Binding of human immunodeficiency virus type 1 gp120 to CXCR4 induces mitochondrial transmembrane depolarization and cytochrome c-mediated apoptosis independently of Fas signaling.

Apoptosis of CD4(+) T lymphocytes, induced by contact between human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp120) and its receptors, could contribute to the cell depletion observed in HIV-infected individuals. CXCR4 appears to play an important role in gp120-induced cell death, but the mechanisms involved in this apoptotic process remain poorly understood. To get insight into the signal transduction pathways connecting CXCR4 to apoptosis following gp120 binding, we used different cell lines expressing wild-type CXCR4 and a truncated form of CD4 that binds gp120 but lacks the ability to transduce signals. The present study demonstrates that (i) the interaction of cell-associated gp120 with CXCR4-expressing target cells triggers a rapid dissipation of the mitochondrial transmembrane potential resulting in the cytosolic release of cytochrome c from the mitochondria to cytosol, concurrent with activation of caspase-9 and -3; (ii) this apoptotic process is independent of Fas signaling; and (iii) cooperation with a CD4 signal is not required. In addition, following coculture with cells expressing gp120, a Fas-independent apoptosis involving mitochondria and caspase activation is also observed in primary umbilical cord blood CD4(+) T lymphocytes expressing high levels of CXCR4. Thus, this gp120-mediated apoptotic pathway may contribute to CD4(+) T-cell depletion in AIDS.

Caspase-dependent apoptosis of cells expressing the chemokine receptor CXCR4 is induced by cell membrane-associated human immunodeficiency virus type 1 envelope glycoprotein (gp120).

Human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins interact with CD4 and chemokine receptors on T cells to deliver signals that trigger either activation, anergy, or apoptosis. However, the molecular mechanisms driving these responses remain poorly understood. In this study we demonstrate that apoptosis is induced upon HIV-1 envelope binding to the chemokine receptor CXCR4. Cells expressing a mutant form of CXCR4 with a C-terminal deletion were also sensitive to HIV-1 envelope-mediated apoptosis, indicating that the cytoplasmic tail of CXCR4 is not required to induce the apoptotic pathway. The specificity of this process was analyzed using several inhibitors of gp120-CD4-CXCR4 interaction. Monoclonal antibodies directed against the gp120-binding site on CD4 (ST4) and against CXCR4 (MAB173) prevented the apoptotic signal in a dose-dependent manner. The cell death program was also inhibited by SDF-1alpha, the natural ligand of CXCR4, and by suramin, a G protein inhibitor that binds with a high affinity to the V3 loop of HIV-1 gp120 envelope protein. These results highlight the role played by gp120-binding on CXCR4 to trigger programmed cell death. Next, we investigated the intracellular signal involved in gp120-induced apoptosis. This cell death program was insensitive to pertussis toxin and did not involve activation of the stress- and apoptosis-related MAP kinases p38(MAPK) and SAPK/JNK but was inhibited by a broad spectrum caspase inhibitor (z-VAD.fmk) and a relatively selective inhibitor of caspase 3 (z-DEVD.fmk). Altogether, our results demonstrate that HIV induces a caspase-dependent apoptotic signaling pathway through CXCR4.

Analysis of the V genes coding for a monospecific human antibody to myosin and functional expression of single chain Fv fragments.

A monospecific human IgM monoclonal antibody (mAb), reactive with myosin from human heart, has been obtained by EBV transformation. This mAb may have a diagnostic potential in the imaging of myocardial necrosis. However, owing to the fact that the molecular mass of an IgM is 900 kDa, a poor diffusion and a slow penetration inside necrotic myocytes could reduce its capacity for scintigraphic detection. In order to alleviate these problems, we constructed the scFv by cloning the VH and VL domains into the pHOG21 vector. Analysis of the V genes proved an unmutated configuration showing that the immortalized B cell issued from the primary IgM repertoire. The expression product in Escherichia coli was a 35 kDa scFv fragment with the antigen-binding specificity of the parental mAb.

Human single-chain Fv fragments from a combinatorial library using the loxP-Cre recombination system.

A human scFv display library has been constructed from peripheral blood lymphocytes of a patient suffering from Hashimoto's thyroiditis. Upon induction of Cre recombinase, the amplified VH and VL genes were recombined via two loxP sites inserted in amplification primers to construct in vitro scFv genes. Either soluble scFvs or scFvs displayed on phage were screened for binding to human thyroglobulin after two pannings with this antigen. Three scFvs were obtained which showed very similar nucleotidic sequences. The VH genes expressed display 96.4% nucleotide sequence homology with the germline VH251 gene, one of the two functional members of the small VH5 family and are mutated in sites already described as "selectively neutral" mutations and the VL genes are close to the germline DPL8 gene. These scFvs bind not only to human thyroglobulin but also to other self and exogenous antigens.

Synthetic peptides derived from the variable regions of an anti-CD4 monoclonal antibody bind to CD4 and inhibit HIV-1 promoter activation in virus-infected cells.

The monoclonal antibody (mAb) ST40, specific for the immunoglobulin complementarity-determining region (CDR) 3-like loop in domain 1 of the CD4 molecule, inhibits human immunodeficiency virus type 1 (HIV-1) promoter activity and viral transcription in HIV-infected cells. To design synthetic peptides from the ST40 paratope that could mimic these biological properties, a set of 220 overlapping 12-mer peptides frameshifted by one residue, corresponding to the deduced ST40 amino acid sequence, was synthesized by the Spot method and tested for binding to recombinant soluble CD4 antigen. Several peptides that included in their sequences amino acids from the CDRs of the antibody and framework residues flanking the CDRs were found to bind soluble CD4. Eleven paratope-derived peptides (termed CM1-CM11) were synthesized in a cyclic and soluble form. All the synthetic peptides showed CD4 binding capacity with affinities ranging from 1.6 to 86.4 nM. Moreover, peptides CM2, CM6, CM7, CM9, and CM11 were able to bind a cyclic peptide corresponding to the CDR3-like loop in domain 1 of CD4 (amino acids 81-92 of CD4). Peptide CM9 from the light chain variable region of mAb ST40 and, to a lesser extent, peptides CM2 and CM11 were able to inhibit HIV-1 promoter long terminal repeat-driven beta-galactosidase gene expression in the HeLa P4 HIV-1 long terminal repeat beta-galactosidase indicator cell line infected with HIV-1. The binding of mAb ST40 to CD4 was also efficiently displaced by peptides CM2, CM9, and CM11. Our results indicate that the information gained from a systematic exploration of the antigen binding capacity of synthetic peptides from immunoglobulin variable sequences can lead to the identification of bioactive paratope-derived peptides of potential pharmacological interest.

Analysis of VH and VL genes of a monospecific human anti-myosin antibody produced by a B cell from the primary repertoire.

Epstein-Barr virus (EBV) transformation of B lymphocytes from a Glanzmann's thrombasthenia patient with a serum antibody to the integrin alpha IIb beta 3, led to the immortalization of a B cell secreting a monospecific IgM monochonal antibody (MAb), B7, reactive with platelet myosin. Analysis of B7 V genes revealed minimally mutated sequences: the immortalized B cell issued from the primary repertoire, with no evidence of an in vivo selection by myosin. The V genes were here compared with sequences of human MAbs available on databases to more clearly understand the monospecificity of the B7 MAb. B7 V genes were closely identical to rearranged V genes in clones with self-specificities, often secreting polyreactive antibodies. In contrast, B7 is an unmutated monoreactive human MAb able to recognize myosin with a high avidity. Comparison of the CDR3H sequence with that of MAbs in databases supports a central role for the CDR3H subdomain in determining monospecificity. Our results suggest the existence of a monospecific autoreactive B cell compartment, besides the well-known polyspecific one, susceptible to be the template of pathogenic autoreactivity, characterized by antibodies of high affinity and specificity.

Anti-digoxin scFv fragments expressed in bacteria and in insect cells have different antigen binding properties.

A gene encoding a single-chain antibody fragment directed against digoxin (named 1C10 scFv) was cloned in two expression systems. For this purpose, a new baculovirus transfer cassette fully compatible with the procaryotic pHEN vector was constructed. Baculovirus production led to higher yield than did Escherichia coli expression. The procaryotic fragment showed variations in the fine specificity profile but an affinity constant nearly identical to that of the 1C10 Fab, whereas the eucaryotic scFv fragment had a lower affinity with a specificity profile identical to original mAb. The half-lives of the digoxin:scFv complexes and the global specificity are compatible with therapeutic use of this antibody fragment.

Affinity of recombinant antibody and antibody fragment binding to human thyroglobulin: potential applications in gene therapy.

Recombinant antibodies and antibody fragments are currently being produced. They can be used in vitro for the structural study of antigen-antibody interactions for instance, but their in vivo production may have applications for gene therapy of certain cancers and severe viral diseases and in developing new animal models of autoimmune disease. We report here these two types of applications using a recombinant antihuman thyroglobulin (hTg) antibody.

In-cell assembly of scFv from human thyroid-infiltrating B cells.

The construction of a large library of single-chain Fv (scFv) antibody fragments involves a random assortment of heavy and light chains. Although useful for the production of recombinant antibodies, this method is not adapted to the study of the autoantibody repertoire formed in vivo during autoimmune diseases. To attain this objective, we describe the use of the in-cell PCR together with Cre-recombination applied, to our knowledge, for the first time to human B cells to obtain in situ pairing of the variable (V) region genes of the immunoglobulin heavy (H) and light (L) chains. Our method is based on amplification and recombination of the VH and VL genes within CD19+ B cells isolated from human thyroid tissue. Nested primers were designed to amplify the known major human VH and VL gene families. After reverse transcription PCR and three rounds of PCR including recombination between VH and VL using the Cre-loxP system, we obtained a unique 800-bp band corresponding in size to scFv fragments. We provide evidence that recombination between VH and VL genes occurred inside the same cell. This in-cell amplification and association procedure is a potentially useful tool for the study of autoantibody gene families and the VH/VL pairing that occurs during the autoimmune process.

In vitro and in vivo secretion of cloned antibodies by genetically modified myogenic cells.

In vivo production of recombinant antibodies by engineered cells may have applications for gene therapy of certain cancers and of certain severe viral diseases. It would also permit the development of new animal models of autoimmune diseases and new approaches for in vivo ablation of specific cell types for fundamental purposes. Using gene transfer of an anti-human thyroglobulin monoclonal antibody, we show here that several cell types permitting autologous grafting of genetically engineered cells are efficiently able to secrete antibodies in vitro. Those cells include skin fibroblasts, hepatocytes, and myogenic cells. We also show that the secreted antibodies display an affinity for the antigen close to that of the parental antibody, with, however, slight differences varying according to the cell type. This indicates that the foldings of antigen combining sites of antibodies produced in B cell- and non-B cell contexts are very similar. Finally, we report that, when implanted in the forelimb of a mouse, genetically modified myogenic cells are able to secrete antibodies for at least 4 months. Taken together, our observations point to the notion that genetic modification of patient cells may be used for long-term antibody-based gene therapies.

Analysis of the individual contributions of immunoglobulin heavy and light chains to the binding of antigen using cell transfection and plasmon resonance analysis.

We have cloned the Tg10 murine monoclonal antibody, which is specific for a human thyroglobulin (hTg) epitope targeted by autoantibodies in several thyroid pathologies. Transfection of COS-7 cells with plasmids expressing Tg10H and -kappa chains combined with surface plasmon resonance analysis (BIAcore) of culture supernatants showed that the entire cloned Tg10 antibody displays an affinity comparable to that of the parental antibody. This approach also permitted determination of the probable role of each chain to the recognition of the cognate epitope due to the ability of COS-7 cells to secrete independently each of the two constituting immunoglobulin chains. Tg10 heavy chain recognizes hTg in the absence of the light chain, but with a ten-fold lower affinity mainly due to an increase in kappaoff. In contrast, the light chain is unable to bind hTg on its own. This suggests that the latter is probably involved in stabilization rather than in initiating the formation of the antibody/antigen complex and that the specificity of Tg10 is mostly, if not exclusively, carried by the heavy chain. The potential applications of combined cell transfection and surface plasmon resonance to our understanding of antigen/antibody interactions are discussed.

Cloning and expression of a single-chain antibody fragment specific for a monomorphic determinant of class I molecules of the human major histocompatibility complex.

B9.12.1 is a monoclonal antibody specific for a monomorphic determinant of human MHC class I molecules. It is currently used for cell typing and is useful for targeting infection of human cells by murine ecotropic retroviruses. We have cloned and expressed it in the form of a single-chain variable fragment (ScFv) that recognizes the same epitope as the parental antibody. Through genetic engineering, this ScFv may be used for developing new cell-typing probes and new retroviral targeting approaches.

Expression in Escherichia coli of soluble and M13 phage-displayed forms of a single-chain antibody fragment specific for digoxin: assessment in a novel drug immunoassay.

A high affinity anti-digoxin single-chain Fv antibody fragment (scFv) was cloned from the mouse 2C2 hybridoma cell line and was functionally expressed both in the Escherichia coli periplasm as a soluble molecule and at the surface of the filamentous M13 bacteriophage as a fusion protein with the gene III minor coat protein. The 2C2 scFv sequence significantly differs from that of all the other anti-digoxin antibodies previously described. The 2C2 scFv shares with its parental monoclonal antibody a high specificity for digoxin, a cross-reactivity with active digoxin metabolites, but none with inactive metabolites. M13 phages displaying the 2C2 scFv at their surface have a high apparent affinity constant for digoxin (6.6 x 10(8) M-1) and were directly used to set up a novel type of immunoenzymatic assay for monitoring digoxin in sera of patients treated for either congestive heart failure or cardiac arrythmias. We thus report for the first time that phages displaying scFv may constitute a large source of important new reagents in the field of immunodiagnosis.