Anti‐HIV‐1 antibody‐dependent cellular cytotoxicity mediated by hyperimmune bovine colostrum IgG

Antibodies with antibody‐dependent cellular cytotoxicity (ADCC) activity play an important role in protection against HIV‐1 infection, but generating sufficient amounts of antibodies to study their protective efficacy is difficult. HIV‐specific IgG can be easily and inexpensively produced in large quantities using bovine colostrum. We previously vaccinated cows with HIV‐1 envelope gp140 and elicited high titers of anti‐gp140‐binding IgG in colostrum. In the present study, we determined whether bovine antibodies would also demonstrate specific cytotoxic activity. We found that bovine IgG bind to Fcγ‐receptors (FcγRs) on human neutrophils, monocytes, and NK cells in a dose‐dependent manner. Antibody‐dependent killing was observed in the presence of anti‐HIV‐1 colostrum IgG but not nonimmune colostrum IgG. Killing was dependent on Fc and FcγR interaction since ADDC activity was not seen with F(ab’)₂ fragments. ADCC activity was primarily mediated by CD14⁺ monocytes with FcγRIIa (CD32a) as the major receptor responsible for monocyte‐mediated ADCC in response to bovine IgG. In conclusion, we demonstrate that bovine anti‐HIV colostrum IgG have robust HIV‐1‐specific ADCC activity and therefore offer a useful source of antibodies able to provide a rapid and potent response against HIV‐1 infection. This could assist the development of novel Ab‐mediated approaches for prevention of HIV‐1 transmission.     

Racially restricted contribution of immunoglobulin Fcγ and Fcγ receptor genotypes to humoral immunity to human epidermal growth factor receptor 2 in breast cancer

Tumour‐associated antigen human epidermal growth factor receptor 2 (HER2) is over‐expressed in 25–30% of breast cancer patients and is associated with poor prognosis. Naturally occurring anti‐HER2 antibody responses have been described in patients with HER2 over‐expressing tumours. There is significant interindividual variability in antibody responsiveness, but the host genetic factors responsible for this variability are poorly understood. The aim of the present investigation was to determine whether immunoglobulin genetic markers [GM (genetic determinants of γ chains)] and Fcγ receptor (FcγR) alleles contribute to the magnitude of natural antibody responsiveness to HER2 in patients with breast cancer. A total of 855 breast cancer patients from Japan and Brazil were genotyped for several GM and FcγR alleles. They were also characterized for immunoglobulin (Ig)G antibodies to HER2. In white subjects (n = 263), GM 23‐carriers had higher levels of anti‐HER2 antibodies than non‐carriers of this allele (p = 0·004). At the GM 5/21 locus, the homozygotes for the GM 5 allele had higher levels of anti‐HER2 antibodies than the other two genotypes (P = 0·0067). In black subjects (n = 42), FcγRIIa‐histidine/histidine homozygotes and FcγRIIIa‐phenylalanine/valine heterozygotes were associated with high antibody responses (P = 0·0071 and 0·0275, respectively). FcγR genotypes in white subjects and GM genotypes in black subjects were not associated with anti‐HER2 antibody responses. No significant associations were found in other study groups. These racially restricted contributions of GM and FcγR genotypes to humoral immunity to HER2 have potential implications for immunotherapy of breast cancer.     

Human Fcγ receptors compete for TGN1412 binding that determines the antibody's effector function

The first‐in‐human clinical trial of the CD28‐specific monoclonal antibody (mAb) TGN1412 resulted in a life‐threatening cytokine release syndrome. Although TGN1412 was designed as IgG4, known for weak Fc:Fcγ receptor (FcγR) interactions, these interactions contributed to TGN1412‐induced T‐cell activation. Using cell lines (TFs) expressing human FcγRI, ‐IIa, ‐IIb, or ‐III, we show that TGN1412 and TGN1412 as IgG1 and IgG2 are bound by FcγRs as it can be deduced from literature. However, upon coculture of TGN1412‐decorated T cells with TFs or human primary blood cells, we observed that binding capacities by FcγRs do not correlate with the strength of the mediated effector function. FcγRIIa and FcγRIIb, showing no or very minor binding to TGN1412, mediated strongest T cell proliferation, while high‐affinity FcγRI, exhibiting strong TGN1412 binding, mediated hardly any T‐cell proliferation. These findings are of biological relevance because we show that FcγRI binds TGN1412, thus prevents binding to FcγRIIa or FcγRIIb, and consequently disables T‐cell proliferation. In line with this, FcγRI^?FcγRII⁺ but not FcγRI⁺FcγRII⁺ monocytes mediate TGN1412‐induced T‐cell proliferation. Collectively, by using TGN1412 as example, our results indicate that binding of monomeric IgG subclasses does not predict the FcγR‐mediated effector function, which has major implications for the design of therapeutic mAbs.     

FcγRIa–γ-chain complexes trigger antibody-dependent cell-mediated cytotoxicity (ADCC) in CD5+ B cell/macrophage IIA1.6 cells

Most receptors for immunoglobulins exist as multi-subunit complexes, with unique ligand binding α-chains, combined with accessory signalling (γ-, β-, or ζ-) chains. The myeloid class I receptor for IgG (FcγRIa) has been shown to be dependent on the FcR γ-chain for surface expression in vivo. In this study we assess the capacity of FcγRIa–γ-chain complexes expressed in IIA1.6 cells to trigger phagocytosis and ADCC. An intact immunoreceptor tyrosine-based activation motif (ITAM) signalling motif proved essential for triggering of biological function via the FcγRIa receptor complex. Both the FcR γ-chain and the FcγRIIa–ITAM proved active in directing phagocytosis of Staphylococcus aureus and ADCC of erythrocytes, triggered by the FcγRIa complex. The capacity of FcγRIa to trigger phagocytic and cytolytic activity by IIA1.6 cells, both considered ‘professional phagocyte’ functions, motivated us to re-evaluate the cell lineage and developmental stage of IIA1.6 cells. Although originally described as mouse B lymphocytes, the IIA1.6 cells proved positive for non-specific esterase activity and expressed the CD5 antigen. These combined characteristics place the IIA1.6 cells within a unique CD5⁺ B cell/macrophage lineage, optimally suited for cell biological analyses of phagocyte receptors.     

FcγRIII (CD16)‐mediated ADCC by NK cells is regulated by monocytes and FcγRII (CD32)

Monocytes are known to engage in reciprocal crosstalk with NK cells but their influence on NK‐cell‐associated antibody‐dependent cellular cytotoxicity (ADCC) is not well understood. We demonstrate that in humans FcγRIII (CD16)‐dependent ADCC by NK cells is considerably enhanced by monocytes, and that this effect is regulated by FcγRII (CD32) crosslinking in healthy individuals. It is known that during HIV‐1 infection, NK cells are known to express low levels of CD16 and exhibit reduced ADCC. We show that immune regulation of CD16‐mediated NK‐cell cytotoxicity by monocytes through CD32 engagement is substantially disturbed in chronic progressive HIV‐1 infection. Expression of activating isoform of CD32 represented a compensatory mechanism for reduced expression of CD16 on NK cells during HIV‐1 infection. As a result, the regulation of NK‐cell‐associated ADCC by monocytes is skewed and eventually constitutes a novel factor that contributes to HIV‐1‐associated immune deficiency, dysregulation and pathogenesis. Our data therefore provide evidence, for the first time, that in humans monocytes act as a rheostat for FcγRIII‐mediated NK‐cell functions maintaining a well‐balanced immune response.     

C‐Reactive Protein Triggers Calcium Signalling in Human Neutrophilic Granulocytes via FcγRIIa in an Allele‐Specific Way

C‐reactive protein (CRP) binds to Fcγ‐receptors, FcγRIIa (CD32) with high affinity and to FcγRIa (CD64) with low affinity. The binding to CD32 has been shown to be allele specific, that is, it binds to R/R131 but not to H/H131. Little is known about the cooperation of CRP and neutrophilic granulocytes (PMNs) in inflammatory reactions. The purpose of the present study was to examine CRP signalling in human PMNs, and whether this signalling is also allele specific. Cytosolic calcium of PMN was measured in a single‐cell digital imaging system. Receptor expression and polymorphism were studied by real‐time RT‐PCR, flow cytometry and standard PCR. C‐reactive protein induced cytosolic calcium signals in PMNs from homozygote R/R131donors, but not in PMNs from heterozygote R/H131 donors. However, after the heterozygote PMNs had been incubated with IFN‐γ (100 U/ml) for 2 h, both the proportion of cells responding and the size of the CRP‐induced calcium signals increased. IFN‐γ increased mRNA expression of CD64 about fivefold and surface protein expression of CD64 about fourfold. The calcium signal elicited by CRP was augmented by PMN adhesion to fibronectin, but almost totally abrogated by sphingosine kinase inhibitors. The signals were partly dependent on calcium influx. In conclusion, calcium signalling instigated by CRP in human PMN is FcγRIIa allele specific, as R/R131 responded to CRP, whereas R/H131 did not. However, increased expression of FcγRIa (CD64), stimulated by IFN‐γ, can augment calcium signalling by CRP in low‐responders. This suggests that the state of the PMNs, as well as the genetic origin, affect sensitivity for CRP.     

Signal regulatory protein α negatively regulates mast‐cell activation following FcεRI aggregation

Mast cells elicit allergic reaction through degranulation and release of proinflammatory mediators after aggregation of the IgE receptor FcεRI. Here we provide evidence to show that signal regulatory protein α (SIRPα), an ITIM‐containing receptor, is an endogenous regulator of IgE‐Ag induced mast‐cell activation. SIRPα expression is promptly reduced in mast cells in response to FcεRI aggregation. Impaired expression of SIRPα in mast cells facilitates FcεRI‐evoked degranulation and de novo synthesis of cytokines (IL‐4, IL‐13, IL‐6, and TNF‐α). We further demonstrate that SIRPα knockdown in mast cells accelerates calcium mobilization and affects cytoskeletal rearrangement (F‐actin disassembly and polymeric tubulin formation) after FcεRI aggregation. Mechanistic studies highlight the prolonged activation of NF‐κB and MAPKs as well as PLC‐γ after FcεRI stimulation as a consequence of the inhibition of SIRPα expression in mast cells. Immunoprecipitation analysis shows that SIRPα knockdown markedly increases IgE‐induced SHP2 interaction with PI3K regulatory subunit PI3Kp85 or IKK‐β in mast cells, indicating that SIRPα may accomplish this through its association and sequestration of SHP2. Collectively, our results strongly indicate that SIRPα is a biological important regulator of FcεRI signaling.     

Meningococcal disease and polymorphism of FcγRIIa (CD32) in late complement component-deficient individuals

Late complement component-deficient (LCCD) individuals lack plasma bactericidal activity and are highly susceptible to meningococcal disease. Phagocytosis plays a significant role in immune defence against meningococci and involves FcγRIIa (CD32) on leucocytes. Two allotypic forms are currently recognized: FcγRIIa-R131 and RIIa-H131. Neutrophils with the IIa-H/H131 allotype are more effective in phagocytosis than IIa-R/R131. We studied the distributions of IIa-R131 and IIa-H131 allotypes among 29 Russian LCCD patients who had suffered from recurrent episodes of meningococcal disease. The distribution of IIa-R/R131 to heterozygous IIa-R/H131 to homozygous IIa-H/H131 genotypes was 0.14:0.29:0.57 for LCCD patients who developed the first episode of disease before 10 years of age. The distribution was 0.21:0.64:0.14 for patients who experienced meningococcal disease above the age of 10 years (χ² = 6, P < 0.05, odds ratio for IIa H/H131 versus R/R131 = 8). Meningococcal disease had a ‘grave’ course in 14 of 31 disease episodes in patients with IIa-R/R131 and IIa-R/H131 allotypes, in contrast to 1 of 18 episodes in patients with IIa-H/H131 allotype (χ² = 7, P < 0.01, odds ratio = 14). We conclude that IIa-H/H131 individuals appear to have a higher acquired antibody-mediated phagocytosis-dependent resistance to meningococcal disease above the age of 10 years. Additionally, effective CD32-mediated phagocytosis may restrict the severity of meningococcal disease in LCCD patients with IIa-H/H131 phenotype.     

FcγRIIa polymorphism and anti-malaria-specific IgG and IgG subclass responses in populations differing in susceptibility to malaria in Burkina Faso

FcγRIIa is known to be polymorphic; and certain variants are associated with different susceptibilities to malaria. Studies involving the Fulani ethnic group reported an ethnic difference in FcγRIIa-R131H genotype frequencies between the Fulani and other sympatric groups. No previous studies have addressed these questions in Burkina Faso. This study aimed to assess the influence of FcγRIIa-R131H polymorphism on anti-falciparum malaria IgG and IgG subclass responses in the Fulani and the Mossi ethnic groups living in Burkina Faso. Healthy adults more than 20 years old belonging to the Mossi or the Fulani ethnic groups were enrolled for the assessment of selected parasitological, immunological and genetic variables in relation to their susceptibility to malaria. The prevalence of the Plasmodium falciparum infection frequency was relatively low in the Fulani ethnic group compared to the Mossi ethnic group. For all tested antigens, the Fulani had higher antibody levels than the Mossi group. In both ethnic groups, a similar distribution of FcγRIIa R131H polymorphism was found. Individuals with the R allele of FcγRIIa had higher antibody levels than those with the H allele. This study confirmed that malaria infection affected less the Fulani group than the Mossi group. FcγRIIa-R131H allele distribution is similar in both ethnic groups, and higher antibody levels are associated with the FcγRIIa R allele compared to the H allele.     

Functional characterization of receptor-type protein tyrosine phosphatase CD148 (HPTPη/DEP-1) in Fcγ receptor IIa signal transduction of human neutrophils

Activation of the 40-kDa low-affinity receptor for IgG (FcγRIIa, CD32) leads to tyrosine phosphorylation, increase of cytosolic free calcium concentration ([Ca²⁺]_i), and production of superoxide anions (O₂^?) in neutrophils (PMN). It has been established that protein tyrosine kinases (PTK) and phosphatases (PTP) are essential for the regulation of intracellular signaling. CD45 is a type I receptor-type protein tyrosine phosphatase (RPTP) with two PTP domains. Recently it has been demonstrated that co-cross-linking of CD45 modulates the signal transduction pathway of FcγRIIa in PMN. In contrast, the functional characteristics of CD148 (HPTPη/DEP-1), a new RPTP with only one PTP domain, is unknown. CD148 is expressed on PMN in slightly lower density than CD45, and in higher density than on lymphocytes. [Ca²⁺]_i measured with fluo-3-loaded PMN by flow cytometry and O₂^? production determined by lucigenin-dependent chemiluminescence were inhibited by co-cross-linking of CD45 with FcγRIIa in comparison to isotype control monoclonal antibody (mAb). In contrast, preincubation with CD148 mAb 143-41 abolished O₂^? generation, but did not inhibit [Ca²⁺]_i rise. In summary, both clustered human RPTP, CD45 and CD148, inhibit FcγRIIa-induced O₂^? production in PMN, but they differ in regulation of [Ca²⁺]_i. Therefore, it is suggested that co-cross-linking of FcγRII with CD45 and CD148 leads to dephosphorylation of different substrates. These distinct functional capacities may be important for differential regulation of FcγR signaling by currently unknown ligands.     

In vivo depletion of CD4+FOXP3+ Treg cells by the PC61 anti‐CD25 monoclonal antibody is mediated by FcγRIII+ phagocytes

Depletion of CD4⁺CD25⁺FoxP3⁺ Treg using PC61 mAb (anti‐murine CD25 rat IgG1) is widely used to characterize Treg function in vivo. However, the mechanism of Treg depletion remains largely unknown. Herein, we report the PC61 mAb's mechanism of action. In peripheral blood, a single injection of PC61 mAb eliminated ～70% of CD4⁺FoxP3⁺ cells with the remaining Treg expressing low or no CD25. Functional blockade of Fcγ receptors with 2.4G2 mAb significantly inhibited PC61 mAb activity. Furthermore, Fcγ receptor (FcγR)III^?/? mice were resistant to Treg depletion. FcγRIII is expressed on immune cells including NK cells and macrophages that are the major effector cells for Ab‐dependent‐cellular‐cytotoxicity and Ab‐dependent‐cellular‐phagocytosis, respectively. Depletion of NK cells had no effect, whereas depletion of phagocytes, including macrophages, by clodronate liposome significantly inhibited Treg depletion. Furthermore, in vitro, PC61 mAb can mediate Ab‐dependent‐cellular‐phagocytosis of CD25⁺ cells by WT or FcγRIIB^?/?, but not FcγRIII^?/?, macrophages. Altogether these data demonstrate the critical role of FcγRIII⁺ phagocytes in mediating Treg depletion by PC61 mAb. This finding may be useful in guiding the development of human Treg targeting therapy.     

Divergent effects of FcγRIIIA ligands on the functional activities of human natural killer cells in vitro

The Fcγ receptor (R)IIIA (CD16) plays an important role in regulating the cytotoxic and non-cytotoxic functions of human natural killer (NK) cells. Some anti-CD 16 monoclonal antibodies (mAb) have been shown to stimulate NK activity, while human monomeric (m) IgG induces dose-dependent inhibition of NK activity. To explore further these interactions mediated via FcγRIIIA, purified NK cells were cultured for 2–3 days in the presence of mIgG, 3G8 mAb, interleukin-2 (IL-2) or a combination of mIgG or 3G8 with IL-2. Binding of mIgG or 3G8 to FcγRIIIA induced divergent effects of functions of cultured NK cells: 3G8 mAb + IL-2 induced dose-dependent inhibition of proliferation attributable to apoptosis; in contrast, mIgG + IL-2 significantly increased NK cell proliferation. Incubation of NK cells in the presence of mIgG up-regulated expression of surface activation markers (CD69, IL-2Rα, ICAM-1), cytotoxicity, cytokine production (IL-1β, IFN-γ and TNF-α) and release of soluble IL-2R. Thus, mIgG binding to FcγRIIIA induced stimulatory signals in human NK cells, leading to up-regulation of IL-2Rα expression, cell proliferation and cytokine release.     

Fcγ receptor II dependency of enhanced presentation of major histocompatibility complex class II peptides by a B cell lymphoma

Here we show that the B cell lymphoma A20.292 is capable of enhanced antigen presentation to CD4⁺ T cells in the presence of specific antibodies. This enhancement was inhibited by anti-Fcγ receptor (R) antibodies, suggesting that it might be due to preferential uptake of the antigen/antibody complex through the FcγRII receptor. However, immunoprecipitation studies revealed that the FcR of A20.292 cells was of the B cell type, FcγRIIb1, which is not thought to be able to internalize antigen/antibody complexes via clathrin-coated pits. It was considered unlikely that A20.292 had an altered form of the B cell FcγR (RIIb1) receptor that enabled internalization, since similar enhancing effects were also observed using an FcγRII^? cell line that had been transfected with FcγRIIb1. To reconcile these findings with the expression of FcγRIIb1, it is postulated that immune complexes are concentrated on the cell surface by the FcγRIIb1 and are thus available for preferential uptake by random fluid-phase endocytosis. This results in more efficient generation of the epitopes recognized by these T cell hybridomas.     

Leishmania donovani‐induced expression of signal regulatory protein α on Kupffer cells enhances hepatic invariant NKT‐cell activation

Signal regulatory protein α (SIRPα) and its cognate ligand CD47 have been documented to have a broad range of cellular functions in development and immunity. Here, we investigated the role of SIRPα–CD47 signalling in invariant NKT (iNKT) cell responses. We found that CD47 was required for the optimal production of IFN‐γ from splenic iNKT cells following exposure to the αGalCer analogue PBS‐57 and in vivo infection of mice with Leishmania donovani. Surprisingly, although SIRPα was undetectable in the liver of uninfected mice, the hepatic iNKT‐cell response to infection was also impaired in CD47^?/? mice. However, we found that SIRPα was rapidly induced on Kupffer cells following L. donovani infection, via a mechanism involving G‐protein‐coupled receptors. Thus, we describe a novel amplification pathway affecting cytokine production by hepatic iNKT cells, which may facilitate the breakdown of hepatic tolerance after infection.     

Detection of Fcγ receptors on human endothelial cells stimulated with cytokines tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ)

This investigation was conducted to detect Fcγ receptors (FcγR) on cytokine-stimulated human endothelial cells (EC) by measuring anti-FcγR MoAb binding with an ELISA. TNF-α and IFN-γ significantly increased the expression of FcγR type II (FcγRII) and type III (FcγRIII) on aortic EC. Simultaneous treatment with both cytokines had a synergistic effect and pretreatment of EC with IFN-γ augmented the effect of TNF-α. The greatest effect was the increase (up to four-to-six-fold) in expression of FcγRII found by the simultaneous treatment of aortic EC with both cytokines. The receptors were expressed on the cell surface and showed receptor capping after incubation at 37°C. This study showed that the inflammatory cytokines TNF-α and IFN-γ enhanced low-affinity FcγR expression on human EC in vitro. The expression of FcγR may contribute to the specific localization of circulating immune complexes on blood vessels in areas of vasculitis.     

The rs150311303 polymorphism in FcγRIIa enhances IgG binding capacity

Fc gamma receptor (FcγR) provides an important link between humoral and cellular immune responses. FcγRIIa-H131R polymorphism has been associated with differential binding to IgG subclasses and susceptibility to severe malaria phenotypes among different populations in the malaria endemic world. In this study, the effect of FCGR2A gene polymorphisms on susceptibility to symptomatic malaria among Ghanaian cohort children was investigated. Blood samples from four hundred and 29 (429) healthy Ghanaian children were genotyped for FCGR2A polymorphisms by direct DNA sequencing. Attributable and relative risks to symptomatic malaria were calculated for the polymorphic variants. Two major FCGR2A polymorphisms, rs1801274A/G (FcγRIIa-H131R) and rs150311303 (FcγRIIa-ins170L), were identified in the study population, and assessment of their risks did not show significant association with susceptibility to symptomatic malaria. The functional significance of these polymorphisms was also examined by evaluating their binding abilities to IgG subclasses using flow cytometric analysis of HEK cells transfected with the FcγRIIa haplotype variants. The binding assay revealed the rs150311303, which was observed only among carriers of the FcγRIIa-131RR genotype for the rs1801274 to consistently enhance binding capacities to all IgG subclasses. Thus, of the three FcγRIIa haplotype variants observed in this study population, the FcγRIIa(RL) haplotype variant was observed to have the highest binding ability to IgG1, IgG3 and IgG4.     

IgG-mediated anaphylaxis via Fcγ receptor in CD40-deficient mice

Anaphylaxis denotes an immediate hypersensitivity reaction to allergen, exclusively mediated by IgE antibodies. However, IgE antibodies do not explain all the syndromes that are encountered. We investigated potent IgG-mediated anaphylaxis in CD40-deficient mice that lack the immunoglobulin class switching for T cell-dependent antigens. Immunization with ovalbumin did not induce either humoral responses of IgG, IgA, and IgE, or systemic anaphylaxis in CD40-deficient mice. Although systemic anaphylaxis by active immunization was not observed in CD40-deficient mice, both passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis assessed by mouse blood pressure monitoring with cervical artery catheterization did take place when antigen-specific IgG was transferred and then antigen challenge given. Further, to investigate the inflammatory pathway of IgG-mediated immediate hypersensitivity reactions, we focused on the Fcγ receptor (FcγR) function. Pretreatment of the mice with the anti-FcγRII/FcγRIII MoAb clearly blocked the response of PCA and passive systemic anaphylaxis, suggesting that they were initiated through FcγR. In conclusion, we directly demonstrate the IgG-mediated anaphylaxis and its triggering mechanism through FcγR in in vivo conditions. In addition to IgE-mediated anaphylaxis, IgG-mediated anaphylaxis should be considered and the blocking of FcγR would provide one of the therapeutic targets for the control of IgG-mediated hypersensitivity diseases.     

Membrane-Spanning Fcγ Receptor III Isoform Expressed on Human Placental Trophoblasts

PROBLEM: Fc receptor for immunoglobulin (FcγR) is an important mediator of immunological functions in the feto-maternal relationship. We have demonstrated by immunohistochemical means that three distinct classes of FcγRS are expressed in the different cell components of the human placenta. METHOD: In this study, FcγRIII isoform expressed on placental trophoblasts (PTs) was investigated by indirect immunofluorescence and cDNA cloning. PTs, isolated from human term placenta by digestion with proteolytic enzyme, were reacted with monoclonal antibodies (MAb) against the Fc-γRs and other surface markers of leukocytes and subjected to flow cytometric analysis. RESULTS: PTs were positively stained with 3G8 and Leul lb against FcγRIII, partially stained with MAb against MHC class I, but not with 32.2 (FcγRI), IV3 (FcγRII), or MAbs against CD4, CD19, or CD56, indicating that only low affinity receptor, FC7RIII, is γexpressed on PTs. The DNA sequence of cloned FcγRIII CDNA from PTs by PCR was identical to that of natural killer (NK) cell isoform, including the position of the stop codon that differs from the granulocyte isoform by several nucleotide substitutions. We further analyzed the susceptibility of PTs against phosphatidylinositol specific phospholipase C (PI-PLC) to determine the structural topology of PT isoform. While the reactivity with 3G8 on PTs was not influenced by treatment with PI-PLC, that on granulocytes was significantly diminished with PI-PLC. CONCLUSIONS: This result confirmed that FcγRIII on PTs is a membrane-spanning molecule, and that it is distinctive from PI anchoring FcγRIII on granulocytes.     

Fcγ receptor-mediated phagocytosis requires tyrosine kinase activity and is ligand independent

Receptors for the invariant chain of immunoglobulins (FcR) define the cellular response to specific antigens. FcγR recognize IgG and so elicit a variety of effector functions including phagocytosis. We are interested in the structural determinants for FcγR-mediated phagocytosis, specifically FcγRI(p135) and FcγRIIa isoforms. The low-affinity receptor, FcγRIIa, is found on macrophages and its cytoplasmic domain contains a tyrosine activation motif which has previously been shown to regulate endocytosis. In contrast, FcγRI has no known signaling motifs, though a functional interaction has recently been demonstrated with the γ chain of the high-affinity receptor for IgE, FcεRI. This accessory molecule has a cytoplasmic tyrosine activation motif implicated in signal transduction. Here we demonstrate that although FcγRI transiently expressed on COS-7 cells is able to rosette opsonized SRBC, it cannot phagocytose them. If the cytoplasmic domain of either γ chain or FcγRIIa replaces that of FcγRI in a chimeric receptor, efficient phagocytosis occurs. This particle ingestion is sensitive to the tyrosine kinase inhibitor genistein. Chimeric receptors where the extracellular domain of either FcγRI or FcγRIIa is replaced with that of CD2, a T cell antigen, indicate that FcγR-mediated phagocytosis is ligand independent. We conclude that phagocytosis is dependent upon close particle apposition, tyrosine kinase activity, and that the process is ligand independent.     

Signal regulatory protein alpha (SIRPα) regulates the homeostasis of CD103+CD11b+ DCs in the intestinal lamina propria

Signal regulatory protein alpha (SIRPα/CD172a) is a conserved transmembrane protein thought to play an inhibitory role in immune function by binding the ubiquitous ligand CD47. SIRPα expression has been used to identify dendritic cell subsets across species and here we examined its expression and function on intestinal DCs in mice. Normal mucosa contains four subsets of DCs based on their expression of CD103 and CD11b and three of these express SIRPα. However, loss of SIRPα signaling in mice leads to a selective reduction in the CD103⁺CD11b⁺ subset of DCs in the small intestine, colon, and among migratory DCs in the mesenteric lymph node. In parallel, these mice have reduced numbers of T_H17 cells in steady‐state intestinal mucosa, and a defective T_H17 response to Citrobacter infection. Identical results were obtained in CD47KO mice. DC precursors from SIRPα mutant mice had an enhanced ability to generate CD103⁺CD11b⁺ DCs in vivo, but CD103⁺CD11b⁺ DCs from mutant mice were more prone to die by apoptosis. These data show a previously unappreciated and crucial role for SIRPα in the homeostasis of CD103⁺CD11b⁺ DCs in the intestine, as well as providing further evidence that this subset of DCs is critical for the development of mucosal T_H17 responses.