Identification of a new European rabbit IgA with a serine-rich hinge region.

In mammals, the most striking IgA system belongs to Lagomorpha. Indeed, 14 IgA subclasses have been identified in European rabbits, 11 of which are expressed. In contrast, most other mammals have only one IgA, or in the case of hominoids, two IgA subclasses. Characteristic features of the mammalian IgA subclasses are the length and amino acid sequence of their hinge regions, which are often rich in Pro, Ser and Thr residues and may also carry Cys residues. Here, we describe a new IgA that was expressed in New Zealand White domestic rabbits of IGHVa1 allotype. This IgA has an extended hinge region containing an intriguing stretch of nine consecutive Ser residues and no Pro or Thr residues, a motif exclusive to this new rabbit IgA. Considering the amino acid properties, this hinge motif may present some advantage over the common IgA hinge by affording novel functional capabilities. We also sequenced for the first time the IgA14 CH2 and CH3 domains and showed that IgA14 and IgA3 are expressed.

Nck-mediated recruitment of BCAP to the BCR regulates the PI(3)K-Akt pathway in B cells.

The adaptor Nck links receptor signaling to cytoskeleton regulation. Here we found that Nck also controlled the phosphatidylinositol-3-OH kinase (PI(3)K)-kinase Akt pathway by recruiting the adaptor BCAP after activation of B cells. Nck bound directly to the B cell antigen receptor (BCR) via the non-immunoreceptor tyrosine-based activation motif (ITAM) phosphorylated tyrosine residue at position 204 in the tail of the immunoglobulin-α component. Genetic ablation of Nck resulted in defective BCR signaling, which led to hampered survival and proliferation of B cells in vivo. Indeed, antibody responses in Nck-deficient mice were also considerably impaired. Thus, we demonstrate a previously unknown adaptor function for Nck in recruiting BCAP to sites of BCR signaling and thereby modulating the PI(3)K-Akt pathway in B cells.

Nonhuman primate IgA: genetic heterogeneity and interactions with CD89.

Nonhuman primates are extremely valuable animal models for a variety of human diseases. However, it is now becoming evident that these models, although widely used, are still uncharacterized. The major role that nonhuman primate species play in AIDS research as well as in the testing of Ab-based therapeutics requires the full characterization of structure and function of their Ab molecules. IgA is the Ab class mostly involved in protection at mucosal surfaces. By binding to its specific Fc receptor CD89, IgA plays additional and poorly understood roles in immunity. Therefore, Ig heavy alpha (IGHA) constant (C) genes were cloned and sequenced in four different species (rhesus macaques, pig-tailed macaques, baboons, and sooty mangabeys). Sequence analysis confirmed the high degree of intraspecies polymorphism present in nonhuman primates. Individual animals were either homozygous or heterozygous for IGHA genes. Highly variable hinge regions were shared by animals of different geographic origins and were present in different combinations in heterozygous animals. Therefore, it appears that although highly heterogeneous, hinge sequences are present only in limited numbers in various nonhuman primate populations. A macaque recombinant IgA molecule was generated and used to assess its interaction with a recombinant macaque CD89. Macaque CD89 was able to bind its native ligand as well as human IgA1 and IgA2. Presence of Ag enhanced macaque IgA binding and blocking of macaque CD89 N-glycosylation reduced CD89 expression. Together, our results suggest that, despite the presence of IgA polymorphism, nonhuman primates appear suitable for studies that involve the IgA/CD89 system.

The B cell receptor promotes B cell activation and proliferation through a non-ITAM tyrosine in the Igalpha cytoplasmic domain.

In addition to the tyrosines of the Igalpha and beta immunoreceptor tyrosine-based activation motifs (ITAMs), the evolutionarily conserved Igalpha non-ITAM tyrosine 204 becomes phosphorylated upon antigen recognition by the B cell receptor (BCR). Here we demonstrate that splenic B cells from mice with a targeted mutation of Igalpha Y204 exhibited an isolated defect in T cell-independent B cell activation, proliferation, and antibody response upon BCR engagement, yet normal BCR capping, antigen internalization, antigen presentation, and T cell-dependent antibody production. Mutant B cells, present in normal numbers, exhibited unimpaired BCR-induced spleen tyrosine kinase (Syk) phosphorylation but reduced B cell linker protein (BLNK) phosphorylation, calcium flux, and nuclear factor kappaB (NFkappaB), c-jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) activation. These results suggest that Igalpha non-ITAM tyrosine 204 promotes a distinct cellular response, namely T-independent B cell proliferation and differentiation via phosphorylation of the adaptor BLNK.

B cell antigen receptor signaling and internalization are mutually exclusive events.

Engagement of the B cell antigen receptor initiates two concurrent processes, signaling and receptor internalization. While both are required for normal humoral immune responses, the relationship between these two processes is unknown. Herein, we demonstrate that following receptor ligation, a small subpopulation of B cell antigen receptors are inductively phosphorylated and selectively retained at the cell surface where they can serve as scaffolds for the assembly of signaling molecules. In contrast, the larger population of non-phosphorylated receptors is rapidly endocytosed. Each receptor can undergo only one of two mutually exclusive fates because the tyrosine-based motifs that mediate signaling when phosphorylated mediate internalization when not phosphorylated. Mathematical modeling indicates that the observed competition between receptor phosphorylation and internalization enhances signaling responses to low avidity ligands.

Neuraminidase treatment abrogates the binding abnormality of IgA1 from IgA nephropathy patients and the differential charge distribution of its alpha-heavy chains.

We have studied the interaction of the Gal-GalNAc-reactive champedak lectin-C with neuraminidase-treated and untreated IgA1 from IgA nephropathy patients. The binding ability of the lectin to untreated IgA1 from IgA nephropathy patients was significantly lower as compared to the untreated IgA1 from normal controls. This differential lectin-binding capacity was abrogated when the experiment was performed on neuraminidase-treated sera. Treatment of the serum IgA1 with neuraminidase also abrogated the differential charge distribution between the alpha-heavy chains of IgA nephropathy patients and normal controls.

Rapid structural characterisation of a murine monoclonal IgA alpha chain: heterogeneity in the oligosaccharide structures at a specific site in samples produced in different bioreactor systems.

A murine hybridoma cell line which secretes a monoclonal IgA antibody, directed against the LPS antigen of Vibrio cholerae, was grown in either a continuous stirred tank reactor, a fluidised bed reactor or a hollow fibre reactor. Different methods were used for the structural characterisation of the IgA alpha chains. A classical approach consisted of Edman sequencing and mass determination of peptides separated by reversed phase HPLC. Alternatively, peptides and glycopeptides from a tryptic digest of each alpha chain were identified directly by MALDI-TOF mass spectrometry. A detailed analysis of the oligosaccharide structures at an unique site on the alpha chain was made by labelling the oligosaccharides released by N-glycosidase F with 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone. After separation by HPLC, mass measurements were made using matrix-assisted laser desorption time of flight mass spectrometry before and after digestion with specific exoglycosidases. The primary structure of the alpha chain of IgA was not affected by different cell culture conditions; in contrast, significant variations could be detected in the pattern of N-linked oligosaccharide structures, most prominently in the degree of sialylation. The efficiency of the analytical techniques in providing quality control of the identity, integrity and consistency of the glycoprotein is shown and discussed.

Characterization of T-cell repertoire of the bone marrow in immune-mediated aplastic anemia: evidence for the involvement of antigen-driven T-cell response in cyclosporine-dependent aplastic anemia.

To determine whether the antigen-driven T-cell response is involved in the pathogenesis of aplastic anemia (AA), we examined the complementarity-determining region 3 (CDR3) size distribution of T-cell receptor (TCR) beta-chain (BV) subfamilies in the bone marrow (BM) of untreated AA patients. AA patients who did not respond to immunosuppressive therapy and those who obtained unmaintained remission early after cyclosporine (CyA) or antithymocyte globulin (ATG) therapy exhibited essentially a normal CDR3 size pattern. In contrast, five patients who needed continuous administration of CyA to maintain remission exhibited a skewed CDR3 size pattern in a number (>40%) of BV subfamilies suggestive of clonal predominance. The skewing of CDR3 size distribution became less pronounced in one of the CyA-dependent patients when the patient achieved unmaintained remission after a 4-year therapy with CyA, whereas it persisted longer than 7 years in the other patient requiring maintenance therapy. Sequencing of BV15 cDNA for which the CDR3 size pattern exhibited apparent clonal predominance in all CyA-dependent patients showed high homology of the amino acid sequence of the CDR3 between two different patients. These findings indicate that antigen-driven expansion of T cells is involved in the pathogenesis of AA characterized by CyA-dependent recovery of hematopoiesis.

Immunochemical, structural and translocating properties of anti-DNA antibodies from (NZBxNZW)F1 mice.

Many monoclonal antibodies (mAb) derived from the spleens of (NZBxNZW)F1 mice react strongly with dsDNA and also other self antigens, although more weakly. When added to cell cultures, these polyreactive anti-DNA mAb penetrate into various cell types and accumulate in the nucleus within a few hours. Almost all anti-DNA mAb bind to cell membrane antigens but the extent of their binding does not directly correspond to their penetration capabilities. Sequence analysis of anti-DNA mAb indicated the use of various germ-line VH families. The complementary-determining regions (CDR) 3 differ but they all contain a relatively high number of tyrosines and positively charged amino acids (lysine and arginine). Haptens (biotin, fluor-escein, oligonucleotides) and macromolecules (peroxidase, IgG) covalently coupled to the mAb or their F(ab')2 and Fab fragments were translocated through the cytoplasm and into the cell nucleus. Furthermore, 61% of peripheral blood lymphocytes were labelled when mice were injected with fluorescein-labelled mAb. Peptides corresponding to CDR2, CDR3 and CDR2 linked to CDR3 (CDR2-3) of several penetrating mAb were prepared and their intracellular translocating capacity was assessed. The CDR2-3 peptide, but not the individual peptides, was able to penetrate cells and could be used as a vector to transport macromolecules. Although all CDR2-3 reacted with dsDNA and other self antigens, each one exhibited a distinct polyreactivity profile.

Relation between the heavy chain complementarity region 3 characteristics and rheumatoid factor binding properties.

Among the rheumatoid factors (RFs), monospecific and polyspecific types can be distinguished. However the molecular basis responsible for their different specificity is not well understood. In a previous report, we have shown that the binding of the majority of the polyspecific antibodies is salt-sensitive. No binding to IgG was observed under high ionic strength (0.3-0.5 M NaCl). This salt-sensitivity was only observed for 18% of the monospecific RFs. Here, we have analyzed 14 RFs representing the 3 different groups (6 salt-insensitive monospecific, 4 salt-sensitive monospecific and 4 salt-sensitive polyspecific RFs). By analysis of the amino acid composition and the distribution of polar and non-polar residues of their heavy chain complementarity-determining region 3 (H-CDR3) in relation to mono/polyspecificity, salt-sensitivity and reactivity against human IgG subclasses, we have identified common structural features responsible for their different binding properties. Salt-sensitive RFs (mono as well as polyspecific antibodies) were characterized by long H-CDR3's (15.3+/-2.7) that contained large numbers of hydrophilic residues such as arginine and serine, while salt-insensitive RFs had more hydrophobic H-CDR3's of smaller length (11.3+/-2.4). In addition, for the monospecific RFs, remarkably similar hydrophilicity H-CDR3 profiles were found that were correlated with their specificity for IgG subclasses. These observations confirm the importance of the H-CDR3 for the binding of RFs to IgG. Furthermore, on the basis of their shorter H-CDR3's and their rather unique H-CDR3 hydrophilicity profiles, it is likely that the majority of the monospecific RFs should be considered as a group of RFs that is independent of the polyspecific RF repertoire.

Nucleotide sequence analysis of a human monoclonal antibody 22-13 reactive with lung tumor-associated antigen.

A human monoclonal antibody (HuMAb) 22-13 (IgG1, kappa) recognizes a cytoplasmic antigen associated primarily with human lung tumors. This study reports the primary nucleotide and deduced amino acid sequences of the rearranged heavy and light chains of the HuMAb 22-13. This HuMAb uses a VH gene member of the V(H)Ia gene family, 51P1 and is productively rearranged with a D-D fusion product of the D(LR)2 and D(XP)2 germ line DH genes and the germ line JH3 gene. HuMAb 22-13 Vkappa belongs to the kappa light chain variable subgroup IIIb family and appears to be derived from the Humkv325 germ line gene and is rearranged with a germ line Jkappa5 gene. The results reveal that production of a HuMAb 22-13 is achieved by rearrangement of the 51P1/Humkv325 germ line variable region gene combination, associated with the autoimmune repertoire and that HuMAb 22-13 has a striking sequence homology to rheumatoid factors (RFs) of the Wa idiotypic family. HuMAb 22-13 and Wa RFs have in common V(H)Ia and VkappaIIIb gene segments, but use different DH, JH and Jkappa gene segments. However, in spite of this structural similarity, HuMAb 22-13 does not display rheumatoid factor activity. Taken together with the reported findings, these data indicate the representation of the shared usage of highly homologous variable region genes in entirely different humoral immune responses in the human system.

Identification of a homolog of the C alpha 3'/hs3 enhancer and of an allelic variant of the 3'IgH/hs1,2 enhancer downstream of the human immunoglobulin alpha 1 gene.

Although four regulatory elements are known downstream of the mouse IgH alpha gene, a single enhancer homologous to hs1,2 has been thus far described downstream of each human alpha gene (Chen, C. and Birshtein, B. K., J. Immunol. 1997. 159: 1310). We characterized a 10-kb region downstream of the human alpha 1 gene. Two B cell-specific regulatory elements homologous to the murine C alpha 3'/hs3 and hs1,2,3' enhancers were found, which are duplicated downstream of alpha 2. The hs1,2 element is in inverted orientation by comparison with a recently reported alpha 1 hs1,2 element: it appears as a common allelic variant carrying an internal tandem repeat insertion and its prevalence in the human population is 60%. As in the mouse, the human hs1,2 enhancer is flanked with long inverted repeats which may have promoted inversion events through homologous recombination. Although the palindromic organization of the region is maintained in human, sequence identity with rodents focuses on core enhancer elements rather than on flanking repeats. Concerted divergence of both sides of the dyad symmetry suggests that inverted repeats are not just evolutionary remnants but rather play an architectural role in the LCR function.

Comparison of CH1 domains in different classes of murine antibodies.

The CH1 domains of antibodies belonging to the following five murine immunoglobulin (Ig) classes IgG1, IgG2a, IgG2b, IgG3 and IgA have been compared. The IgG CH1 domain structures are, as would be expected, similar overall, but show local conformational variations. When compared with IgG CH1 domain structures, the IgA CH1 domain displays several significant structural differences, which are a consequence of insertions/ deletions and specific structural constraints. In regions of structural differences in the IgG CH1 domains, the spatial correspondence of residues is not reflected by conventional (Kabat) sequence number. Thus the sequence alignment and numbering for CH1 domains has been revised to be consistent with the three-dimensional alignments.

The efficiency of cysteine-mediated intracellular retention determines the differential fate of secretory IgA and IgM in B and plasma cells.

Previous studies on IgM secretion demonstrated a role for the mu chain C-terminal cysteine (Cys575) in preventing the transport of unpolymerized subunits along the secretory pathway. The sequence homology between the C-terminal tailpieces of mu and alpha heavy chains prompted us to investigate the role of cysteine-mediated, retention in the control of IgA secretion during B cell development. Similar to IgM, IgA are not secreted by B lymphocytes: the retention mechanism can be reversed by the reducing agent 2-mercaptoethanol, suggesting that disulfide interchange reactions are involved in the quality control of both IgM and IgA. Yet, alpha 2L2 subunits, but not mu2L2, are secreted constitutively by plasma cells. We demonstrate that the differential retention of IgM and IgA subunits by myeloma transfectants is mainly due to the presence of an acidic residue upstream the alpha chain C-terminal cysteine. The regulation of polymeric Ig secretion during B cell development provides an example of how thiol-mediated quality control can be modulated according to the aminoacidic context surrounding the critical cysteine and to the cell type.

Selective transport of IgA. Cellular and molecular aspects.

The principal characteristic immunoglobulin of mucosal surfaces, secretory immunoglobulin A (S-IgA), is the product of two different types of cell present in mucosal and glandular tissues. Submucosal plasma cells, which are generated largely within the common mucosal immune system, synthesize predominantly polymeric, J chain-containing IgA, which is selectively bound by polymeric immunoglobulin receptor or secretory component (SC) on the basolateral surfaces of mucosal and glandular epithelial cells. The molecular and cellular events involved in SC expression, its intravesicular transport together with its polymeric IgA ligand to the apical surface of the epithelial cell, during which IgA becomes covalently linked to SC, and the proteolytic cleavage of SC from the apical membrane to release S-IgA into the lumen have been elucidated. Additional receptors and mechanisms for the uptake, catabolism, and transport of IgA exist, especially in the liver. The biologic significance of IgA transport lies in the secretion of large quantities of S-IgA antibodies for the protection of huge areas of mucosal surfaces and for the provision of passive immunity to suckling infants, and in the immune elimination of antigenic materials by hepatobiliary transport.