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S E Cullen

Publications and source records attributed to S E Cullen.

At least 37 records · Page 2Linked to original sources
PubMed1984

Antigen presentation by the BCL1 murine B cell line: in vitro stimulation by LPS.

We examined the antigen-presenting capacity of BCL1 tumor cells, which are capable of differentiating in vitro with respect to immunoglobulin synthesis/secretion under the influence of LPS. In vivo passaged BCL1 cells depleted of host cell contamination either by positive selection employing panning with anti-lambda reagents, or by elimination of latex-ingesting adherent cells, are capable of MHC-restricted antigen presentation to a GAT-immune T cell line. The BCL1 cells act as antigen-presenting cells when freshly explanted, but gradual loss of this function occurs, and cells cultured for 3.5 days cannot present antigen unless LPS is included during the culture period. BCL1 cells are equivalently Ia+ after the culture period with or without LPS stimulation. Other B cell lines capable of antigen presentation appear to express this trait constitutively, and the in vivo passaged BCL1 line is therefore unique among B cell lines in having antigen-presenting cell function that can be modulated. The data suggest that freshly explanted or LPS-cultured BCL1 cells are heterogeneous with respect to antigen-presenting capacity, and the basis for this heterogeneity is being sought. BCL1 offers an opportunity to study requirements for antigen presentation by B cells.

Animals↗
PubMed1983

Identification of a new component in the murine Ia molecular complex.

In this report, we describe a previously unidentified component in the murine Ia antigen complex. SDS-PAGE analysis of anti-Ia immunoprecipitates prepared from spleen cells biosynthetically labeled with 35S-sulfate showed no detectable incorporation of 35SO4 into alpha, beta, or Ii chains but did not reveal the presence of a novel sulfate-bearing molecule of considerable molecular weight heterogeneity (46-69-kdaltons). The 46-69-kdalton molecule could be precipitated with monoclonal antibodies specific for I-A, I-E, and Ii glycoproteins but was not seen in control precipitates, nor in association with IgG or class I MHC molecules. Preliminary biochemical characterization indicated that the 46-69-kdalton product is extremely polydisperse, both in charge and apparent molecular weight, is sensitive to proteases, and bears the sulfate moiety on a large pronase-resistant structure. These results suggested this component might be a proteoglycan. Definitive identification of this component as a proteoglycan was accomplished by selective enzymatic degradation experiments which showed that the sulfate-bearing component of the 46-69-kdalton molecule is chondroitin 6-sulfate.

Animals↗
PubMed1983

Disulfide structure of murine Ia alloantigens.

The tryptic (T) and T insoluble chymotryptic (TIC) peptide maps from 35S-cysteine (Cys) labeled, disulfide-linked I-Ak dimer were compared to those from 35S-Cys labeled I-Ak alpha and beta chains which were not covalently linked. These comparisons indicated that the alpha and beta chains found in the covalent I-Ak dimer were not a specialized subset of I-A alpha and beta chains. Furthermore, these data, along with the knowledge that alkylation of spleen cells prior to and during detergent solubilization prevents the formation of disulfide-linked I-Ak dimer, indicate that covalent dimer formation is an inefficient and artifactual process. Comparison of the T and TIC peptide maps of reduced and nonreduced 35S-Cys labeled I-Ak alpha and beta chains suggests that the I-Ak alpha chain contains one intrachain disulfide bond, whereas the I-Ak beta chain contains two intrachain disulfide bonds. Examination of the T and TIC peptide maps of the reduced and nonreduced 35S-Cys labeled I-Ak dimer identifies the Cys-containing peptides which are involved in the formation of the artifactual I-Ak dimer interchain (alpha-beta) disulfide bond. Comparison of 35S-Cys labeled I-Ak and I-Ek alpha and beta chains by T and TIC peptide mapping reveals considerably more homology between the two alpha-chains and between the two beta-chains than is observed using other 3H-amino acid precursors, thus indicating that the I-Ak and I-Ek alloantigens are homologous in their amino acid sequences adjacent to the Cys resides. The reasons for the inability to induce formation of interchain (alpha-beta) disulfide bonds in I-Ek molecules are discussed.

Animals↗
PubMed1982

Analysis of murine Ia antigen glycosylation by lectin affinity chromatography. I-Ak alpha chain subspecies and beta chains are each glycosylated differently.

Murine Ia antigens consist of two glycosylated polypeptide chains, the alpha chain and the beta chain. We have used lectin affinity chromatography to confirm previous work in our laboratory that three distinct, differentially glycosylated I-Ak alpha chains (alpha 1, alpha 2, and alpha 3) exist and to compare the carbohydrate of the alpha chain with that of the beta chain. Glycopeptides derived from pronase digestion of [3H]mannose-labeled I-Ak alpha 1, alpha 2, alpha 3, and beta chains were sequentially passed over columns of immobilized concanavalin A, Lens culinaris lectin, phytohemagglutinin-E, and phytohemagglutinin-L in a prescribed manner to generate a lectin affinity profile, which, in turn, allowed assignment of a minimal oligosaccharide structure for each glycopeptide studied. The lectin affinity profile for each chain was unique. The alpha 1, alpha 2, and beta chains each possess complex-type N-linked oligosaccharides, although the branching pattern and specific sugar residues found on each differ. The alpha 3 chain, on the other hand, possesses predominantly high mannose or hybrid-type N-linked oligosaccharides. Lectin affinity analysis of glycopeptides derived from pronase digestion of high pressure liquid chromatography-isolated tryptic-chymotryptic fragments from alpha 2 and alpha 3 and tryptic fragments from beta revealed that specific minimal oligosaccharide structures were associated with particular fragments. In addition, although tryptic-chymotryptic peptide maps of alpha 2 and alpha 3 were similar, alpha 2 fragments bear predominantly complex-type N-linked oligosaccharides, whereas homologous alpha 3 fragments bear high mannose or hybrid-type N-linked oligosaccharides. Possible explanations of the oligosaccharide heterogeneity are discussed.

Animals↗
PubMed1982

Murine I-Ak alpha-chain subspecies with glycosylation differences.

In isolating homogeneous populations of murine Ia antigens by SDS-PAGE we have found that immunoprecipitated I-Ak molecules from whole spleen possess an electrophoretically heterogeneous population of alpha-chains. SDS-PAGE of 3H-mannose-labeled I-Ak immunoprecipitates revealed that the alpha-chain peak was divided into three regions (alpha 1, alpha 2, and alpha 3), each of which migrated as a distinct individual band when isolated and reelectrophoresed. Only alpha 1 and alpha 2 labeled with 3H-fucose. Nonequilibrium pH gradient electrophoresis excluded the possibility that any of the species was invariant chain, but rather suggested that the subspecies were structurally related and that alpha 3 has fewer sialic acid residues than alpha 1 and alpha 2. Sialic acid differences between alpha 1, alpha 2, and alpha 3 were further suggested by SDS-PAGE and by peptide mapping of neuraminidase-treated immunoprecipitates. Tryptic peptide maps of 3H-leucine-labeled alpha 1, alpha 2, and alpha 3 were identical, indicating that the polypeptide backbone of the molecules is the same for all subspecies. Our results suggest that alpha 1 and alpha 2 have fucose and sialic acid and thus possess complex type oligosaccharides, whereas alpha 3 lacks these sugars and possess high mannose-type saccharides. The potential relationships of the alpha-chain subspecies is discussed. In addition, we demonstrate the usefulness of high-pressure liquid chromatography in the isolation of glycopeptides for their subsequent analysis.U

Animals↗
PubMed1981

A variant alpha-chain in an Ia molecule from the I-A subregion of the mouse major histocompatibility complex: a possible intragenic recombination.

The recombinant MHC haplotype t1, present in mouse strain A.TL and B10.TL, encodes an I-A molecule with a variant alpha-chain that was recognized when tryptic peptide digests from t1 and the nominal parental haplotype (a1 or k) were compared by double-label reverse phase HPLC. The t1 I-A alpha-chain possesses a tryptic peptide absent from a1 and k I-A alpha-chains, and lacks several peptides present in a1 and k. The peptide that differs in t1 is not a mannose-bearing peptide, and thus its altered mobility is probably not due to carbohydrate modification. The alteration in t1 could result from mutation or intragenic recombination, but the unique t1 peptide migrates in a position identical to a peptide found in haplotype s, a partner in the (s X a1) cross that generated the t1 recombinant. If this apparently shared peptide indicates an intragenic recombination, this places the I-A alpha-gene centromeric to the other I-A subregion gene, and together with the data of Jones, suggests a gene order of I-A alpha, I-A beta, I-E beta (A alpha, A beta, Ae).

Animals↗
PubMed1981

Differential glycosylation of murine B cell and spleen adherent cell Ia antigens.

Examination of I-A and I-E molecules from purified B cells and splenic adherent cells from various haplotypes revealed a consistent difference in isoelectric focusing (IEF) that has been localized to the alpha-chain. alpa-Chains from B cells appeared heterogeneous and contained acidic IEF bands absent from the adherent cell I-A molecules. No difference in Ia beta-chain or H-2 IEF patterns was observed when B cell and adherent cell preparatios were compared when B cell and adherent cell preparations were compared. I-A alpha-chain preparations from the 2 cell sources showed no differences in 3H-leucine-labeled tryptic peptides separated by reverse-phase high-pressure liquid chromatography. Digestion with neuraminidase, kinetics of labeling, and subcellular distribution indicated that the extra acidic IEF bands in B cell Ia represent a mature, more heavily sialated form of alpha-chain that is not present in adherent cells. The selective differential glycosylation of B cell and adherent cell Ia could have some relation to the function of those cell types or to cell type-specific recognition of those cells.

Animals↗
PubMed1980

Identity of molecules bearing murine Ia alloantigenic specificities Ia.7 and Ia.22: evidence for a single type of I-E molecule in homozygotes.

In homozygous mice bearing I regions derived from haplotype k, only a single type of Ia molecule bearing the alloantigenic specificities Ia.7 and Ia.22 was found using techniques of sequential immune precipitation and tryptic peptide analysis. As suggested at the fourth Ir Gene Workshop (Sachs 1978), Ia.7 is considered here to be an antigenic determinant associated with I-E-subregion-encoded molecules, i.e., it is excluded from the I-C subregion. The I-C subregion is currently defined mainly by functional traits. It is now known that the I-E molecules are composed of an alpha chain encoded in the I-E subregion, and a beta chain encoded in the I-A subregion. Since the I-C subregion is not involved with the determination of these Ia molecules, and since in homozygotes there is apparently only a single type of molecule bearing both specificities Ia.7 and Ia.22, the term "I-E/C" molecule should probably be dropped in favor of the simpler designation I-E.

Animals↗
PubMed1980

Structural relation of murine "third locus" (H-2L) major histocompatibility antigens to the products of H-2K and H-2D loci.

The primary structures of the allelic products of H-2L, the murine "third locus" major histocompatibility antigen, were compared with the structures of the H-2K and H-2D antigens by tryptic-peptide analysis. The H-2Ld,H-2Lq, H-2Dd, H-2Dq and H-2Kd molecules were radiolabeled and isolated from spleen cell detergent lysates with specific alloantisera. Comparisons of the tryptic maps of the H-2L, H-2D, and H-2K molecules of the H-2d haplotype demonstrated that H-2Ld molecules were as different from H-2Dd molecules as H-2Dd molecules were from H-2Kd molecules. In contrast, the H-2Lq and H-2Dq molecules were found to be extremely similar and a second peptide separation method was required to discern structural differences between the 2 molecules. Comparisons of the allelic products H-2Ld and H-2Lq indicated possible structural differences despite the lack of a known serologic difference. These results, while raising questions about the genetic evolution of the H-2L locus relative to the H-2K and H-2D loci, demonstrate that the structure of the H-2L molecule is distinct from that of the H-2K and H-2D molecules. These structural analyses further substantiate the functional studies that define the H-2L molecule as the "third" major histocompatibility molecule of the mouse.

Alleles↗
PubMed1979

Insertion of Ia and H-2 alloantigens into model membranes.

The study of immune phenomena dependent on the major histocompatibility complex (MHC) would be greatly simplified by the use of MHC antigen-containing liposomes in various functional systems. Towards this end, we have constructed unilamellar phosphatidylcholine liposomes containing H-2 and Ia antigens. These molecules were not simply trapped within the aqueous compartment of the liposome as assessed by their accessibility to papain digestion. They were shown to be integrally inserted in the liposome bilayer because they could not be dissociated from the liposome with high salt and EDTA concentrations but could be solubilized by detergent. A sensitive radioimmunoassay showed that the Ia molecules were antigenically active in the liposome environment. Both Ia and H-2 antigens could be immunoprecipitated from detergent-solubilized liposomes. By comparing liposome-associated Ia activity in the presence and absence of detergent and by showing accessibility of the Ia antigens to papain, it was concluded that the majority of Ia antigens were exposed on the external surface of the liposome. These results suggest that the orientation of MHC antigens in liposomes closely parallels their natural orientation in the cell membrane.

Antigens, Surface↗
PubMed1978

Immune response-associated antigens on mouse leukemia cells. I. Detection of Ia antigens on GRSL cells.

The expression of immune response-associated (Ia) antigens on the surface of mouse strain GR (H-2dx) ascites leukemia (GRSL) cell lines was studied by cytotoxic tests, immunofluorescence, and immunoprecipitation assays. Ia expression varied among the three GRSL cells lines (GRSL 2, GRSL 14, and GRSL 15) studied by cytotoxic assay. GRSL 14 cells showed the strongest expression of Ia antigens among these three cell lines. A time-course study of tumor growth in mice revealed that Ia antigens on the tumor cells demonstrated the strongest expression 10 days after injection of GRSL cells into GR mice, and that subsequently it decreased until the death of the animal. Cells treated with neuraminidase exhibited more readily detectable Ia antigens, expecially in the late stages of leukemia, which suggested that Ia antigens had been masked by sialic acid. Immunoprecipitation studies revealed that Ia molecules on the leukemia cell had the same molecular weight as those on the normal lymphocytes. Immunofluorescence studies disclosed that Ia antigens were distributed diffusely on the surface of the tumor cells.

Animals↗
PubMed1978

Labeling characteristics and separation of Ia antigen subunits.

Experiments with biosynthetic incorporation of 3H-amino acids into murine and guinea pig Ia antigens have indicated that these antigens consist of two polypeptide chains of 33,000 and 25,000 daltons, respectively, occasionally linked by disulfide bonds into a 58,000 dalton molecule. In contrast, studies with lactoperoxidase-catalyzed radioiodination have indicated that these Ia antigens consist of only a single chain of 25,000 daltons. We therefore undertook a study to explore the basis of these discrepant results. Since 3H-tyrosine labeled both chains well, the lack of tyrosine residues in the 33,000 dalton chain could not be the explanation for the lack of radioiodination. However, by partially purifying the Ia antigen preparation with Lens culinaris (lentil) lectin affinity chromatography before immunoprecipitation and by increasing the resolution of analysis by using discontinuous-SDS polyacrylamide gel electrophoresis, it was possible to show that the 33,000 dalton chain was in fact radioiodinated, though still poorly so relative to the 25,000 dalton chain, and that a radioiodinated 58,000 dalton molecule could be detected. These experiments suggest that the 25,000 dalton chain is more exposed to the external cellular environment, and thus more readily iodinated by lactoperoxidase. In addition, the studies indicate that the choice of labeling method, purification procedures, and analytical methods must be taken into account when interpreting experimental results.

Animals↗
PubMed1978

Murine Ia antigens: identification and mapping of Ia.23 and further definition of the I-E subregion.

The Ia.23 specificity is a private specificity of the H-2d haplotype which we have assigned to the I-E subregion. It is found on the same Ia molecule that bears Ia.7, a public specificity previously assigned to the I-C subregion of haplotypes H-2d, H-2k, H-2p and H-2r. We suggest a reassignment of specificity Ia.7 to the I-E subregion to simplify the interpretation of the data presented in this paper and elsewhere, and to permit an orderly description of that part of the I region to the right of I-J.

Animals↗
PubMed1978

Isolation and analysis of H-2 and Ia alloantigens from wild mouse strains.

Immunoprecipitation and SDS-PAGE analysis were used to examine H-2 and Ia antigens from mouse strains with wild-derived MHC haplotypes. Antisera raised against the wild-derived strains contained anti-H-2 and anti-Ia antibodies which precipitated antigen molecules readily distinguishable by a single assay procedure. The antibodies to wild strains were cross-reactive with standard laboratory haplotypes. Evidence supporting the similar genetic organization of wild and standard haplotypes was found, including isolation of separate H-2K and H-2D molecules from a wild-derived strain, and isolation of two separate Ia molecules from a wild-derived strain.

Animals↗
PubMed1977

Mutation in a new H-2-associated histocompatibility gene closely linked to H-2D.

Sequential precipitations of soluble BALB/c antigen with antisera detecting private and public H-2 specificities indicated three distinct classes of molecules of 45,000 mol wt. However, only two of these classes of molecules were detectable in antigen from the loss mutant, BALB/c-H-2db. The class of molecules, detectable in the wild-type strain but missing in the mutant, does not bear private specificities but does react with an antiserum detecting H-2 public specificities. Absorption in mutant mice of the antiserum to public specificities, left antibodies specific for the antigen detectable in BALB/c but not BALB/c-H-2db. Genetic mapping studies using this specific antiserum indicated that the antigenic loss of this mutant is in a gene which maps in or close to the H-2D region, separable from the H-2K, S, G, Qa-2, and Tla regions.

Animals↗
PubMed1977

Immunochemical evidence for an additional H-2 region closely linked to H-2D.

Anti-H-2 reagents have been tested on solubilized spleen cell preparations in combinations expected to be specific for D region products. Two different types of molecules were detected. One showed the expected reactivity with both antisera to private and antisera to public specificities. However, an additional molecule was detected which reacted only with antisera to public specificities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis migration patterns indicated that both products have a similar molecular size of approximately 45,000 daltons. The data therefore present chemical evidence for the existence of a third H-2-associated gene product of 45,000 mol wt in addition to the classical H-2K and H-2D antigens.

Animals↗