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

Stenger, Elizabeth O.; Rosborough, Brian R.; Mathews, Lisa; Ma, Huihui; Mapara, Markus; Thomson, Angus W.; Turnquist, Heth R.

doi:10.1182/blood.V120.21.4110.4110

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-12^hi 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-Ifngr1^tm1Agt/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)-Ifngr1^tm1Agt/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<0.05). Neutralization of IFN-γ in co-cultures containing LPS-stimulated RAPA-DC decreased levels of apoptosis to those of LPS-exposed CTR-DC (18.5±1.3% versus 14.7±3.6%; NS). IFN-γ-R^−/− CD4⁺ T cells exposed to LPS-stimulated IL-12^hi RAPA-DC exhibited decreased apoptosis when compared to wild-type CD4⁺ T cells (WT 15.7±3.6% versus IFN-γ-R^−/− 4.9±1.1%; p<0.05) (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<0.05). Depletion of IFN-γ did not impact on the T_reg-enriching capacity (CD4^hiFoxP3⁺/CD4^hiFoxP3⁻) of RAPA-DC (24.2±0.4% versus 29.2±6.2% with anti-IFN-γ mAb; p=NS). Further, whereas CTR-DC and LPS-exposed CTR-DC did not prolong survival, IL-12^hi RAPA-DC significantly prolonged survival from GVHD (median survival in days: GVHD, 17.5 d; RAPA-DC, 27 d, p=NS compared to GVHD; RAPA-DC + LPS, 35 d, p<0.01; syngeneic control, >50 d, p<0.01) (Figure 3). In contrast, CTR-DC had comparable GVHD mortality (21 d, p=NS).

Conclusions:

Increased apoptosis induced by LPS-stimulated IL-12^hi 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-12^hi 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.

Figure 1: