Comparison of CD28‐B7.1 and B7.2 functional interaction in resting human T cells: Phosphatidylinositol 3‐kinase association to CD28 and cytokine production

Abstract

CD28 is a 44kDa homodimer present on T cells providing an important costimulatory signal for T cell proliferation, cytokine production and cytokine receptor expression. CD28 activation is mediated by interaction with its counter-receptors, B7.1/CD80 and B7.2/B70/CD86. The biochemical basis of these co-stimulatory signals are still poorly understood, particularly in resting T cells. However, various biochemical pathways such as tyrosine phosphorylation, phospholipase C, sphingomyelinase and phosphatidylinositol 3-kinase (PI3-K) activation have been reported to play a role in CD28 signaling in tumor T cell lines and CD28-transfected cells or pre-activated T cells. In addition, recent reports propose that CD28-B7.1 and B7.2 interaction could be involved in the production of Th1 and Th2 cytokines, respectively, but the putative biochemical basis for these different functions is still unknown. We have analyzed the functional and molecular consequences of CD28 activation by B7.1 and B7.2 in human resting T cells. We demonstrate in this report that both CD28-B7.1 and CD28-B7.2 interactions induce the association of PI3-K to CD28 in the CD4 subpopulation, whereas it was barely detectable in CD8 cells. This association involves the binding of the src homology domain 2 (SH2) of p85 to tyrosine-phosphorylated CD28 and does not require pre-activation by CD3-T cell receptor. Worthmannin, a specific inhibitor of PI3-K enzymatic activity within the nanomolar range also inhibits the interleukin-2 production induced by costimulation mediated by either the B7.1- and B7.2-transfected cells or CD28 monoclonal antibodies. The only slight difference between B7.1 and B7.2 costimulation is the IC50 of worthmannin being 25 and 110 nM, respectively, which could suggest differences in their activation of the T cell PI3-K.

Novel Role of Phosphatidylinositol 3-Kinase in CD28-mediated Costimulation

“…Truitt et al (17) also found that wortmannin did not inhibit CD28-dependent IL-2 production from freshly isolated murine CD4 ϩ T cells. On the contrary, wortmannin was reported to inhibit CD28-mediated costimulation of IL-2 in human primary T cells (13)(14)(15). One of the possible explanations for the apparent discrepancies among these reports may be the nature of the cell used in the respective studies.…”

“…Since the T cell responses that are induced by CD28-mediated cosignaling overlap the reported functions of PI3K in lymphocyte activation, it is conceivable that PI3K may be the critical signaling molecule in T cell costimulation. For example, wortmannin, a potent inhibitor of PI3K, is reported to inhibit CD28-dependent IL-2 production in human peripheral T cells (13)(14)(15). Furthermore, CD28 mutants, which are unable to bind to PI3K, demonstrate that PI3K is required for CD28-mediated IL-2 production in mouse T cell hybridoma cell lines (4,16).…”

CD80 and CD86 Are Not Equivalent in Their Ability to Induce the Tyrosine Phosphorylation of CD28

“…Using purified human T cells, Olive and colleagues (39) also demonstrated that both CD80 and CD86 induced the SH2-dependent association of CD28 with PI3-K. However, they further demonstrated that wortmannin, a specific inhibitor of lipid kinase activity of PI3-K via binding to the p110 subunit, inhibited CD80 costimulation of IL-2 production more efficiently than CD86 costimulation (IC 50 25 and 110 nM, respectively) (39). This suggested that the dependence on PI3 lipid kinase activity of CD80 and CD86 stimulation differed, an observation consistent with our results.…”

Quantitative analysis detects ubiquitous expression of apoptotic regulators in B cell non-Hodgkin’s lymphomas

“…27,28 Band intensity was quantified by densitometry using a Biolmage analyzer (Biolmage, Palo Alto, CA, USA). Normalized level of expression of a specific protein was given as the ratio (optical density of the specific protein/optical density of p 85), and standardized results were expressed in arbitrary units.…”