T cell receptor-independent CD4 signalling: CD4–MHC class II interactions regulate intracellular calcium and cyclic AMP Wenhong Zhou a , Rolf Ko ¨nig a,b, * a Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, TX 77555-1070, USA b Sealy Center for Molecular Science, The University of Texas Medical Branch, Galveston, TX 77555-1070, USA Received 11 October 2002; accepted 21 January 2003 Abstract CD4 is a coreceptor on T helper (Th) cells that interacts with MHC class II molecules (MHCII). The mechanisms mediating the effects of CD4 on responses by T helper cells to stimulation of the antigen-specific T cell receptor (TCR) are still poorly understood. Here, we demonstrate T cell costimulation via CD4 signalling independent of T cell receptor-mediated signals. Incubation of T helper cells with peptide mimetics of the CD4-binding region on the MHC class II h2 domain caused intracellular calcium mobilization in the absence of antigen or other T cell receptor stimuli. Engagement of CD4 by peptide mimetics or wild-type MHC class II, but not by mutant MHC class II molecules incapable of engaging CD4, inhibited the T cell receptor-mediated increase in cyclic AMP (cAMP) concentrations in T helper cells. CD4-mediated signals activated cyclic AMP phosphodiesterases (PDEs) and inhibited adenylyl cyclase. Full activation and clonal expansion of antigen-stimulated T helper cells required the CD4-mediated regulation of cyclic AMP. Our results suggest a costimulatory mechanism of CD4 function that acts on the second messengers, calcium and cyclic AMP. D 2003 Elsevier Science Inc. All rights reserved. Keywords: T lymphocytes; Activation; Signal transduction; Phosphodiesterase; Adenylyl cyclas 1. Introduction T helper (Th) cells regulate adaptive immune responses by promoting and directing the differentiation and activation of B cells and cytotoxic CD8 + T cells [1,2]. Activation of Th cells is critical for the clearance of pathogenic infections [3,4] and for the destruction of tumor cells [5]. Th cells also participate in the pathogenesis of many autoimmune disor- ders [6,7] and in transplant rejection [8]. Th cells recognize antigen (Ag) in the context of MHC class II molecules (MHCII) and express the CD4 coreceptor on their cell surface [9], which interacts with nonpolymorphic regions of MHCII [10,11]. The association of the Ag-specific T cell receptor (TCR) and CD4 with the same MHCII during Ag stimulation initiates Th cell activation [12]. One of the earliest Ag-induced signalling events is the mobilization of calcium ions (Ca 2+ ), which is essential for Th cell activation [13]. This second messenger activates several cytosolic enzymes, initiating downstream signalling cascades [14]. However, Ag stimulation also generates signals through the TCR that antagonize Th cell activation. For example, TCR-mediated signals activate the cyclic AMP (cAMP)-dependent protein kinase A (PKA) [15,16]. PKA initiates a signalling pathway that inhibits Ag-induced T cell proliferation and cytokine production [17,18]. There- fore, to achieve full activation of Th cells, TCR-mediated signals must be modified. Interactions between CD4 and MHCII increase Ag- induced Th cell proliferation and cytokine production [19,20]. Initially, it was thought that CD4 functioned as an adhesion molecule, enhancing contact between Th cells and Ag-presenting cells (APCs) [9]. However, CD4 and MHCII interact with extremely low affinity [21,22], and soluble CD4 0898-6568/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0898-6568(03)00037-8 Abbreviations: Ag, antigen; APC, antigen-presenting cell; Ca 2+ , calcium ions; cAMP, cyclic AMP; CD4-IP, CD4-interacting peptide; DMSO, dimethylsulfoxide; FACS, fluorescence-activated cell sort; FITC, fluorescein isothiocyanate; iPDE, inhibitor of phosphodiesterase; IBMX, 3- isobutyl-1-methylxanthine; MHCII, MHC class II molecules; Ova323, ovalbumin peptide 323– 339; OvaT, ovalbumin peptides derived by trypsin treatment; PDE, phosphodiesterase; PHA, phytohemagglutinin; PI3, phosphoinositide 3; PKA, protein kinase A; RPE, R-phycoerythrin; TCA, trichloroacetic acid; Th, T helper; TCR, T cell receptor. * Corresponding author. Department of Microbiology and Immunol- ogy, The University of Texas Medical Branch, Medical Research Building, Room 3.142H, Mechanic and 11th Street, Mail Route 1070, Galveston, TX 77555-1070, USA. Tel.: +1-409-747-0395; +fax: +1-409-747-6869. E-mail address: rokonig@utmb.edu (R. Ko ¨nig). www.elsevier.com/locate/cellsig Cellular Signalling 15 (2003) 751 – 762