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J R Lamb

Publications and source records attributed to J R Lamb.

At least 91 records · Page 5Linked to original sources
PubMed1993

In vivo clonal dominance and limited T-cell receptor usage in human CD4+ T-cell recognition of house dust mite allergens.

Sensitivity to house dust mite antigens in atopic individuals is a major cause of allergic diseases, ranging from asthma to rhinitis and dermatitis. We have studied the T-cell receptor (TCR) usage of house-dust-mite-specific CD4+ T-cell clones isolated from an atopic individual, by using the anchored polymerase chain reaction, and have analyzed the peripheral TCR repertoire of the same individual. Several T-cell clones had identified TCRs at the sequence level, despite the fact that they had been independently isolated, in some cases, in different years. These data suggest the presence in vivo of long-lived T-cell clones. We have also shown that junctional sequences identical to these clones are present in peripheral blood T cells taken 6 years after the isolation of the T-cell clones. The analysis of TCR genes used by the panel of clones reveals oligoclonality, with the variable (V) region gene segments V alpha 8 and V beta 3 being dominant, although there is minimal conservation of junctional sequences. The results have implications for understanding the TCR recognition of an environmental aeroallergen and the life span of T-cell clones in vivo during a chronic immune response.

Allergens↗
PubMed1993

Analysis of human T cell responses to the group II allergen of Dermatophagoides species: localization of major antigenic sites.

BACKGROUND: IgE antibodies reactive with the group II allergens of Dermatophagoides species (house dust mite [HDM]) are a major component of the allergic immune response in HDM-allergic atopic individuals. METHODS: Here we demonstrate, with the use of overlapping synthetic peptides of the group II allergen of Dermatophagoides pteronyssinus (Der p II), that polyclonal T cells isolated from the majority of atopic HDM-allergic individuals and healthy nonatopic control subjects respond to Der p II and that T-cell epitopes are present in all regions of the protein. RESULTS: From comparison of peptide-specific T-cell proliferation in both groups of individuals, it appears that together peptides 61-86 and 78-104 are the most frequently recognized (16 of 18 individuals). We also observed that nine of the 18 individuals responded to T-cell epitopes in the region 11-50, and with Der p II-reactive T-cell clones, three distinct T-cell epitopes were mapped within the sequence 11-35. Also, with the use of T-cell clones, two additional epitopes were identified at residues 81-96 and 91-101. CONCLUSIONS: These results suggest that T-cell epitopes located in these regions (11-50 and 61-104) are immunodominant. The value of this information in the potential application of Der p II peptides to desensitize HDM allergic responses is discussed.

Amino Acid Sequence↗
PubMed1993

Analysis of T cell repertoire and function in mice transgenic for the human V beta 3 TCR.

We have constructed mice containing the human V beta 3 TCR gene from the influenza virus haemagglutinin specific human CD4+ T cell clone HA1.7. Similar cell yields were obtained from transgenic and non-transgenic lymphoid tissue, with normal levels of T cells and with no unusual bias of the CD4 or CD8 subpopulations. Immunostaining and FACS analysis of transgenic thymocytes, spleen, and mesenteric lymph nodes revealed that the majority of T cells expressed the human V beta 3 TCR on the cell surface. Small numbers of cells expressing murine TCR beta chain were also detected. Polymerase chain reaction analysis revealed that an extensive V alpha TCR repertoire was used in the human V beta 3 transgenic mice. Lymphocytes from the spleen and mesenteric lymph nodes of transgenic mice were assessed for functional activity in vitro. Isolated cells were stimulated with mitogen or superantigen, as well as directly through the TCR-CD3 complex, and their ability to proliferate and secrete lymphokines analysed. Cells from transgenic mice responded well after stimulation with phytohaemagglutinin, concanavalin A, anti-CD3 antibody, anti-CD3 antibody with phorbol ester, and Staphylococcus aureus enterotoxin B, and also showed alloreactivity in a mixed lymphocyte reaction. Minimal levels of response were detected after stimulation with murine TCR beta antibody. Together, these data suggest that a human TCR beta chain is able to associate with a murine TCR alpha chain, to form a fully functional surface TCR-CD3 complex.

Animals↗
PubMed1993

Clonal analysis of the atopic immune response to the group 2 allergen of Dermatophagoides spp.: identification of HLA-DR and -DQ restricted T cell epitopes.

The group 2 allergens of Dermatophagoides spp. (house dust mite, HDM) are a major immunological target for IgE antibodies in the allergic immune response of HDM atopic individuals. In this report the heterogeneity of the T cell repertoire reactive with the group 2 allergen of Dermatophagoides pteronyssinus (Der p 2) of a HDM allergic individual was investigated using overlapping synthetic peptides. By clonal analysis four distinct T cell epitopes were identified, located within residues 16-31, 22-40, 82-100, and 111-129. The importance of these epitopes was confirmed by investigation of the peripheral T cell repertoire, with the polyclonal T cell response to Der p 2 failing to show marked variations in epitope specificity over time. Serological inhibition studies and the use of Epstein-Barr virus transformed B cell lines characterized for their expression of HLA-D region gene products demonstrated that recognition of peptides 16-31 and 111-129 was restricted by HLA-DQ (DQB1*0301), whereas peptide 82-100 is recognized in association with HLA-DR (DRB1*1101). Peptide 22-40 was presented by both HLA-DRB1*1101 and -DQB1*0301 class II molecules. The potential application of these findings lies in the design of peptide-based immunotherapeutics for the management of HDM allergic disease.

Amino Acid Sequence↗
PubMed1993

CD28 mRNA rapidly decays when activated T cells are functionally anergized with specific peptide.

The induction of non-responsiveness in specific clones of T cells in vivo might be expected to reverse immunologically mediated disease processes. With this goal in mind, experiments in vitro with cloned T cells have investigated the mechanisms of induction of anergy. Resting T cells can be functionally inactivated in vitro by high doses of appropriate peptide in either the presence or absence of antigen presenting cells. During the induction of anergy, the modulation of the surface phenotype of T cells is similar to that of cells proliferating in response to an immunogenic stimulus. The amount of T cell receptor at the cell surface is down regulated, whereas the CD2 and CD25 receptors are increased in density. In contrast, CD28 has been identified as a membrane protein that is differentially regulated during activation and the induction of non-responsiveness. Ligation of CD28 provides an efficient costimulatory signal for activation of T cells. In this report, we show that not only resting cells, but also fully activated T cells can be rendered non-responsive and that this process is accompanied by profound downregulation of CD28, both at the level of cytoplasmic mRNA and surface expression of the mature protein. This observation anticipates clinical intervention in immunologically mediated disease, where the target T cells are more likely to be activated than in a resting state.

Antigens, CD↗
PubMed1993

House dust mite allergy: from T-cell epitopes to immunotherapy.

CD4+ T-lymphocytes induce and regulate allergic inflammatory responses to common environmental aeroallergens derived from Dermatophagoides spp. (house dust mite, HDM), which cause clinical symptoms in approximately 10% of the population. Definition of the molecular structure of HDM proteins combined with the ability to isolate monoclonal populations of human CD4+ T-cells representative of the 'interleukin-4 (IL-4) dominant' functional phenotype, which support immunoglobulin E (IgE) synthesis, has allowed T-cell recognition of HDM to be examined in detail. The results of these investigations demonstrated extensive heterogeneity in both the antigen and HLA class II restriction specificity of the HDM reactive T-cell repertoire. Furthermore, long-lived clones of T-cells with oligoclonality in T-cell antigen receptor (TcR) usage, driven by chronic stimulation with HDM, have been identified in human peripheral blood. The presentation of specific peptides and superantigens under conditions that induce T-cell non-responsiveness has provided an in vitro model for analysing the mechanisms of CD4+ T-cell targeted immunotherapy. It appears that the mechanisms underlying T-cell anergy are accompanied by a transient downregulation of TcR and CD28 and mediated by a shift in the cytokine profile from that of the 'IL-4 dominant' to the 'interferon-gamma (IFN-gamma) dominant' functional phenotype of CD4+ T-cells. In parallel, using a murine model, it has been demonstrated that administration of an immunodominant peptide via the mucosal surfaces of the respiratory and alimentary tracts may tolerize an established response to intact HDM proteins. The potential application of these models in the development of novel approaches to immunotherapy is discussed.

Allergens↗
PubMed1993

Human in vitro experimental model for CD4+ T cell targeted immunotherapy to house dust mite.

CD4+ T cells play a critical role in the initiation and potentiation of the allergic immune response. Development of allergen-mediated hyposensitization therapies directed at this cell type requires two key steps: (1) identification of the epitopes that are crucial for activating the T-cell response and (2) definition of the specificity of the HLA-D region molecules that restrict T-cell recognition of these epitopes. We have located the major T-cell determinants of the group II allergen of the house dust mite species Dermatophagoides pteronyssinus (Der p II). We have also identified a T-cell clone for which the HLA-DP*0401 allele restricts recognition of the group I allergen residues of house dust mite. This clone overproduces IL-4 and IL-5, cytokines that promote IgE synthesis. When these cells are rendered nonresponsive by incubation with a supraoptimal concentration of their allergen peptide determinant, they lose their ability to secrete IL-4 but maintain interferon gamma production. This modified pattern favors a switch away from the pathway of IgE synthesis to that of IgG synthesis. These findings suggest that the use of selected peptides in vaccines may allow the redirection of allergic immune responses.

Allergens↗
PubMed1992

Major histocompatibility complex independent clonal T cell anergy by direct interaction of Staphylococcus aureus enterotoxin B with the T cell antigen receptor.

The Staphylococcal enterotoxin superantigens stimulate vigorous responses in T cells bearing certain T cell antigen receptor (TCR) V beta regions. In addition to activation, these superantigens also impart negative signals to T cells resulting in a profound state of unresponsiveness or anergy. The Staphylococcus aureus enterotoxins (SE) B and C2 bind to a closely related site on major histocompatibility complex (MHC) human leukocyte antigen (HLA)-DR1 molecules. Only SEB, however, interacts with the TCR V beta 3 region of HA1.7, a human HLA-DR1 restricted T cell clone specific for influenza haemagglutinin. In competition experiments, we demonstrated that the induction of anergy in HA1.7 by SEB is unaffected by the presence of SEC2. These results suggest that SEB-induced anergy is MHC independent and involves a direct interaction between the TCR and SEB. To resolve definitively whether SEB binds directly to T cells in the absence of MHC class II molecules, the cDNAs encoding the HA1.7 TCR were transfected into an MHC class II-negative human T cell line. The addition of SEB to these transfectants resulted in the downregulation of cell surface TCR expression, an increase in the concentration of intracellular calcium ions, the production of lymphokines, and reduced responsiveness to a subsequent challenge with SEB. We conclude that SEB interacts directly with the TCR in the absence of cointeraction with MHC class II molecules, and that this interaction may induce anergy in HA1.7.

Amino Acid Sequence↗
PubMed1992

Uncoupling of cytokine mRNA expression and protein secretion during the induction phase of T cell anergy.

The CD4+ T cell clone HA1.7 may be made specifically nonresponsive, or anergic, to its cognate Ag, an influenza hemagglutinin peptide (HA), by pretreatment with the superantigen Staphylococcus aureus enterotoxin B or with high concentrations of HA itself. We compare the patterns of mRNA expression and protein production of selected T cell cytokines during the first 24 h after treatments that induce anergy in HA1.7 and during the same period after treatments that simulate normal cellular activation. The cytokines examined include TNF-alpha, IL-8/neutrophil activating protein-1 and the RANTES/SIS cytokines, a family of small secreted proteins with inflammatory and potential antiproliferative and leukocyte regulating activities. Messenger RNA for TNF-alpha, human MIP-1 alpha, human MIP-1 beta, and IL-8 are all induced during the development of clonal anergy in HA1.7, and these levels are significantly higher than those seen during activation of the clone using an anti-CD3 antibody and IL-2. These high levels of mRNA also persist longer than those seen after anti-CD3 and IL-2 activation. However, the increased levels of mRNA are not typically accompanied by increased protein secretion. In all cases but one, the amount of cytokine secreted by HA1.7 cells was greater after anti-CD3 and IL-2 treatments than after anergy-inducing treatments. Thus, the induction of T cell anergy in HA1.7 does not appear to require a general inhibition of T cell cytokine mRNA expression, and, in fact, anergy treatments appear to superinduce certain cytokine transcripts, but anergy-specific posttranscriptional mechanisms may exist by which cytokine release is regulated.

Animals↗
PubMed1992

Localization of the immunologic activity in the superantigen Staphylococcal enterotoxin B using truncated recombinant fusion proteins.

The exotoxins of certain strains of Staphylococcus aureus strains are able both to stimulate potent proliferation and induce anergy in T lymphocytes expressing the appropriate T cell Ag receptor V beta gene elements. Although T cell activation by the S. aureus enterotoxins requires the presence of accessory cells bearing class II Ag of the MHC, unlike the peptide fragments of nominal Ag, they contact the external surfaces of both the class II MHC and TCR molecules. This paper investigates the immunologically active domains of S. aureus enterotoxin B (SEB) using truncated fragments of rSEB expressed as a fusion protein with protein A. The results of the experiments reported here indicate that the minimal fragment of SEB able to stimulate and induce anergy in hemagglutinin-reactive human T cells expressing V beta 3.1 gene elements is located in the amino-terminal portion of the molecule within residues 1-138. Deletion of the first 30 amino acid residues renders rSEB unable to stimulate T cells expressing V beta 3.1, whereas polyclonal T cells still respond to this molecule. This implies that the stimulation of several TCR-V beta families may be caused by the interaction with different regions of the toxin. The localization of immunologically active sites in the bacterial enterotoxins is needed to investigate both their biology and potential application as immunomodulatory agents.

Antigens, Bacterial↗
PubMed1992

Peptide-induced nonresponsiveness of HLA-DP restricted human T cells reactive with Dermatophagoides spp. (house dust mite).

The activation of CD4+ T lymphocytes, which play a central role in allergic inflammation, depends on the recognition of allergen-derived peptides in association with major histocompatibility complex class II gene products. In this report we demonstrate, at a clonal level, that a component of the T-cell repertoire reactive with Dermatophagoides spp. (house dust mite) in atopic individuals, is restricted by HLA-DP class II molecules. This supports the recent results emerging from genetic epidemiologic studies that indicate positive associations between the HLA-DP phenotype and immune responsiveness to a variety of common allergens. Our findings also reveal that the T cells restricted by HLA-DP recognize a species-specific epitope located in the group I allergen of Dermatophagoides pteronyssinus (residues 101-119). Furthermore, we report that the pretreatment of the T cells restricted by HLA-DP with the Der p I peptide renders them nonresponsive to an immunogenic challenge with house dust mite allergen, and the loss of antigen-dependent proliferation is associated with downregulation of membrane expression of the T-cell antigen receptor. The ability to functionally inactivate T cells restricted by HLA-DP, as well as those that recognize allergen in association with HLA-DR class II molecules, suggests that desensitization with allergen-derived peptides may have therapeutic potential in the management of allergic diseases irrespective of their HLA class II association.

Animals↗
PubMed1992

Generation of monoclonal antibodies against a human T cell receptor beta chain expressed in transgenic mice.

The generation of a panel of monoclonal antibodies specific for different variable (V) regions of human T cell receptors will be of great importance in the study of T cell-mediated diseases. However, relatively few such reagents exist, due in part to the poor immunogenicity of TcRs on the surface of human T cells. We have employed a strategy in which T cells from a transgenic mouse line expressing a human V beta 3 C beta 1 TcR were used to immunise syngeneic conventional mice to generate two monoclonal antibodies specific for human T cell receptors. Binding of antibody JOVI.3, which stained approximately 5% of human peripheral blood CD3 positive T cells, correlated with the expression of the human TcR V beta 3 gene segment. Antibody JOVI.1 recognised a determinant on the majority of TcRs, staining 50-75% of peripheral blood T cells and T cell lines expressing different V beta regions. Some TcRs, however, failed to react with this antibody. Both antibodies immunoprecipitated detergent-solubilised TcR molecules and were capable of inducing proliferation of peripheral blood T cells.

Animals↗
PubMed1992

Analysis of the basis of resistance and susceptibility of CD4+ T cells to human immunodeficiency virus (HIV)-gp120 induced anergy.

The resistance and susceptibility of T cells to human immunodeficiency virus (HIV)-gp120 induced anergy was examined. Antigen-dependent proliferation of polyclonal T cells was markedly inhibited by gp120, whereas from the analysis of monoclonal populations, T cells resistant to the effects of gp120 could be identified. Similarly, exposure of monoclonal T cells to gp120 in the absence of accessory cells, also demonstrated that some T cells could resist the induction of anergy. Loss of antigen recognition was associated with phenotypic modulation of CD3 and CD28, which was not observed in T cells resistant to functional inactivation by gp120. Modulation of CD4 was not related to induction of anergy in the monoclonal T cells examined in this study. Inhibition of T-cell responses by anti-CD4 antibodies was compared to that by gp120. Anti-CD4 antibodies, which cross-compete with gp120 for binding to CD4, inhibited the response to antigen of monoclonal T cells. In contrast, no tolerogenic signals were delivered by pretreating T cells with the anti-CD4 antibodies in the absence of accessory cells, indicating that inhibition was due to abrogation of the interaction of CD4 with major histocompatibility complex (MHC) class II molecules expressed on accessory cells. Although the free CD4-binding region peptide of gp120 could inhibit polyclonal T-cell responses, only the carrier-bound peptide was able to modulate cloned T cells, suggesting a conformational requirement for functional inactivation through engagement of CD4. The results reported here using clonal CD4+ T-cell populations demonstrate that effects of gp120 on antigen-dependent proliferation are not uniform, and that therapeutic intervention might be directed at T-cell populations identified as susceptible to HIV-gp120 induced anergy.

Antigens, CD↗
PubMed1992

The requirements for activation of an antigen specific T-cell clone via the CD3 complex.

We have studied the requirements for the activation of the haemagglutinin peptide specific cloned T-cell HA1.7 by anti-CD3 antibodies and the mechanism of modulation of the CD3 complex. Cross-linking of the complex either by a soluble antibody second layer or an immobilized matrix of antibody is necessary for induction of a proliferative response. This proliferative response is totally dependent on exogenously added IL-2. Modulation of the cross-linked CD3 complex is by patching, capping and internalization of the antigen and is not essential for the proliferative response.

Animals↗
PubMed1992

Detection of in vivo production of tumour necrosis factor-alpha by human thyroid epithelial cells.

We have established previously that human thyroid epithelial cells (TEC) from patients with autoimmune thyroiditis are able to synthesize cytokines, such as interleukin-1 (IL-1) and interleukin-6 (IL-6). This paper examines TEC in sections from autoimmune thyroiditis for the in vivo production of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) using the combined techniques of in situ hybridization and immunohistochemistry. Thyroid tissue from patients with Graves' disease, Hashimoto's disease and non-toxic goitre was examined and both mRNA and the protein of TNF-alpha were detected in TEC on frozen sections. Representative figures of only Graves' samples are illustrated in this paper. In contrast, using the same methods, IFN-gamma was detected only in the infiltrating cells and not in TEC of thyroid tissue from the patients.

Epithelium↗
PubMed1991

Effect of natural polymorphism at residue 86 of the HLA-DR beta chain on peptide binding.

Class I and class II MHC glycoproteins are highly polymorphic molecules that bind antigenic peptides and present them on cell surfaces for recognition by T lymphocytes. Even though MHC polymorphism has long been known to affect both peptide binding and recognition by the TCR, the role of individual amino acids of MHC proteins in these interactions is poorly understood. To examine the effect of a small number of amino acid residues on T cell stimulation, B lymphoblastoid cell lines homozygous for the closely related DR1 subtypes, Dw1 and Dw20, and the DR4 subtypes, Dw4 and Dw14, were compared for their ability to present an immunogenic influenza hemagglutinin peptide (HA307-319) to an Ag-specific, DR1,4-restricted T cell clone. B cell lines expressing DR1 Dw20 and DR4 Dw14 presented HA307-319 much less efficiently than DR1 Dw1 and DR4 Dw4 and bound a biotinylated analogue of the same peptide less well. Analysis of DRB1 gene sequences suggested that polymorphism at residue 86 had a major effect on peptide binding. Differences in binding of a set of HA307-319 analogues biotinylated at each residue to cells expressing DR1 Dw1 and DR1 Dw20 suggested that the polymorphism affected the interactions of many peptide residues with the class II molecule. In inhibition assays, DR1 Dw1 and DR4 Dw4 were shown to differ from DR1 Dw20 and DR4 Dw14 in their length requirements for peptide binding. Using a larger panel of homozygous B cell lines expressing many class II haplotypes, a Ser-309 substituted HA307-319 analogue was shown to bind to most B cell lines expressing Val-86 containing alleles (including DR1 Dw20 and DR4 Dw14) but failed to bind most B cell lines expressing Gly-86 alleles (including DR1 Dw1 and DR4 Dw4). The results indicated that polymorphism at residue 86 influenced the specificity and affinity of peptide binding and affected the conformation of peptide-DR protein complexes without completely eliminating T cell recognition.

Alleles↗
PubMed1991

An in vitro model of peptide-mediated immunomodulation of the human T cell response to Dermatophagoides spp (house dust mite).

Allergic sensitivity of Dermatophagoides spp (house dust mites) is mediated by specific IgE antibody, the production of which requires the presence of CD4+ helper T cells. Attempts to hyposensitize this response in allergic individuals have depended on the administration of extracts of specific allergen. However, the ability of peptides derived from unrelated antigens to inhibit specific immune responses offers an alternative approach to therapy. We have addressed this question by examining the ability of a nonstimulatory peptide analogue derived from influenza virus hemagglutinin to modulate T cell recognition of house dust mite. The peptide inhibited the response of mite-specific CD4+ T cell clones restricted by either the HLA-DRAB1 or DRAB3 gene products. Furthermore, mite-induced polyclonal T cell responses were negatively modulated by the peptide, whereas recognition of common recall antigens remained intact. The inhibitory effects were mediated at the level of the antigen-presenting cell, since no inhibition of mitogen or anti-CD3 antibody-driven T cell proliferation was observed. In direct binding assays, the peptide analogue bound to selected HLA-DR molecules expressed on the membrane of antigen-presenting cells, with specificity predominantly for those class II proteins capable of restricting house dust mite-allergen T cell recognition.

Adult↗