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C Milstein

Publications and source records attributed to C Milstein.

At least 91 records · Page 5Linked to original sources
PubMed1990

Lack of somatic mutation in a kappa light chain transgene.

Analysis of mice transgenic for immunoglobulin genes should allow definition of the cis-acting DNA sequences required to target somatic mutation to antibody V genes. We have looked for mutations in a chimeric kappa transgene encoding a V region specific for the hapten 2-phenyloxazolone (phOx) linked to a rat C kappa gene. Two independent lines of transgenic mice were hyperimmunized with phOx and splenic hybridomas established. In B cells that had been selected by antigen and which used mouse anti-phOx genes, the endogenous sequences were found to be mutated whereas the transgene remained unchanged. These results suggest either that (a) if the transgene is a "passenger" gene expressed at a low level, transgene mutation is a rare event, or that (b) sequences far from the kappa coding region are necessary to direct somatic mutation.

Amino Acid Sequence↗
PubMed1990

Expression of CD1 in the mouse thymus.

The CD1 antigens are a family of differentiation antigens found predominantly, but not exclusively, in the human thymus. Although three antigens (CD1a-c) are described by monoclonal antibodies, five genes (CD1A-E) are found in the human genome. The cloning of the mouse CD1 genes (Bradbury, A., Belt, K.T., Nery, T.M., Milstein, C. and Calabi, F., EMBO J. 1988. 7:3081) demonstrated the presence of homologues to human CD1D, but not to any of the other human CD1 genes. In this work we have examined the expression of mouse CD1D mRNA in the thymus and shown that it is predominantly cortical, as is the expression of the CD1 antigens in man. Somewhat surprisingly, we also find that most CD1D mRNA in the mouse thymus is unspliced. Despite this, we have also been able to show, using a polyclonal antiserum directed against a bacterial fusion protein, the existence of the expected protein product.

Animals↗
PubMed1990

Three-dimensional structure determination of an anti-2-phenyloxazolone antibody: the role of somatic mutation and heavy/light chain pairing in the maturation of an immune response.

The three-dimensional structure of the Fab fragment of an anti-2-phenyloxazolone monoclonal antibody (NQ10/12.5) in its native and complexed forms has been determined at 2.8 and 3.0 A resolution, respectively. Identification of hapten-contacting residues has allowed us to evaluate the contribution of individual somatic point mutations to maturation of the immune response. In particular, amino acid residues 34 and 36 of the light chain, which are frequently mutated in antibodies with increased affinity for 2-phenyloxazolone, are shown to interact directly with the hapten. We propose that the strict maintenance of certain amino acid sequences at the potentially highly variable VL-JL and VH-D-JH junctions observed among anti-2-phenyloxazolone antibodies is due largely to structural constraints related to antigen recognition. Finally, the three-dimensional model of NQ10/12.5, which uses the typical light chain of primary response anti-2-phenyloxazolone antibodies but a different heavy chain, allows an understanding of how, by preserving key contact residues, a given heavy chain may be replaced by another, apparently unrelated one, without loss of hapten binding activity and why the V kappa Ox1 germline gene is so frequently selected amongst the other known members of this family.

Amino Acid Sequence↗
PubMed1989

A physical map linking the five CD1 human thymocyte differentiation antigen genes.

Human CD1 is a family of thymocyte differentiation antigens which consist of heavy chains with mol. wts between 43 and 49 kd binding to beta 2 microglobulin. They are distant relatives of the major histocompatibility complex (MHC) class I and II products. Five human CD1 genes have been described. Three (CD1A, -B and -C) code for the serologically defined CD1a, -b and -c antigens. The protein products of the other two genes, CD1D and CD1E, remain unknown. All CD1 genes are located on chromosome 1 and hence are independent of the MHC locus. In this paper, the tight linkage of the CD1 genes has been established by pulse field gel electrophoresis, cosmid cloning and walking techniques. The 190 kb of DNA linking all five CD1 genes has been spanned by 14 overlapping cosmids. The order of the genes in the CD1 complex is CD1D-CD1A-CD1C-CD1B-CD1E, and, with the exception of CD1B, they are arranged in the same transcriptional orientation. The genes are evenly spaced in the complex except for the distance between CD1D and CD1A, which is two to three times greater than the average.

Antigens, CD↗
PubMed1989

Two steps in the intracellular transport of IgD are sensitive to energy depletion.

The secretory pathway of murine IgD can be dissected by the use of carbonylcyanide m-chlorophenylhydrazone (CCCP), which inhibits two distinct steps of intracellular transport. The newly synthesized IgD that accumulates at the first step contains high mannose type oligosaccharides which are partially trimmed. The IgD arrested at this step is less processed than the IgD arrested by treatment with monensin. The properties of this biosynthetic intermediate are consistent with inhibition of Ig passage from the endoplasmic reticulum to the Golgi complex. A second CCCP-sensitive step exists in the biosynthesis of IgD, and is characterized by delta-chains that are resistant to endoglycosidase H and contain galactose. This indicates that this second step occurs during or after the passage through the trans-Golgi compartment. The galactose-containing oligosaccharides of the delta-chains arrested at this step do not contain fucose (as do mature, secreted delta-chains). Fucosylation is not inhibited by CCCP, nor is the secretion of fucose-containing delta-chains. These results show that terminal sugars are added to secretory IgD in at least two transport compartments, separable by their sensitivity to CCCP. The inhibition of the secretory pathway at both steps is reversible; upon removal of the drug the arrested IgD is processed normally and is secreted. The sensitivity to CCCP probably reflects transport steps that are sensitive to even partial depletion of ATP, because treatments with other inhibitors of oxidative phosphorylation yield similarly arrested Ig molecules. Thus, by using the protonophore CCCP, we demonstrate two energy-requiring steps in IgD transport which seem to be at two transitions in the secretory pathway. One step is during the passage from the endoplasmic reticulum to the mid-Golgi compartment and the other step is during Ig passage through the trans-Golgi, or subsequent transport to the cell surface.

Adenosine Triphosphate↗
PubMed1989

Two classes of CD1 genes.

Herein, we report the DNA sequence of two human CD1 genes, R2 and R3, distinct from those encoding the CD1a, -b and -c antigens. Both genes appear to have an exon/intron structure analogous to the previously analyzed CD1 genes and to be functional on the basis of their sequence. Analysis of the variability patterns, potential intramolecular interactions and predicted secondary structure profile on an alignment of all known CD1 alpha chains suggest some shared structural features with major histocompatibility complex class I molecules in the alpha 1 domains but substantial differences in the alpha 2 domains. Sequence comparison shows that, while R2 is most related to CD1a, -b and -c, albeit to a somewhat lower degree than the latter are to themselves, R3 is more homologous to mouse than to human CD1, suggesting the existence of two functional classes within the CD1 gene family. We propose to retain the non-committal R2 and R3 names until the putative antigens have been identified and their tissue distribution has been established.

Amino Acid Sequence↗
PubMed1989

The rabbit CD1 and the evolutionary conservation of the CD1 gene family.

A comparison of the genes encoding the CD1 leucocyte differentiation antigens in man and mouse shows important differences which prompted us to analyze the CD1 genes of the rabbit. We have found that the rabbit genome contains multiple CD1 loci. Upon cloning and sequencing, one of these loci was found to encode the known rabbit CD1-like antigen (R-Ta) and to be closely related to the human CD1b gene, which is absent in the mouse, while a second rabbit gene is closely related to both the human R3 and the mouse CD1 genes. The data reinforce the notion of the existence of two classes of CD1 genes, one of which is conserved in all species, while the other, albeit also evolutionarily old, has been deleted in mice as well as in other rodents.

Amino Acid Sequence↗
PubMed1989

Phorbol esters potentiate the induction of class I HLA expression by interferon alpha.

We have studied the effect of phorbol esters on the induction of class I histocompatibility antigen (HLA) expression by interferons (IFNs) in the T-cell line MOLT-4 and in the MOLT-4 mutant YHHH. Addition of IFN-alpha to phorbol 12,13-dibutyrate-pretreated MOLT-4 cells causes a greater than 20-fold increase in the expression of class I HLA, as compared to a 4- to 7-fold IFN-alpha-induced increase in control cells. Pretreatment with phorbol 12,13-dibutyrate does not alter the class I HLA response to IFN-gamma or the responses of other IFN-induced genes. This effect of phorbol 12,13-dibutyrate reproduces in MOLT-4 cells the phenotype of the mutant YHHH, which also displays a selective enhanced class I HLA response to IFN-alpha. Pretreatment of YHHH with phorbol 12,13-dibutyrate does not affect any of the responses induced by IFN. These findings suggest the existence of a phorbol ester-sensitive factor, inducible in MOLT-4 and constitutively expressed or modified in YHHH, which operates in the pathway of induction of class I HLA by IFN-alpha but not in the pathway used by IFN-gamma.

Drug Synergism↗
PubMed1989

Characterisation of the first monoclonal antibody against the pronase resistant core of the Alzheimer PHF.

A monoclonal antibody, NOAL 6.423 has been produced which unequivocally identifies a protein which is a constituent of the pronase resistant core of the Alzheimer PHF. Although the site recognised by this antibody is contained somewhere within the highly conserved triple repeat region of the tau molecule, no experiments have succeeded so far in demonstrating NOAL 6.423 reactivity in any brain proteins other than those derived from PHFs. A site which can be recognised by NOAL 6.423 cannot be generated in preparations of normal porcine tau by simple proteolytic cleavage with pronase. Likewise reactivity with the PHF peptide recognised by NOAL 6.423 is not altered by alkaline phosphatase. The unusual properties of NOAL 6.423 are consistent with the possibility that its recognition site is not determined solely by the primary structure of the conserved region of the tau molecule.

Alzheimer Disease↗
PubMed1988

Reshaping human antibodies: grafting an antilysozyme activity.

The production of therapeutic human monoclonal antibodies by hybridoma technology has proved difficult, and this has prompted the "humanizing" of mouse monoclonal antibodies by recombinant DNA techniques. It was shown previously that the binding site for a small hapten could be grafted from the heavy-chain variable domain of a mouse antibody to that of a human myeloma protein by transplanting the hypervariable loops. It is now shown that a large binding site for a protein antigen (lysozyme) can also be transplanted from mouse to human heavy chain. The success of such constructions may be facilitated by an induced-fit mechanism.

Animals↗
PubMed1988

Differential expression and interferon response of HLA class I genes in thymocyte lines and response variants.

cDNA clones which seem to represent the alleles of HLA-A and -B expressed by the thymoma MOLT-4 have been isolated and used as locus-specific probes to measure the corresponding mRNA levels in MOLT-4 and other human thymocyte lines, and the effect of interferon (IFN)-alpha and -gamma on these levels. It is shown in MOLT-4, and in its derived line YHHH, that HLA-B mRNA levels are undetectable before treatment but respond to IFN-alpha and -gamma more markedly than those of HLA-A. This differential induction is best shown with YHHH, which is hypersensitive to IFN-alpha, where the HLA-B mRNA levels increase to a level threefold those of HLA-A. Other thymocyte lines tested also showed preferential induction by IFN-alpha of HLA-B, although the basal levels of HLA-A and -B tended to be similar. The effect of the altered ratio of HLA-A to -B mRNA on surface expression of the antigens and the correlation between basal level expression and inducibility are discussed.

Antigenic Variation↗
PubMed1988

Sensitive and high resolution in situ hybridization to human chromosomes using biotin labelled probes: assignment of the human thymocyte CD1 antigen genes to chromosome 1.

A method for in situ hybridization originally developed for mapping genes in the nematode, Caenorhabditis elegans has been adapted for high resolution cytological mapping of genes in the human. The probe DNAs are labelled by incorporation of biotin dUTP and the site of hybridization detected by immunofluorescence. For the accurate assignment of the hybridization signal to chromosome bands, visualized by staining with Hoechst 33258, a heterologous ribosomal DNA probe is also included in the hybridization reaction. These rDNA signals are used as fiducial markers when aligning the two fluorescent images. We demonstrate the method by assignment of the human thymocyte CD1 antigen genes to human chromosome 1q22-23.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1988

Mouse CD1 is distinct from and co-exists with TL in the same thymus.

Human CD1 antigens have a similar tissue distribution and overall structure to (mouse) TL. However recent data from human CD1 suggest that the mouse homologue is not TL. Since no human TL has been conclusively demonstrated, we have analysed the murine CD1 genes. Two closely linked genes are found in a tail to tail orientation and the limited polymorphism found shows that, as in humans, the CD1 genes are not linked to the MHC. Both genes are found to be equally transcribed in the thymus, but differentially in other cell types. The expression in liver, especially, does not parallel CD1 in humans. This demonstrates conclusively that CD1 and TL are distinct and can co-exist in the same thymus. It is paradoxical that despite the structural similarity between mouse and human CD1, the tissue distribution of human CD1 is closer to TL. The possibility of a functional convergence between MHC molecules and CD1 is discussed.

Amino Acid Sequence↗
PubMed1988

Isolation of a fragment of tau derived from the core of the paired helical filament of Alzheimer disease.

A substantially enriched preparation of Alzheimer paired helical filaments (PHFs) has been used as a starting point for biochemical studies. Pronase treatment, which strips off adhering proteins, leaves a resistant core that is structurally intact. This has been used to raise a monoclonal antibody that decorates the filament core. The antibody has been used to follow the extraction of two peptide fragments (9.5 and 12 kDa) by immunoblotting. The link between the PHF as a morphological entity and these peptides has been established independently by photoaffinity labeling with a chemical ligand to the PHF core. Sequence analysis of these peptides was used to design oligonucleotide probes for cloning a cognate cDNA, which leads to its identification as human microtubule-associated tau protein. The sequencing of the 9.5- and 12-kDa peptides shows they are derived from a conserved region of tau containing three repeating segments. Since these fragments have been copurified with the Pronase-resistant core and are only released by subsequent steps, the corresponding part of the tau molecule must be tightly bound in the PHF core.

Alzheimer Disease↗
PubMed1987

Regulation of membrane IgM expression in secretory B cells: translational and post-translational events.

IgM secreting cells express little or no membrane IgM. This is not always due to absence of the relevant mRNA. To investigate the synthesis and processing of membrane (micron) and secreted (microseconds) polypeptides in secretory B cells, myeloma cells were transfected either with a plasmid containing an intact mu gene or with one only capable of directing micron (not microseconds) mRNA synthesis. Although myeloma transfectants could make abundant levels of micron mRNA, they did not express IgM on the cell surface. In the myeloma host, micron mRNA is translated some 5-fold less efficiently than microseconds mRNA. However, this translational control does not totally preclude micron synthesis, indicating post-translational regulatory events. No difference between micron and microseconds chains could be detected in their rate of assembly with light chains or in their stability, although both types of heavy chain were degraded more rapidly when synthesized in the absence of light chain, or when the hydrophobic nature of the leader sequence was destroyed by site-directed mutagenesis. However, whereas intracellular microseconds chains in IgM-secreting plasmacytoma were found to be concentrated in the Golgi, the micron chains were mainly located in the endoplasmic reticulum. Retention in the endoplasmic reticulum is also observed for both micron and microseconds when synthesized in the absence of light chain. We propose that it is the expansion of the endoplasmic reticulum that accompanies B cell to plasma cell differentiation which is in part responsible for the down-regulation of surface IgM expression. Such a mechanism may also affect the expression of other surface proteins.

Animals↗
PubMed1987

The induction of class I HLA by interferon-alpha is independent of the cell cycle, but the expression is enhanced by a G1/S block.

The induction of class I HLA expression by interferon-alpha (IFN-alpha) was studied in lymphoid cells arrested or traversing different stages of the cell cycle. Exponential cultures of MOLT-4 cells and the MOLT-4 cell variant YHHH were treated with the cell cycle inhibitors aphidicolin and colcemid to obtain cell populations arrested in G1/S and G2/M, respectively, and also cells traversing from S to M and vice versa. Cytofluorimetry with the monoclonal antibody YTH/76.3 (which specifically detects those class I molecules which are most susceptible to IFN-alpha induction) was used to quantitate the class I HLA response to IFN-alpha. The results showed that the response to IFN-alpha is not restricted to a given stage of the cell cycle. These studies also revealed that when the cells were arrested at G1/S, the absolute level of class I HLA expression was enhanced 2-3-fold, both in the presence or absence of either IFN-alpha or IFN-gamma. Therefore, even when absolute levels changed, the ratio of IFN-induced expression to basal expression remained constant at all cell cycle stages. The level of expression of another surface antigen (the CD1 antigen HTA-1) was not affected by the G1/S block. The results were confirmed by dot blot hybridization of poly(A)+ RNA using cDNA-specific probes. These findings suggest that the effect of IFN-alpha is continuous throughout the cell cycle but that a G1-dependent event determines the extent of class I HLA expression, and leads to a synergistic superinduction by IFN in G1/S-arrested cells.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1987

Activation of memory and virgin B cell clones in hyperimmune animals.

To study the long-term memory response, BALB/c mice were allowed to rest for over a year after a secondary immunization with the hapten 2-phenyl-oxazol-5-one (phOx). For the tertiary immunization two different protocols were used. In one protocol mice were injected i.v. and 3 days later spleen cells were fused to a nonproducing hybridoma line. PhOx-specific hybridomas were established and the sequence of the heavy and light chain mRNA was determined. This tertiary response resembled the diversity pattern of the secondary response with a further increase both in somatic mutations and in the average dissociation constant. The high number of somatic mutations demonstrates the persistence of memory B cell clones over a long time period. In the second protocol mice were boosted with an i.p. injection of alumprecipitated antigen phOx and 7 or 14 days later spleen cells were fused. Sequence analysis of heavy and light chain mRNA showed that these tertiary response antibody molecules had surprisingly few somatic mutations, indicating an activation of virgin B cell clones in these hyperimmunized animals. The maturation of these newly stimulated B cell clones seems to follow somewhat similar rules to those found for the primary response. It appears therefore that the two immunization protocols reflect the response of memory and virgin B cells, respectively.

Amino Acid Sequence↗