Biomedical subjects

R A Lake

Publications and source records attributed to R A Lake.

At least 19 recordsLinked to original sources

PubMed2000

Structure of the Mesothelin/MPF gene and characterization of its promoter.

Mesothelin is a cell-surface antigen of unknown function that is expressed in a highly tissue-specific manner. Expression of the protein is limited to mesothelium, the tissue forming the pleural, pericardial, and peritoneal membranes. The purpose of this study was to characterize the gene and identify elements responsible for its transcription. The gene contains an 1884-bp open reading frame encoded by 15 exons occupying 8 kb of human chromosome 16. An 1850-bp region of genomic DNA at the 5' end of the gene encompassing the proposed transcriptional start site was cloned. This region lacks a TATA box and other regulatory elements such as SP1 sites, which are commonly found in promoters. Transient transfection analyses demonstrated that mesothelium-specific control elements are present within the 1.85-kb region. Minimal constitutive promoter elements were localized to a 317-bp region. Tissue-specific enhancer elements upstream of the minimal promoter were found to activate transcription from the homologous and a heterologous promoter in a position- and orientation-independent manner.

Blotting, Northern↗

PubMed2000

Replication-restricted vaccinia as a cytokine gene therapy vector in cancer: persistent transgene expression despite antibody generation.

BACKGROUND: As antitumoral immunity requires the generation of local immunity directed against tissue proteins, we attempted to recreate within tumors the same environment found within tissues affected by autoimmune diseases (i.e., prolonged cytokine expression). Vaccinia virus (VV) has not been widely used as a cytokine gene therapy vector because of presumed high immunogenicity that would likely make repeated injections impossible; therefore, we modified it by inserting the cytokine gene into the thymidine kinase region, rendering it replication-restricted. The cytokine chosen was human interleukin-2 (IL-2); a molecule with powerful antitumoral effects. METHODS: Six patients with the treatment-resistant tumor malignant mesothelioma received intratumoral (i.t.) VV-IL-2 therapy for 12 weeks by injection of 10(7) plaque-forming units of VV-IL-2 per dose. Serial tumor biopsies, sputum, urine, and blood samples were tested for VV-IL-2 mRNA expression; VV culture and T-cell infiltrates were evaluated by immunohistochemistry. Patients and contacts of patients were monitored for changes in VV immunoglobulin G (IgG) levels and clinical evidence of VV infection. RESULTS: VV-IL-2 was not excreted and was only cultured in one patient from tumor biopsies. A T-cell infiltrate was detected in 50% of tumor biopsies. VV-IL-2 mRNA expression was highest on days 1-3 postinjection and was detected for up to 3 weeks after each injection even though VV IgG levels rose in all patients. No significant toxicities, infection of patient contacts, or tumor regressions were observed. CONCLUSIONS: I.t. VV-IL-2 administration is safe, is associated with minimal toxicity, and results in i.t. expression of VV-IL-2 for up to 3 weeks postinjection regardless of the level of anti-VV IgG titers generated. This suggests that VV may be a good vector for repeated cytokine gene therapy of solid human cancer.

Adult↗

PubMed2000

Partial T cell activation with an altered superantigenic ligand.

T cells have the capacity to respond to ligands as full, weak, partial or null agonists, or indeed as antagonists. In the present paper, it is reported that staphylococcal enterotoxin B (SEB) mutated in a T cell receptor (TCR) contact site (SEBDelta61Y) behaves as an altered ligand for a T cell clone (AC20) that expresses the Vbeta17 TCR. The T cells were partially activated by SEBDelta61Y, as shown by TCR down-modulation and up-regulation of the IL-2 receptor. However, these cells did not secrete IL-2, IL-3, IL-4 or IFN-gamma, nor did they proliferate. Analysis of intracellular protein tyrosine phosphorylation after cellular activation provided further evidence that SEBDelta61Y could transduce a signal via the Vbeta17 TCR. The events following receptor ligation were clearly different when the T cells were stimulated with SEB or SEBDelta61Y, manifested as both quantitatively and qualitatively different patterns of phosphorylation of intracellular substrates. In contrast, only quantitative differences were apparent when a transfectant expressing the same alpha/beta TCR was stimulated with the different superantigens. Together, these results provide the first demonstration that altered TCR ligands are not restricted to peptides substituted at secondary TCR contact residues. Rather, an altered superantigenic ligand mutated in the TCR binding site can behave as a partial agonist.

Binding Sites↗

PubMed2000

Serologic responses in patients with malignant mesothelioma: evidence for both public and private specificities.

Malignant mesothelioma (MM) is a pulmonary malignancy that appears to be immunogenic based on a large number of studies in both animals and humans. This notion is supported by our recent demonstration using Western blot analysis of immunoglobulin G antibodies reactive with a variety of autoantigens in many patients with MM. In view of the enormous potential of such antigens in early diagnosis, immunotherapy, and vaccination of at-risk individuals, it was essential to identify these antigens. We therefore applied the SEREX technique (serologic identification by recombinant expression cloning), using a serum pool from six patients as the probe against an expressed complementary DNA library derived from a cloned MM cell line. We screened over one million recombinants and obtained sequence information on eight antigens that had provoked immunoglobulin heavy chain class switching, presumably as a consequence of T-cell recognition. Six of these antigens were identifiable (U2AF[65], Siah binding protein, topoisomerase IIbeta, ZFM1, mIre1, and pendulin), and of the others, one was found as a single EST from a myotube library (Jemm-1); the other (Jemm-2) was not represented in any EST database even as a weak homolog. Consistent with our previous findings, each of the characterizable antigens would be expected to be associated with the cell nucleus. Each of the autoantibody specificities was uniquely associated with a single patient with the exception of antibodies to TOPIIbeta and U2AF(65). We found 13 of 14 (93%) patients with MM had antibodies to TOPIIbeta and two of 14 (14%) patients had antibodies to U2AF(65). The number of serum reactivities, taken as a measure of the complexity of the immune response, correlates with patient survival and with an index of systemic inflammation. These data suggest that a broader range of serologic reactivities reflects a more active host response to the presence of tumor.

Animals↗

PubMed1999

Tumor antigens are constitutively presented in the draining lymph nodes.

Tumor growth is rarely associated with a strong specific CTL response, suggesting that the immune system is ignorant of the presence of tumor because the Ags are not readily available to or are sequestered from potential effector cells. We studied the in vivo activation of naive TCR transgenic hemagglutinin (HA)-specific CD8+ T cells adoptively transferred into mice bearing HA-expressing tumor using 5,6-carboxy-succinimidyl-fluorescein-ester labeling, which allows the identification of proliferating HA-specific T cells. We demonstrate that tumor Ags are constitutively presented in the lymph nodes draining tumors and are powerfully mitogenic for responding T cells despite the absence of anti-tumor CTL responses. Importantly, this proliferative signal occurs throughout tumor growth and is still detectable 6 mo after tumor inoculation when tumor is not palpable. These results provide the first evidence that there is constitutive presentation of tumor Ags in draining lymph nodes.

Animals↗

PubMed1999

T-cell receptor transgenic analysis of tumor-specific CD8 and CD4 responses in the eradication of solid tumors.

The role of tumor-specific CD8 and CD4 lymphocytes in rejecting solid tumors has been difficult to determine because of the lack of models in which tumor antigen, specific CD8 cells, and specific CD4 cells can be monitored and controlled. To investigate the minimal components required for the induction and maintenance of CTL activity sufficient to reject a solid tumor in vivo, we transfected the influenza hemagglutinin (HA) gene into a nonimmunogenic class I+/class II- murine malignant mesothelioma (MM) tumor line to generate an endogenous tumor antigen and used TCR transgenic mice with class I- or class II-restricted specificities for HA as sources of naive, tumor-specific T cells. The data show that the presence of a strong tumor antigen is not in itself sufficient to induce an effective CTL response, nor does the presence of a high frequency of precursor cells guarantee tumor rejection. We also show that tumor-specific CD4 cells, when CTL numbers are suboptimal, greatly enhance the eradication of tumor, confirming the importance of antigen-presenting cell presentation of tumor antigens to class II-restricted cells. These data confirm that T-cell receptor transgenic cells, combined with nominal tumor antigen transfection, represent powerful tools to analyze tumor-specific T-cell responses.

Animals↗

PubMed1999

MHC multimerization, antigen expression and the induction of APC amnesia in the developing immune response.

Class II multimer formation is an important event in immune recognition. Not only is multimerization a prerequisite for T cell activation, but it is a signal to APC. In the present article, we propose that multimerization can result in the specific removal of ligand complexes from the cell surface of the APC, an event which may influence the overall pattern of T cell reactivity.

Animals↗

PubMed1999

Cross-presentation of tumour antigens: evaluation of threshold, duration, distribution and regulation.

The development of technology to measure antigen presentation in the secondary lymphoid system has provided the opportunity of analysing components of the host antitumour immune response that have, until now, been unavailable for study. In particular, this technology has enabled us to evaluate threshold levels of tumour antigen required for cross-presentation in draining lymph nodes, the duration of this antigen presentation and processes that regulate tumour antigen presentation. Thus, we have been able to dissect out the relationship between antigen presentation and the resultant development of effector function in class I-restricted T cells, as well as the role of regulatory CD4 cells. We have also used this technology to evaluate the effects of antitumour therapy on local antigen cross-presentation.

Adoptive Transfer↗

PubMed1998

Sera from patients with malignant mesothelioma can contain autoantibodies.

Malignant mesothelioma (MM) is resistant to all conventional forms of therapy though there is considerable evidence from clinical trials and animal models of the disease that an immune response can be elicited to the tumour. In order to define those target antigens expressed by MM cells which might provide a focus for an effective immune response we tested patients' sera for the presence of MM autoantibodies by Western blot analysis. Eight of 29 (28%) patients with MM had serum antibodies of the IgG class in high titre and each antiserum recognised different protein antigens. In those individuals where sequential samples were available, the antibody titre increased with the progression of the disease though the number of target antigens remained constant. Sera from the eight patients were studied further: six of the antigen complexes were expressed at least partially in the nucleus; two showed some specificity for the tumour in that they discriminated antigens that were highly expressed in all human MM cell lines, but were not expressed in a human SV40 transformed mesothelial line; four of the antisera recognised a homologue in mouse tissue and each of these had a different pattern of expression. Collectively, these antisera define a subset of nuclear autoantigens that are over-expressed in dividing cells.

Adult↗

PubMed1997

Tandem peptide epitopes facilitate CD4-dependent activation of T cell clones.

Peptides that consist of two tandemly repeated epitopes joined by a flexible linker have an increased affinity for class II molecules and are more potent at inducing proliferation of T cell clones than monomeric epitopes. The increase in potency of peptides with two epitopes for individual T cell clones is proportional to the relative CD4 dependence of the clones. We show that epitope dimers activate T cell clones that respond sub-optimally to monomeric epitopes presented by APC from HIV-infected donors. We hypothesize that HIV+ APC normally fail to stimulate the clones because virally encoded gp 120 sequesters CD4 from the activation complex, but epitope dimers overcome this effect because they are better able to recruit CD4. The alpha beta heterodimer of human class II (HLA-DR1) is further ordered as a dimer of heterodimers (superdimer) at least in its crystal form. Since class II molecules have an open-ended antigen binding groove, the superdimer is theoretically permissive of stable binding of two peptide epitopes linked in tandem. Our data support a role for the MHC class II dimer of heterodimers in amplifying the proliferative response of T cells to antigen by dint of the superdimers having a higher affinity for CD4 than the nominal class II alpha beta heterodimers.

Antigen-Presenting Cells↗

PubMed1997

A function for CD2 on murine B cells?

CD2 is expressed on murine B cells, probably as a result of a chromosomal translocation event during speciation. There are no activating antibodies to mouse CD2, but when activated non-specifically, only T cells up-regulate their expression of CD2. We investigated the expression and function of CD2 on B cells using mice transgenic for human CD2 under the control of a modified version of the autologous promoter/enhancer that included the immunoglobulin enhancer. This construction directs expression of human CD2 to the B cell compartment as well as to the T cell compartment. In this paper, we confirm that activating pairs of anti-CD2 monoclonal antibodies are mitogenic for mouse T cells transgenic for human CD2. In contrast, mouse B cells that express similar amounts of human CD2 are not stimulated to proliferate by equivalent doses of these antibodies. We were also unable to show any functional consequence for these B cells as a result of CD2 ligation.

Adjuvants, Immunologic↗

PubMed1996

A T cell clone with three potential TCR alpha chain rearrangements expresses only one receptor combination at the cell surface.

In determining the T cell receptor (TcR) usage of various T cell clones that recognize peptide antigens derived from allergens, a particular clone (AC20) was found, that apparently expressed three different species of mRNA encoding alpha chains. The logical conclusion that the cells were not clonal was refuted by the finding of only a single beta chain rearrangement. One of the alpha chains (V alpha20), was not in frame, but two V alpha8 transcripts of different lengths were both potentially translatable. Sequence analysis suggested that the shorter transcript was generated by a secondary splice event from the longer, through the use of a splice donor sequence encoded by the J alpha38 gene segment. The efficiency of excision of the intervening sequence is such that approximately equal amounts of the long and short transcripts occur in the steady state pool of mRNA. This phenomenon has been reported previously in TcR alpha rearrangements, but it has never been made clear whether these truncated chains can form a functional TcR. Reconstitution of a TcR negative cell line with these transcripts showed that only the full length alpha chain was able to pair efficiently with the beta chain to generate a functional receptor at the cell surface.

Amino Acid Sequence↗

PubMed1996

Defective antigen processing associated with familial disseminated mycobacteriosis.

To gain insights into a possible immune defect predisposing to disseminated mycobacteria infection, we studied three of six surviving children with disseminated Mycobacterium avium complex infection, who had no recognized form of immunodeficiency. We used mycobacteria isolated from the patients and diphtheria, tetanus and pertussis vaccine (DTP) to study antigen-specific T lymphocyte responses. We observed that interferon-gamma (IFN-gamma) production by T cells in response to antigens (both mycobacteria and DTP) in these patients with disseminated infection was greatly impaired. This defect did not seem to be the result of T cell unresponsiveness, as phytohaemagglutinin (PHA) stimulation was able to induce high levels of IFN-gamma comparable to those seen in control patients with localized infection. Further experiments showed that peripheral blood mononuclear cells (PBMC) from patients with disseminated infection were able to present influenza haemagglutinin (HA) peptides to specific T cell clones. However, this ability was lost when the whole HA protein was used as source of antigen. Taken together, these observations support the notion that the primary immune defect in these patients with disseminated mycobacterial infection rests in the antigen-processing functions of their antigen-presenting cells (APC). These findings may provide clues to the wider problem of susceptibility to mycobacteria and other intracellular pathogens and have implications in designing therapy for these patients.

Antigen Presentation↗

PubMed1996

Regulation of cytokine and chemokine transcription in a human TH2 type T-cell clone during the induction phase of anergy.

BACKGROUND: Selected cytokines produced by allergen specific CD4+ T cells from atopic individuals contribute to both the specific and non-specific effector mechanisms of the allergic immune response. The chemokine family of cytokines and tumour necrosis factor (TNF)-alpha are leucocyte regulatory and proinflammatory molecules. The chemokines include interleukin (IL)-8 and the RANTES/SIS cytokines. OBJECTIVE: There has been no systematic survey of chemokine production in T-cell subtypes. Because of their wide range of biological properties, it might be expected that they would be closely regulated by T cells. This paper illustrates one way (through the characterization of T-cell clones) these questions might be addressed. METHODS: Northern blot analysis was used to quantitate steady state transcription of selected cytokine genes and enzyme linked immunosorbent assay (ELISA) was used to quantitate soluble product. RESULTS: mRNA expression of the chemokines (IL-8, HuMIP-1 alpha and HuMIP-1 beta) and TNF alpha is upregulated in TH2-like cloned house dust mite reactive human CD4+ T cells under conditions of activation and during the induction phase of anergy. Although the development of anergy superinduces mRNA for both IL-8 and TNF alpha, protein production is low compared with that released during activation. In contrast, RANTES, a chemoattractant for CD4+/CD45RO+ memory T cells, eosinophils and basophils, is constitutively expressed at the RNA level by the T cells and not modulated by signals of activation and anergy induction. The production of IL-2, IL-4 and IL-5 mRNA and proteins during the induction of anergy peaks at 2 h after stimulation, whereas the kinetics following activation of the T cells is delayed in comparison. CONCLUSION: These data show that the induction of the anergic state coincides with post-transcriptional regulation of selected cytokine genes. Further study of these phenomena will impact on our understanding of the mechanisms of induction of anergy and the regulation of allergic immune responses in desensitization.

Animals↗

PubMed1996

Oligoclonal V gene usage by T lymphocytes in bronchoalveolar lavage fluid from sarcoidosis patients.

The T-cell antigen receptor (TCR) repertoire was examined in lymphocytes isolated from the lungs and blood of 12 sarcoidosis patients and nine control patients. This analysis, by polymerase chain reaction (PCR), examined the variable (V)-domain genes of both the alpha and beta chains of the TCR. This is the first study to examine the usage of all known V alpha gene segments in sarcoidosis. A similar degree of diversity was observed in the TCR repertoire in the lungs and blood of the sarcoidosis patients. However, 11 of the 12 sarcoidosis patients showed an increased use of particular TCR V alpha and V beta genes in lung T cells as compared with blood. The pattern of TCR V gene bias in the lung T cells was specific for each patient. The clonality of selected V genes was examined by determining the third complementarity-determining region (CDR3) length polymorphism of particular PCR products. The majority of lung T cells with biased TCR V gene segments were oligoclonal. Altogether, these results suggest oligoclonal expansion of lung T cells in response to a local antigenic stimulus, with additional nonspecific T-cell accumulation. The variability in the V gene segments used by the expanded T-cell subsets in different sarcoidosis patients may reflect different epitopes or antigens being recognized in the lung, as well as variations in major histocompatibility complex (MHC) haplotype between the patients.

Adult↗

PubMed1995

The high mobility group transcription factor, SOX4, transactivates the human CD2 enhancer.

A strong T cell-specific enhancer is located 3' to the human CD2 gene. Six sequences within this enhancer are bound by proteins present in T cell nuclear extracts. These sequences share homology with sequences bound by several transcription factors involved in T cell- and lymphoid-specific transcription. The results presented here demonstrate that the human T cell-specific transcription factor, SOX4, is able to bind to one of these regions; further, SOX4 transactivates transcription of a reporter gene via three tandem copies of this sequence. The binding of SOX4 to this site is not via a canonical HMG protein binding sequence, identifying a novel class of binding site for this protein. A second sequence within the CD2 enhancer closely resembles the IL-2 NF-AT site. We show that it is bound by the ets-related factor, Elf1. However, unlike the IL-2 NF-AT sequence, the CD2 NF-AT-like sequence is unable to confer transcriptional inducibility on a reporter gene. Consistent with this result, we show that the observed increase in expression of CD2 protein on the cell surface following T cell activation is a post-transcriptional event.

Antigens, CD↗

PubMed1995

Differential induction of the NF-AT complex during restimulation and the induction of T-cell anergy.

Stimulation of human CD4+ T-cell clones through the T-cell receptor (TcR) by high doses of specific peptide results in the induction of a long-lived state of nonresponsiveness that has been called anergy. During the induction of anergy, T cells are phenotypically similar to cells responding to an immunogenic stimulus. The amount of TcR at the cell surface is downmodulated, whereas the CD2 and CD25 receptors are increased. When restimulated, however, anergic T cells fail to up-regulate transcription of the IL-2 gene and in consequence do not produce IL-2. In this study, we have compared the ability of various transcription factors to bind to their appropriate site on DNA. Factors were isolated from the nuclei of T cells that were in the induction phase of anergy or were undergoing activation. The pattern of binding activity in restimulated T cells is consistent with the pattern that has previously been shown to regulate T-cell-specific expression of the IL-2 and the beta chain of the TcR genes. The measured binding to a TCF-1 site is the same in the nuclei of resting, activated, and anergized cells. The inducible factors NK-kappa B, beta E2, CD28RC, and AP-1 are not expressed in resting cells and are twofold lower in anergized as compared with activated cells. In contrast, anergic T cells express approximately eightfold lower amounts of NF-AT, a member of the class of inducible factors that regulates IL-2 gene transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1995

The domain structure and functional relationships in the bacterial superantigen, SEB.

Staphylococcal enterotoxin B (SEB) has three essential biological properties that appear to be mediated by particular domains of the protein. As a superantigen, SEB triggers the T cell receptor (TcR) of a selected subset of T cells where the beta chain is encoded by particular V beta gene products. T cell triggering is generally dependent upon the ability of SEB to form a ternary complex with the TcR and MHC class II molecules and this interaction is relatively unrestricted by class II polymorphism. The third property of SEB is its potent toxicity; a few nanograms are sufficient to cause vomiting and violent diarrhoea in primates. In this study, we confirm the importance of the amino terminal domain of SEB for stimulation of human T cells. It is clear that the first 138 residues are the minimal mitogenic component of the toxin, and deletion of just seven more residues abrogates all activity. A mutant molecule with an internal deletion of these seven residues (SEB delta 131-138) was mitogenic suggesting that the region can confer stability to the rest of the protein, but does not include contact sites for either class II or the TcR. We further show that the disulphide loop is not essential for T cell recognition and that each of our mutants binds class II molecules with reduced affinity and is subtly variant in the pattern of TcR that it stimulates.

Amino Acid Sequence↗