Arsen Zakarian

Duration and Strength of Extracellular Signal-Regulated Kinase Signals Are Altered During Positive Versus Negative Thymocyte Selection

During thymocyte development, high-affinity/avidity TCR engagement leads to the induction of negative selection and apoptosis, while lower TCR affinity-avidity interactions lead to positive selection and survival. To elucidate how these extracellular interactions are translated into intracellular signals that distinguish between positive and negative selection, we developed a culture system in which naive double-positive thymocytes were either induced to differentiate along the CD8+ lineage pathway or were triggered for clonal deletion. Using this system, we show that sustained low level activation of extracellular signal-regulated kinases (ERKs) promotes positive selection, whereas strong but transient ERK activation is coupled with negatively selecting stimuli. Importantly, similar ERK activation profiles were demonstrated during positive selection for strong agonist ligands presented at low concentrations or weak agonist ligands. This is consistent with the affinity/avidity model and a role for strong or weak agonists during positive selection. Surprisingly, the addition of a pharmacological inhibitor which blocks ERK activation prevented the induction of negative selection. These data suggest that the duration and strength of the TCR signal is involved in discriminating between positive and negative selection.

TNF receptor 1 (TNFR1) and CD95 are not required for T cell deletion after virus infection but contribute to peptide-induced deletion under limited conditions.

Deletion of mature T cells maintains cellular homeostasis and is involved in the maintenance of self tolerance to some peripheral self antigens. Previous studies have presented conflicting evidence for a role of the tumor necrosis factor receptor (TNFR) family member CD95 (Fas) in peripheral T cell deletion using CD95‐deficient mice. To evaluate cooperation between CD95 and another TNFR family molecule, TNFR1, we generated mice deficient for both CD95 and TNFR1. We showed that TNFR1 and CD95 do not contribute to the decline of antigen‐specific cytotoxic T lymphocytes after virus infection. Using TNFR1 / CD95‐deficient mice expressing the P14 TCR specific for a lymphocytic choriomeningitis virus‐derived peptide (p33) we showed that deletion of p33‐specific CD8+ T cells following high dose p33 administration is also normal. However, in non‐TCR‐transgenic TNFR1 / CD95‐deficient mice treated with the same p33 regimen, tolerance induction was defective. These data indicate that TNFR1 and CD95 are dispensable for deletion of antigen‐specific T cells after viral infection. However, under certain conditions, both TNFR1 and CD95 appear to cooperate in CD8+ T cell deletion.

Degree of ERK activation influences both positive and negative thymocyte selection.

Considerable evidence suggests that the ERK pathway is required for positive but not negative thymocyte selection. Here, we report that ERK is highly activated in double‐positive (DP) thymocytes expressing an MHC class I‐restricted TCR (P14) in response to negatively selecting conditions, whereas ligands that trigger positive selection induced weaker ERK activation. Biochemical evidence also shows that death by neglect is associated with a further reduction in ERK activation. These findings are consistent with the affinity / avidity model of thymocyte selection. To further examine the role of ERK in negative selection we used the MEK‐1 inhitibor, PD98059, a specific pharmacological inhibitor of the ERK pathway. Biochemical data demonstrated a reduction of ERK activity by PD98059 in the presence of the negatively selecting ligand. Analysis of P14 TCR‐transgenic fetal thymic lobes cultured with PD98059 under negatively selecting conditions showed impaired clonal deletion of DP thymocytes and a concomitant increase in positive selection of functional mature, TCRhi transgenic T cells. This demonstrates that altering ERK activity switched negative to positive selection. Contrary to previous reports that show an exclusive role for ERK signaling in positive selection, our data demonstrate that negative selection is also sensitive to the degree of ERK activation.

Inhibition of TCR triggering by a spectrum of altered peptide ligands suggests the mechanism for TCR antagonism

Understanding the parameters involved in T cell activation has been complicated by the discovery of partial T cell agonists. Altered peptide ligands (APL) have recently shown that different subsets of T cell responses can be selectively activated by certain peptides, which define a hierarchy of T cell activation. For cytotoxic T cells, this hierarchy ranges from sensitizing target cells for lysis through proliferation to effector cell induction. The degree of TCR down‐regulation mediated by APL‐MHC interactions correlates well with the induction of specific T cell effector functions. This suggests that the potential agonist response induced by a given peptide occurs at different triggering thresholds. To examine the relative agonist and antagonist functions of different peptides, we have investigated the ability of lymphocytic choriomeningitis virus glycoprotein‐derived APL to induce or inhibit a range of effector functions in naive CD8+ T cells. By this, we have defined a hierarchy of peptides that display a range of properties from strong agonist to no agonist function. At each level, peptides that were ranked lower in this hierarchy were able to interfere or antagonize the induction of effector functions by higher ranking peptides. We have therefore shown that this spectrum of peptides ranging from strong to no agonist function has an inverse gradient from strong antagonist to no antagonist function. Moreover, the ability of the different peptides to inhibit TCR internalization correlated with their ranking within the hierarchy. These findings support the model that antagonists are effectively preventing TCR oligomerization and functional TCR triggering.

IDENTIFICATION OF A CROSS-REACTIVE SELF LIGAND IN VIRUS-MEDIATED AUTOIMMUNITY

Molecular mimicry has been considered to be one of the potential mechanisms underlying the induction of autoimmune diseases. Using a TCR‐transgenic model specific for lymphocytic choriomeningitis virus (LCMV) we have examined the potential for cross‐reactive recognition of tissue‐restricted self peptides. Several peptides were identified that were able to cross‐react with the TCR‐transgenic virus‐specific T cells in vitro. One peptide was derived from dopamine β‐mono‐oxygenase, an enzyme expressed in the adrenal medulla. Interestingly, after activation of the transgenic T cells with LCMV glycoprotein peptides or viruses, infiltration of the adrenal medulla was detected in conjunction with alterations in dopamine metabolism. However, complete destruction of the adrenal medulla was not observed. This suggests that molecular mimicry may be sufficient for self recognition and infiltration, but other factors clearly contribute to chronic autoimmune disease.

Enhanced T cell responses contribute to the genetic predisposition of CD8-mediated spontaneous autoimmunity.

A number of factors have been demonstrated to influence the induction of pathogenic autoimmune responses, including the loss of regulatory T cells. To assess the contribution of regulatory T cells in CD8+ T cell‐mediated autoimmunity, RIP‐gp/P14 double‐transgenic mice expressing the lymphocytic choriomeningitis virus (LCMV) glycoprotein (gp) on pancreatic β‐islet cells, together with T cells expressing an LCMV‐gp‐specific T cell receptor (TCR), were crossed to RAG 2‐deficient mice. The loss of potentially regulatory T cells, however, did not contribute to diabetes induction. Surprisingly, both RIP‐gp/P14‐RAG+/– and RIP‐gp/P14‐RAG–/– developed spontaneous disease, suggesting an influence of the 129 genetic background on disease susceptibility. Further studies demonstrated that disease susceptibility was not due to nonspecific T cell activation, nor to enhanced cross‐presentation of LCMV‐gp, nor to decreased expression levels of the negative regulatory molecule CD5. Disease susceptibility did associate, however, with enhanced T cell responses. Thus, T cell hyperactivity combined with various genetic factors may predispose an individual to autoimmunity.