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Biomedical subjects

E O Long

Publications and source records attributed to E O Long.

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

An endogenous processing pathway in vaccinia virus-infected cells for presentation of cytoplasmic antigens to class II-restricted T cells.

The recognition of virus-infected cells by class I MHC-restricted cytotoxic T cells requires endogenous processing of antigen for presentation. It is still unclear whether endogenous processing of antigen can be utilized by class II MHC molecules for presentation. To test this possibility, a human B cell line expressing HLA-A2 and HLA-DR1 was infected with a recombinant vaccinia virus expressing the Influenza A virus M1 matrix protein (VAC-M1) and was assayed for lysis by different M1-specific cytolytic T cell lines, restricted by either HLA-A2 or by HLA-DR1. Class II-restricted lysis of VAC-M1-infected cells did occur. This lysis required de novo M1 synthesis and was not due to exogenous antigen. Several properties of the endogenous processing pathway for class II-restricted presentation were different from those of the pathway utilized by class I molecules. First, class II-mediated recognition of VAC-M1 infected cells was less efficient, requiring higher doses of virus and longer infection times, than the class I-mediated recognition. Second, chloroquine completely blocked presentation of endogenous M1 to class II-restricted T cells but had no effect on the class I-restricted presentation. Third, the class II-restricted presentation of M1 was only mildly affected by Brefeldin A, a drug that prevents transport from the endoplasmic reticulum to the Golgi, whereas the class I-restricted presentation of M1 was completely abrogated by this drug. These data demonstrate the existence of an endogenous processing pathway for the presentation of cytosolic antigen by class II molecules and show that this pathway is distinct from the one used for presentation by class I molecules.

Anti-Bacterial Agents↗
PubMed1990

The alpha 1 domain of the HLA-DR molecule is essential for high-affinity binding of the toxic shock syndrome toxin-1.

Several exoproteins from the bacterium Staphylococcus aureus are highly potent polyclonal activators of T cells in the presence of cells bearing class II antigens of the major histocompatibility complex (MHC). These toxins, including the toxic shock syndrome toxin (TSST-1), act at nanomolar concentrations, bind directly to class II molecules, and do not require the processing typical of nominal antigen. Each toxin is capable of stimulating a subpopulation of peripheral T lymphocytes bearing particular V beta sequences as part of their alpha beta T-cell receptors. It is not known how these so-called 'superantigens' bind to class II and how this binding stimulates T cells. In this study, the different affinities of TSST-1 for human class II molecules DR and DP were exploited to define the region of a class II molecule necessary for high-affinity binding. Using chimaeric alpha- and beta-chains of DR and DP expressed at the surface of transfected murine fibroblasts and a binding assay with TSST-1, it was shown that the alpha 1 domain of DR is essential for high-affinity binding, and further that TSST-1 binding did not prevent subsequent binding of a DR-restricted antigenic peptide. This is compatible with a model of superantigen making external contacts with both class II and T cell receptor, and suggests that the V beta portion of the T-cell receptor interacts with the nonpolymorphic alpha-chain of DR.

Animals↗
PubMed1990

Fine specificity and HLA restriction of myelin basic protein-specific cytotoxic T cell lines from multiple sclerosis patients and healthy individuals.

Myelin basic protein (MBP) is a candidate Ag for the autoimmune process believed to be involved in the pathogenesis of multiple sclerosis (MS). To investigate the fine specificity and HLA restriction of human MBP-specific CTL, long term T cell lines (TCL) were established from 22 MS patients and 16 healthy individuals by repeated antigenic restimulation. By using this approach, MBP-specific cytotoxic TCL were generated from 81% of the lines from MS patients and 69% of those from controls. TCL from both groups expressed the CD3+, CD4+, CD8- phenotype and secreted substantial amounts of IFN-gamma. By using large enzymatic and small synthetic peptides of MBP, TCL were primarily specific for the C-terminal part of the molecule and to a lesser extent for the N-terminal portion. Two regions of the molecule, MBP peptide 87-106 and MBP peptide 154-172, were recognized by the majority of the polyspecific lines and by four and three of 14 monospecific TCL, respectively. These highly immunogenic regions are of interest because they include sequences encephalitogenic in other species. The HLA restriction of each line was determined by using antibody blocking as well as various target cells including EBV-transformed B cells, homozygous typing cells, and fibroblasts transfected with cDNA for DR-alpha and DR-beta genes. All TCL were restricted by HLA-DR Ag. Several HLA-DR molecules restricted multiple cathepsin D-derived and synthetic MBP peptides, including the regions of peptides 87-106 and 154-172 which, respectively, were recognized in conjunction with four and three HLA-DR types. Three of these HLA-DR types are overrepresented in MS patients in different geographic regions. Together, these findings suggest that the MBP-specific cytotoxic T cell response, although not sufficient for disease, may be important for the pathogenesis of MS.

Amino Acid Sequence↗
PubMed1990

Structural requirements for pairing of alpha and beta chains in HLA-DR and HLA-DP molecules.

To test for the assembly of human MHC class II molecules having an alpha chain from one isotype (HLA-DR, -DQ, or -DP) and the beta chain of another (mixed isotypic pairs), murine fibroblasts were transfected with expressible cDNAs encoding the different class II alpha and beta chains. A rapid and efficient transient transfection system was developed using a polyoma virus-based vector. Typically, 30-50% of cells transfected using this system expressed high levels of class II molecules on their surface, but only with matched isotypic pairs. Biochemical analysis of cells transfected with matched or mixed isotypic pairs of the DR and DP molecules revealed that only matched chains could pair efficiently inside the cell. Thus, the lack of expression of the two mixed isotypic pairs is due to inefficient primary assembly of the class II molecule and not to a processing or transport defect. To define what region of the beta chains controlled their assembly with alpha chains, a series of chimeric cDNA molecules containing both DR and DP beta chain sequences were constructed. Expression of these chimeric beta chains with DR and DP alpha chains was determined by cytofluorimetry and biochemical analysis. Both alpha chains paired with beta chains in which only the beta 1 domain was isotypically matched. In contrast, the pattern of expression of chimeras made at other points within the beta 1 domain was different for DR and DP. These data show that different areas of primary sequence are important for the assembly of different human class II isotypes, and suggest that HLA-DR and -DP molecules have different secondary or tertiary structures in their NH2-terminal domains.

Animals↗
PubMed1989

Transcription of unrearranged T cell receptor delta genes in CD3- major histocompatibility complex-unrestricted cytotoxic cells.

Transcription of the T cell receptor (TcR) delta gene was detected in clones and in enriched populations of CD3- major histocompatibility complex-unrestricted cytotoxic cells. The transcripts were derived, at least in part, from unrearranged TcR delta genes, as shown by hybridization to a synthetic oligonucleotide probe corresponding to germ-line sequences just upstream of J delta 1. Transcripts from unrearranged TcR delta genes are similar in size to those from productively rearranged genes. The fact that the bulk of delta transcripts was derived from unrearranged genes was supported by the observation that TcR delta gene rearrangements were not detected in DNA isolated from an enriched CD3- cytotoxic cell population. Transcription of the TcR delta gene in CD3- cytotoxic cells is consistent with the notion that these cells belong to a T cell lineage.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1989

Class II MHC molecules and the HIV gp 120 envelope protein interact with functionally distinct regions of the CD4 molecule.

A murine T cell hybridoma with a receptor specific for the class I molecule H-2 Dd was transfected with an expressible cDNA for human CD4. Expression of the human class II MHC molecule HLA-DP on Dd-positive murine fibroblasts resulted in a greatly enhanced response of the CD4-positive T cell hybridoma, measured either by lymphokine production or by rosette formation. Inhibition of these functional assays with anti-CD4 monoclonal antibodies implicated the two amino-terminal domains of CD4 in an interaction with the HLA-DP molecule. This interaction was blocked by incubation with recombinant gp120 envelope protein of HIV. In contrast, recombinant soluble CD4 did not inhibit and was able to prevent the inhibition by gp120. Anti-CD4 antibody blocking experiments clearly indicated that distinct regions of CD4 interact respectively with gp120 and with class II MHC molecules.

Animals↗
PubMed1989

Pathways of viral antigen processing and presentation to CTL: defined by the mode of virus entry?

Processing requirements for antigen presentation to cytotoxic T lymphocytes (CTL) vary among viruses and between major histocompatibility complex (MHC) class I- and class II-restricted responses to the same virus. In this article, Eric Long and Steven Jacobson argue that the mode of virus entry may define processing pathways and that the invariant chain associated with MHC class II molecules may account for the distinct processing requirements for MHC class I- and class II-restricted CTL.

Animals↗
PubMed1989

HLA class II-restricted presentation of cytoplasmic measles virus antigens to cytotoxic T cells.

To analyze the nature of the HLA class II-restricted cytotoxic T-lymphocyte (CTL) response to measles virus, murine fibroblasts were transfected with expressible cDNA clones for human HLA-DR antigen and for measles virus matrix or nucleocapsid proteins. DR-positive murine fibroblasts transfected with measles virus matrix or nucleocapsid genes were lysed by class II-restricted measles virus-specific CTL lines. Lysis was as efficient as with infected autologous B-cell lines, even though the measles virus cytoplasmic proteins were undetectable by antibodies in the transfected target cells. These results demonstrate that cytoplasmic viral antigens can be presented to CTL in the context of HLA class II antigens and that measles virus matrix and nucleocapsid proteins contribute to class II-restricted measles virus-specific CTL responses. These results also show that endogenously synthesized measles virus proteins can be efficiently presented by class II antigens. The implications of these findings for measles virus pathogenesis and for antigen processing are discussed.

Animals↗
PubMed1988

Specific lysis of allogeneic cells after activation of CD3- lymphocytes in mixed lymphocyte culture.

Human CD3- lymphocyte populations were obtained by treating peripheral blood lymphocytes with mAbs directed to CD3, CD4, and CD8 surface antigens. The resulting populations were cultured with irradiated allogeneic cells; at day 4, 100 U/ml IL-2 were added and cultures continued for an additional 10 d. The resulting populations were CD3-CD2+CD7+ and displayed cytolytic activity against PHA-induced blast cells bearing the stimulating alloantigens but not against autologous or unrelated allogeneic blast cells. When CD3- populations were cultured with irradiated autologous cells, no cytolytic activity could be detected either against autologous or allogeneic blast cells. On the other hand, K562 target cells were lysed by both MLC-derived CD3- cell populations regardless of the origin (autologous or allogeneic) of the stimulating cells. CD3- clones were further derived from MLC-stimulated CD3- populations. These clones displayed a cytolytic pattern similar to the original MLC populations as only specific PHA blasts could be lysed. These clones did not express detectable surface TCR-alpha/beta or -gamma/delta molecules and lacked productive mRNA for TCR alpha and beta chains, while small amounts of TCR-gamma mRNA were detectable in one of four clones tested. Also mRNA for CD3 gamma and delta chains were undetectable in all clones, however, CD3 epsilon mRNA was consistently present.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1988

Structural and functional studies of HLA-DR restricted antigen recognition by human helper T lymphocyte clones by using transfected murine cell lines.

Murine L cells expressing the products of transfected HLA-DR1 genes functioned as APC for two influenza-specific, human Th cell clones with comparable efficiency to a DR1-expressing human lymphoblastoid cell line. In order to investigate the restriction specificity of the two Th clones, a transfectant expressing the species-mismatched MHC class II dimer DR1:I-E was tested as an APC. Both T cells showed no loss of Ag sensitivity due to substitution of the murine chain. One of the Th clones, TLC 72, showed even greater degeneracy by responding to Ag in the context of I-Ek. Taking into account the lower level of MHC class II expression on the I-Ek transfectant, there is remarkably little loss of efficiency of Ag-induced T cell activation due to the substitution of I-E for DR as restriction element. The Ag-specific responses of both clones were inhibited by anti-CD4 antibody when DR-transfected L cells or human lymphoblastoid cells were used as APC. This inhibition was also seen when Ag was presented to TLC72 by the I-Ek-expressing transfectant. Whether this inhibition is the result of negative signaling or of blocking an interaction between human CD4 and I-Ek is discussed. Similarly the inhibitory effects of mAb against the T cell accessory molecule LFA/1 were the same for both clones when either the transfectants or the lymphoblastoid cell line were used as APC, suggesting that L cells may express a molecule that is capable of acting as a ligand for human LFA/1. The results presented here further illustrate the value of transfectants in analyzing T cell recognition and accessory cell requirements. The patterns of degeneracy of MHC restriction exhibited by these clones provides a platform for a more detailed analysis of key residues involved in MHC class II-restricted T cell Ag recognition.

Amino Acid Sequence↗
PubMed1988

CD3-negative lymphokine-activated cytotoxic cells express the CD3 epsilon gene.

The expression of genes encoding different polypeptide chains of the TCR-CD3 complex was analyzed in a panel of cloned MHC-unrestricted cytotoxic cells. The clones were derived from CD3+ and CD3- human PBL. After expansion in rIL-2, all clones were able to lyse the NK-sensitive target cell line K562. In contrast, lysis of fresh tumor cells was achieved almost exclusively by CD3- clones. To test whether a known TCR-CD3 complex may be involved in MHC-unrestricted cytotoxicity, total RNA from nine CD3+ and 11 CD3- clones was isolated and hybridized with DNA probes for the TCR alpha-, beta-, and gamma-chains and for the CD3 gamma-, delta-, and epsilon-chains. TCR gamma transcripts were present at high levels in CD3+CD4- CD8- clones but were undetectable in all CD3- clones. Lysis of fresh tumor cells is an activity which can be independent of the TCR alpha beta and TCR gamma complexes because the CD3- clones did not express these TCR genes. Interestingly, all CD3- clones expressed CD3 epsilon transcripts, but not CD3 gamma- or delta-transcripts. CD3- lymphokine-activated cytotoxic cells may therefore be derived from immature T cells which do not yet express a complete CD3 complex. The CD3 epsilon chain, if expressed in CD3- cells in association with other molecules, could be involved in the activation and lytic function of these MHC-unrestricted cytotoxic cells.

Antigens, Surface↗
PubMed1988

Structural model of HLA-DR1 restricted T cell antigen recognition.

Two human helper T cell determinants in influenza have been identified, one in the hemagglutinin and the other in the matrix protein (M1). Both were shown to be DR1 restricted by using transfected L cells to present antigen. Comparison of the sequences of the two peptides revealed a similar pattern that could account for their DR1 specificity if the peptides adopt a helical conformation. The model was supported by the demonstration that hybrid peptides, composed of the amino acids that interact with DR1 from one determinant and the residues that interact with the T cell receptor from the other, were recognized by each clone. The generality of the motif was confirmed by the finding that DR1 individuals respond to a ragweed peptide containing the defined pattern.

Allergens↗
PubMed1988

Presentation of influenza hemagglutinin peptide in the presence of limited allostimulation by HLA-DR1 transfected human fibroblasts.

A human fibroblast expressing HLA-DR1 antigen on its surface was generated by transfection with DR alpha and DR beta cDNAs. The ability of this transfected fibroblast line to process and to present antigens was analyzed by using human T-lymphocyte clones (TLCs) specific for HLA-DR1 alloantigen or restricted by DR1 in their recognition of influenza virus. TLC responses were measured in proliferative assays and were tested for blocking by monoclonal antibodies specific for MHC antigens. Two TLCs specific for a discrete segment (aa 306-320) of the influenza hemagglutinin molecule responded to the antigen added in peptide form but not as intact virion. The transfected fibroblast line thus appears unable to process antigen properly. A DR1-alloreactive TLC was able to respond to the transfected fibroblast. However, 24 other DR1-alloreactive TLCs and oligoclonal T-cell lines were unable to respond. These results suggest either that the conformation of the DR1 molecule on a transfected fibroblast allows peptide presentation but not allorecognition, or that self antigen processing and subsequent presentation by MHC antigens is necessary for allorecognition.

Antibodies, Monoclonal↗
PubMed1988

Antigen presentation to HLA class II-restricted measles virus-specific T-cell clones can occur in the absence of the invariant chain.

A human fibroblast line expressing HLA-DR1 antigen on its surface was generated by transfection with DR alpha and DR beta cDNAs. Expression of the invariant chain gene was not detectable in the transfected fibroblasts and was not induced by infection with measles virus. Lysis of measles virus-infected cells occurred with DR1- but not with DR4-restricted measles virus-specific cytotoxic T-lymphocyte (CTL) clones and was inhibited by a monoclonal antibody specific for DR antigen. Therefore, the invariant chain is not required for DR-restricted presentation of measles virus antigens by this fibroblast line. Transfected fibroblasts were lysed as efficiently as an autologous B-cell line even though they expressed much less surface DR antigen. Lysis of both the transfected fibroblasts and the B-cell line was insensitive to treatment with chloroquine. These results demonstrate that expression of a DR alpha beta heterodimer at the surface of this fibroblast line is necessary and sufficient for presentation of measles virus antigens to specific CTL clones.

Antigen-Presenting Cells↗
PubMed1987

Expression patterns of MHC class II genes in rabbit tissues indicate close homology to human counterparts.

Genomic clones corresponding to five distinct major histocompatibility complex class II alpha-chains have been described for the rabbit; four of these encode complete, potentially functional alpha-chains. Hybridization analysis and preliminary sequence analysis indicate that one of these clones is structurally related to HLA-DP alpha, one to -DR alpha, one to -DQ alpha and one to -DZ alpha. Probes specific for the four class II genes were used to screen RNA samples from normal rabbit tissues to determine which of these genes are transcribed and whether expression of any particular gene is tissue specific. All four of the genes are transcribed, but there are variations in the levels of expression, in tissue distribution, and in transcript size. The highest levels of RLA-DR alpha, -DQ alpha, and -DP alpha transcription were found in lymphoid tissues. Lower levels of transcription were also detectable in several nonlymphoid tissues. Transcripts observed were about 1.3 kb, a size expected for these class II alpha-chain genes based on experience with their human homologues. The RLA-DZ alpha probe corresponding to HLA-DZ alpha hybridized weakly with a band of 3.6 kb; its expression could be detected only in lymphoid tissues. The size of the DZ alpha transcript, its tissue distribution, and partial sequence data confirm its homology with the human gene DZ alpha. In blots of total cellular RNA, a probe for a recently described human beta-chain, DO beta, hybridized to a transcript of about 1.3 kb in lymphoid tissues. These data indicate that RNA transcripts corresponding to all HLA class II loci described to date can be detected in rabbit tissues.

Animals↗
PubMed1986

Two forms of the Ia antigen-associated invariant chain result from alternative initiations at two in-phase AUGs.

The Ia antigen-associated invariant chain (In) exists in humans as two major related forms, p33 and p35. The mRNA for In contains two in-phase AUGs, at positions 8 and 56 from the cap site. Cells transfected with a full-length cDNA clone in an expression vector synthesize both p33 and p35. Cell-free translation of mRNA synthesized in vitro from cDNA also produces both forms. When the first ATG is deleted from the cDNA clone, only the smallest form of In is produced. Mutations introduced at the second ATG lead to synthesis of the large form only. The alternative use of two in-phase AUGs on a unique mRNA is thus responsible for the synthesis of p33 and p35. This is the first documented example of such a mechanism in nonviral systems.

HLA-D Antigens↗