Fc alpha R could be co-expressed on Con A blasts, as is the case with T2D4 Fc gamma R(+), Fc alpha R(+) T hybridoma cells, which are known to produce IgG-binding factor(s) (IgG BF) and IgA BF. The relationship between Fc gamma R and Fc alpha R on a single cell was studied by using monoclonal anti-Fc gamma R antibody (2. 4G2 ). The reactivity of 2. 4G2 antibody with T cell Fc gamma R was proved by the inhibition of EA gamma rosette formation by Con A blasts or T2D4 cells. The addition of 2. 4G2 monoclonal antibody, however, did not affect the induction of Fc alpha R on Con A blasts by IgA. Furthermore, the binding of IgA to Fc alpha R already expressed on L5178Y T lymphoma cell line cells was not inhibited by the monoclonal antibody. The results confirmed that Fc alpha R are distinct from Fc gamma R co-expressed on the same Con A blasts, and that the expression of Fc alpha R on Fc gamma R(+) T cells and their production of suppressive IgA BF may be induced by the binding of IgA to Fc alpha R.     

Cynomolgus and pigtail macaque IgG subclasses: characterization of IGHG genes and computational analysis of IgG/Fc receptor binding affinity

Macaques are the most widely used experimental nonhuman primate (NHP) species. Rhesus (Macaca mulatta, Macmul), cynomolgus (Macaca fascicularis, Macfas), and pigtail (Macaca nemestrina, Macnem) macaques continue to be popular models for vaccine and infectious diseases research, especially HIV infection and AIDS, and for the development of antibody-based therapeutic strategies. Increased understanding of the immune system of these species is necessary for their optimal use as models of human infections and intervention. In the past few years, the antibody/Fc receptor system has been characterized in a stepwise manner in these species. We have continued this characterization by identifying the four IG heavy gamma (IGHG) genes of Macfas and Macnem in this study. Our results show that these genes share a high degree of similarity with those from other NHP species, while presenting consistent differences when compared to human IGHG genes. Furthermore, comparison of Macfas IGHG genes with those described in other studies suggests the existence of polymorphism. Using sequence- and structure-based computational tools, we performed in silico analysis on multiple polymorphic Macfas IgG and their interactions with human IgG Fc receptors (FcγR), thus predicting that Macfas IGHG polymorphisms influence IgG protein stability and/or binding affinity towards FcγR. The presence of macaque IGHG polymorphisms and macaque/human amino acid changes at locations potentially involved in antibody functional properties indicate the need for cautious design and data interpretation of studies in these models, possibly requiring the characterization of antibody/Fc receptor interactions at the individual level.