Structure of a human IgA1 Fab fragment at 1.55†Šresolution: potential effect of the constant domains on antigen-affinity modulation.

Despite being the most abundant class of immunoglobulins in humans and playing central roles in the adaptive immune response, high-resolution structural data are still lacking for the antigen-binding region of human isotype A antibodies (IgAs). The crystal structures of a human Fab fragment of IgA1 in three different crystal forms are now reported. The three-dimensional organization is similar to those of other Fab classes, but FabA1 seems to be more rigid, being constrained by a hydrophobic core in the interface between the variable and constant domains of the heavy chain (VH-CH1) as well as by a disulfide bridge that connects the light and heavy chains, influencing the relative heavy/light-chain orientation. The crystal structure of the same antibody but with a G-isotype CH1 which is reported to display different antigen affinity has also been solved. The differential structural features reveal plausible mechanisms for constant/variable-domain long-distance effects whereby antibody class switching could alter antigen affinity.

Impact of IgA constant domain on HIV-1 neutralizing function of monoclonal antibody F425A1g8.

With the majority of HIV infections resulting from mucosal transmission, induction of an effective mucosal immune response is thought to be pivotal in preventing transmission. HIV-specific IgA, but not IgG, has been detected in the genital tract, seminal fluid, urethral swabs, urine, and vaginal wash samples of HIV-negative sex workers and HIV-status discordant couples. Purified mucosal and plasma IgA from some individuals with highly exposed, persistently seronegative status can neutralize infection and present cross-clade neutralization activity, though present at low levels. We generated a CD4-induced human mAb, F425A1g8, and characterized the impact of its isotype variants on HIV neutralizing activity. The result showed that, in contrast to little neutralization by the F425A1g8 IgG1 in the absence of sCD4, the IgA1 variant of the Ab displayed significant independent neutralization activity against a range of HIV clade B isolates in the absence of sCD4. Studies of the neutralizing function of IgA isotypes, and the functional relationship between different antigenic epitopes and IgA Abs, may also suggest strategies for the intervention of virus transmission and spread within the mucosa of the host, as well as serve to inform the design of vaccine strategies that may be more effective at preventing mucosal transmission. This research clearly suggests that IgA isotype, because of its unique molecular structure, may play an important role in HIV neutralization.

Antibody C region influences TGN1412-like functional activity in vitro.

The unexpected outcome of the clinical trial of the superagonistic CD28 mAb TGN1412 (IgG4κ) continues to stimulate interest. We show that TGN1412 binds similarly to human and cynomolgus macaque FcγR, eliminating the possibility that differences in Fc-mediated interactions with FcγR contributed to the failure of preclinical testing in macaques to predict toxicity in humans. The influence of the Fc domain and C region structure on the in vitro functional activity of TGN1412 was investigated using F(ab')(2) and Fab fragments derived from TGN1412 recovered from the trial and recombinant TGN1412 subclass variants and mutants. Superagonistic activity, as measured by cytokine release and proliferation, was assessed by exposing PBMCs to immobilized mAbs/fragments or to aqueous mAbs/fragments in the presence of HUVEC monolayers. Removing the Fc generally curtailed or abolished PBMC activation. However, eliminating detectable FcγR-binding of the IgG4 by mutation (L235E) did not abrogate activity. Stabilizing the "wild-type" IgG4 hinge (S228P) enhanced activity without increasing FcγR binding, which could only partially be explained by inhibition of Fab arm-exchange. Subclass switching the IgG4 mAb to IgG1 decreased activity, whereas switching to IgG2 markedly increased activity. We conclude that the C region strongly influences in vitro CD28-mediated superagonistic signaling. Superagonism requires an intact Fc, as shown by the absence of activity of TGN1412 Fab and F(ab')(2) fragments, but, notably, appears to be relatively independent of FcγR-binding properties. We propose that the Fc, potentially through restricting flexibility, maintains a favorable V region conformation to allow superagonistic activity. These findings have important implications for Ab design strategies.

Molecular and functional characterization of cynomolgus monkey IgG subclasses.

Studies for vaccine and human therapeutic Ab development in cynomolgus monkeys (cynos) are influenced by immune responses, with Ab responses playing a significant role in efficacy and immunogenicity. Understanding the nature of cyno humoral immune responses and characterizing the predominant cyno IgG types produced and the Fc-FcγR interactions could provide insight into the immunomodulatory effects of vaccines. Anti-drug Ab responses against human IgG therapeutic candidates in cynos may affect efficacy and safety assessments because of the formation of immune complexes. There is, however, limited information on the structure and function of cyno IgG subclasses and how they compare with human IgG subclasses in Fc-dependent effector functions. To analyze the functional nature of cyno IgG subclasses, we cloned four cyno IgG C regions by using their sequence similarity to other primate IgGs. The four clones, cyno (cy)IGG1, cyIGG2, cyIGG3, cyIGG4, were then used to construct chimeric Abs. The sequence features of cyno IgG subclasses were compared with those of rhesus monkey and human IgG. Our data show that rhesus monkey and cyno IgG C regions are generally highly conserved, with differences in the hinge and hinge-proximal CH2 regions. Fc-dependent effector functions of cyno IgG subclasses were assessed in vitro with a variety of binding and functional assays. Our findings demonstrate distinctive functional properties of cyno IgG subclasses. It is notable that human IgG1 was less potent than cyno IgG1 in cyno FcγR binding and effector functions, with the differences emphasizing the need to carefully interpret preclinical data obtained with human IgG1 therapeutics.

The constant region of the membrane immunoglobulin mediates B cell-receptor clustering and signaling in response to membrane antigens.

B cells are activated in vivo after the B cell receptors (BCRs) bind to antigens captured on the surfaces of antigen-presenting cells. Antigen binding results in BCR microclustering and signaling; however, the molecular nature of the signaling-active BCR clusters is not well understood. Using single-molecule imaging techniques, we provide evidence that within microclusters, the binding of monovalent membrane antigens results in the assembly of immobile signaling-active BCR oligomers. The oligomerization depends on interactions between the membrane-proximal Cmicro4 domains of the membrane immunoglobulin that are both necessary and sufficient for assembly. Antigen-bound BCRs that lacked the Cmicro4 domain failed to cluster and signal, and conversely, Cmicro4 domains alone clustered spontaneously and activated B cells. These results support a unique mechanism for the initiation of BCR signaling in which antigen binding induces a conformational change in the Fc portion of the BCR, revealing an interface that promotes BCR clustering.

Silencing of the immunoglobulin heavy chain locus by removal of all eight constant-region genes in a 200-kb region.

Silencing or removal of individual C (constant)-region genes and/or adjacent control sequences did not generate fully deficient Ig (immunoglobulin)- mice. A reason is that different C genes share many functional tasks and most importantly are individually capable of ensuring lymphocyte differentiation. Nevertheless, incomplete arrests in B-cell development were found, most pronounced at the onset of H-chain expression. Here we show that removal of 200 kb accommodating all C genes, Cmu-Cdelta-Cgamma3-Cgamma1-Cgamma2b-Cgamma2a-Cepsilon-Calpha, stops antibody production. For this two loxP targeting constructs were introduced into the most 5' C gene and the distal alpha 3' enhancer. Cre-loxP-mediated in vivo deletion was accompanied by extensive germ-line mosaicism, which could be separated by breeding. Homozygous C-gene deletion mice did not express Ig H or L chains and flow cytometry revealed a complete block in B-cell development. However, C-gene removal did not affect DNA rearrangement processes following locus activation, as recombination efficacy appears to be similar to what is found in normal mice.

Costimulatory molecule immune enhancement in a plasmid vaccine model is regulated in part through the Ig constant-like domain of CD80/86.

There is great interest in understanding the role of costimulatory molecules in immune activation. In both the influenza and HIV DNA immunization models, several groups have reported that coimmunization of mice with plasmids encoding immunogen and CD86, but not CD80, effectively boosts Ag-specific T cell activation. This difference in immune priming provided an opportunity to examine the functional importance of different regions of the B.7 molecules in immune activation. To examine this issue, we developed a series of chimeric CD80 and CD86 constructs as well as deletion mutants, and examined their immune activating potential in the DNA vaccine model. We demonstrate that the lack of an Ig constant-like region in the CD80 molecule is critically important to the enhanced immune activation observed. CD80 C-domain deletion mutants induce a highly inflammatory Ag-specific cellular response when administered as part of a plasmid vaccine. The data suggest that the constant-like domains, likely through intermolecular interactions, are critically important for immune regulation during costimulation and that engineered CD80/86 molecules represent more potent costimulatory molecules and may improve vaccine adjuvant efficacy.

Duplication of primate and rodent B7-H3 immunoglobulin V- and C-like domains: divergent history of functional redundancy and exon loss.

B7-H3 is a novel protein structurally related to the B7 family of ligands by the presence of a single set of immunoglobulin-V-like and immunoglobulin-C-like (VC) domains. By multiplex PCR, the dominantly expressed form of human B7-H3 was found to be a splice variant containing tandemly duplicated VC domains (VCVC). In contrast, mouse B7-H3 cDNA contained only one single VC form due to an exon structure corresponding to V-(pseudoexon C)-(pseudoexon V)-C. Comparisons of human, monkey, mouse, and hamster genomic B7-H3 reveal that primates, but not rodents, exhibited a higher degree of intramolecular sequence similarity between VC duplications than between molecules. Both VC and VCVC forms of human B7-H3 inhibited CD4(+) T cell proliferation and downregulated cytokine production upon TCR activation. These results suggest independent, but convergent, paths of B7-H3 active domain duplication followed by divergent histories of exon degeneration in rodents and exon maintenance by humans.

Immunoglobulin KM genes in Guillain-Barré syndrome.

Guillain-Barré syndrome is associated with antecedent Campylobacter jejuni infection. Only a minority of the infected individuals, however, develops the disease, implying a role for genetic factors in conferring susceptibility. To determine the role of immunoglobulin KM genes (genetic markers of the constant region of kappa chains) in the etiology of this syndrome, we genotyped 83 patients and 196 healthy controls from Norway for KM1 and KM3 alleles by polymerase chain reaction-restriction fragment length polymorphism. The frequency of KM3 homozygotes was significantly increased in patients compared with controls (86.7% vs. 74%, P=0.01, odds ratio=2.3). Conversely, the frequency of KM1/KM3 heterozygotes was significantly decreased in patients compared with controls (13.3% vs. 26%, P=0.01, odds ratio=0.4). These results suggest that KM genes may be relevant to the etiology of Guillain-Barré syndrome.

A point mutation in the constant region of Ig lambda1 prevents normal B cell development due to defective BCR signaling.

Surface expression of B cell antigen receptors (BCRs) containing Ig and Igalpha/Igbeta generates signals required to transit discrete developmental checkpoints. The mechanism by which BCR components collaborate to initiate signals is still unclear. The expression of Iglambda1 in SJL mice is 50-fold lower than in other strains. Here, we demonstrate by gene targeting that a point mutation, which changes a glycine to a valine in the lambda1 constant region, is responsible for this defect. In vitro experiments show that Ig receptors bearing this mutation, while expressed normally, are deficient in signaling. These findings reveal a direct involvement of the Ig light chain (IgL) in B cell signaling and development beyond the requirement of light chains for BCR assembly.

Hybrid IgA2/IgG1 antibodies with tailor-made effector functions.

Immunoglobulin (Ig) A and IgG are the principal immune effector molecules at mucosal surfaces and in blood, respectively. Mucosal IgA is polymeric and bound to secretory component, whereas serum IgG is monomeric. We have now produced IgA2/IgG1 hybrid antibodies that combine the properties of IgA and IgG. Antibodies with Calpha3 at the end of the IgG H chain resemble IgA and form polymers with J chain that bind the polymeric Ig receptor. Like IgG, the hybrid proteins activated complement and bound FcgammaRI and protein A. Though the hybrid proteins contained both Cgamma2 and Cgamma3, they have a short in vivo half-life. Surprisingly, this decreased half-life correlated with a higher avidity than that of IgG for murine FcRn. Interestingly, antibodies with Calpha1 replacing Cgamma1 were resistant to extremes of pH, suggesting that Calpha1 increases antibody stability. These results provide insights into engineering antibodies with novel combinations of effector functions.

Conventional and surrogate light chains differentially regulate Ig mu and Dmu heavy chain maturation and surface expression.

Positive selection of precursor (pre-) B cells by Ig membrane mu H chains (mum HC) and counterselection mediated by the truncated HC Dmu depend on the ability of each HC to form a pre-B cell receptor (pre-BCR) signaling complex with the surrogate L chain (SLC) components lambda5 and Vpre-B. To better understand how pre-BCR signaling output is determined by its Ig components and the SLC, we investigated the regulation of pre-BCR surface expression and HC secretory maturation in a new nonlymphoid system. We took this approach as a means to distinguish B-lineage-specific effects from pre-BCR-intrinsic properties that may influence these aspects of pre-BCR homeostasis necessary for signaling. As in pre-B cells, the SLC in nonlymphoid cells supported only a limited degree of mum HC maturation and low pre-BCR surface expression levels compared with conventional LCs, indicating that this was due to an intrinsic property of the SLC. We identified the non-Ig region of lambda5 as harboring the restrictive activity responsible for this phenotype. This property of lambda5 was also evident with Dmu, but the overall SLC- and L chain-dependent requirements for Dmu maturation and surface expression were markedly different from those for mum. Surprisingly, Dmu was modified in an unusual manner that was only dependent on Vpre-B. These results establish a novel function of lambda5 in limiting surface pre-BCR levels and reveal biochemical properties of Ig molecules that may underlie the diverse consequences of pre-BCR signaling in vivo by different HCs.

Regulation of germline promoters by the two human Ig heavy chain 3' alpha enhancers.

The human IgH 3' enhancers, located downstream of each of the two Calpha genes, modulate germline (GL) transcription of the IgH genes by influencing the activity of promoter-enhancer complexes upstream of the switch and intervening (I) regions. The regulation of GL alpha1 and alpha2 promoters by different human 3' enhancer fragments was investigated in cell lines representing various developmental stages. Both alpha1HS1,2 and alpha2HS1,2 fragments show equally strong enhancer activity on the GL alpha1 and alpha2 promoters in both orientations when transiently transfected into a number of mature B cell line (DG75, CL-01, and HS Sultan). However, there is no activity in a human pre-B cell line (NALM-6) nor a human T cell line (Jurkat). HS3 shows no enhancer activity by itself in any of the cell lines, whereas a modest effect is noted using HS4 in the three mature B cell lines. However, the combination of the alpha2HS3-HS1,2-HS4 fragments, which together form a potential locus control region, displays a markedly stronger enhancer activity than the individual fragments with a differential effect on the alpha1 and alpha2 promoters as compared with the gamma3 promoter. Our results suggest that the human GL alpha promoter may be regulated by two independent pathways. One pathway is induced by TGF-beta1 which directs IgA isotype switch through activation of the GL alpha promoter and no TGF-beta1-responsive elements are present in the different 3' enhancer fragments. The other route is through the human 3' enhancer regions that cis-up-regulate the GL alpha promoter activity in mature B cells.

Inhibition of complement-mediated immune hemolysis by peptides derived from the constant domain of immunoglobulin.

High-dose administration of intravenous immunoglobulin is reported to be useful for inhibiting complement-dependent immune cytolysis. We have found that, among the proposed C1q-binding sites of the Fc portion of human IgG1, only residues 282-292 inhibited pig red blood cell lysis by human serum. Moreover, a hexadecemeric multiple antigen peptide of residues 282-292 from IgG showed significantly greater activity in suppressing complement-mediated immune cytolysis and can be used in place of high-dose intravenous immunoglobulin, which is extracted from donors and thus is expensive.

Targeted deletion of the IgA constant region in mice leads to IgA deficiency with alterations in expression of other Ig isotypes.

A murine model of IgA deficiency has been established by targeted deletion of the IgA switch and constant regions in embryonic stem cells. B cells from IgA-deficient mice were incapable of producing IgA in vitro in response to TGF-beta. IgA-deficient mice expressed higher levels of IgM and IgG in serum and gastrointestinal secretions and decreased levels of IgE in serum and pulmonary secretions. Expression of IgG subclasses was complex, with the most consistent finding being an increase in IgG2b and a decrease in IgG3 in serum and secretions. No detectable IgA Abs were observed following mucosal immunization against influenza; however, compared with those in wild-type mice, increased levels of IgM Abs were seen in both serum and secretions. Development of lymphoid tissues as well as T and B lymphocyte function appeared normal otherwise. Peyer's patches in IgA-deficient mice were well developed with prominent germinal centers despite the absence of IgA in these germinal centers or intestinal lamina propria. Lymphocytes from IgA-deficient mice responded to T and B cell mitogens comparable to those of wild-type mice, while T cells from IgA-deficient mice produced comparable levels of IFN-gamma and IL-4 mRNA and protein. In conclusion, mice with targeted deletion of the IgA switch and constant regions are completely deficient in IgA and exhibit altered expression of other Ig isotypes, notably IgM, IgG2b, IgG3, and IgE, but otherwise have normal lymphocyte development, proliferative responses, and cytokine production.

Role of maternal Ig in the induction of C kappa-specific CD8+ T cell tolerance.

Although the influence of maternal Ig on the B cell repertoire and subsequent Ab response has been extensively studied, much less attention has been devoted to their effects on T cell responses of the offspring. To address this question, we have studied the influence of maternal kappa-positive Ig (Ig kappa) on the C kappa-specific CD8+ T cell response of kappa knock-out (kappa-/-) pups resulting from various crosses and foster nursings. These systems allowed control of physiologic transmission of Ig kappa at defined periods of ontogeny. Our data show that conventional transfer of maternal Ig via the placenta plus colostrum/milk or adoptive transfer via only the colostrum/milk were the most efficient at tolerizing C kappa-specific CD8+ responses. Surprisingly, tolerance was not detected in kappa-/- pups born to kappa+/- females obtained by cesarean delivery and suckled by kappa-/- mothers (transplacental supply only). Tolerance, which was strong until 5 wk of age, was reversible and waned with the decrease of Ig kappa serum concentration. Depletion of CD4+ T cells at the time of C kappa peptide immunization abolished the tolerance of C kappa-specific CD8+ T cells. These data suggest that an oral supply of Ig is very efficient at inducing and maintaining tolerance of C kappa-specific CD8+ T cells, at least for several weeks after birth, and that suppression rather than deletion is responsible for this tolerance. In addition, they strengthen the view that tolerance of CD8+ T cells to a soluble Ag is never permanently acquired even if it is present in large quantities during ontogeny.

Domain-switched mouse IgM/IgG2b hybrids indicate individual roles for C mu 2, C mu 3, and C mu 4 domains in the regulation of the interaction of IgM with complement C1q.

Although polymeric IgM and monomeric IgG are potent activators of the classical complement pathway, previous studies have indicated that monomeric IgM is inactive. To understand this and to examine the roles of the individual mu domains in complement activation, we created a set of IgM/IgG2b mouse chimeric Abs in which homologous domains of both Abs have been interchanged, either singly or together with adjacent domains. The monomer subunits (H2L2) of the resulting chimeras were analyzed for their capacities to bind C1q and to initiate complement-mediated lysis (CML) of haptenated erythrocytes. When C gamma 2 was flanked by C mu 4, the inherent C1q-binding activity of the C gamma 2 domain was lost. This demonstrates that C mu 4 can suppress the C1q-binding activity of the adjacent C gamma 2 domain, and suggests that C mu 4 may exert a similar effect on the C mu 3 domain in the IgM monomer subunit. When C mu 3 was located in an IgG2b background and potentially freed from the constraints imposed by the IgM background, the monomer was not able to bind C1q or initiate CML. This suggests that these activities are not expressed inherently in the C mu 3 domain. The transplantation of C mu 3 together with C mu 4 into the IgG background permitted polymer formation. This polymer was able to bind C1q, although neither the monomer nor the polymer forms were active in CML; conversely, all IgM polymers with a transplanted C gamma 2 domain were active in both C1q binding and CML, and demonstrated apparent Kd values similar to that of wild-type IgM.

The IL-2 receptor gamma c chain does not function as a subunit shared by the IL-4 and IL-13 receptors. Implication for the structure of the IL-4 receptor.

The IL-2 receptor (IL-2R) gamma c subunit is also a component of the receptors for IL-4, IL-7, IL-9, and IL-15. The IL-4R and IL-13R appear to share a common subunit, and gamma c was proposed to be this shared subunit. In this study, we have assessed the relative contribution of gamma c to the mouse IL-4R and IL-13R. The MC/9 mast cell line constitutively expresses gamma c and proliferates to IL-4 and IL-13, but only the response to IL-4 was blocked by anti-gamma c mAbs. After transfection of the IL-4- and IL-13-responsive gamma c-negative B9 plasmacytoma with full length (m gamma) or cytoplasmic-tailless gamma c cDNA (m gamma t), only the proliferative response to IL-4 was affected by the surface expression of these gamma c molecules. The inability of m gamma or m gamma t expression to affect IL-13-induced proliferation by B9 indicates that gamma c does not obviously contribute to the IL-13R and does not function as the shared subunit of the IL-4R and IL-13R. This study suggests that there are two distinct IL-4R, one of which is independent of gamma c.

Effect of altered CH2-associated carbohydrate structure on the functional properties and in vivo fate of chimeric mouse-human immunoglobulin G1.

Immunoglobulin G (IgG) molecules are glycosylated in CH2 at Asn297; the N-linked carbohydrates attached there have been shown to contribute to antibody (Ab) stability and various effector functions. The carbohydrate attached to the IgG constant region is a complex biantennary structure. Alterations in the structure of oligosaccharide have been associated with human diseases such as rheumatoid arthritis and osteoarthritis. To study the effects of altered carbohydrate structure on Ab effector function, we have used gene transfection techniques to produce mouse-human chimeric IgG1 Abs in the Chinese hamster ovary (CHO) cell line Lec 1, which is incapable of processing the high-mannose intermediate through the terminal glycosylation steps. We also produced IgG1 Abs in Pro-5, the wild-type CHO cell line that is the parent of Lec 1. The Pro-5-produced Ab (IgG1-Pro-5) was similar to IgG1-My 1, a myeloma-produced IgG1 Ab of the same specificity, in its biologic properties such as serum half-life, ability to effect complement-mediated cytolysis, and affinity for Fc gamma RI. Although the Lec 1-produced Ab, IgG1-Lec 1, was properly assembled and retained antigen specificity, it was incapable of complement-mediated hemolysis and was substantially deficient in complement consumption, C1q binding, and C1 activation. IgG1-Lec 1 also showed reduced but significant affinity for Fc gamma R1 receptors. The in vivo half-life of IgG1-Lec 1 was shorter than that of either the myeloma- or Pro-5-produced counterpart, with more being cleared during the alpha-phase and with more rapid clearance during the beta-phase. Clearance of IgG1-Lec 1 could be inhibited by the administration of yeast-derived mannan. Thus the uptake of IgG1-Lec 1 appears to be accelerated by the presence of terminally mannosylated oligosaccharide. Therefore, certain Ab functions as well as the in vivo fate of the protein are dramatically affected by altered carbohydrate structure. Expression of Igs in cell lines with defined glycosylation mutations is shown to be a useful technique for investigating the contribution of carbohydrate structure to Ab function.

Mechanism of lymphocyte function-associated molecule 3-Ig fusion proteins inhibition of T cell responses. Structure/function analysis in vitro and in human CD2 transgenic mice.

Soluble ligands specific for cell surface molecules involved in APC-T cell interactions can signal cells and modulate immune responses. Recently, we reported that LFA3TIP, a fusion protein comprised of the first LFA-3 extracellular domain fused to the hinge, CH2, and CH3 regions of a human IgG1 inhibits proliferation of human T cells in vitro. We report herein the cell-based mechanism(s) of LFA3TIP in inhibition by studying the effects of structurally altered LFA3-Ig fusion proteins on proliferation of human PBL in vitro and on responses of mice transgenic for human CD2. We show that LFA3TIP inhibition requires expression of both the LFA-3 and CH2 domains of the fusion protein that bind CD2 and Fc gamma RI or Fc gamma RIII, respectively. LFA3TIP forms an intracellular Fc gamma R/CD2 bridge and directs cytolysis of CD2+ cells by freshly drawn human PBL in vitro as well as the non-C-mediated depletion of peripheral T cells of human CD2 transgenic mice. The cell-based mechanism(s) of LFA3TIP inhibition are discussed.