Scott S. Stuckman

T-cell activation and receptor downmodulation precede deletion induced by mucosally administered antigen

The fate of antigen-specific T cells was characterized in myelin basic protein (MBP) T-cell receptor (TCR) transgenic (Tg) mice after oral administration of MBP. Peripheral Th cells are immediately activated in vivo, as indicated by upregulation of CD69 and increased cytokine responses (Th1 and Th2). Concurrently, surface TCR expression diminishes and internal TCR levels increase. When challenged for experimental autoimmune encephalomyelitis during TCR downmodulation, Tg mice are protected from disease. To characterize Th cells at later times after antigen feeding, it was necessary to prevent thymic release of naive Tg cells. Therefore, adult Tg mice were thymectomized before treatment. TCR expression returns in thymectomized Tg mice 3 days after MBP feeding and then ultimately declines in conjunction with MBP-specific proliferation and cytokine responses (Th1-type and Th2-type). The decline correlates with an increase in apoptosis. Collectively, these results demonstrate that a high dose of fed antigen induces early T-cell activation and TCR downmodulation, followed by an intermediate stage of anergy and subsequent deletion.

Alterations in Immune Cell Phenotype and Function after Experimental Spinal Cord Injury

Traumatic injury to the spinal cord initiates a cascade of inflammatory-mediated injury and repair processes within the nervous system. In parallel, spinal injury could influence peripheral mechanisms of host defense (e.g., wound healing, antibody production) by altering lymphocyte phenotype and function. The goal of this study was to evaluate the physiological impact of spinal contusion injury on phenotypic and functional indices of lymphocyte activation. A flow cytometric time-course analysis of lymphocytes isolated from lymph node and spleen revealed an increase in CD4+ and a decrease in CD8+ lymphocytes during the first week post injury. The functional potential of lymphocytes was also evaluated based on their ability to proliferate in the presence of a biologically relevant antigen (myelin basic protein, MBP) or a lymphocyte mitogen. The data revealed increased proliferation to MBP by 3 days postinjury in lymphocytes isolated from lymph node but not spleen. By 1 week postinjury, increased proliferation to mitogen was noted in both the lymph node and the spleen suggesting a general increase in lymphocyte reactivity during this time interval. Circulating corticosterone (CORT), an endogenous glucocorticoid with significant effects on lymphocyte phenotype and function, was elevated within 24 h after spinal cord injury (SCI) and remained above control levels throughout the duration of our studies (up to 1 month postinjury). The present data suggest injury-associated changes in immune cell phenotype and function paralleled by the activation of the hypothalamic-pituitary-adrenal (HPA) axis.

Suppression of Experimental Autoimmune Encephalomyelitis Using Peptide Mimics of CD28

The B7:CD28/CTLA-4 costimulatory pathway plays a critical role in regulating the immune response and thus provides an ideal target for therapeutic manipulation of autoimmune disease. Previous studies have shown that blockade of CD28 signaling by mAbs can both prevent and exacerbate experimental autoimmune encephalomyelitis (EAE). In this study, we have designed two CD28 peptide mimics that selectively block B7:CD28 interactions. By surface plasmon resonance, both the end group-blocked CD28 peptide (EL-CD28) and its retro-inverso isomer (RI-CD28) compete effectively with the extracellular domain of CD28 for binding to B7-1. Both the CD28 peptide mimics inhibited expansion of encephalitogenic T cells in vitro. A single administration of EL-CD28 or RI-CD28 peptide significantly reduced disease severity in EAE. Importantly, we show that either CD28 peptide mimic administered during acute disease dramatically improved clinical signs of EAE, suppressing ongoing disease. The ratio of CD80:CD86 expression was significantly lower on CD4+ and F4/80+ spleen cells in CD28 peptide-treated mice. Peripheral deletion of Ag-specific CD4+ T cells occurs following in vivo blockade of CD28 with synthetic CD28 peptides.

Neuroendocrine modulation of chronic relapsing experimental autoimmune encephalomyelitis: A critical role for the hypothalamic-pituitary-adrenal axis

Murine relapsing EAE can be profoundly suppressed by restraint stress (RST) administered beginning prior to neuroantigen immunization. This study determined what hormone pathway(s) mediate disease suppression. Our results showed that nadolol (NAD), a beta2-adrenergic antagonist, did not reverse the RST-induced suppression of EAE. However, administration of either RU486 or aminoglutethimide, which block the action of peripheral glucocorticoids, resulted in a partial reversal of EAE suppression. Administration of exogenous corticosterone mimicked the effects of RST, in terms of suppression of EAE, decrease in lymphoid cell numbers and decrease in Thl cytokine production. Therefore, the HPA axis plays a more profound role in the RST-induced suppression of EAE than does the sympathetic nervous system.

Potassium, but not atropine-stimulated cortical acetylcholine efflux, is reduced in aged rats

Using in vivo microdialysis, cortical acetylcholine (ACh) efflux was measured in freely moving Brown Norway/Fischer 344 F1 rats, aged 4 or 22 months. The effects of local, intracortical perfusion of atropine (1.0 or 100.0 microM) via the dialysis probe were compared to local K+ (100.0 mM) stimulation in the presence of elevated extracellular Ca2+ (2.5 mM). Basal cortical ACh efflux in aged rats was similar to that of young animals. Administration of atropine (1.0 or 100.0 microM) via the cortical dialysis probe substantially increased cortical ACh efflux, but did not differentially stimulate ACh efflux in young and aged rats. In contrast, ACh efflux stimulated locally with K+ and Ca2+ was significantly reduced in aged rats relative to young adults. The implications of the dissociable effects of K(+)-depolarization and muscarinic blockade for local regulation of cortical ACh efflux in aged animals are discussed.

A Retro-Inverso Peptide Mimic of CD28 Encompassing the MYPPPY Motif Adopts a Polyproline Type II Helix and Inhibits Encephalitogenic T Cells In Vitro

Complete activation of T cells requires two signals: an Ag-specific signal delivered via the TCR by the peptide-MHC complex and a second costimulatory signal largely provided by B7:CD28/CTLA-4 interactions. Previous studies have shown that B7 blockade can either ameliorate experimental autoimmune encephalomyelitis by interfering with CD28 signaling or exacerbate the disease by concomitant blockade of CTLA-4 interaction. Therefore, we developed a functional CD28 mimic to selectively block B7:CD28 interactions. The design, synthesis, and structural and functional properties of the CD28 free peptide, the end group-blocked CD28 peptide, and its retro-inverso isomer are shown. The synthetic T cell-costimulatory receptor peptides fold into a polyproline type II helical structure commonly seen in regions of globular proteins involved in transient protein-protein interactions. The binding determinants of CD28 can be transferred onto a short peptide mimic of its ligand-binding region. The CD28 peptide mimics effectively block the expansion of encephalitogenic T cells in vitro suggesting the potential usefulness of the peptides for the treatment of autoimmune disease conditions requiring down-regulation of T cell responses.