Mice were purchased from Jackson Laboratory and bred in-house. All animal procedures performed were approved by the Institutional Animal Care and Use Committee. Femurs and tibias were harvested from C57BL/6 [wild-type (WT)] mice between 8 and 14 weeks of age to generate bone marrow-derived DC. WT mice were also used as recipients for in vivo adoptive transfer experiments. Transgenic B6.Cg-Tg(TcraTcrb)425Cbn/J (OT-II) mice were used as a source of naive CD4⁺ T cells responsive to ovalbumin (OVA_323–339).

Bone marrow-derived DC were generated as described by a modified protocol of Inaba et al. (22). Briefly, bone marrow cells were cultured in IMDM (Thermo Fisher Scientific, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific), 2 mM L-glutamine (Thermo Fisher Scientific), 100 U/ml penicillin/streptomycin (Thermo Fisher Scientific), and 20 ng/ml GM-CSF for 8 days in culture. On day 6 (of the 8-day culture), cells were purified for a homogenous DC population using CD11c microbeads (Miltenyi Biotec, Auburn, CA, USA) for positive selection. AIF1 was knocked down using an ECM 830 (BTX, Holliston, MA, USA) square wave electroporator with 1 nmol (6.65 µg) of siRNA oligos in 4 mm gap cuvettes with the following settings: 310 V, 10 ms, 1 pulse. AIF1 siRNA (siAIF1) sequence used: 5′-GGCAAGAGAUCUGCCAUCUUG-3′ (Thermo Fisher Scientific, Grand Island, NY, USA). Scrambled siRNA served as controls (siControl): 5′-GGGCTCTACGCAGGCATTTAA-3′. Additionally, studies used silencer pre-designed siRNA 73668 targeting AIF1 purchased from Thermo Fisher Scientific: 3′-GGUGAAGUACAUGGAGUUU-5′. After electroporation of siRNA on day 6 in CD11c⁺-sorted DC, cells were placed back into culture. On day 7, 24 h after siRNA transfection, DC were matured with 250 ng/ml of LPS (or other TLR agonists) for an additional 24 h. On day 8, these siRNA transfected mature DC were used to assess immunophenotype and prime naïve CD4⁺ OT-II T cells. For all in vivo studies, DC were adoptively transferred into mice 24 h after siRNA transfection to compensate for the trafficking time required to enter the draining lymph nodes and prime T cell responses.

For isolation of naïve CD4⁺ T cells from OT-II mice, CD8⁺ cytotoxic T cells and MHC class II⁺ antigen presenting cells were depleted by negative selection from spleen and lymph nodes using primary antibodies to CD8 and MHC class II (BioLegend, San Diego, CA, USA) followed by secondary labeling with anti-rat IgG magnetic microbeads (Qiagen, Hilden, Germany). Cells were then depleted by passing through a magnetic column. The approach yielded 96 ± 2.1% purity of CD4⁺ T cells. These naïve CD4⁺ T cells were cultured with 1.0, 0.3, or 0.1 µg/ml of OVA peptide (ISQAVHAAHAEINEAGR)-_323–339-pulsed siAIF1 or siControl LPS-matured DC at a ratio of 10:1, respectively. Peptides were purchased from AnaSpec (Fremont, CA, USA). Scrambled non-specific peptides served as controls for some experiments, with the following sequences: VAAGIAQAHESIREHAN and IENHQIAGAAERSAAVH.

OVA_323–339-pulsed siAIF1 or siControl mature DC stimulated OT-II CD4⁺ T cells were harvested at the 24 h time point to evaluate early activation markers CD69, CD62L, and CD25; antibodies purchased from BioLegend. For proliferation assays, CD4⁺ T cells pre-labeled with 2.5 µM CFSE (Thermo Fisher Scientific) were cultured with OVA_323–339-pulsed siAIF1 or siControl DC for 96 h. Cells were co-stained with antibodies to IL-2 (BioLegend) for intracellular cytokine detection after fixation and permeabilization. For polarization experiments, OVA_323–339-pulsed siAIF1 or siControl DC were cultured with CD4⁺ T cells for 12–14 days with re-stimulation on day 5 using respective peptide-pulsed siAIF1 or siControl DC supplemented with 200 U/ml of IL-2. T cell cytokine responses were then evaluated by stimulation with 20 ng/ml PMA and 1 µg/ml ionomycin for 4 h in the presence of 10 µg/ml of brefeldin A prior to fixation, permeabilization, and intracellular staining of IFNγ, IL-4, IL-17A, and IL-10. For Treg phenotype, cells were stained 12–14 days after initial priming by OVA_323–339-pulsed siAIF1 or siControl DC for CD25, Foxp3, CD27, CTLA-4, and CD44. These cells were not stimulated with mitogens prior to immunophenotyping. All antibodies purchased from BioLegend. Cells were then acquired by a flow cytometric analyzer.

OT-II T cells were expanded for 12–14 days by siAIF1 or siControl DC pulsed with OVA peptide. After expansion, these T cells were then labeled with Cell Tracker Violet dye (Thermo Fisher Scientific). These labeled cells are referred to as suppressors. Next, naïve CD4⁺ or CD8⁺ T cells were isolated from WT mice by negative depletion and confirmed CD25⁻ by flow cytometric analyses. Briefly, the CD4⁺CD25⁻ or CD8⁺CD25⁻ T cells were labeled with 2.5 µM CFSE. These CFSE-labeled naïve T cells are the responders. The purpose of labeling with different dyes was to distinguish suppressor from the responder populations. The suppressor and responder T cells were then cultured together at a 3:1, 1:1, 1:3, and 1:10 ratio, respectively, prior to stimulation with anti-CD3/CD28-coated microbeads (Dynabeads; Thermo Fisher Scientific). Cells were incubated for 72–96 h prior to collection, staining, and analysis of responder T cell proliferation using a modified approach by Collison and Vignali (23).

CD4⁺ T cells were isolated from OT-II mice and labeled with CFSE. Next, 5 × 10⁶ of these CFSE-labeled OT-II CD4⁺ T cells were intravenous injected into WT mice, as described by Moon et al. (24). Next, 2 × 10⁶ control (siControl) or AIF1 knockdown (siAIF1) DC pulsed with OVA_323–339 were subcutaneously (s.q.) injected once at 6 h or twice at 6 and 12 h after the transfer of the CFSE-labeled OT-II cells. For these studies, DC were treated with siRNA 24 h prior to adoptive transfer, with LPS stimulation at the 12 h time point. Finally, the DC were incubated with 5 µg/ml of OVA_323–339 peptide in the last 4 h prior to adoptive transfer. Subcutaneous injection sites were the scruff of the neck and/or the upper thigh-just above the hind leg. The draining lymph nodes nearest to the s.q. injection were harvested 3.5 or 5.5 days post-injection to assess CD4⁺ T cell proliferation and effector responses. In each experiment, the same lymph nodes were collected: axillary, brachial, inguinal, lumbar, and superficial cervical. The tissues were then disassociated into single suspension, washed, fixed, and stained for intracellular assessment of cytokines.

After various time points of DC or DC-T cell co-culture incubation periods, cells were spun down and supernatants collected. Mouse Inflammation Kit Cytometric Bead Array (CBA; BD Biosciences) and Legendplex Mouse Th Cytokine Panel (BioLegend) were used to detect cytokine levels in the supernatant; CBA kit analyzed IL-12p70, IL-10, IL-6, TNFα, MCP-1, and Legendplex was used to analyze IL-17A, IL-4, and IFNγ. Median fluorescence intensity values were normalized to cytokine concentration based on internal standard controls following manufacturer recommended protocols.

Lymph nodes and spleens from WT mice were harvested and immediately fixed in 3% paraformaldehyde in PBS overnight at 4°C. After fixation, tissues were placed in 10% sucrose for 1 h prior to cryosectioning at 25 and 50 µm. Next, sections were permeabilized and stained with CD11c-Alexa488 (BioLegend), rabbit monoclonal AIF1 (EPR16588 or EPR178847; Abcam, Cambridge, MA, USA), and DAPI. Secondary anti-rabbit Alexa-594 was used to detect AIF1. Sections were then imaged using a three-channel wide-field fluorescence microscope and analyzed by ImageJ.

Cell lysates were prepared by incubating Nonidet P-40 cell lysis buffer (Amresco, Solon, OH, USA) with cells for 30 min before high-speed centrifugation. Lysates were collected and ran in 15% gels using vertical gel electrophoresis. Protein content was transferred from gels to nitrocellulose blots using the Pierce Power Blotter (Thermo Fisher Scientific). GAPDH served as a loading control. After primary antibody staining, secondary antibodies conjugated to fluorochromes were used for visualizing protein bands on the Odyssey imaging system (LI-COR, Lincoln, NE, USA). Image Studio 5.2 software (LI-COR) was used to calculate relative fluorescence intensities.

Cell surface staining was performed in PBS supplemented with EDTA and 2.5% FBS (FACS buffer). Single cell suspensions were washed with FACS buffer two to three times prior to staining with fluorochrome tagged-antibodies. Cells were stained for 15 min at 4°C with 10 µl of a 10 µg/ml working concentration per 2 × 10⁵ cells. Cells were then washed and fixed for 20 min in 3% paraformaldehyde at 4°C. For intracellular staining, fixed cells were permeabilized with 0.2% saponin in PBS for 1 h. Next, primary antibodies were added and cells incubated for 1 h. For primary unconjugated antibodies, secondary-tagged fluorochrome-labeled antibodies were prepared. These secondary antibodies were diluted to 1:1,000–1:3,000 working concentrations and 10 µl were added per 2 × 10⁵ cells. Cells were allowed to incubate for 1 h or overnight followed by extensive washing. Samples were acquired using a BD FACSVerse flow cytometric analyzer. Datasets were analyzed using FlowJo v10 (TreeStar, Ashland, OR, USA). Respective isotype controls and/or fluorochrome-labeled isotype controls were used in all assays. Gating strategies were established based on respective isotype controls.

GraphPad Prism v6.0 (GraphPad Software, La Jolla, CA, USA) was used to determine statistical significance. Student unpaired two-tailed t-test was used to evaluate the significance of two groups. One-way or two-way analysis of variance was used to evaluate the significance between the means of three or more independent groups. A p-value ≤0.05 was considered statistically significant; *<0.05, **<0.01, and ns, non-significant. Error bars for all figures indicate SEs.

To assess expression of AIF1 in DC, both lymph nodes and spleens were harvested from WT mice and evaluated for co-expression with CD11c⁺ subsets. WT mice were injected with either PBS or an LPS and IFNγ cocktail for 24 h. Spleen and lymph node tissues were then isolated, cryosectioned, and stained. Fluorescence microscopy revealed co-localization of AIF1 with CD11c⁺ cells in both lymph nodes and the spleen (Figures 1A,B). Next, total cells isolated from the lymph nodes or the spleen were analyzed by flow cytometry for expression of AIF1. Live cells were gated on MHC class II and evaluated for co-expression of CD11c and AIF1. Results revealed high expression of AIF1 in MHC class II⁺CD11c⁺ DC from both lymph nodes and the spleen (Figures 1C,D). Finally, LPS and IFNγ stimulation resulted in significant increase in AIF1 expression in these MHC class II⁺CD11c⁺ subsets.

Bone marrow-derived DC were generated and sorted to yield a pure population of CD11c⁺ subsets (Figure S1A in Supplementary Material). Transfection of siRNA oligonucleotides targeting AIF1 (siAIF1) was used to knockdown the gene expression in CD11c⁺ DC. Scrambled non-targeting siControl. Results yielded a consistent 70–75% knockdown of AIF1 from endogenous levels as measured by western blot analyses (Figure 2A). Flow cytometric analyses confirmed that siRNA targeted knockdown of AIF1 in the CD11c⁺ DC reduced expression from 60.6 down to 16.0% 48 h post-transfection (Figure 2B). Results also revealed that optimal siRNA knockdown of AIF1 occurred during the 48–72 h time point post-transfection and that sustained depression of AIF1 lasted up to 5 days in culture (Figure S1B in Supplementary Material). Knockdown using siRNA (both in-house and pre-design oligonucleotides) did not yield any major differences in viability in comparison with controls (Figure 2C). Phenotypic characterization of LPS-matured CD11c⁺ DC transfected with siAIF1 showed no significant differences in expression of CD80, CD86, CD83, CD40, 33D1, CD11b, MHC class I, MHC class II, and F4/80 compared with siControl treated (Figure 2D). Furthermore, no significant differences were found in secretion of the pro-inflammatory cytokines TNFα, IL-6, IL-12p70, IFNγ, IL-10, and MCP-1 (Figure 2E). These observations were recapitulated using other TLR agonists (i.e., TLR3-Poly I:C, TLR5-Flagellin, TLR9-CpG; data not shown).

Antigen presentation capacity of DC was evaluated upon AIF1 knockdown. In these studies, siControl vs. siAIF1 LPS-matured DC pulsed with OVA_323–339 peptide was cultured with naïve OT-II CD4⁺ T cells. These isolated T cells were confirmed to be naïve CD4⁺ CD25⁻ subsets (Figure S1C in Supplementary Material). After 24 h, impaired early activation responses were observed in cognate T cells primed by siAIF1 DC compared with siControl, as measured by reduced CD25 (63.4–36.3%) and CD69 (63.7–34.1%) expression and restrained down regulation of CD62L (32.4–52.3%) (Figure 3A). Supernatant harvested at 72 h of culture was also assessed. These studies found decreased levels of IL-2, TNFα, and IFNγ in the CD4⁺ T cells primed by AIF1 knockdown DC compared with siControl (Figure 3B); the no stimulation group contained T cells without the presence of OVA peptide-pulsed DC. Lastly, CFSE-labeled CD4⁺ T cell responders were primed by siControl or siAIF1 DC at varying concentrations of peptide (1.0, 0.3, and 0.1 μg/ml) and allowed to incubate for 96 h prior to measurement. Cells were then harvested and co-stained with antibodies targeting IL-2. The results revealed a significant reduction in proliferative capacity of the siAIF1 DC primed T cells compared with the siControl group (Figure 3C); non-stimulated CFSE-labeled T cells served as internal controls. There was a significant impairment in T cell proliferative capacity in the siAIF1 cohort, reducing proliferation by as much as 50% at 0.1 µg/ml of OVA peptide vs. siControl DC groups. T cell responses were found to be antigen dose dependent, where higher levels of peptide stimuli could override the phenotype observed in the siAIF1 DC stimulated groups. To confirm antigen-specificity, scrambled OVA peptide was employed as an additional internal control. No proliferation occurred in the scrambled OVA control from either siAIF1 or siControl DC stimulated groups (Figure S1D in Supplementary Material).

For evaluating polarization responses, T helper cell-associated cytokine expression was measured 12 days after initial stimulation by OVA-pulsed siAIF1 vs. siControl DC. These investigations revealed restrained IFNγ from 23.7 to 2.6% expression in siControl vs. siAIF1, respectively, and IL-17A from 6.6 to 2.2% (upon PMA/ionomycin re-stimulation in the presence of brefeldin A; Figures 4A,B). Additionally, in DC primed CD4⁺ T cells, there was a concomitant threefold increase in IL-10 production from 17.5 to 48.8% in siControl vs. siAIF1 DC primed CD4⁺ T cells, respectively (Figure 4B). The datasets are representative of three independent experiments. Intracellular cytokine flow analysis results were further validated using multiplex cytometric bead array assays. Cytokines were measured from supernatant collected 4 h after the PMA/ionomycin re-stimulation. IFNγ, IL-4, and IL-17A cytokine expressions were decreased in siAIF1 group compared with siControl, whereas IL-10 expression was markedly increased (Figure 4C). These findings suggest that AIF1 expression in DC plays an immunomodulatory role in promoting pro-inflammatory responses in T helper cells.

Due to increased IL-10 and decreased levels of IFNγ and IL-17A, the studies next measured Treg generation. siAIF1 DC expanded OT-II CD4⁺ T cells resulted in a threefold increase in CD25⁺Foxp3⁺ subsets (7.35–22.2%) compared with siControl DC by day 14 (Figure 5A). Further evaluation found a consistent, albeit moderate, increase in CD27 and CTLA-4, as well as a decrease in CD44 in the siAIF1 group compared with siControl (Figure 5B). This corroborated literature reports that Foxp3⁺ cells are largely present within the CD27⁺ population and that CTLA-4 is associated with attenuation of antigen presenting cells via B7 co-stimulatory engagement (25–27).

Subsequent experiments assessed whether these T cells expanded by siAIF1 DC were functionally suppressive. Briefly, total OT-II CD4⁺ T cells expanded from siControl or siAIF1 DC were harvested, washed, and labeled with a violet marker; this group is termed the suppressors. These suppressors were then counted and cultured with newly isolated naïve WT CD4⁺ or CD8⁺ T cells labeled with CFSE at approximately a 3:1, 1:1, and 1:3 ratio; these CFSE-labeled naïve WT T cells are termed the responders (Figure 5C). This approach allowed distinguishing between the two populations to distinctly assess proliferation of each respective group. Upon anti-CD3/CD28 stimulation, both CFSE-labeled CD4⁺ and CD8⁺ T cell responders cultured with siAIF1 expanded suppressors were restrained in proliferation after 72 h in culture (Figures 5D,E). In three independent experiments, results identified markedly reduced proliferation of responder T cells in the presence of siAIF1 expanded suppressor T cells compared with that of the siControl group. This confirmed that the siAIF1 expanded CD4⁺ Tregs were functionally suppressive in their ability to restrain neighbor T cell expansion.

In vivo adoptive cell transfer studies were performed to corroborate in vitro results. Briefly, CFSE-labeled OT-II CD4⁺ T cells were adoptively transferred into WT recipient mice. Approximately 10% of CFSE⁺ OT-II cells were identified in the lymph nodes 36 h after adoptive transfer into the WT recipients (Figure 6A). siAIF1 or siControl OVA_323–339-pulsed LPS-matured DC were then injected subcutaneously either once (1×) at 6 h or twice (2×) at the 6 and 12 h time points after initial transfer of the CFSE-labeled OT-II CD4⁺ T cells into the WT recipients. After 5.5 days, the draining lymph nodes of recipient mice were then analyzed for proliferation and co-expression of IL-2 and IFNγ from the adoptively transferred CFSE-labeled OT-II T cells. For the mice cohort receiving adoptively transferred DC twice (at 6 and 12 h time points), results showed restrained T cell proliferation, as assessed by CFSE dilution assays, from 37.0% in the siControl to 12.6% in the siAIF1 transfected groups (Figure 6B). Adoptive transfer of LPS-matured siAIF1 or siControl DC into the WT recipients without OVA peptide pulsing served as control (Figure 6C). For the mice cohort receiving adoptively transferred DC once (at 6 h) after initial transfer of the CFSE-labeled OT-II cells, proliferation decreased from 28.0% for siControl to 10.4% for the siAIF1 cohort (Figure 6D). Adoptively transferred siAIF1 or siControl DC without pulsing with OVA peptide elicited no T cell expansion (Figure 6E). These results describe a novel role of AIF1 in governing DC antigen presentation capacity for directing T cell proliferation and effector responses.