Ig Variable Domain Research Articles

Surface Ig variable domain glycosylation affects autoantigen binding and acts as threshold for human autoreactive B cell activation.

The hallmark autoantibodies in rheumatoid arthritis are characterized by variable domain glycans (VDGs). Their abundant occurrence results from the selective introduction of N-linked glycosylation sites during somatic hypermutation, and their presence is predictive for disease development. However, the functional consequences of VDGs on autoreactive B cells remain elusive. Combining crystallography, glycobiology, and functional B cell assays allowed us to dissect key characteristics of VDGs on human B cell biology. Crystal structures showed that VDGs are positioned in the vicinity of the antigen-binding pocket, and dynamic modeling combined with binding assays elucidated their impact on binding. We found that VDG-expressing B cell receptors stay longer on the B cell surface and that VDGs enhance B cell activation. These results provide a rationale on how the acquisition of VDGs might contribute to the breach of tolerance of autoreactive B cells in a major human autoimmune disease.

TET3 dioxygenase modulates gene conversion at the avian immunoglobulin variable region via demethylation of non-CpG sites in pseudogene templates.

Diversification of the avian primary immunoglobulin (Ig) repertoire is achieved in developing B cells by somatic hypermutation (SHM) and gene conversion (GCV). GCV is a type of homologous recombination that unidirectionally transfers segments of Ig pseudogenes to Ig variable domains. It is regulated by epigenetic mechanisms like histone modifications, but the role of DNA methylation remains unclear. Here, we demonstrate that the chicken B‐cell line DT40 lacking TET3, a member of the TET (Ten‐eleven translocation) family dioxygenases that facilitate DNA demethylation, exhibited a marked reduction in GCV activity in Ig variable regions. This was accompanied by a drop in the bulk levels of 5‐hydroxymethylcytosine, an oxidized derivative of 5‐methylcytosine, whereas TET1‐deficient or TET2‐deficient DT40 strains did not exhibit such effects. Deletion of TET3 caused little effects on the expression of proteins required for SHM and GCV, but induced hypermethylation in some Ig pseudogene templates. Notably, the enhanced methylation occurred preferably on non‐CpG cytosines. Disruption of both TET1 and TET3 significantly inhibited the expression of activation‐induced cytidine deaminase (AID), an essential player in Ig diversification. These results uncover unique roles of TET proteins in avian Ig diversification, highlighting the potential importance of TET3 in maintaining hypomethylation In Ig pseudogenes.

Ancient Use of Ig Variable Domains Contributes Significantly to the TCRδ Repertoire.

The loci encoding B and T cell Ag receptors are generally distinct in commonly studied mammals, with each receptor's gene segments limited to intralocus, cis chromosomal rearrangements. The nurse shark (Ginglymostoma cirratum) represents the oldest vertebrate class, the cartilaginous fish, with adaptive immunity provided via Ig and TCR lineages, and is one species among a growing number of taxa employing Ig-TCRδ rearrangements that blend these distinct lineages. Analysis of the nurse shark Ig-TCRδ repertoire found that these rearrangements possess CDR3 characteristics highly similar to canonical TCRδ rearrangements. Furthermore, the Ig-TCRδ rearrangements are expressed with TCRγ, canonically found in the TCRδ heterodimer. We also quantified BCR and TCR transcripts in the thymus for BCR (IgHV-IgHC), chimeric (IgHV-TCRδC), and canonical (TCRδV-TCRδC) transcripts, finding equivalent expression levels in both thymus and spleen. We also characterized the nurse shark TCRαδ locus with a targeted bacterial artifical chromosome sequencing approach and found that the TCRδ locus houses a complex of V segments from multiple lineages. An IgH-like V segment, nestled within the nurse shark TCRδ translocus, grouped with IgHV-like rearrangements we found expressed with TCRδ (but not IgH) rearrangements in our phylogenetic analysis. This distinct lineage of TCRδ-associated IgH-like V segments was termed "TAILVs." Our data illustrate a dynamic TCRδ repertoire employing TCRδVs, NARTCRVs, bona fide trans-rearrangements from shark IgH clusters, and a novel lineage in the TCRδ-associated Ig-like V segments.

Determinants in the Ig Variable Domain of Human HAVCR1 (TIM-1) Are Required To Enhance Hepatitis C Virus Entry.

Hepatitis C virus (HCV) is the leading cause of chronic hepatitis in humans. Several host molecules participate in HCV cell entry, but this process remains unclear. The complete unraveling of the HCV entry process is important to further understand viral pathogenesis and develop therapeutics. Human hepatitis A virus (HAV) cellular receptor 1 (HAVCR1), CD365, also known as TIM-1, functions as a phospholipid receptor involved in cell entry of several enveloped viruses. Here, we studied the role of HAVCR1 in HCV infection. HAVCR1 antibody inhibited entry in a dose-dependent manner. HAVCR1 soluble constructs neutralized HCV, which did not require the HAVCR1 mucinlike region and was abrogated by a mutation of N to A at position 94 (N94A) in the Ig variable (IgV) domain phospholipid-binding pocket, indicating a direct interaction of the HAVCR1 IgV domain with HCV virions. However, knockout of HAVCR1 in Huh7 cells reduced but did not prevent HCV growth. Interestingly, the mouse HAVCR1 ortholog, also a phospholipid receptor, did not enhance infection and a soluble form failed to neutralize HCV, although replacement of the mouse IgV domain with the human HAVCR1 IgV domain restored the enhancement of HCV infection. Mutations in the cytoplasmic tail revealed that direct HAVCR1 signaling is not required to enhance HCV infection. Our data show that the phospholipid-binding function and other determinant(s) in the IgV domain of human HAVCR1 enhance HCV infection. Although the exact mechanism is not known, it is possible that HAVCR1 facilitates entry by stabilizing or enhancing attachment, leading to direct interactions with specific receptors, such as CD81.IMPORTANCE Hepatitis C virus (HCV) enters cells through a multifaceted process. We identified the human hepatitis A virus cellular receptor 1 (HAVCR1), CD365, also known as TIM-1, as a facilitator of HCV entry. Antibody blocking and silencing or knockout of HAVCR1 in hepatoma cells reduced HCV entry. Our findings that the interaction of HAVCR1 with HCV early during infection enhances entry but is not required for infection support the hypothesis that HAVCR1 facilitates entry by stabilizing or enhancing virus binding to the cell surface membrane and allowing the correct virus-receptor positioning for interaction with the main HCV receptors. Furthermore, our data show that in addition to the phospholipid-binding function of HAVCR1, the enhancement of HCV infection involves other determinants in the IgV domain of HAVCR1. These findings expand the repertoire of molecules that HCV uses for cell entry, adding to the already complex mechanism of HCV infection and pathogenesis.

Myelin Oligodendrocyte Glycoprotein and Multiple Sclerosis.

Myelin oligodendrocyte glycoprotein (MOG) is located on the external surface of myelin, a membranous component of the central nervous system (CNS) that forms the insulating lipid layer around neurons. The major MOG splicing variant (a1 transcript) encodes a transmembrane protein with an extracellular domain of an Ig variable (IgV) fold. MOG IgV domains from the same or different cells dimerize and contribute to the organization and maintenance of the myelin sheath in neurons. The encepalitogenic T cells recognize MOG and its immunodominant epitopes (epitopes 1-22, 35-55 and 92-106 located at the dimer interface) as foreign antigens and cause the destruction of myelin (demyelination) leading to the clinical condition known as multiple sclerosis (MS). Recognition of the antigen takes place in the context of the trimolecular complex formed by HLA, MOGpeptides and TCR. Understanding the role of MOG in MS. We have reviewed herein, the genomic organization of the human MOG gene, the structural characteristics of the MOG protein, the involvement of MOG in MS and clinical studies for the treatment of MS based on MOG peptide analogues. Conjugates of antigenic MOG peptides to mannan and combinations of antigenic MOG and other peptides chemically linked to cells of the immune system may modify the immune response, alleviating in some cases the symptoms of MS.

Minimal Residual Disease in Patients with Follicular Lymphoma Treated with Obinutuzumab or Rituximab As First-Line Induction Immunochemotherapy and Maintenance in the Phase 3 GALLIUM Study

Minimal Residual Disease in Patients with Follicular Lymphoma Treated with Obinutuzumab or Rituximab As First-Line Induction Immunochemotherapy and Maintenance in the Phase 3 GALLIUM Study

Glycosylation Alters Dimerization Properties of a Cell-surface Signaling Protein, Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1)

Human carcinoembryonic antigen-related cell adhesion molecule 1 (C?/Au: EACAM1) is a cell-surface signaling molecule involved in cell adhesion, proliferation, and immune response. It is also implicated in cancer angiogenesis, progression, and metastasis. This diverse set of effects likely arises as a result of the numerous homophilic and heterophilic interactions that CEACAM1 can have with itself and other molecules. Its N-terminal Ig variable (IgV) domain has been suggested to be a principal player in these interactions. Previous crystal structures of the β-sandwich-like IgV domain have been produced using Escherichia coli-expressed material, which lacks native glycosylation. These have led to distinctly different proposals for dimer interfaces, one involving interactions of ABED β-strands and the other involving GFCC'C″ β-strands, with the former burying one prominent glycosylation site. These structures raise questions as to which form may exist in solution and what the effect of glycosylation may have on this form. Here, we use NMR cross-correlation measurements to examine the effect of glycosylation on CEACAM1-IgV dimerization and use residual dipolar coupling (RDC) measurements to characterize the solution structure of the non-glycosylated form. Our findings demonstrate that even addition of a single N-linked GlcNAc at potential glycosylation sites inhibits dimer formation. Surprisingly, RDC data collected on E. coli expressed material in solution indicate that a dimer using the non-glycosylated GFCC'C″ interface is preferred even in the absence of glycosylation. The results open new questions about what other factors may facilitate dimerization of CEACAM1 in vivo, and what roles glycosylation may play in heterophylic interactions.

Analysis of Minimal Residual Disease in Follicular Lymphoma Patients in Gadolin, a Phase III Study of Obinutuzumab Plus Bendamustine Versus Bendamustine in Relapsed/Refractory Indolent Non-Hodgkin Lymphoma

BackgroundMinimal residual disease (MRD) response after first-line treatment of follicular lymphoma (FL) is likely to predict clinical course. The prognostic relevance of MRD in relapsed/refractory (r/r) FL remains unclear. We report MRD assessment with respect to clinical outcomes in r/r FL pts in GADOLIN (NCT01059630). GADOLIN is an open-label, multicenter, randomized phase III study of pts with r/r indolent NHL (refractory to rituximab) to investigate the efficacy and safety of obinutuzumab (G) plus bendamustine (B) followed by G maintenance vs B alone. A significant improvement in PFS in the G-B arm was reported (Sehn L, et al. ASCO 2015).MethodsMRD was analyzed by t(14;18) and/or Ig variable domain allele-specific RQ-PCR in pts with a clonal marker detectable at diagnosis in peripheral blood (PB) or bone marrow (BM) by consensus PCR. (Assessment of Ig rearrangements allows detection of a lymphoma marker in pts with a t(14;18) breakpoint not detected by generic PCR and avoids false positive signals, as a low level of the translocation can be detected in some healthy individuals.) Assays were designed with a sensitivity of 10-5 and accepted for MRD assessment when a sensitivity of ≤10-4 was reached. Results were evaluated according to ESG-MRD criteria. MRD status was analyzed at interim staging (C4, D1) and after end of induction (EOI), and defined as negative (-ve) if RQ-PCR and subsequent nested PCR produced a -ve result, i.e. achieving an MRD response. PFS was measured from the EOI date.Results321 of 396 randomized pts were diagnosed with FL. Baseline samples (PB and/or BM) were available for 285 of the 304 FL pts who had completed induction at the clinical cut-off date (1 Sep 2014; FL-ITT population). A clonal marker was detected in 183 (64%) of these pts; 128/183 (70%) had a RQ-PCR assay fulfilling the sensitivity criteria. EOI samples were available for 93 pts (biomarker-evaluable population) and 64 had a PB sample at mid-induction to assess MRD-response kinetics. The distribution of age, stage, and FLIPI in the biomarker-evaluable population was similar to the non-evaluable population of the B arm, while there was an enrichment of younger age, advanced-stage disease, and high risk FLIPI in the biomarker-evaluable population of the G-B arm.MRD response was analyzed in 93 pts at EOI and was significantly higher in pts receiving G-B induction, with 42/51 (82%) achieving MRD -ve status compared with 18/42 pts (43%) in the B arm (p<0.0001, Chi-Squared). At mid-induction, 30/39 (77%) pts in the G-B arm achieved early MRD -ve status vs 10/25 (40%) in the B arm (p=0.0029; Table). MRD response was associated with clinical CR; 2/33 (6%) MRD positive (+ve) pts vs 17/60 (28%) MRD -ve pts achieved a CR. Moreover, 39/63 pts with partial remission were MRD -ve.Pts in the G-B arm who were MRD -ve at EOI had an improved PFS at 24 mo post-EOI (74%; median PFS not reached) compared with the B arm (21%; median PFS, 7.6 mo). PFS for MRD non-responders was comparably poor in both treatment arms; all pts progressed before 24 mo with a post-EOI median PFS of 5.4 mo (G-B) and 3.0 mo (B; Figure).DiscussionOur results suggest that G significantly contributes to the depth of response to B vs B alone during induction treatment and support the notion that MRD status at EOI treatment is a sensitive marker of efficacy in the setting of r/r FL. MRD response identifies a prognostically favorable group of pts that appear to benefit from treatment with G-B at relapse. The improved PFS outcome suggests that these pts also benefit from G maintenance. Pts without an MRD response had a very poor prognosis, irrespective of treatment arm. Future analyses will assess MRD kinetics during maintenance and follow-up. [Display omitted] [Display omitted] DisclosuresOff Label Use: Obinutuzumab (GA101; Gazyva/Gazyvaro) is a CD20-directed cytolytic antibody and is indicated, in combination with chlorambucil, for the treatment of patients with previously untreated chronic lymphocytic leukemia. This abstract reports on bendamustine with or without obinutuzumab in patients with CD20+ R-ref, indolent non-Hodgkin lymphoma.. Belada:Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding. Danesi:Roche: Employment. Fingerle-Rowson:Roche: Employment, Equity Ownership. Gribben:Celgene: Consultancy, Honoraria; Gilead: Honoraria; Roche/Genentech: Honoraria; Pharmacyclics: Honoraria; Janssen: Honoraria. Harbron:Roche: Employment, Equity Ownership; AstraZeneca: Equity Ownership. Kahl:Roche/Genentech: Consultancy; Seattle Genetics: Consultancy; Millennium: Consultancy; Cell Therapeutics: Consultancy; Celgene: Consultancy; Infinity: Consultancy; Pharmacyclics: Consultancy; Juno: Consultancy. Mundt:Roche/Genentech: Employment. Sehn:Roche/Genentech: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Lundbeck: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria. Cheson:Teva: Research Funding; Gilead: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Roche/Genentech: Consultancy, Research Funding; Spectrum: Consultancy; Astellas: Consultancy; Ascenta: Research Funding; AstraZeneca: Consultancy; MedImmune: Research Funding.

P2-351: Exceptional catalytic hydrolysis of amyloid-β peptide by immunoglobulin light chain variable domain constructs

Immunoglobulins (Igs) that bind amyloid-β peptides (Aβ) are under consideration for passive immunotherapy of Alzheimer disease (AD). Catalytic Igs that hydrolyze Aβ are candidate second generation therapeutic agents by virtue of their ability to inactivate multiple Aβ molecules per Ig molecule. We have described Aβ hydrolysis via a nucleophilic mechanism by intact Igs and recombinant Ig fragments. However, homogeneous Igs with catalytic rate constants sufficient for therapy have not been generated until now. In this study, we identified single chain Ig variable domain constructs (IgVs) with catalytic activity sufficient for AD therapy from a human IgV library composed of single chain Fv clones (scFvs; these constructs mimic the VL-VH paired structure of physiological Igs) along with a few unusual IgVs generated by cloning errors. Aβ hydrolysis by the IgVs was monitored by radioassay and mass spectrometry. Mutations (4–6/IgV molecule) were introduced by error prone replication. Specific nucleophilic IgVs displayed on phages were selected by covalent binding to an electrophilic Aβ analog. Screening and phage selection procedures identified rare catalystic IgVs with exceptional hydrolytic activity directed at the His14-Gln15 bond of Aβ. Irrelevant proteins were not hydrolyzed. The activities were sufficient to ensure hydrolysis of 105 Aβ molecules/catalyst molecule over 10 days, corresponding to the approximate half-life of full-length IgG in circulation. The IgVs had unusual structures containing heterodimeric VL segments (IgVL2) or an unpaired, intact VL segment linked to a truncated VH peptide (IgVL). Repair of the aberrant VH segment in an IgVL clone yielded scFv constructs with substantially reduced Aβ hydrolyzing activity. Random mutagenesis of an IgVL2 construct followed by covalent phage selection permitted isolation of clones with ∼40-fold improved Aβ hydrolyzing activity. An electrophilic hapten reacted irreversibly with the IgVs, suggesting that the Aβ hydrolysis reaction occurs via a nucleophilic catalytic mechanism. Molecular modeling suggested the presence of serine protease-like catalytic sites in the VL domains. Unlike physiological Igs, the IgVL scaffold supports exceptional Aβ hydrolytic activity and represents a new molecular format suitable for development of catalytic AD immunotherapy.

The PD-1/PD-L1 complex resembles the antigen-binding Fv domains of antibodies and T cell receptors.

Signaling through the programmed death 1 (PD-1) inhibitory receptor upon binding its ligand, PD-L1, suppresses immune responses against autoantigens and tumors and plays an important role in the maintenance of peripheral immune tolerance. Release from PD-1 inhibitory signaling revives "exhausted" virus-specific T cells in chronic viral infections. Here we present the crystal structure of murine PD-1 in complex with human PD-L1. PD-1 and PD-L1 interact through the conserved front and side of their Ig variable (IgV) domains, as do the IgV domains of antibodies and T cell receptors. This places the loops at the ends of the IgV domains on the same side of the PD-1/PD-L1 complex, forming a surface that is similar to the antigen-binding surface of antibodies and T cell receptors. Mapping conserved residues allowed the identification of residues that are important in forming the PD-1/PD-L1 interface. Based on the structure, we show that some reported loss-of-binding mutations involve the PD-1/PD-L1 interaction but that others compromise protein folding. The PD-1/PD-L1 interaction described here may be blocked by antibodies or by designed small-molecule drugs to lower inhibitory signaling that results in a stronger immune response. The immune receptor-like loops offer a new surface for further study and potentially the design of molecules that would affect PD-1/PD-L1 complex formation and thereby modulate the immune response.

The crystal structure of myelin oligodendrocyte glycoprotein, a key autoantigen in multiple sclerosis.

Myelin oligodendrocyte glycoprotein (MOG) is a key CNS-specific autoantigen for primary demyelination in multiple sclerosis. Although the disease-inducing role of MOG has been established, its precise function in the CNS remains obscure. To gain new insights into the physiological and immunopathological role of MOG, we determined the 1.8-A crystal structure of the MOG extracellular domain (MOGED). MOGED adopts a classical Ig (Ig variable domain) fold that was observed to form an antiparallel head-to-tail dimer. A dimeric form of native MOG was observed, and MOGED was also shown to dimerize in solution, consistent with the view of MOG acting as a homophilic adhesion receptor. The MOG35-55 peptide, a major encephalitogenic determinant recognized by both T cells and demyelinating autoantibodies, is partly occluded within the dimer interface. The structure of this key autoantigen suggests a relationship between the dimeric form of MOG within the myelin sheath and a breakdown of immunological tolerance to MOG that is observed in multiple sclerosis.

Role of Native-State Topology in the Stabilization of Intracellular Antibodies

The role played by the geometric position of each amino acid in the folding process of the immunoglobulin (Ig) variable domain is identified and measured through molecular dynamics simulations of models based on the topology of its native state. This measure allows identifying the parts of the protein that, for geometrical reasons, when mutated, would result in relevant protein stability changes. Simulations were performed without considering the covalent disulfide bond present in most of the Ig domains. The results are in good agreement with site-directed mutagenesis experiments on the folding of intracellular antibodies in which the disulfide bond does not form. We also found agreement with data on amino acid conservation in the Ig variable domain sequences. This indicates a new way for a rational approach to the design of intracellular antibodies more resistant to the suppression of the disulfide bond that occurs in the cytoplasm.

CD47 Signals T Cell Death

Activation-induced death of T cells regulates immune responses and is considered to involve apoptosis induced by ligation of Fas and TNF receptors. The role of other receptors in signaling T cell death is less clear. In this study we demonstrate that activation of specific epitopes on the Ig variable domain of CD47 rapidly induces apoptosis of T cells. A new mAb, Ad22, to this site induces apoptosis of Jurkat cells and CD3epsilon-stimulated PBMC, as determined by morphological changes, phosphatidylserine exposure on the cell surface, uptake of propidium iodide, and true counts by flow cytometry. In contrast, apoptosis was not observed following culture with anti-CD47 mAbs 2D3 or B6H12 directed to a distant or closely adjacent region, respectively. CD47-mediated cell death was independent of CD3, CD4, CD45, or p56lck involvement as demonstrated by studies with variant Jurkat cell lines deficient in these signaling pathways. However, coligation of CD3epsilon and CD47 enhanced phosphatidylserine externalization on Jurkat cells with functional CD3. Furthermore, normal T cells required preactivation to respond with CD47-induced apoptosis. CD47-mediated cell death appeared to proceed independent of Fas or TNF receptor signaling and did not involve characteristic DNA fragmentation or requirement for IL-1beta-converting enzyme-like proteases or CPP32. Taken together, our data demonstrate that under appropriate conditions, CD47 activation results in very rapid T cell death, apparently mediated by a novel apoptotic pathway. Thus, CD47 may be critically involved in controlling the fate of activated T cells.

Integrin-associated protein immunoglobulin domain is necessary for efficient vitronectin bead binding.

Integrin-associated protein (IAP/CD47) is physically associated with the alpha v beta 3 vitronectin (Vn) receptor and a functionally and immunologically related integrin on neutrophils (PMN) and monocytes. Anti-IAP antibodies inhibit multiple phagocyte functions, including Arg-Gly-Asp (RGD)-initiated activation of phagocytosis, chemotaxis, and respiratory burst; PMN adhesion to entactin; and PMN transendothelial and transepithelial migration at a step subsequent to tight intercellular adhesion. Anti-IAP antibodies also inhibit binding of Vn-coated particles to many cells expressing alpha v beta 3. However, prior studies with anti-IAP did not directly address IAP function because they could not distinguish between IAP blockade and antibody-induced signaling effects on cells. To better determine the function of IAP, we have characterized and used an IAP-deficient human cell line. Despite expressing alpha v integrins, these cells do not bind Vn-coated particles unless transfected with IAP expression constructs. Increasing the level of alpha v beta 3 expression or increasing Vn density on the particle does not overcome the requirement for IAP. All known splice variants of IAP restore Vn particle binding equivalently. Indeed, the membrane-anchored IAP Ig variable domain suffices to mediate Vn particle binding in this system, while the multiply membrane-spanning and cytoplasmic domains are dispensable. In all cases, adhesion to a Vn-coated surface and fibronectin particle binding through alpha 5 beta 1 fibronectin receptors are independent of IAP expression. These data demonstrate that some alpha v integrin ligand-binding functions are IAP independent, whereas others require IAP, presumably through direct physical interaction between its Ig domain and the integrin.

Refinement and Analysis of the Structure of the First Two Domains of Human CD4

The structure of a fragment of human CD4 containing two immunoglobulin (Ig)-like domains has been determined by X-ray crystallography and refined at 2·2 Å resolution. The structure determination involved iterative building and simulated-annealing refinement, beginning with a partial model. Comparison of domain 1 with an Ig variable domain shows that CD4 has a long and prominent CDR2-like loop (the C′C″ corner) and shortened CC′ and FG loops (which mediate dimerization in IgV modules). Comparison of domain 2 with Ig modules and domain 1 shows that it can be described as a truncated Ig V domain, in which strands C″ and D are deleted. The intersheet disulfide in domain 2 is absent, and there is an altered packing of the two β-sheets together with a remodeling of the hydrophobic core. The interface between domains 1 and 2 is a lap joint with an extensive hydrophobic surface. The key features of domain 1 that contribute to the interface are found at corresponding positions in domain 2, leading us to propose that the contact between domains 2 and 3 will resemble the one between domains 1 and 2.

Molecular cloning of a novel member of the immunoglobulin gene superfamily homologous to the polymeric immunoglobulin receptor.

The CMRF35 monoclonal antibody recognizes a cell membrane antigen present on the surface of monocytes, neutrophils, a proportion of peripheral blood T and B lymphocytes and lymphocytic cell lines. Initial studies with CMRF35 suggested an unusual pattern of serological reactivity which did not correspond to any of the known leukocyte differentiation antigen clusters. We describe here the cloning and sequencing of a cDNA encoding the CMRF35 antigen by means of expression in COS cells and immunoselection with the CMRF35 monoclonal antibody. The cDNA encodes a novel integral membrane glycoprotein of 224 amino acids that represents a new member of the immunoglobulin (Ig) gene superfamily. The molecule comprises (a) a single extracellular Ig variable domain remarkably similar to the Fc receptor for polymeric IgA and IgM, (b) a membrane-proximal domain containing a high proportion of proline, serine and threonine residues that was predicted to be heavily O-glycosylated, (c) an unusual transmembrane anchor that contained a glutamic acid and a proline residue and (d) a short cytoplasmic tail. Transcripts encoding the CMRF35 protein were detected in early monocytic cell lines, in peripheral blood T cells and in some B lymphoblastoid cell lines, confirming the results of immunocytological staining. However, the level of CMRF35 expression on peripheral blood T cells was shown to decrease in response to mitogenic stimulation. The likelihood that the CMRF35 antigen shares a common evolutionary ancestor with the poly Ig Fc receptor is discussed.

Mutations in CD8 that affect interactions with HLA class I and monoclonal anti-CD8 antibodies.

The T cell co-receptor, CD8, binds to the alpha 3 domain of HLA class I (Salter, R.D., R.J. Benjamin, P.K. Wesley, S.E. Buxton, T.P.J. Garrett, C. Clayberger, A.M. Krensky, A.M. Norman, D.R. Littman, and P. Parham. 1990. Nature [Lond.]. 345:41; Connolly, J.M., T.A. Potter, E.M. Wormstall, and T.H. Hansen. 1988. J. Exp. Med. 168:325; and Potter, T.A., T.V. Rajan, R.F. Dick II, and J.A. Bluestone. 1989. Nature [Lond.]. 337:73). To identify regions of CD8 that are important for binding to HLA class I, we performed a mutational analysis of the CD8 molecule in the immunoglobulin (Ig)-like variable domain. Our mutational analysis was based on our finding that using a cell-cell adhesion assay murine CD8 (Lyt-2) did not bind to human class I. Since the interaction of human CD8 with HLA class I is species specific, we substituted nonconservative amino acids from mouse CD8 and analyzed the ability of the mutated CD8 molecules expressed in COS 7 cells to bind HLA class I-bearing B lymphoblastoid cells, UC. Mutants with the greatest effect on binding were located in a portion of the molecule homologous to the first and second hypervariable regions of an antibody combining site. In addition, a panel of 12 anti-CD8 monoclonal antibodies were used to stain the 10 CD8 mutants, and amino acids that affected antibody binding were localized on the crystal structure of the Bence-Jones homodimer, REI. Support for an Ig-like structure of CD8 can be found in the pattern of substitutions affecting antibody binding. This work supports the similar tertiary structure of the CD8 alpha-terminal domain and an Ig variable domain.

Recent studies of the interaction of rabbit dimeric IgA with its polymeric immunoglobulin receptor

Rabbit secretory components (SC) constitute a highly heteregeneous population of glycoprotein molecules that are present in secretions as free or bound forms to polymeric immunoglobulins (Ig). Two SC families are known, one of high molecular weight (⋍ 80 Kd) composed of five (perhaps six) domains related to Ig variable domains, and one of low molecular weight (⋍ 55 Kd) An account of our most recent experimental data is reviewed in this article. We have shown: 1. 1) that both the high and low Mr SC families possess the same relative avidity for binding to dimeric IgA of the g-sublanguage; 2. 2) that the first NH 2-terminal domain of SC derived from the high and low Mr polypeptides is necessary and sufficient for efficient non-covalent binding to dimeric IgA of the g-sublanguage; 3. 3) that the low Mr SC polypeptide derives from the high Mr SC by the internal deletion of the entire second and third domains, suggesting that these domains are not involved in the binding reaction with polymeric Ig; 4. 4) that the heterogeneity of rabbit secretory components is, in large part, due to the expression of several polymorphic forms (allotypes) susceptible to be recognized by specific alloantisera; the biochemical characterization of the three known SC allotypes (t61, t62 and t63) reveals that t62 and t63 are structurally very similar to each other and markedly divergent from the t61 homologue; 5. 5) that by using non-cross-reactive alloantisera, the major immunodominant allotopes are confined within the COOH-terminal domains 3, 4 and 5 of SC; 6. 6) that the location of the residues involved in the attachment of the carbohydrate unit within domain 1 varies according to the allotype: t61 is N-linked glycosylated at position 70, whereas about 75 % of t62 molecules are devoid of sugars; the remaining 25 % of t62 molecules are glycosylated at residue position 90; these oligosaccharide chain units are linked to asparagine residues in the acceptor site consensus sequence, Asn-X-Thr/Ser; 7. 7) that the presence of the carbohydrate unit in domain 1 is not required for efficient binding of this domain to polymeric Ig: indeed, after enzymatic deglycosylation, domain 1 exhibits a relative binding avidity which is indistinguishable from that of the native glycosylated domain 1.