FcγR Binding and ADCC Activity of Human IgG Allotypes.

Antibody dependent cellular cytotoxicity (ADCC) is an Fc-dependent effector function of IgG important for anti-viral immunity and anti-tumor therapies. NK-cell mediated ADCC is mainly triggered by IgG-subclasses IgG1 and IgG3 through the IgG-Fc-receptor (FcγR) IIIa. Polymorphisms in the immunoglobulin gamma heavy chain gene likely form a layer of variation in the strength of the ADCC-response, but this has never been studied in detail. We produced all 27 known IgG allotypes and assessed FcγRIIIa binding and ADCC activity. While all IgG1, IgG2, and IgG4 allotypes behaved similarly within subclass, large allotype-specific variation was found for IgG3. ADCC capacity was affected by residues 291, 292, and 296 in the CH2 domain through altered affinity or avidity for FcγRIIIa. Furthermore, allotypic variation in hinge length affected ADCC, likely through altered proximity at the immunological synapse. Thus, these functional differences between IgG allotypes have important implications for therapeutic applications and susceptibility to infectious-, allo- or auto-immune diseases.

Novel Approach to Cell Surface Discrimination Between KIR2DL1 Subtypes and KIR2DS1 Identifies Hierarchies in NK Repertoire, Education, and Tolerance.

Cumulative activating and inhibitory receptor signaling controls the functional output of individual natural killer (NK) cells. Investigation of how competing signals impact response, however, has been hampered by the lack of available antibodies capable of distinguishing inhibitory and activating receptors with highly homologous ectodomains. Utilizing a novel combination of monoclonal antibodies with specificity for discrete inhibitory KIR2DL1 and activating KIR2DS1 allotypes found among 230 healthy donors, we investigated allele-specific receptor expression and function driven by KIR2DL1 and KIR2DS1 alleles. We found that co-expression of the HLA-C2 ligand diminishes KIR2DL1, but not KIR2DS1, cell surface staining, but does not impact the respective frequencies of KIR2DL1- and KIR2DS1-expressing cells within the NK repertoire. We can distinguish by flow cytometry NK cell populations expressing the most common KIR2DL1-C245 allotypes from those expressing the most common KIR2DL1-R245 allotypes, and we show that the informative differential binding anti-KIR2DL1/S1 clone 1127B is determined by amino acid residue T154. Although both KIR2DL1-C245 and KIR2DL1-R245 subtypes can be co-expressed in the same cell, NK cells preferentially express one or the other. Cells expressing KIR2DL1-C245 exhibited a lower KIR2DL1 cell surface density and lower missing-self reactivity in comparison to cells expressing KIR2DL1-R245. We found no difference, however, in sensitivity to inhibition or cell surface stability between the two KIR2DL1 isoforms, and both demonstrated similar expansion among NKG2C+ KIR2DL1+ NK cells in HCMV-seropositive healthy individuals. In addition to cell surface density of KIR2DL1, copy number of cognate HLA-C2 hierarchically impacted the effector capacity of both KIR2DL1+ cells and the tolerization of KIR2DS1+ NK cells. HLA-C2 tolerization of KIR2DS1+ NK cells could be overridden, however, by education via co-expressed self-specific inhibitory receptors, such as the heterodimer CD94/NKG2A. Our results demonstrate that effector function of NK cells expressing KIR2DL1 or KIR2DS1 is highly influenced by genetic variability and is calibrated by co-expression of additional NK receptors and cognate HLA-C2 ligands. These findings define the molecular conditions under which NK cells are activated or inhibited, potentially informing selection of donors for adoptive NK therapies.

O-glycosylated IgA rheumatoid factor induces IgA deposits and glomerulonephritis.

Structural aberrations of O-linked glycans present in the IgA1 hinge region are associated with IgA nephropathy, but their contribution to its pathogenesis remains incompletely understood. In this study, mice implanted with hybridoma secreting 6-19 IgA anti-IgG2a rheumatoid factor, but not 46-42 IgA rheumatoid factor bearing the same IgA allotype, developed mesangial deposits consisting of IgA, IgG2a, and C3. Studies in immunoglobulin- and C3-deficient mice revealed that the development of these glomerular lesions required the formation of IgA-IgG2a immune complexes and subsequent activation of complement. The proportion of polymeric and monomeric forms, the IgG2a-binding affinity, and the serum levels of IgA-IgG2a immune complexes were similar between 6-19 IgA- and 46-42 IgA-injected mice. In contrast, the analysis of oligosaccharide structures revealed highly galactosylated O-linked glycans in the hinge region of 6-19 IgA and poorly O-glycosylated in the hinge region of 46-42 IgA. Furthermore, the structure of N-linked glycans in the CH1 domain was the complex type in 6-19 IgA and the hybrid type in 46-42 IgA. In summary, this study demonstrates the presence of O-linked glycans in the hinge region of mouse IgA and suggests that 6-19 IgA rheumatoid factor-induced GN could serve as an experimental model for IgA nephropathy.

Interaction between immunoglobulin allotypes and NK receptor genes in diabetes post-hepatitis C virus infection.

Genetic interactions between natural killer (NK) cells immunoglobulin-like receptor (KIR) genes and immunoglobulin allotypes have been previously reported in type 2 diabetes mellitus (DM) patients. Puerto Rican Americans with a history of intravenous drug use who developed DM following HCV infection (n=32) were compared to individuals infected with HCV without diabetes (n=121) and to DM non-infected individuals (n=95). Subjects were genotyped for KIRs and immunoglobulin allotypes. We found interactions of immunoglobulin allotypes KM3/KM3 with NK inhibitory receptors 2DL3/2DL3, 2DL1 in the absence of 2DS4 associated with susceptibility to DM in HCV infected individuals. These data suggest the possibility that a subset of patients with HCV could have an immune-mediated component contributing to the development of DM.

Incomplete assembly of IgA2m(2) in Chinese hamster ovary cells.

Myeloma and Chinese hamster ovary (CHO) cells are frequently used for the production of recombinant antibodies. With increasing interest in producing recombinant IgA for protection against infectious agents, it is essential to characterize the IgA produced in these cells. Here we show that while myeloma cells secrete IgA2m(2) predominantly as H(2)L(2), CHO cells secrete H(2)L and H(2) in addition to fully assembled H(2)L(2). When the CHO cells also synthesize J chain and secretory component (SC), polymeric IgA and secretory IgA in which SC is disulfide bonded to the polymeric IgA are produced. Blocking cysteines on purified IgA2m(2) protein by alkylating with iodoacetamide stabilizes the disulfide bonds between the H and L chains suggesting that the disulfide bonds between H and L chains are unstable. Taken together our results suggest that the covalent assembly of IgA2m(2) is different in myeloma and CHO cells.

Interaction of KIR genes and G1M immunoglobulin allotypes confer susceptibility to type 2 diabetes in Puerto Rican Americans.

The susceptibility to type 2 diabetes (T2D) involves genetic factors. We studied the distribution of KIR and MHC class I ligands phenotype and genotype frequencies, as well as immunoglobulin KM and GM allotype frequencies in a group of patients (N = 95) with T2D and ethnically matched healthy controls (N = 74) with Puerto Rican ethnic background. We found a slight increase of the 2DL3/2DL3 homozygous genotype in T2D. Moreover, the association between 2DL3/2DL3 genotype was significant in the presence of 2DS4 (pC = 0.01). Also, we observed an epistatic effect of the interaction of 2DL3/2DL3, 2DS4 with allele z of G1M in T2D (pC = 0.004, OR = 3.60, 95% CI, 1.62-8.10). This genetic interaction between KIR and G1M allotypes, associated with T2D, was also significant by multiple logistic regression analysis (p < 0.0001, OR = 4.90, 95% CI, 2.12-11.3). We did not detect population stratification using unlinked short tandem repeat (STR) markers, demonstrating that the patients and controls were ethnically matched. Hence, we have demonstrated in this study an epistatic interaction between KIR genes and the G1M allotype that influences the susceptibility to T2D in Puerto Rican Americans. Our findings are important for understanding the autoimmune or innate immune inflammatory-mediated mechanisms involved in the pathogenesis of T2D.

Fc receptor homolog 3 is a novel immunoregulatory marker of marginal zone and B1 B cells.

Two members of the recently identified FcR homolog (FcRH) family in mice demonstrate preferential B cell expression. One of these, FcRH3, encodes a type I transmembrane protein with five extracellular Ig domains and a cytoplasmic tail with a consensus ITIM and a noncanonical ITAM. Analysis of full-length cDNAs from five different mouse strains defines two FcRH3 alleles. A panel of FcRH3-specific mAbs was generated to define its expression pattern and functional potential on B lineage cells. Although poorly detected on the majority of bone marrow or peripheral blood cells, FcRH3 was readily identified on splenic marginal zone (MZ) and MZ precursor B cells, but not on the bulk of newly formed B cells, follicular B cells, germinal center B cells, and plasma cells. In the peritoneal cavity, FcRH3 was found on B1 cells, and not on the majority of B2 cells. Consistent with its possession of an ITIM and ITAM-like sequence, FcRH3 was tyrosine phosphorylated following pervanadate treatment, and its coligation with the BCR inhibited calcium mobilization. These results suggest FcRH3 is a novel immunoregulatory marker of MZ and B1 B lineage cells.

Positive selection of the peripheral B cell repertoire in gut-associated lymphoid tissues.

Gut-associated lymphoid tissues (GALTs) interact with intestinal microflora to drive GALT development and diversify the primary antibody repertoire; however, the molecular mechanisms that link these events remain elusive. Alicia rabbits provide an excellent model to investigate the relationship between GALT, intestinal microflora, and modulation of the antibody repertoire. Most B cells in neonatal Alicia rabbits express V(H)n allotype immunoglobulin (Ig)M. Within weeks, the number of V(H)n B cells decreases, whereas V(H)a allotype B cells increase in number and become predominant. We hypothesized that the repertoire shift from V(H)n to V(H)a B cells results from interactions between GALT and intestinal microflora. To test this hypothesis, we surgically removed organized GALT from newborn Alicia pups and ligated the appendix to sequester it from intestinal microflora. Flow cytometry and nucleotide sequence analyses revealed that the V(H)n to V(H)a repertoire shift did not occur, demonstrating the requirement for interactions between GALT and intestinal microflora in the selective expansion of V(H)a B cells. By comparing amino acid sequences of V(H)n and V(H)a Ig, we identified a putative V(H) ligand binding site for a bacterial or endogenous B cell superantigen. We propose that interaction of such a superantigen with V(H)a B cells results in their selective expansion.

Cysteine residues required for the attachment of the light chain in human IgA2.

In humans, there are two subclasses of IgA, IgA1 and IgA2, with IgA2 existing as three allotypes, IgA2m(1), IgA2m(2) and IgA2(n). In IgA1, Cys(133) in C(H)1 forms the disulfide bond to the L chain. Our previous studies indicated that in IgA2 lacking Cys(133), a disulfide bond forms between the alpha-chain and the L chain when Cys(220) is followed by Arg(221), but not when Cys(220) is followed by Pro(221), suggesting that the Cys in C(H)1 might be involved in disulfide bonding to the L chain. However, here we show that covalent assembly of the H and L chains in IgA2(n) requires hinge-proximal Cys(241) and Cys(242) in C(H)2 and not Cys(196) or Cys(220) in C(H)1. Using pulse-chase experiments, we have demonstrated that wild-type IgA2(n) with Arg(221) and Cys(241) and Cys(242) assembles through a disulfide-bonded HL intermediate. In contrast, the major intermediate for IgA2 m(1) with Pro(221) assembly was H(2) even though both Cys(241) and Cys(242) were present. Only a small fraction of IgA2 m(1) assembles through disulfide-bonded HL. Overall, our studies indicate that for IgA2 covalent assembly of the H and L chains requires the hinge-proximal cysteines in C(H)2 and that the structure of C(H)1 influences the efficiency with which this covalent bond forms.

The herpes simplex virus type 1 Fc receptor discriminates between IgG1 allotypes.

Herpes simplex virus type 1 (HSV-1) expresses a complex of two virally encoded glycoproteins, gE and gl, which is capable of binding nonimmune human IgG. The gE-gl complex has thus become known as an Fc receptor (FcR), which reportedly binds human IgG subclasses in the order IgG4 > IgG1 > or = IgG2 and does not bind IgG3 from many individuals. There is, however, allelic variation in the genes encoding the human IgG1 heavy chain constant region and this gives rise to allotypes of IgG1. Using recombinant monoclonal IgG molecules of known isotype and mutants thereof we have unexpectedly discovered that the HSV-1 FcR discriminates between IgG1 allotypes. This is evidence of functional differences between IgG1 allotypes that may account for their distribution in populations. Furthermore, these findings suggest HSV-1 FcR binding sites on the IgG molecule some distance from the proposed binding site in the CH2-CH3 domain interface.

The N-glycans determine the differential blood clearance and hepatic uptake of human immunoglobulin (Ig)A1 and IgA2 isotypes.

Human immunoglobulin (Ig)A exists in blood as two isotypes, IgA1 and IgA2, with IgA2 present as three allotypes: IgA2m(1), IgA2m(2), and IgA2m(n). We now demonstrate that recombinant, chimeric IgA1 and IgA2 differ in their pharmacokinetic properties. The major pathway for the clearance of all IgA2 allotypes is the liver. Liver-mediated uptake is through the asialoglycoprotein receptor (ASGR), since clearance can be blocked by injection of excess galactose-Ficoll ligand and suppressed in ASGR-deficient mice. In contrast, only a small percentage of IgA1 is cleared through this pathway. The clearance of IgA1 lacking the hinge region with its associated O-linked carbohydrate was more rapid than that of wild-type IgA1. IgA1 and IgA2 that are not rapidly eliminated by the ASGR are both removed through an undefined ASGR-independent pathway with half-lives of 14 and 10 h, respectively. The rapid clearance of IgA2 but not IgA1 through the liver may in part explain why the serum levels of IgA1 are greater than those of IgA2. In addition, dysfunction of the ASGR or altered N-linked glycosylation, but not O-glycans, that affects recognition by this receptor may account for the elevated serum IgA seen in liver disease and IgA nephropathy.

Tritrichomonas foetus extracellular cysteine proteinase cleavage of bovine IgG2 allotypes.

Bovine trichomoniasis is a sexually transmitted disease associated with reproductive failure. Systemic immunization results in protective IgG antibodies in uterine and vaginal secretions. Because bovine IgG2 is a better opsonin than IgG1, it is potentially important in defense. Yet, Tritrichomonas foetus extracellular cysteine proteinase (TFECP) cleaves bovine IgG2, evading protective IgG2 responses. Variations in resistance of the 2 IgG2 allotypes to digestion may explain inherited differences in protection. To address this hypothesis, TFECP was incubated with both IgG2 allotypes at different concentrations and times. The digestion products were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, stained, and quantitated by image analysis. IgG2a was digested faster by TFECP than IgG2b. Differences in the sizes and numbers of digestion products were observed, but the presence of bands the size of Fc and Fd fragments indicated that both allotypes were cleaved at the hinge. Cysteine in the digestion mixture reduced the antibody molecules and increased the rate of digestion, but IgG2a was still more susceptible to cleavage than IgG2b in the absence of cysteine. Thus, not only reduced H chains can be cleaved by cysteine proteinase secreted by T. foetus but also intact functional antibody molecules. Because parasites may evade protective antibody responses by cleaving IgG2, animals with the more resistant IgG2b allotype may be better protected by immunization than animals with the more readily digested IgG2a allotype.

The B cell receptor, but not the pre-B cell receptor, mediates arrest of B cell differentiation.

B cell development in organ cultures of fetal liver from mice at day 14 of gestation resembles in kinetics and cell numbers generated the one observed in vivo. This development in vitro can be blocked by an IL-7 receptor-specific monoclonal antibody. Monoclonal antibodies specific for the pre-B cell receptor, i. e. for VpreB, lambda5, or muH chains, do not perturb B cell development in these organ cultures up to and including the CD25+ small pre-BII cell stage. However, muH chain-specific antibodies inhibit the appearance of the subsequent surface IgM+ immature B cells. In organ cultures of muH chain allotype heterozygous (muHa x muHb)F1 fetal livers a dose-dependent inhibition by allotype-specific monoclonal antibodies of sIgM+ immature B cells expressing the corresponding, but not the other, allotype was observed. By combining cell sorting with limiting dilution analysis of lipopolysaccharide-reactive cells, the probable target cell of this muH chain-specific inhibition was identified as an IgM+, CD23-immature B cell. Hence, engagement of the pre-B cell receptor by specific antibodies does not influence B cell development, while engagement of the B cell receptor on immature B cells does.

In vivo T helper cell response to retro-inverso peptidomimetics.

Peptide analogues containing reversed peptide bonds between each residue along the peptide sequence (retro-inverso modification) have been analyzed for their antigenic and in vivo immunogenic properties in the MHC II and Th cell response context. Two antigenic peptides were selected for this study, namely peptide 103-115 of poliovirus VP1, which is involved in the production of Abs that neutralize the infectivity of the virus, and peptide 435-446 from the third constant region of mouse heavy chain IgG2a allopeptide gamma 2ab, which mimics a corneal Ag implicated in autoimmune keratitis. In a competition assay performed in vitro using reference hybridomas of known MHC class II restriction, both retro-inverso analogues bound (although more weakly in our test) to I-Ad and/or I-Ed class II molecules. However, in both cases, this lower affinity was apparently largely compensated in vivo, as a T cell response (with IL-2 secretion), equivalent to that obtained with the wild-type peptides, was observed following immunization of BALB/c mice with the retro-inverso analogues. Moreover, these T cells proliferated and produced IL-2 in response to the cognate peptides. It is concluded that the T cell receptors of T cells primed in vivo with the retro-inverso analogues readily cross-react with parent and retro-inverso analogue-MHC complexes. The approach of using pseudopeptides containing changes involving the backbone, and not the orientation of side chains, may thus be promising to design potent immunogens for class II-restricted T cells.

Requirement for a beta 2-microglobulin-associated Fc receptor for acquisition of maternal IgG by fetal and neonatal mice.

There is considerable evidence to suggest that an FcR similar in structure to class I MHC Ags, neonatal Fc receptor (FcRn), transports IgG across the intestinal epithelium of suckling mice. However, this has not previously been shown definitively, nor has it been shown whether FcRn is the only, or even the major, IgG transporter in the neonatal mouse gut. We report here that neonatal mice homozygous for a targeted disruption of the beta 2microglobulin (beta 2m) gene, which encodes one subunit of FcRn, had reduced FcRn alpha-chain at the lumenal plasma membrane of intestinal cells. These mice had strikingly lower serum IgG levels during the first month after birth than littermates that possessed functional FcRn. Furthermore, we found by fostering mice on mothers with a different IgG allotype that all of the IgG in sera of beta 2m-/- mice was endogenous, and that none was obtained from milk. We conclude that FcRn is the only transporter of IgG from mother to young in the mouse. The onset of IgG synthesis in mice that received no milk IgG lagged behind that in siblings with normal IgG transport, suggesting that maternal IgG stimulates Ab production in the neonate. We noted no difference between the IgG concentrations in the milk of beta 2m-/- and beta 2m+/- mice, indicating that FcRn is not involved in the secretion of IgG into milk.

Immunoglobulin allotypes (GM and KM) and their interactions with HLA antigens in autoimmune diseases: a review.

GM and KM immunoglobulin (Ig) allotypes and their interactions with HLA antigens have been analyzed in various autoimmune diseases: multiple sclerosis, rheumatoid arthritis, insulin-dependent diabetes mellitus (IDDM), systemic lupus erythematosus, coeliac disease, Crohn's disease, Graves' disease, atrophic thyroiditis, Hashimoto's thyroiditis, myasthenia gravis, chronic active hepatitis, alopecia areata, uveitis, vitiligo, Turner's syndrome, glomerular nephritis, Berger's disease and idiopathic dilated cardiomyopathy. This review reports published results about associations or linkages, as well as the origins of the populations, the numbers of patients and controls tested. The possible role of Ig polymorphisms in the physiopathology of autoimmune diseases is discussed. Ig allotypes and statistical methods used to analyse the HLA and Ig data are also described.

Herpesvirus serology, aberrant specific immunoglobulin G2 and G3 subclass patterns and Gm allotypes in individuals with low levels of IgG3.

One objective of this study was to determine whether IgG3-deficient individuals have an increased frequency of reactivated herpesvirus infections. Serum titres to Epstein-Barr virus (EBV) and human herpesvirus-6 were examined in 10 healthy and in 10 symptomatic persons with serum IgG3 < 0.1 g/l. Atypical titres were found in 16% of the IgG3-deficient individuals. Reactivations of these viruses thus do not seem common in IgG3 deficiency. Antigen-specific IgG responses were also determined. A lowered frequency of IgG3 to an EBV-derived peptide was found only in symptomatic, IgG3-deficient individuals. Levels of IgG2 to a bacterial polysaccharide were lowered in the same group, despite normal serum levels of total IgG2. A functional IgG2 deficiency may contribute to symptoms in IgG3 deficiency. The G3(g) allotype, known to be associated with low total IgG3, dominated in IgG3-deficient persons (13/17) independently of presence or absence of symptoms. A linkage of G3(g) to the G2(n) negative allotype, associated with low IgG2, was equally common irrespective of symptoms. G3(g) and absence of G2(n) seem to be one prerequisite for most of IgG3 deficiency combined with low specific IgG2.

Association of immunoglobulin Km and Gm allotypes with specific antinuclear antibodies and disease susceptibility among connective tissue disease patients.

The distribution of the immunoglobulin Km(1) and Gm phenotypes was examined in patients with connective tissue diseases, including systemic lupus erythematosus, mixed connective tissue disease, and scleroderma, whose sera were characterized for antibodies against nuclear antigens and polypeptides of U small nuclear ribonucleoproteins. We found a strong association between Km(1) phenotype and susceptibility to systemic lupus erythematosus (P less than 0.00001, relative risk = 17). We also found a positive association between the Km(1) phenotype and the presence of anti-double-stranded DNA antibodies. The presence of certain immunoglobulin genes or gene families may have a role in susceptibility to the development of autoantibodies and/or of connective tissue disease.

Jacalin, the human IgA1 and IgD precipitating lectin, also binds IgA2 of both allotypes.

The lectin jacalin from jackfruit seeds shows a human IgA-subclass specificity by gel precipitation and Western blotting. However, its reactivity with IgA2 is a matter of controversy. We further studied the immunoglobulin isotype specificity of jacalin by affinity chromatography with myeloma sera and by inhibition of jacalin binding to solid-phase IgA1 by purified monoclonal immunoglobulins. The lectin proved to bind IgA2 of both allotypes with a lower apparent affinity than for IgA1 and IgD.

HLA and immunoglobulin allotypes in mixed connective tissue disease.

A group of patients with mixed connective tissue disease (MCTD) were HLA and immunoglobulin allotyped. We found that the incidence of DR4 in the patient group was increased compared with that in the normal controls, but the increase was restricted to the subgroup of patients with arthritis. The age at onset of MCTD was lower in patients with DR4 and higher in patients with DR2 compared with patients who did not have these antigens. A1, B8, and DR3 were more frequent, but not significantly so, in the MCTD patient group. We also found that there was a significant perturbation of the Gm allotype frequencies in patients with MCTD.