Characterization of a plant-produced recombinant human secretory IgA with broad neutralizing activity against HIV

Recombinant Secretory IgA (SIgA) complexes have the potential to improve antibody-based passive immunotherapeutic approaches to combat many mucosal pathogens. In this report, we describe the expression, purification and characterization of a human SIgA format of the broadly neutralizing anti-HIV monoclonal antibody (mAb) 2G12, using both transgenic tobacco plants and transient expression in Nicotiana benthamiana as expression hosts (P2G12 SIgA). The resulting heterodecameric complexes accumulated in intracellular compartments in leaf tissue, including the vacuole. SIgA complexes could not be detected in the apoplast. Maximum yields of antibody were 15.2 μg/g leaf fresh mass (LFM) in transgenic tobacco and 25 μg/g LFM after transient expression, and assembly of SIgA complexes was superior in transgenic tobacco. Protein L purified antibody specifically bound HIV gp140 and neutralised tier 2 and tier 3 HIV isolates. Glycoanalysis revealed predominantly high mannose structures present on most N-glycosylation sites, with limited evidence for complex glycosylation or processing to paucimannosidic forms. O-glycan structures were not identified. Functionally, P2G12 SIgA, but not IgG, effectively aggregated HIV virions. Binding of P2G12 SIgA was observed to CD209 / DC-SIGN, but not to CD89 / FcalphaR on a monocyte cell line. Furthermore, P2G12 SIgA demonstrated enhanced stability in mucosal secretions in comparison to P2G12 IgG mAb.     

HIV-specific functional antibody responses in breast milk mirror those in plasma and are primarily mediated by IgG antibodies

Despite months of mucosal virus exposure, the majority of breastfed infants born to HIV-infected mothers do not become infected, raising the possibility that immune factors in milk inhibit mucosal transmission of HIV. HIV Envelope (Env)-specific antibodies are present in the milk of HIV-infected mothers, but little is known about their virus-specific functions. In this study, HIV Env-specific antibody binding, autologous and heterologous virus neutralization, and antibody-dependent cell cytotoxicity (ADCC) responses were measured in the milk and plasma of 41 HIV-infected lactating women. Although IgA is the predominant antibody isotype in milk, HIV Env-specific IgG responses were higher in magnitude than HIV Env-specific IgA responses in milk. The concentrations of anti-HIV gp120 IgG in milk and plasma were directly correlated (r = 0.75; P < 0.0001), yet the response in milk was 2 logarithm units lower than in plasma. Similarly, heterologous virus neutralization (r = 0.39; P = 0.010) and ADCC activity (r = 0.64; P < 0.0001) in milk were directly correlated with that in the systemic compartment but were 2 log units lower in magnitude. Autologous neutralization was rarely detected in milk. Milk heterologous virus neutralization titers correlated with HIV gp120 Env-binding IgG responses but not with IgA responses (r = 0.71 and P < 0.0001, and r = 0.17 and P = 0.30). Moreover, IgGs purified from milk and plasma had equal neutralizing potencies against a tier 1 virus (r = 0.65; P < 0.0001), whereas only 1 out of 35 tested non-IgG milk fractions had detectable neutralization. These results suggest that plasma-derived IgG antibodies mediate the majority of the low-level HIV neutralization and ADCC activity in breast milk.     

Dectin-1 Is Essential for Reverse Transcytosis of Glycosylated SIgA-Antigen Complexes by Intestinal M Cells

Intestinal microfold (M) cells possess a high transcytosis capacity and are able to transport a broad range of materials including particulate antigens, soluble macromolecules, and pathogens from the intestinal lumen to inductive sites of the mucosal immune system. M cells are also the primary pathway for delivery of secretory IgA (SIgA) to the gut-associated lymphoid tissue. However, although the consequences of SIgA uptake by M cells are now well known and described, the mechanisms whereby SIgA is selectively bound and taken up remain poorly understood. Here we first demonstrate that both the Cα1 region and glycosylation, more particularly sialic acid residues, are involved in M cell–mediated reverse transcytosis. Second, we found that SIgA is taken up by M cells via the Dectin-1 receptor, with the possible involvement of Siglec-5 acting as a co-receptor. Third, we establish that transcytosed SIgA is taken up by mucosal CX3CR1⁺ dendritic cells (DCs) via the DC-SIGN receptor. Fourth, we show that mucosal and systemic antibody responses against the HIV p24-SIgA complexes administered orally is strictly dependent on the expression of Dectin-1. Having deciphered the mechanisms leading to specific targeting of SIgA-based Ag complexes paves the way to the use of such a vehicle for mucosal vaccination against various infectious diseases.     

Design,synthesis and application of benzyl‐sulfonate biomimetic affinity adsorbents for monoclonal antibody purification from transgenic corn

The human anti‐human immunodeficiency virus (HIV) antibody 2G12 (mAb 2G12) is one of the most broadly neutralizing antibodies against HIV that recognizes a unique epitope on the surface glycoprotein gp120. In the present work, a limited affinity‐ligand library was synthesized and evaluated for its ability to bind and purify recombinant mAb 2G12 expressed in transgenic corn. The affinity ligands were structural fragments of polysulfonate triazine dye Cibacron Blue 3GA (CB3GA) and represent novel lead scaffolds for designing synthetic affinity ligands. Solid phase chemistry was used to synthesize variants of CB3GA lead ligand. One immobilized ligand, bearing 4‐aminobenzyl sulfonic acid (4ABS) linked on two chlorine atoms of the triazine ring (4ABS‐Trz‐4ABS), displayed high affinity for mAb 2G12. Absorption equilibrium, 3D molecular modelling and molecular dynamics simulation studies were carried out to provide a detailed picture of the 4ABS‐Trz‐4ABS interaction with mAb 2G12. This biomimetic affinity ligand was exploited for the development of a facile two‐step purification protocol for mAb 2G12. In the first step of the procedure, mAb 2G12 was purified on an S‐Sepharose FF cation exchanger, and in the second step, mAb 2G12 was purified using affinity chromatography on 4ABS‐Trz‐4ABS affinity adsorbent. Analysis of the antibody preparation by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and enzyme‐linked immunosorbent assay showed that the mAb 2G12 was fully active and of sufficient purity suitable for analytical applications. Copyright © 2013 John Wiley & Sons, Ltd.     

Application of a PEG/salt aqueous two-phase partition system for the recovery of monoclonal antibodies from unclarified transgenic tobacco extract

Aqueous two-phase partition systems (ATPS) have been widely used for the separation of a large variety of biomolecules. In the present report, the application of a polyethylene glycol/phosphate (PEG/phosphate) ATPS for the separation of anti-HIV monoclonal antibodies 2G12 (mAb 2G12) and 4E10 (mAb 4E10) from unclarified transgenic tobacco crude extract was investigated. Optimal conditions that favor opposite phase partitioning of plant debris/mAb as well as high recovery and purification were found to be 13.1% w/w (PEG 1500), 12.5% w/w (phosphate) at pH 5 with a phase ratio of 1.3 and 8.25% w/w unclarified tobacco extract load. Under these conditions, mAb 2G12 and mAb 4E10 were partitioned at the bottom phosphate phase with 85 and 84% yield and 2.4- and 2.1-fold purification, respectively. The proposed ATPS was successfully integrated in an affinity-based purification protocol, using Protein A, yielding antibodies of high purity and yield. In this study, ATPS was shown to be suitable for initial protein recovery and partial purification of mAb from unclarified transgenic tobacco crude extract.     

CD4 epitope masking by gp120/anti-gp120 antibody complexes. A potential mechanism for CD4+ cell function down-regulation in AIDS patients.

The in vitro suppressive effect of gp120 and gp120/anti-gp120 antibody is well known but not yet proven to operate in vivo. We report findings consistent with the presence of gp120/anti-gp120 antibody complexes on CD4+ lymphocytes from HIV-infected patients with advanced disease. PBMC from most AIDS patients showed selective masking of the CD4 epitope associated with the gp120 binding site; immunoprecipitation of PBMC with anti-CD4 mAb disclosed high amounts of IgG bound to CD4 receptors. Antibodies against HIV env proteins, but not other HIV products or CD4 Ag, were detected in purified CD4+ cell culture supernatants; in vitro culture was associated with normalization of both CD4 expression in PBMC and the lymphocyte proliferative response to anti-CD3. gp120 presence could not be directly demonstrated, but findings strongly suggested that CD4+ lymphocytes from most HIV-infected patients with advanced disease were covered with gp120/anti-gp120 antibody complexes, which are responsible for down-regulation of surface CD4 expression as well as functional lymphocyte impairment; this event may represent an important mechanism in the pathogenesis of HIV-associated immunodeficiency.     

Cytokines as Adjuvants for the Induction of Anti-Human Immunodeficiency Virus Peptide Immunoglobulin G (IgG) and IgA Antibodies in Serum and Mucosal Secretions after Nasal Immunization

Safe and potent new adjuvants are needed for vaccines that are administered to mucosal surfaces. This study was performed to determine if interleukin-1alpha (IL-1alpha) combined with other proinflammatory cytokines provided mucosal adjuvant activity for induction of systemic and mucosal anti-human immunodeficiency virus (HIV) peptide antibody when intranasally administered with an HIV peptide immunogen. Nasal immunization of BALB/c mice with 10 microg of an HIV env peptide immunogen with IL-1alpha, IL-12, and IL-18 on days 0, 7, 14, and 28 induced peak serum anti-HIV peptide immunoglobulin G1 (IgG1) and IgA titers of 1:131,072 and 1:7,131, respectively (P = 0.05 versus no adjuvant). The use of cholera toxin (CT) as a mucosal adjuvant induced serum IgG1 and IgA titers of 1:32,768 and 1:776, respectively. The adjuvant combination of IL-1alpha, IL-12, and IL-18 induced anti-HIV peptide IgA titers of 1:1,176, 1:7,131, and 1:4,705 in saliva, fecal extracts and vaginal lavage, respectively. Titers induced by the use of CT as an adjuvant were 1:223, 1:1,176, and 1:675, respectively. These results indicate that the proinflammatory cytokines IL-1alpha, IL-12, and IL-18 can replace CT as a mucosal adjuvant for antibody induction and are important candidates for use as mucosal adjuvants with HIV and other vaccines.     

Assembly, secretion, and vacuolar delivery of a hybrid immunoglobulin in plants

Secretory immunoglobulin (Ig) A is a decameric Ig composed of four alpha-heavy chains, four light chains, a joining (J) chain, and a secretory component (SC). The heavy and light chains form two tetrameric Ig molecules that are joined by the J chain and associate with the SC. Expression of a secretory monoclonal antibody in tobacco (Nicotiana tabacum) has been described: this molecule (secretory IgA/G [SIgA/G]) was modified by having a hybrid heavy chain sequence consisting of IgG gamma-chain domains linked to constant region domains of an IgA alpha-chain. In tobacco, about 70% of the protein assembles to its final, decameric structure. We show here that SIgA/G assembly and secretion are slow, with only approximately 10% of the newly synthesized molecules being secreted after 24 h and the bulk probably remaining in the endoplasmic reticulum. In addition, a proportion of SIgA/G is delivered to the vacuole as at least partially assembled molecules by a process that is blocked by the membrane traffic inhibitor brefeldin A. Neither the SC nor the J chain are responsible for vacuolar delivery, because IgA/G tetramers have the same fate. The parent IgG tetrameric molecule, containing wild-type gamma-heavy chains, is instead secreted rapidly and efficiently. This strongly suggests that intracellular retention and vacuolar delivery of IgA/G is due to the alpha-domains present in the hybrid alpha/gamma-heavy chains and indicates that the plant secretory system may partially deliver to the vacuole recombinant proteins expected to be secreted.     

抗禽流感病毒H5N1 分泌型IgA 在中国仓鼠卵巢细胞中的表达

分泌型IgA (SIgA) 在机体的粘膜免疫中具有重要作用,在外分泌道中比单体IgA和IgG抗体具有更好的抗感染活性。为了表达抗禽流感病毒H5N1人-鼠嵌合分泌型IgA抗体,首先以本室先前构建的稳定表达IgA的中国仓鼠卵巢细胞 (CHO) 细胞系为基础,共转染分泌片和J链表达质粒,然后用抗生素Zeocin选择阳性克隆细胞,利用倍比稀释的方法筛选分泌SIgA的单克隆细胞,通过Western blotting分析培养上清中SIgA的表达情况。结果表明,在CHO细胞中成功表达了SIgA抗体,上述研究为研制分泌型     

Plant-derived mouse IgG monoclonal antibody fused to KDEL endoplasmic reticulum-retention signal is N-glycosylated homogeneously throughout the plant with mostly high-mannose-type N-glycans

Plants are potential hosts for the expression of recombinant glycoproteins intended for therapeutic purposes. However, N-glycans of mammalian glycoproteins produced in transgenic plants differ from their natural counterparts. The use of the endoplasmic reticulum (ER)-retention signal has been proposed to restrict glycosylation of plantibodies to only high-mannose-type N-glycans. Furthermore, little is known about the influence of plant development and growth conditions on N-linked glycosylation. Here, we report a detailed N-glycosylation profiling study of CB.Hep1, a mouse IgG2b monoclonal antibody (mAb) against hepatitis B surface antigen (HBsAg) currently expressed in tobacco plants (Nicotiana tabacum L.). The KDEL ER-retention signal was fused to the C-terminal of both light and heavy chains. The structures of the N-linked glycans of this mAb produced in transgenic tobacco plants at various growth stages were analysed by high-performance liquid chromatography (HPLC) profiling techniques and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and compared with those of murine origin. The high-mannose-type oligosaccharides accounted for more than 80% of the total N-glycans, with Man7GlcNAc2 being the most abundant species. Some complex N-glycans bearing xylose and small amounts of oligosaccharides with both xylose and fucose were identified. No appreciable differences were detected when comparing glycosylation at different leaf ages, e.g. from seedling leaves up to 8 weeks old and top or basal leaves of mature plants, or between leaves, stems and whole plants. A strict retention of glycoproteins to ER by the use of the tetrapeptide KDEL was not sufficient, even though the majority of the resulting N-glycosylation was of the high-mannose type. It is highly likely to be dependent on other factors, which are most probably protein specific.     

Intranasal vaccination using interleukin-12 and cholera toxin subunit B as adjuvants to enhance mucosal and systemic immunity to human immunodeficiency virus type 1 glycoproteins

We have investigated the induction of protective mucosal immunity to human immunodeficiency virus type 1 (HIV-1) isolate 89.6 by intranasal (i.n.) immunization of mice with gp120 and gp140 together with interleukin-12 (IL-12) and cholera toxin subunit B (CTB) as adjuvants. It was found that both IL-12 and CTB were required to elicit mucosal antibody responses and that i.n. immunization resulted in increased total, immunoglobulin G1 (IgG1), and IgG2a anti-HIV-1 antibody levels in serum; increased total, IgG1, IgG2a, and IgA antibody expression in bronchoalveolar lavage fluids; and increased IgA antibody levels in vaginal washes. Levels of anti-HIV-1 antibodies in both sera and secretions were higher in groups immunized with gp140 than in those immunized with gp120. However, only gp120-specific mucosal antibodies demonstrated neutralizing activity against HIV-1 89.6. Taken together, the results show that IL-12 and CTB act synergistically to enhance both systemic and local mucosal antibody responses to HIV-1 glycoproteins and that even though gp140 induces higher antibody titers than gp120, only gp120-specific mucosal antibodies interfere with virus infectivity.     

Generation of transgenic plants expressing plasma membrane-bound antibodies to the environmental pollutant microcystin-LR

In this paper we describe the engineering and regeneration of transgenic tobacco plants expressing a recombinant plasma membrane-retained antibody specific to microcystin-LR (MC-LR), the environmental toxin pollutant produced by cyanobacteria. The antibody was created by a genetic fusion of the antigen binding regions of the microcystin-specific single chain antibody, 3A8, with the constant regions from the murine IgG1κ, Guy’s 13, including a membrane retention sequence at the C-terminal end of the antibody heavy chain. The antibody produced in the leaves was shown to be functional by binding to MC-LR in an ELISA with antibody yields in transgenic plant leaves reaching a maximum of 1.2 μg g⁻¹ leaf f.wt (0.005% total soluble protein). Antibody-MC-LR complexes formed in leaves after addition of MC-LR to hydroponic medium around the roots of transgenic plant cultures.     

Site‐specific proteolytic degradation of IgG monoclonal antibodies expressed in tobacco plants

Plants are promising hosts for the production of monoclonal antibodies (mAbs). However, proteolytic degradation of antibodies produced both in stable transgenic plants and using transient expression systems is still a major issue for efficient high‐yield recombinant protein accumulation. In this work, we have performed a detailed study of the degradation profiles of two human IgG1 mAbs produced in plants: an anti‐HIV mAb 2G12 and a tumour‐targeting mAb H10. Even though they use different light chains (κ and λ, respectively), the fragmentation pattern of both antibodies was similar. The majority of Ig fragments result from proteolytic degradation, but there are only a limited number of plant proteolytic cleavage events in the immunoglobulin light and heavy chains. All of the cleavage sites identified were in the proximity of interdomain regions and occurred at each interdomain site, with the exception of the V_L/C_L interface in mAb H10 λ light chain. Cleavage site sequences were analysed, and residue patterns characteristic of proteolytic enzymes substrates were identified. The results of this work help to define common degradation events in plant‐produced mAbs and raise the possibility of predicting antibody degradation patterns ‘a priori’ and designing novel stabilization strategies by site‐specific mutagenesis.     

Reduced virus infectivity inN. tabacum secreting a TMV-specific full-size antibody

We developed a new concept of controlling plant virus infection based on the expression and secretion of full-size antibodies in plants. The neotope-specific anti-Tobacco Mosaic Virus (anti-TMV) antibody mAb24 has a high affinity towards epitopes present only on the surface of intact tobacco mosaic virions. The infectivity of the virus is inhibited almost completely if TMV is adsorbedin vitro at ratios as low as 300 antibody molecules per virion prior to inoculation. Cloned full-size cDNAs of mAb24 heavy and light chains were integrated into the plant expression vector pSS in tandem array and used for transformation ofNicotiana tabacum. The resulting transgenic tobacco plants expressed heavy- and light-chains of mAb24 which were assembled into functional antibodies and exported to the intercellular space. TMV specificity and affinity of the plant-produced antibody (mAb24-P) was not altered when compared to the original murine mAb24. F₁ progenies segregated 3:1 with respect to antibody secretion and showed up to two-fold higher expression levels compared to the F₀ plants. Upon infection with TMV F₁ plants producing mAb24-P showed a reduction of necrotic lesion numbers which is correlated with the amount of antibody produced in transgenic plants.The nucleotide sequence data reported appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers X67210 (heavy-chain cDNA) and X67211 (light-chain cDNA).     

Considerations for extraction of monoclonal antibodies targeted to different subcellular compartments in transgenic tobacco plants

Monoclonal antibody production from transgenic tobacco plants offers many advantages over other heterologous production systems, creating the prospect of production at a scale that will allow new prophylactic and therapeutic applications in global human and animal health. However, information on the major processing factors to consider for large-scale purification of antibodies from transgenic plants is currently limited, and is in urgent need of attention. The purpose of this project was to investigate methods for the initial extraction of recombinant immunoglobulin G (IgG) antibodies from transgenic tobacco leaf tissue. Three different transgenic plant lines were studied in order to establish the parameters for optimal extraction of monoclonal antibodies that accumulate in the apoplasm, at the plasma membrane or within the endoplasmic reticulum. For each transgenic line, seven techniques for physical extraction were compared. The factors that determine the optimal extraction of antibodies from plants have a direct influence on the initial choice of expression strategy, and so must be considered at an early stage. The use of small-scale techniques that are applicable to large-scale purification was a particularly important consideration. The optimal extraction technique varied with the target location of IgG in the plant cell, and the dependence of antibody yield on the physical extraction methodology employed, the pH of the extraction buffer and the extraction temperature was demonstrated in each case. The addition of detergent to the extraction buffer may improve the yield, but this was found to be dependent on the site of accumulation of IgG within the plant cell.     

A fluorescence polarization assay to quantify biotin and biotin-binding proteins in whole plant extracts using Alexa-Fluor 594 biocytin

A high-throughput fluorescence polarization assay has been developed for the detection of biotin and biotin-binding proteins in whole leaf extracts. Various groups are investigating the insecticidal properties of avidin and other biotin-binding proteins expressed in leaves of transgenic plants. The methods commonly used to quantify biotin and avidin in leaf extracts are enzyme-linked immunosorbent assay (ELISA) and Western blotting. Here we describe a homogeneous fluorescence polarization (FP) method that quantifies transgenic avidin in whole leaf extract by the simple addition of the fluorescent avidin ligand Alexa-Fluor 594 biocytin (AFB). The FP assay exploits the fact that AFB excites and emits in regions of the spectrum that are relatively free of background fluorescence in leaf extract. Transgenic leaf avidin can be quantified within 1-2 h by the FP method, in comparison with 1-2 days for ELISA and Western blotting. The FP method can also measure the amount of biotin in control leaves, not expressing avidin. Functional avidin levels of 1.54 μM (26.1 μg/g leaf tissue) were detected in tobacco leaves expressing vacuole-targeted avidin. Control leaves had biotin levels of around 0.74 μM (∼0.18 μg/g leaf tissue). Reagent costs are minimal: typically AFB is used at concentrations of 1-10 nM, avidin is used at 1-100 nM, and sample volumes are 20 μL in 384-well microplates.     

Simultaneous blockade of Fc gamma receptors and indirect labeling of mouse lymphocytes by the selective detection of allotype-restricted epitopes on the kappa chain of rat monoclonal antibodies

BACKGROUND: Incubation of mouse hemopoietic cells with rat monoclonal antibodies (mAbs) of the IgG class sometimes results in Fc-region mediated binding of immunoglobulins by Fc-receptors. This unwanted binding can be prevented by preincubation of target cells with the rat anti-mouse anti-CD16/32 (2.4G2) mAb. METHODS: To avoid the cross-reactivity of fluorochrome-conjugated secondary anti-rat antibodies with the Fc-receptor blocking 2.4G2, direct fluorochrome-conjugated immunoglobulins need to be used. However, we report that a mouse mAb (MRC OX12) with a strong rat Igk(a) allotype preference can be used for flow cytometric measurements in conjunction with unlabeled rat mAbs with the simultaneous blockade of Fc gamma receptors by the 2.4G2 mAb. Results and Discussion This lack of reactivity of OX12 against the 2.4G2 mAb is remarkable, as it could efficiently detect another Sprague-Dawley-derived rat mAb. This staining procedure (unlabeled rat mAb of the appropriate strain detected by OX12 mAb in the presence of 2.4G2 IgG) is an attractive alternative to using direct antibody conjugates, while satisfying the need for an effective Fc gamma-receptor blockade.     

Expression of cholera toxin B-proinsulin fusion protein in lettuce and tobacco chloroplasts--oral administration protects against development of insulitis in non-obese diabetic mice

Lettuce and tobacco chloroplast transgenic lines expressing the cholera toxin B subunit–human proinsulin (CTB-Pins) fusion protein were generated. CTB-Pins accumulated up to ~16% of total soluble protein (TSP) in tobacco and up to ~2.5% of TSP in lettuce. Eight milligrams of powdered tobacco leaf material expressing CTB-Pins or, as negative controls, CTB–green fluorescent protein (CTB-GFP) or interferon–GFP (IFN-GFP), or untransformed leaf, were administered orally, each week for 7 weeks, to 5-week-old female non-obese diabetic (NOD) mice. The pancreas of CTB-Pins-treated mice showed decreased infiltration of cells characteristic of lymphocytes (insulitis); insulin-producing β-cells in the pancreatic islets of CTB-Pins-treated mice were significantly preserved, with lower blood or urine glucose levels, by contrast with the few β-cells remaining in the pancreatic islets of the negative controls. Increased expression of immunosuppressive cytokines, such as interleukin-4 and interleukin-10 (IL-4 and IL-10), was observed in the pancreas of CTB-Pins-treated NOD mice. Serum levels of immunoglobulin G1 (IgG1), but not IgG2a, were elevated in CTB-Pins-treated mice. Taken together, T-helper 2 (Th2) lymphocyte-mediated oral tolerance is a likely mechanism for the prevention of pancreatic insulitis and the preservation of insulin-producing β-cells. This is the first report of expression of a therapeutic protein in transgenic chloroplasts of an edible crop. Transplastomic lettuce plants expressing CTB-Pins grew normally and transgenes were maternally inherited in T₁ progeny. This opens up the possibility for the low-cost production and delivery of human therapeutic proteins, and a strategy for the treatment of various other autoimmune diseases.     

Modulation of mouse complement receptors 1 and 2 suppresses antibody responses in vivo

A mAb, 7G6, that binds to mouse CR1 and CR2 and down-modulates their expression on splenic B cells in vivo, was used to determine whether a decrease in CR1 and CR2 expression affects antibody responses to different T-dependent and T-independent Ag. Injection of mice with the mAb 7G6 prior to immunization with FITC haptenated Salmonella typhimurium (SH5771), Salmonella montevideo (SH5770), SRBC, or Ficoll dramatically decreased subsequent antibody responses to FITC. Although both IgM and IgG primary antibody responses were affected similarly, the antibody levels were most inhibited during early phases of the response. In contrast, down-modulation of the CR did not affect memory antibody responses, because injection of mice with 7G6 before a second immunization with FITC-SH5771 had no effect on subsequent anti-FITC antibody production. Moreover, polyclonal in vivo activation of the mouse immune system by anti-mouse IgD antibodies was not affected by previous administration of 7G6, because anti-IgD-induced increases in Ia expression and serum IgG1 levels were not affected. Taken together, these observations suggest that CR1 and CR2 may play an important role in enhancing primary antibody responses to many T-dependent and T-independent Ag and may contribute to a host's response to naturally occurring antigens such as bacteria.     

Mac-1 (CD11b/CD18) as accessory molecule for Fc alpha R (CD89) binding of IgA

IgA, the principal ligand for FcalphaRI, exists in serum as monomeric IgA and at mucosal sites as secretory IgA (SIgA). SIgA consists of dimeric IgA linked by joining chain and secretory components. Human polymorphonuclear leukocytes (PMN) and mouse PMN transgenic for human FcalphaRI exhibited spreading and elicited respiratory burst activity upon interaction with either serum or SIgA. However, PMN devoid of the beta(2) integrin Mac-1 (Mac-1(-/-)) were unable to bind SIgA, despite expression of FcalphaRI. Consistent with this, serum IgA stimulated Mac-1(-/-) PMN oxygen radical production, in contrast to SIgA. Binding studies showed the secretory component, by itself, to interact with Mac-1-expressing PMN, but not with Mac-1(-/-) PMN. These data demonstrate an essential role for Mac-1 in establishing SIgA-FcalphaRI interactions.