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Biomedical subjects

A Traunecker

Publications and source records attributed to A Traunecker.

At least 19 recordsLinked to original sources
PubMed1992

Specific low-affinity recognition of major histocompatibility complex plus peptide by soluble T-cell receptor.

The T-cell receptor is necessary and sufficient for recognition of peptides presented by major histocompatibility complex molecules. Other adhesion molecules, like CD4 or CD8, play an auxiliary role in antigen recognition by T cells. Here we analyse T-cell receptor (TCR) binding using a soluble rather than a cell-bound receptor molecule. A TCR-immunoglobulin chimaera is constructed with the variable and the first constant regions of both the TCR alpha- and beta-chains linked to the immunoglobulin light-chain constant regions. This soluble TCR is expressed, assembled and secreted as an alpha beta heterodimer by a myeloma cell line transfected with the recombinant genes. Furthermore, the soluble TCR is biologically active: it specifically inhibits antigen-dependent activation of the relevant T-cell clones and thus discriminates between proper and irrelevant peptides presented by major histocompatibility complex molecules.

Animals↗
PubMed1992

Activated human T cells express a ligand for the human B cell-associated antigen CD40 which participates in T cell-dependent activation of B lymphocytes.

To identify the ligand for the B cell-associated antigen CD40, we constructed a chimeric immunoglobulin molecule where the extracellular portion of the CD40 protein replaced the normal immunoglobulin variable region. No binding was detected on resting peripheral blood T cells. However, following T cell activation with phorbol esters and ionomycin, the chimeric protein bound specifically to activated human T cells and precipitated a 35-kDa protein from such cells. The induction of the CD40 ligand was detectable on the cell surface after 1 h, with maximal expression after 8 h of stimulation. The T cells expressing CD40 ligand were predominantly CD4 positive, although a proportion of CD8-positive cells also expressed the protein. There was no particular correlation with CD45 phenotype. Finally, we found that soluble CD40 inhibited T-dependent B cell proliferation. The results are discussed in the context of cognate interactions between B and T cells.

Adult↗
PubMed1992

Janusin: new molecular design for bispecific reagents.

It is well established that soluble CD4 (sCD4) inhibits HIV infection in vitro, regardless of the virus strain or genetic variant. Most effective molecules, thus far, based on sCD4 are those in which CD4 is combined with immunoglobulin constant regions (CD4-IgG or CD4-IgM). Such molecules maintained HIV-gp120 specificity mediated by CD4 and also antibody effector functions such as complement activation, Fc receptor binding, long serum half-life or transport across the placental barrier. We have now developed sCD4 molecules which are even more potent anti-HIV reagents. These molecules are based on the principle of bispecific antibodies and they have properties capable of retargeting cytotoxic T lymphocytes onto HIV-infected cells and inducing efficient killing. CD4 combined with anti-human CD3 (FvCD3) single-chain combining site has been produced (CD4-FvCD3-JANUSIN). This molecule shows the expected biological activities, namely, binding to the 2 ligands, human CD3 and gp120, also efficiently retargeting CTLs of any specificity onto HIV-infected cells. In addition, several advantages over classical bispecific antibodies can be achieved: only one polypeptide, not a mixture containing the desired product, is produced, thus simplifying the purification process. In addition, Janusin designs do not contain the Ig Fc portion, which could mediate illegitimate retargeting of T-cells. In addition to CD4-FvCD3-JANUSIN, receptor-Fv, Fv-Fv or ligand-Fv Janusins can be produced.

Antibodies↗
PubMed1991

Development of lymphocytes in interleukin 7-transgenic mice.

We have developed and established mouse transgenic lines in which the mouse interleukin 7 gene was targeted for expression in the lymphoid cell compartment. Northern blot analysis indicate that the transgene is expressed in bone marrow (BM), spleen and thymus, but not in kidney, liver, brain or heart. Both the frequency and absolute numbers of B cell precursors and mature B lymphocytes are increased in the BM and spleen of the transgenic mice. Although there is no expansion of the pro-T lymphocyte population in the BM, the number of all major subsets of thymocytes and peripheral T lymphocytes is increased in the majority of the transgenic mice analyzed. The B and T cell lymphocytes in the transgenic mice are functionally competent. In contrast, the number of granulocytes and macrophages in the BM of transgenic mice is similar to that in control non-transgenic littermates. Our results indicate that interleukin 7 plays an important role in vivo in the development of B and T lymphocytes.

Animals↗
PubMed1991

Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on HIV infected cells.

The human immunodeficiency virus type 1 (HIV-1) uses cell surface CD4 as a receptor to infect susceptible cells. Therefore, different forms of soluble CD4 (sCD4) molecules have been developed recently for potential therapeutic purposes. Here we describe a novel design of sCD4 molecules which exploit cytotoxic T cells as their effector function. The principle of bispecific antibodies was exploited and further developed to create new bispecific reagents which could retarget cytotoxic T cells of any specificity and thus, induce killing of HIV-1 infected cells. The most advanced molecules, Janusins, contain in one polypeptide chain the first two N-terminal CD4 domains and single chain combining site against the human CD3 complex (FvCD3).

Amino Acid Sequence↗
PubMed1991

Surface expression of the beta T cell receptor (TCR) chain in the absence of other TCR or CD3 proteins on immature T cells.

T cell receptor (TCR) beta genes are rearranged prior to TCR alpha genes. A productively rearranged TCR beta gene suppresses further V beta gene rearrangement. Here we show that in beta TCR transgenic mice the TCR beta-chain can be expressed on the surface of immature CD4-8- thymocytes, but not on mature T cells, in the absence of any other known TCR chain and proteins of the CD3 complex. Analysis by NEPHGE and SDS-PAGE showed that at least some beta TCR exists on the surface as a large disulfide-linked complex with unknown acidic molecules. The introduction of the beta TCR gene into scid mice resulted in the expression of the beta TCR on the cell surface of thymocytes and induced the expression of CD4 and CD8 co-receptors as well as transcription of the alpha TCR locus.

Animals↗
PubMed1989

Production and secretion of recombinant soluble CD3 polypeptides by myeloma-derived transfectant clones.

Soluble forms of three human CD3 proteins have been produced by recombinant DNA techniques. The extracellular domain of CD3-gamma, -delta or -epsilon has been linked to the constant region of mouse immunoglobulin kappa light chain to form gamma-kappa, delta-kappa and epsilon-kappa chimaeric proteins. These are secreted by mouse myeloma-derived transfectant cell lines and are immunoprecipitable by CD3- or kappa-specific polyclonal antisera. Yields of 100-500 micrograms secreted recombinant proteins per litre of culture medium were obtained, which could be purified by anti-kappa affinity chromatography. The production of soluble CD3 illustrates the applicability of this technology to a loosely associated protein complex.

Antigens, Differentiation↗
PubMed1989

Highly efficient neutralization of HIV with recombinant CD4-immunoglobulin molecules.

The human immunodeficiency virus type 1 (HIV-1) exploits the cell surface CD4 molecule to initiate the infection which can lead, eventually, to acquired immunodeficiency syndrome (AIDS). The HIV-1 envelope protein, gp120, interacts specifically with CD4 and soluble CD4 molecules have been shown to inhibit HIV infectivity in vitro. Effective inhibition in vivo may, however, require more potent reagents. We describe here the generation of molecules which combine the specificity of CD4 and the effector functions of different immunoglobulin subclasses. Replacing the VH and CH1 domains of either mouse gamma 2a or mu heavy chains with the first two N-terminal domains of CD4 results in molecules that are secreted in the absence of any immunoglobulin light chains. We find that the pentameric CD4-IgM chimaera is at least 1,000-fold more active than its dimeric CD4-IgG counterpart in syncytium inhibition assays and that effector functions, such as the binding of Fc receptors and the first component of the complement cascade (Clq), are retained. Similar chimaeric molecules, combining CD4 with human IgG were recently described by Capon et al., but these included the CH1 domain and did not bind Clq. Deletion of the CH1 domain may allow the association and secretion of heavy chains in the absence of light chains, and we suggest that the basic design of our constructs may be generally and usefully applied.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Solubilizing the T-cell receptor--problems in solution.

Recombinant DNA technology has been central in answering some of the most important questions in immunology and has recently helped to define the complex of membrane associated proteins on T-cell surfaces responsible for antigen recognition. Here André Traunecker and colleagues describe attempts to facilitate the analysis of this complex using genetic engineering to produce solubilized T-cell receptor and associated molecules.

Animals↗
PubMed1988

Soluble CD4 molecules neutralize human immunodeficiency virus type 1.

Human immunodeficiency virus (HIV) infection can bring about total collapse of the immune system by infecting helper T lymphocytes which express CD4, the molecule which mediates interaction between the cell surface and viral envelope glycoprotein gp120 (refs 3-10). HIV apparently escapes the effects of neutralizing antibodies in vivo by generating new variants which must still interact with CD4 to maintain a cycle of infection. One route to block HIV infection, therefore, could use solubilized CD4 protein to inhibit attachment of the virus to its target cell. We have used recombinant DNA techniques to generate soluble forms of CD4, and show here that these are potent inhibitors of HIV infection in vitro.

Animals↗
PubMed1987

A new mouse TCR V gamma gene that shows remarkable evolutionary conservation.

We have identified a new mouse T-cell receptor V gamma gene segment, V gamma 4.4, which frequently undergoes rearrangements in AKR thymomas, and at a lower frequency in fetal thymocytes. V gamma 4.4 is the fourth and the most 5' V gene segment in the gamma 4 cluster, being 7.3 kb from V gamma 4.3. Surprisingly, V gamma 4.4 is more homologous to eight human V gamma genes than to the other mouse V gamma genes. It has only a 38% nucleotide and 21% amino acid sequence homology to the most homologous mouse V gamma gene (V gamma 4.1), whereas these homologies to the human V gamma 8 gene are as high as 68% and 48% respectively.

Animals↗
PubMed1986

Rearrangements of T cell receptor loci can be found only rarely in B lymphoid cells.

We have studied the rearrangement status of the T cell receptor genes in 64 B lymphoid cell lines, and we found that, unlike the immunoglobulin heavy chain genes in T lymphocytes, T cell receptor beta and related gamma chain genes are almost always in germ-line configuration in B lymphoid cells. The only exception was a myeloma MOPC511 (IgA, chi) which contained all T cell receptor genes, beta 1, beta 2, gamma 1, gamma 2, gamma 3 and alpha, in rearranged configuration in both homologous chromosomes. This exception supports the concept that all immunoglobulin and T cell receptor genes exploit the same recombinase to build their complete variable regions. Obviously, in MOPC511 cells the regulation, which confers the tissue specificity i.e. T vs. B lymphocytes, has failed.

B-Lymphocytes↗
PubMed1986

A novel approach for preparing anti-T cell receptor constant region antibodies.

To obtain antibodies against the individual chains of the T cell receptor (TCR) complex, we have produced chimeric proteins containing domains from immunoglobulin (Ig) and TCR polypeptide chains. Basically, the Ig light chains were used as carriers for the TCR constant (C) region domains. The exons which encode the main body of the C regions of the alpha, beta and the related gamma polypeptide chains were "engineered" into the intronic region between the rearranged Ig variable (V) region and C kappa region genes. All three chimeric genes were expressed in myeloma cells, and the proteins of expected apparent molecular weight were produced. Secreted proteins containing the C beta domain were purified from the culture supernatant by using anti-kappa antibody affinity columns, and two rabbits were then immunized with the purified protein. Both rabbits produced antibodies able to immunoprecipitate the heterodimeric TCR protein.

Animals↗
PubMed1986

Normal T cell development is possible without 'functional' gamma chain genes.

The T cell-specific gamma gene family is organized into four V, J and C gene segments containing clusters (gamma 1, gamma 2, gamma 3, gamma 4) in germline DNA. We found that the V, J and C elements of gamma 2 are physically linked on a stretch of 6 kb of DNA while those of gamma 3 are found within a 15-kb region. Rearrangements take place only within the clusters, explaining the rigid rearrangement patterns seen in T lymphocytes. New V gamma, J gamma and C gamma gene segments were discovered and characterized allowing the better understanding of the potential germline diversity of the gamma gene family. No correlation with T cell function, i.e. cytolytic or helper, and the type of the productive gamma rearrangement could be established. In contrast we found that functional T cell clones have been able to mature without any functional gamma chain genes.

Animals↗
PubMed1984

Somatic mutation creates diversity in the major group of mouse immunoglobulin kappa light chains.

Using a cloned cDNA of a mouse immunoglobulin kappa light chain synthesized in a myeloma MOPC321 (V kappa-21 subgroup C) as a probe we could detect 13 germ line V kappa gene segments. 11 of these were isolated. Using a set of overlapping cloned segments, we showed that nine of these germ line V kappa genes are arranged in two linkage clusters and that they all have the same transcriptional orientation (11, 12, 22). These two clusters occupy 90 and 30 kb of chromosomal DNA and contain six and three V kappa's, respectively. We determined the complete nucleotide sequences of five germ line V kappa's and showed that three of them encode the prototype sequence of V kappa-21 subgroups B, C, and E. None of these five germ line V kappa's encodes the variant amino acid sequences of known V kappa-21 subgroups. We thus conclude that, as in the lambda 1 light chains, the variant V regions are encoded by gene segments derived by a few somatic mutations from the corresponding germ line DNA. Such somatic mutations are not restricted to sequences encoding the hypervariable regions: they also occur in sequences encoding framework regions.

Amino Acid Sequence↗
PubMed1984

Different ways to modify monoclonal antibodies.

In this paper we summarize experiments which were undertaken to create altered antibody molecules. Three different approaches were used. Established hybridoma lines were re-hybridized to mouse spleen cells to generate arrays of secondary hybridomas which express one particular heavy chain and one specificity together with a multitude of different light chains. In such hybrids the influence of light chains to the antibody combining site and the influence of affinity to antibody effector functions can be studied. Another way to obtain altered antibodies was the selection of cells producing less lytic IgM. With this technique we obtained (among many other variants) a series of mu-deletion products which were used to map the fine specificity of rat anti-mouse mu monoclonal antibodies. Both the anti-mu antibodies and the deletion variants were used to assign the Clq binding to the fourth C mu-domain demonstrating the power of mutant IgM in the structure-function analysis. In a third series of experiments we show the feasibility of generating new antibody combining sites by the methods of molecular genetics. The variable region gene of a heavy chain was placed in front of a kappa-constant region gene. The plasmid construct was transferred into mouse myeloma lines which stably express a variable heavy-constant light chain protein. Upon fusion with a light chain producing line, chimaeric light chain dimers with a functional antibody combining site were secreted. These experiments demonstrate that new series of man-made antibody molecules can be made in the future.

Animals↗