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The T-cell costimulatory receptors, CD28 and the inducible costimulator (ICOS), are required for the generation of follicular B helper T cells (TFH) and germinal center (GC) reaction. A common signal transducer used by CD28 and ICOS is the phosphoinositide 3-kinase (PI3K). Although it is known that CD28-mediated PI3K activation is dispensable for GC reaction, the role of ICOS-driven PI3K signaling has not been defined. We show here that knock-in mice that selectively lost the ability to activate PI3K through ICOS had severe defects in TFH generation, GC reaction, antibody class switch, and antibody affinity maturation. In preactivated CD4⁺ T cells, ICOS delivered a potent PI3K signal that was critical for the induction of the key TFH cytokines, IL-21 and IL-4. Under the same settings, CD28 was unable to activate PI3K but supported a robust secondary expansion of T cells. Thus, our results demonstrate a nonredundant function of ICOS-PI3K pathway in the generation of TFH and suggest that CD28 and ICOS play differential roles during a multistep process of TFH differentiation.

The T-cell costimulatory receptors, CD28 and the inducible costimulator (ICOS), are required for the generation of follicular B helper T cells (TFH) and germinal center (GC) reaction. A common signal transducer used by CD28 and ICOS is the phosphoinositide 3-kinase (PI3K). Although it is known that CD28-mediated PI3K activation is dispensable for GC reaction, the role of ICOS-driven PI3K signaling has not been defined. We show here that knock-in mice that selectively lost the ability to activate PI3K through ICOS had severe defects in TFH generation, GC reaction, antibody class switch, and antibody affinity maturation. In preactivated CD4+ T cells, ICOS delivered a potent PI3K signal that was critical for the induction of the key TFH cytokines, IL-21 and IL-4. Under the same settings, CD28 was unable to activate PI3K but supported a robust secondary expansion of T cells. Thus, our results demonstrate a nonredundant function of ICOS-PI3K pathway in the generation of TFH and suggest that CD28 and ICOS play differential roles during a multistep process of TFH differentiation.

Endogenous opioids play an important role in a variety of physiological processes and mediate their actions through at least three distinct subtypes of cell surface receptors: mu, kappa and delta. The mu opioid receptor is considered to be the major target for the production of analgesia and the development of drug tolerance and dependence. Receptor desensitization has been considered to play important role in opioid tolerance. However, understanding mu opioid receptor desensitization mechanisms using rat brain tissues or cell lines has been difficult because of the presence of multiple subtypes of opioid receptor. This thesis documents the mechanisms of desensitization of the mu opioid receptor expressed in Chinese hamster ovary cells without interference caused by the presence of other opioid receptor subtypes. In this cell line, the mu opioid receptor was desensitized after 1hr exposure to the opioid peptide, DAMGO, largely by the loss of receptors from the cell surface without affecting the affinity or proportion of agonist-detected high affinity sites. Using site-directed mutagenesis, the structural determinants for this agonist-induced functional desensitization was determined to be threonine 394 preceded by an acidic amino acid in the cytoplasmic tail, presumably serving as a primary phosphorylation site for G protein coupled receptor kinase. However, mu opioid receptor down-regulation with an agonist treatment appears to be governed by two distinct pathways. One was G protein-dependent, GRK-dependent, and the other was G protein-independent but tyrosine kinase-dependent. In addition, the mu opioid receptor was shown to be a direct substrate for protein tyrosine kinase. Unlike other prototypic G protein coupled receptor, G protein coupling to the mu opioid receptor was tighter and resistant to agonist-induced G protein uncoupling from the mu opioid receptor. The persistent G protein coupling property of the mu opioid receptor with agonist exposure is partially determined by the intracellular regions of the receptor with contributions from multiple transmembrane domains. Taken together, these findings establish that the agonist-induced mu opioid receptor desensitization is mediated by receptor down-regulation rather than G protein uncoupling from the receptor and that multiple domains of the receptor contribute to its tight coupling to G protein and resistance to agonist-induced dissociation.

The electrophysiological action of adenosine on spinal dorsal horn neurons in culture was studied. Adenosine (100-500$\\mu$ M) was found to have no effect on the resting membrane potential during current-clamp recordings. However, adenosine caused a 50-100% increase in the rate of action potential discharge evoked by depolarizing intracellular current pulses. This effect was reversible and reproducible. To investigate the underlying mechanisms the effect of adenosine on voltage-activated K $\\sp{+}$currents was examined in voltage-clamp experiments. Depolarizing voltage steps evoked a rapidly-activating transient outward current beginning at near$-$ 60 mV and a sustained outward current at$-$ 20 mV. Adenosine reduced the amplitude of the transient outward current but had no effect on the sustained current. The transient outward current appeared to be an A-current because it was blocked by 4-aminopyridine but was insensitive to tetraethylammonium. These results suggest a novel effect of adenosine to reduce accommodation by depressing an A-current in dorsal horn neurons.