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W R Heath

Publications and source records attributed to W R Heath.

At least 73 records · Page 4Linked to original sources
PubMed1994

T cell receptor antagonist peptides induce positive selection.

We have used organ culture of fetal thymic lobes from T cell receptor (TCR) transgenic beta 2M(-/-) mice to study the role of peptides in positive selection. The TCR used was from a CD8+ T cell specific for ovalbumin 257-264 in the context of Kb. Several peptides with the ability to induce positive selection were identified. These peptide-selected thymocytes have the same phenotype as mature CD8+ T cells and can respond to antigen. Those peptides with the ability to induce positive selection were all variants of the antigenic peptide and were identified as TCR antagonist peptides for this receptor. One peptide tested, E1, induced positive selection on the beta 2M(-/-) background but negative selection on the beta 2M(+/-) background. These results show that the process of positive selection is exquisitely peptide specific and sensitive to extremely low ligand density and support the notion that low efficacy ligands mediate positive selection.

Amino Acid Sequence↗
PubMed1994

Peptide antagonists that promote positive selection are inefficient at T cell activation and thymocyte deletion.

We set out to determine whether thymocytes from T cell receptor (TCR) transgenic animals specific for a class I-restricted determinant from ovalbumin (OVA) showed the same fine specificity for antigen-driven deletion in single-cell suspension culture as required for mature T cell activation. The transgenic TCR is specific for the Kb-restricted peptide OVA257-264 (SIINFEKL) which is known to have four TCR contact residues at position 1, 4, 6, and 7 from the crystal structure of this fragment in complex with Kb. OVA257-264 analogs systematically substituted at each of these positions were assayed for their ability to promote immature double-positive thymocyte deletion or mature T cell activation of a cytotoxic T lymphocyte line derived from this transgenic mouse. In the absence of additional antigen-presenting cells, single-cell thymocyte suspensions showed that the specificity for double-positive thymocyte deletion and mature T cell activation was virtually identical, demonstrating a limited cross-reactivity with a number of variants having conservative substitutions at these exposed residues. These peptides were considerably more efficient at both thymic deletion and mature T cell activation than a number of non-conservative substitution analogs known to act as antagonists of OVA257-264 and capable of selecting transgenic T cells in thymic organ culture. Therefore, both peripheral T cell activation and thymic deletion have an overall similar pattern of peptide specificity which differs from that required for positive selection. This suggests that a subset of major histocompatibility complex-presented peptides could promote positive selection without causing either thymic deletion or peripheral activation of those selected T cells.

Amino Acid Sequence↗
PubMed1993

Avidity for antigen can influence the helper dependence of CD8+ T lymphocytes.

To examine the influence of avidity on the helper dependence of CD8+ T cells, purified CD8+ T cells from transgenic mice expressing a TCR specific for H-2Kb were tested for their responsiveness to spleen cells expressing different densities of H-2Kb. High avidity interactions resulted in the induction of strong proliferative responses by purified CD8+ T cells. These cells synthesized their own IL-2 and IL-2R. By contrast, low avidity interactions failed to induce proliferation or detectable IL-2 synthesis, although IL-2R were induced and addition of rIL-2 induced a proliferative response. These results are consistent with a model in which TCR avidity for Ag can determine the helper dependence of a CD8+ T cell.

Animals↗
PubMed1993

Failure of a protective major histocompatibility complex class II molecule to delete autoreactive T cells in autoimmune diabetes.

The association of major histocompatibility complex genes with autoimmune diseases is firmly established, but the mechanisms by which these genes confer resistance or susceptibility remain controversial. The controversy extends to the nonobese diabetic (NOD) mouse that develops disease similar to human insulin-dependent diabetes mellitus. The transgenic incorporation of certain class II major histocompatibility complex genes protects NOD mice from diabetes, and clonal deletion or functional silencing of autoreactive T cells has been proposed as the mechanism by which these molecules provide protection. We show that neither thymic deletion nor anergy of autoreactive T cells occurs in NOD mice transgenic for I-Ak. Autoreactive T cells are present, functional, and can transfer diabetes to appropriate NOD-recipient mice.

Animals↗
PubMed1993

Expression of two alpha chains on the surface of T cells in T cell receptor transgenic mice.

CD8+ T cells taken directly from mice expressing a Kb-specific T cell receptor (TCR) transgene expressed the transgenic TCR in a bimodal profile as detected by flow cytometric analysis using a clonotype-specific monoclonal antibody. Those cells expressing the lower density of the transgenic TCR expressed the transgenic beta chain and two different alpha chains on their surface. One alpha chain was the product of the alpha transgene, whereas the other was derived by endogenous rearrangement. This report provides the first demonstration that T cells isolated directly from mice may express two different TCR clonotypes on their surface. The potential consequences of this finding for studies using TCR transgenic mice and for the induction of autoimmunity are discussed.

Animals↗
PubMed1992

Autoimmune diabetes as a consequence of locally produced interleukin-2.

During cell differentiation in the thymus, self-reactive T cells can be generated. The majority of these seem to be deleted after intrathymic encounter with the relevant autoantigen. As all self antigens are unlikely to be present in the thymus, some autoreactive T cells may escape censorship. Here we study the fate of these cells using transgenic mice expressing the class I molecule H-2Kb (Kb) in the insulin-producing beta-cells of the pancreas. These mice were crossed with mice transgenic for genes encoding a Kb-specific T-cell antigen receptor (TCR) which could be detected using a clonotype-specific monoclonal antibody. Although T cells expressing the highest level of transgenic TCR were deleted intrathymically in double-transgenic mice, Kb-specific T cells were detected in the periphery. These cells caused the rejection of Kb-expressing skin grafts, but ignored islet Kb antigens even after priming. But when double-transgenic mice were crossed with transgenic mice expressing the lymphokine interleukin-2 in the pancreatic beta-cells, there was a rapid onset of diabetes. These results indicate that autoreactive T cells that ignore self antigens may cause autoimmune diabetes when provided with exogenous 'help' in the form of interleukin-2.

Animals↗
PubMed1991

Alloreactive T cells discriminate among a diverse set of endogenous peptides.

Previous studies have demonstrated that class I major histocompatibility complex (MHC) molecules are occupied by peptides of endogenously synthesized self proteins. Since graft rejection appears to be mediated by the normal occurrence of high frequencies of cytolytic T lymphocytes (CTLs) specific for allogeneic MHC molecules, it is important to know if such CTLs recognize specific MHC-peptide complexes (as opposed to the MHC molecule per se) and, if so, whether allorecognition is the result of the recognition of a limited spectrum of antigenic determinants or, alternatively, the recognition of a diverse array of MHC-self peptide complexes. This issue has been examined using a mutant cell line, T2Kb, that expresses class I molecules devoid of endogenously derived peptides. This cell line was not recognized by Kb-specific alloreactive CTLs. However, upon exposure to peptides derived by cyanogen bromide cleavage of cytoplasmic proteins these cells became sensitized for recognition and lysis by a majority of the CTL clones examined. Reverse-phase HPLC fractionation of the heterogeneous cell-derived peptides revealed that individual CTL clones were specific for different peptide antigen(s). Thus, the high frequency of alloreactive T cells that is responsible for graft rejection appears to represent the sum of numerous T-cell clones specific for a diverse array of endogenous peptide antigens presented in the context of allogeneic class I molecules.

Animals↗
PubMed1991

Cell-type-specific recognition of allogeneic cells by alloreactive cytotoxic T cells: a consequence of peptide-dependent allorecognition.

Recently, we provided the first direct evidence that the determinant recognized by some alloreactive cytotoxic T lymphocytes (CTL) clones included an endogenous peptide antigen(s), which could be derived by cyanogen bromide cleavage of cytoplasmic proteins. The studies outlined in this report provide three important findings which confirm and extend our previous results. First, the description of peptide-dependent Kb-specific alloreactive CTL clones is now extended to clones isolated from several responders primed with allogeneic EL4 tumor cells. This indicates that the previously reported results were not unique to the original EL4-primed responder. Second, we establish that peptide-dependent Kb-specific alloreactive CTL clones can be identified after priming with normal allogeneic spleen cells. Thus, induction of peptide-dependent CTL clones is not a unique property of EL4 tumor cells. These results suggest that peptide plays an important role in formation of the allogeneic determinants recognized during graft rejection. Third, these studies reveal that a number of alloreactive CTL clones exhibit cell-type-specific recognition of allogeneic murine target cells. This is consistent with the possibility that some CTL clones recognize peptide antigens that are limited in their tissue distribution. The mechanisms and implications of these findings are discussed.

Animals↗
PubMed1989

Peptide-dependent recognition of H-2Kb by alloreactive cytotoxic T lymphocytes.

Antigen-specific T lymphocytes appear to recognize foreign antigens in the form of peptide fragments presented within the antigen-binding groove of class I or class II molecules encoded by the major histocompatibility complex (MHC). Alloreactive T cells also show specificity for MHC molecules, and various reports suggest that residues of the MHC molecules constitute at least part of the ligand to which alloreactive T-cell receptors bind. The X-ray crystal structure of the human MHC class I molecule, HLA-A2, has provided evidence to strengthen the argument that MHC-bound self-peptide might also contribute to such recognition. We now provide direct evidence for this, showing that at least some alloreactive cytotoxic T lymphocyte clones recognize peptide fragments derived from cytoplasmic proteins. We reasoned that if self-peptides were involved in allorecognition, then the sequence of some of these peptides could vary between species, resulting in species-restricted distribution of the relevant ligand(s). Several alloreactive cytotoxic T lymphocyte clones specific for H-2Kb, expressed by the murine cell line EL4, did not lyse a human-cell transfectant expressing the H-2Kb molecule (Jurkat-Kb cells). However, these clones were able to lyse Jurkat-Kb cells sensitized by preincubation with an EL4 cytoplasmic extract cleaved by cyanogen bromide. The sensitizing activity from this extract was destroyed by protease and appeared to be due to a peptide consisting of 10 to 15 amino acids.

Animals↗
PubMed1989

Species-restricted interactions between CD8 and the alpha 3 domain of class I influence the magnitude of the xenogeneic response.

As compared with the vigorous T cell response normally observed against allogeneic MHC molecules, T cells recognize xenogeneic MHC molecules poorly. To define structural features of the MHC molecule important for such species-specific recognition, HLA-A2(A2)-specific murine CTL were examined for their recognition of transfected cell lines expressing the class I molecules A2 or A2/H-2Kb(A2/Kb). A2/Kb is a chimeric molecule consisting of the alpha 1 and alpha 2 domains of A2 and the alpha 3, transmembrane, and cytoplasmic regions of Kb. The majority of CTL clones showed enhanced recognition of transfected cell lines expressing this chimeric molecule. Enhanced recognition was shown to correlate with sensitivity of the CTL clones to inhibition by anti-CD8 antibody. These results suggested that CD8 may interact with class I in a species-specific manner, and that suboptimal CD8 interaction with the alpha 3 domain of xenogeneic molecules may be an important contribution to poor xenoreactivity. This conclusion was supported by the capacity of A2/Kb, but not A2 human cell transfectants, to induce a primary in vitro CTL xenoresponse specific for A2.

Animals↗
PubMed1989

Mapping of epitopes recognized by alloreactive cytotoxic T lymphocytes using inhibition by MHC peptides.

To identify epitopes recognized by alloreactive CTL we have examined H-2Kb-specific CTL for their recognition of synthetic peptides with sequences derived from the native Kb class I molecule. Consecutive nested peptides spanning the immunogenic alpha 1 and alpha 2 domains of Kb were tested for their capacity to inhibit CTL clones in their recognition of cells expressing the native Kb molecule. Inhibition by these peptides was found to be an extremely rare event. One peptide (Kb.111-122) did inhibit recognition by one particular CTL clone, clone 13. Upon further investigation it was observed that clone 13 also recognized peptide Kb.111-122 when presented in the context of the syngeneic MHC molecule, Kd. Considering that residues 111 to 122 are located at the base of the antigen groove, and clone 13 is able to recognize Kb.111-122 when presented by syngeneic target cells, we suggest that inhibition of this CTL clone may be due to MHC restricted, self-presentation of peptide rather than to direct binding of free peptide to the TCR. Taken together, these results suggest inhibition of allospecific CTL by MHC peptides is a rare event at least for Kb recognition. Furthermore, they demonstrate the need for caution when interpreting inhibition by peptide as evidence for recognition by the TCR of the corresponding region on the native molecule.

Amino Acid Sequence↗
PubMed1989

Consequences of self-presentation of peptide antigen by cytolytic T lymphocytes.

We have used H-2Db-restricted CTL clones specific for peptide 365 to 380 of the influenza nucleoprotein to seek evidence for interaction between the TCR and peptide Ag. Preincubation of these CTL with peptide 365 to 380 resulted in inhibition of target cell lysis. In addition, CTL lysed allogeneic targets in the presence of soluble peptide Ag. Investigation of the basis of these two phenomena revealed a requirement for expression of H-2Db molecules by the effector cells. Either preincubation with anti-Db mAb or the use of chimera-derived H-2d CTL specific for Db plus peptide ablated both peptide-dependent inhibition and lysis of allogeneic cells, suggesting these activities are a consequence of self-presentation of peptide Ag by CTL. Lysis of allogeneic cells appears to represent bystander lysis by CTL in response to recognition of peptide on other effector cells. Lysis inhibition is attributable to a highly potent form of cold target inhibition in which CTL serve as their own cold targets.

Animals↗
PubMed1989

Therapeutic and side-effect profile of a selective and reversible MAO-A inhibitor, brofaromine. Results of dose-finding trials in depressed patients.

Brofaromine (CGP 11 305 A), a new reversible and selective MAO-A inhibitor, was studied in two multicentre, (Trial A and Trial B) double-blind, dose-finding trials in a total of 124 depressed in-patients. Doses of 25, 50 and 75 mg bid were compared, to determine which was the most effective. The duration of the trials was four weeks. The comparative drugs were nomifensine (100 mg/day) and tranylcypromine (20 mg/day). The majority of patients in the Trial A was classified as "endogenous" depression. Diagnosis of depression was based on DSM-III or ICD-9 criteria. Conversely, most of the patients in Trial B were "non-endogenous" depressives. In "endogenous" depression, a statistically significant linear dose-response relationship was found in all the efficacy variables assessed. The most effective dose was 150 mg/day. This dose gave a mean drop of 25.3 +/- 11.9 (S.D.) points in the total Hamilton Depression Rating Scale (HAMD) scores and provided successful treatment in 83% of the patients treated, success being defined as a drop of at least 50% in the initial HAMD score at the end of the trial period. In "non-endogenous" depression, no statistical difference was found between the four treatment groups in any of the efficacy variables assessed. Response rate in all brofaromine groups averaged 59% (tranylcypromine group 60%). Tolerability was good in 90% or more of the brofaromine patients in both trials, regardless of the dose administered. The side effects reported most frequently were sleep disturbances, nausea, and headaches.

Adult↗