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Biomedical subjects

E K Bikoff

Publications and source records attributed to E K Bikoff.

30 records · Page 2Linked to original sources

Formation of complexes between self-peptides and MHC class II molecules in cells defective for presentation of exogenous protein antigens.

A vertebrate immune response is initiated by the presentation of foreign protein Ag to MHC class II-restricted T lymphocytes by specialized APC. Presentation of self-peptides in association with MHC class II molecules is also necessary for the induction of T cell tolerance. It is important to understand whether functionally divergent APC are responsible for delivering these distinct signals to class II-restricted T cells. Here we examine the ability of I-Ad surface molecules expressed in diverse cell types to stimulate I-Ad-restricted T cells. Recipients included J558L myeloma cells and EL4 lymphoma cells expressing barely detectable or undetectable levels of Ii chain mRNA. This allowed us to examine the influence of Ii expression on the presentation of intracellular Ag and thus test the hypothesis that Ii chain is necessary to prevent access of self-peptides to newly synthesized class II molecules. Ii chain expression did not restore the ability of transformants to process and present soluble protein Ag. A striking result was the finding that cells showing a defect in the exogenous class II presentation pathway were capable of functioning as stimulators when they expressed intracellular secreted but not signal-less V-CH3b Ag. Thus, so-called professional APC that can capture and process exogenous protein Ag may express a specialized set of proteins not required for the presentation of self-peptides.

Animals↗

MHC class I surface expression in embryo-derived cell lines inducible with peptide or interferon.

It has long been recognized that the absence of expression of products of the major histocompatibility complex (MHC) during early development might allow the fetus to escape recognition by maternal lymphocytes. In addition to the MHC class I heavy chain and beta 2-microglobulin, antigenic peptide is an essential structural component of the class I molecule. Indeed, there is evidence that MHC-linked genes encoding peptide transporter molecules and possibly components of a proteolytic complex are necessary for MHC class I assembly and stability at the cell surface. Here we demonstrate that embryonic cells in general show a defect in MHC class I assembly. Surface expression was rescued in the presence of an appropriate antigenic peptide, or by treatment with interferon. Consistent with this, HAM1 messenger RNA was not constitutively expressed, but was inducible by interferon, and during differentiation in vitro. Thus, tolerance of the fetal allograft may in part be controlled at the level of peptide-dependent MHC class I assembly.

Animals↗

Distinct patterns of expression of MHC class I and beta 2-microglobulin transcripts at early stages of mouse development.

The class I surface Ag recognized during tissue graft rejection are composed of a 40- to 45-kDa H chain and a 12-kDa L chain, beta 2-microglobulin (beta 2m). Regulation of MHC gene expression during early development is thought to play an essential role in maternal tolerance of the fetal allograft. Here we used in situ hybridization techniques to characterize the temporal and spatial pattern of expression of MHC class I mRNA in the developing mouse embryo. MHC class I transcripts were initially detected at day 9.5 postcoitus in the primary and secondary trophoblast giant cell populations. At this stage, none of the remaining fetal components of the developing placenta expresses MHC class I mRNA. By contrast, the outer zone of trophoblast giant cells is the only trophectoderm-derived tissue in the developing placenta that does not express detectable levels of beta 2m mRNA. These findings indicate that the onset of MHC class I and beta 2m gene expression in early postimplantation stage embryos is not coordinately regulated. In addition, we made use of pluripotent embryonic stem (ES) cells to study developmental regulation of MHC class I and beta 2m gene expression in vitro. ES cells can be induced to form endoderm-like cells by the technique of suspension culture. In contrast to retinoic acid-treated F9 cells, the endoderm derived from ES cells expressed high levels of beta 2m mRNA, but no detectable MHC class I transcripts. These findings are consistent with results of our in situ hybridization experiments showing high levels of expression of beta 2m mRNA, and extremely weak expression of MHC class I transcripts in derivatives of the visceral endoderm in vivo.

Animals↗

Class II-restricted IgG2ab-specific T cells recognize a signal-minus form of the V-CH3b antigen.

To study the question when and where self peptides become associated with major histocompatibility complex class II molecules for tolerance induction, we recently developed a system in which the intracellular site(s) of antigen expression could be manipulated using gene cloning techniques. We previously constructed a truncated IgGa gene comprising a variable (V) domain and the CH3 domain (not including the membrane exons) from the IgG2ab heavy (H) chain. The secreted form of the V-CH3b protein was expressed at high levels under control of the Ig H chain enhancer in Ia+ B lymphoma cells and was efficiently recognized by class II-restricted IgG2ab-specific T cell hybrids. Here we describe a modified V-CH3b gene construct in which the sequences encoding the signal peptide were deleted. A strong argument can be made that the signal-less V-CH3b protein is predominantly expressed in the cytosol. We show that transfected L cell lines expressing the signal-less form of the V-CH3b protein can stimulate class II-restricted IgG2ab-specific T cells. Cell mixing experiments indicate that this response cannot be due to passive uptake of soluble antigenic peptides released into culture supernatants. These experiments demonstrate that a cytoplasmic protein having no obvious means of reaching the cell surface can be presented to class II-restricted T cells.

Animals↗

Defective assembly of class I major histocompatibility complex molecules in an embryonic cell line.

Developmentally regulated expression of the products of the major histocompatibility complex (MHC) is thought to play a key role in maternal tolerance of the fetal allograft. Here we analyze a cell line (EE2H3), derived from early post-implantation-stage mouse embryos, that is defective for MHC class I assembly. To follow expression of a single well-defined class I product, we introduced the H-2Dd gene under control of the human beta-actin promoter. We found that the transfected EE2H3 cells expressed abundant levels of H-2Dd heavy chains and beta 2-microglobulin protein, but only small amounts of H-2Dd surface protein. Surface expression was rescued by the addition of an appropriate antigenic peptide, or by culturing the cells at low temperature. The phenotype exhibited by EE2H3 is thus remarkably similar to that described for class I-negative somatic cell variants selected using antibodies and complement. However, a striking difference was that surface expression in H-2Dd-transfected EE2H3 cells was markedly enhanced in response to treatment with interferon. Thus, we have identified a novel class I assembly-defective cell line. Considering that EE2H3 was established from primary cultures of mouse embryo cells without immunoselection, and is therefore likely to represent a cell population normally present in post-implantation-stage embryos, these findings raise the possibility that expression of class I surface antigens during early development may in part be controlled post-translationally at the level of MHC class I assembly.

Animals↗

Analysis of beta 2-microglobulin gene expression in the developing mouse embryo and placenta.

The major transplantation (MHC) Ag in the mouse are cell-surface glycoproteins composed of a 40- to 45-kDa H chain and a 12-kDa L chain, beta 2-microglobulin (beta 2 m). Regulation of MHC gene expression during early development is thought to play a critical role in maternal tolerance of the fetal allograft. We have used in situ hybridization techniques to characterize the temporal and spatial pattern of expression of the beta 2 m gene in the developing mouse embryo. beta 2 m mRNA is first detected in the extra-embryonic derivatives of the early primitive streak stage embryo where it is specifically restricted to the tissues of the ectoplacental cone and chorion. Expression of the beta 2 m gene continues in these tissues during formation of the definitive placenta. beta 2 m transcripts were also detected in the tissues of the visceral yolk sac starting at 10.5 days. Comparable levels of expression were observed in both the visceral yolk sac endoderm and the extra-embryonic mesoderm. These results clearly indicate that the onset of beta 2 m gene expression is not restricted to early hemopoietic cells. In the embryo-proper, significant levels of beta 2 m mRNA were not detected until day 9.5. At this stage, we observed a strong signal over the liver rudiment. The remaining somatic tissues expressed low levels of transcripts. Similar results were obtained at later stages of development, i.e., the fetal liver was consistently the most strongly positive organ. Overall these in situ hybridization studies define the pattern of beta 2 m gene transcription and thus potential sites of surface expression of class I proteins in specific cell lineages in the early postimplantation stage embryo.

Animals↗

Presentation of IgG2a antigens to class II-restricted T cells by stably transfected B lymphoma cells.

Here we describe a panel of BALB/c T cells specific for IgG2a of the b allotype in association with I-Ad. We used DNA-mediated gene transfer techniques to localize antigenic determinants recognized by responding T cells. Initially a truncated IgG2aa gene comprising a variable domain and the CH3 domain (not including the membrane exons) from the BALB/c IgG2aa heavy chain was introduced into myeloma cells. The V-CH3 protein was expressed at high levels under control of the Ig heavy chain enhancer. Secretion of the V-CH3 protein did not require assembly of H-H dimers or an association with light chains. To generate stably transfected B cell lines that would stimulate our class II-restricted T cells, we replaced most of the BALB/c IgG2aa CH3 exon with CH3 coding sequences from a C57BL/6 IgG2ab cDNA clone and introduced these constructs into Ia+ B lymphoma cells. The IgG2ab CH3-transfected B cells were recognized by BALB/c Igh-1b-specific T cell hybrids in the absence of exogenous antigen. Experiments using glutaraldehyde-fixed cells as stimulators indicate that presentation of the secreted form of V-IgG2ab CH3 requires processing. We found that a significant fraction of the endogenously synthesized V-IgG2ab CH3 protein was, however, present as already processed antigen.

Animals↗

T cell recognition of endogenous IgG2a expressed in B lymphoma cells.

We recently characterized a panel of C57BL/6J T cell clones specific for IgG2a of the a allotype in association with I-Ab. Several of the clones gave surprisingly strong responses in the presence of normal spleen cells from several H-2b strains, including C3H.SW, A.BY, D1.LP and BALB.B, without the addition of exogenous antigen. Experiments using Igh-congenic mouse strains demonstrated that this response was directed towards shared allotypic determinants expressed on endogenously synthesized IgG2a molecules in various strains. Here we present evidence that the cell(s) responsible for this stimulation are conventional low density splenic accessory cells. Presentation of endogenously synthesized IgG2a by this population of dendritic cells, macrophages, and/or activated B cells was chloroquine sensitive. Thus, we conclude that this response is probably directed towards secreted IgG2a molecules that are internalized, processed and re-expressed at the cell surface in association with class II molecules. We also tested the ability of T cell clone B61-34 to respond in the presence of the B lymphoma cell line M12.C3.A2. A strong response was again observed in the absence of exogenous antigen. Northern gel analysis of M12.C3.A2 messenger RNA provided evidence that these cells synthesize IgG2a in both a secreted and a membrane form. The response directed towards endogenous IgG2a expressed in M12.C3.A2 lymphoma cells is chloroquine resistant and considerably more efficient than that stimulated by IgG2a added as exogenous soluble antigen. These results demonstrate for the first time that B cells have the capacity to present antigenic determinants expressed on endogenously synthesized immunoglobulins to class II-restricted T cells. The implications of these findings with respect to network models of immune regulation are discussed.

Animals↗

Homologous monoclonal antibodies specific for alpha 1-3 dextran stimulate polyclonal proliferation of Lyb-5+ B cells.

In this paper we describe the polyclonal blast response induced in nonimmune murine splenocytes by two homologous alpha 1-3 dextran-binding myeloma protein, J558 and MOPC104E. This stimulation appears to be independent of MHC or IgCh gene complex control, and proceeds entirely in a T-dependent fashion. The responding B cell population appears to belong to the more mature Lyb-5+ subset. This response was elicited by several independently prepared batches of J558 and MOPC104E, each of which was conclusively shown to be free of endotoxin contamination. Experiments are presented that suggest that this stimulation is being mediated via the Fab portion of these two myeloma proteins, in particular, a shared IdX, rather than through their Fc.

Animals↗

Tolerance for self IG at the level of the Ly1+ T cell.

Experiments presented in this report demonstrate that specificity of the Ly1+ T cell proliferative response to NP-modified Ig is controlled by Igh-C-linked genes. In addition, we describe the mechanism whereby Igh-C-encoded molecules influence Ly1+ T cell activity. We show that Igh-C-linked control of T cell responses to NP-modified Ig is a secondary consequence of naturally acquired tolerance for self Ig. Unresponsiveness to self Ig is not due to a defect expressed functionally at the level of the antigen-presenting cell, nor is it associated with active suppression. These results suggest that tolerance for self Ig at the level of the Ly1+ T cell is due to functional deletion of Ly1+ T cell clones specific for self Ig. The possibility is considered that regulatory effects mediated by passively administered antibodies may in part be due to induction of Ly1+ T cell tolerance for self Ig.

Animals↗

In vitro synthesis of transfer RNA. I. Purification of required components.

We have described an in vitro system in which active su+III tRNATyr is synthesized from a phi80psu++III DNA template. Using this system, we have identified four essential components that are required for synthesis of tRNA. The first of these is DNA-dependent RNA polymerase. It has been shown that a crude preparation of DNA-dependent RNA polymerase synthesizes su++III tRNATyr precursor similar to that which has been isolated in vivo, and that this preparation is capable of supporting high levels of tRNA synthesis. With purified DNA-dependent RNA polymerase, the su++III tRNATyr precursor was not observed as a transcription product and tRNA synthesis was below detetable levels. On this basis, a second essential component for tRNA synthesis was identified. This fraction, designated Fraction V, in combination with purified RNA polymerase, catalyzes the synthesis of precursor tRNA. The third component is a ribonuclease (RNase P III), which specifically catalyzes the removal of the extra nucleotides present at the 3' terminus of the tRNA precursor. In the absence of this fraction, the in vitro synthesized su++III tRNATyr is slightly larger than 4 S and contains additional nucleotides beyond the normal --CCAOH 3 terminus of the mature tRNA. The fourth essential component required is a fraction containing RNase P, a previously identified endonuclease which specifically catalyzes the removal of the 5' extra nucleotides present on tRNA precursors.

Chromatography, Gel↗

In vitro synthesis of transfer RNA. II. Identification of required enzymatic activities.

We have shown that the synthesis of active su+III tRNATyr from a phi80psu+III DNA template requires the action of four distinct enzymatic activities. The first of these, DNA-dependent RNA polymerase, catalyzes the formation of a large molecular weight transcript, initiating synthesis at a specific site 41 nucleotides proximal to the 5' end of the su+III tRNATyr structural gene and continuing at least 100 nucleotides beyond the 3' terminus of the su+III tRNATyr sequence. The second required component, designated Fraction V, allows purified DNA-DEPENDENT RNA polymerase to function in tRNA synthesis. We have shown that this fraction contains an endonuclease that together with DNA-dependent RNA polymerase is responsible for the synthesis of su+III tRNATyr "precursor". Thus, su+III tRNATyr precursor is not itself the primary transcription product of the su+III tRNATyr gene, but rather, it arises as a result of post-transcriptional cleavage of a much larger transcript by the action of the nuclease present in Fraction V. The third enzymatic activity required for synthesis of active su+III tRNATyr is a ribonuclease (RNase P III) that specifically catalyzes the removal of the 3' extra nucleotides from the su+III tRNATyr precursor. The fourth activity required for synthesis of tRNA is a previously identified endonuclease, RNase P, that specifically catalyzes the removal of the 5' extra nucleotides from tRNA precursors. The properties of RNase P purified according to the procedure developed in this laboratory have been compared with those of the enzyme purified from ribosomes according to the procedure described by Robertson et al. (Robertson, H.D., Altman, S., and Smith, F.D. (1972) J.Biol. Chem. 247, 5243-5251.).

Coliphages↗