Oligonucleotide-mediated inhibition of CD28 expression induces human T cell hyporesponsiveness and manifests impaired contact hypersensitivity in mice.

Ligation of CD28 provides a costimulatory signal essential for Ag-mediated T cell activation via the TCR. Blocking CD28 ligation can inhibit cytokine expression and elicits a state of T cell hyporesponsiveness. In this study, we examined the effect of inhibiting CD28 expression on in vitro and in vivo T cell responses. To address this, we have synthesized a series of G-rich phosphorothioate oligonucleotides that inhibited activation-induced transcription and cell surface expression of CD28 on human T cells. CD28 blockade was selective, as expression of other activation-induced receptors was unaffected by oligonucleotide treatment. Using strategic changes to base composition, we identified a minimal 12-mer sequence, containing two sets of four contiguous guanosines separated by 3 to 5 bases, which conferred activity in vitro. Furthermore, inhibition of CD28 expression mediated by one representative active oligonucleotide, GR1, resulted in a concomitant dose-dependent diminution of anti-CD3/PMA-induced cytokine (IL-2, IFN-gamma, IL-8) production. Inhibition of IL-2 synthesis was dependent on CD28 expression, as GR1 failed to abrogate activated IL-2 production in a CD28-deficient T cell line, HUT 78. The inhibitory activity of GR1 reduced T cell proliferative responses in MLR and induced Ag-specific T cell hyporesponsiveness to alloantigens. Finally, s.c. administration of GR1 impaired in vivo contact hypersensitivity responses in mice and was associated with substantially decreased CD28 and IFN-gamma mRNA expression in lymph node cells. Collectively, our studies show the tolerogenic potential of oligonucleotide-mediated CD28 inhibition on T cell activation, in vitro and in vivo.