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. Author manuscript; available in PMC: 2020 Jan 1.
Published in final edited form as: J Leukoc Biol. 2018 Dec 4;105(1):123–130. doi: 10.1002/JLB.1A0118-010RR

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

Diana M Elizondo 1, Temesgen E Andargie 1, Naomi Haddock 1, Ricardo L Louzada da Silva 2, Tatiana Rodrigues de Moura 2, Michael W Lipscomb 1,*
PMCID: PMC6310075  NIHMSID: NIHMS994721  PMID: 30512224

Abstract

Allograft Inflammatory Factor-1 (AIF1) is a cytoplasmic scaffold protein that contains Ca2+ binding EF-hand and PDZ interaction domains important for mediating intracellular signaling complexes in immune cells. The protein plays a dominant role in both macrophage- and dendritic cell (DC)-mediated inflammatory responses. This study now reports that AIF1 expression in DC is important in directing CD8+ T cell effector responses. Silencing AIF1 expression in murine CD11c+ DC suppressed antigen-specific CD8+ T cell activation, marked by reduced CXCR3, IFNγ and Granzyme B expression, and restrained proliferation. These primed CD8+ T cells had impaired cytotoxic killing of target cells in vitro. In turn, studies identified that AIF1 silencing in DC robustly expanded IL-10 producing CD8+ CD122+ PD-1+ regulatory T cells that suppressed neighboring immune effector responses through both IL-10 and PD-1-dependent mechanisms. In vivo studies recapitulated bystander suppression of antigen-responsive CD4+ T cells by the CD8+ Tregs expanded from the AIF1 silenced DC. These studies further 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.

Keywords: dendritic cells, Tregs, tolerogenic, programmed death-1, suppression, cytotoxic T cells

GRAPHICAL ABSTRACT

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Introduction

Dendritic cells (DC) are professional antigen presenting cells that direct T cell activation, proliferation and polarization [1, 2]. In addition to directing immunity, DC also play prominent roles in modulating peripheral tolerance by inducing anergy in responder T cells and/or directing fates towards T regulatory (Treg) states [3].

CD8+ T cells are restricted to MHC class I and are required for detecting presentation of intracellular pathogen-derived antigens. Recently, identification of CD8+ T regulatory cells has been shown to limit exacerbated innate and adaptive immune responses [4, 5]. These cells, identified as expressing both PD-1 (programmed death-1; CD279) and CD122 (IL-2Rβ chain), have been shown to be important in producing IL-10 and suppressing neighboring T cell responses [6]. Initially, CD122+ subsets identified a regulatory pool of CD8+ T cells [7–9]. Critically, PD-1 has been shown to be potent for modulating immune effector responses vs. establishing tolerance in disease settings [10–12]. The unique presence of PD-1 on CD8+ T cell subsets has helped to distinguish and identify effector and memory subsets from that of regulatory pools [6, 11, 13].

Allograft Inflammatory Factor-1 (AIF1), also known as ionized calcium-binding adapter molecule 1, is a 17 kD interferon gamma-inducible calcium binding EF-hand protein [14]. For immune cells, the gene is largely restricted to myeloid subsets and is responsible for mounting immune responses [15]. 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.

In this study, silencing of AIF1 in DC restrained CD8+ T cells effector functions and, in turn, redirected them to functionally suppressive IL-10 producing CD122+ PD-1+ regulatory T cells. This investigation provides additional evidence that AIF1 expression in DC is instrumental in governing adaptive immune responses and that its loss leads to tolerance.

Materials and Methods

Mice

Mice were purchased from Jackson Laboratory and bred in-house in pathogen free settings at Howard University. All animal procedures performed were approved by the Institutional Animal Care and Use Committee. C57BL/6 (wild type; WT) mice were used as a source of bone marrow and as recipients for in vivo adoptive transfer experiments. Transgenic C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-I) and B6.Cg-Tg(TcraTcrb)425Cbn/J (OT-II) mice were used as a source of CD8+ T cells that recognize ovalbumin peptide residues 257–264 (OVA257–264; SIINFEKL) and CD4+ T cells that recognize residues 323–339 (OVA323–339), respectively.

Flow Cytometry and Antibodies

Cell surface staining was performed in PBS supplemented with 0.2 µg/ml EDTA and 2.5% FBS (FACS buffer). Single cell suspensions were washed with FACS buffer 2–3 times prior to staining with fluorochrome tagged-antibodies. Cells were stained for 15 minutes 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 minutes in 3% paraformaldehyde (Sigma-Aldrich, St. Louis MO) 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. The following antibodies were purchased from BioLegend: CD122 (TM-beta1), PD-1/CD279 (29F.1A12), IL-10 (JES5–16E3), IFNγ (XMG1.2), Granzyme B (QA16A02), CXCR3 (CXCR3–173), CD62L (MEL-14). CD31 (390) was purchased from BD Biosciences (San Diego CA) and AIF1 (EPR16588) from Abcam (Cambridge MA). 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 or Accuri C6 flow cytometric analyzer. Datasets were analyzed using FlowJo v10 (TreeStar, Ashland OR). Respective isotype controls and/or fluorochrome-labeled isotype controls were used in all assays. Gating strategies were established based on respective isotype controls. For proliferation assays, gates were established based on unstimulated labeled cells.

Generation of bone marrow-derived dendritic cells and siRNA knockdown

Femurs and tibias were harvested from C57BL/6 (wild type; WT) mice between 10–16 weeks of age to generate bone marrow-derived DC, as described by a modified protocol of Inaba K et al [19]. Briefly, bone marrow cells were cultured in IMDM (Thermo Fisher Scientific, Grand Island NY) 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 7 days in culture. 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 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’. After electroporation of siRNA on day 5 in CD11c+-sorted DC, cells were placed back into culture. 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].

Isolation of CD8+ T cells for in vitro stimulation and proliferation assays

For isolation of naïve CD8+ T cells from OT-I mice, CD4+ T cells and MHC class II+ antigen presenting cells were depleted by negative selection from spleen and lymph nodes using primary antibodies to CD4 and MHC class II (BioLegend; San Diego CA) followed by secondary labeling with anti-rat IgG magnetic microbeads (Qiagen; Hilden Germany). Cells were then depleted by passing through a magnetic column. This resulted in a purity greater than 97% for CD8+ CD44- CD62L+ cells. These isolated naïve CD8+ T cells were cultured with SIINFEKL peptide- or OVA protein-pulsed siAIF1 or siControl LPS-matured DC at a ratio of 10:1, respectively. MOG protein and scrambled non-specific peptides served as controls. Cells were pulsed for 5 h at 37˚C. All OVA protein and peptides were purchased from AnaSpec (Fremont CA). SIINFEKL peptide was used at 0.1 or 0.3 µg/ml and OVA protein at 20 µg/ml.

SIINFEKL peptide- or OVA protein-pulsed siAIF1 or siControl mature DC stimulated OT-I CD8+ T cells were harvested at the 48, 72 and 96 h time point to evaluate CXCR3, Granzyme B, IFNγ, and IL-10 by flow cytometric analyses; antibodies purchased from BioLegend. Supernatant was additionally collected to assess IFNγ or IL-10 production by ELISA using kits purchased from BioLegend. For proliferation assays, CD8+ T cells pre-labeled with 1 µM Cell Trace Far Red dye (Thermo Fisher Scientific) were cultured with SIINFEKL peptide-pulsed siAIF1 or siControl DC for 72 or 96 h. Cells were co-stained with antibodies to IL-10 or IFNγ for intracellular cytokine detection after fixation and permeabilization. For Treg phenotype, cells at either day 5, 6 or 7 were harvested prior to staining for CD122, CD279 (PD-1) and CD8. All antibodies purchased from Biolegend. Cells were then acquired on a flow cytometric analyzer. In some experiments, CD8+ T cells primed by siAIF1 or siControl DC pulsed with either OVA protein or SIINFEKL peptide were collected and plated at equal numbers. Cells were then stimulated with 20 ng/ml of PMA and 1 μg of ionomycin for 5 h at 37˚C. Supernatant was collected and assayed for presence of IFNγ or IL-10.

In vitro cytotoxic CD8+ T cell killing assays

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. The SIINFEKL peptide-pulsed group was labeled with 0.05 μM CFSE. The control scrambled peptide-pulsed was labeled with 0.5 μM CFSE. The two groups were mixed together at approximately a 1:1 ratio prior to plating with the CD8+ T cells expanded from either siControl or siAIF1 DC. The approach is modified from works by Durward et al [21]. Decrease in frequency of the SIINFEKL peptide-pulsed 0.05 μM CFSE labeled target cells was used as a measurement of antigen-specific cytotoxic T cell killing. No SIINFEKL peptide or scrambled control peptide served as internal controls.

CD8+ T regulatory cell in vitro suppression assays

OT-I CD8+ T cells were expanded for 7 days by siAIF1 or siControl DC. These expanded CD8+ T cells are the suppressors. Next, naïve CD4+ T cells were isolated from WT mice by negative depletion and labeled with 2.5 μM CFSE. These CFSE-labeled naïve CD4+ T cells are the responders. The suppressor and responder T cells were then cultured together at a 1:1, 1:3, 1:10 or 1:30 ratio, respectively, in the presence of either IgG controls or neutralizing antibodies to IL-10 or PD-1. To stimulate proliferation of the naïve CD4+ T cell responders, OVA323–339 pulsed-antigen presenting cells were added to the culture. Cells were incubated for 72 or 96 h prior to collection, staining, and analysis of responder CD4+ T cell proliferation. The experiment is a modified approach by Collison et al [22].

In vivo CD8+ T regulatory suppression assays

CD4+ T cells were isolated from OT-II mice, suspended to 5 × 106 per ml and labeled with 2.5 μM of CFSE. Additionally, 5 × 106 day 7-expanded CD8+ T cells from either siControl or siAIF1 DC were collected. These CFSE-labeled OT-II CD4+ T cells and expanded OT-I CD8+ T cells were then mixed together at a 1:1 ratio and re-suspended in 250 μl of PBS. Cells were then intravenous injected into WT recipient mice, as described by Moon et al [23]. Injection of OT-II CD4+ T cells-only served as internal control. 12 h later, the WT recipient mice were then intraperitoneal injected with 10 μg of OVA323–339 peptide in 200 μl of PBS. Mice were sacrificed 3 and 5 days post-treatment. Spleen and lymph nodes were harvested and disassociated into single cell suspension, washed, and fixed. Cells were then analyzed for CFSE-labeled CD4+ T cell proliferation. The in vivo suppression technique is modified from the approach by Workman et al. [24].

ELISA assays

After specific time points of cell incubations, supernatant was collected and stored at −80°C until use. IL-10 (BioLegend), TGFβ (BioLegend) and IFNγ (BD Biosciences) ELISA kits were used following manufacturers’ recommended protocols to measure cytokine expression. Absorbance values were collected and quantified using the Synergy HT (BioTek, Winnoski, VT, USA) multimode microplate reader.

Statistical Analysis

GraphPad Prism v7.0 (GraphPad Software, La Jolla, CA) was used to determine statistical significance. Student unpaired two-tailed t test was used to evaluate the significance of two groups. A p value ≤ 0.05 was considered statistically significant; * = <0.05, ** = <0.01, and ns = non-significant. Error bars for all figures indicate standard deviation.

Results

Loss of AIF1 in DC impairs antigen-specific CD8+ T cell responses

Previous studies have shown that AIF1 expression in dendritic cells (DC) modulates immunity in CD4+ T cell subsets [18]. As a follow up, these new investigations set to delineate whether AIF1 expression in DC governs antigen-specific CD8+ T cell responses. Knockdown of AIF1 by siRNA (siAIF1) in DC resulted in significant impairment of cytotoxic CD8+ T lymphocyte effector functions. Both SIINFEKL-peptide and OVA protein-pulsed siAIF1 DC primed with OT-I CD8+ T cells had reduced expression of Granzyme B and CXCR3 in comparison to control DC that received scrambled non-targeting siRNA oligos (siControl; Figure 1A). For SIINFEKL peptide, there was a dramatic reduction in CXCR3+ Granzyme B+ CD8+ T cells from 62.7% ±3.9 in the siControl treated group down to 17.7% ±1.1 in the siAIF1 groups. The same trend was observed upon pulsing with OVA protein, with a reduction from 24.3% ±2.1 in the siControl to that of 3.9% ±0.4 in siAIF1 cohorts. With reduced effector responses observed in the siAIF1 group, the studies next measured proliferative capacity coupled with expression of IFNγ, which is a hallmark cytokine for cytotoxic T cell effector responses. AIF1 knockdown DC restrained proliferation of responder CD8+ T cells (Figure 1B). Importantly, those T cells that were able to be expanded in the siAIF1 DC cohort had significantly lower IFNγ expression. Levels were reduced to 8.8% ±0.6 in SIINFEKL peptide- and 4.6% ±1.4 in OVA protein-primed groups, in comparison to 34.8% ±2.3 and 19.4% ±1.1, respectively, in the siControl groups. Lastly, the studies next collected siAIF1 DC- or siControl DC expanded CD8+ T cells and plated them at equal numbers. Cells were then re-stimulated with mitogen for 5 h. Results revealed significant restrain in ability to produce IFNγ from siAIF1 DC expanded CD8+ T cells in comparison to siControl DC in both peptide and protein priming conditions (Figure 1C).

Figure 1. AIF1 knockdown in DC restrains antigen-specific T cell effector responses.

Figure 1.

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Naïve CD8+ T cells were primed by either SIINFEKL peptide- or OVA protein-pulsed control DC (siControl) or AIF1 knockdown DC (siAIF1). (A) CD8+ T cells evaluated for co-expression of CXCR3 and Granzyme B by flow cytometric analyses. (B) CD8+ T cells were pre-labeled with Cell Trace Far Red dye prior to culture with peptide or protein-pulsed siControl or siAIF1 DC. Proliferation and co-expression of IFNγ are assessed in the CD8+ T cell responder subsets 96 h after priming by DC. (C) Expanded CD8+ T cells from siControl or siAIF1 DC were plated at equal numbers prior to stimulation with PMA/ionomycin mitogen cocktail for 4 h. Supernatant was harvested and assessed for IFNγ production by ELISA. The lighter shade box [●] represents siControl and the darker filled [■] siAIF1. Data is representative of four independent experiments.

CD8+ T cells expanded by DC silenced for AIF1 have impaired ability to kill target cells

In vitro killing assays were next performed to corroborate impaired effector responses. CD4+ T cells from wild type mice pulsed with SIINFEKL peptide were utilized as targets for cell killing. Robust killing of the SIINFEKL peptide-pulsed target cells, but not co-cultured scrambled control peptide-pulsed, was observed by the CD8+ cytotoxic T cells primed by siControl DC (Figure 2A). The siControl cohort had decreased target numbers down from 45.6% ±0.6 in the internal control group to 24.4% ±1.3 in SIINFEKL and to 27.9% ±2.0 in OVA protein groups. The internal control group is target cells not pulsed with SIINFEKL peptide or OVA protein. However, restrained killing was observed by the CD8+ T cells stimulated by siAIF1 DC, where reduction from the internal control was down only to 35.6% ±3.3 in SIINFEKL peptide and 42.0% ±2.7 in OVA protein groups. Varying the concentration of target-to-effector ratio revealed that roughly 10 fold more siAIF1 DC expanded CD8+ T cells were required to yield the same killing efficacy as the siControl group (Figure 2B).

Figure 2. Silencing of AIF1 in DC abrogates expanded CD8+ T cell killing capacity in vitro.

Figure 2.

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(A) 0.05 μM CFSE-labeled SIINFEKL-peptide pulsed target cells and 0.5 μM CFSE-labeled control-peptide pulsed control cells were combined at a 1:1 ratio. This mixture was then cultured with CD8+ T cells expanded from either SIINFEKL peptide- or OVA protein-pulsed siControl or siAIF1 DC for 24 h. Histogram plots show the region labeled target as the SIINFEKL-peptide pulsed group measured for killing; decreased levels are a measure of killing efficacy. Non-labeled region marker is control peptide-pulsed. The Internal Control histogram plot represents the 0.05 μM and 0.5μM CFSE-labeled cells cultured without SIINFEKL or control peptide. Bar graph shows percentages of the target cells from the histogram plots. Data is representative of three independent experiments. (B) Bar graph displays percent of target cells at varying target-to-effector ratios of [●] siControl or [■] siAIF1 DC expanded CD8+ T cells from either SIINFEKL peptide- (left graph) or OVA protein-pulsed cohorts (right graph). Cells were cultured at 30:1, 10:1, 3:1 and 1:1. Data is representative of three independent experiments.

DC silenced for AIF1 expand IL-10 producing CD122+ PD1+ CD8+ T cells

With restrained cytotoxic T cell effector responses, the studies next set to evaluate generation of CD8+ T regulatory subsets. Several reports have shown that CD8+ CD122+ T cell subsets can serve to both maintain homeostasis and restrain exacerbated immune pathologies [7, 8, 25]. These CD8+ T regulatory cells exerted suppression through IL-10 and PD-1 dominant mechanisms. In assessing onset of regulatory fates, siAIF1 DC pulsed with either SIINFEKL peptide or OVA protein resulted in increased levels of CD122+ PD-1+ subsets expanded from naïve CD8+ T cells (Figure 3A). In siControl DC, levels of CD122+ PD-1+ observed were 15.8% ±0.9 and 17.3% ±0.6 for SIINFEKL-peptide and OVA protein-pulsed groups, respectively. However, in the siAIF1 DC, studies reproducibly found higher levels of CD122+ PD-1+ populations, with 42.9% ±3.4 expanded in the SIINFEKL peptide and 46.5% ±4.1 in the OVA protein groups. In addition to CD8+ T cells expanded by siAIF1 DC having reduced proliferation capacity, there was also a greater proportion of IL-10 production in the few proliferating subsets when compared to subsets stimulated by siControl DC (Figure 3B). In the SIINFEKL peptide cohort, the siControl DC had 55.0% ±5.1 IL-10+ proliferative subsets compared to 32.1% ±2.9 that were negative for IL-10. However, in the siAIF1 DC cohort, 26.7% ±1.9 were IL-10+ and only 7.1% ±0.5 were IL-10 negative. Similar results were seen in the OVA protein expanded population. This would suggest that the siAIF1 DC expanded CD8+ T cells had a reduced overall proliferation capacity, but that the majority of the few dividing cells were producing IL-10. To further investigate IL-10 production, total CD8+ T cells expanded from either siControl or siAIF1 DC were collected and plated at equal numbers after 7 days. Cells were then stimulated with mitogens prior to assessing total release of IL-10 into the supernatant. At equal cell number plating, CD8+ T cells expanded from siAIF1 DC produced several fold higher levels of IL-10 in comparison to the siControl group (Figure 3C).

Figure 3. Expansion of IL-10 producing PD-1+ CD8+ T cell subsets by AIF1 knockdown DC.

Figure 3.

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(A) SIINFEKL peptide- or OVA protein-pulsed siControl or siAIF1 DC expanded CD8+ T cells were assessed for IL-2Rβ (CD122) and PD-1 (CD279) co-expression. (B) The CD8+ T cells labeled with Cell Trace Far Red were evaluated on day 4 for proliferation and co-expression of IL-10 upon priming by siControl or siAIF1 DC in presence of SIINFEKL peptide or OVA protein. (C) siControl or siAIF1 DC expanded CD8+ T cells were plated at equal numbers and assessed for IL-10 production by ELISA after PMA/ionomycin mitogen stimulation. Datasets are representative of four independent experiments.

AIF1 knockdown DC primed CD8+ T cells suppress neighboring CD4+ T cell expansion

Regulatory function of the CD8+ T cells expanded by siAIF1 DC was next assessed by evaluating ability to suppress neighbor CD4+ T cell proliferation in vitro. OVA protein pulsed siControl or siAIF1 DC expanded CD8+ T cells were cultured with OT-II isolated CFSE-labeled CD4+ T cells at varying ratios. Next, antigen presenting cells pulsed with OVA323–339 peptide were used to stimulate the CFSE-labeled CD4+ T cells in the co-culture; the OT-I CD8+ T cells are unresponsive to OVA323–339 peptide. Absence of the siControl or siAIF1 DC expanded CD8+ T cells served as internal controls. Results revealed approximately a 50% reduction in CD4+ T cell proliferation, from 77.8% ±3.9 in the siControl to 37.4% ±2.8 in the siAIF1 cohort (Figure 4A). To further delineate the potential role of IL-10 or PD-1 in mediating suppression, neutralizing antibodies were added during the co-culture. Neutralizing either IL-10 or PD-1 alleviated the suppressive function of the siAIF1 expanded CD8+ T cells on the proliferating responder CD4+ T cell cells. However, no significant differences were observed upon addition of neutralizing antibodies to IL-10 or PD-1 in the siControl DC groups. Lastly, varying concentrations of CFSE-labeled CD4+ T cell responders with siControl or siAIF1 DC expanded CD8+ T cells were measured (Figure 4B). As the ratio of CD8+ T cells from the siAIF1 DC cohort was reduced, the effective suppression of CD4+ T cell responder proliferation was proportionally limited.

Figure 4. AIF1 knockdown DC expanded CD8+ T cell subsets suppress proliferation of neighbor CD4+ T cells.

Figure 4.

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OT-I derived CD8+ T cells activated by siControl or siAIF1 DC were cultured with CFSE-labeled responder naïve CD4+ T cells derived from the OT-II mouse at a 1:1 ratio. (A) Antigen presenting cells pulsed with OVA323–339 peptide were then used to stimulate the OT-II CFSE-labeled CD4+ T cells responders in the presence of the expanded CD8+ T cells. Cells were cultured in the presence of neutralizing antibodies to IL-10 or PD-1. Respective IgG isotype antibodies were used as controls. No presence of CD8+ T cells with IgG isotype antibody addition served as a baseline for measuring proliferation capacity of the responder OT-II CD4+ T cells. Cells were collected after 96 h of stimulation and proliferation was analyzed by flow cytometry. (B) Varying ratios of suppressor CD8+ T cells expanded from either siAIF1 or siControl DC to the responders CFSE-labeled OT-II CD4+ T cells were added. Ratios were at 1:1, 1:3, 1:10, or 1:30 of CFSE-labeled responder CD4+ T cells to the CD8+ T cell suppressors, respectively. OVA323–339-pulsed antigen presenting cells were used to prime the responder CD4+ T cells. Cells were cultured in the presence of neutralizing antibodies to [■] IL-10 or [▲] PD-1. [●] IgG isotype antibody served as internal controls. Data is representative of three independent experiments.

Adoptive transfer of AIF1 knockdown DC restrains T effector responses in vivo

In vivo studies were next performed to recapture in vitro observed suppressive functions of AIF1 knockdown DC expanded CD8+ T cells. Expanded siAIF1 or siControl DC primed CD8+ T cells were adoptively transferred into WT recipient mice along with freshly isolated CFSE-labeled CD4+ OT-II T cells. After transfer of the respective expanded CD8+ T cells and CFSE-labeled OT-II CD4+ T cells, recipient mice were then injected with OVA323–339 peptide coupled with LPS as an adjuvant. Evaluation of cellular responses several days later revealed that the CFSE-labeled CD4+ T cells had reduced ability to proliferate in vivo (Figure 5A and 5B). Although there was an impaired proliferative capacity of the responder CD4+ T cells, these adoptive transfer studies did not identify conversion into CD25+ Foxp3+ or IL-10+ CD4+ T regulatory cell subsets (data not shown).

Figure 5. In vivo suppression of T cell responses by siAIF1 DC expanded CD8+ T cells.

Figure 5.

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OVA protein-pulsed siControl or siAIF1 DC expanded OT-I CD8+ T cells from in vitro cultures were adoptively transferred into WT recipient mice along with OT-II CFSE-labeled CD4+ T cells. After 12 h, the WT recipient mice were i.p. injected with OVA323–339 peptide and adjuvant. Lymph nodes and spleens were collected at day 3 or 5 post-treatment. (A) CD4+ CD8- CFSE+ subsets were evaluated for proliferation. (B) Graph of proliferation of CD4+ CD8- CFSE+ subsets from three mice. Data is representative of two independent experiments.

4. Discussion

Loss of AIF1 expression in DC redirects T cell effector responses to that of regulatory fates. In the case of CD8+ T cells, AIF1 knockdown DC restrained ability to upregulate Granzyme B concomitant with CXCR3 expression in comparison to controls. Interestingly, there were less changes in the CXCR3+ Granzyme B- and CXCR3- Granzyme B+ populations. Reports have shown that murine CD8+ CD122+ Tregs express CXCR3 [6]. This may suggest that these IL-10-producing CD8+ Tregs express CXCR3 as a means of controlling the magnitude of inflammatory reactions at the site of inflammation. Although Granzyme B has been shown to be important for CD4+ regulatory functions [26], the functional role of Granzyme B in CD8+ Tregs does remain unclear. However, without expression of CXCR3, it would suggest that these subsets would be retained in lymph nodes and thus be unable to exert effector functions at infection sites.

Silenced AIF1 expression in DC induced functionally suppressive IL-10-producing CD8+ T regulatory subsets. In vitro studies revealed a large increase in CD122+ PD-1+ CD8+ T cells expanded from AIF1 knockdown DC. This corroborates literature defining of regulatory CD8+ T cells from that of effector and/or memory pools [6]. Most strikingly, the expanded CD8+ T cells from AIF1 knockdown DC had higher levels of IL-10 from the limited proliferating cells. In the control DC expanded cohort, IL-10 production began to lower at the later dividing stages of CD8+ T cells. However, in the AIF1 knockdown DC primed CD8+ T cells, IL-10 levels remained high throughout the rounds of cell division. This would suggest that AIF1 knockdown DC limited and/or selectively supported proliferation of IL-10-producing subsets. Furthermore, the levels of IFNγ were found to be inversely proportional to IL-10 in the control vs. AIF1 knockdown DC expanded CD8+ T cells. Taken together, AIF1 knockdown DC redirect naïve CD8+ T cells from effector towards a regulatory fate. Overtly higher concentrations of antigen, particularly above 1 µg/ml of SIINFEKL, overrode the imprint of naïve CD8+ T cell conversion into the IL-10+ regulatory fate. This may suggest that excessively high levels of antigen can direct immunostimulatory responses in absence of AIF1 exerted effects during priming events, which is an important requirement during high pathogen load in the host. Given that MAPK p38 phosphorylation is impaired in macrophages silenced for AIF1 [27], this could suggest that AIF1 acts similarly through p38 in DC to help direct and support downstream TLR- and cytokine-signaling activities to promote adaptive immune responses [28].

AIF1 knockdown DC expanded IL-10 producing CD8+ T cells that exerted dominant suppression of neighboring CD4+ T cell proliferation. The studies used naïve CD4+ T cells from an OT-II mouse as the responders. As the CD8+ T cells were derived from the OT-I strain, they would not be activated by presence of OVA323–339 peptide. This allowed the system to specifically measure CFSE-labeled CD4+ OT-II T cell proliferation capacities in the presence of the siAIF1 DC expanded CD8+ OT-I T cells. OVA323–339-pulsed antigen presenting cells stimulated proliferation of responder CD4+ T cells in the absence of CD8+ T cells or with CD8+ T cells expanded from siControl DC. However, in the presence of CD8+ T cells expanded from DC silenced for AIF1 expression, a significant reduction in proliferation was observed. It is important to note that there was no conversion to CD4+ CD25+ Foxp3+ T regulatory cell subsets upon co-culture with CD8+ Tregs expanded from siAIF1 DC.

IL-10 and PD-1 expression by CD8+ T cell subsets directly suppress immune responses. PD ligand-1 (PD-L1 or B7-H1) is expressed on both DC and T cells [29]. Engagement of PD-1 and PD-L1 abrogates effector functions [10, 30]. Similarly, IL-10 is a potent suppressor of T cell effector responses and the antigen presentation capacity of DC [31, 32]. Neutralization of these molecules in vitro confirmed that each play a major suppressive role by the CD8+ T regulatory subsets expanded by siAIF1 DC. Rescued proliferation capacity was largely seen upon neutralization of IL-10 or PD-1 in the siAIF1 DC expanded CD8+ T cell cohort. This suggests that both IL-10 and PD-1 expression are involved in suppressing bystander antigen-specific CD4+ T cell proliferation responses in vitro. However, it remains unclear in these studies whether neutralization indirectly inhibited the CD8+ T cell influence on the antigen presenting cells bearing the OVA323–339 peptide or if it directly restrains CD4+ T cell proliferation capacity through cell-cell engagement. Furthermore, the kinetics of the study may also suggest that the pre-activated state of CD8+ T regulatory cells expanded by siAIF1 DC are able to more robustly suppress the naïve CD4+ T cells undergoing initial priming by the antigen presenting cells. This tracks with why the studies are able to identify suppression at low ratios of CD4+ T effector to CD8+ T regulatory co-cultures. This is most likely exerted through IL-10 cytokine production by the CD8+ Tregs in the co-culture, but can also encompass PD-1 and PDL-1 interactions between the APC and T cells. Thus, as literature as noted, it would argue that the expanded CD8+ Tregs from the AIF1 knockdown DC exert both mechanisms to ensure adequate suppression of immune responses. However, it is unclear which of the two, IL-10 or PD-1, exert a more dominant mechanism. Differences in antibody affinity, molecule/ligand concentrations, and receptor expression levels are variable for in vitro conditions. Future studies warrant usage of IL-10−/− or PD-1−/− mouse models to clearly delineate the role each have in respect to functional roles of CD8+ T regulatory subsets expanded by AIF1-deficient DC.

ACKNOWLEDGEMENTS

This work was funded, in part, by the U.S. National Institutes of Health (Grant #SC1GM127207 and #SC2GM103741), Department of Defense (Grant #W911NF-14–1-0123), and National Science Foundation (Grant #1428768). The authors are grateful to Franklin Ampy, Clarence M. Lee and Winston Anderson for assistance with statistical analyses and revision of the work.

Abbreviations

AIF1

Allograft Inflammatory Factor-1

DC

dendritic cell

H

hour

siRNA

small interfering RNA

siAIF1

AIF1 siRNA knockdown group

siControl

Scrambled control siRNA knockdown group

Treg

T regulatory cell

WT

wild type

Footnotes

DISCLOSURES

The authors declare no conflicts of interests

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