Inhibition of Allograft Inflammatory Factor-1 in Dendritic Cells Restrains CD4+ T Cell Effector Responses and Induces CD25+Foxp3+ T Regulatory Subsets

Abstract

Allograft inflammatory factor-1 (AIF1) is a cytoplasmic scaffold protein shown to influence immune responses in macrophages and microglial cells. The protein contains Ca2+ binding EF-hand and PDZ interaction domains important for mediating intracellular signaling complexes. This study now reports that AIF1 is expressed in CD11c+ dendritic cells (DC) and silencing of expression restrains induction of antigen-specific CD4+ T cell effector responses. AIF1 knockdown in murine DC resulted in impaired T cell proliferation and skewed polarization away from T helper type 1 and 17 fates. In turn, there was a parallel expansion of IL-10-producing and CD25+Foxp3+ T regulatory subsets. These studies are the first to demonstrate that AIF1 expression in DC serves as a potent governor of cognate T cell responses and presents a novel target for engineering tolerogenic DC-based immunotherapies.

IL-10 producing CD8+ CD122+ PD-1+ regulatory T cells are expanded by dendritic cells silenced for Allograft Inflammatory Factor-1

“…On day 6, 24 h after siRNA transfection, DC were matured with 250 ng/ml of LPS and cultured for an additional 24 h. On day 7, the siRNA transfected mature DC were used to prime naïve CD8 + OT-I T cells. Flow cytometric analyses confirmed routine transfection with siRNA to yield greater than 70% knockdown in CD11c + DC, as previously reported [18]. …”

“…On day 5 (of the 7 day culture), cells were purified for a homogenous DC population using CD11c microbeads (Miltenyi Biotec, Auburn CA). The approach yielded greater than 96% purity, as previously described [18]. AIF1 was knocked down using an ECM 830 (BTX, Holliston MA) square wave electroporator with 1 nanomole (nmol) of siRNA oligos in 4 mm gap cuvettes in 200 μl of Opti-MEM (Thermo Fisher Scientific) with the following settings: 310 V, 10 ms, 1 pulse.…”

“…AIF1 was knocked down using an ECM 830 (BTX, Holliston MA) square wave electroporator with 1 nanomole (nmol) of siRNA oligos in 4 mm gap cuvettes in 200 μl of Opti-MEM (Thermo Fisher Scientific) with the following settings: 310 V, 10 ms, 1 pulse. AIF1 siRNA (siAIF1) sequence used: ‘5-GGCAAGAGAUCUGCCAUCUUG-3’ (Thermo Fisher Scientific, Grand Island NY), as previously described [18, 20]. Scrambled siRNA served as controls (siControl): ‘5-GGGCTCTACGCAGGCATTTAA-3’.…”

“…siAIF1 or siControl DC expanded OT-I CD8 + T cells were collected, washed, and plated with target CFSE labeled-CD4 + T cells isolated from WT mice using negative depletion approaches, as previously described [18, 20]. Briefly, the WT CD4 + T cells were split into two equal groups: SIINFEKL peptide-pulsed and scrambled control peptide-pulsed.…”

“…Dysregulation of AIF1 has been largely associated with both neuroinflammatory- and autoimmune-related disorders [16, 17]. Recent reports have identified that AIF1 expression in DC directly modulates CD4 + T cell responses [18], but no study has delineated the role in governing CD8 + T cell responses.…”