Sulfur mustard-increased proteolysis followingin vitro andin vivo exposures

The pathologic mechanisms underlying sulfur mustard (HD)-induced skin vesication are as yet undefined. Papirmeister et al. (1985) postulate enhanced proteolytic activity as a proximate cause of HD-induced cutaneous injury. Using a chromogenic peptide substrate assay, we previously reported that in vitro exposure of cell cultures to HD enhances proteolytic activity. We have continued our investigation of HD-increased proteolytic activity in vitro and have expanded our studies to include an in vivo animal model for HD exposure. In vitro exposure of human peripheral blood lymphocytes (PBL) to HD demonstrated that the increase in proteolytic activity is both time- and temperature-dependent. Using a panel of 10 protease substrates, we established that, the HD-increased proteolysis was markedly different from that generated by plasminogen activator. The hairless guinea pig is an animal model used for the study of HD-induced dermal pathology. When control and HD-exposed PBL and hairless guinea pig skin where examined, similarities in their protease substrate reactivities were observed. HD-exposed hairless guinea pig skin biopsies demonstrated increased proteolytic activity that was time-dependent. The HD-increased proteolytic response was similar in both in vitro and in vivo studies and may be useful for elucidating both the mechanism of HD-induced vesication and potential treatment compounds.Abbreviations CPSPA chromogenic peptide substrate protease assay - HD sulfur mustard - PBL human peripheral blood lymphocytes - pNA p-nitroaniline In conducting the research described in this report, the investigators adhered to the Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, revised 1985.The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.     

IL-4 decreases Fc gamma R membrane expression and Fc gamma R-mediated cytotoxic activity of human monocytes

Monocytes can express three classes of FcR for IgG: Fc gamma RI, Fc gamma RII, and Fc gamma RIII (CD64, CD32, and CD16, respectively) of which the Fc gamma RIII is expressed after prolonged culture. Fc gamma R expression is regulated by IFN-gamma. Because IFN-gamma and IL-4 have antagonistic effects on the expression of the FcR for IgE on human monocytes, we studied the effect of IL-4 on Fc gamma R expression and function. We show that IL-4 down-regulates Fc gamma RI, Fc gamma RII, and Fc gamma RIII expression of cultured monocytes and inhibits IFN-gamma enhanced Fc gamma RI expression. Exposure of monocytes to IL-4 for 40 h resulted in a dose-dependent decrease of the expression of all three Fc gamma R that persisted throughout the whole culture period (7 days). Anti-IL-4 antibodies completely reversed the IL-4 effect. In addition the impaired Fc gamma R expression correlated directly with reduced Fc gamma R-mediated function because monocytes cultured in the presence of IL-4 have a reduced capacity to lyse human E opsonized with human IgG anti-D or mouse antiglycophorin A antibodies. These observations, together with the previous finding that IL-4 induces Fc epsilon RIIb expression on monocytes, indicate that IL-4 and IFN-gamma may control the Fc gamma R-mediated immune response by differentially regulating Fc gamma R expression.     

Activation of cloned human natural killer cells via Fc gamma RIII

The Fc gamma RIII (CD16) Ag on human NK cells involved in antibody-dependent cellular cytotoxicity has been demonstrated to be an important activation structure. The present studies were carried out to further characterize the functional role of the CD16 Ag and the mechanisms whereby cytotoxicity is activated by using human NK clones. In phenotypic studies Fc gamma RIII was found to be expressed heterogeneously on various human cloned NK cells. Expression on CD3- and CD3+ clones varied with the donor and mAb used for detection. Functional data demonstrated that cytotoxicity against NK-resistant target cells can be induced in CD3-CD16+ NK clones and CD3+CD16+ clones with NK activity when various CD16 mAb were used. CD16 antibodies but not reactive isotype control antibodies induced cytotoxicity. In contrast to complete CD16 antibodies F(ab')2 fragments were not able to activate the cytotoxic mechanism. Both an antibody against FcR on the target cell (Fc gamma RII) and a CD11a antibody blocked induction of cytotoxicity. These results suggest that three steps are critical for activation of CD16+ cells via Fc gamma RIII: 1) specific binding of CD16 antibodies to Fc gamma RIII on effector cells irrespective of the epitope recognized; 2) cross-linking of effector cell CD16 Ag through binding of the Fc site of CD16 antibodies via corresponding FcR on the target cell membrane; and 3) interaction of CD11a/18 molecules with the target cell membrane.     

Fc gamma RII/CD32-negative human Langerhans cells may be responsible for the immunostimulatory activity of freshly isolated epidermal cells.

Cultured murine and human epidermal Langerhans cells (LC) undergo a phenotypical and functional maturation process. In fact, they loose Fc gamma RII and Birbeck granules, increase HLA-DR expression, and become potent accessory cells for allogeneic MLR. However, resident/freshly isolated human epidermal LC represent a phenotypically heterogeneous cell population. Indeed, a subset of CD1a+ LC lacks Birbeck granules, is Fc gamma RII/CD32-, and strongly expresses HLA-DR and the RFD1 antigen that is considered to be specific for interdigitating cells. In the present study the functional capacity of this Fc gamma RII/CD32- CD1a+ LC subset was investigated in MLR assays by comparing the stimulatory activity of freshly isolated crude epidermal cells (EC) with that of freshly isolated EC depleted in CD1a+ or in Fc gamma RII+ cells. Thereby, we observed that crude EC stimulated allogeneic PBMC while the removal of CD1a+ cells abrogated this stimulation. However, crude EC depleted in Fc gamma RII/CD32+ cells still exhibited a stimulatory capacity that was at least equal to that of crude EC. Taken together, these data suggest that among resident human epidermal LC there exists a subset of phenotypically and functionally more differentiated cells that may be solely responsible for the stimulatory capacity of freshly isolated crude EC.     

Differential regulation of the low affinity Fc receptor for IgE (Fc epsilon R2/CD23) and the IL-2 receptor (Tac/p55) on eosinophilic leukemia cell line (EoL-1 and EoL-3)

Two types of activation Ag, low affinity FcR for IgE (Fc epsilon R2)/CD23 and IL-2R (Tac/p55), were expressed and differently regulated on human eosinophilic leukemia cell lines (EoL-1 and EoL-3). Because the binding of IgE on EoL-3 cells was completely inhibited by H107 (anti-Fc epsilon R2/CD23 mAb) but not by irrelevant mAb, essentially all the low affinity Fc epsilon R2 on EoL-3 seemed to be the Fc epsilon R2/CD23 molecules. Both IL-4 and IFN-gamma enhanced the surface expression of Fc epsilon R2, whereas IL-1, IL-2, and IL-5 showed no effects, as determined by surface staining with anti-Fc epsilon R2 antibody (H107). In contrast to Fc epsilon R2 up-regulation, IL-4 and IFN-gamma showed a differential effect on the regulation of IL-2R (Tac/p55). Whereas IFN-gamma up-regulated the receptor expression of IL-2R/Tac, IL-4 did not. The result suggests that these lymphokines are involved in the different aspects of the activation pathway of the eosinophils. The possible role of Fc epsilon R2 and IL-2R on the function of eosinophils in allergic reaction is discussed.     

An HIV-1 gp120 envelope human monoclonal antibody that recognizes a C1 conformational epitope mediates potent antibody-dependent cellular cytotoxicity (ADCC) activity and defines a common ADCC epitope in human HIV-1 serum

Among nonneutralizing HIV-1 envelope antibodies (Abs), those capable of mediating antibody-dependent cellular cytotoxicity (ADCC) activity have been postulated to be important for control of HIV-1 infection. ADCC-mediating Ab must recognize HIV-1 antigens expressed on the membrane of infected cells and bind the Fcγ receptor (FcR) of the effector cell population. However, the precise targets of serum ADCC antibody are poorly characterized. The human monoclonal antibody (MAb) A32 is a nonneutralizing antibody isolated from an HIV-1 chronically infected person. We investigated the ability of MAb A32 to recognize HIV-1 envelope expressed on the surface of CD4(+) T cells infected with primary and laboratory-adapted strains of HIV-1, as well as its ability to mediate ADCC activity. The MAb A32 epitope was expressed on the surface of HIV-1-infected CD4(+) T cells earlier than the CD4-inducible (CD4i) epitope bound by MAb 17b and the gp120 carbohydrate epitope bound by MAb 2G12. Importantly, MAb A32 was a potent mediator of ADCC activity. Finally, an A32 Fab fragment blocked the majority of ADCC-mediating Ab activity in plasma of subjects chronically infected with HIV-1. These data demonstrate that the epitope defined by MAb A32 is a major target on gp120 for plasma ADCC activity.     

IgE-mediated activation of NK cells through Fc gamma RIII

NK cells express Fc gamma RIII (CD16), which is responsible for IgG-dependent cell cytotoxicity and for production of several cytokines and chemokines. Whereas Fc gamma RIII on NK cells is composed of both Fc gamma RIII alpha and FcR gamma chains, that on mast cells is distinct from NK cells and made of Fc gamma RIII alpha, FcR beta, and FcR gamma. Mast cells show degranulation and release several mediators, which cause anaphylactic responses upon cross-linking of Fc gamma RIII as well as Fc epsilon RI with aggregated IgE. In this paper, we examined whether IgE activates NK cells through Fc gamma RIII on their cell surface. We found that NK cells produce several cytokines and chemokines related to an allergic reaction upon IgE stimulation. Furthermore, NK cells exhibited cytotoxicity against IgE-coated target cells in an Fc gamma RIII-dependent manner. These effects of IgE through Fc gamma RIII were not observed in NK cells from FcR gamma-deficient mice lacking Fc gamma RIII expression. Collectively, these results demonstrate that NK cells can be activated with IgE through Fc gamma RIII and exhibit both cytokine/chemokine production and Ab-dependent cell cytotoxicity. These data imply that not only mast cells but also NK cells may contribute to IgE-mediated allergic responses.     

Human Fc epsilon RIalpha-specific human single-chain Fv (scFv) antibody with antagonistic activity toward IgE/Fc epsilon RIalpha-binding

The alpha-chain of Fc epsilon RI (Fc epsilon RIalpha) plays a critical role in the binding of IgE to Fc epsilon RI. A fully human antibody interfering with this interaction may be useful for the prevention of IgE-mediated allergic diseases. Here, we describe the successful isolation of a human single-chain Fv antibody specific to human Fc epsilon RIalpha using human antibody phage display libraries. Using the non-immune phage antibody libraries constructed from peripheral blood lymphocyte cDNA from 20 healthy subjects, we isolated three phage clones (designated as FcR epsilon 27, FcR epsilon 51, and FcR epsilon 70) through two rounds of biopanning selection. The purified soluble scFv, FcR epsilon 51, inhibited the binding of IgE to recombinant Fc epsilon RIalpha, although both FcR epsilon 27 and FcR epsilon 70 showed fine binding specificity to Fc epsilon RIalpha. Since FcR epsilon 51 was determined to be a monomer by HPLC, BIAcore analysis was performed. The dissociation constant of FcR epsilon 51 to Fc epsilon RIalpha was estimated to be 20 nM, i.e., fortyfold lower than that of IgE binding to Fc epsilon RIalpha (K(d) = 0.5 nM). With these characteristics, FcR epsilon 51 exhibited inhibitory activity on the release of histamine from passively sensitized human peripheral blood mononuclear cells.     

Functions of the various IgG Fc receptors in mediating killing of Toxoplasma gondii.

The three types of IgG FcR (Fc gamma RI, Fc gamma RII, Fc gamma RIII) on human leukocytes play an important role in elimination of antibody-coated infectious agents. To further understand the role of the different Fc gamma R in mediating this killing, we examined the ability of human myeloid and lymphoid cells to kill the protozoan Toxoplasma gondii in the presence of antitoxoplasma IgG or bispecific antibodies. Although human myeloid cells (monocytes, macrophages, neutrophils, and eosinophils) all lysed unsensitized T. gondii, killing by these cells was significantly enhanced by opsonization with antitoxoplasma rabbit IgG. Human lymphocytes, however, did not lyse T. gondii unless the parasites were coated with antibody. The role of antibody and Fc gamma R in mediating ADCC of T. gondii was then examined using bispecific antibodies made by chemically cross-linking Fab fragments of antitoxoplasma antibodies to Fab fragments of antibodies specific for human leukocyte surface Ag, including Fc gamma R. Thus, simultaneous binding of these bispecifics to parasites and effector cells allowed an evaluation of killing when T. gondii were targeted to each Ag independently. Bispecifics which targeted T. gondii to Fc gamma RI, II or III enhanced lysis by monocytes. However, similar results were obtained with bispecifics targeting T. gondii to non-Fc gamma R Ag (CD11b or beta 2-microglobulin) on monocytes. Likewise, polymorphonuclear leukocytes mediated significantly more lysis in the presence of bispecifics linking T. gondii to Fc gamma RII, Fc gamma RIII, or the two non-Fc gamma R Ag CD11b and beta 2-microglobulin. Thus, although human myeloid cells did not require antibody-Fc gamma R triggering to kill T. gondii, antibody appeared to enhance lysis by capturing and directing the parasites to the effector cell surface. Human lymphocytes, in contrast, mediated significant lysis of T. gondii only in the presence of bispecifics targeting T. gondii to Fc gamma RIII, indicating a requirement for specific triggering of Fc gamma RIII for killing by large granular lymphocytes. Consequently, using bispecifics to compare targeting to specific Ag, both non-Fc gamma R and Fc gamma R, allowed determination of the role of antibody-Fc gamma R interactions in T. gondii killing. In addition, these studies demonstrate the potential of bispecifics in determining the role of specific Ag in killing of or infection by pathogens.     

Involvement of a metalloprotease in spontaneous and phorbol ester-induced release of natural killer cell-associated Fc gamma RIII (CD16-II).

Two genes encode the CD16 low affinity IgG FcR. CD16-I (Fc gamma RIII-1) is expressed on PMN as a phosphatidylinositol-glycan anchored glycoprotein. CD16-II (Fc gamma RIII-2) is expressed on NK cells and macrophages as a transmembrane glycoprotein associated with CD3 zeta or Fc epsilon RI-gamma. NK cells spontaneously release soluble CD16-II from the cell surface and this is enhanced by activation with phorbol ester. In this study, we demonstrate that a metalloprotease is involved in the spontaneous and PMA-induced release of CD16-II from NK cells. 1,10-phenanthroline, an inhibitor of Zn(2+)-dependent metalloproteases, efficiently inhibits CD16-II release. 1,7-phenanthroline, an inactive analogue that doesn't chelate Zn2+ or other divalent metal cations, and inhibitors of serine proteases do not affect spontaneous or PMA-induced release of CD16-II. Murine P815 mastocytoma cells transfected with human CD16-II cDNA shed membrane CD16, and 1,10-phenanthroline inhibits this process. P815 transfectants expressing CD16-II molecules with truncated cytoplasmic domains also release soluble receptors, indicating that the cytoplasmic segment of CD16-II is not required for interaction with the protease or the cytoskeleton. By contrast, 1,10-phenanthroline does not inhibit PMA-induced release of CD16-I glycoprotein from PMN, indicating a different mechanism of release for this phosphatidylinositol-glycan anchored molecule. Prior studies have demonstrated that NK cells are activated via the inositol phosphate pathway after engagement of CD16-II by immune complexes or Ig-coated tumor cell targets. A membrane metalloprotease with substrate specificity for CD16-II that is activated by PKC stimulation may provide a mechanism for releasing the immune complex or target from the effector cells and halting signal transduction.     

Impact of linker and conjugation chemistry on antigen binding,Fc receptor binding and thermal stability of model antibody-drug conjugates

Antibody-drug conjugates (ADCs) with biotin as a model cargo tethered to IgG1 mAbs via different linkers and conjugation methods were prepared and tested for thermostability and ability to bind target antigen and Fc receptor. Most conjugates demonstrated decreased thermostability relative to unconjugated antibody, based on DSC, with carbohydrate and amine coupled ADCs showing the least effect compared with thiol coupled conjugates. A strong correlation between biotin-load and loss of stability is observed with thiol conjugation to one IgG scaffold, but the stability of a second IgG scaffold is relatively insensitive to biotin load. The same correlation for amine coupling was less significant. Binding of antibody to antigen and Fc receptor was investigated using surface plasmon resonance. None of the conjugates exhibited altered antigen affinity. Fc receptor FcγIIb (CD32b) interactions were investigated using captured antibody conjugate. Protein G and Protein A, known inhibitors of Fc receptor (FcR) binding to IgG, were also used to extend the analysis of the impact of conjugation on Fc receptor binding. H10_NPEG4 was the only conjugate to show significant negative impact to FcR binding, which is likely due to higher biotin-load compared with the other ADCs. The ADC aHIS_NLC and aHIS_TPEG8 demonstrated some loss in affinity for FcR, but to much lower extent. The general insensitivity of target binding and effector function of the IgG1 platform to conjugation highlight their utility. The observed changes in thermostability require consideration for the choice of conjugation chemistry, depending on the system being pursued and particular application of the conjugate.     

The inhibition of T cell proliferative responses by crosslinking CD7 and IgM-Fc receptors.

Previous work from our laboratory described a human T cell soluble ligand that inhibited T cell proliferative responses to mitogen and alloantigen by interacting with CD7 and/or the receptor for the IgM-Fc portion (FcR mu) on T cells. In this report, we used mouse anti-human CD7 monoclonal antibodies (mAb) and purified human IgM (HIgM) to substitute for the human ligand and examined the possible involvement of these receptors in the inhibition of T cell proliferation. Preincubation of human T cells with mouse anti-CD7 mAb, HIgM, mouse anti-human IgM (MAH IgM) alone, or any of these combinations as a primary antibody did not inhibit mitogen- or alloantigen-induced T cell replication. Similar effects were seen with the pretreatment of T cells with an irrelevant negative control primary mAb or a secondary-step goat anti-mouse immunoglobulin (GAM Ig), goat anti-human IgM-Fc (GAH Fc mu), or both. In contrast, the pretreatment of T cells with anti-CD7 and/or HIgM followed by the appropriate secondary-step crosslinking antibody significantly reduced their proliferative responses to mitogen and alloantigen. Similarly, crosslinking of CD7 and FcR mu on human transformed T cell lines inhibited their spontaneous proliferation. The inhibitory effect of crosslinking CD7 and FcR mu was not due to cytotoxic effects of these antibodies and appears to be temperature sensitive. These findings suggest that crosslinking CD7 and/or FcR mu appears to have a novel role in down-regulating T cell proliferation.     

TOSO, the Fcmicro receptor, is highly expressed on chronic lymphocytic leukemia B cells, internalizes upon IgM binding, shuttles to the lysosome, and is downregulated in response to TLR activation

TOSO/FAIM3 recently has been identified as the long-sought-after FcR for IgM (FcμR). FcμR is expressed on human CD19(+) B cells, CD4(+)/CD8(+) T cells, and CD56(+)/CD3(-) NK cells and has been shown to be overexpressed in chronic lymphocytic leukemia (CLL) cells. CLL is a malignancy of mature IgM(+) B lymphocytes that display features of polyreactive, partially anergized B cells related to memory B cells. In this article, we report that FcμR is O-glycosylated in its extracellular domain and identify the major sites of O-glycosylation. By using immunofluorescence confocal microscopy, we found that FcμR localized to the cell membrane but also found that large pools of FcμR accumulate in the trans-Golgi network. Aggregation of FcμR on CLL cells by IgM prompted rapid internalization of both IgM and FcμR, reaching half-maximal internalization of cell-bound IgM within 1 min. Upon internalization, FcμR transported IgM through the endocytic pathway to the lysosome, where it was degraded. Using a series of FcμR deletion mutants, we identified a proline-rich domain essential for cell surface expression of FcμR and a second domain, containing a YXXΦ motif, that controls internalization. Although it has been reported that BCR activation increases FcμR expression, we found that activation of TLRs strongly downregulated FcμR at both the mRNA and protein levels. Through internalization of IgM bound immune complexes, FcμR may play a role in immune surveillance and contribute to B cell activation. In addition, FcμR deserves study as a potential pathway for the delivery of therapeutic Ab-drug conjugates into CLL cells.     

A subset of human dendritic cells expresses IgA Fc receptor (CD89), which mediates internalization and activation upon cross-linking by IgA complexes

Immature dendritic cells (DC) sample Ags within nonlymphoid tissues and acquire exogenous proteins/pathogens via scavenger receptors or Ig FcR such as Fc gamma R and Fc epsilon R. IgA is present in a significant proportion among serum Ig and is the main isotype in mucosae, where DC are numerous. We found that a functional Fc alpha R (CD89) was expressed in situ and in vitro on interstitial-type DC but not on Langerhans cell-type DC. Interstitial-type DC expressed CD89 as a 50- to 75-kDa glycoprotein with a 32-kDa protein core, which was down-regulated upon addition of TGF-beta 1. DC, Fc alpha R specifically, bound IgA1 and IgA2. Cross-linking of CD89 on DC triggered endocytosis in time-dependent manner. In addition, internalization of polymeric IgA complexes induced the production of IL-10 and DC activation, as reflected by up-regulation of CD86 costimulatory molecules, class II MHC expression, and increased allostimulatory activity. Therefore, interstitial-type DC may use Fc alpha R-mediated Ag sampling in the subepithelium to check tissue integrity while Langerhans cells inside epithelial layers may neglect IgA immune complexes.     

MLC-conditioned media stimulate an increase in Fc receptors on human macrophages

Macrophage Fc receptors (FcR) are essential for antibody-dependent cellular cytotoxicity and for optimal phagocytosis of opsonized particulate antigens. Culture in the presence of conditioned medium from mixed leukocyte cultures (MLC-CM) resulted in a dose- and time-dependent increase (up to 10-fold) in FcR-dependent binding of 125I-labeled IgG1 to promyelocytic HL-60 cells, macrophage-like U-937 cells, and normal cultured human monocytes. FcR increase in HL-60 cells was blocked by cycloheximide (100 microM) and was accompanied by a slight decrease in binding affinity. Since cell volume did not change, the increase in FcR probably represents an increase in the surface density of FcR sites. MLC-CM prepared with or without serum were equally effective in augmenting FcR sites, whereas only serum-containing MLC-CM caused morphologic change of U-937 and HL-60 cells.     

Effective targeting of pathogens to neutrophils via chimeric surfactant protein D/anti-CD89 protein

Targeting of specific pathogens to FcRs on immune effector cells by using bispecific Abs was reported to result in effective killing of the pathogens, both in vitro and in vivo. Instead of targeting a specific pathogen to an FcR, we assessed whether a broad spectrum of pathogens can be targeted to an FcR using surfactant protein D (SP-D). SP-D is a collectin that binds a great variety of pathogens via its carbohydrate recognition domain. A recombinant trimeric fragment of SP-D (rfSP-D), consisting of the carbohydrate recognition domain and neck domain of human SP-D, was chemically cross-linked to the Fab' of an Ab directed against the human Fc alpha RI (CD89). In vitro, the chimeric rfSP-D/anti-CD89 protein enhanced uptake of Escherichia coli, Candida albicans, and influenza A virus by human neutrophils. Blocking of the interaction between rfSP-D/anti-CD89 and either the pathogen or CD89 abolished its stimulatory effect on pathogen uptake by neutrophils. In addition, rfSP-D/anti-CD89 stimulated killing of E. coli and C. albicans by neutrophils and enhanced neutrophil activation by influenza A virus. In conclusion, rfSP-D/anti-CD89 effectively targeted three structurally unrelated pathogens to neutrophils. (Col)lectin-based chimeric proteins may thus offer promise for therapy of infectious disease.     

Human epidermal Langerhans cells express only the 40-kilodalton Fc gamma receptor (FcRII)

In man, three distinct classes of receptors for the Fc fragments of IgG (FcRI, II, III) have been defined. The FcRI has a Mr of about 72 kDa, binds human IgG-coated E, and is recognized by mAb such as 32. The FcRII has a Mr of 40 kDa, binds murine IgG1-coated E, and reacts with the mAb IV.3 and CIKM5, which recognize CDw32 moieties. Lastly, the FcRIII has a Mr of 50 to 70 kDa and is recognized by anti-CD16 mAb. In the present study we have shown that i) only murine IgG1-coated E form rosettes with 49 +/- 1.5% (mean +/- SEM, n = 9) of CD1a+ epidermal cells (EC) (which represent Langerhans and indeterminate cells) and that ii) the mAb anti-FcRII CIKM5 prevents this rosette formation. Among the mAb reacting with the three different types of FcR, only those recognizing FcRII i) stain about 55 +/- 1.5% (mean +/- SEM, n = 9) of the CD1a+ EC and ii) reveal the presence of dendritic cells in epidermal sheets obtained by suction blister. Under the electron microscope i) apparently all the cells forming rosettes or reacting with the gold-labeled anti-FcRII mAb (CIKM5 or the F(ab) fragment of IV.3) contained Birbeck granules and ii) the gold-labeled mAb were internalized in unfixed Langerhans cells by receptor-mediated endocytosis and accumulated in lysosomes. Labeling by the anti-FcRII mAb of the CD1a+ cells in suspension disappears after 48 h of culture. All these observations strongly suggest that CD1a+ EC express only the FcRII. This conclusion was confirmed by immunoprecipitation experiments, whereas no specific immunoprecipitate was noted with the anti-FcRI or anti-FcRIII mAb, the anti-FcRII mAb immunoprecipitated a protein of Mr 40 kDa.     

Characterization of IgG FcR-mediated proliferation of human T cells induced by mouse and human anti-CD3 monoclonal antibodies. Identification of a functional polymorphism to human IgG2 anti-CD3.

T cell activation induced by mouse anti-CD3 mAb has shown to be dependent on the Ig isotype of these antibodies. A study of isotype dependency of human antibodies, however, seems more relevant to human effector systems, especially in view of the availability of humanized antibodies for clinical applications. We constructed a panel of mouse and mouse/human chimeric anti-CD3 mAb, which differ only in their CH region and hence have identical binding sites and affinity. By using these antibodies, we now studied their ability to induce T cell proliferation in human PBMC and analyzed the classes of IgG FcR involved in these responses. The human (h)IgG1, hIgG3, and hIgG4, as well as mouse (m)IgG2a and mIgG3 anti-CD3 mAb induced an Fc gamma RI (CD64)-dependent T cell proliferation in all donors. Activation with hIgG2 and mIgG1 anti-CD3 mAb was observed to be mediated via the low affinity Fc gamma RII (CD32). It was found that leukocytes in a normal donor population display a functional polymorphism with respect to hIgG2 anti-CD3 responsiveness. This polymorphism was found to be inversely related to the previously defined Fc gamma RII-polymorphism to mIgG1 anti-CD3 mAb. Monocytes expressing the Fc gamma RII mIgG1 low responder (LR) allele support hIgG2 anti-CD3 induced T cell proliferation efficiently, whereas cells homozygous for the Fc gamma RII mIgG1 high responder (HR) allele do not. This observation could be confirmed in T cell activation studies using hFc gamma RIIa-transfected mouse fibroblasts, expressing either the mIgG1 anti-CD3 HR or LR Fc gamma RII-encoding cDNA.