Reconstitution of STAT4 Restores Defective Interferon-gamma Production and Allows Normal IFN-gamma-Dependent Responses after Autologous Stem Cell Transplantation.

Abstract

Production of IFN-gamma is critical for optimal antitumor immune responses in several preclinical animal models. Patients with lymphoma who have undergone autologous hematopoietic stem cell transplantation exhibit profoundly diminished IFN-gamma production in vivo during IL-12 therapy (Clin Cancer Res. 2002; 8: 3383). Furthermore, post-transplant patient peripheral blood mononuclear cells (PBMCs) stimulated directly in vitro with IL-12 secrete little if any IFN-gamma. To determine if restoration of IFN-gamma production would be sufficient to reconstitute IFN-gamma-dependent responses after transplantation, the integrity of IFN-gamma signaling was assessed in post-transplant patient PBMCs. Compared to control subject PBMCs, post-transplant patient PBMCs expressed equivalent levels of CD119 (IFN-gamma receptor alpha) and STAT1. Moreover, tyrosine phosphorylation of STAT1 in response to IFN-gamma did not differ in post-transplant patient as compared to control PBMCs. Thus, IFN-gamma signaling is intact after transplantation. Our prior studies have shown that STAT4 protein levels are decreased by ~97% in post-transplant patient PBMCs, whereas levels of Jak2, Tyk2, and STAT3 are similar to control PBMCs (Blood 2005; 106: 963). We wished to demonstrate directly that STAT4 deficiency is the mechanism of defective IFN-gamma production after autologous transplantation. Enriched CD4+ T cells were isolated from post-transplant patient and control PBMCs and cultured under conditions that promote Th1 differentiation. STAT4 deficiency persisted in post-transplant patient CD4+ T cells cultured under Th1 conditions, whereas these cells expressed normal levels of T-bet. Compared to control Th1 cells, post-transplant patient CD4+ T cells cultured under Th1 conditions exhibited markedly reduced IFN-gamma production after restimulation with CD3 mAb. These results are consistent with expected impairment of Th1 differentiation due to profound STAT4 deficiency. STAT4 cDNA was transiently transfected into Th1-cultured CD4+ T cells from post-transplant patients. After stimulation with CD3 mAb plus IL-12, the amounts of IFN-gamma secreted by STAT4-transfected patient cells were equivalent to or higher than those secreted by control subject cells. These data indicate that reconstitution of STAT4 expression is sufficient to restore IFN-gamma production by post-transplant patient PBMCs. As IFN-gamma signaling is normal post-transplant, circumventing STAT4 deficiency should therefore restore IFN-gamma-dependent antitumor immune responses after autologous transplantation. Future studies will determine the molecular mechanisms of STAT4 deficiency after transplantation and develop clinically feasible methods to circumvent this deficiency.