Biomedical subjects

E O Long

Publications and source records attributed to E O Long.

At least 73 records · Page 4Linked to original sources

PubMed1994

Invariant chain prevents the HLA-DR-restricted presentation of a cytosolic peptide.

Three properties of the HLA-DR-associated invariant chain (Ii) may contribute to the distinction between the class I and class II Ag presentation pathways. First, Ii prevents peptide binding to alpha beta 1 Ii complexes. Second, Ii promotes assembly of class II alpha beta heterodimers and their transport out of the endoplasmic reticulum. Third, Ii provides a targeting signal for the transport of class II molecules to endocytic compartments. However, it is not known whether Ii can prevent class II-restricted T cell recognition of endogenous peptides transported into the endoplasmic reticulum. In addition, recent evidence has indicated that, in the absence of Ii, newly synthesized class II molecules cannot form stable complexes with peptides. In this study, transfected human fibroblast cells expressing HLA-DR1 alone or with an excess of Ii were tested for their ability to present a DR1-restricted epitope of the influenza virus matrix protein produced as a short cytosolic peptide by use of an episomal expression vector. Presentation to a DR1-restricted T cell clone was very efficient in cells expressing class II molecules without Ii, but not in cells expressing class II and Ii. The inhibition by Ii was specific for the endogenous cytosolic peptide, because the same epitope processed from exogenous influenza virus particles was presented only by cells expressing class II with Ii. Ii did not inhibit the HLA-A2-restricted presentation of another cytosolic peptide. Thus, T cells can detect a cytosolic peptide loaded onto class II alpha beta heterodimers, and Ii prevents such endogenous peptide presentation.

Amino Acid Sequence↗

PubMed1994

Two processing pathways for the MHC class II-restricted presentation of exogenous influenza virus antigen.

The natural Ag influenza virus A was used to test the requirements for the HLA-DR1-restricted presentation of the epitopes 18-29 in the matrix protein and 307-318 in the hemagglutinin protein. CD4+ cytotoxic T cell clones of similar efficiency were used to detect presentation of these two epitopes. Presentation of the matrix epitope by APC pulsed with either inactivated virus particles or purified soluble protein followed the classical pathway in that 1) it required invariant chain expression, 2) it was blocked by inhibition of protein synthesis, and 3) it was dependent on a function(s) encoded in the MHC class II region. These characteristics suggest that peptides corresponding to the matrix epitope can only load onto newly synthesized class II molecules that were targeted to a processing compartment by the invariant chain. In contrast, presentation of the hemagglutinin epitope processed from virus particles followed a different pathway. First, presentation of hemagglutinin was independent of invariant chain expression. Second, a human B lymphoblastoid cell line in which protein synthesis was inhibited for 9 h was still able to present hemagglutinin even at very low doses of Ag. Third, a DR1-transfected mutant B cell line missing the MHC class II region was able to present hemagglutinin. Thus, mature class II alpha beta molecules can acquire immunogenic peptides derived from intact natural Ags for presentation to CD4+ T cells. This pathway may be useful for the binding of peptides derived from Ags that are rapidly degraded upon uptake into APC.

Antigen Presentation↗

PubMed1993

Presentation of cytosolic antigen by HLA-DR requires a function encoded in the class II region of the MHC.

The processing pathway for the MHC class II-restricted presentation of endogenous cytosolic Ag is distinct from the class I pathway since a cytosolic form of the influenza virus A hemagglutinin, expressed by a recombinant vaccinia virus, was presented by HLA-DR in a B cell mutant lacking the TAP1 subunit of the transporter for Ag presentation (TAP). In this report, two additional B cell mutants have been used to define the requirements of this TAP1-independent processing pathway. The first mutant, .61, lacks expression of both TAP1 and TAP2 genes, and of both LMP2 and LMP7 genes encoding proteasome subunits. As expected, class I-restricted presentation of the influenza virus matrix protein was totally deficient in mutant .61. In contrast, class II-restricted presentation of both the natural cytosolic matrix and the engineered cytosolic hemagglutinin proteins was functional in mutant .61. Thus, presentation of cytosolic Ag by class II molecules is independent of both TAP subunits and of the two MHC-encoded proteasome subunits. However, this endogenous processing pathway is dependent on at least one other function encoded in the class II region of the MHC as demonstrated with the second mutant, .174, in which a large deletion eliminates all expressed class II genes. Mutant .174 transfected with HLA-DR1 genes was previously shown to be defective in the presentation of exogenous Ag but normal in the presentation of short exogenous peptides. We show here that .174(DR1) is also defective in the presentation of cytosolic matrix and hemagglutinin proteins. This similar requirement for the class II-restricted presentation of either cytosolic Ag or internalized exogenous Ag suggests that both forms of Ag are ultimately targeted to the same cellular compartment for association with class II molecules.

Antigen Presentation↗

PubMed1993

Cell surface HLA-DR-invariant chain complexes are targeted to endosomes by rapid internalization.

Class II molecules of the major histocompatibility complex (MHC) bind peptides derived from protein antigens delivered into endocytic compartments and present these peptides to CD4+ T cells. The precursors to functional MHC class II molecules loaded with peptides are complexes of the invariant chain associated with class II alpha beta heterodimers. Targeting of newly synthesized MHC class II molecules to endosomes is mediated by the invariant chain, but the intracellular transport route is not known. This study demonstrates that in a human B-cell line a large population of MHC class II-invariant chain complexes reaches endosomes by rapid internalization from the cell surface. Quantitation of cell surface MHC class II-invariant chain complexes and of their surface half-life revealed that 3000 complexes internalized per minute into endosomes. This highly efficient endocytosis was mediated by the cytoplasmic tail of the invariant chain. After internalization, the invariant chain dissociated from the MHC class II-invariant chain complexes. This pathway may represent an important mechanism for loading class II molecules with immunogenic peptides from several endocytic compartments.

B-Lymphocytes↗

PubMed1993

Recognition of virus-infected cells by natural killer cell clones is controlled by polymorphic target cell elements.

Natural killer (NK) cells provide a first line of defense against viral infections. The mechanisms by which NK cells recognize and eliminate infected cells are still largely unknown. To test whether target cell elements contribute to NK cell recognition of virus-infected cells, human NK cells were cloned from two unrelated donors and assayed for their ability to kill normal autologous or allogeneic cells before and after infection by human herpesvirus 6 (HHV-6), a T-lymphotropic herpesvirus. Of 132 NK clones isolated from donor 1, all displayed strong cytolytic activity against the NK-sensitive cell line K562, none killed uninfected autologous T cells, and 65 (49%) killed autologous T cells infected with HHV-6. A panel of representative NK clones from donors 1 and 2 was tested on targets obtained from four donors. A wide heterogeneity was observed in the specificity of lysis of infected target cells among the NK clones. Some clones killed none, some killed only one, and others killed more than one of the different HHV-6-infected target cells. Killing of infected targets was not due to complete absence of class I molecules because class I surface levels were only partially affected by HHV-6 infection. Thus, target cell recognition is not controlled by the effector NK cell alone, but also by polymorphic elements on the target cell that restrict NK cell recognition. Furthermore, NK clones from different donors display a variable range of specificities in their recognition of infected target cells.

Clone Cells↗

PubMed1993

Infection of natural killer cells by human herpesvirus 6.

Natural killer (NK) cells are a functionally defined subset of non-T, non-B lymphocytes of bone marrow origin, which induce lysis of selected target cells, including neoplastic and virus-infected cells. The NK cell function provides an important mechanism of primary defence against viruses in vivo, as demonstrated by the occurrence of multiple herpesvirus infections in patients congenitally lacking NK cells. Here we show that functionally competent CD3- NK clones can be productively infected by human herpesvirus 6 (HHV-6), a T-lymphotropic DNA virus that may play a role in the acquired immunodeficiency syndrome (AIDS) and in the chronic fatigue syndrome, two disorders associated with a defective NK cell activity. The infection is cytopathic and induces de novo expression of CD4, an antigen not expressed within the NK lineage, thereby predisposing NK cells to infection by human immunodeficiency virus type 1 (HIV-1). These results provide evidence that a herpesvirus can directly target and kill NK cells, a potential strategy to suppress the natural anti-viral immunity of the host.

Adult↗

PubMed1992

Distinct binding sites on HLA-DR for invariant chain and staphylococcal enterotoxins.

During biosynthesis, class II molecules of the major histocompatibility complex exist as complexes of the polymorphic alpha and beta chains in association with trimers of the invariant chain (Ii). The nonpolymorphic Ii contains sequences necessary for proper targeting of class II to endosomal compartments, where Ii is degraded. Ii also prevents the premature association of antigenic peptides with class II molecules. It is not known whether the effect of Ii on peptide binding extends to other ligands of class II, specifically exogenous superantigens. Cells expressing a mutant Ii molecule stably associated with HLA-DR at the cell surface were tested for their ability to interact with staphylococcal toxins. Most toxins (staphylococcal enterotoxins A-E and exfoliative toxin) were found to bind to cells expressing this alpha beta Ii complex with levels comparable to cells expressing only alpha beta chains at the cell surface. Cells expressing surface alpha beta Ii complexes stimulated polyclonal populations of peripheral blood T cells in association with these toxins. Binding of toxic shock syndrome toxin (TSST) and subsequent stimulation of T cells were reduced by the presence of the Ii. This reduction was not due to an alteration in the repertoire of T cells responding to TSST in the presence of Ii. Data from experiments with a T-cell clone suggest that interactions between class II molecules and T-cell antigen receptors occur during staphylococcal enterotoxin-mediated stimulation and that surface Ii does not interfere with such interactions.

Antigens, Bacterial↗

PubMed1992

MHC class II deletion mutant expresses normal levels of transgene encoded class II molecules that have abnormal conformation and impaired antigen presentation ability.

Successive transfers of HLA-DR alpha and beta genes restored expression of HLA-DR antigens to human B-lymphoblastoid cell line, LCL .174, from which all known expressible class II genes are deleted. While transferent cells displayed large amounts of DR on their surfaces, transgene-encoded DR3 molecules lacked a conformation-dependent epitope. DR1-restricted CTL lysis of DR1-expressing transferents pulsed with native influenza virus proteins was greatly reduced; the same cells were efficiently lysed in the presence of CTL-recognized influenza peptides. The properties of DR-expressing transferents of .174 suggest they are defective in producing peptides from exogenous proteins or in forming DR/peptide complexes. Comparison with other DR-expressing deletion mutants indicates that at least one gene in an approximately 230 kb DNA segment between the DQ1 and Ring 7 loci is needed for normal DR-mediated processing and presentation. Production of DR3 molecules having the conformation-dependent 16.23 epitope and efficient DR1-restricted presentation of influenza viral epitopes occurred in a B cell line that has a mutation specifically eliminating expression of the TAP1 transporter gene, which is in the approximately 230 kb interval and is needed for production of HLA class I/peptide complexes.

Antibodies, Monoclonal↗

PubMed1992

Processing pathways for presentation of cytosolic antigen to MHC class II-restricted T cells.

Antigens presented to CD4+ T cells derive primarily from exogenous proteins that are processed into peptides capable of binding to class II major histocompatibility complex (MHC) molecules in an endocytic compartment. In contrast, antigens presented to CD8+ T cells derive mostly from proteins processed in the cytosol, and peptide loading onto class I MHC molecules in an early exocytic compartment is dependent on a transporter for antigen presentation encoded in the class II MHC region. Endogenous cytosolic antigen can also be presented by class II molecules. Here we show that, unlike class I-restricted recognition of antigen, HLA-DR1-restricted recognition of cytosolic antigen occurs in mutant cells without a transporter for antigen presentation. In contrast, DR1-restricted recognition of a short cytosolic peptide is dependent on such a transporter. Thus helper T-cell epitopes can be generated from cytosolic antigens by several mechanisms, one of which is distinct from the classical class I pathway.

Amino Acid Sequence↗

PubMed1992

Sequences in both class II major histocompatibility complex alpha and beta chains contribute to the binding of the superantigen toxic shock syndrome toxin 1.

Class II major histocompatibility complex (MHC) molecules present peptides derived from processed antigen to antigen-specific CD4-positive T cells. In addition, class II molecules bind with high affinity another class of antigens, termed superantigens. T cell stimulation by superantigens depends almost exclusively on the V beta segment expressed by the T cell receptor (TCR). Mapping of the superantigen binding site on class II molecules should provide valuable information on how MHC and TCR molecules interact. Recombinant mouse I-A class II molecules expressed on transfected L cells were analyzed for their ability to bind the toxic shock syndrome toxin 1. Polymorphic residues in the alpha helices of both the alpha and beta chains of I-A contributed to quantitative toxin binding, suggesting that the toxin binds to either a combinatorial or a conformational site on class II MHC molecules.

Animals↗

PubMed1992

Identification of HLA-DR1 beta chain residues critical for binding staphylococcal enterotoxins A and E.

Superantigens are thought to make external contacts with major histocompatibility complex (MHC) class II molecules and with the V beta portion of a T cell antigen receptor (TCR), thereby stimulating entire families of T cells. The precise mapping of superantigen binding sites on class II molecules may provide valuable information on how TCR and MHC molecules interact. Two bacterial superantigens, staphylococcal enterotoxins A and E (SEA/SEE) bind well to most HLA-DR alleles, but poorly to HLA-DRw53. The sequences responsible for this binding were localized to the putative alpha helix of the DR beta chain by measuring toxin binding to a panel of chimeric class II molecules expressed on transfected cells. Binding of SEA/SEE to the DRw14 (Dw9) molecule suggested that the conserved histidine 81 in the beta chain of most DR molecules was important, whereas the tyrosine 81 in the DRw53 beta chain was detrimental for high-affinity binding. To prove this, reciprocal point mutations were introduced in the DR1 and DRw53 beta chains. Mutation of histidine 81 in the DR1 beta chain to tyrosine reduced SEA/SEE binding, but did not prevent recognition of two DR1-restricted peptides by six of eight antigen-specific T cell lines. Conversely, introduction to histidine at position 81 in the DRw53 beta chain restored normal levels of SEA/SEE binding. These data suggest that a binding site of SEA and SEE lies on the outer face of the beta chain alpha helix, pointing away from the antigen-binding groove.

Amino Acid Sequence↗

PubMed1992

Stable surface expression of invariant chain prevents peptide presentation by HLA-DR.

Class II major histocompatibility complex (MHC) molecules are cell surface glycoproteins that bind and present immunogenic peptides to T cells. Intracellularly, class II molecules associate with a polypeptide referred to as the invariant (Ii) chain. Ii is proteolytically degraded and dissociates from the class II complex prior to cell surface expression of the mature class II alpha beta heterodimer. Using human fibroblasts transfected with HLA-DR1 and Ii cDNAs, we now demonstrate that truncation of the cytoplasmic domain of Ii results in the failure of Ii to dissociate from the alpha beta Ii complex and leads to stable expression of class II alpha beta Ii complexes on the cell surface. Furthermore, biochemical analysis and peptide presentation assays demonstrated that transfectants with stable surface alpha beta Ii complexes expressed very few free alpha beta heterodimers at the surface and were very inefficient in their ability to present immunogenic peptides to T cells. These results support the hypothesis that the cytoplasmic domain of Ii is responsible for endosomal targeting of alpha beta Ii and directly demonstrate that association with Ii interferes with the antigen presentation function of class II molecules.

Antigens, Differentiation, B-Lymphocyte↗

PubMed1992

Antigen processing for presentation to CD4+ T cells.

The immune system surveys the organism for the presence of foreign or abnormal structures. An important role in the immune response is assumed by T lymphocytes that recognize foreign antigen while tolerating self-proteins. T lymphocytes can recognize only peptide fragments that are presented to them by molecules of the major histocompatibility complex (MHC). Antigen processing for presentation to T cells involves distinct cellular compartments where peptides and MHC molecules interact. Whereas class I MHC molecules (recognized by CD8+ cytotoxic T cells) acquire peptides in an early biosynthetic compartment, class II molecules (recognized by CD4+ helper T cells) acquire peptides most efficiently in an endocytic compartment. It has emerged recently that the class II processing compartment can be fed not only from the outside with exogenous antigen but also from endogenous sources, including membrane-associated and cytosolic proteins. The potential sources of proteins that can trigger a helper T cell response during viral infections and that can induce self-tolerance are thus much wider than previously anticipated.

Animals↗

PubMed1991

A myelin basic protein peptide is recognized by cytotoxic T cells in the context of four HLA-DR types associated with multiple sclerosis.

We have examined previously the peptide specificity of the T cell response to myelin basic protein (MBP) in patients with multiple sclerosis (MS) and healthy controls, and demonstrated that an epitope spanning amino acids 87-106 was frequently recognized. Because this region is encephalitogenic in some experimental animals, it has been postulated that the response to the epitope may have relevance to MS. In this study, the fine specificity of this response is studied using four well-characterized, monospecific T cell lines from three MS patients and an identical twin of a patient. Each of the lines recognized a peptide with the same core sequence, amino acids 89-99, although the responses were affected to various degrees by truncations at the COOH- or NH2 terminal ends of the 87-106 epitope. Importantly, the epitope was recognized in conjunction with four different HLA-DR molecules. Also, the T cell receptor beta chain usage was heterogeneous, and each line expressed a different VDJ sequence. The four HLA-DR molecules restricting the response to this epitope have been shown to be overrepresented in MS populations in various geographic areas, suggesting that the response to this region of the MBP molecule may be relevant to the pathogenesis of MS. These findings may have important implications in designing therapeutic strategies for the disease.

Amino Acid Sequence↗

PubMed1991

An unusual form of alternative splicing in the HLA-DNA gene.

The HLA-DNA gene of the human major histocompatibility complex (MHC) produces RNA transcripts of unusual size compared to the 1.3 kilobase (kb) transcripts of other class II genes. Several cDNA clones derived from HLA-DNA transcripts were isolated, including a full-length 3.5 kb cDNA named DN1. Sequence analysis of DN1 demonstrated that the 3.5 kb transcript is fully spliced, contains a long 3' untranslated region, and encodes an apparently normal class II alpha chain. Two other cDNA clones, DN2 and DN7, revealed an alternative splice that resulted from the use of a splice acceptor upstream of the second exon encoding the alpha 1 domain. The translation product of this alternative transcript would share the signal sequence of the DN alpha chain but would otherwise be unrelated. Removal of the signal sequence would yield a highly basic peptide of 42 amino acids. RNA was prepared by in vitro transcription of DN1 and DN2, and was used to direct protein synthesis in a rabbit reticulocyte system. The product of DN1 had an apparent relative mass of 25,000, whereas the product of DN2 was a peptide with a relative mass of about 6000. The alternatively spliced RNA represents about 1/10 of the transcripts from the HLA-DNA gene in the cells tested, which included transformed B-cell lines as well as normal blood cells and thymus cells.

Amino Acid Sequence↗

PubMed1991

Efficient cDNA expression vectors for stable and transient expression of HLA-DR in transfected fibroblast and lymphoid cells.

cDNA expression vectors with several useful features were constructed. First, the long terminal repeat of Rous sarcoma virus was used as a promoter to obtain high levels of expression in various cells of human and mouse origin. Second, cis-linked expression units that confer resistance either to mycophenolic acid or the neomycin analog G418 were inserted to facitate the isolation of transfected cells expressing the cDNA of interest. Third, by replicating in simian COS cells, these vectors can be used for efficient transient expression. cDNA fragments encoding the DR alpha or DR beta chains of human class II major histocompatibility complex antigens were inserted into these vectors and high levels of cell surface HLA-DR antigen were obtained after cotransfection into mouse and human fibroblasts. These vectors were also successfully used to correct the inability of a class II-negative B cell line, derived from a patient with a congenital immunodeficiency, to present peptide antigen to DR-restricted T cells.

Animals↗

PubMed1991

Activated CD3- CD16+ natural killer cells express a subset of the lymphokine genes induced in activated alpha beta + and gamma delta + T cells.

In this study we analysed the potential of highly purified polyclonal TcR alpha beta+, TcR gamma delta + and CD3- NK cells, to produce lymphokines in response to mitogenic stimulation. RNA hybridizations were performed to detect with high sensitivity the induction of multiple lymphokine genes. Upon stimulation with lectin and phorbol ester TcR gamma delta + lymphocytes expressed the same set of lymphokine genes as the TcR alpha beta + lymphocytes expressed the same set of lymphokine genes as the TcR alpha beta + lymphocytes, which included IL-2, -3, -4, -5, GM-CSF, TNF alpha and beta, IFN gamma. In contrast, a more limited set of lymphokine genes (GM-CSF, TNF alpha and beta, IFN gamma) was induced in activated CD3- NK cells, thus indicating that this subpopulation of cells may display different regulatory functions, with respect to CD3+ T lymphocytes.

Antigens, Differentiation↗

PubMed1990

HLA-DR2a is the dominant restriction molecule for the cytotoxic T cell response to myelin basic protein in DR2Dw2 individuals.

The HLA-DR2 restriction of the T cell response to myelin basic protein (MBP) was studied using murine L cells transfected with DRalpha and either DR2a or DR2b beta-chain cDNA. DR2a and DR2b represent the two isotypic DRbeta chains expressed in DR2Dw2 haplotypes. Eleven MBP-specific cytolytic T cell lines derived from patients with multiple sclerosis were isolated. Two of these cell lines recognized MBP-pulsed DR2-expressing L cell transfectants and four of them could only recognize the L cells if the adhesion molecule ICAM-1 was expressed in addition to HLA-DR2. Five of the six lines were restricted by HLA-DR2a; one line recognized Ag in conjunction with DR2b, but only if ICAM-1 was coexpressed. The remaining five lines did not lyse MBP-pulsed L cells. The ability of the DR2b molecules on transfected cells to stimulate T cells was confirmed with DR2b-allospecific T cell clones. Although five MBP-specific lines were restricted by DR2a, they recognized different parts of the MBP molecule, as demonstrated by the presentation of shorter peptides. Thus, our results suggest that DR2a is a dominant restriction molecule in MBP-specific responses by DR2+ MS patients. The results also indicate that the reported heterogeneity in MBP epitopes recognized by DR2-restricted T cells, may not be due to the use of different restriction elements but rather to the binding of different MBP peptides to DR2a molecules.

Animals↗