Biomedical subjects

W A Jefferies

Publications and source records attributed to W A Jefferies.

At least 37 records · Page 2Linked to original sources

PubMed1993

Mycobacteria-macrophage interactions. Macrophage phenotype determines the nonopsonic binding of Mycobacterium tuberculosis to murine macrophages.

During tuberculosis, host defenses may be determined, in part, by the capacity of resident, elicited, and activated macrophages to bind and ingest Mycobacterium tuberculosis. We have investigated the mechanism by which macrophages bind M. tuberculosis and other mycobacteria in a serum-free system. The extent of binding of M. tuberculosis to macrophages was dependent on the phenotype of the macrophage; thioglycollate-elicited and immune-activated macrophages bound mycobacteria poorly, whereas resident macrophages bound mycobacteria efficiently. Within 'freshly' explanted macrophage populations (from 2 to 24 h in vitro) poor binding of mycobacteria correlated with poor binding of C3bi-coated particles, but not with variations in the level of complement receptor 3 (CR3) expression. Induction of C3bi-coated particle binding in thioglycollate-elicited macrophages by PMA was not accompanied by enhanced M. tuberculosis binding. Inhibition of M. tuberculosis binding by resident macrophages could only be achieved using a mAb recognizing an epitope within CR3 distinct from that which recognizes C3bi. Our results suggest that nonopsonic binding of M. tuberculosis is mediated by a site within CR3, which is distinct from the C3bi binding site. In addition, we show a variation in the capacity of different macrophage phenotypes to bind mycobacteria nonopsonically. These data suggest that heterogeneity in macrophage-mediated clearance of M. tuberculosis may be a significant factor in the progression of tuberculosis.

Animals↗

PubMed1993

IFN-gamma-induced recognition of the antigen-processing variant CMT.64 by cytolytic T cells can be replaced by sequential addition of beta 2 microglobulin and antigenic peptides.

The present study describes the functional nature of the MHC class I determinants expressed in CMT.64 cells and was undertaken to define and further analyze the deficiency in the cell line CMT.64 in the hope of elucidating the relative functional importance of constituent parameters in the recognition of these cells by CTL. We show that induction of Kb in CMT.64 cells with IFN-gamma results in molecules capable of presenting VSV epitopes to the appropriate CTL. However, cells untreated with IFN-gamma and infected with VSV are not recognized by VSV-specific CTL. This study reveals that beta 2 m3 is synthesized in limiting amounts in uninduced CMT.64 and becomes highly expressed after IFN-gamma induction. Thus, the limiting amount of beta 2 m expressed in uninduced cells may partially explain the inability of the cells to present viral components of CTL recognition. This concept is reinforced by the experiments identifying two functional effects upon the addition of immunogenic peptides to uninduced CMT.64 cells: at high peptide concentrations in excess of 5 nM, CMT.64 cells are recognized efficiently after 5 min of incubation; at the limiting peptide concentration of 500 pM, uninduced CMT.64 cells are only recognized providing beta 2m is added before or simultaneously with the antigenic peptide. BFA, an inhibitor of protein transport, and emetine, an inhibitor of protein synthesis, were used to show that at high peptide concentrations, 25 microM, recognition takes place after the peptide has stabilized the limited amount of newly arriving MHC/beta 2m complexes, devoid of peptides, at the cell surface of uninduced CMT.64 cells. These experiments thereby exclude the possibility that peptides are taken up into CMT.64 cells for assembly, transport and surface expression of functional MHC/beta 2m/peptide complexes. In summary, our data expands previous research showing the importance of exogenous beta 2m in sensitizing cells for CTL recognition with peptides added exogenously. These functional experiments also imply that the concentration of endogenous beta 2m may regulate the amount of MHC class I expressed at the cell surface and receptive to exogenous peptides. Finally, the phenotype of CMT.64 cells we describe provides evidence of the complexity of the Ag-presenting capacity of this cell line not previously identified in other studies on these cells, thus revising our understanding of the Ag-processing deficiency in these cells.

Animals↗

PubMed1993

Differential recruitment of viral and allo-epitopes into the MHC class I antigen processing pathway of a novel mutant of Ltk- cells. HSV/MHC class I restriction/immune recognition/antigen processing/antigen presentation/influenza virus.

It is generally thought that the Ag processing pathways for endogenously synthesized proteins are the same for allo and viral Ag processing. However, this view does not take into consideration the diversity of specialized maturation and assembly pathways for viruses. In particular, viral assembly that takes place within intracellular membranes may require unique Ag processing steps. In this study we sought to assess this possibility. Hence, we describe the CTL response against a murine Ltk- cell derivative, gro29, which was previously shown to be defective in the propagation of herpes simplex virus type 1 (HSV-1). In HSV-1-infected gro29 cells, viral polypeptides are synthesized in normal amounts and viral assembly takes place. However, transport of the assembled particles is defective in these cells, resulting in the accumulation of noninfectious virus in cytoplasmic vesicles, and a reduction in the release of viral particles by at least 2000-fold. We show that the rate of transport of individual endogenous proteins through the organelles of the secretory pathway is also impaired, but only by roughly 50%, suggesting that the defect in this cell line affects the transport of particles to a greater extent than the transport of individual proteins. It is also shown that allogeneic and influenza A- specific CTL responses are indistinguishable between gro29 and Ltk- cells, as is the response against target cells pretreated with a influenza A derived synthetic peptide. By contrast, HSV-1-infected gro29 cells are approximately eightfold less sensitive than infected Ltk- cells to lysis by HSV-1-specific CTL. This illustrates that in contrast to the allogeneic and influenza specific responses, the recruitment of herpes virus-specific Ag into the Ag-processing pathway is dependent on a cellular function that is also required for viral maturation and egress. We believe that this is the first demonstration of this phenomenon.

Animals↗

PubMed1992

Characterization of the specificity and genetic restriction of human CD4+ cytotoxic T cell clones reactive to capsid antigen of rubella virus.

Using 11 overlapping synthetic peptides covering more than 95% of the amino acid sequence of capsid protein of rubella virus, 7 CD4+ T cell clones (R10, R11, R18, A2, A10, A11, and A12) isolated from 2 rubella seropositive donors reacted strongly to rubella capsid peptides C6 (residues 119-152), C9 (residues 205-233), or C11 (residues 255-280), respectively, in both proliferation and cytotoxicity assay. Truncated peptides C6E (residues 125-139), C9B (residues 205-216), and C11E (residues 260-272) were shown to be involved directly to the T cell determinants of C6, C9, and C11, respectively. Genetic restriction of these T cell clones was analyzed by using human cell lines with various HLA-DR phenotypes as targets and/or antigen-presenting cells in cytotoxicity assay and/or proliferation assays. The results indicated that the recognition of peptide C6 by T cell clones (R11 and R18) was associated with DRw9 molecule, while the HLA restriction element of the responses of other T cell clones (A2 and A11, A10, and A12) that reacted with peptide C9 or C11 was DR4 molecule. However, there may be a cross-recognition by the T cell clone (A12) between DR1 and DR4 subtypes.

Amino Acid Sequence↗

PubMed1992

Identification of T-cell epitopes on E2 protein of rubella virus, as recognized by human T-cell lines and clones.

T-cell epitopes on the E2 protein of rubella virus were studied by using 15 overlapping synthetic peptides covering the E2 protein sequence. The most frequently recognized epitopes on E2 were E2-4 (residues 54 to 74), with 5 of 10 tested T-cell lines responding to it. Two CD4+ cytotoxic T-cell cloned isolated from one T-cell line responded strongly in proliferation assays with peptide E2-4 and were cytotoxic to target cells presenting the E2-4 determinant. Truncated peptides contained within the E2-4 peptide sequence were used to define the T-cell determinants. Results indicated that amino acid residues 54 to 65 were directly involved. Human cell lines with different HLA phenotypes were tested for the capacity to present the antigenic determinants. The results suggested that recognition of peptide E2-4 by T-cell clones was associated with HLA DR7.

Amino Acid Sequence↗

PubMed1992

Latent adenoviral infection in the pathogenesis of chronic airways obstruction.

Childhood infection of the respiratory tract has been proposed as an independent risk factor in the pathogenesis of the chronic obstructive pulmonary disease (COPD) that develops in cigarette smokers. The present study examines adult lung tissue for latent adenoviral DNA because many of its 41 serotypes cause childhood respiratory disease and the virus is known to persist in other tissues in a latent form. Lung tissue resected for solitary nodules from 20 patients with airways obstruction and 20 patients without airways obstruction, matched for age, sex, and smoking history, were compared to determine whether adenoviral DNA is more commonly found in patients with COPD. The polymerase chain reaction (PCR) was used to examine two widely separated segments of the adenoviral genome. In situ hybridization (ISH) was performed using a probe covering the entire viral genome to determine the types of cell infected by the virus. The PCR analysis showed that a 675 base pair target sequence of the E1A region of the adenovirus was present in most of the lungs studied with greater copy numbers in the smokers with airways obstruction in both paraffin-embedded (p less than 0.002) and frozen lung tissue (p less than 0.016), whereas the E3/19K region showed no difference between the groups. When sufficient copy numbers were present to localize the DNA by ISH it was found in epithelial cells of the smokers who had airways obstruction. These data are consistent with a current model of adenoviral integration into host DNA and suggest that the E1A region of the adenovirus may contribute to the pathogenesis of COPD.

Adenovirus Infections, Human↗

PubMed1991

Adenovirus infection inhibits the phosphorylation of major histocompatibility complex class I proteins.

Major histocompatibility complex (MHC) class I molecules act as peptide receptors to direct the recognition of foreign antigens by cytolytic T cells. The cell surface expression and trafficking of these peptide receptors is thought to be controlled by the conformation of the MHC molecule and possibly by the phosphorylation of the cytoplasmic portion of the heavy chain protein. It is of some interest that adenoviruses (Ads) have evolved proteins that interfere with the expression of MHC molecules. One of these proteins, called E3/19k, binds to newly synthesized MHC molecules in the rough endoplasmic reticulum (RER) and inhibits their trafficking to the cell surface. Here we show that during the infection of a human cell line with Ad2, the phosphorylation of the endogenous MHC molecules is inhibited. We also observe that the phosphorylation of the endogenous HLA molecules is grossly impaired in a human cell line transfected with the Ad2 EcoRI D fragment containing the E3/19k gene. We conclude that the E3/19k protein inhibits the phosphorylation of the MHC heavy chains and that this may be one of the important functions of this protein in infected cells. In addition, we show that a mutant of the E3/19k protein, which lacks an RER retention signal but which retains its ability to bind to HLA molecules, does not inhibit the phosphorylation of HLA molecules and that phosphorylated molecules are not Endo H sensitive. This suggests that HLA molecules are phosphorylated after leaving the medial-Golgi compartment, thus providing the most compelling evidence yet that HLA molecules are phosphorylated at or near the cell surface. Finally, to our knowledge, this is the first study under which the phosphorylation of MHC molecules is shown to be altered and may have some relevance for other pathogenic conditions.

Adenoviridae Infections↗

PubMed1990

E3/19K from adenovirus 2 is an immunosubversive protein that binds to a structural motif regulating the intracellular transport of major histocompatibility complex class I proteins.

We have previously expressed in transgenic mice a chimeric H-2Kd/Kk protein called C31, which contains the extracellular alpha 1 domain of Kd, whereas the rest of the molecule is of Kk origin. This molecule functions as a restriction element for alloreactive and influenza A-specific cytotoxic T lymphocytes (CTL) but is only weakly expressed at the cell surface of splenocytes. Here, we show that the low cell surface expression is the result of slow intracellular transport and processing of the C31 protein. A set of hybrid molecules between Kd and Kk were used to localize the regions in major histocompatibility complex (MHC) molecules that are important for their intracellular transport and to further localize the structures responsible for binding to the adenovirus 2 E3/19K protein. This protein appears to be an important mediator of adenovirus persistence. It acts by binding to the immaturely glycosylated forms of MHC class I proteins in the endoplasmic reticulum (ER), preventing their passage to the cell surface and thereby reducing the recognition of infected cells by virus-specific T cells. We find the surprising result that intracellular transport and E3/19K binding are controlled primarily by the first half of the second domain of Kd, thus localizing these phenomena to the five polymorphic residues in this region of the Kd protein. This result implies that the E3/19K protein may act by inhibiting peptide binding or by disrupting the oligomerization of MHC class I molecules required for transport out of the ER. Alternatively, the E3/19K protein may inhibit the function of a positively acting transport molecule necessary for cell surface expression of MHC class I molecules.

Adenovirus E3 Proteins↗

PubMed1988

Cytolytic T cells recognize a chimeric MHC class I antigen expressed in influenza A infected transgenic mice.

A chimeric H-2Kd/Kk gene, called pC31, contains the extracellular alpha 1 domain of Kd origin whereas the rest of the molecule is of Kk origin. Disruption of the syngeneic alpha 1-alpha 2 structure results in a total abrogation of the function of the C31 protein as a restriction element for H-2Kd and Kk restricted T cells during virus infection. In an attempt to obtain information on the functional polymorphism of MHC class I antigens as restriction elements, we have introduced the pC31 gene into the germ line of C3H/He mice (H-2k). The pC31 gene was transcribed in all tissues examined and the expression pattern paralleled the endogenous H-2Kk gene. However, the mRNA for the transgene was approximately 10-times more abundant, which was reflected in an elevated expression of the C31 protein in transgenic splenocytes. Most of the C31 antigen was found intracellularly. The C31 antigen could condition transgenic cytotoxic T lymphocytes in a specific manner during influenza A virus infection and functioned as the restricting element during T cell lysis of the infected cells. These results suggest that entire exons may be exchanged between MHC class I genes and that this exchange can generate novel and functional restriction elements.

Animals↗

PubMed1987

Species heterogeneity in macrophage expression of the CD4 antigen.

The CD4 antigen is expressed on T cells of all mammalian species examined and appears to play an important role in the response of T cells to antigen. In humans, the molecule acts as a receptor for the AIDS virus. Previous studies have demonstrated that M phi in the rat and human also express the CD4 antigen, which is indistinguishable from that on T cells. In this paper we demonstrate by FACS analysis, Northern blot hybridization, and immunoperoxidase labeling that, in striking contrast to the rat and human, mouse M phi do not express the CD4 (L3T4) antigen. This species heterogeneity indicates that T cells and M phi regulate CD4 antigen expression independently and that CD4 may not be essential for M phi function.

Animals↗

PubMed1987

The MRC OX-44 antigen marks a functionally relevant subset among rat thymocytes.

A monoclonal antibody called MRC OX-44 is described that labels all myeloid cells and peripheral lymphoid cells but only 12% of thymocytes. The OX-44+ thymic cells include most if not all cells found in the medulla but only a small fraction of the cortical cells. Together with CD4 and CD8 antigens, seven subsets of thymic cell were defined and it was notable that most CD4- CD8- cells were OX-44+ whereas almost all CD4+ CD8+ cells were OX-44-. In functional tests, the OX-44+ cells accounted for all proliferation by thymocytes when stimulated by allogeneic spleen cells or concanavalin A plus growth factors and OX-44- cells were completely negative in these assays. Also, in tests for thymopoiesis after intra-thymic injection of cells, all activity was OX-44+. It seems possible that the OX-44+ set may include all functionally relevant cells in the rat thymus.

Animals↗

PubMed1987

Expression of the W6/32 HLA epitope by cells of rat, mouse, human and other species: critical dependence on the interaction of specific MHC heavy chains with human or bovine beta 2-microglobulin.

The HLA class I epitope W6/32 is conformationally dependent on both heavy chain and beta 2-microglobulin (beta 2M). Previously, the W6/32 epitope has been detected in humans and other primates as well as from bovine sources. Two controversial reports suggest the W6/32 epitope is constitutively expressed by either normal or transformed murine cells expressing the Db allele. Here we show that the appearance of the W6/32 epitope in murine cells results from the association of either the Db or Kd gene products with either bovine or human beta 2M. We use congenic mouse strains and hybrid H-2 class I genes between Db and Kb to map the W6/32 epitope to particular amino acid residues in the alpha 2 domain. Subsequently, we show that beta 2M exchange is not confined to murine or human cells in vitro but can be detected after beta 2M injection into a mouse. The data presented suggests that beta 2M exchange takes place at the cell surface under physiological conditions and indicates that MHC class I heavy chains are in an equilibrium between the bound and unbound form of beta 2M.

Amino Acid Sequence↗

PubMed1987

Antigens of activated rat T lymphocytes including a molecule of 50,000 Mr detected only on CD4 positive T blasts.

Mouse monoclonal antibodies (MAbs) have been prepared against rat T cell blasts. One MAb called MRC OX-40 recognized an antigen that differed from any previously described in that its expression was detected only on T blasts that also expressed the CD4 antigen. The OX-40 MAb did not detect an activation determinant of CD2 or CD4 molecules but recognized a distinct chain of mol. wt 50,000. The OX-40 MAb augmented T cell proliferation at late stages on in vitro responses. Other MAbs without obvious counterparts in other species were MRC OX-48 and MRC OX-49,50 which recognized cell surface molecules of mol. wts of about 95,000 and 90,000, respectively. The OX-48 antigen was not expressed on resting lymphocytes but was found on a subset of T and B blasts and also on other leucocytes. The OX-49,50 antigen was found on most haemopoietic cells but was expressed at greatly increased levels after lymphocyte activation and this was also the case for MRC OX-47 antigen which is of unknown Mr. The MRC OX-39 MAb was found to bind the rat IL-2 receptor; expression of this antigen was detected on thymic dendritic cells as well as on T blasts. The phenotype of rat T blasts compared to resting cells was also examined and changes in expression of L-CA, Thy-1, OX-2 and CD8 antigens were seen in addition to the changes found with the above MAbs.

Animals↗

PubMed1987

Peptide and nucleotide sequences of rat CD4 (W3/25) antigen: evidence for derivation from a structure with four immunoglobulin-related domains.

The rat W3/25 antigen was the first marker antigen of helper T lymphocytes to be identified. Subsequently, the human OKT4 antigen (now called CD4) was described, and cell distribution and functional data suggested that W3/25 and OKT4 antigens were homologous. This is now confirmed by the matching of peptide sequences from W3/25 antigen with sequence predicted from rat cDNA clones detected by cross-hybridization with a cDNA probe for human CD4. Analysis of the two sequences suggests an evolutionary origin from a structure with four immunoglobulin-related domains, although only domain 1 at the NH2 terminus meets the standard criteria for an immunoglobulin-related sequence. CD4 domains 2 and 4 contain disulfide bonds but seem like truncated immunoglobulin domains, whereas domain 3 may have a pattern of beta-strands like an immunoglobulin variable domain, but without the disulfide bond.

Amino Acid Sequence↗

PubMed1986

Monoclonal antibodies against a rat leucocyte antigen block antigen-induced T-cell responses via an effect on accessory cells.

The MRC OX-45 and OX-46 mouse monoclonal antibodies recognize a rat cell surface glycoprotein of 45,000 MW that is present on a wide variety of haematopoietic cells and on endothelial cells. MRC OX-45 IgG or F(ab')2 blocked the primary mixed lymphocyte response (MLR) and the secondary response of T lymphocytes to the soluble antigen DNP-BGG. In contrast, the antibodies had no effect on the cytotoxic activity of specific (CTL) or non-specific (NK) killer cells or on proliferative responses stimulated by lectins or oxidative mitogenesis. The inhibitory effect was at the level of stimulator cells rather than responders since mouse anti-rat xenogeneic MLRs were inhibited but rat anti-mouse responses were unaffected. However, the effect was not a direct one because inhibition was seen when irradiated spleen cells were used as stimulators but not when cell populations highly enriched for dendritic cells were used. In the latter case, inhibition potentiated by antibody could be restored if a peritoneal cell population enriched for macrophages was added back to the cultures. The inhibitory effects of these monoclonal antibodies seem most likely to be due to potentiation of nonspecific suppression by macrophages.

Animals↗

PubMed1985

Authentic T helper CD4 (W3/25) antigen on rat peritoneal macrophages.

The rat W3/25 antigen that appears to be equivalent to human CD4 (T4) antigen is expressed on thymocytes and T helper cells and plays a role in the response of T helper cells to antigen. The W3/25 and anti-T4 antibodies also label macrophages. In this paper we examine whether the macrophage antigen is the same as that on T cells. New monoclonal antibodies against the rat CD4 antigen, MRC OX-35 through OX-38, are described, all of which label peritoneal macrophages from normal and athymic rats. The molecular weight of W3/25 antigen on macrophages is indistinguishable from that on T cells. We conclude that macrophages express authentic CD4 (W3/25) antigen. Another new monoclonal antibody, MRC OX-34, labels an antigen of 50-54,000 mol wt that is expressed on rat T but not B cells or peritoneal macrophages. It was used to control for the presence of any T cell products in immunoprecipitation from rat macrophage extracts.

Animals↗

PubMed1985

Analysis of lymphopoietic stem cells with a monoclonal antibody to the rat transferrin receptor.

A mouse monoclonal IgG2a antibody, designated MRC OX-26, is shown to be specific for the rat transferrin receptor, but does not block transferrin binding. The antibody labelled a myeloma, three leukaemia cell lines and normal dividing cells of various types, but also bound to a number of nondividing normal tissues. No labelling of lymphopoietic stem cells could be detected, even though approximately 25% of bone marrow and over 95% of fetal liver cells were clearly labelled.

Animals↗

PubMedDate not supplied

Transferrin receptor on endothelium of brain capillaries.

The blood/brain barrier prevents the passive diffusion of proteins and metabolites from cerebral blood vessels into tissue spaces around neuronal and glial cells. To provide nutrients for these cells, transport mechanisms must exist and indeed have been demonstrated for metabolites. We now show that monoclonal antibodies against rat and human transferrin receptors label blood capillaries in the brain but not in other tissues. In the rat this labelling occurs after injection of antibody into the blood, thus the receptors seem to be accessible at the endothelial surface. It is possible that transferrin receptors are expressed on these cells to allow transport of transferrin (and thus iron) into brain tissues.

Animals↗