T cell adhesion primes antigen receptor-induced calcium responses through a transient rise in adenosine 3',5'-cyclic monophosphate.

It is well established that sustained increases in cyclic AMP (cAMP) such as those triggered by forskolin inhibit T cell activation. We describe here an unexpected phenomenon: in T cells, a transient cAMP increase triggered by the interaction with a dendritic cell strongly potentiates T cell receptor (TCR) signaling. We discovered this effect by examining the molecular basis of the adhesion-dependent sensitization of T cells. T cell adhesion caused extracellular-signal-regulated kinase (ERK) activation, which was necessary for the sensitization process. T cell sensitization could be mimicked in suspended cells by the uncaging of caged cAMP upon ultraviolet illumination. Calcium responses occurring in T cells upon interaction with dendritic cells were strongly inhibited when protein kinase A activation was blocked. Thus, whereas sustained cAMP increases are well known to inhibit TCR signaling, transient cAMP increases occurring physiologically upon formation of an immunological synapse facilitate antigen detection.

Fluorometric studies of ligand-induced conformational changes of CD38.

The lymphoid surface antigen CD38 is a NAD(+)-glycohydrolase that also catalyzes the transformation of NAD(+) into cyclic ADP-ribose, a calcium mobilizing second messenger. In addition, ligation of CD38 by antibodies triggers signaling in lymphoid cells. Since the cytoplasmic tail of CD38 is dispensable for this latter property, we have previously proposed that CD38-mediated receptor signal transduction might be regulated by its conformational state. We have now examined the molecular changes of this protein during its interaction with NAD(+) by measuring the intrinsic fluorescence of CD38. We have shown that addition of the substrate produced a dramatic decrease in the fluorescence of the catalytically active recombinant soluble ectodomain of murine CD38. Analysis of this event revealed that the catalytic cycle involves a state of the enzyme that is characterized by a low fluorescence which, upon substrate turnover, reverts to the initial high intrinsic fluorescence level. In contrast, non-hydrolyzable substrates trap CD38 in its altered low fluorescence state. Studies with the hydrophilic quencher potassium iodide revealed that the tryptophan residues that are mainly involved in the observed changes in fluorescence, are remote from the active site. Similar data were also obtained with human CD38, indicating that studies of intrinsic fluorescence will be useful in monitoring the transconformation of CD38 from different species. Together, these data demonstrate that CD38 undergoes a reversible conformational change after substrate binding, and suggest a mechanism by which this change could alter interactions with different cell-surface partners.

T cell adhesion lowers the threshold for antigen detection.

Antigen detection and initiation of TCR signaling only occur, under physiological conditions, when T cells are adherent, and not in suspension. We show here that T cell adhesion causes an increase in the Ca(2+) content of intracellular stores and of the amount of phosphatidylinositol 4,5-bisphosphate in the plasma membrane, and enhances TCR-induced Ca(2+) signaling. This priming can be observed in freshly isolated T cells, in activated T cells, and in some T cell lines. Stimulation of T cells by specific monomeric MHC-peptide complexes only triggers Ca(2+) responses after T cell adhesion. This solves a controversial issue concerning the minimum valency of activatory TCR ligands. Adhesion-induced T cell priming not only occurs upon binding to artificial substrates such as immobilized ligands, but also upon interaction with dendritic cells. Thus, this phenomenon is likely to contribute to the high sensitivity of antigen detection by T cells in secondary lymphoid organs.

G protein-dependent CCR5 signaling is not required for efficient infection of primary T lymphocytes and macrophages by R5 human immunodeficiency virus type 1 isolates.

The requirement of human immunodeficiency virus (HIV)-induced CCR5 activation for infection by R5 HIV type 1 (HIV-1) strains remains controversial. Ectopic CCR5 expression in CD4(+)-transformed cells or pharmacological inhibition of G(alpha)i proteins coupled to CCR5 left unsolved whether CCR5-dependent cell activation is necessary for the HIV life cycle. In this study, we investigated the role played by HIV-induced CCR5-dependent cell signaling during infection of primary CD4-expressing leukocytes. Using lentiviral vectors, we restored CCR5 expression in T lymphocytes and macrophages from individuals carrying the homozygous 32-bp deletion of the CCR5 gene (ccr5 Delta32/Delta32). Expression of wild-type (wt) CCR5 in ccr5 Delta32/Delta32 cells permitted infection by R5 HIV isolates. We assessed the capacity of a CCR5 derivative carrying a mutated DRY motif (CCR5-R126N) in the second intracellular loop to work as an HIV-1 coreceptor. The R126N mutation is known to disable G protein coupling and agonist-induced signal transduction through CCR5 and other G protein-coupled receptors. Despite its inability to promote either intracellular calcium mobilization or cell chemotaxis, the inactive CCR5-R126N mutant provided full coreceptor function to several R5 HIV-1 isolates in primary cells as efficiently as wt CCR5. We conclude that in a primary, CCR5-reconstituted CD4(+) cell environment, G protein signaling is dispensable for R5 HIV-1 isolates to actively infect primary CD4(+) T lymphocytes or macrophages.