NIH Public Access Author Manuscript J Autoimmun. Author manuscript; available in PMC 2012 December 1. NIH-PA Author Manuscript Published in final edited form as: J Autoimmun. 2011 December ; 37(4): 328–341. doi:10.1016/j.jaut.2011.09.005. Tolerogen-induced interferon-producing killer dendritic cells (IKDCs) protect against EAE Eduardo Huarte1, Agnieszka Rynda-Apple1, Carol Riccardi1, Jerod A. Skyberg1, Sarah Golden1, MaryClare F. Rollins1, Andrew Ramstead1, Larissa O. Jackiw1, Massimo Maddaloni1, and David W. Pascual1,2 1Department of Immunology and Infectious Diseases, Montana State University, 960 Technology Blvd., Bozeman, Montana 59718, USA Abstract NIH-PA Author Manuscript Natural killer (NK) cells and dendritic cells (DCs) have been shown to link the innate and adaptive immune systems. Likewise, a new innate cell subset, interferon-producing killer DCs (IKDCs), shares phenotypic and functional characteristics with both DCs and NK cells. Here, we show IKDCs play an essential role in the resolution of experimental autoimmune encephalomyelitis (EAE) upon treatment with the tolerizing agent, myelin oligodendrocyte glycoprotein (MOG), genetically fused to reovirus protein σ1 (termed MOG-pσ1). Activated IKDCs were recruited subsequent MOG-pσ1 treatment of EAE, and disease resolution was abated upon NK1.1 cell depletion. These IKDCs were able to kill activated CD4+ T cells and mature dendritic DCs, thus, contributing to EAE remission. In addition, IKDCs were responsible for MOG-pσ1-mediated MOG-specific regulatory T cell recruitment to the CNS. The IKDCs induced by MOG-pσ1 expressed elevated levels of HVEM for interactions with cognate ligand-positive cells: LIGHT+ NK and Teff cells and BTLA+ B cells. Further characterization revealed these activated IKDCs being MHC class IIhigh, and upon their adoptive transfer (CD11c+NK1.1+MHC class IIhigh), IKDCs, but not CD11c+NK1.1+MHC class IIintermediate/low (unactivated) cells, conferred protection against EAE. These activated IKDCs showed enhanced CD107a, PD-L1, and granzyme B expression and could present OVA, unlike unactivated IKDCs. Thus, these results demonstrate the interventional potency induced HVEM+ IKDCs to resolve autoimmune disease. Keywords NIH-PA Author Manuscript EAE; tolerance; Treg cells; IKDC; HVEM © 2011 Elsevier Ltd. All rights reserved. 2 Corresponding author: David W. Pascual, Ph.D., Dept. of Immunology and Infectious Diseases, Montana State University, P.O. Box 173610, Bozeman, MT 59717-3610, USA. Phone: (406) 994-6244; Fax: (406) 994-4303; dpascual@montana.edu. Conflict of interest The authors have no financial conflict of interest. Authorship contribution E.H. and D.W.P. conceptualized and designed the experiments, analyzed the data, and prepared the manuscript; E.H., A.R-A., C.R., J.S., and M-C. R. performed the experiments and provided intellectual contributions; S.G., A.R-A., A.R., and M.M. designed and constructed the protein constructs; L. O. J. performed the cell-sorting experiments. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Huarte et al. Page 2 1. Introduction NIH-PA Author Manuscript The maintenance of immunological self-tolerance is an active process, requiring the participation of both the adaptive and innate immune systems. However, most efforts in studying tolerance have focused on the adaptive rather than on the innate arm of immunity; consequently, the innate immune system’s contribution to tolerance is not as well understood, particularly the role of NK cells on autoimmunity [1, 2]. This regulatory role is suggested by NK cells’ ability to recognize and efficiently lyse activated T cells [3], as well as act as a source of proinflammatory cytokines (IFN- , GM-CSF, and TNF-α) and chemokines (CCL3, CCL4, and CCL5) [4]. Previous studies suggest the existence of a cell subset, termed bitypic NK/DCs [5] or NKDCs [6], that shares phenotypical and functional features of both NK cells and DCs, despite the lack of an unambiguous set of phenotypic markers or a defined transcription factor. More recently, a new cell subset, referred to as interferon-producing killer dendritic cells (IKDCs) [7, 8], has been defined that shares features of both NK and DCs. Phenotypically, IKDCs have been originally described as CD3−, CD19−, NK1.1+, CD49b+, CD11cintermediate, B220+, Gr-1−, and MHC class IIintermediate/high [7, 8]. NIH-PA Author Manuscript Functionally, IKDCs exhibit both cytolytic activity and MHC class II Ag presentation and therefore have been postulated as a bridge between the adaptive and innate arms of the immune system [7, 8]. It is still controversial whether IKDCs represent a separate lineage of DCs or merely a state of activation of NK cells, because poor Ag presentation capabilities by IKDCs is the heart of this debate. On one hand, IKDCs are able to present peptides to lymphocytes, but fail to process and present Ag to induce CD4+ T cell proliferation [7, 9], suggesting IKDCs are activated NK cells. This point of view is accepted by several groups [10–12] and supported by data showing human NK cells having limited Ag presentation capacity once activated [13]. On the other hand, IKDCs may originate from different lymphoid precursors [14] and can become bona fide Ag-presenting cells after encountering virus [15] or tumor cells [16, 17], thus, supporting the notion of IKDCs as an independent cell lineage that truly shares phenotypic and functional features of NK cells and DCs. NIH-PA Author Manuscript Despite the recent focus on IKDCs’ involvement in cancer [8, 15, 16] or viral infections [14], little or no information is available on the role IKDCs play in inflammatory diseases. Here, we show for the first time, to our knowledge, that IKDCs contribute to the resolution of EAE. Stimulation of IKDCs is noted after mucosal administration of an auto-Ag, myelin oligodendrocyte glycoprotein, fused to reovirus type 3 protein sigma 1 (termed MOG-pσ1). EAE is a T cell-dependent inflammatory disease principally mediated by IL-17 and is highly reproducible in susceptible rodent strains after immunization with TCR-reactive peptides and mimics some aspects of human multiple sclerosis (MS) [18]. Past efforts to induce modulation of autoimmunity by delivering auto-Ags have proven successful [19]; however, such efforts are hampered by requiring large doses of Ag or repeated administration. To overcome these barriers, we have found single dose tolerance or immunotherapy can be accomplished by genetically fusing Ags [20] or auto-Ags [21, 22] to (pσ1). Mucosal pσ1delivered tolerogens have been found to induce IL-10-producing conventional regulatory T (Treg) cells, as well as IL-4-producing Th2 cells, and when either subset is adoptively transferred, EAE could be reduced in an Ag-dependent fashion. Immunotherapy with MOGpσ1 requires MOG to be coupled to pσ1, since pσ1 alone, pσ1 plus rMOG, or rMOG alone is unable to reverse EAE. Most notably, MOG-pσ1 could reverse EAE clinical disease within 24 h of treatment [22]. Although enhanced numbers of Treg cells have been found to infiltrate the CNS after MOGpσ1 treatment, understanding how this is accomplished within such a short time frame suggests possible innate cell involvement. In this context, the role of NK cells in the J Autoimmun. Author manuscript; available in PMC 2012 December 1. Huarte et al. Page 3 NIH-PA Author Manuscript development of EAE has received considerable attention in recent years. NK1.1+ cells have been shown to be protective during the course of EAE [23,24], since depletion of NK cells with an anti-NK1.1 mAb leads to EAE exacerbation [25,26], which could be due to their cytotoxic effects upon activated autoreactive CD4+ T cells or DCs [3]. Consequently, an enrichment of NK cells in the CNS of diseased animals leads to amelioration of EAE [26], while blocking the NK cell migration to the CNS exacerbates EAE [27, 28]. A protective effect of NK cells could also be attributed to the production of neuroprotective factors, such as the brain-derived neurotropic factor (BDNF) and neurotropin 3 (NT-3) [29]. Additionally, NK cell-produced IL-5 seems to play a protective role in MS [30], and MS patients present lower levels of NK cells [31], thus, suggesting a regulatory role for human NK cells. However, a consensus has yet to be reached over NK cells’ role in the pathogenesis and development of EAE [32,33], since other groups have reported EAE amelioration after NK cell depletion [34] Interestingly, an immunoregulatory subset of human NK cells based on their expression of CD56 and IL-2Rα levels has been proposed [35]. Supporting this hypothesis, the reduction of the clinical MS relapse rate during pregnancy is associated with an increase in the CD56bright (regulatory) subpopulation [36]. However, a similar phenotype is still lacking in murine models [33], and thus further studies are warranted in order to clarify the role of NK cells’ involvement in EAE and other autoimmune diseases. NIH-PA Author Manuscript NIH-PA Author Manuscript Herein we show IKDCs play a central role in the resolution of EAE following oral administration of MOG-pσ1. MOG-pσ1 induced an increase in the number of CD11c+NK1.1+ cells, but not conventional NK cells, in the periphery, as well as in the CNS of mice with EAE. CD11c+NK1.1+ cells were also able to lyse both DCs and activated CD4+ T cells and, after MOG-pσ1 treatment, induced a robust proliferation by transgenic OVA TCR CD4+ T cells, thus, fulfilling the definition of IKDCs and effectively providing a link between the adaptive and innate immune systems. Moreover, IKDCS from MOG-pσ1treated mice showed a more activated phenotype than those from untreated controls, with an increase in the expression level of herpesvirus entry mediator (HVEM) and reduced expression of the inhibitory molecule NKG2A, allowing the lysis of CD4+ T cells [37]. Interestingly, NKG2A+ IKDCs were responsible for producing IL-10, a cytokine shown to have a pivotal role in the amelioration of EAE after MOG-pσ1 treatment [22]. In the absence of NK1.1+ cells, MOG-pσ1 treatment failed to recruit Treg cells into the CNS of diseased mice with a consequential loss of clinical efficacy. In addition, IKDCs were involved in the recruitment of activated (CD69+) MOG-specific Treg cells into the CNS. Importantly, MOGpσ1 treatment also upregulated the level of expression of MHC class II molecules by CNSinfiltrating IKDCs. These MHC class IIHi IKDCs presented a more activated phenotype than their MHC class IIintermediate/low counterparts and showed enhanced expression of CD80, HVEM, granzyme B, CD107a (lysosomal associated membrane protein-1, LAMP-1) [38], and PD-L1 (programmed cell death 1 ligand, B7-H1) [39,40]. Adoptive transfer of these activated MHC class IIHi IKDCs, but not CD11c+NK1.1+MHC classintermediate/low cells, dramatically improved the clinical resolution of EAE, underscoring the critical role played by IKDCs in treating EAE. 2. Materials and methods 2.1. Preparation of pσ1, MOG-pσ1, and MOG/GFP-pσ1 MOG-pσ1 and pσ1 were prepared, as previously described [22]. To enable detection of MOG-pσ1 binding, MOG-pσ1 was genetically modified to incorporate GFP. Briefly, MOG was removed with EcoRI from the MOG-pσ1 plasmid, and the remnant backbone was treated with calf intestine phosphatase and filled with a new EcoRI-SalI MOG and a SalIEcoRI GFP fragments via a tripartite ligation. Recombinant clones were analyzed by DNA sequencing. The final construct for MOG/GFP-pσ1 was transformed into Pichia pastoris via electroporation (ECM630 BTX Harvard apparatus). Protein expression in yeast and protein J Autoimmun. Author manuscript; available in PMC 2012 December 1. Huarte et al. Page 4 NIH-PA Author Manuscript purification were performed, as previously described [22]. Proteins were assessed for purity and quality by Coomassie-stained polyacrylamide gels and by Western blot analysis, using an anti-His-tag mAb (Invitrogen, Carlsbad, CA). 2.2. Mice Female 6–8 wk old C57BL/6 mice (Frederick Cancer Research Facility, National Cancer Institute, Frederick, MD) were used throughout the study. Breeding colonies of transgenic FoxP3-GFP, IL10−/−, and OT-II mice (The Jackson Laboratory, Bar Harbor, ME) were maintained at Montana State University Animal Resources Center under pathogen-free conditions in individual ventilated cages under HEPA-filtered barrier conditions and were fed sterile food and water ad libitum. All animal care and procedures were in compliance with institutional policies for animal health and well-being. 2.3. EAE induction and MOG-pσ1-based therapies NIH-PA Author Manuscript For EAE induction, mice were challenged s.c. in the flank with 200 μg of MOG35–55 peptide (Biosynthesis, Inc., Lewisville, TX) in 100 μl of IFA (Sigma-Aldrich, St. Louis, MO) containing 400 μg killed Mycobacterium tuberculosis (Difco Laboratories, Detroit, MI) on day 0. On days 0 and 2, mice were treated i.p. with 200 ng of Bordetella pertussis toxin (List Biological Laboratories, Campbell, CA). Mice were monitored and scored daily for disease progression: 0, normal; 1, a limp tail; 2, hind limb weakness; 3, hind limb paresis; 4, quadriplegia; 5, moribund state. When indicated, mice were given a single oral 50 μg dose of MOG-pσ1. 2.4. In vivo NK cell depletion Mice were i.v injected with 500 μg/ml of anti-NK1.1 mAb (PK136; Bio X Cell, West Lebanon, NH) at the time of or 7 days after EAE challenge. Mice were subsequently treated weekly i.p. until day 21 p.ch. 2.5. Isolation of mononuclear cells from CNS NIH-PA Author Manuscript Mice were perfused through the left cardiac ventricle with cold PBS. The forebrain and cerebellum were dissected, and the spinal cords (SCs) flushed out with PBS by hydrostatic pressure and digested with 10 ml HBSS containing 500 U/ml of collagenase (SigmaAldrich) for 60–90 min at 37°C with shaking. The tissues were then homogenized in cold HBSS buffer (Sigma-Aldrich) with the plunger of a syringe, filtered through a 70 μm cell strainer to obtain a single cell suspension, and centrifuged. Cell pellets from 4–5 mice were pooled, resuspended in 70% Percoll (Sigma-Aldrich), and underlaid with 30% Percoll. Mononuclear cells were isolated from the 30/70 interphase after gradient density centrifugation, washed, and stained for flow cytometry. 2.6. Tetramer staining Tetramer-based enrichment was performed similar to that previously described [41]. Briefly, CNS-infiltrating lymphocytes and splenocytes were first stained with MOG42–55 -APC or OVA329–337-APC tetramer (NIH Tetramer Core Facility, Emory Univ., Atlanta, GA) for 1h at room temperature in PBS containing 2% of FBS, 2% rat serum, and Fc block (BD Pharmingen). Cells were then stained with anti-APC magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer instructions and then passed over a magnetized column (Miltenyi Biotec) to separate bound and unbound fractions. The resulting fractions were then stained for extracellular markers and acquired fluorescence using a LSR-II flow cytometer (Becton Dickinson, San Jose, CA) and analyzed with Flow Jo software (Tree Star Inc., Ashland, OR). J Autoimmun. Author manuscript; available in PMC 2012 December 1. Huarte et al. Page 5 2.7. Cytokine ELISAs NIH-PA Author Manuscript Spleens were removed 18 – 21 days after EAE induction from PBS and MOG-pσ1 treated mice. Bead-isolated CD25+CD4+ or CD25−CD4+ T cells (>95% purity) were cultured for 4 days in the presence of 1:1 ratio of T cell-depleted splenic feeder cells with or without MOG35–55 restimulation. Capture ELISA was employed to quantify the levels of IFN- , IL-10, IL-17, and TGF- , as previously described [22]. 2.8. Characterization and isolation of IKDCs NIH-PA Author Manuscript Splenic and lymph node (LN) cells including mesenteric LNs (MLNs) and head and neck LNs (HNLNs) were stained with biotinylated or fluorochrome-conjugated mAbs against B220 (clone RA3-6B2), CD1d (1B1), CD3 (145-2C11), CD25 (PC61), CD5 (53-7.3), CD11c (HL3), and CD49b (Ha1/29), all from BD Pharmingen. CD4 (GK1.5), CD107a (1D4B), NKG2D (clone C7), and TRAIL (N2B2) mAbs were obtained from Biolegend (San Diego, CA). NK1.1 (PK136), NKG2A (16A11), HVEM (LH1), BTLA (8F4), MHC-II (M5/114.15.2), and CD11b (M1/70) mAbs were obtained from eBioscience; anti-PD-L1 mAb was from Serotec; and anti-LIGHT mAb was from R & D Systems. Intracellular staining for FoxP3, IFN- , granzyme B, and IL-10 was accomplished using FITC-, PE-Cy5-, or PE-labeled mAbs (eBioscience), and PE- or APC-anti-IFN- or -anti IL-10 mAbs (BD Pharmingen). Cells were acquired using a LSR-II flow cytometer (Becton Dickinson) and analyzed with Flow Jo software (Tree Star Inc.). Cell-sorting was accomplished using a FACSAria (Becton Dickinson), as previously described [16]. Briefly, spleens and LNs were stained for CD3, CD11c, B220, MHC class II, CD11b, and NK1.1, and three different populations were sorted based on the expression of CD11c, NK1.1, and MHC class II. IKDCs were confirmed to be B220+ and CD3−. Cell purity exceeded 95%. 2.9. MOG/GFP-pσ1 binding assays Splenic cells from naïve or EAE mice were incubated at 4°C with 20 μg/ml of MOG/GFPpσ1 for 30 min, washed, and stained with mAbs to NK1.1 (clone PK136, eBioscience); CD11c (HL3, BD Pharmingen); B220 (RA3-6B2, BD Pharmingen); and HVEM (LH1, eBioscience) for another 20 min before being analyzed by flow cytometry. 2.10. Adoptive transfer C57BL/6 mice were challenged with MOG35–55, as described above, and 15 days later, they were orally treated with 50 μg of MOG-pσ1. Twenty-four h later, spleens and LNs were combined and sorted, as indicated above. 1×105 CD11c+NK1.1+ MHC class IIhigh IKDCs or CD11c+NK1.1+ MHC class IIlow cells were adoptively transferred i.v. into C57BL/6 mice already MOG35–55-challenged 7 days earlier. NIH-PA Author Manuscript 2.11. Proliferation assay Briefly, 1 × 105 purified OT-II CD4+ T cells were stimulated with OVA323–339 (2.0 μg/ml) or OVA (1 mg/ml) in the presence of irradiated (3000 rad) NK cells, DCs, or IKDCs as a source of Ag-presenting cells for 4 days. When indicated, Ag-presenting cells were pretreated with B16F1 melanoma cells (ATCC, Manassas, VA) at a ratio of 10:1 or 10 μg/ ml of MOG-pσ1 for 30 min at 4°C. One μCi per well of [3H]-thymidine was added during the last 12 h of incubation. The cells were then harvested, and incorporated [3H]-thymidine was counted using a Beckman LS 6500 scintillation counter. 2.12. 51Chromium release assay Cytotoxic assays were performed using DCs, CD4+ T cells, or YAC-1 lymphoma cells (ATCC) as target cells. Briefly, 1 H 106 target cells were incubated with 50 μCi of 51Cr for 2 h at 37°C. Labeled cells were washed three times and then incubated with varying J Autoimmun. Author manuscript; available in PMC 2012 December 1. Huarte et al. Page 6 NIH-PA Author Manuscript numbers of effector cells. For concanamycin A (CMA; Wako Bioproducts, Richmond, VA) treatment, 100 nM of CMA was added to desired wells. After 4 h incubation, 100 μl of each supernatant was harvested and radioactivity counted using a Beckman LS 6500 scintillation counter. Specific lysis was calculated by measuring the level of released 51Cr from target cells corrected for spontaneous release/total 51Cr release obtained by detergent (5% Triton X-100) lysis × 100. Spontaneous lysis was measured from wells containing only labeled target cell [42]. 2.13. Statistical analysis The ANOVA followed by posthoc Tukey test was applied to show differences in clinical scores in treated versus PBS mice. The student t test was used to evaluate the differences between percentages of positive cells between groups, and P-values < 0.05 are indicated. 3. Results 3.1. MOG-pσ1 abates EAE, and this therapeutic effect is abrogated upon NK1.1 mAb depletion NIH-PA Author Manuscript Mucosal application of the fusion protein, MOG-pσ1, is effective against EAE in both prophylactic and therapeutic regimens. In fact, a single oral dose at the peak of disease resulted in significant 3-fold reduction in clinical scores within 24 h after treatment (Fig. 1A). This resolution in EAE was accompanied by 3-fold increase in the number of NK1.1+ cells in the spleens of treated mice (Fig. 1B). To investigate further if MOG-pσ1’s protective effect was mediated by NK1.1+ cells, mice were treated with a depleting PK136 mAb [24], starting at 0 or 7 days after EAE induction (Fig. 1C). Mice continued to receive weekly PK136 mAb treatments until day 21 p.ch, and NK1.1+ cell depletion significantly augmented EAE for either treatment. Furthermore, MOG-pσ1’s protective effect was abated in mice depleted of their NK1.1+ cells (Fig. 1D). These data confirmed previous reports that highlighted the relevance of NK cells for EAE clearance [25, 26] and indicated MOG-pσ1’s clinical benefits were, at least partially, dependent on NK1.1+ cells. 3.2. NK1.1 cell depletion abrogates MOG-pσ1-mediated recruitment of FoxP3+ Treg cells NIH-PA Author Manuscript Confirming our previous finding that MOG-pσ1 treatment induced an up-regulation in the number of FoxP3+ Treg cells in the spleens of treated mice (Fig. 1E and F; [22]), we questioned whether this increase was NK cell-dependent. Mice with EAE were NK1.1 mAbdepleted at the peak of disease (day 18) and, on day 20, were treated with MOG-pσ1 or PBS. Mice previously treated with the depleting mAb showed a reduced number of FoxP3+ CD25+CD4+ T cells in their LNs (Fig. 1G), while there was no effect on the number of Treg cells on the spleens (Supplementary Fig. S1). Moreover, CNS-infiltrating BM-derived cells showed a significantly lower percentage of FoxP3+ Treg cells in GFP-FoxP3 reporter mice depleted of NK1.1 cells when compared with PBS-treated mice (Fig. 1H), further implicating the significance for NK1.1+ cells or their subsets in Treg cell recruitment. 3.3. Induced Treg cells are MOG-specific Since conventional staining for MOG class II tetramers fails to detect low avidity Agspecific CD4+ T cells [43], a cell enrichment step [41] was performed to enable detection of MOG-specific Treg cells. Splenocytes from EAE mice (day 15 p.ch, score 1–2) were stained with OVA329–337 or MOG42–55 tetramers prior to magnetic purification, and only the (column) bound fraction was stained with MOG42–55 tetramer (Fig. 2A). Subsequent analysis of splenocytes and CNS-infiltrating lymphocytes from severely sick (day 18, score 3–4) FoxP3-GFP reporter mice, the majority of CNS-infiltrating FoxP3+ CD4+ T cells in the bound fraction were tetramer-specific (Fig 2B-C), as opposed to CD8+ T cells (data not J Autoimmun. Author manuscript; available in PMC 2012 December 1. Huarte et al. Page 7 NIH-PA Author Manuscript shown). Thus, MOG-pσ1 treatment enhanced the absolute number of tetramer-specific CNSinfiltrating Treg cells (Fig. 2C). Moreover, most of the FoxP3+ tetramer-specific SCinfiltrating lymphocytes appeared activated since these were CD69+ (Fig. 2D), unlike naive tetramer-negative CD25+CD4+ T cells (Fig. 2D). Thus, this analysis demonstrates MOGpσ1 treatment enhances the recruitment of MOG-specific Treg cells into the CNS. To determine the responsible cytokines induced by MOG-pσ1, cytokine analyses were performed on purified splenic CD25−CD4+ and CD25+CD4+ T cells isolated at the peak of the clinical EAE (around day 20 p.ch) from mice treated with MOG-pσ1 or PBS 24h earlier. Treg cells from MOG-pσ1-treated mice showed significant elevations in IL-10 when compared to Treg cells from diseased PBS-treated mice (p < 0.01) with concomitant reductions in IFN- and IL-17 (p < 0.01; Fig. 2E). There were no significant changes in TGF- production (Fig. 2E). These results show MOG-pσ1-stimulated Treg cells protect via IL-10 with concomitant inhibition of IL-17and IFN- in a MOG-specific fashion. 3.4. MOG-pσ1 increases the number of CD11c+NK1.1+ cells NIH-PA Author Manuscript Further analysis of the NK cell phenotype revealed that MOG-pσ1 treatment of EAE mice enhanced the numbers of CD11c+NK1.1+ cells, but not conventional CD11c−NK1.1+ NK cells, in their spleens relative to PBS-treated mice (Fig. 3A). This population of cells expressed high levels of surface B220, MHC class II, and CD49b (Fig. 3A), as others have found [7, 8]. Stimulation with MOG35–55 showed these cells were IFN- + (Fig. 3A), again, in concordance with these original studies. Given that MOG-pσ1 treatment enhanced CD11c+NK1.1+ cells in diseased mice, we queried if MOG-pσ1 treatment in the absence of EAE could induce CD11c+NK1.1+ cells in naïve mice. While naïve mice showed almost no CD11c+NK1.1+ cells, oral MOG-pσ1 treatment augmented the presence of these cells in spleens and MLNs (Fig. 3B). However, this increase in CD11c+NK1.1+ cells was transient, and mice challenged with EAE 40 days earlier showed no increased numbers of this subset. Interestingly, EAE mice treated with MOG-pσ1 had negligible amount of CD11c+NK1.1+ cells in the MLNs (Fig. 3B), while ~1.0% of CD45Hi CD11b+ BM-derived cells infiltrating the CNS were CD11c+NK1.1+ cells versus ~0.3% found in the PBS-treated EAE mice (Fig. 3C). A modest increase in the percentage of CD11c+NK1.1+ in the bone marrow of EAE versus naive mice (p < 0.05) was also found, although MOG-pσ1 intervention failed to further increase this percentage (Supplementary Fig. S2). 3.5. CD11c+NK1.1+ cells are phenotypically compatible with IKDCs and produce IL-10 NIH-PA Author Manuscript To determine if the CD11c+NK1.1+ cells were indeed IKDCs, a more detailed phenotypic and functional analysis was performed. CD11c+NK1.1+ cells expressed elevated levels of both CD80 and CD86 in spleens (Fig. 4A, left panels) and MLNs (Fig. 4A, right panels). Strikingly, although SC-infiltrating CD11c+NK1.1+ cells were MHC class IIintermediate/low, MOG-pσ1 treatment induced a dramatic up-regulation of MHC class II (Fig. 4B). Both conventional CD11c− NK1.1+ NK cells and CD11c+NK1.1+ cells co-expressed the NK cell markers, NKG2D, NKG2A, and TRAIL (Fig. 4C and data not shown), while CD11c+NK1.1− DCs were negative for these NK molecules (Fig. 4C). Although MOG-pσ1 treatment appeared to have no effect on the expression of the stimulatory molecule, NKG2D, CNS-infiltrating CD11c+NK1.1+ cells in MOG-pσ1-treated mice showed reduced expression of the inhibitory molecule, NKG2A (Fig. 4D). However, since MOG-pσ1 treated animals showed increased numbers of SC-infiltrating IKDCs, the total number of NKG2A+ IKDCs was greater in MOG-pσ1-treated than PBS-treated mice (Fig. 4D). Interestingly, while DCs and conventional CD11c− NK1.1+ cells did not produce IL-10, approximately 50% of CD11c+NK1.1+ cells produced IL-10 two weeks after EAE induction (Fig. 4E). After MOG-pσ1 treatment, IL-10 production by NK1.1+ cells was restricted to the NKG2A+ subset (Fig. 4F). J Autoimmun. Author manuscript; available in PMC 2012 December 1. Huarte et al. Page 8 3.6. CD11c+NK1.1+ cells are functionally compatible with IKDCs and can present peptide Ags NIH-PA Author Manuscript To determine if the CD11c+NK1.1+ cells were functionally compatible with the definition for IKDCs, their cytolytic activity and Ag presentation capacities were analyzed. In addition to lysing NK cells’ targets, YAC-1 cells, the CD11c+NK1.1+ cells effectively lysed activated CD4+ T cells and mature DCs (Fig. 5A). Supporting this notion for IKDCs, their cytolytic activity was inhibited by > 50% in the presence of concanamycin A (CMA), indicating their mechanism of killing is perforin-dependent [44] (Fig. 5B). NIH-PA Author Manuscript To assess their capacity as Ag-presenting cells, CD11c+NK1.1− DCs, CD11c−NK1.1+ NK cells, or CD11c+NK1.1+ cells were isolated from naive mice, mice just starting to show clinical symptoms of EAE (day 12, score 1), or from severely sick mice (day 21, score 3). Subsequently, the individual subsets were pulsed with OVA or OVA323–339 to activate OTII CD4+ T cells. Although CD11c+NK1.1+ cells were able to present OVA323–339 peptide, they failed to induce strong OT-II cell proliferation when pulsed with intact OVA (Fig. 5C), unlike conventional DCs. However, the previously observed up-regulation of MHC class II molecules after MOG-pσ1 treatment (Fig. 4B) prompted us to investigate whether MOGpσ1 treatment had any effect on their ability to process and present Ag. Indeed, when the CD11c+NK1.1+ cells were incubated with OVA protein in the presence of MOG-pσ1, they were able to stimulate the proliferation of OT-II CD4+ T cells (Fig. 5D), albeit not as efficiently as conventional DCs isolated from the same mice, thus, fulfilling their functional definition as Ag-presenting cells. Similarly, treatment of CD11c+NK1.1+ cells with B16F1 melanoma cells prior to OVA incubation also enhanced specific CD4+ T cell proliferation, consistent with that previously reported [17]. Thus, the observed CD11c+NK1.1+ subset truly shares both phenotypical and functional characteristics of NK cells and DCs and consequently is considered IKDCs. 3.7. MOG-pσ1 treatment up-regulates HVEM expression by NK1.1+ cells NIH-PA Author Manuscript Since IKDCs were enhanced in the spleen and CNS following MOG-pσ1 treatment of EAE mice, we queried if MOG-pσ1 treatment had any impact upon the number of CNSinfiltrating IKDCs. Interestingly, while a significant proportion of NK1.1+ cells were positive for the co-stimulatory molecule HVEM, CD4+ T cells were mostly positive for the HVEM ligand, LIGHT [48,49] (Fig. 6A), while B220+ B cells were positive for the coinhibitory molecule BTLA (B and T lymphocyte attenuator; data not shown), another HVEM ligand [45]. Interestingly, B220+CD5+ regulatory B cells, but not B220+CD5− inflammatory B cells [46], were also LIGHT+ (Fig. 6B). Furthermore, a significant increase was found in the number of HVEM+ IKDCs (both in the SCs and spleens) of MOG-pσ1treated mice (Fig. 6C and D). A kinetic analysis of HVEM expression of splenic IKDCs following EAE induction showed a rapid up-regulation of HVEM, peaking around day 14. Such up-regulation of HVEM levels was not evident in IL-10−/− mice, although these mice showed aggravated disease (average maximum score 4.5 versus 2.5 in wt mice (data not shown), further suggesting the relevance of HVEM+ NK1.1+ cells in the resolution of EAE (Fig. 6D). Overall, these data suggest an important role for HVEM expression by IKDCs, either directly acting as an activator of IKDCs or indirectly as an inhibitor of BTLA/ LIGHT+ CD4+ T or B cells. Consequently, the observed up-regulation of HVEM by IKDCs after MOG-pσ1 treatment may provide a rationale for the observed clinical improvement. 3.8. HVEM+ IKDCs bind MOG-pσ1 To assess whether MOG-pσ1 directly interacts with IKDCs, we took advantage of a MOG/ GFP-pσ1 construct to perform a cell binding study. Splenocytes from naïve mice, mice with EAE (day 21, score 2–2.5), or mice recovering from EAE (day 40, score 1) were incubated with MOG/GFP-pσ1 or PBS. Interestingly, only HVEM+ NK1.1+ cells were able to bind J Autoimmun. Author manuscript; available in PMC 2012 December 1. Huarte et al. Page 9 NIH-PA Author Manuscript MOG/GFP-pσ1 protein. Consequently, NK1.1+ cells derived from day 21 EAE mice showed the greatest binding (61.5 ∀ 5.2 of total NK1.1+ cells vs. 6.3 ∀ 1.8 in the naïve group) (Fig. 7A and B). To assess whether MOG-pσ1 binding to HVEM+ NK1.1+ cells was mediated via MOG or pσ1, binding assays were performed in the presence of an anti-HVEM mAb (20 μg/ml), recombinant MOG, or increasing concentrations of recombinant pσ1 (20 and 100 μg/ml). Only pσ1 was able to significantly reduce the binding of MOG-pσ1 to the HVEM+ NK1.1+ cells (Fig. 7B), showing that, although HVEM is an activation marker on NK cells, it does not directly bind to MOG-pσ1. A GFP-pσ1 construct (lacking MOG) was also able to effectively bind to HVEM+ NK1.1+ cells, further confirming pσ1 responsible for the binding (data not shown). Further analysis of the subpopulation bound to MOG/GFP-pσ1 showed that CD11c+ NK1.1+ IKDCs, but not CD11c+NK1.1− DCs or CD11c−NK1.1+ NK cells, were able to bind MOG/GFP-pσ1 protein (Fig. 7C). Again, MOG-pσ1 binding was restricted to the HVEM+ subpopulation (Fig. 7C), further suggesting the importance of the HVEM+ IKDC population in the MOG-pσ1 associated clinical resolution of EAE. 3.9. Adoptive transfer of IKDCs protects against EAE NIH-PA Author Manuscript To further investigate the role of IKDCs in EAE, a FACS-sorting strategy was optimized to allow isolation of IKDCs from NK cells and DCs from MOG-challenged mice (day 15 p.ch). CD11c+NK1.1+ IKDCs were further subdivided into MHC class IIHi IKDCs or MHC class IIintermediate/low cells (Fig. 8A) [15,33]. As previously reported [17], about a third of the CD11c+NK1.1+MHC class IIHi subset was also CD11b+ in contrast to the CD11b− CD11c+ NK1.1+MHC class IIintermediate/low cells (Fig. 8B). Interestingly, the MHC class IIHi subset also expressed higher levels of CD80 than their MHC class IIintermediate/low counterparts (Fig. 8B). As expected, the vast majority of DCs was MHC class IIhigh, while CD11c−NK1.1+ (NK) cells were MHC class II-negative (Fig. 8C). When FACS-sorted activated MHC class IIhigh IKDCs versus unactivated IKDCs and were adoptively transferred into mice challenged with MOG35–55 7 days earlier, only MHC class IIHi IKDCs were able to significantly reduce EAE (Fig. 8D). Interestingly, mice adoptively transferred with unactivated IKDCs showed slightly more severe disease than the PBS control group during the first 20 days of the disease, and in fact they failed to resolve EAE (Fig. 8D). These data demonstrate MHC class IIHi IKDCs, not MHC class IIintermediate/low cells, are essential for dampening EAE, providing a clinical benefit mediated by MOG-pσ1 intervention. 3.10. Activated IKDCs up-regulate HVEM, CD107a, PDL-1, and granzyme B and act as Agpresenting cells NIH-PA Author Manuscript Since MOG-pσ1 exclusively bound to HVEM+ IKDCs (Fig. 7), we queried whether MHC class IIHi IKDCs overexpressed HVEM. Indeed, MHC class IIHi IKDCs showed significantly higher levels of HVEM when compared to MHC class IIintermediate/low cells (Fig. 9A), thus, providing a rationale for MOG-pσ1’s therapeutic effects. When IKDCs and NK cells were cultured with the NK target cell line YAC-1, a profound up-regulation in CD107a, PD-L1, and granzyme B, by the MHC class IIHi IKDCs, not NK cells, further suggested a critical role for IKDCs in EAE recovery (Fig. 9B and C). Interestingly, both PDL1 and CD107a levels were much higher for IKDCs than on NK cells (Fig. 9C). However, when IKDCs were cultured with YAC-1 cells in the presence of mAbs specific for either BTLA or HVEM, this up-regulation was abolished, again suggesting the protective role of IKDCs was mediated by this HVEM/BTLA interaction (Fig. 9C). To assess whether activated MHC class IIHi IKDCs were responsible for Ag presentation, as shown above (Fig. 4D), NK1.1+ CD11c−, NK1.1− CD11c+, NK1.1+ CD11c+ MHC class J Autoimmun. Author manuscript; available in PMC 2012 December 1. Huarte et al. Page 10 NIH-PA Author Manuscript IIHi, and NK1.1+ CD11c+ MHC class IIintermediate/low cells were purified from EAE mice (day18 p.ch), and cells were pulsed with OVA overnight prior to being co-cultured with OTII CD4+ T cells. Only conventional DCs (NK1.1− CD11c+) and activated IKDCs (NK1.1+ CD11c+ MHC IIHi) were able to induce a strong proliferation of CD4+ T cells (Fig. 9D), suggesting that only activated IKDCs (NK1.1+ CD11c+ MHC class IIHi) show the effector function required for protection against EAE. 4. Discussion NIH-PA Author Manuscript MS is a neurodegenerative disease of the CNS that involves infiltration of activated inflammatory cells, damaging both myelin and axons, and is the most prevalent cause of paralysis in young adults [32]. EAE, a rodent model for MS, shares many of the same features [47] of this neurodegenerative disease and is T cell-mediated. Although MS has been studied for more than 150 years, an effective and safe therapy for MS remains elusive [48]. However, IFN- 1a treatment has proven to be therapeutic for MS, enhancing both the number and activity of Treg cells and the percentage of CD56bright NK cells [32, 49], a subset associated with clinical remissions [50] and reduced brain MRI lesions [51, 52]. However, up to 50% of patients do not respond to type I IFNs [53]. Moreover, both EAE models and clinical trials have shown that IFN- treatment reduces clinical symptoms in Th1 cell-driven disease, but worsens Th17 cell-induced disease by a mechanism that remains unclear [54,55]. Altogether, the available data indicate the relevance of IFNs in MS and possibly the importance of these described IKDCs, a source of IFNs. Other therapies, such as glatiramer acetate [56, 57] or mAbs, Rituximab [58], Daclizumab [59] and Alemtuzumab [60], directed against CD20, CD25, and CD52, respectively, are currently under clinical investigation and have promising efficacy, although some safety concerns remain [48]. NIH-PA Author Manuscript IKDCs resemble both NK cells and DCs and were originally described as CD11cint B220+ NK1.1+ CD3− CD19− Gr-1− cells [7,8]. However, acceptance of this novel lymphoid subset as a specific entity has yet to be widely accepted, partially, because of its lack of a unique cell surface marker or a defined transcriptional factor. Consequently, reliance on shared phenotypic markers, as well as functional attributes, is required to characterize IKDCs. In this regard, IKDCs are found to exert potent anti-tumor activity through the secretion of IFN- and direct killing of tumor cells. Although some controversy remains over their Ag presentation capacity (and if consequently these truly constitute a separate lineage of DCs or a mere state of activation of NK cells), recent data [17] indicate that at least a subset of IKDCs (the CD11b+ fraction) is licensed by tumor cells to acquire DC-like function by gaining the ability to process and present Ags to naive T cells. Consequently, it was found IKDCs could present OVA or OVA323–339 to OT-II T cells, but, unlike conventional DCs, failed to stimulate strong proliferative CD4+ T cell responses to intact OVA. However, MOG-pσ1 treatment enhanced MHC class II levels on IKDCs, suggesting such treatment may enhance their ability to present Ag. Since B16F1 melanoma cells can stimulate CD11b+ IKDCs to process and present Ag [17], it was indeed found treatment of CD11c+NK1.1+ cells with either B16F1 or MOG-pσ1 significantly enhanced Ag presentation. This collective evidence shows that MOG-pσ1-induced IKDCs share both phenotypical and functional attributes of both DC and NK cells, suggesting these are truly IKDCs. A marked increase in the number of CD11c+NK1.1+ cells in MOG-pσ1-treated EAE mice was observed. These IKDCs (predominately B220+ CD11b+ CD49b+) produced IFNfollowing MOG35–55 restimulation, yet could also produce IL-10, a potent immunomodulatory cytokine. It has been previously reported that co-production of IFNand IL-10 by NK cells dampens inflammatory responses [61], further implicating the relevance of these cells in EAE remission. In addition, CD11c+NK1.1+ cells were also able J Autoimmun. Author manuscript; available in PMC 2012 December 1. Huarte et al. Page 11 NIH-PA Author Manuscript to efficiently lyse YAC-1 cells, suggesting these are NK cells, and also, albeit with a lower avidity, lyse activated CD4+ T cells and mature DCs, as previously reported [3]. While reduced expression of the NK cell inhibitory molecule, NKG2A, was observed after MOGpσ1 treatment, the total number of NKG2A+ IKDCs in the CNS was markedly enhanced, and IL-10 production was limited to these NKG2A+ cells. Given these findings, it is possible that IKDC cytolytic and regulatory activities may contribute to resolving EAE. NIH-PA Author Manuscript Upon evaluating the activation status of IKDCs, a dramatic up-regulation of HVEM was found after EAE induction, and these cells showed enhanced MOG-pσ1 binding, suggesting a possible relationship between HVEM expression and the capacity of IKDCs to become responsive to MOG-pσ1 treatment. HVEM belongs to the tumor necrosis factor receptor family and regulates host immune responses to various pathogens and autoiummune diseases upon interactions with one of several ligands. Aside from binding herpesvirus gD protein, HVEM is able to bind Ig superfamily members, BTLA and CD160, and TNF superfamily ligands, LIGHT and LTα [45]. Of these, HVEM interactions with BTLA and LIGHT are of especial interest, since LIGHT is expressed by the majority of CD4+ T cells and regulatory B220+CD5+ B cells [46] in our system, and virtually all of the B cells are positive for BTLA. Engagement of HVEM by trans interaction with BTLA or LIGHT has been reported to induce co-stimulatory signaling and promote cell survival via activation of NF-κB [62]and IFN- production and to increase anti-tumor activity of NK cells [63]. On the other hand, HVEM signaling through BTLA leads to inactivation mediated by SH2 domain-containing protein tyrosine phosphatases 1 and 2 [64]. Due to this dual capacity of delivering both costimulatory and inhibitory signals, HVEM is often considered a molecular switch of the immune system [45,65]. In our case, expression of HVEM by IKDCs may lead to inactivation of BTLA-expressing CD4+ T lymphocytes, as well as IKDC activation through interaction with LIGHT+ CD4+ T cells or BTLA-expressing B cells. As previously suggested, cis interactions between double positive HVEM and BTLA IKDCs may also be occurring [rev. in 45]. A significant proportion of regulatory B220+CD5+ B cells are also positive for LIGHT and therefore may be implicated in the activation of HVEM+ IKDCs. Additional studies are currently under way to further elucidate the complex role of HVEM/ BTLA/LIGHT in EAE. NIH-PA Author Manuscript This work also shows Treg cells play a critical role in the resolution of EAE following MOG-pσ1 treatment, thus, confirming our previous findings. Analysis of the CNSinfiltrating Treg cells showed these to be MOG-specific following staining with MHC class II tetramers, and to be more activated evident by the increased numbers of CD69+ Treg cells present in the CNS in MOG-pσ1-treated mice than in EAE diseased mice. More importantly, this beneficial impact by MOG-pσ1 is dependent on NK1.1+ cells, since mice treated with a depleting anti-NK1.1 mAb failed to recruit Treg cells and resulted in no clinical improvement. One possible explanation for this finding is the loss of DCs via cytolysis by MOG-pσ1-activated IKDCs, since cross-talk between DCs and Treg cells has been wellestablished [66]. IKDCs express high levels of NKG2A, which is consistent with this hypothesis [67]. Alternatively, there could be direct interaction between NK and Treg cells through BTLA/HVEM ligation or NK cell-dependent CCL22 secretion [68]. Additional studies are warranted to determine how cells interact to resolve autoimmunity. MHC class II levels in conjunction with NK1.1 and CD11c have previously been considered the only unambiguous marker for IKDCs [15,33]. Here we show MHC class IIHi CD11c+NK1.1+ activated IKDCs, but not the unactivated MHC class IIintermediate/low CD11c+NK1.1+ cells, are able to induce a strong CD4+ T cell proliferation and, more importantly, prevent EAE development after adoptive transfer into MOG-challenged mice, further indicating the relevance of this population of cells in the development of autoimmune disorders. Since activated IKDCs up-regulate HVEM, they are an obvious J Autoimmun. Author manuscript; available in PMC 2012 December 1. Huarte et al. Page 12 NIH-PA Author Manuscript candidate to mediate MOG-pσ1’s effects. Interestingly, IKDCs also up-regulate the effector molecules granzyme B, CD107a, and PD-L1. During the last few years, PD-1/PD-L1 interaction has emerged as a critical regulator of EAE and other autoimmune diseases [39,69]. The observed PD-L1 up-regulation may contribute to the clinical effect through the inhibition of PD-1+ encephalitogenic CD4+ T cells [69]. To the best of our knowledge, this is the first work in which IKDCs are demonstrated to be protective in autoimmune diseases, thus, opening the door to future clinical interventions. However, further studies are needed in order to unambiguously dissect all the multiple proposed mechanisms of action (IL-10 production, HVEM up-regulation, Treg cell recruitment, cytolytic activity, PD-1 ligation, and Ag presentation) and their relative clinical relevance. Supplementary Material Refer to Web version on PubMed Central for supplementary material. Acknowledgments NIH-PA Author Manuscript We thank the NIH Tetramer Core Facility for kindly providing us with MHC class II tetramers used in this study and Ms. Nancy Kommers for her assistance in preparing the manuscript. This work is supported by U.S. Public Health Service Grant R01 AI-078938 and, in part, by Montana Agricultural Station and U.S. Department of Agriculture Formula Funds. 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Mailloux AW, Young MR. NK-dependent increases in CCL22 secretion selectively recruits regulatory T cells to the tumor microenvironment. J Immunol. 2009; 182:2753–65. [PubMed: 19234170] 69. Schreiner B, Bailey SL, Shin T, Chen L, Miller SD. PD-1 ligands expressed on myeloid-derived APC in the CNS regulate T-cell responses in EAE. Eur J Immunol. 2008; 38:2706–17. [PubMed: 18825752] NIH-PA Author Manuscript NIH-PA Author Manuscript J Autoimmun. Author manuscript; available in PMC 2012 December 1. Huarte et al. Page 17 Highlights NIH-PA Author Manuscript • IKDCs are recruited to CNS subsequent MOG-pσ1 treatment of EAE mice. • IKDCs stimulate and recruit activated MOG-specific Treg cells to the CNS. • IKDCs are able to kill activated CD4+ T cells and mature dendritic cells. • IKDCs express elevated levels of HVEM, while CD4 T cells and B cells express LIGHT and BTLA • Adoptive transfer of MHC-IIhigh IKDCs confers protection against EAE. NIH-PA Author Manuscript NIH-PA Author Manuscript J Autoimmun. Author manuscript; available in PMC 2012 December 1. Huarte et al. Page 18 NIH-PA Author Manuscript NIH-PA Author Manuscript Fig. 1. NIH-PA Author Manuscript MOG-pσ1 reduces EAE by recruiting Treg cells in a NK1.1+ cell-dependent fashion. A. C57BL/6 mice were orally treated with 50 μg of MOG-pσ1 or with PBS 20 days after MOG35–55-induced EAE. MOG-pσ1-treated mice showed improved resolution of clinical disease 24 h after treatment. The average score of 5 mice per group is depicted. *P < 0.05 versus PBS-treated group. A representative experiment of 8 is shown. B. Naïve C57BL/6 mice (left) or those induced with EAE were treated 18 days later with PBS (center) or 50 μg of MOG-pσ1 (right), and the percentage of NK1.1 cells in the spleens was evaluated 24 h later. C. C57BL/6 mice were treated weekly with PK136 depleting mAb starting at the time of EAE induction or 7 days later and monitored for disease course. D. C57BL/6 mice, either depleted or not of NK1.1+ cells by PK136 mAb treatment, were then orally dosed with MOG-pσ1 or PBS at the time of EAE induction, and monitored for disease course: *P < 0.05 versus PBS. E and F. EAE was induced in GFP-FoxP3 reporter mice and 18 days later orally dosed with 50 μg of MOG-pσ1 or PBS. Twenty-four hours later, mice were sacrificed, and spleens were removed and cultured for 4 days in the presence of MOG35–55 and feeder cells before being analyzed by flow cytometry. F. The average percentage of splenic FoxP3+ CD25+CD4+ T cells is shown after treatment with PBS (open bar) or MOGpσ1 (black bar). G. LNs from C57BL/6 mice with EAE, depleted of NK1.1 cells (right panel), or not (left panel), were stained for CD4 and CD25 24 h after treatment with MOGpσ1. The number of CD25+CD4+ T cells is shown. H. Evaluation of FoxP3 expression by CNS-infiltrating BM-derived cells from GFP-FoxP3 reporter mice with EAE treated with PBS (left panel) or PK136 mAb (right panel) is shown. J Autoimmun. Author manuscript; available in PMC 2012 December 1. Huarte et al. Page 19 NIH-PA Author Manuscript NIH-PA Author Manuscript Fig. 2. NIH-PA Author Manuscript MOG-pσ1 treatment increases the number of MOG-specific Treg cells. A. Splenocytes from C57BL/6 EAE mice were incubated with APC-OVA329–337 (upper panels) or MOG42–55 tetramer (lower panels) and then reacted with magnetic anti-APC-beads. Magnetically bound (right panels) and unbound cell fractions (left panels) were then stained for CD4, CD11b, and CD45. A representative experiment of 3 is shown. B. Spleens and CNSinfiltrating lymphocytes from GFP-FoxP3 reporter mice were isolated, stained with APCMOG tetramer, and again divided into bound and unbound cell fractions. Histograms representing tetramer staining for both fractions are shown. C. Absolute number of MOGspecific Treg cells in the bound fraction of indicated groups is shown. *P < 0.05 versus PBStreated mice. D. SC-infiltrating lymphocytes were purified from GFP-FoxP3 EAE mice and separated into MOG-tetramer bound and unbound fractions. Cells were then stained for CD4, CD11b, CD45, and CD69, and the percentage of CD69+ tetramer-specific cells is shown for FoxP3+CD4+ and FoxP3−CD4+ T cells in the bound fraction. E. CD25+CD4+ and CD25−CD4+ T cells were purified from PBS- or MOG-pσ1-treated mice 15 days after EAE induction, and were restimulated in vitro with MOG35–55 in the presence of irradiated APCs. Cytokine levels (mean ∀ SEM from triplicate cultures) in culture supernatants were measured by cytokine-specific ELISAs 4 days after culture, and values are corrected for cytokine levels produced by unstimulated cells. *P < 0.05 versus PBS-treated mice. J Autoimmun. Author manuscript; available in PMC 2012 December 1. Huarte et al. Page 20 NIH-PA Author Manuscript Fig. 3. NIH-PA Author Manuscript MOG-pσ1 increases the infiltration of CD11c+NK1.1+ IKDCs. A. Lymphocytes from PBSor MOG-pσ1-treated EAE mice were isolated, restimulated with MOG35–55, and stained for expression of NK1.1, CD11c, MHC class II, B220, CD49b, and intracellular IFN- . *P < 0.05 versus PBS-treated mice. A representative experiment of three is depicted. B. Percentages of CD11c+NK1.1+ cells in spleens and MLNs of PBS- or MOG-pσ1-treated PBS-treated or EAE mice (3–5/group) are shown. Mice recovering from EAE (day 40) are also included. C. PBS- or MOG-pσ1-treated mice, as well as recovering mice (day 40 p.ch), and CD45Hi CD11b+ BM-derived cells were evaluated for percentage of CD11c+NK1.1+ cells infiltrating the spinal cord (SC). *P < 0.05 versus PBS-treated mice. A representative experiment of three is depicted. NIH-PA Author Manuscript J Autoimmun. Author manuscript; available in PMC 2012 December 1. Huarte et al. Page 21 NIH-PA Author Manuscript NIH-PA Author Manuscript Fig. 4. CD11c+NK1.1+ cells are phenotypically IKDCs, and the level of NKG2A decreases after MOG-pσ1 treatment. C57BL/6 mice were treated with MOG-pσ1 or PBS 20 days after induction of EAE, and 24 h later lymphocytes from spinal cord (SC), spleens, and LNs were purified and stained for analysis by flow cytometry. A. CD80 and CD86 staining was shown for DCs and CD11c+NK1.1+ cells in spleens (left panels) and MLNs (right panels); staining with an isotype control mAb is also shown (filled histogram). B. Levels of expression of MHC class II by SC-infiltrating CD11c+NK1.1+ cells are shown for PBS- and MOG-pσ1treated mice. C. Percentages of NKG2A+ and TRAIL+ NK cells, DCs, and CD11c+NK1.1+ cells obtained from spleens of EAE mice are shown. *P < 0.05, **P < 0.010 versus DCs. D. NKG2A expression is reduced in SC-infiltrating NK1.1+ cells following MOG-pσ1 treatment (data depicted are representative of 5 mice/group), but absolute number of CNSinfiltrating NKG2A+ IKDCs is overall enhanced. *P < 0.05 versus PBS-treated mice. E. At the peak of disease, intracellular IL-10 staining was analyzed for CD11c+NK1.1−, CD11c+NK1.1+, and CD11c−NK1.1+ subsets. F. IL-10 is predominantly produced by NKG2A+ IKDC subset induced by MOG-pσ1 treatment. Data are of 3 mice/group. NIH-PA Author Manuscript J Autoimmun. Author manuscript; available in PMC 2012 December 1. Huarte et al. Page 22 NIH-PA Author Manuscript Fig. 5. NIH-PA Author Manuscript NIH-PA Author Manuscript CD11c+NK1.1+ cells are functionally IKDCs and able to kill CD4+ T cells and DCs and stimulate the proliferation of OT-II cells. A. Splenic and LN CD11c+NK1.1+ cells from EAE mice were cell-sorted and cultured for 3 days in the presence of IL-2 before performing a 51Cr release assay against autologous CD3/CD28 activated CD4+ T cells, bone marrowderived DCs, or YAC-1 cells at the indicated effector: target (E/T) ratios. B. 51Cr release assay using CD11c+NK1.1+ effector cells against YAC-1 target cells in the presence of concanamycin A (CMA) or control media was performed at various E/T ratios. *P ≤ 0.05 versus CMA. C. DCs and NK cells, as well as CD11c+NK1.1+ cells, were cell-sorted from splenocytes obtained from C57BL/6 mice 10 or 20 days (clinical scores 1 and 3, respectively) after EAE induction and co-cultured with OT-II CD4+ T cells and OVA protein. As positive control, NK cells, DCs, and CD11c+NK1.1+ cells from naive mice were pulsed with OVA323–339 and compared to cultures stimulated in the absence of Ag. The mean of triplicate cultures ∀ SD is depicted and is representative from three experiments; *P < 0.05 depicts significant differences for OVA presentation by CD11c+ DCs versus CD11c+NK1.1+ cells. D. CD11c+NK1.1+ cells and DCs were cell-sorted from C57BL/6 mice at the peak of the disease and preincubated or not with B16F1 melanoma cells or MOG-pσ1 plus OVA protein before the co-culture with purified OT-II CD+ T cells. The mean of triplicate cultures ∀ SD representative of two experiments is shown; *P < 0.05, **P < 0.010 versus OVA-pulsed CD11c+NK1.1+ cells. J Autoimmun. Author manuscript; available in PMC 2012 December 1. Huarte et al. Page 23 NIH-PA Author Manuscript Fig. 6. NIH-PA Author Manuscript MOG-pσ1 treatment induces an up-regulation of HVEM in NK1.1+ cells. A. C57BL/6 mice were treated with MOG-pσ1 or PBS 20 days after EAE induction, and 24 h after treatment, spleens and SC cells were stained for CD4, NK1.1, HVEM, LIGHT, and BTLA. Percentages of infiltrating CD4+ T cells and IKDCs in the SC of PBS- or MOG-pσ1-treated mice are shown. Staining by an isotype control mAb is shown as filled histogram; * P ≤ 05. A representative experiment of three is depicted. B. EAE was induced in C57BL/6 mice, and 20 days later, splenic, LN, and SC lymphocytes were purified and analyzed by flow cytometry for CD5, CD19, HVEM, LIGHT, and BTLA expression. Gating strategy is shown on the left-hand side. Histograms and percentages of positive cells are shown on at the righthand side. Staining by an isotype control mAb is shown as filled histogram. A representative mouse out of five is shown. C. A histogram depicts HVEM levels for splenic NK1.1+ cells from naïve and PBS- or MOG-pσ1-treated EAE mice. A representative experiment of three is shown. D. Percentages of splenic CD11c+NK1.1+ IKDCs expressing HVEM at different time points after EAE induction are shown. Percentages of HVEM+ IKDCs from IL-10−/− mice at day 14 after EAE induction are also shown. *P < 0.05, **P ≤ 0.01 versus naive mice. NIH-PA Author Manuscript J Autoimmun. Author manuscript; available in PMC 2012 December 1. Huarte et al. Page 24 NIH-PA Author Manuscript NIH-PA Author Manuscript Fig. 7. HVEM+ IKDCs bind MOG-pσ1. A. Splenic and LN lymphocytes from naïve- or EAEchallenged mice 10 or 40 days earlier were tested for their ability to bind MOG/GFP-pσ1 following 4 days restimulation with MOG35–55 and IL-2. Negative control cells were stained with MOG-pσ1 instead of MOG/GFP-pσ1. Data depict cells gated on NK1.1+ cells (representative of four mice/group). B. Percentages of NK1.1 HVEM+ cells binding to MOG/GFP-pσ1 were assessed. Anti- HVEM mAb (20 μg/ml), recombinant MOG (20 μg/ ml), or recombinant pσ1 (20 and 100 μg/ml) was added when indicated. Each column represents the mean ± SD from 4 mice/group. **P ≤ 0.01 versus negative control (NC); ‡P < 0.01 versus d21 MOG/GPF-pσ1 staining. (C) Splenocytes from a mouse (representative of 3 mice) at the peak of the disease were gated on CD11c+ DCs, CD11c+NK1.1+ cells (IKDCs), and NK1.1+ CD11c− NK cells and analyzed for MOG/GFP-pσ1 binding and HVEM expression. NIH-PA Author Manuscript J Autoimmun. Author manuscript; available in PMC 2012 December 1. Huarte et al. Page 25 NIH-PA Author Manuscript Fig. 8. NIH-PA Author Manuscript Adoptive transfer of IKDCs protects against EAE. A. C57BL/6 mice were challenged with MOG and 15 days later treated with 50 μg of MOG-pσ1. Twenty-four hours later, mice were sacrificed, and their spleens and LNs were combined and stained with CD11c, NK1.1, MHC class II, and B220. CD11c+NK1.1+ cells were sorted based on their MHC class II expression. B. MHC class IIHi- and MHC class IIlow-sorted cells were analyzed for CD11b and CD80 expression. The purity of the sorting is shown on the inserted panels. C. CD11c+ DC and NK1.1+ cells were analyzed for B220 and MHC-class II expression. As expected, only CD11c+ cells expressed high levels of MHC class II. D. 1×105 CD11c+NK1.1+MHC class IIhi cells (activated IKDCs) or CD11c+NK1.1+MHC class IIlow (unactivated) cells were adoptively transferred (AT) into C57BL/6 mice previously induced with EAE 7 days earlier and monitored for disease course. Average score of five mice is shown. *P < 0.05, **P < 0.010 versus PBS. NIH-PA Author Manuscript J Autoimmun. Author manuscript; available in PMC 2012 December 1. Huarte et al. Page 26 NIH-PA Author Manuscript NIH-PA Author Manuscript Fig. 9. Activated IKDCs, but not NK cells, up-regulate HVEM, CD107a, and PDL-1. A. C57BL/6 mice were challenged with MOG, and 15 days later their splenocytes were analyzed for HVEM expression. Only CD11c+NK1.1+MHC class IIHi IKDCs, but not CD11c+NK1.1+MHC class intermediate/low cells, up-regulated HVEM expression. Left panel shows a representative histogram. Right panel shows the individual MFI values and the mean for 8 mice/group. B and C. IKDCs and NK cells were cultured in the presence or absence of YAC-1 cells and anti-BTLA and anti-HVEM mAbs, and CD107a, PD-L1, and granzyme B expression were measured by flow cytometry. YAC-1 cell co-culture increases CD107a degranulation and PD-L1 overexpression by IKDCs, and anti-BTLA or anti-HVEM mAb treatment blocks such up-regulation. **P ≤ 0.01 versus PBS-treated mice; †P <0.01 versus NK cells. D. NK1.1+ CD11c−, NK1.1− CD11c+, NK1.1+ CD11c+ MHC class IIHi, and NK1.1+ CD11c+ MHC class IIintermediate/low cells (2×104) were pulsed with OVA overnight and then co-cultured with 1×105 OT-II CD4+ T cells, and 3H-thymidine incorporation was measured after 3 days of culture. *P < 0.05, **P ≤ 0.01 versus negative control (NC) group (without Ag-presenting cells). NIH-PA Author Manuscript J Autoimmun. Author manuscript; available in PMC 2012 December 1. View publication stats