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Biomedical subjects

D Plaksin

Publications and source records attributed to D Plaksin.

18 recordsLinked to original sources
PubMed2000

Antibodies directed against the MHC-I molecule H-2Dd complexed with an antigenic peptide: similarities to a T cell receptor with the same specificity.

alphabeta TCRs, which use an Ab-like structure to form a combining site, recognize molecular complexes consisting of peptides bound to MHC class I (MHC-I) or class II (MHC-II) molecules. To explore the similarities and differences between Ab and T cell recognition of similar structures, we have isolated two mAbs, KP14 and KP15, that specifically bind H-2D(d) complexed with an HIV envelope gp160-derived peptide, P18-I10. These Abs are MHC and peptide specific. Fine specificity of mAb binding was analyzed using a panel of synthetic peptides, revealing similarities between the mAb and a cloned TCR with the same specificity. These two mAbs used the same V(H) and J(H) gene segments, but different D, Vkappa, and Jkappa genes. Administered in vivo, mAb KP15 blocked the induction of CTL specific for recombinant vaccinia virus-encoded gp160, indicating its ability to bind endogenously generated MHC/peptide complexes. Analysis of the fine specificity of these mAbs in the context of their encoded amino acid sequences and the known three-dimensional structure of the H-2D(d)/P18-I10 complex suggests that they bind in an orientation similar to that of the TCR. Thus, the plasticity of the B cell receptor repertoire and the structural similarities among BCR and TCR allow Abs to effectively mimic alphabeta TCRs. Such mAbs may be useful in the therapeutic modulation of immune responses against infectious agents or harmful self Ags as well as in tracing steps in Ag processing.

Amino Acid Sequence↗
PubMed1999

NK and CTL recognition of a single chain H-2Dd molecule: distinct sites of H-2Dd interact with NK and TCR.

We generated transgenic mice expressing a single-chain beta2-microglobulin (beta2m)-H-2Dd. The cell-surface beta2m-H-2Dd molecule was expressed on a beta2m-deficient background and reacted with appropriate mAbs. It was of the expected m.w. and directed the normal development of CD8+ T cells in the thymus of a broad TCR repertoire. It also presented both exogenously provided and endogenous peptide Ags to effector CD8+ T cells. In tests of NK cell education and function, it failed to reveal any interaction with NK cells, suggesting that the site of the interaction of NK receptors with H-2Dd was disrupted. Thus, the sites of TCR and NK receptor interaction with H-2Dd are distinct, an observation consistent with independent modes of TCR and NK receptor evolution and function.

Animals↗
PubMed1999

An antibody single-domain phage display library of a native heavy chain variable region: isolation of functional single-domain VH molecules with a unique interface.

To develop very small antibody-derived recognition units for experimental, medical, and drug design purposes, a heavy chain variable region (VH) single-domain phage-display library was designed and constructed. The scaffold that was used for library construction was a native sequence of a monoclonal antibody with a unique VH/VL interface. There was no need to modify any residues in the VL interface to avoid non-specific binding of VH domain. The library repertoire, consisting of 4x10(8)independent clones, was generated by the randomization of nine amino acid residues in complementary determining region 3. The library was screened by binding to protein antigens, and individual clones were isolated. The VH genes encoding for specific binding clones were rescued and large amounts of soluble and stable single-domain VH protein were made from insoluble inclusion bodies by in vitro refolding and purification. Biochemical and biophysical characterization of the VH protein revealed a highly specific, correctly folded, and stable monomeric molecule. Binding studies demonstrated an affinity of 20 nM. The properties of these molecules make them attractive for clinical, industrial, and research applications, as well as a step toward improvement in the design of small molecules that are based on the hypervariable loops of antibodies.

Amino Acid Sequence↗
PubMed1999

The X-ray crystal structure of a Valpha2.6Jalpha38 mouse T cell receptor domain at 2.5 A resolution: alternate modes of dimerization and crystal packing.

We describe here the structure of a murine T cell receptor (TCR) Valpha2.6Jalpha38 (TCRAV2S6J38) domain, derived from a T cell hybridoma with specificity for the H-2Ddmajor histocompatibility complex class I molecule bound to a decamer peptide, P18-I10, from the HIV envelope glycoprotein gp120, determined by X-ray crystallography at 2.5 A resolution. Unlike other TCR Valpha domains that have been studied in isolation, this one does not dimerize in solution at concentrations below 1 mM, and the crystal fails to show dimer contacts that are likely to be physiological. In comparison to other Valpha domains, this Valpha2.6 shows great similarity in the packing of its core residues, and exhibits the same immunoglobulin-like fold characteristic of other TCR Valpha domains. There is good electron density in all three complementarity-determining regions (CDRs), where the differences between this Valpha domain and others are most pronounced, in particular in CDR3. Examination of crystal contacts reveals an association of Valpha domains distinct from those previously seen. Comparison with other Valpha domain structures reveals variability in all loop regions, as well as in the first beta strand where placement and configuration of a proline residue at position 6, 7, 8, or 9 affects the backbone structure. The great variation in CDR3 conformations among TCR structures is consistent with an evolving view that CDR3 of TCR plays a plastic role in the interaction of the TCR with the MHC/peptide complex as well as with CDR3 of the paired TCR chain.

Amino Acid Sequence↗
PubMed1997

Rigidification of the alpha2 helix of an MHC class I molecule by a valine to proline mutation in position 165 does not prevent peptide-specific antigen presentation.

Although classical MHC class I glycoproteins bind peptide Ags for display at the cell surface, some MHC class I-related molecules such as the neonatal Fc receptor (FcRn) execute their function without binding peptide ligands. The three-dimensional structure of the FcRn suggested that a substitution of the conserved valine at position 165 of the alpha2 helix by proline contributed to a kink in the position of this helix relative to the alpha1 helix, and resulted in closing of the potential peptide-binding cleft. To test the contribution of proline 165 to the occlusion of the cleft and the binding of potential antigenic peptides, we introduced this mutation into the classical murine MHC class I molecule, H-2Dd, and characterized the ability of such a mutant to present peptide Ags to either a peptide-specific, H-2Dd-restricted T cell hybridoma (B4.2.3), or an allospecific, peptide-dependent, T cell hybridoma (3DT52.5.8). We show that the V165P mutation, expressed at the cell surface either in H-2Dd or in a single chain membrane version of H-2Dd, fails to eliminate recognition of the peptide/MHC complexes by two different T cells. Evaluation of a panel of synthetic substituted peptides suggests that subtle differences in the fine specificity of presentation can be discerned. Thus, the proline substitution at position 165 of FcRn and some other class I-like molecules is not the sole cause of the lack of peptide presentation.

Animals↗
PubMed1997

A three-domain T cell receptor is biologically active and specifically stains cell surface MHC/peptide complexes.

We have expressed in bacteria a single-chain T cell receptor (scTCR) with specificity for an HIV gp120-derived peptide bound to the murine MHC-I molecule, H-2Dd. This scTCR consists of V alpha covalently linked to the VbetaCbeta domains that was solubilized, refolded, and purified in high yield. Specific binding of the scTCR to MHC/peptide complexes was demonstrated by surface plasmon resonance, with a Kd of 2 to 8 x 10(-6) M. This scTCR specifically inhibited T cell activation, and stained cell surface MHC/peptide complexes as measured by cytofluorimetry. The preservation of binding specificity by such a three-domain scTCR suggests that this structure is sufficient for specific MHC/peptide recognition and that this strategy will be of general use as applied to other TCR.

Amino Acid Sequence↗
PubMed1996

A T cell receptor V alpha domain expressed in bacteria: does it dimerize in solution?

To evaluate the potential for dimerization through a particular T cell receptor (TCR) domain, we have cloned the cDNA encoding a TCR V alpha from a hybridoma with specificity for the human immunodeficiency virus (HIV) envelope glycoprotein 120-derived peptide P18-110 (RGPGRAFVTI) bound to the murine major histocompatibility complex (MHC) class I molecule, H-2Dd. This cDNA was then expressed in a bacterial vector, and protein, as inclusion bodies, was solubilized, refolded, and purified to homogeneity. Yield of the refolded material was from 10 to 50 mg per liter of bacterial culture, the protein was soluble at concentrations as high as 25 mg/ml, and it retained a high level of reactivity with an anti-V alpha 2 monoclonal antibody. This domain was monomeric both by size exclusion gel chromatography and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Circular dichroism spectra indicated that the folded V alpha domain had secondary structure similar to that of single immunoglobulin or TCR domains, consisting largely of beta sheet. Conditions for crystallization were established, and at least two crystal geometries were observed: hexagonal bipyramids that failed to diffract beyond approximately 6 A, and orthorhombic crystals that diffracted to 2.5 A. The dimerization of the V alpha domain was investigated further by solution nuclear magnetic resonance spectroscopy, which indicated that dimeric and monomeric forms of the protein were about equally populated at a concentration of 1 mM. Thus, models of TCR-mediated T cell activation that invoke TCR dimerization must consider that some V alpha domains have little tendency to form homodimers or multimers.

Amino Acid Sequence↗
PubMed1996

Highly sensitive immunoassays for detection of barley stripe mosaic virus and beet necrotic yellow vein virus.

Enzyme immunoassays based on the use of monoclonal antibodies (MAbs) were developed for the detection of the barley stripe mosaic virus (BSMV) and the beet necrotic yellow vein virus (BNYVV). Assays employing conjugates of MAbs to horseradish peroxidase (HRP) were compared to systems with biotinylated MAbs and streptavidin conjugated either to monomeric HRP or to HRP homopolymers with different polymerisation degrees including those of 20, 40 and 80. In the ELISA with streptavidin-polymeric HRP conjugates the assay detection limit was about 12-25 times as good as in the monoclonal double antibody sandwich ELISA. The system for BSMV detection with polymeric HRP was sufficiently sensitive to detect a single infected seed among more than 10(4) healthy ones. The assay detection limit was 1 ng/ml. The immunoenzyme system for BNYVV allowed virus detection in 1:12000 diluted extracts from BNYVV-infected leaves.

Animals↗
PubMed1996

Studying interactions involving the T-cell antigen receptor by surface plasmon resonance.

T-lymphocyte activation is initiated by the interaction of the alpha beta TCR with a complex consisting of a class I or class II MHC-encoded molecule and an antigenic peptide, displayed on the surface of an antigen-presenting cell. Real-time binding measurements using surface plasmon resonance have revealed kinetic and equilibrium parameters for the interactions between purified MHC molecules and peptides, between TCR and MHC-peptide complexes, and between TRC and superantigens. The MHC-peptide interaction is characterized by its high affinity and long half-life, the TCR-MHC/peptide interaction by its low affinity and short half-life, and the TCR-superantigen interaction by its low-to-moderate affinity, which is dependent on the particular superantigen involved. The consistent finding is that both MHC-peptide complexes and superantigens interact with TCR with a low affinity attributable to rapid dissociation. That an MHC-peptide complex that encounters a single TCR only briefly can still deliver the necessary activation signals offers a mechanistic conundrum for which several solutions have been proposed.

Animals↗
PubMed1994

Effective anti-metastatic melanoma vaccination with tumor cells transfected with MHC genes and/or infected with Newcastle disease virus (NDV).

The therapeutic efficacy of active immunization with B16-F10.9 melanoma cells transfected with syngeneic major histocompatibility complex (MHC) class-I genes, modified by infection with Newcastle Disease virus (NDV) or modified by both treatments, was compared. B16-F10.9 tumor-bearing mice were treated at various stages of tumor growth and metastasis with irradiated, modified tumor-cell vaccines. Irradiated tumor cells and H-2Db transfectants did not stimulate anti-tumor immunity while H-2Kb transfectants and NDV-modified F10.9 cells showing low and high expression of MHC class-I genes efficiently prevented metastasis of small established tumors. NDV-modified parental-cell vaccines functioned optimally and improved overall survival by about 60%, also at early stages of metastasis establishment. A synergistic effect of H-2Kb expression and virus modification on rejection of micrometastases was observed in mice bearing advanced tumors. Postoperative vaccination of mice carrying multiple metastases with NDV-modified vaccines caused significant, but incomplete, reduction of metastatic tumor load. The therapeutic effect of NDV-modified tumor vaccines was dependent on multiple immune mechanisms. Depletion of CD8, CD4 or NK cells by in vivo treatment with monoclonal antibodies reversed the immunotherapeutic effects of the vaccine. Thus, tumor xenogenization and gene modification may act synergistically to vaccinate against advanced tumors, while single modalities can effectively vaccinate against metastasis at early stages of tumor growth.

Animals↗
PubMed1994

c-fos and c-jun overexpression in malignant cells reduces their tumorigenic and metastatic potential, and affects their MHC class I gene expression.

Reduced co-expression of the c-fos and c-jun protooncogenes has been correlated with the down regulation of H-2K class I major histocompatibility antigens in high-metastatic cell lines from the Lewis lung carcinoma, B16 melanoma and the K1735 melanoma. Transfection of c-jun and c-fos genes into the high metastatic clones D122 (3LL) and F10.9 (B16 melanoma) resulted in activation of H-2 class I gene expression. D122 transfectants expressing high levels of c-jun and c-fos and F10.9 transfectants expressing high levels of c-fos exhibited markedly reduced tumorigenicity and were of low metastatic potential. In contrast, transfection of junB into the low metastatic, high H-2Kb, Db expressor clone A9 (3LL), reduced MHC class I gene expression, and converted the parental low, into high-metastatic cells. The data demonstrate the involvement of genes from the fos and jun family in regulation of MHC class I expression and consequently in regulation of immunogenicity and metastatic competence of tumor cells.

Animals↗
PubMed1993

KBF1 (p50 NF-kappa B homodimer) acts as a repressor of H-2Kb gene expression in metastatic tumor cells.

Downregulation of major histocompatibility complex class I expression is causally related to high malignancy and low immunogenicity of certain murine tumors. In this study, we have analyzed the roles of the nuclear factors KBF1/p50 and p65 in regulation of class I expression in high and low metastatic tumor cells. Low class I-expressing cells show at higher levels of KBF1/p50 and NF-kappa B (p50/p65) binding activity than high class I-expressing cells. However, an excess of KBF1 over NF-kappa B is observed in low expressing cells, while an excess of NF-kappa B over KBF1 is observed in high expressing cells. Stable transfection of a p65 expression vector into low class I-expressing cells activated H-2 transcription and cell surface expression, while stable transfection of p50 expression vector into high expressing cells suppressed H-2Kb transcription and cell surface expression. Our studies suggest that KBF1 has the potential of downregulating class I gene expression, whereas dimers containing the p65 subunit are activators of class I gene expression.

Animals↗
PubMed1992

H-2Db gene transfer into highly metastatic D122 cells results in tumor rejection in allogeneic recipients, but does not affect metastasis in syngeneic recipients. Implications for mechanisms of allorejection.

Highly metastatic, weakly immunogenic Lewis lung carcinoma clones express very low levels of H-2Kb and moderate levels of H-2Db class-I major histocompatibility complex antigens. These cells metastasize spontaneously in mice with C57BL/6 genetic background possessing the H-2Db locus, and grow as local tumors across allogeneic barriers. Transfection of the H-2Db genes into the highly metastatic clone D122 did not alter the growth or metastatic capacity of these cells in syngeneic mice. However, these cells were rejected in allogeneic mice. Transfection of the H-2Kd or H-2Kk genes into D122 elicited a CTL population that cross-reacted with cells bearing native H-2Db antigens. These data suggest that overlapping allo-CTL populations are induced by a native alloantigen and by alloantigen peptides presented through self class-I molecules.

Animals↗
PubMed1989

Abolishment of metastasis formation by murine tumor cells transfected with "foreign" H-2K genes.

High metastatic, low immunogenic Lewis lung carcinoma clones express low levels of H-2Kb major histocompatibility complex antigens. These cells metastasize spontaneously in mice with C57BL/6 genetic background possessing the H-2Db locus. Transfection of different H-2K genes abrogates metastasis in H-2K, H-2D compatible mice and in C57BL/6 recipients. The transfected cells are potent inducers of H-2K-restricted and alloreactive cytotoxic lymphocytes that kill H-2K-positive cells and cross-react with parental nontransfected cells.

Animals↗
PubMed1988

The reversal of the metastatic phenotype by gene transfer.

When studying the function of MHC-restricted immune responses in controlling metastatic growth we discovered that highly metastatic clones of mouse tumours express the H-2D but lack expression of the H-2K gene of the MHC system. The de novo expression of the H-2K antigen, after H-2K gene transfection, resulted in the reversal of a metastatic to a non-metastatic phenotype. This reversal was causally related to the acquisition of H-2K-restricted immunogenic properties. Immunization with H-2K-transfected cells, after surgical removal of the local tumour, abolished or significantly reduced the growth of metastases. We subsequently observed that H-2K expression is correlated with expression of the c-fos oncogenes. Transfection of H-2K-negative cells with v-fos or c-fos genes resulted in the expression of H-2K. Our studies suggest that one of the main functions of the c-fos proto-oncogenes is control of the expression of the MHC genes. Searching for additional molecular properties which characterize the metastatic phenotype, we observed that the metastatic clones of each of our lung-metastasizing tumours expresses an fms-related oncogene. This was correlated with a membrane-bound tyrosine kinase, which has the properties of growth factor receptors. We examine the possibility that our fms-like gene codes for this protein kinase, which represents a receptor for a local growth factor that controls metastatic growth in the lung.

Animals↗
PubMed1988

Reversal of the metastatic phenotype in Lewis lung carcinoma cells after transfection with syngeneic H-2Kb gene.

High metastatic clones of the murine 3LL carcinoma express greatly reduced levels of H-2Kb major histocompatibility complex class I antigens, while low metastatic clones of the same tumor express high levels of H-2Kb. Induced expression of this antigen after transfection with the H-2Kb gene resulted in conversion of a metastatic to a non- or low-metastatic phenotype. Unlike the parental cells, transfected cells are potent inducers of H-2Kb-restricted syngeneic cytotoxic lymphocytes that kill the Kb-positive clones and cross-react with parental nontransfected cells. Preimmunization of mice with Kb-positive transfectants conferred protection against metastatic spread of malignant cells. Moreover, immunotherapy of metastasis was achieved by immunization with the H-2Kb-transfected cells of animals already carrying a growing local tumor of the parental cells.

Animals↗
PubMed1987

Expression of major histocompatibility class I genes in differentiating leukemic cells is temporally related to activation of c-fos proto-oncogene.

The relationship between the expression of the c-fos proto-oncogene and the expression of the class I major histocompatibility (MHC) antigens during the early stages of induced differentiation in three different leukemic cell lines was examined. In the U937 histiocytic lymphoma line TPA induced an increase in mRNA and cell surface MHC expression which followed induction of c-fos. In contrast, in the murine erythro-leukemia cell line, DMSO induced declining constitutive c-fos levels that were accompanied by declining mRNA and cell surface MHC expression. In the pluripotent HL60 promyelocytic line induction of macrophage differentiation with TPA led to c-fos induction and rising MHC levels, whereas induction of granulocyte differentiation with DMSO did not induce c-fos expression and was followed by declining MHC levels. Taken together, the results suggest that the c-fos proto-oncogene might be involved in the control of class I MHC antigen expression during differentiation.

Cell Differentiation↗