Biomedical subjects

K Heeg

Publications and source records attributed to K Heeg.

At least 55 records · Page 3Linked to original sources

PubMed1995

Induction of responsiveness in superantigen-induced anergic T cells. Role of ligand density and costimulatory signals.

The bacterial superantigen staphylococcal enterotoxin B (SEB) induces in vivo a state of anergy defined by the inability of V beta 8+ CD4+ T cells to produce IL-2 upon restimulation in vitro. However, restimulation in vivo triggers a burst of acutely released lymphokines including IL-2 and TNF, paralleled by up-regulation of lymphokine-specific mRNA. Since anergy as defined in vitro appears not to operate in vivo, we analyzed parameters able to induce responsiveness in anergic T cells. We show here that in vitro stimulation of anergic T cells with competent Ag-presenting cells induces responsiveness, provided the APC (activated B cells or dendritic cells) present high concentrations of SEB. Crosslinking of CD28 molecules on anergic T cells could substitute the requirement for competent APC. Quantitation of TCR threshold by determining the SEB concentrations able to trigger half-maximal T cell responses revealed that anergic and normal T cells exhibited the same TCR threshold for the expression of functional IL-2 receptors (IL-2R), yet the TCR threshold for induction of IL-2 production was 10- to 100-fold elevated in anergic T cells. TCR threshold for normal and anergic T cells was further dependent on the type of APC, i.e., costimulus-competent APC required 100-fold less SEB. The results indicate that extrinsic factors such as ligand concentration and costimulus competence of APC can overcome the heightened TCR threshold of anergic T cells, thus reverting anergy into responsiveness.

Animals↗

PubMed1995

Differential effects of the immunosuppressive agents cyclosporine and leflunomide in vivo. Leflunomide blocks clonal T cell expansion yet allows production of lymphokines and manifestation of T cell-mediated shock.

The effects of leflunomide and CsA on immune responses in vitro and in vivo were investigated. Like CsA, leflunomide inhibited mitogen- or antigen-driven T cell proliferation in vitro. However, leflunomide impaired neither the capability of T cells to produce IL-2 and IL-4, nor the expression of IL-2R, that is, the acquisition of competence. In contrast to CsA, the IL-2-driven growth of secondary T cells was blocked by leflunomide. Cell cycle analyses revealed that activated T cells did not enter S phase of the cell cycle in the presence of leflunomide. Next, the effects of leflunomide and CsA on the T cell response toward the bacterial superantigen (SAg) staphylococcal enterotoxin B (SEB) were analyzed in vivo. SEB-induced early deletion (apoptosis) of a fraction of SEB-reactive V beta 8+ T cells and IL-2R expression were not impaired by either CsA nor leflunomide. On the other hand, both CsA and leflunomide prevented V beta 8-selective clonal T cell expansion and generation of SEB-specific cytolytic activity. In contrast to CsA, leflunomide treatment permitted in vivo SEB-induced production of T cell-derived lymphokines (IL-2 and TNF). Further, leflunomide failed to protect D-galactosamine-sensitized mice from SEB-induced, T cell-mediated lethal shock, whereas CsA was fully protective. Manifestation of SEB-induced T cell anergy was not impaired by leflunomide. Our results provide evidence that CsA and leflunomide differ significantly in their functional properties to suppress immune responses in that both agents inhibit T cell functions linked to clonal expansion, while leflunomide does not inhibit lymphokine secretion and thus permits lymphokine-mediated immune functions.

Animals↗

PubMed1995

Bacterial superantigens induce T cell unresponsiveness in B cell-deficient mice.

Bacterial superantigens such as staphylococcal enterotoxin B (SEB) cause in vivo a profoud and long-lasting state of unresponsiveness in ligand-reactive T cells. To test whether presentation of SEB by small resting B cells to ligand-reactive T cells is essential for the induction of T cell unresponsiveness, we analyzed the effect of SEB in B cell-deficient mice. We observed T cell deletion and T cell unresponsiveness in both B cell-deficient mice and control mice. We conclude that presentation of SEB by resting B cells is not a prerequisite for the induction of T cell unresponsiveness in vivo.

Animals↗

PubMed1995

In vivo CTL induction with point-substituted ovalbumin peptides: immunogenicity correlates with peptide-induced MHC class I stability.

Class I molecules are conformationally sensitive to peptide binding, prolonging the complex's half-life on the surface of the cell. By making a series of H2-Kb anchor motif amino acid point substitutions in the ovalbumin 257-264 octamer, we were able to analyse subtle changes in peptide binding, Kb stabilization and in vivo immunogenicity. The cell line RMA-S was used to determine peptide-dependent Kb stabilization under equilibrium and non-equilibrium binding conditions. Sixteen conservative and non-conservative amino acid substitutions were made at positions 3, 5 or 8 of the peptide. At 37 degrees C, Kb stabilization was differentially affected by these substitutions, with several substitutions severely affecting Kb surface expression. When the substituted peptides were used as immunogens to prime cytotoxic T lymphocytes (CTL) in vivo, each peptide's ability to stabilize Kb directly correlated with the intensity of specific CTL activation. We conclude that peptide class I stabilization is an important influencing factor in determining cell surface steady-state expression of these peptides and thus the breadth of CTL recruitment. These concepts may relate the phenomenon of immunodominance to cell surface-presented peptide steady-state levels and may also aid in peptide vaccine design.

Amino Acid Sequence↗

PubMed1995

Superantigen-reactive T cells that display an anergic phenotype in vitro appear functional in vivo.

Clonal deletion and/or inactivation establishes tolerance to self antigens. Endogenous and exogenous (bacterial) superantigens, like the staphylococcal enterotoxins, induce ligand-specific clonal anergy in vivo and thus are believed to mirror aspects of post-thymic tolerance mechanisms in mature peripheral T cells. Here we analyzed the level of anergy of ligand-responsive V beta 8+ T cells from staphylococcal enterotoxin B (SEB)-primed mice in vivo and in vitro. Upon in vitro restimulation with SEB, CD4+V beta 8+ and CD8+V beta 8+ T cells failed to produce IL-2. However, functional IL-2 receptors were triggered, since supplementation with IL-2 induced clonal growth in virtually all CD4+V beta 8+ and CD8+V beta 8+ T cells as determined by limiting dilution analyses. Thus in vitro unresponsiveness of lymphocytes from SEB-primed mice reflects the inability of SEB-reactive V beta 8+ T cells to produce IL-2. Surprisingly, anergy as defined in vitro was at variance with that in vivo. Following further challenge with SEB, systemic and acute lymphokine production (including IL-2 and tumor necrosis factor) occurred with almost identical peak values and kinetics to primary in vivo responses, and D-galactosamine-sensitized mice succumbed to lethal shock. Polymerase chain reaction analyses revealed that CD4+V beta 8+ expressed IL-2-specific mRNA in vivo upon restimulation with SEB. While lymphokine production and expression of the IL-2 receptor was similar to the response to in vivo primary stimulation, only CD8+V beta 8+ T cells expanded clonally upon reintroduction of SEB in vivo. Hence primed V beta 8+ T cells challenged with SEB display in vitro anergy yet in vivo responsiveness, at least in part. We conclude that the state of anergy is reversible, dependent upon the quality of activation signals provided in in vivo rather than in in vitro culture conditions.

Animals↗

PubMed1995

Identification of H-2Kb binding and immunogenic peptides from human papilloma virus tumour antigens E6 and E7.

Peptides can be used to induce MHC class I restricted cytotoxic T cells (CTL) through in vivo immunization. This approach may enable the development of peptide vaccination schemes for immunization against viral infection in humans. Human papillomavirus (HPV) is one of a few viruses associated with human cancer and the development of an anti-cancer vaccine seems possible. As a model approach, we searched the E6 and E7 proteins of the human papillomavirus type 16 for possible murine MHC class I restricted peptide epitopes. We utilized the mouse H2-Kb peptide binding motif which consists of phenylalanine or tyrosine at position five and leucine at the carboxy-terminus with the modification that leucine could be replaced by other aliphatic but non-aromatic amino acids. Four peptide sequences from E6 and two from E7 were selected. These peptides were tested for their ability to bind and stabilize Kb and for their immunogenicity in vivo. It was shown that one peptide from E6, E6.1 (50-57), bound Kb, but was not able to prime mice in vivo. In contrast, the two selected E7 peptides E7.1 (21-28) and E7.2 (48-55) bound Kb and were immunogenic in vivo. The peptide induced CTL lysed syngeneic EL-4 cells transfected with the open reading frame of E7 but not vector only transfectants. This implies that both peptides were naturally processed and presented by Kb on the surface of target cells. MHC class I peptide binding motifs therefore appear to be an effective and useful tool to predict peptide epitopes of proteins associated with cancer.

Amino Acid Sequence↗

PubMed1995

Superantigens: the paradox of T-cell activation versus inactivation.

Superantigens are interesting for several reasons. They exhibit unusual T-cell activation characteristics, they allow to follow in vivo ligand-reactive T cells, they cause T-cell-mediated shock symptoms and they may be causally involved in certain human diseases. This review focuses on the paradox of T-cell activation versus T-cell inactivation as caused by bacterial superantigens in vivo. T-cell activation leads to the acute release of toxic concentrations of lymphokines including tumor necrosis factor alpha/beta, gamma-interferon, interleukin (IL)-2, IL-4 and IL-10. T-cell inactivation mirrors induction of anergy, T-cell receptor downregulation, and that of CD2, CD4 and CD8 cell surface molecules, as well as of apoptosis. Two waves of apoptosis cripple the T-cell repertoire of superantigen-reactive T cells, an immediate one being induced within 24 h and a late one occurring at days 3-4. In between, clonal expansion of anergic ligand-reactive T cells takes place.

Down-Regulation↗

PubMed1995

Sensitization of MHC class I-restricted T cells to exogenous proteins: evidence for an alternative class I-restricted antigen presentation pathway.

Immunization with exogenous proteins usually fails to immunize CD8+ T cells in vivo. Here we report that chicken ovalbumin (OVA) denatured by heat or sodium dodecyl sulphate (SDS) effectively induced CD8+ cytolytic T cells in vivo. The cytolytic T-lymphocyte (CTL) population generated recognized syngeneic target cells pulsed with the immunodominant OVA peptide (257-264) or transfected with the OVA protein-encoding gene. To analyse the mechanisms of how denatured OVA enters the class I-restricted pathway of antigen presentation, we took advantage of the fact that denatured OVA sensitizes target cells in vitro for lysis by OVA-specific CTL. We found that neither inhibition of protein synthesis (by cycloheximide) nor blocking of transport via the Golgi apparatus (by brefeldin A) interfered with the class I-restricted presentation of denatured OVA in vitro. In addition, transporter associated with antigen presentation (TAP)-dependent transport into the endoplasmic reticulum (ER) was not required for effective presentation, as TAP-deficient cells (RMA-S) could be sensitized effectively by denatured OVA for recognition by class I-restricted CTL. In contrast, class I-restricted presentation of denatured OVA was sensitive to lysosomotropic agents (NH4Cl, vinblastine and leupeptin), indicating that endosomal-like compartments are involved in the presentation of denatured OVA. Sensitization was inhibited at low temperature, yet took place in the presence of sucrose and in the absence of K+, indicating that denatured OVA enters the cell via fluid-phase endocytosis. Hence the results provide further evidence for an alternative class I-restricted pathway of antigen presentation for exogenous proteins. As that pathway seems to be effective in vivo, it offers a new and effective way of vaccination of CD8+ CTL.

Amino Acid Sequence↗

PubMed1995

Peptide engineering allows cytotoxic T-cell vaccination against human papilloma virus tumour antigen, E6.

Major histocompatibility complex (MHC) class I allele-specific binding motifs have proved useful in predicting cytotoxic T-cell epitopes from immunogenic proteins. In a search of the E6 protein from human papilloma virus type 16 utilizing the Kb binding motif, we discovered four potential binding peptides. One peptide, E6.1 (sequence 50-57, YDFAFRDL), was poor in its ability to stabilize empty Kb on RMA-S cells, with a t1/2 = 33 min versus 30 min for empty Kb. This peptide subsequently proved to be non-immunogenic upon mouse in vivo vaccination. It was hypothesized that an isoleucine for aspartate substitution at position 2 would improve Kb stabilization kinetics and therefore immunogenic potential. The engineered peptide E6.1 I2 increased the Kb t1/2 to 100 min and was immunogenic upon in vivo vaccination. Cytolytic T lymphocytes (CTL) raised with the E6.1 I2 peptide responded to cells pulsed with either the wild-type peptide or the engineered peptide, implying a blindness to the substitution. More striking, these CTL also lysed a syngeneic cell line transfected with the E6 gene, implying that the E6.1 peptide was processed and presented. These data demonstrate that subimmunogenic peptides can be engineered to improve binding kinetics, which in turn improves immunogenicity. Provided that poor binding peptides are processed, the induction threshold for CTL activation can be achieved with engineered peptides, thus allowing for the kill of wild-type target cells. This approach may prove relevant to the design of subunit vaccines to virally induced tumours.

Animals↗

PubMed1994

Exogenous superantigens acutely trigger distinct levels of peripheral T cell tolerance/immunosuppression: dose-response relationship.

Ligand-specific immunosuppression requires an understanding of the parameters that control peripheral T cell tolerance. T cell receptor (TcR) transgenic mice offer a clear advantage for studying post-thymic tolerance mechanisms in vivo that are operational in a monoclonal T cell population with preselected antigen specificity. Yet it is unclear whether the rules defined in monoclonal T cells of genetically manipulated mice reflect those operative in clonally diverse peripheral T cells of normal mice. To analyze acute tolerance mechanisms in unselected peripheral T cells, we challenged normal mice with the superantigen staphylococcal enterotoxin B (SEB) and analyzed ligand-reactive V beta 8+ T cells for TcR-triggered tolerance mechanisms such as anergy, TcR down-regulation, or apoptosis. Upon challenge with graded doses of SEB (0.001-10 micrograms) V beta 8+ T cells become anergic within 6-16 h. Importantly, a dosage effect of SEB in regard to the level of anergy induced was observed. Anergy induced by low concentrations of SEB (0.001-0.1 microgram) is transient and is overcome by clonal growth, while higher concentrations of SEB (0.1-10 micrograms) cause long-lasting anergy resistant to cell cycle progression. At high SEB concentrations (1-10 mg) about 50% of the anergic V beta 8+ T cells additionally down-regulate their TcR-CD3 complex, followed by a loss of CD2, CD4, CD8 accessory molecules. In parallel, T cell phenotype-negative but genotypically V beta 8+ T cells are generated. The T cell phenotype-negative cells reacquire their V beta 8+ T cell phenotype upon culture in vitro. In vivo, a subset of V beta 8+ cells, defined by an intermediate stage of TcR down-regulation, i.e. V beta 8lowCD3+ cells, but not T cell phenotype-negative cells are selectively programmed for apoptosis, which occurs within 1 h. These data suggest that SEB triggers distinct tolerance pathways which operate in a hierarchical fashion in clonally diverse ligand-reactive T cells. Specifically, the results illustrate the power of exogenous superantigens to exploit these distinct tolerance pathways, thereby achieving distinct levels of immunosuppression.

Animals↗

PubMed1994

The immunodominant peptide from listeriolysin in Quil A liposomes vaccinates CD8+ cytolytic T cells and confers protection to infection.

Cytolytic T-cell vaccination with immunodominant MHC class I-restricted peptides contained within Quil A liposomes has been previously demonstrated. In recent years, Quil A has been under consideration for use as an adjuvant in humans. We assessed the possible use of peptide inoculation in the context of Quil A liposomes to be protective in a mouse model. Listeria monocytogenes was used as the challenge pathogen and the previously identified listeriolysin 91-99 peptide as the immunogen. The listeriolysin 91-99 Quil A liposome inoculum showed significant enhancement of survival after challenge with up to 10 times the L. monocytogenes LD50.

Adjuvants, Immunologic↗

PubMed1994

Vaccination with immunodominant peptides encapsulated in Quil A-containing liposomes induces peptide-specific primary CD8+ cytotoxic T cells.

Immunostimulating complexes (ISCOMs), containing lipids, the saponin Quil A, and proteinaceous antigens, have been proven to vaccinate effectively CD8+ cytolytic T cells in vivo. However, conventional ISCOM technology is restricted to hydrophobic proteins or fatty acid-derivatized proteins or peptides. We therefore analysed whether Quil A-containing liposomes are an effective vehicle to shuttle hydrophilic proteins or peptides into the MHC class I pathway of antigen presentation resulting in the in vivo induction of antigen-specific cytolytic T cells (CTL). Liposomes were formed by a lipid dry-down method followed by resuspension with an aqueous solution containing protein/peptide and Quil A and then an extrusion step. Quil A-containing liposomes are an effective means to elicit a CD8+ CTL response to peptide antigen in vivo. CTL could be raised in C57B1/6 mice against ovalbumin (OVA) peptide 257-264 and vesicular stomatitis virus nucleoprotein 52-59, as well as in Balb/c mice against listeriolysin peptide 91-99 and cytomegalovirus pp89 168-176, demonstrating the versatility of this approach. The elicited response was peptide-specific, peptide dose-dependent and Quil A was necessary. Vaccination with liposomes entrapping the whole ovalbumin molecule or an extended (OVA) peptide 254-276 also yielded a CTL responsive to the immunodominant OVA peptide 256-264, implying cellular internalization and correct processing. Thus Quil A-containing liposomes appear to be a versatile vehicle to vaccinate CD8+ T cells in vivo; in addition, they could rapidly enhance the understanding of subunit vaccines and rules of antigen processing and peptide-MHC class I binding.

Adjuvants, Immunologic↗

PubMed1994

Superantigen-induced anergy of V beta 8+ CD4+ T cells induces functional but non-proliferative T cells in vivo.

The response profile of staphylococcal enterotoxin B (SEB)-primed murine V beta 8+ CD4+ and V beta 8+ CD8+ T cells was analysed upon rechallenge in vitro. While in vitro responses to secondary stimulation with SEB were reduced to background levels, the in vivo reactivity after rechallenge with SEB was retained, in that SEB-primed mice succumbed to lethal T-cell shock, lymphokines [interleukin-1 (IL-1), IL-2, Il-4, IL-6, IL-10, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha)], and lymphokine-specific mRNA accumulation could be detected in V beta 8+ CD4+ and V beta 8+ CD8+ T cells. However, V beta 8+ CD4+ T cells failed to enter the cell cycle. While the phenotype of V beta 8+ CD8+ T cells was indistinguishable from that of their counterparts from naive mice, V beta 8+ CD4+ T cells exhibited in vivo an unusual phenotype as non-proliferative but functional T cells. We conclude that in vitro-defined anergy does not disclose the functional abilities of ligand-reactive V beta 8+ T cells in vivo, and that priming with superantigen (SAg) induces in vivo a differentiation of SEB-reactive V beta 8+ CD4+ T cells into a non-proliferative but functional phenotype.

Animals↗

PubMed1993

Bacterial superantigens induce rapid and T cell receptor V beta-selective down-regulation of L-selectin (gp90Mel-14) in vivo.

Upon challenge of mice with bacterial superantigens such as staphylococcal enterotoxin B, several facets of TCR V beta-selective acute T-cell alterations can be observed, which include acute T cell priming, and systemic lymphokine release followed by ligand-specific unresponsiveness. Prompted by experiments showing that stimulation of T cells by phorbol esters in vitro results in rapid shedding of the L-selectin homing receptor, we investigated the expression of adhesion molecules on superantigen-responsive T cells in vivo. Here we show that bacterial superantigens cause TCR V beta-specific loss of L-selectin. Down-regulation of L-selectin was selective, since the expression of other lymphocyte surface receptors was not changed. L-Selectin down-regulation represents a superantigen-induced immediate cell surface alteration and was not observed on T cells stimulated by TCR-specific antibodies. Loss of expression was almost complete within 30 min, and recovered 50 h after challenge. The results suggest that acute loss of L-selectin is a hallmark of T cell activation by bacterial superantigens that may result in profound changes of T lymphocyte recirculation pathways.

Animals↗

PubMed1993

Acquired resistance to superantigen-induced T cell shock. V beta selective T cell unresponsiveness unfolds directly from a transient state of hyperreactivity.

TCR V beta selective T cell activation and systemic release of T cell-derived lymphokines causing lethal shock in D-galactosamine (D-Gal)-sensitized mice depicts only one facet of in vivo challenge with the superantigen staphylococcal enterotoxin B (SEB). An immediate second major aspect represents the induction of peripheral unresponsiveness in SEB-reactive V beta 8+ T cells. SEB causes in vivo within 4 h resistance to an otherwise lethal challenge with SEB plus D-Gal, as well as to a challenge with the heterologous ligand toxic shock syndrome toxin 1 plus D-Gal. Contrary to the first challenge, no serum-borne IL-2 and TNF are discernible during the second challenge. On the other hand, kinetic analyses in vitro of LN cells draining the site of the first in vivo challenge indicate that SEB-reactive T cells develop via a transient state of hyperreactivity into a profound state of ligand-specific unresponsiveness. Yet unresponsive V beta 8+ T cells express IL-2R and are responsive to the growth-promoting effect of IL-2. Cyclosporin A does not impair sequential induction of hyperreactivity and unresponsiveness with concomitant IL-2R expression, but effectively blocks systemic IL-2 and TNF release during the initial hyperreactive phase. Taken together, the in vitro data imply that ligand-specific hyperreactivity followed immediately by ligand-specific unresponsiveness represents a hallmark of in vivo challenge with the superantigen SEB. The in vivo data suggest the existence of additional suppressive elements masking the ligand specificity of the state of unresponsiveness induced by SEB.

Animals↗

PubMed1993

Primary in vivo responses to ovalbumin. Probing the predictive value of the Kb binding motif.

CD8+ cytolytic T cells recognize Ag presented by MHC class I molecules on the surface of target cells. It is known that presenting cells process nascent protein into peptides of approximately eight to nine amino acids which bind to the peptide groove of MHC class I and are transported to the cell surface. Recently, several laboratories have postulated that each MHC class I haplotype has a binding motif of at least two amino acids nested within the peptide. One such motif is XXXXF/YXXL which binds to the mouse MHC class I molecule, H2-Kb, and can be found in the known antigenic peptide from OVA at amino acids 257-264. By using the motif to scan OVA five peptides were found that fit this pattern, OVA 11-18, OVA 55-62, OVA 107-114, OVA 176-183, and OVA 257-264. Binding studies revealed that three out of the five peptides (OVA 55-62, OVA 176-183, and OVA 257-264) bind to MHC class I. To test the natural antigenicity of the predicted peptides, C57BL/6 mice were immunized with OVA containing immunostimulating complexes to elicit a MHC class I-driven response to naturally processed OVA. The cytolytic potential of the responding T cell population was tested in vitro by using EL-4 cells preincubated with the predicted synthetic peptides as targets. The known antigenic peptide OVA 257-264 elicited a strong response; however, OVA 176-183 was also recognized while the remaining three were not recognized. The CTL response did not strictly correlate with the ability of the selected peptides to bind Kb, for example, OVA 55-62 was able to bind Kb efficiently, yet elicited no cytolytic response. In addition, the plasticity of the peptide-binding motif was probed by making amino acid substitutions, and as a result the motif proved to be more flexible than previously suspected. This represents the first report of a Kb-associated CTL epitope within OVA other than OVA 257-264. It also demonstrates the predictive quality of the Kb-binding motif; however, not all predicted peptides were recognized by primary OVA-induced CTL, implying more rules of processing and binding are needed.

Amino Acid Sequence↗

PubMed1993

Quantitative analysis of lymphokine mRNA expression by a nonradioactive method using PCR and anion exchange chromatography.

Amplification of DNA by the polymerase chain reaction (PCR) has become an efficient tool in the study of gene expression. We describe the use of HPLC anion exchange chromatography to quantitate PCR products amplified from cDNA. The technique circumvents the use of both radioactivity and gel electrophoresis. We show that the method permits accurate quantitation of the gene product of interest and provides a clear separation of specific and non-specific products. The technique was applied to quantitate TNF-beta mRNA levels in unstimulated and stimulated mouse T cells.

Animals↗

PubMed1993

Clonal deletion as direct consequence of an in vivo T cell response to bacterial superantigen.

To date clonal deletion of peripheral mature T cells is restricted to in vivo model systems characterized by prolonged exposure of mice to antigens and clonal T cell expansion preceding clonal deletion. Here we describe that upon challenge of mice with the superantigen staphylococcal enterotoxin B two immediate events become imposed on ligand-reactive V beta 8+ T cells in lymph node cells draining the local site of injection. First, and within hours V beta selective clonal deletion is initiated via an apoptotic process. Second, the remaining V beta 8+ T cells first develop a profound state of ligand-specific unresponsiveness and subsequently initiate clonal in vivo growth. It is suggested that the dichotomy of events observed reflects a direct consequence of T cell receptor occupancy in the context of inappropriate signalling.

Animals↗