Faculty Bibliography

A set of mouse IgAs containing amino acids differing amongst the six alpha-chain allotypes was constructed by mutating an S107-IgA plasmid and transfecting it into a non-producer myeloma cell line along with a kappa-chain plasmid. The secreted IgAs were examined for their possession of a covalent bond between alpha- and light (L: )-chains and for their ability to bind to three anti-allotypic monoclonal antibodies, HIS-M2, HY-15, and HY-16. IgA of the Igh-2 ( a ) allotype was found to be unique in its total lack of a covalent bond between alpha and L: -chains, formation of which apparently depends on the presence of an 'extra' Cys in the hinges of all of the other five allotypes. The allotypic epitopes are associated with identifiable amino acids in the Calpha1 region of the molecule. Binding to HIS-M2 requires Ala 216 whereas binding to HY-15 requires Pro 216 and Asp 222. Binding to Hy-16 requires Arg 183 and either Pro 216 or Ser 216 but not Ala 216. However, binding to HY-16 by all of the IgAs produced by transfectants is impaired by defective glycosylation in the transfected myeloma and is only revealed after deglycosylation

Six IgA allotypes are serologically identifiable in inbred mice. The sequences of the PCR-amplified C alpha 1, C alpha 2 and C alpha 3 exons from the genomic DNA of mice of four previously unsequenced allotypes already have been compared with those of BALB/c and of a wild mouse, Mus pahari, in the literature. Sporadic differences, including several that may encode the known allotypic determinants, are found throughout the three exons, but major differences occur in the hinge. The hinge is longest in DBA/2 ( Igh-2(c)) mice, having an extra codon compared with that of BALB/c ( Igh-2(a)) and B10.A ( Igh-2(b)) mice. It is two codons shorter in CE ( Igh-2(f)) and four shorter in M. pahari, AKR and NZB (both Igh-2(d)) mice, but the position of the missing codons in the latter two strains is offset from that in M. pahari. The hinges in BALB/c ( Igh-2(a)) and DBA/2 ( Igh-2(c)) differ most from each other and from the other three allotypes, which are fairly closely related. Both BALB/c and DBA/2 have O-linked glycosylation sites, but they are in different positions in the hinge. Compared with BALB/c ( Igh-2(a)), B10.A( Igh-2(b)) has two extra Cys residues in the hinge, while DBA/2 ( Igh-2(c)), AKR/NZB ( Igh-2(d)) and CE ( Igh-2(f)) each have one. The differences in hinge length may have arisen by mismatching of highly repetitive portions of its sequence during meiotic recombination. Possible effects of the differences in hinge length and composition on the behavior of the mouse IgA allotypes are discussed

The T560 B lymphoma produces polyreactive IgG2a with the features of natural autoantibody. All T560 cells bear and secrete IgG2a but a small fraction spontaneously co-express IgA. Cells secreting IgA alone cannot be detected. IgA secretion is enhanced by interaction of T560 cells either with activated T cells and cognate antigen, or with LPS, but not with cytokines, including IL-5 and TGF-beta. IgA and IgG2a mRNAs have identical V186.2. DFL 16 and JH1 sequences from framework 2 through JH1. PCR analysis reveals that previous recombination events have led to deletion of the mu, gamma3, gamma1, gamma2b constant region genes from both the productive and the unproductive chromosome but the former has retained gamma2a, epsilon and alpha, the latter only alpha. Digestion-circularization (DC)-PCR experiments provide formal proof of DNA recombination between Ca and the intron upstream of Cmu. Evidently, the productive chromosome has switched only as far as gamma2a, the unproductive all the way to the alpha constant region gene. The unproductive allele is transcriptionally active as evidenced by the presence of mRNA encoding Calphal inappropriately spliced to a cryptic splice site in the downstream intron of DQ52 (eliminated from the productive chromosome). A specific RT-PCR using oligonucleotide primers derived from the upstream initiation site of the Ialpha exon and from Calpha1 discloses that T560 cells contain alpha-germ line mRNA, presumably transcribed from the Ialpha-region of the productive chromosome, spliced to Calpha. Treatment with LPS stops production of these spliced transcripts suggesting that it may promote either DNA recombination in cells spontaneously transcribing Ialpha or a change in splicing such that Ialpha sequence is no longer joined to Calpha. Verification of the DC-PCR product by sequencing reveals that the T560 and B10.A IgA (Ig2b allotype) hinge is different from the BALB/c IgA (Ig2a allotype) hinge: it has two extra Cys and has eliminated the first Thr, a potential glycosylation site in BALB/c IgA

T560, a mouse B lymphoma that originated in gut-associated lymphoid tissue, expresses receptors that bind dimeric IgA and IgM in a mutually inhibitory manner but have little affinity for monomeric IgA. Evidence presented in this paper indicates that the receptor is poly-Ig receptor (pIgR) known in humans and domestic cattle to bind both IgA and IgM. The evidence includes the demonstration that binding of IgM is J chain dependent, and that pIg-precipitated receptor has an appropriate Mr of 116-120 kDa and can be detected on immunoblots with specific rabbit anti-mouse pIgR. Overlapping RT-PCR performed using template mRNA from T560 cells and oligonucleotide primer pairs designed from the published sequence of mouse liver pIgR indicate that T560 cells express mRNA virtually identical with that of the epithelial cell pIgR throughout its external, transmembrane, and intracytoplasmic coding regions. Studies using mutant IgAs suggest that the Calpha2 domain of dimeric IgA is not involved in high-affinity binding to the T560 pIgR. Inasmuch as this mouse B cell pIgR binds IgM better than IgA, it is similar to human pIgR and differs from rat, mouse, and rabbit epithelial cell pIgRs that bind IgA but not IgM. Possible explanations for this difference are discussed. All clones of T560 contain some cells that spontaneously secrete both IgG2a and IgA, but all of the IgA recoverable from the medium and from cell lysates is monomeric; it cannot be converted to secretory IgA by T560 cells

In mice undergoing a graft-versus-host (GVH) reaction, donor T cells responding to the host's MHC antigens induce polyclonal activation of the host's B cells and secretion of their antibodies and autoantibodies. T560, a CD5- B lymphoma that arose in the gut-associated lymphoid tissue (GALT) of a (B10 x B10.H2aH4(b)pWts) F1 hybrid mouse that had been injected with parental B10.H2aH4b splenocytes, is of particular interest because it produces switched, heavily mutated, but, nevertheless, polyreactive immunoglobulin. T560 bears and contains IgG2a but switches to IgA spontaneously. The T560 Ig variable region is encoded by a V186.2-related VH gene, juxtaposed to DFL 16 and J(H)1, and by a Vkappa gene of the Vkappa 4/5 group juxtaposed to Jkappa1. Both VH and VK are heavily mutated. The IgA binds to polystyrene, to p-azophenyl-phosphorylcholine (PC)-conjugated keyhole limpet hemocyanin (KLH) (PC-KLH), to 2,4,6 trinitrophenylated (TNP)-KLH and to human TNF-beta but not to KLH, human TNF-alpha, or any of several other Ags tested. Hapten inhibition experiments indicate that the polystyrene, PC- and TNP-binding sites do not overlap. The switched isotypes and heavy load of somatic mutations found in the T560 IgG2a/IgA suggest that T cell-dependant somatic selection of the T560 precursor B cell may have been superimposed on polyclonal B cell activation originally associated with the GVH

The specificity and properties of a novel IgA receptor expressed on the surface of a tissue culture-adapted B cell lymphoma, T560, that originated in murine gut-associated lymphoid tissue, have been explored. Like the IgA receptors of murine T and splenic B cells studied by others, the T560 IgA receptor is trypsin sensitive and neuraminidase resistant and is up-regulated on T560 cells by exposing them overnight to high concentrations of polymeric IgA. Unlike them, the T560 IgA receptor is inhibited by low concentrations of IgM and high concentrations of IgG2a and IgG2b, binds at pH 4.0 but not at pH 8.0, is down-regulated by activation of protein kinase C and is sensitive to phosphatidylinositol-specific phospholipase C, indicating that it is glycosyl phosphatidylinositol-linked to the cell membrane. It is not a cell-bound form of galactosyl transferase, does not appear to bind to Ig through carbohydrate residues and does not react specifically with antibody to secretory component. It may be a completely new, cross-reactive receptor, perhaps related in some way to the polymeric Ig receptor or to the receptor for IgA expressed on the apical surface of Peyer's patch M cells, which is known to cross-react with IgG. Alternatively, it may be homologous to the highly IgA-specific Fc alpha R of T cells but, perhaps because of its glycosyl phosphatidylinositol linker, may have an ability to move and interact with other Ig receptors on the cell surface such that Ig bound to them are cross-inhibitory