IL-12hi Rapamycin-Conditioned Dendritic Cells Mediate IFN-γ-Dependent Apoptosis of Alloreactive CD4+ T Cells and Limit Graft-Versus-Host Diseas

Abstract Abstract 4110 Introduction: Rapamycin (RAPA) inhibits the serine/threonine kinase mammalian Target of Rapamycin to profoundly modulate immune cell function. Dendritic cells (DC) exposed to RAPA (RAPA-DC) exhibit tolerogenic properties, including promotion of experimental cardiac allograft survival. RAPA-DC enrich for CD4+FoxP3+ regulatory T cells and induce alloreactive T cell apoptosis and anergy. Yet, paradoxically, RAPA-DC secrete increased IL-12, which is central for the generation of IFN-γ+CD4+ T helper type 1 cells. IFN-γ can be pro-apoptotic, and IL-12-driven IFN-γ inhibits graft-versus-host disease (GVHD) following hematopoietic stem cell transplantation. We hypothesized that IL-12hi RAPA-DC, unlike control (CTR)-DC, would be effective in the prevention of GVHD by supporting IFN-γ-mediated apoptosis of alloreactive T cells. Methods: DC were generated from C57BL/6 (H2-b) or B6.129S7-Ifngr1tm1Agt/J (IFN-γ-R−/− [H2-b]) bone marrow (BM) in 7 day (d) culture in the absence (CTR-DC) or presence of RAPA (RAPA-DC). CTR- and RAPA-DC cultures remained either untreated or were stimulated via TLR4 with LPS (100 ng/ml) for an additional 18 hours. Washed and CD11c+ purified DC from these groups were then used as stimulators of allogeneic CD4+ T cells (BALB/c [H2-d] or CByJ.129S7(B6)-Ifngr1tm1Agt/J [IFN-γ-R−/− {H2-d}]) in 5 d mixed leukocyte reaction (MLR) with or without anti-IFN-γ antibody. T cell apoptosis was quantified using an Annexin V Apoptosis Detection kit. The capacity of DC to prevent GVHD was assessed in irradiated BALB/c mice reconstituted with 5×106 T cell-depleted B6 BM on d0. Recipient mice received 1×106 CD11c+ BALB/c DC (CTR-, RAPA-, or LPS-exposed DC) on d0 and 1×106 B6 T cells on d1. Mice were monitored daily, and moribund mice or those with >20% weight loss were euthanized. Results: Compared to CTR-DC, especially when LPS-stimulated, RAPA-DC induced increased apoptosis (Annexin V+7-AAD+) of alloreactive CD4+ T cells in MLR (13.9±1.6% versus 7.1±0.2%; +LPS: 30.6±3.4% versus 14.7±3.6%; both p Figure 1 ). There was no difference in the extent of apoptosis of alloreactive CD4+ T cells induced by IFN-γ-R−/− RAPA-DC compared to WT RAPA-DC (10.6±7.6% versus 12.4±7.6% respectively; +LPS: 30.1±16.3% versus 27.3±12.0%; both p=NS) ( Figure 2 ). The addition of anti-IFN-γ mAb to LPS-stimulated IFN-γ-R−/− RAPA-DC significantly decreased levels of CD4+ T cell apoptosis comparable to LPS-stimulated WT CTR-DC (18.0±10.3% versus 22.0±6.3%, p 50 d, p Figure 3 ). In contrast, CTR-DC had comparable GVHD mortality (21 d, p=NS). Conclusions: Increased apoptosis induced by LPS-stimulated IL-12hi RAPA-DC is mediated directly on CD4+ allogeneic T cells via IFN-γ and not through actions on DC. Thus, increased IL-12 may support IFN-γ-mediated activation-induced cell death while sparing regulatory T cells and may underlie the capacity of LPS-exposed RAPA-DC to prevent GVHD. IL-12hi human RAPA-DC, generated with the aid of endotoxin-free, synthetic TLR4 agonists, may offer a means to harness the demonstrated capacity of both IL-12 and tolerogenic DC to prevent GVHD following hematopoietic stem cell transplant. Download : Download high-res image (24KB) Download : Download full-size image Figure 1: . Download : Download high-res image (30KB) Download : Download full-size image Figure 2: . Download : Download high-res image (22KB) Download : Download full-size image Figure 3: . Disclosures: No relevant conflicts of interest to declare.