Th1 Polarization of CD4 T Cells by Toll-Like Receptor 3-Activated Human Microglia

J Neuropathol Exp Neurol Vol. 66, No. 9 Copyright Ó 2007 by the American Association of Neuropathologists, Inc. September 2007 pp. 848Y859

ORIGINAL ARTICLE

+ Th1 Polarization of CD4 T Cells by Toll-Like Receptor 3-Activated Human Microglia

Carolyn S. Jack, BSc, Nathalie Arbour, PhD, Manon Blain, Ute-Christiane Meier, PhD, Alexandre Prat, PhD, MD, and Jack P. Antel, MD Downloaded from https://academic.oup.com/jnen/article/66/9/848/2916936 by guest on 29 September 2021

INTRODUCTION Abstract Microglia are long-lived cells derived from bone Toll-like receptors (TLRs) are expressed by human microglia and marrow precursors that populate the CNS early during translate environmental cues into distinct activation programs. We development. Because of their innate immune cell character, addressed the impact of TLR ligation on the capacity of human namely their capacity to express antigen-presenting and + microglia to activate and polarize CD4 Tcellresponses.As costimulatory molecules (1Y4), microglia have the potential microglia exist under distinct states of activation, we examined both to regulate T cell activation in the brain. Reactivation of ramified and ameboid microglia isolated from adult and fetal CNS, self-specific T cells within the CNS upon local autoantigen respectively. In vitro, ligation of TLR3 significantly increased major encounter is increasingly recognized as central to both histocompatibility complex and costimulatory molecule expression multiple sclerosis (MS) and its animal model, experimental on adult microglia and induced high levels of interferon->, autoimmune encephalomyelitis (5Y7). Brain-infiltrating interleukin-12p40, and interleukin-23. TLR4 and, in particular, CD4+ T cells require major histocompatibility complex TLR2 had a more limited capacity to induce such responses. (MHC) class II antigen presentation by in situ antigen- + Coculturing allogeneic CD4 T cells with microglia preactivated presenting cells (APCs). During the course of neuroinflam- with TLR3 did not increase T cell proliferation above basal levels but mation these APCs may also include macrophages and F consistently led to elevated levels of interferon- secretion and Th1 populations of dendritic cells (DCs); however, the functional polarization. Fetal microglial TLR3 responses were comparable; in role that human microglia can play with regard to T cell contrast, TLR2 and TLR4 decreased major histocompatibility population expansion and/or induction of cytokine and + complex class II expression on fetal cells and reduced CD4 Tcell chemokine secretion remains ill defined. In particular, the proliferation to levels below those found in untreated cocultures. All capacity for human microglia to respond to environmental 3 TLRs induced comparable interleukin-6 secretion by microglia. activation signals and to subsequently regulate T cell Our findings illustrate how activation of human microglia via TLRs, activation in the CNS has not been clarified. particularly TLR3, can change the profile of local CNS immune Human microglia in normal-appearing white matter + responses by translating Th1 polarizing signals to CD4 Tcells. have been found to express high levels of MHC as well as Key Words: CNS, Cytokines, Interferon-F, Innate immunity, costimulatory molecules, particularly with respect to their Multiple sclerosis. rodent counterparts raised under clean laboratory conditions (8). Adult microglia are highly ramified and generally thought to exist in a resting state, although this state has recently been shown to involve dynamic branch process motility in rodents (9). In contrast, the macrophage-like, phagocytic, or scavenger Bamoeboid[ microglia found in the From the Neuroimmunology Unit (CSJ, MB, JPA), Montreal Neurological developing human brain represent a reactive state; further- Institute, McGill University, Montreal, Quebec, Canada; Neuroimmu- more, this state of activation is also found at sites of CNS nology Research Laboratory (NA, AP), Research CenterYCentre Hospitalier Universitaire de Montreál, University of Montreal, Montreal, damage in the adult brain (10). The gamut of microglial Quebec, Canada; and Neuroimmunology Group, Department of Neuro- activation states is increasingly recognized to extend beyond science (U-CM), Institute of Molecular and Cellular Science, Barts and these 2 polar extremities and to encompass a broad range of The London, Queen Mary’s School of Medicine and Dentistry, London, tailored responses to signaling inputs. Toll-like receptors England. (TLRs) have emerged as central players in the recognition Send correspondence and reprint requests to: Jack P. Antel, MD, Neuro- immunology Unit, Room 111, Montreal Neurological Institute, McGill of, and response to both stranger and danger signals by University, Montreal, QC, Canada; E-mail: [email protected] APCs, including microglia (11Y13). Each TLR recognizes a This work was funded by the Canadian Institutes of Health Research. C.S.J. distinct class of ligands and can lend specificity to the innate is supported by a studentship from the Multiple Sclerosis Society of immune cellular response by inducing distinct signaling Canada (MSSC). N.A. holds a Senior Research Fellowship from the Y Canadian Institutes of Health Research. A.P. holds a Donald Paty career pathways (14 16). TLR3 signaling uses the adaptor molecule award from the MSSC and is a junior scholar from the Fonds de la TRIF (15, 16), whereas TLR2 requires MyD88, and TLR4 is Recherche en Sante´ du Que´bec. unique in that it can use both sets of adaptor-dependent

848 J Neuropathol Exp Neurol Volume 66, Number 9, September 2007

Copyright @ 2007 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited. J Neuropathol Exp Neurol Volume 66, Number 9, September 2007 Microglial TLR3-Mediated Th1 Polarization pathways. TRIF signaling can lead to the production of type I Nonadherent oligodendrocytes were washed off, and the interferon (IFN). remaining adherent microglia were plated and used within Most studies on autoimmune responses in MS have 2 to 5 days. Our previous studies have shown that our in + involved IFN-F-secreting CD4 T helper (Th)1 cells (17); vitro cultures of microglia have minimal contaminating cells + however, the newly identified Th17 CD4 T cell subset has (G5% and usually G1%), as determined using immunocyto- been shown to have a central pathogenic role in the animal chemistry (20, 21). This small percentage of cells is generally model, experimental autoimmune encephalomyelitis (18). found to be of the astrocytic lineage (glial fibrillary acidic + The polarization of infiltrating CD4 T helper cells into Th1 protein-positive). In our fluorescence-activated cell sorter or Th17 cells is contingent on local APC-provided cytokines, analysis 85% to 90% of cells were determined to be microglia such as interleukin (IL)-12 for Th1 and transforming growth based on CD14 or CD68 staining (as described below). We factor-A, IL-6, and IL-23 for Th17. The role for type I IFN in did not detect a distinct population of myeloid cells bearing high maturing APCs and in driving Th1-type responses is also markers of either perivascular microglia (CD45 ,MHC increasingly recognized. Thus, the capacity for microglia to class IIhigh) or DCs; however, we cannot rule out the Downloaded from https://academic.oup.com/jnen/article/66/9/848/2916936 by guest on 29 September 2021 sense and then translate CNS environmental cues to possibility that there may be a small percentage (1%Y2%) T lymphocytes is important in inducing not only the of these cells present. expansion of cell populations but also CD4+ T cell polarization to phenotypes with pathogenic potential. Isolation and Characterization of Human We have previously shown that human microglia Fetal Microglia mount distinct activation programs tailored to environmental Human CNS tissue (cerebral hemispheres) from cues via TLRs (11). In particular, TLR3 has the greatest fetuses at 18 to 23 weeks of gestation was obtained from capacity to induce the Th1-polarizing cytokine IL-12p70 the Human Fetal Tissue Repository (Albert Einstein College (11). This study addresses the functional consequence to of Medicine, Bronx, NY), following Canadian Institutes cells of the adaptive immune system downstream of TLR- for Health Research-approved guidelines. As previously mediated polarization of human microglial responses. We described, fetal CNS was dissociated with trypsin and demonstrate the different capacities for TLRs 2, 3, or 4 to DNase I followed by mechanical dissociation (22). After regulate antigen-presenting and costimulatory molecule washing, the cell suspension was plated at a concentration of expression, cytokine secretion profiles of microglia, and the 5to10Â 106 cells/mL in flasks in Dulbecco_s modified + consequent activation and polarization of CD4 T cells. We Eagle_s medium supplemented with 5% FCS, antibiotics, find that TLR-mediated regulation of MHC expression and glutamine, and glucose. The media was changed after 1 T cell proliferation differs between adult ramified and fetal week of culture; after 2 weeks, floating ameboid microglia ameboid microglia, in particular after TLR2 and TLR4 were harvested by removal of the supernatant after the gentle ligation. TLR3-mediated activation of both ramified and shaking of the flasks. Microglia were then cultured at 2.5 Â ameboid microglia was consistently found to have the 105 cells/mL in snap-cap round-bottom tubes (Becton Dick- greatest capacity to translate Th1-polarizing signals to inson Biosciences, Mississauga, ON, Canada). CD4+ T helper cells. Microglial Activation with Toll-Like Receptor Ligands for Functional Assays MATERIALS AND METHODS Poly(inosinic acid):poly(cytidylic acid) (PIC), a synthetic double-stranded RNA, (50 Kg/mL, Amersham Phar- Isolation and Characterization of Human macia Biotech), lipopolysaccharide (LPS) from Escherichia Adult Microglia coli serotype 0127:B8 (100 ng/mL, Sigma, Oakville, ON, Normal-appearing white matter was obtained from Canada), and palmityl-3-cysteine-serine-lysine-4 (PAM) young adult patients undergoing partial temporal lobe (100 ng/mL) (Invitrogen, San Diego CA), synthetic lipo- resection for nontumor/noninfection-related intractable epi- protein, were used as ligands for TLR3, TLR4, and TLR2, lepsy. The protocol was approved by an institutional review respectively, to stimulate microglia. For allogeneic coculture board according to the guidelines of the Canadian Institutes with CD4+ T cells, adult ramified microglia were plated in for Health Research. Primary microglia were isolated as 24-well plates (2.5 Â 105, 1.25 Â 105, or 6.25 Â 104 cells/ previously described, on the basis of the differential mL/well), and fetal ameboid microglia were cultured in adherence of glial subtypes to tissue culture plates (19). snap-cap round-bottom polypropylene tubes (2.5 Â 105 cells/ Briefly, brain tissue was dissociated enzymatically with mL/well). Microglia were preactivated for 24 or 48 hours in trypsin and DNAse (both from Invitrogen, Burlington, ON, the presence of the TLR ligands above. Canada) and mechanically by passage through a nylon mesh. Levels of Interleukin-12p40, Interleukin-23, A mixed glial cell suspension (oligodendrocytes and micro- > glia) was obtained by separation on a 30% Percoll gradient and Interferon- in Microglial Supernatants or (Amersham Pharmacia Biotech, Montreal, QC, Canada). Interferon-F, Interleukin-5, and Interleukin-6 The mixed cell population was cultured for 2 subsequent in Microglia overnight periods in tissue culture flasks in minimal essential T cell coculture supernatants, were assessed using medium supplemented with 5% fetal calf serum (FCS), the appropriate ELISA kits (BD Biosciences, Mississauga, antibiotics, L-glutamine and glucose (all from Invitrogen). ON, Canada) according to the manufacturer’s directions.

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Plates were read using a Multiscan EX 96-well plate 40 cycles of 15 seconds at 95-C and 1 minute at 60-C) in a reader (Thermo Labsystems, Franklin, MA) at a 450-nm volume of 25 KL with 1Â TaqMan Universal Master Mix wavelength. (Applied Biosystems). Primers and TaqMan FAM-labeled MGB probes for p40, p35, and p19 (Assays-on Demand), as Flow Cytometric Analysis of well as the primers and VIC/TAMRA probe for 18S, were Microglial Maturation from Applied Biosystems. Amplification of 18S was used as an endogenous control to account for variability in the amount Ramified adult microglia were detached using trypsin- of RNA transcribed as described previously (11). mRNA EDTA (Sigma); ameboid fetal microglia were obtained by quantification was extrapolated from standard curves, gen- pipetting from snap-cap round-bottom polypropylene tubes. Fc receptors were blocked overnight with normal mouse erated by making serial 10-fold dilutions of cDNA, and plotting C immunoglobulins at 3 Kg/KL (Dako Diagnostics Canada, T versus arbitrary levels of input cDNA. Mature human monocyte-derived DCs, a cell type found to have Mississauga, ON, Canada) and 10% human serum (Sigma) Downloaded from https://academic.oup.com/jnen/article/66/9/848/2916936 by guest on 29 September 2021 high expression of p19, p35, and p40, were generated in PBS supplemented with 1% FCS and 0.1% sodium azide + (Sigma). The following anti-human monoclonal antibodies according to published protocols (23, 24). Briefly, CD14 from BD Biosciences were used to characterize microglial cells isolated from peripheral blood mononuclear cells were maturation: fluorescein isothiocyanate (FITC)-conjugated cultured with granulocyte-macrophage colony-stimulating anti-HLA-DRDPDQ (all MHC class II molecules), APC- factor (100 ng/mL) and IL-4 (20 ng/mL) for 6 days, conjugated anti-HLA-DR (MHC class II), phycoerythrin followed by a 24-hour stimulation with LPS (100 ng/mL) before RNA extraction. cDNA generated from these 93% (PE) or PECy5-conjugated anti-HLA-ABC (all MHC class I j to 95% pure mature DCs (CD14 , CD83+, MHC class IIhigh, molecules), PE- or PECy5-conjugated anti-CD80, PE- or high + + APC-conjugated anti-CD86, and FITC-conjugated anti- MHC class I , CD80 , and CD86 ) was used to generate CD40. FITC-conjugated anti-CD14 and/or FITC-conjugated all standard curves. Thus, mRNA expression is expressed anti-CD68 (BD Biosciences) were used to verify microglial as arbitrary units of the gene in question (p19, p35, or purities. Isotypes matched for concentration of the primary p40) normalized to 18S, relative to expression in human antibodies were used for all stainings, under each condition. monocyte-derived DCs. For intracellular staining, microglia were fixed and permea- bilized in 4% (w/v) paraformaldehyde (Sigma) with 0.1% Isolation and Labeling of Human (w/v) saponin (Sigma) in Hanks_ balanced salt solution CD4+ T Lymphocytes (Invitrogen) and then incubated with antibody in 0.1% (w/v) CD4+ T lymphocytes were isolated from venous saponin, 1% fetal bovine serum, and 0.1% (w/v) NaN3 PBS, blood drawn from normal consenting donors in agreement followed by 2 washes and finally resuspended in 1% (v/v) with institutional guidelines, as described previously (25). fetal bovine serum and 0.1% (w/v) NaN3 PBS. Cells were Briefly, peripheral blood mononuclear cells were isolated acquired on a FACS Calibur (Becton Dickinson) and using Ficoll-Hypaque (Amersham Pharmacia Biotech, analyzed using FlowJo software (Treestar, Ashland, OR). Uppsala, Sweden) density gradient centrifugation, and CD4+ Delta mean fluorescence intensity (MFI) was determined T lymphocytes were subsequently purified using MACS as the difference between the MFI of the specific stain and (Miltenyi Biotec, Toronto, ON, Canada) anti-CD4+ beads the MFI for the appropriate isotype control in the same (95%Y97% pure CD4+CD3+ cells, as assessed by flow sample. cytometry). The cells were subsequently labeled using carboxyfluorescein succinimidyl ester (CFSE) (Molecular RNA Isolation, Reverse-Transcription, Probes [now Invitrogen]) in serum-free media for 10 minutes and Quantitative Real-Time Polymerase at 37-C. CFSE was quenched with FCS, cells were washed Chain Reaction twice, and CFSE incorporation was checked by light micros- copy. CFSE-labeled CD4+ T cells were plated at 1 million Total RNA was isolated from adult microglia after cells/mL/well of 24-well plates in RPMI 1640 supplemented lysis with TRIzol (Invitrogen) using the Qiagen RNeasy with 10% FCS, antibiotics, and glutamine and cocultured Mini Kit (Qiagen, Mississauga, ON, Canada) according to for 6 days with preactivated microglia. T cells were then the manufacturer_s instructions. For microglia, Qiagen harvested by vigorous pipetting and surface stained using Minelute columns were used to concentrate RNA in a small anti-human PE-conjugated CD3 and APC-conjugated anti- volume (12 KL). All RNA samples isolated were treated CD4 (BD Biosciences) and analyzed for CFSE dilution by with DNase (Qiagen). Total RNA (G3 Kg) was used for flow cytometry, performed as described above. reverse transcription. Briefly, cDNA was generated using random hexaprimers (Roche, Laval, QC, Canada) with the Moloney murine leukemia virus-RT enzyme (Invitrogen) at Cell Death Assays 42-C. Expression levels for p40, p35, and p19 were CD4+ cell death was determined by flow cytometry determined by quantitative polymerase chain reaction after 7-amino-actinomycin D (7AAD) (BD Biosciences) (qPCR) using the ABI PRISM 7700 Sequence Detection staining of unfixed CD4+ T cells isolated after 6 days in System (Applied Biosystems, Foster City, CA). Cycling allogeneic cocultures. Percent cell death was quantified as was performed according to default temperature settings the percentage of 7AAD+ CD4+ cells and expressed as fold (2 minutes at 50-C and 10 minutes at 95-C, followed by increase in cell death over the untreated control cocultures.

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Copyright @ 2007 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited. J Neuropathol Exp Neurol Volume 66, Number 9, September 2007 Microglial TLR3-Mediated Th1 Polarization Downloaded from https://academic.oup.com/jnen/article/66/9/848/2916936 by guest on 29 September 2021

FIGURE 1. Toll-like receptor (TLR) signaling increases surface major histocompatability complex (MHC) class I and II expression on human adult ramified microglia. Primary cultures of microglia isolated from adult brain were exposed for 24 hours to poly(inosinic acid):poly(cytidylic acid) (PIC) at 50 Kg/mL (TLR3), lipopolysaccharide (LPS) at 100 ng/mL (TLR4), and palmityl-3- cysteine-serine-lysine-4 (PAM) at 100 ng/mL (TLR2), and surface expressions of MHC class II (A, B) and MHC class I (C) were determined by flow cytometry. (A) Representative staining showing the shift in mean fluorescence intensity (MFI) for MHC class II over isotype control (CTRL) for 1 donor under basal (untreated, first panel), PIC (second panel), LPS (third panel), or PAM- treated conditions (last panel). (B) Pooled data from 4 different donors demonstrating surface MHC class II upregulation above baseline levels after incubation with TLR ligands (CTRL vs PIC, p = 0.07; CTRL vs LPS, p = 0.07). (C) Pooled MFI data from 4 donors for MHC class I upregulation (CTRL vs PIC, p = 0.02; CTRL vs LPS, p = 0.07).

Statistical Analyses expression data obtained from 4 different human microglial Data handling and analysis (Student t-test) were per- donors are presented in Figure 1B; the large error bars formed using Prism 3.0 (GraphPad Software, San Diego, CA). are indicative of a wide range in basal expression among donors. The pooled data demonstrate a strong trend toward TLR3- and TLR4-mediated upregulation of surface MHC RESULTS class II expression, whereas TLR2 signaling was relatively less potent (Fig. 1B). A similar pattern of upregulation Toll-Like Receptor Signaling Increases Major was observed for MHC class I, as shown in Figure 1C Histocompatability Complex and Costimulatory (control [CTRL] vs PIC, p = 0.02; CTRL vs LPS, p = 0.07; Molecule Expression on Adult Microglia n = 4 donors). The TLR-mediated MHC upregulation As increased expression of MHC and costimulatory profile at 48 hours was comparable to that seen at 24 molecules in the brain is strongly correlated with the hours, suggesting sustained TLR-ligation effects (data not presence of neuroinflammation in situ, we first sought to shown). determine whether TLR-mediated activation of human Basal expression of the costimulatory molecules CD80 microglia can upregulate the surface expression of such and CD40 was near the limit of detection on adult microglia, molecules. We used the synthetic double-stranded PIC, LPS, whereas CD86 was consistently expressed (Fig. 2). All and the synthetic triacylated lipoprotein PAM to ligate 3 costimulatory molecules were significantly upregulated by TLR3, TLR4, and TLR2, respectively, as each uses distinct TLR3 ligation: CD80 (Fig. 2A; CTRL vs PIC, p = 0.03, n = intracellular signaling pathways. Although surface MHC 3 donors), CD86 (Fig. 2B; CTRL vs PIC, p = 0.02, n = 3 class II expression on human adult microglia is relatively donors), and CD40 (Fig. 2C; CTRL vs PIC, p = 0.03, n = 3). high under basal conditions, TLR ligation can further Surface costimulatory molecule expression remained highly upregulate expression (Fig. 1A, B). Pooled MHC class II elevated downstream of TLR3 activation at 48 hours (data

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Copyright @ 2007 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited. Jack et al J Neuropathol Exp Neurol Volume 66, Number 9, September 2007 not shown). TLR4 signaling led to a trend toward CD80 and TLR2 pathway was comparatively less effective than that CD86 upregulation at 24 hours; however, in contrast to via the TLR3 or TLR4 at promoting the expression of both TLR3 signaling, this effect was not sustained to 48 hours MHC and costimulatory molecules on the human adult (Fig. 2A, B; data not shown). Overall, activation via the microglia.

Microglial Polarizing Cytokines Are Regulated by Toll-Like Receptor Signaling We have previously shown that microglial activation responses, including IFN-A mRNA expression and IL-12p70 secretion, are not homogeneous but rather can be tailored to the TLR signal (11). IL-12 and IL-23 share a p40 subunit; p40 dimerizes with either p35 to generate IL-12 or with p19 Downloaded from https://academic.oup.com/jnen/article/66/9/848/2916936 by guest on 29 September 2021 to generate IL-23. To dissect the ability for TLR signaling to regulate the expression of these molecules, qPCR analysis was conducted after the 6-hour exposure of microglia to the ligands for TLR2, TLR3, or TLR4 (Fig. 3). Microglial p35, p19, and p40 mRNA expression was quantified relative to expression in mature human monocyte-derived DCs, which are known to produce these cytokines. Any value greater than 1 (Fig. 3AYC) indicates expression above that in human DCs and vice versa. We found significantly higher levels of IL-12p35 mRNA downstream of TLR3 signaling (p = 0.02, n = 6 donors), with a 25-fold increase in the mean over baseline expression. LPS also induced IL-12p35 but to a lesser extent than the TLR3 ligand (p = 0.07; n = 6 donors; Fig. 3A). In contrast, TLR4 signaling was more effective than that of TLR3 at inducing IL-23p19 at the mRNA level, resulting in a 40-fold increase over basal expression (Fig. 3B; CTRL vs LPS, p = 0.03, n = 6 donors). Whereas both TLR3- and TLR2-induced p19 was higher than baseline (on average, 13- and 8-fold, respectively), the increase was not significant in the pooled analysis because of the range in expression levels among the human donors. Both TLR3 and TLR4 induced significant levels of the common p40 subunit at the mRNA level (Fig. 3C; CTRL vs PIC, p = 0.03; CTRL vs LPS, p = 0.002; n = 6 donors). We next investigated the expression of these polarizing cytokines at the protein level after 24-hour stimulation of microglia. In agreement with our results at the mRNA level, supernatants from TLR3- and TLR4-stimulated microglia were found to contain significant levels of p40, although TLR3 induced higher levels than TLR4, as shown in Figure 4A (CTRL vs PIC, p = 0.001; CTRL vs LPS, p = 0.02; n = 7 donors). In contrast to our mRNA results, secretion levels of IL-23 protein, as determined using an ELISA detecting the p19 subunit, were highest in response to TLR3 signaling (Fig. 4B, CTRL vs PIC, p = 0.02, n = 9 FIGURE 2. Toll-like receptor (TLR) signaling increases co- donors). TLR4 signaling induced significant but lower levels stimulatory molecule expression on adult microglia. Primary of the IL-23 protein (p = 0.01, n = 9) and there was a trend cultures of microglia isolated from adult brain were exposed toward increased IL-23 downstream of TLR2. to ligands for TLR3, TLR4, or TLR2 (poly(inosinic acid): We next investigated the secretion of type I interfer- K poly(cytidylic acid) [PIC] 50 g/mL, lipopolysaccharide [LPS] ons. IFN-A and select IFN-> genes such as IFN->4 are 100 ng/mL, and palmityl-3-cysteine-serine-lysine-4 (PAM) known to be triggered rapidly after TLR3 ligation and, in an 100 ng/mL, respectively) for 24 hours, and surface expression autocrine manner, boost the subsequent prolonged IFN-> of costimulatory molecules was determined by flow cytom- response. To substantiate our previous observations demon- etry, as described previously. Results are pooled delta mean A fluorescence intensity data from 3 donors: (A) CD80 (control strating TLR3-mediated IFN- mRNA expression (11), we [CTRL] vs PIC, p = 0.03); (B) CD86 (CTRL vs PIC, p = 0.02); measured IFN-> at the protein level in culture supernatants (C) CD40 (CTRL vs PIC, p = 0.03). *, p G 0.05. from microglia exposed to TLR ligands for 24 hours. As

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FIGURE 3. Differential increase of interleukin (IL)-12p35, IL-12p40, and IL-23p19 mRNA expression in human adult microglia after Toll-like receptor (TLR) ligation. Primary cultures of adult microglia were activated for 6 hours with FIGURE 4. Modulation of polarizing cytokine secretion TLR ligands (poly(inosinic acid):poly(cytidylic acid) [PIC], proﬁles downstream of Toll-like receptor (TLR) signaling in lipopolysaccharide [LPS], or palmityl-3-cysteine-serine-lysine- human adult microglia. Adult microglia were activated for 24 4 (PAM), as described previously). mRNA expression levels hours with TLR ligands (poly(inosinic acid):poly(cytidylic acid) were quantiﬁed by real-time polymerase chain reaction [PIC], lipopolysaccharide [LPS], or palmityl-3-cysteine-serine- relative to the endogenous reference 18S RNA. Data are lysine-4 (PAM), as described previously) at which time point expressed as arbitrary units and represent pooled data from 6 supernatants were collected. Frozen supernatants were sub- (control [CTRL], PIC, and LPS) or 4 (PAM) individual microglial sequently assayed for protein secretion by ELISA. (A) p40 donors. (A) Upregulation of p35 mRNA expression (CTRL vs secretion by TLR-activated microglia (control [CTRL] vs PIC, PIC, p = 0.02, CTRL vs LPS, p = 0.07). (B) Upregulation of p19 p = 0.001; CTRL vs LPS, p = 0.02; n = 7 donors). (B) IL-23 mRNA expression (CTRL vs LPS, p = 0.03). (C) Upregulation secretion (CTRL vs PIC, p = 0.02; CTRL vs LPS, p = 0.01; n = 9 of p40 expression (CTRL vs PIC, p = 0.03; CTRL vs LPS, p = donors). (C) IFN-> secretion (CTRL vs PIC, p = 0.02, n = 7 0.002). **, p G 0.01. donors).

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shown in Figure 4C, TLR3 induced high-level IFN-> production (792.2 T 245.4 pg/mL, p = 0.02, n = 7 donors), whereas this response was absent downstream of either TLR2 or TLR4 signaling.

Toll-Like Receptor-Mediated Activation of Microglia Modulates Functional CD4+ T Cell Responses We next tested the capacity for TLR-activated human microglia to enhance allogeneic CD4+ T cell proliferation in a 6-day CFSE-based assay. A representative histogram illustrating CD4+ T cell CFSE fluorescence after coculture Downloaded from https://academic.oup.com/jnen/article/66/9/848/2916936 by guest on 29 September 2021 with microglia is shown versus T cells alone in Figure 5A. The percentage of CFSE-low, proliferating CD4+ T cells pooled from multiple experiments (n = 4, n = 9, and n = 5 for microglia; T cell ratios of 1:16, 1:8, and 1:4, respectively) is shown in Figure 5B. Regardless of microglial TLR preactivation, CD4+ T cell proliferation levels were equiv- alent to those found in nonactivated cocultures; furthermore, such results were consistent at 3 different microglia/CD4+ T cell ratios, ruling out any alloantigen dose effect. Because activation can also lead to increased T cell death, we measured cell death in these allogeneic cocultures (Fig. 5C). Notably, pretreatment of human microglia with TLR3 led to significantly higher levels of CD4+ T cell death relative to untreated cultures, as determined by increased 7AAD incorporation (p = 0.02, n = 6). To determine whether TLR-triggered microglial activation profiles can subsequently polarize the adaptive cellular compartment, we tested for cytokines associated with distinct T cell subsets (i.e. IFN-F and IL-17) in the supernatant of allogeneic CD4+ T cells cocultured with preactivated microglia. In agreement with the observed capacity for signaling via the TLR3 pathway to mature microglia, preactivation with PIC was consistently found to + favor Th1 polarization after coculture with CD4 T cells (Fig. 6A; CTRL vs PIC, p = 0.04, n = 9). This increased IFN-F response was not paralleled by an increase in IL-5 FIGURE 5. Toll-like receptor (TLR) 3-mediated activation of secretion (Fig. 6B), reflecting a polarization of the cellular + human adult microglia does not boost allogeneic CD4 Tcell response toward that of a Th1-type. A trend toward increased proliferation but increases T cell death. Carboxyfluorescein Th2-type cytokine secretion was seen in TLR2 preactivated succinimidyl ester (CFSE)-labeled allogeneic CD4+ T cells were cocultures, on the basis of levels of IL-5 secretion (Fig. 6B: added to preactivated (for 24 hours, with TLR ligands as de- CTRL vs PAM, p = 0.09; PIC vs PAM, p = 0.07; n = 9) scribed previously) cultures of microglia and cocultured for and paralleled by levels of IL-13 secretion (CTRL, 48.5 T 6 days. T cell proliferation was determined by CFSE dilution 25.7 pg/mL [n = 4]; PIC, 17.1 T 14.7 pg/mL [n = 5]; LPS, using flow cytometry. (A) Representative histogram for gated T T + 30.6 22.7 pg/mL [n = 5]; and PAM, 136.6 51.3 pg/mL CD4 T cells, illustrating the shift in CFSE fluorescence for T cells [n = 5]; PIC vs PAM, p = 0.04). cocultured with microglia (thick black line) versus T cells alone (shaded). The gate determining the percentage of divided CD4+ Despite high levels of IL-23 secretion downstream of T cells is illustrated. (B) Pooled data illustrating the percentage TLR3 microglial activation we were unable to detect IL-17 of total T cells having undergone cell division in culture (CFSE- in the allogeneic coculture supernatant (data not shown). low), using 3 different microglia to T cell ratios (1:4, 1:8, and This finding may reflect the capacity for high levels of IFN-F 1:16; n = 4, n = 9, and n = 5, respectively). No significant in- to negatively regulate the production of this cytokine. crease in proliferation above control was observed under Signaling via all 3 TLRs led to a strong tendency + any TLR stimulation. (C) CD4 T cell death, as determined toward a significant increase in IL-6 secretion in the by 7-aminoactinomycin D (7-AAD) staining using flow cytom- cocultures (Fig. 6C; CTRL vs PIC, p = 0.05; CTRL vs etry in the same 6-day cocultures. The change in the percentage + + LPS, p = 0.07; CTRL vs PAM, p = 0.03), indicating that, in of 7AAD CD4 cells (relative to untreated cultures) is expressed contrast to the previously polarized responses, all 3 TLRs as fold/control (control [CTRL] vs PIC, p = 0.02, n = 6). induced the general activation of microglia.

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isolated from the human fetal brain, which exist in an Bameboid[ activation state, in contrast to the ramified nature of their adult counterparts. We first tested the capacity for TLR signaling to regulate the expression of MHC molecules on the surface of fetal microglia, cultured in round-bottom non-tissue culture-treated tubes to retain ameboid morphology. Fetal microglia are similar to their adult counterparts in their relatively high basal levels of MHC class II expression (adult microglia: mean delta MFI = 64.8 T 31.5, n = 9 donors; fetal microglia: mean delta MFI = 33.69 T 11.3, n = 11 donors). Fetal microglia also parallel adult microglia in the profile of their IL-6 response to TLR ligation (IL-6 secretion levels: baseline 1.8 T 1.5 ng/mL; TLR3, 19.7 T Downloaded from https://academic.oup.com/jnen/article/66/9/848/2916936 by guest on 29 September 2021 0.2 ng/mL; TLR4, 19.9 T 10.0 ng/mL; and TLR2, 17.3 T 7.0 ng/mL; n = 2 fetal donors) consistent with our previously reported findings with adult microglia (11). In contrast, the regulation of surface MHC class II expression on the fetal cells diverged from that of adult ramified microglia. Only TLR3 signaling led to an increase in MHC class II expression above basal levels at 24 hours (Fig. 7A; CTRL vs PIC, p = 0.07, n = 6 donors). Although elevated expression levels persisted for TLR3, ligation of either TLR2 or TLR4 actually led to significantly down- regulated MHC class II expression relative to baseline after a 48-hour treatment (Fig. 7A; CTRL vs LPS, p = 0.05; CTRL vs PAM, p = 0.04; n = 4). This decreased surface expression did not extend to MHC class I regulation; as shown in Figure 7A; increased MHC class I expression is evident downstream of all TLRs, both at 24 hours (CTRL vs PIC, p = 0.02; CTRL vs LPS, p = 0.05; n = 5 donors) and at 48 hours (n = 2 donors). Regulation of the costimulatory molecules CD80, CD86, and CD40 in fetal microglia was comparable to that observed on the adult cells (as shown previously in Fig. 2), as TLR3 ligation exclusively led to highly elevated and sustained levels of expression of these molecules (data not shown). We assessed the capacity of fetal microglia to stimulate allogeneic CD4+ T cells after TLR ligation using a CFSE FIGURE 6. Toll-like receptor (TLR) 3-activated human adult assay similar to that previously described, but conducting microglia can subsequently polarize allogeneic CD4+ T helper the studies in round-bottom tubes to conserve ameboid mor- cells. Carboxyfluorescein succinimidyl ester (CFSE)-labeled phology. Both TLR2- and TLR4-preactivated microglia allogeneic CD4+ T helper cells and 24-hour preactivated reduced T cell proliferation to levels below those found in microglia (TLR ligands, as described previously), were cocul- basal microglia-T cell cocultures (Fig. 7B). However, when tured for 6 days. T cell polarization was determined by Th1 polarization was tested in the cellular supernatants of measuring T cell cytokines (interferon [IFN]-F and interleukin these cocultures, we consistently found that TLR3-triggered [IL]-5) in the culture supernatant by ELISA. Microglial activa- microglial activation was uniquely able to induce a Th1-type tion was determined by measuring myeloid cytokine (IL-6) profile, leading to high levels of IFN-F (Fig. 7C) in a manner secretion. (A) Elevated IFN-F secretion (control [CTRL] vs poly(inosinic acid):poly(cytidylic acid) [PIC], p = 0.04, CTRL vs analogous to that found with the adult microglia. lipopolysaccharide [LPS], p = 0.1; n = 9). (B) IL-5 secretion (CTRL vs palmityl-3-cysteine-serine-lysine-4 [PAM], p = 0.09). DISCUSSION (C) Comparable IL-6 secretion downstream of all 3 TLRs TLR signaling can influence the acute innate inflam- (CTRL vs PIC, p = 0.05; CTRL vs LPS, p = 0.07; CTRL vs PAM, matory response as well as the consequent adaptive immune p = 0.03; n = 8). response (16, 26Y29). Recent findings have highlighted a role for TLRs in the activation of autoreactive T cells (30, 31) and the ability for TLRs to trigger the switch from Toll-Like Receptor-Mediated Polarization of autoreactivity to autoimmunity (32, 33). Further studies have Fetal Microglial Activation Programs emphasized the importance of T cell reactivation within the We next determined whether the capacity for TLR3 to target organ in determining clinical outcome of autoimmune induce a Th1-polarizing program extended to microglia inflammation (5, 6, 32). In this study, we demonstrate the

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FIGURE 7. Comparison of Toll-like receptor (TLR) 2, TLR3, and TLR4-mediated regulation of the antigen-presenting cell (APC) capacity of human fetal microglia and downstream effects on CD4+ T helper cells. (A, B) Purified cultures of microglia isolated from fetal brains were exposed in round-bottom polypropylene tubes (amoeboid morphology) to the ligands for TLR3, TLR4, or TLR2 (poly(inosinic acid):poly(cytidylic acid) [PIC] 50 Kg/mL, lipopolysaccharide (LPS) 100 ng/mL, or palmityl-3-cysteine-serine- lysine-4 [PAM] 100 Kg/mL, respectively) for 24 or 48 hours. Surface expression of major histocompatability complex (MHC) class II and MHC class I was determined by flow cytometry, as described previously. (A) MHC class II surface expression (24 hours: control [CTRL] vs PIC, p = 0.07, n = 6; 48 hours: CTRL vs LPS, p = 0.05; CTRL vs PAM, p = 0.04, n = 4); (B) MHC class I surface expression (24 hours: CTRL vs PIC, p = 0.02; CTRL vs LPS, p = 0.05). (C) Allogeneic carboxyfluorescein succinimidyl ester (CFSE)- labeled CD4+ T helper cells were added to preactivated microglia and cocultured for 6 days in round-bottom tubes. Decreased T cell proliferation is observed downstream of TLR4 or TLR2 stimulation, as assayed by CFSE dilution (CTRL vs LPS, p = 0.07; CTRL vs PAM, p = 0.08; p = 7). Six-day allogeneic coculture supernatants were tested by ELISA for levels of T cell interferon-F secretion. capacity for individual TLRs to trigger microglial activation sion varied from donor to donor, probably reflecting pre- programs that instruct divergent adaptive immune responses. vious environmental exposures. Here we have shown that We found that the upregulation of MHC and costimulatory the expression of these molecules can be further upregulated molecule expression and the profile of polarizing cytokine by TLR-triggered signaling in adult microglia. TLR signal- secretion by human adult microglia is TLR ligand-specific. ing can also enhance the expression of CD80, CD86, and TLR3 led to more significant and sustained increases in CD40. TLR3 induced highly elevated and sustained surface expression than either TLR2 or TLR4. The fetal ameboid expression of all the above molecules. TLR4 upregulated microglial response to signaling downstream of TLR2 and all but CD40 (Fig. 1), but the increased expression levels TLR4 was found to diverge from that of their ramified adult observed were not as persistent as those downstream of counterparts with respect to both MHC class II expression TLR3. In contrast, TLR2 was only minimally effective at and the subsequent induction of CD4+ T helper cell pro- enhancing surface expression of these molecules. liferation. In contrast, strong Th1-polarizing responses were We found that MHC molecule regulation in the adult consistently and preferentially induced by TLR3 signaling in microglia was distinct from that observed in the macrophage- both adult and fetal microglia. like fetal ameboid microglia. Whereas TLR3 ligation again Adult microglia isolated from normal-appearing white led to highly elevated and persistent MHC expression on fetal matter expressed relatively high basal levels of MHC class I microglia, prolonged (48 hours) TLR2 or TLR4 signaling led and II, as reported previously (3, 34Y37), and such expres- to significantly reduced levels of surface MHC class II

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Copyright @ 2007 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited. J Neuropathol Exp Neurol Volume 66, Number 9, September 2007 Microglial TLR3-Mediated Th1 Polarization relative to baseline expression (Fig. 7). MHC class I as well as TLR3-TRIF-mediated polarization of immune responses may the costimulatory molecules CD80, CD86, and CD40 were be due to direct signaling to the T cells themselves; however, regulated in a manner more analogous to that recorded for the type I IFN has also been found to play a role in boosting the ramified adult microglia (Figs. 2 and 6; data not shown). This IL-12 response, particularly before T cell feedback to APCs disparity between ramified and ameboid microglial MHC (47, 60). IL-12 production by DCs is ordinarily tightly class II mirrors the discrepant regulation of this same controlled, requiring a second signal via CD40 (24, 61), molecule in DCs versus macrophages (38); whereas TLR2 lending significance to our finding that TLR3 triggers a robust ligands have been shown to increase MHC class II expression polarization program in human microglia that includes the by DCs, the same ligands have been shown to inhibit IFN-F- production of IL-12 and the upregulation of CD40. dependent class II expression and antigen-processing activity We documented the capacity for TLR-mediated signal- of macrophages (26, 27, 29, 38Y44). ing in adult microglia to induce CD4+ T cell polarization TLR-mediated activation of adult microglia regulated using allogeneic cocultures. We have previously shown that the production of IL-12, IL-23, and type I IFN, all of which human adult microglia have the functional capacity to Downloaded from https://academic.oup.com/jnen/article/66/9/848/2916936 by guest on 29 September 2021 are cytokines known to contribute to T cell polarization support autologous T cell proliferative responses to recall (45Y47). Both TLR3 and TLR4 ligation induced significant antigens (3). Because of the limitations in obtaining autolo- levels of p40 mRNA, the common subunit of IL-12 and gous blood from patients corresponding to the CNS tissue IL-23; however, TLR3 triggered greater p40 protein accu- and in having an antigen to which all individuals will mulation. TLR3 signaling also led to high levels of IL-12p35 respond, the alloactivation system provides us with a mRNA, in line with our previous report showing TLR3- practical approach to monitoring the regulation of T cell triggered IL-12p70 secretion (11). Although TLR4 induced responses. In our assay system, we found no overall increase higher levels of IL-23p19 mRNA than TLR3 (Fig. 2), this in the levels of T cell proliferation at any T cell to microglia profile was not conserved at the protein level, as TLR3 led to ratio, as determined by CFSE, after preactivation of microglia higher levels of protein accumulation. This may reflect the with any of the TLR ligands tested (Fig. 5). However, fact that because of limited tissue availability, our analysis at preactivation with TLR3 led to a significant increase in T cell the mRNA level only consisted of a 6-hour snapshot, death, as determined by levels of 7AAD incorporation by highlighting the importance of analyzing the protein accu- CD4+ T cells cocultured with adult microglia, consistent with mulation of these key innate polarizing cytokines in human the observation that robust T cell activation can also lead to APC responses. In a recent study by Li et al (48), TLR4- increased T cell death (62, 63). Pre-exposure of adult mediated activation was the most effective means to induce microglia to PIC also led to significantly higher levels of in vitro IL-23 in microglia isolated from 8- to 10-week-old IFN-F secretion by the allogeneic CD4+ T cells (Fig. 6A). human fetal brains. The reason for the discrepancy with our Low-level Th2-type cytokine secretion (IL-5 and IL-13) was findings is unclear, but it may reflect either the different age observed in the TLR2-preactivated microglia-T cell cocul- of the material and/or the protocol used for isolation. This tures, which contrasts with TLR3 preactivated cocultures in same study showed that microglia/macrophages are a which Th2 responses were consistently absent. significant source of IL-23 in MS brain lesions. The capacity to increase T cell activation via polarized Type I IFN is another key polarizing cytokine linking cytokine secretion, unaccompanied by any increase in innate and adaptive immune responses. IFN->/A can aug- proliferation, parallels results reported by Napolitani et al ment APC function and upregulate costimulatory molecule (24) using human monocyte-derived DCs. We observed expression (27, 49, 50) as well as direct T cell polarization similar PIC-induced CD4+ T cell responses using fetal toward Th1-type responses (49, 51Y53).We have previously microglia. Although such dissociated responses (prolifera- reported that human adult microglia express high levels tion vs cytokine secretion) have also been described in of mRNA for IFN-A after exposure to the TLR3 ligand rodent microglia, this strong TLR3-mediated polarization PIC whereas, by comparison, TLR4 induces only a mini- response was not detected in these studies (24, 64). Despite mal signal (11). IFN->/A secretion occurs as a sequential high levels of IL-23 downstream of TLR3, no IL-17 was event; initially, the rapid phase response comprises primarily detected in our system using adult microglia. This may IFN-A (and IFN->4) (16, 54, 55). Autocrine/paracrine IFN-A reflect the inhibitory effect of such high levels of IFN-F (65). signaling subsequently induces interferon regulatory factor-7 TLR2- and TLR4-mediated activation, observed to signifi- activation, leading to the induction of the full gamut of IFN-> cantly downregulate MHC class II expression in the fetal genes (16). We report here that TLR3 induced a robust and microglia, only reduced CD4+ T helper cell proliferation significant secretion of IFN-> protein and that this response levels in the fetal microglia-T cell cocultures. In contrast, all was completely lacking downstream of both TLR2 and TLR4. 3 TLRs triggered higher level IL-6 secretion, again indicat- TLR4 has been previously reported to induce IFN-A but not ing the capacity for all 3 TLRs to induce generalized IFN-> in peripheral cells (16, 56). Signaling via TLR3 has microglial activation programs. been found to promote proinflammatory immune responses in In this study we examined TLR-mediated activation of various settings and, when compared with TLR4, has been microglia and polarization of the subsequent adaptive found to mediate a more potent antiviral response (57). immune response downstream of signaling induced by Furthermore, the capacity for TLR3 to activate and polarize exogenous ligands. As TLR3 is highly expressed in human immune responses is often found to be contingent on type I glial cells (11), endogenous TLR3 ligands, such as mRNA IFN signaling (32, 56, 58, 59). The role of type I IFN in the released from dying or damaged cells (15, 16, 66Y68) may

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Copyright @ 2007 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited. Jack et al J Neuropathol Exp Neurol Volume 66, Number 9, September 2007 also induce these polarizing immune responses. We 12. Olson JK, Miller SD. Microglia initiate central nervous system innate hypothesize that such a pathologic mechanism could occur and adaptive immune responses through multiple TLRs. J Immunol Y in human autoimmune and viral-mediated demyelinating 2004;173:3916 24 13. Bsibsi M, Ravid R, Gveric D, et al. Broad expression of Toll-like disorders. This study models the differential capacity for receptors in the human central nervous system. J Neuropathol Exp TLR ligands to modify microglial activation programs. Neurol 2002;61:1013Y21 Because double-stranded RNA is generated during the life 14. Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive cycle of many viruses, TLR3-mediated polarization of local immune responses. Nat Immunol 2004;5:987Y95 15. Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate CNS immune activation may be a contributing factor during immunity. Cell 2006;124:783Y801 CNS viral replication. One prevailing hypothesis for MS 16. Honda K, Taniguchi T. IRFs: Master regulators of signalling by is that a common infection may precipitate, trigger, or Toll-like receptors and cytosolic pattern-recognition receptors. Nat otherwise contribute to relapses. Viruses with neurotropic Rev Immunol 2006;6:644Y58 potential may generate ligands for microglial-mediated 17. Sospedra M, Martin R. Immunology of multiple sclerosis. Annu Rev Immunol 2005;23:683Y747 Downloaded from https://academic.oup.com/jnen/article/66/9/848/2916936 by guest on 29 September 2021 activation and polarization of brain immunity. Furthermore, 18. Bailey SL, Schreiner B, McMahon EJ, et al. CNS myeloid DCs additional endogenous ligands may exist, such as the presenting endogenous myelin peptides ’preferentially’ polarize CD4+ Y recently identified TLR3 protein agonist, stathmin (69). TH-17 cells in relapsing EAE. Nat Immunol 2007;8:172 80 Our finding that TLR3 signaling in microglia favors a 19. Yong VW, Antel JP. Culture of glial cells from human brain biopsies. In: Fedoroff S, Richardson E, eds. Protocols for Neural Cell Culture. polarized proinflammatory adaptive immune response raises Totowa, NJ: Humana Press, Inc., 1992:81Y96 the distinct possibility that endogenous TLR3 ligands could 20. Pouly S, Becher B, Blain M, et al. Expression of a homologue of rat potentiate neuroinflammation. NG2 on human microglia. Glia 1999;27:259Y68 21. Williams K Jr, Ulvestad E, Cragg L, et al. Induction of primary T cell responses by human glial cells. J Neurosci Res 1993;36:382Y90 ACKNOWLEDGMENTS 22. D’Souza S, Alinauskas K, McCrea E, et al. Differential susceptibility of The authors are grateful to Ellie McCrea, Igal Ifergan, human CNS-derived cell populations to TNF-dependent and independent immune-mediated injury. J Neurosci 1995;15:7293Y7300 and Hania Ke´bir for technical assistance and for contribu- + tions, both intellectual and technical, from Philippe Saikali, 23. Xia CQ, Kao KJ. Monocyte-derived CD1a dendritic cells generated in two different culture systems: Immunophenotypic and functional Veronique Miron, Cornelia Podjaski, and Peter Darlington. comparison. Scand J Immunol 2003;57:324Y32 The authors are also very grateful for the contributions of 24. Napolitani G, Rinaldi A, Bertoni F, et al. Selected Toll-like receptor Dr. AndreÓlivier and Dr. Jeffery Hall, who provided adult agonist combinations synergistically trigger a T helper type 1-polarizing human CNS tissues, and to Dr. Bradford Poulos, Director of program in dendritic cells. Nat Immunol 2005;6:769Y76 25. Seguin R, Moditi Z, Rotondo R, et al. 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