-Interacting Satellite Glial Cells in Human Trigeminal Ganglia Have an APC Phenotype

This information is current as Monique van Velzen, Jon D. Laman, Alex KleinJan, of September 28, 2021. Angelique Poot, Albert D. M. E. Osterhaus and Georges M. G. M. Verjans J Immunol 2009; 183:2456-2461; Prepublished online 27 July 2009;

doi: 10.4049/jimmunol.0900890 Downloaded from http://www.jimmunol.org/content/183/4/2456

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Neuron-Interacting Satellite Glial Cells in Human Trigeminal Ganglia Have an APC Phenotype1

Monique van Velzen,* Jon D. Laman,†‡ Alex KleinJan,§ Angelique Poot,* Albert D. M. E. Osterhaus,* and Georges M. G. M. Verjans2*

Satellite glial cells (SGC) in sensory ganglia tightly envelop the neuronal cell body to form discrete anatomical units. This type of glial cell is considered neuroectoderm-derived and provides physical support to neuron somata. There are scattered hints in the literature suggesting that SGC have an immune-related function within sensory ganglia. In this study, we addressed the hypothesis of human trigeminal ganglia (40 ؍ that SGC are tissue-resident APC. The immune phenotype and function of a large series (n (TG) were assessed by detailed flow cytometry, in situ analyses, and functional in vitro assays. Human TG-resident SGC (TG- SGC) uniformly expressed the common leukocyte marker CD45, albeit at lower levels compared with infiltrating T cells, and the markers CD14, CD68, and CD11b. In addition, TG-SGC expressed the myeloid dendritic cell (DC) marker CD11c, Downloaded from the T cell costimulatory molecules CD40, CD54, CD80, and CD86 and MHC class II. However, the mature DC marker CD83 was absent on TG-SGC. Functionally, TG-SGC phagocytosed fluorescent bacteria, but were unable to induce an allogeneic MLR. Finally, TG-infiltrating T cells expressed the T cell inhibitory molecules CD94/NKG2A and PD-1, and the interacting TG-SGC expressed the cognate ligands HLA-E and PD-L1, respectively. In conclusion, the data demonstrate that human TG-SGC have a unique leukocyte phenotype, with features of both and immature myeloid DC, indicating that they have a role as TG-resident APC with potential T cell modulatory properties. The Journal of Immunology, 2009, 183: 2456–2461. http://www.jimmunol.org/

ensory ganglia are part of the peripheral . geminal (TG), and reactivates intermittently (6). Re- They contain cell bodies of sensory establishing cent studies in mice and humans emphasized the importance of S the connection between the periphery and CNS. Sensory infiltrating T cells to control latent HSV infections in sensory ganglia lack a blood- barrier and enclose a high number of ganglia (7–9). Virus-specific T cells are directly juxtaposed to satellite glial cells (SGC)3 (1–3). SGC are considered to be neu- latently infected neurons, produce and cytolytic ef- roectoderm-derived and involved in the maintenance of sensory fector molecules, but do not induce neuronal damage (7, 8, neuron homeostasis by regulating extracellular ion and nutrient 10–12). Current data suggest that the neurons themselves or by guest on September 28, 2021 levels within sensory ganglia (2). In contrast to CNS-resident glial hitherto unrecognized resident cells in latently infected sensory cells, like and , SGC have a distinct interaction ganglia induce and coordinate this nonpathogenic chronic T cell with neurons (2, 3). They directly associate with the neuronal response (8, 10–12). , so that each neuronal cell body is completely surrounded by In this study, we addressed the hypothesis that SGC are tissue- a sheet of several SGC providing physical support and a protective resident APC. The availability of a series of fresh postmortem barrier (3). The numerous fine invaginations between the neuron human TG specimens enabled us to combine ex vivo and in situ and SGC sheath illustrate their intimate association (2, 3). Upon analyses for the phenotypic and functional characterization of hu- mechanical injury to sensory neurons, SGC undergo morphologi- man TG-resident SGC (TG-SGC). cal changes, proliferate, and up-regulate a variety of growth fac- tors, cytokines, and the glial marker glial fibrillary acidic protein Materials and Methods (2, 4, 5). Clinical specimens Human ␣-herpesviruses, like HSV, are a common threat to Heparinized peripheral blood and TG specimens, i.e., left and right TG, human sensory ganglia. HSV establishes a lifelong latent infec- were obtained from 40 subjects (median age 79 years, range 41–94 tion in neurons within sensory ganglia, predominantly the tri- years) at autopsy with a mean postmortem interval of 6 h (range 2.5– 15.5 h). The TG tissue panel consisted of 34 donors with a CNS disease (mainly Alzheimer’s disease and Parkinson’s disease) and six donors

† ‡ without evidence of CNS disease. The cause of death was not related to *Department of Virology, Department of Immunology, MS Center ErasMS, and ␣-herpesvirus infections. No significant differences in the immunolog- §Pulmonary Medicine, Erasmus Medical Center, Rotterdam, The Netherlands ical parameters analyzed were detected between donors with or without Received for publication May 26, 2009. Accepted for publication June 9, 2009. a history of CNS disease (data not shown). Specimens were either snap- The costs of publication of this article were defrayed in part by the payment of page frozen (n ϭ 23) or transferred to tubes (n ϭ 17) containing culture charges. This article must therefore be hereby marked advertisement in accordance medium consisting of RPMI 1640 (Lonza) supplemented with heat- with 18 U.S.C. Section 1734 solely to indicate this fact. inactivated 10% FBS (Greiner) and antibiotics. Written informed con- 1 This study was supported in part by the International Consortium on Anti-Virals (to sent from the donor or next of kin was obtained. The local ethical M.v.V.) and the Dutch MS Research Foundation (to J.D.L.). committees approved the study, which was conducted according to the tenets of the Declaration of Helsinki. 2 Address correspondence and reprint requests to Dr. Georges M.G.M. Verjans, De- partment of Virology, Room Ee1720a, Erasmus Medical Center, s-Gravendijkwal Generation of TG single cell suspensions 230, 3015 CE Rotterdam, the Netherlands. E-mail address: [email protected] 3 Abbreviations used in this paper: SGC, ; DC, dendritic cell; TG, Generation of single cell suspensions from human TG was performed es- trigeminal ganglia; PD, programmed death; PD-L1, PD ligand l. sentially as previously described (12). In brief, the TG were fragmented and subsequently treated with Liberase Blendzyme 3 (0.2 U/ml, Roche) at Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 37°C for 1 h. Dispersed cells were filtered through a 70-␮m pore size cell www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900890 The Journal of Immunology 2457 strainer (BD Biosciences), and the flow-through was collected in PBS 488 nm argon, and 561 nm diode laser to detect DAPI, fluorescein, and containing 1% FBS. From the same donor, PBMC were isolated from Alexa Fluor 610-PE, respectively. heparinized peripheral blood (ϳ4 ml per donor) by density gradient centrifugation on Ficoll-Hypaque (12). Donor PBMC and TG single cell Allogeneic MLR assay suspensions were directly used for phenotypic and functional analyses. CD14-enriched TG-SGC, peripheral blood-derived monocytes and mature Flow cytometry dendritic cells (DC) were used as stimulator cells in allogeneic MLR as- says. Due to the low number of monocytes recovered from TG donors’ Donor-matched PBMC and TG cells were subjected to multicolor flow cyto- PBMC, mature DC were generated from peripheral blood samples of metric analyses using the following fluorochrome-conjugated mAbs: CD3- healthy blood donors (n ϭ 2). To obtain mature DC, CD14-enriched pe- allophycocyanin (UCHT1; DakoCytomation), CD11b-PE (Bear-1; Beckman ripheral blood-derived monocytes were cultured with IL-4 and GM-CSF Coulter), CD11c-allophycocyanin (S-HCL3; BD Biosciences), CD14-FITC for 6 days to generate immature monocyte-derived DC, and subsequently (TU¨ K4; DakoCytomation), CD40-FITC (5C3; BD Biosciences), CD45-PerCP matured with a mixture as previously described (13, 14). The (2D1; BD Biosciences), CD54-FITC (6.5B5; DakoCytomation), CD68-PE mature DC phenotype, characterized by high CD80, CD83, and CD86 ex- (Y1/82A; BD Biosciences), CD80-FITC (MAB104; Beckman Coulter), pression (15), was confirmed by flow cytometry (data not shown). The CD83-allophycocyanin (HB15e; BD Biosciences), CD86-PE (FUN-1; effector cells, i.e., allogeneic peripheral blood T cells, were labeled with BD Biosciences), HLA-DR-PerCP (L243; BD Biosciences), CD94- CFSE (Invitrogen) at a final concentration of 0.5 ␮M. The stimulator cells FITC (DX22; eBioscience), NKG2A-allophycocyanin (131411; eBio- were cocultured with effector cells at a ratio of 1:10 at 37°C. At day 7, cells science), programmed death (PD)-1-PE (MIH4; eBioscience), and PD were harvested for flow cytometric analyses. Cells were stained with CD3- ligand 1 (PD-L1)-PE (MIH1; eBioscience). Cells were labeled accord- allophycocyanin (UCHT1; DakoCytomation) to discriminate between T ing to the manufacturers’ instructions and appropriate isotype- and flu- cells and stimulator cells. orochrome-matched unrelated mAbs were included as negative con- trols. Cells and data were analyzed on a BD FACSCalibur flow Results Downloaded from cytometer and BD CellQuest Pro software (BD Biosciences). Human TG-SGC express typical macrophage markers In situ analyses We have previously shown that TG-SGC uniformly express MHC Snap-frozen TG were embedded in Tissue Tek OCT compound (Sakura) class II, suggesting that they have a role as APC (12). Tissue- and cut into 6-␮m sections on a Leica CM 3050S cryostat. Sections were resident APC, including macrophages and DC, express the com- fixed in acetone for 10 min and incubated with the following unconjugated mon leukocyte marker CD45 enabling their distinction from stro- mAbs according to the manufacturer’s instructions: CD11b (ICRF44; BD mal cells like fibroblasts. Paired TG-derived cells and PBMC http://www.jimmunol.org/ Biosciences), CD11c (B-ly6; BD Biosciences), CD14 (TU¨ K4; DakoCyto- mation), CD16 (3G8; BD Biosciences), CD40 (5D12; Pangenetics), CD45 samples were assayed for CD45 expression. In contrast to PBMC, ϩ (2B11ϩPD7/26; DakoCytomation), CD54 (LB-2; BD Biosciences), CD64 the TG-derived CD45 cell pool included two distinct cell popu- (32.2; DakoCytomation), CD68 (KP1; DakoCytomation), CD80 (M24; In- lations: CD45high and CD45low cells (Fig. 1A). Whereas the nogenetics), CD83 (Hb15a; Beckman Coulter), CD86 (1G10; Pangenetics), CD45high cells consisted mainly of T cells (data not shown), all CD94 (HP-3B1; Immunotech), HLA-E (4D12) by gift from D. E. Geraghty low (Fred Hutchinson Cancer Research Center, Seattle, WA), PD-1 (MIH4; CD45 cells expressed the monocyte/macrophage marker CD14 eBioscience), and PD-L1 (MIH1; eBioscience). Primary mAb were visualized (Fig. 1B and Table I). In situ analyses showed that CD14 was using the avidin-biotin system (DakoCytomation) and AEC (3-amino-9-eth- expressed by TG-SGC (Fig. 1B). As hinted upon by a previous ylcarbazole; Sigma-Aldrich) as substrate, and sections were counterstained report (8), the macrophage-specific marker CD68 was expressed with hematoxylin (Sigma-Aldrich), examined under a Zeiss Axioskop, and intracellularly (Fig. 1C and Table I), but not at the cell surface of by guest on September 28, 2021 photographed using a Nikon DC-U1 camera. For each donor and each marker, three sections and three fields per section were analyzed. Human tonsil sec- TG-SGC (data not shown). Additionally, TG-SGC selectively ex- tions were used as positive control tissue, and appropriate isotype and conju- pressed Ag uptake receptors like CD11b and CD11c (Fig. 1, D and gate-negative control stainings were included. E, and Table I), as well as CD16 and CD64 (data not shown). In For double stainings, sections were fixed in acetone, and endogenous situ double stainings confirmed the flow cytometry data and dem- peroxidase activity and endogenous biotin were blocked before incubation with the first primary Ab CD14 (TU¨ K4) or CD45 (2B11ϩPD7/26). The onstrated coexpression of CD14, CD45 and CD11c on TG-SGC first mAb was detected using an avidin-biotin-HRP system (Biogenex). (Fig. 2). Before substrate incubation, sections were incubated with normal mouse serum (10%) and a CD11c-PE mAb (B-ly6), which was visualized using an Human TG-SGC have an immature myeloid DC phenotype anti-PE secondary Ab (AbD Serotec) and an alkaline phosphatase-conju- The complement receptor CD11c is commonly used as a marker to gated tertiary Ab (Sigma-Aldrich). Slides were first developed with Fast ϩ blue substrate, followed by incubation with AEC substrate solution. discriminate between myeloid (DC; CD11c ) and plasmacytoid (CD11cϪ) DC (16). Maturation of myeloid DC is characterized by Enrichment of peripheral blood- and TG-derived cell up-regulation or induction of surface markers like MHC class II populations and the costimulatory molecules CD80, CD83, and CD86 essential Monocytes and TG-SGC were isolated using anti-CD14 microbeads and a for T cell interaction and stimulation (15). Surface expression of MACS magnetic separator (Miltenyi Biotec) according to the manufactur- CD83 is considered characteristic for functionally mature DC (17). er’s instructions. T cells were isolated from PBMC of healthy blood donors The expression of CD11c and MHC class II on TG-SGC using anti-CD3 microbeads (Miltenyi Biotec). Flow cytometry confirmed that the enriched cell fractions contained Ͼ85% CD14ϩ cells and Ͼ95% prompted us to determine the expression of additional DC markers. CD3ϩ cells, respectively (data not shown). Whereas the TG-SGC expressed both CD80 and CD86 (Fig. 1, F and G and Table I), the mature DC marker CD83 could not be Phagocytosis assay detected (Fig. 1H and Table I). Furthermore, TG-SGC coexpressed TG single cell suspensions were incubated with fluorescein-labeled MHC class II and the T cell adhesion molecule CD54 (Fig. 1I). Escherichia coli K-12 strain bioparticles (Invitrogen) in a cell-to-par- Except for CD40, all markers determined were expressed uni- ticle ratio of 1:100 according to the manufacturer’s instructions. After formly on all TG-SGC. Whereas all TG-SGC of the TG donors incubation at 37°C for 2 h, cells were washed extensively and subjected ϭ ϩ to flow cytometry or used for immunocytological analyses. For the lat- (n 4) analyzed were CD40 by flow cytometry, in situ analyses ter procedure, E. coli-treated TG-SGC were enriched using anti-CD14 revealed interdonor variation of CD40 expression on TG-SGC. beads, spun down onto glass slides, fixed with 4% paraformaldehyde Two of six TG donors analyzed showed weak but positive CD40 and stained with Alexa Fluor 610-PE-conjugated anti-CD68 mAb (KP1; staining on TG-SGC, which was occasionally associated with T DakoCytomation). Cytospins were mounted in ProLong Gold anti-fade reagent with DAPI (4Ј,6-diamidino-2-phenylindole; Invitrogen) and an- cell clusters (Fig. 1J). The discrepancies observed could be due to alyzed on a confocal laser-scanning microscope (LSM510 Meta; Zeiss). the use of two different anti-CD40 mAbs in the separate assays, Pictures were made using multitrack recording with a 405 nm diode, and, in case of the differential CD40 expression observed in the in 2458 HUMAN SGC REPRESENT TISSUE-RESIDENT APC

FIGURE 2. Human TG-resident SGC express APC markers. Human TG single cell suspensions, and frozen TG biopsy specimens, were ana- lyzed for the markers CD45 and CD11c (A), and CD14 and CD11c (B) in cytometry analysis (n ϭ 14 donors) and double-color in situ analysis (n ϭ 6 donors) on consecutive sections, respectively. CD45low and CD45high cells are arbitrarily green and blue in all dot plots, respec- tively. The number for each quadrant in the dot plot represents cells expressing the indicated marker. Slides were developed with AEC and Fast blue resulting in red and blue staining patterns, respectively. A double positive cell, stained purple, is enlarged for experiment (far Downloaded from right). Original magnification is ϫ200.

Human TG-SGC phagocytose bacterial particles

A critical role of macrophages is to phagocytose cellular debris http://www.jimmunol.org/ and pathogens. Because the TG-SGC have a macrophage pheno- type, we determined their capability to phagocytose bacterial par- ticles. Whole TG cell suspensions were incubated with fluoresce- in-conjugated E. coli after which the phagocytic cell type was identified by flow cytometry. Bacteria were predominantly asso- FIGURE 1. Human TG-resident SGC express macrophage- and DC- ciated with the CD45low TG cells, identified in the experiment as specific markers. A, Dot plots of paired TG cells (left) and PBMC (right) TG-SGC (Fig. 3A). Because this assay does not discriminate be- stained for CD45 to demonstrate that human TG harbor a unique cell pop- tween membrane bound and internalized bacteria, the E. coli- ulation expressing CD45 at low levels (gate R2). CD45low and CD45high by guest on September 28, 2021 cells are arbitrarily green and blue in all dot plots, respectively. Subsequent treated TG-SGC were isolated using anti-CD14 magnetic beads panels show representative ex vivo flow cytometric analysis (left)(n ϭ 14 and subsequently subjected to immunocytology. Confocal laser donors) and in situ analysis (right)(n ϭ 6 donors) of the expression of scanning microscopy revealed that the bacteria colocalized with CD14 (B), CD68 (C), CD11b (D), CD11c (E), CD80 (F), CD86 (G), CD83 the late endosome marker CD68 (Fig. 3A), demonstrating that TG- (H), CD54 (I), and CD40 (J) detected. The number for each quadrant in dot SGC have actively phagocytosed the bacteria. plot represents the percentage of cells expressing the indicated marker defined on matched isotype control mAb stainings. Sections were devel- Human TG-SGC are unable to induce an allogeneic MLR oped with AEC (bright red precipitate) and counterstained with hematox- Although immature myeloid DC primarily function as phagocytes, ylin (blue nuclei). Original magnifications are ϫ200 (B–E, and J) and DC maturation is associated with up-regulation of costimulatory ϫ400 (F–I). and MHC molecules, secretion of cytokines, down-regulation of phagocytic capacity, and increased ability to induce T cell situ analyses, may be attributed to unknown TG donor-specific char- responses (15). It is well established that mature DC are potent acteristics. Similar to the other markers analyzed, CD40 expression stimulators of an allogeneic MLR, a characteristic that distin- did not correlate with the presence of interacting T cells or latent guishes them from other APC (18). Because TG-SGC expressed ␣-herpesvirus infection (data not shown). Table II presents a compar- a myeloid DC phenotype, they were used as stimulator cells in ative overview of the phenotype of human TG-SGC. allogeneic MLR assays. From the same donor, peripheral blood- derived CD14ϩ monocytes and CD14ϩ TG-SGC were cocul- tured with CFSE-labeled allogeneic T cells. In contrast to ma- Table I. Marker expression on CD45low human TG-SGC ture monocyte-derived DC, both monocytes and TG-SGC were unable to induce T cell proliferation (Fig. 3B), indicating that Percentage of Positive human TG-SGC resemble immature myeloid DC both pheno- Marker TG-SGC Ϯ SDa No. of Donors typically and functionally. Ϯ CD14 95.9 4.7 9 Human TG-infiltrating T cells express T cell inhibitory CD68 97.3 Ϯ 1.1 2 CD11b 92.3 Ϯ 6.6 2 molecules and TG-SGC the respective ligands Ϯ ϩ CD11c 88.5 8.3 8 Although neuron-interacting CD8 T cells express cytolytic mol- CD80 82.3 Ϯ 18.5 5 CD86 94.8 Ϯ 5.6 6 ecules, like perforin and granzyme B, neuronal damage is not ob- CD83 6.3 Ϯ 4.1 3 served in type-1 HSV latently infected TG, suggesting that the CD40 91.9 Ϯ 7.2 4 cytolytic activity of the CD8ϩ T cells is inhibited (7–12, 19). Re- a Data represent the average of TG-SGC that express the indicated marker deter- cently, Suvas et al. (19) have shown that the NK inhibitory mol- ϩ mined by flow cytometry. ecule complex CD94/NKG2A prevents CD8 T cell-mediated TG The Journal of Immunology 2459

Table II. Comparison of phenotype and functional characteristics of TG-SGC to other human APCa

Macrophageb Immature DCb Mature DCb CNS Microgliab TG-SGC

Phenotype CD14 and CD68 ϩϪϪϩc ϩ CD16 and CD64 ϩϩϪϩϩ CD11b and CD11c ϩϩϩϩϩ MHC class II ϩϩϩϩϩϩ CD45 High High High Low Low CD40 and CD54 ϩϩϩϩϩϩ CD80 and CD86 ϩϩϩϩϩϩ CD83 ϪϪϩϩc Ϫ Function Phagocytosis ϩϩϪϩϩ Allogeneic MLR ϪϪϩϩc Ϫ

a Results indicate the presence (ϩ), intensity (ϩϩ; high and low), or absence (Ϫ) of the markers or functional characteristics indicated. b Data previously described (14, 24, 28). c Upon stimulation with LPS, microglia express CD14 and CD83, and are able to induce an allogeneic MLR (24). Downloaded from neuron destruction in mice. Whereas the majority of the TG-infil- CD94 expression colocalized with CD3 within neuron-interacting trating HSV-specific CD8ϩ T cells expressed CD94/NKG2A, both T cell clusters (Fig. 4A). neurons and CD11bϩ cells expressed the cognate ligand Qa-1b In addition to NK inhibitory molecules, several studies have (19). Analogous to the mouse, human TG-infiltrating T cells co- indicated that the molecule PD-1 and its ligand PD-L1 negatively expressed CD94 and NKG2A (Fig. 4A). Moreover, the frequency regulate T cell effector functions (21–25). Both CD4ϩ and CD8ϩ of CD94/NKG2Aϩ T cells in TG (mean frequency 13 Ϯ 4%) was TG-infiltrating T cells expressed PD-1, but percentages and ex- http://www.jimmunol.org/ higher compared with peripheral blood (mean frequency 3 Ϯ 1%), pression levels did not differ between donor-matched TG-derived suggesting selective infiltration or differentiation of T cells to ex- T cells (mean 29 Ϯ 7%) and peripheral blood T cells (mean 35 Ϯ press CD94/NKG2A locally. The cognate receptor HLA-E (20) 12%) (Fig. 4B). However, in situ analyses revealed that neuron- was expressed throughout the TG tissue, including TG-SGC, and interacting T cell clusters tended to have a higher PD-1 expression, compared with scattered T cells (Fig. 4B). Notably, PD-L1 expres- sion was confined to TG-SGC and appeared to be higher on TG- SGC in proximity to the T cell clusters (Fig. 4C).

Discussion by guest on September 28, 2021 For decades, SGC have been regarded as nursing cells providing physical support to neuron somata in sensory ganglia. The current study demonstrates that human TG-SGC have phenotypic and

FIGURE 3. Human TG-resident SGC share functional characteristics with macrophages and immature myeloid DC. A, Human TG-SGC were incubated with fluorescein-conjugated bacteria to determine their phago- cytic function by flow cytometry (left) and confocal laser scanning micros- copy (right). CD45low and CD45high cells are arbitrarily green and blue in the dot plot, respectively (left). Cytospins of CD14-enriched TG-SGC treated with fluorescein-conjugated bacteria (bacteria in green) were stained for CD68 (late endosomes in red) and DAPI (cellular nuclei in FIGURE 4. Human TG-infiltrating T cells express inhibitory molecules. blue) and examined by confocal laser scanning microscopy (right). B, Dot A, Dot plot of ex vivo flow cytometry (left) on CD94 and NKG2A expres- plots of a representative allogeneic MLR using mature monocyte-derived sion on gated T cells, and in situ analyses of CD3, CD94, and HLA-E on DC generated from peripheral blood-derived monocytes of a healthy blood consecutive sections. B and C, Dot plots of ex vivo flow cytometric (top) donor (DC, left), and CD14-enriched peripheral blood monocytes (PB and in situ analyses (bottom) of CD3 and PD-1 (B), and CD3 and PD-L1 CD14ϩ, middle), and CD14-enriched TG-SGC (TG CD14ϩ, right) recov- (C) on consecutive sections. The number for each quadrant in dot plot ered from the same TG donor, hereby used as stimulator cells in combi- represents the percentage of cells expressing the indicated marker defined nation with CFSE-labeled allogeneic T cells. The percentage indicates the on matched isotype control mAb stainings. Dot plots in A and B are gated frequency of T cells that proliferated upon incubation at 37°C for 7 days. on CD3ϩ cells. Representative data from six TG donors are shown. Sec- Results are representative of two experiments performed on two TG tions were developed with AEC (bright red precipitate) and counterstained donors. with hematoxylin (blue nuclei). Original magnification is ϫ200. 2460 HUMAN SGC REPRESENT TISSUE-RESIDENT APC functional APC properties. Two main findings are reported. First, acterized by up-regulation of MHC class II and CD68 (41, 42). human TG-SGC have a unique leukocyte phenotype, with features Consequently, the discrepancy between both studies may in part be of both macrophages and immature myeloid DC. Second, TG-in- attributed to the relatively high age of the TG donors analyzed in filtrating T cells expressed the T cell inhibitory molecules CD94/ this study. NKG2A and PD-1, and the interacting TG-SGC expressed the cog- It is generally established that TG-infiltrating CD8ϩ T cells in- nate ligands HLA-E and PD-L1, respectively. hibit HSV-1 reactivation by means of IFN-␥ and cytolytic effector Current knowledge on CNS-resident glial cells advocate their molecules (7, 8, 12, 43, 44). Nevertheless, the latently infected role as critical participants in the healthy and diseased by neurons encountered are not damaged, suggesting that cytolytic T maintaining axonal integrity and myelination, providing nutrients, cell effector functions are inhibited (8, 12, 43). The expression of controlling formation and function, and immune regula- CD94/NKG2A on human TG-infiltrating T cells is consistent with tion (26–30). Whereas macroglia, like astrocytes and oligodendro- a previous study on mouse TG, demonstrating that blocking the cytes, are derived from the neuroectoderm (31), microglia express CD94-NKG2A/Qa-1b interaction in ex vivo TG cultures resulted several leukocyte cell markers implicating their origin from my- in neuronal cell lysis (19). CD94 expression in human TG was eloid progenitor cells (26, 32). Microglia are the main CNS-resi- selectively expressed by T cells interacting with neuronal somata, dent APC that constantly sense and sample the brain environment suggesting an analogous role of the CD94-NKG2A/HLA-E inter- and coordinate immune responses in response to danger signals action in human latently infected TG. Notably, Qa-1b was ex- (26–28). They resemble macrophages and immature myeloid DC pressed by neurons, but also CD11bϩ cells in mouse TG (19). The and have been implicated in neurodegenerative disorders like CD11bϩ Qa-1bϩ cells may represent the effector DC that are func- multiple sclerosis (27, 29, 30). Both human and rodent micro- tionally involved in controlling local T cell responses in HSV-1 Downloaded from express low levels of the membrane molecule CD45, a latently infected mouse sensory ganglia (38). marker commonly used to distinguish microglia (CD45low) In addition to CD94/NKG2A, the data on human TG suggest the from stromal cells and macroglia (both CD45Ϫ) and infiltrating involvement of the T cell inhibitory molecule PD-1. Human TG- lymphocytes (CD45high) (33, 34). infiltrating T cells and TG-SGC expressed PD-1 and PD-L1, re- In contrast to CNS glial cells, the immune function of peripheral spectively. Notably, the expression of both markers appeared to be

nervous system resident SGC is poorly defined. Our data demon- higher within neuron-interacting T cell clusters. IFN stimulation http://www.jimmunol.org/ strate that human TG-SGC closely resemble CNS microglia both up-regulates PD-1 and PD-L1 expression on receptive cells (22, phenotypically and functionally (Table II). Microglia and TG-SGC 45). Consequently, the differential PD-1 and PD-L1 expression are CD45low, and express similar macrophage- and DC-associated observed may be attributed to IFN-␥ secreted by activated T cells markers and T cell costimulatory molecules (Figs. 1 and 2, and recognizing the latent virus. Functional studies are mandatory to Table II) (26). Furthermore, both cell types actively phagocytose investigate the role of both the HLA-E/CD94-NKG2A and PD-1/ bioparticles and are unable to induce primary T cell responses (Fig. PD-L1 pathway to inhibit cytolytic T cell effector function in hu- 3) (35, 36). Hitherto, peripheral nervous system-resident SGC have man HSV-1 latently infected TG. Moreover, elucidation of the T been considered to be neuroectoderm-derived (2, 3, 37). The cur- cell inhibitory mechanisms used in the peripheral nervous system rent study challenges this concept, suggesting that human TG-SGC may provide tools for the development of future therapeutic inter- by guest on September 28, 2021 arise from myeloid progenitors analogous to microglia (26, 32). vention strategies to counteract undue cell damage associated with Recent data obtained by the Carbone group (38) support this T cell-mediated chronic diseases. hypothesis. The authors studied the local effector cells involved in In conclusion, the data presented in this study show that human maintaining virus-specific CD8ϩ T cell responses that control TG-resident SGC have a unique leukocyte phenotype, sharing HSV-1 latency in sensory ganglia of experimentally infected mice. properties with macrophages and immature myeloid DC. We hy- It was shown that CD8ϩ T cell homeostasis was depending on a pothesize that TG-SGC are tissue-resident APC involved in sens- tripartite interaction that includes infiltrating CD4ϩ T cells and ing the local environment and the control of local T cell responses recruited DC. The effector DC originated from circulating mono- to protect the irreplaceable neuronal somata in TG. cytes and expressed high levels of CD11b, CD11c, MHC class II, and F4/80. In situ analyses showed that the CD11cϩ DC were Acknowledgments occasionally found in close proximity to CD8ϩ T cells, but more We thank the Netherlands Brain Bank team for their efforts to provide the strikingly they appeared to surround the neuronal somata (38). The human TG specimens and are indebted to the donors who agreed to provide comparable phenotype and anatomic localization of murine sen- TG specimens for research purposes. We also thank D. E. Geraghty (Fred sory ganglia-resident DC and human TG-SGC suggest that they Hutchinson Cancer Research Center, Seattle, WA) for providing the anti- HLA-E mAb 4D12. The authors acknowledge discussions within the Eu- represent the same cell type. This local APC may present the cog- ϩ ropean Cooperation in Science and Technology (COST) Action BM0603 nate HSV-1 Ags to infiltrating virus-specific CD8 T cells. Studies Inflammation in Brain Disease Neurinfnet, and networking support in mice support this notion (39, 40). Alternatively, HSV-1-specific from COST. CD8ϩ T cells may penetrate the SGC sheet to interact directly with the latently infected neurons (7). Because neurons do not express Disclosures ϩ MHC class II, infiltrating virus-specific CD4 T cells most likely The authors have no financial conflict of interest. interact with TG-SGC. In contrast to a previous study on human TG, the majority of the References investigated markers analyzed in this study were uniformly ex- 1. Bechmann, I., I. Galea, and V. H. Perry. 2007. What is the blood-brain barrier (not)? Trends Immunol. 28: 5–11. pressed by TG-SGC of the TG donors studied. This finding was 2. Hanani, M. 2005. Satellite glial cells in sensory ganglia: from form to function. irrespective of the HSV status of the donor and varicella zoster Brain Res. Rev. 48: 457–476. virus serostatus, and the presence of infiltrated T cells (data not 3. Pannese, E. 1981. The satellite cells of the sensory ganglia. Adv. Anat. Embryol. Cell Biol. 65: 1–111. shown). A major difference between the preceding and present 4. Elson, K., A. Simmons, and P. Speck. 2004. Satellite cell proliferation in murine study is the median age of the TG donors analyzed: 29 vs 79 years, sensory ganglia in response to scarification of the skin. Glia 45: 105–109. 5. Lee, S. E., H. Shen, G. Taglialatela, J. M. Chung, and K. Chung. 1998. Expres- respectively (8). Animal studies have demonstrated that aging in- sion of nerve growth factor in the after peripheral nerve duces the transition of naive microglia into an activated state, char- injury. Brain Res. 796: 99–106. The Journal of Immunology 2461

6. Roizman, B., D. Knipe, and R. J. Whitley. Fields’ Virology, 5th ed. D. Knipe, 25. Jurado, J. O., I. B. Alvarez, V. Pasquinelli, G. J. Martinez, M. F. Quiroga, and P. Howley, eds. Philadelphia, Pa. Lippincott Williams & Wilkins; 2007: E. Abbate, R. M. Musella, H. E. Chuluyan, and V. E. Garcia. 2008. Programmed 2501–2601. death (PD)-1:PD-ligand 1/PD-ligand 2 pathway inhibits T cell effector functions 7. Khanna, K. M., R. H. Bonneau, P. R. Kinchington, and R. L. Hendricks. 2003. during human tuberculosis. J. Immunol. 181: 116–125. virus-specific memory CD8ϩ T cells are selectively activated and 26. Guillemin, G. J., and B. J. Brew. 2004. Microglia, macrophages, perivascular retained in latently infected sensory ganglia. Immunity 18: 593–603. macrophages, and pericytes: a review of function and identification. J. Leukocyte 8. Theil, D., T. Derfuss, I. Paripovic, S. Herberger, E. Meinl, O. Schueler, Biol. 75: 388–397. M. Strupp, V. Arbusow, and T. Brandt. 2003. Latent herpesvirus infection in 27. Hanisch, U. K., and H. Kettenmann. 2007. Microglia: active sensor and versatile human trigeminal ganglia causes chronic immune response. Am. J. Pathol. 163: effector cells in the normal and pathologic brain. Nat. Neurosci. 10: 1387–1394. 2179–2184. 28. Nimmerjahn, A., F. Kirchhoff, and F. Helmchen. 2005. Resting microglial cells 9. van Lint, A. L., L. Kleinert, S. R. Clarke, A. Stock, W. R. Heath, and are highly dynamic surveillants of brain parenchyma in vivo. Science 308: F. R. Carbone. 2005. Latent infection with herpes simplex virus is associated with 1314–1318. ϩ ongoing CD8 T-cell stimulation by parenchymal cells within sensory ganglia. 29. Streit, W. J. 2004. Microglia and Alzheimer’s disease pathogenesis. J. Neurosci. J. Virol. 79: 14843–14851. Res. 77: 1–8. 10. Khanna, K. M., A. J. Lepisto, V. Decman, and R. L. Hendricks. 2004. Immune 30. Ulvestad, E., K. Williams, R. Bjerkvig, K. Tiekotter, J. Antel, and R. Matre. control of herpes simplex virus during latency. Curr. Opin. Immunol. 16: 1994. Human microglial cells have phenotypic and functional characteristics in 463–469. ϩ common with both macrophages and dendritic antigen-presenting cells. J. Leu- 11. Liu, T., K. M. Khanna, X. Chen, D. J. Fink, and R. L. Hendricks. 2000. CD8 kocyte Biol. 56: 732–740. T cells can block herpes simplex virus type 1 (HSV-1) reactivation from latency 31. Dong, Y., and E. N. Benveniste. 2001. Immune function of astrocytes. Glia 36: in sensory neurons. J. Exp. Med. 191: 1459–1466. 180–190. 12. Verjans, G. M., R. Q. Hintzen, J. M. van Dun, A. Poot, J. C. Milikan, 32. Chan, W. Y., S. Kohsaka, and P. Rezaie. 2007. The origin and cell lineage of J. D. Laman, A. W. Langerak, P. R. Kinchington, and A. D. Osterhaus. 2007. microglia: new concepts. Brain. Res. Rev. 53: 344–354. Selective retention of herpes simplex virus-specific T cells in latently infected 33. Becher, B., and J. P. Antel. 1996. Comparison of phenotypic and functional human trigeminal ganglia. Proc. Natl. Acad. Sci. USA 104: 3496–3501. properties of immediately ex vivo and cultured human adult microglia. Glia 18: 13. Sallusto, F., and A. Lanzavecchia. 1994. Efficient presentation of soluble antigen

1–10. Downloaded from by cultured human dendritic cells is maintained by granulocyte/macrophage col- 34. Greter, M., F. L. Heppner, M. P. Lemos, B. M. Odermatt, N. Goebels, T. Laufer, ony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis R. J. Noelle, and B. Becher. 2005. Dendritic cells permit immune invasion of the factor ␣. J. Exp. Med. 179: 1109–1118. CNS in an animal model of multiple sclerosis. Nat. Med. 11: 328–334. 14. Allard, S. D., K. Pletinckx, K. Breckpot, C. Heirman, A. Bonehill, A. Michiels, 35. Carson, M. J., C. R. Reilly, J. G. Sutcliffe, and D. Lo. 1998. Mature microglia C. A. van Baalen, R. A. Gruters, A. D. Osterhaus, P. Lacor, et al. 2008. Func- resemble immature antigen-presenting cells. Glia 22: 72–85. tional T-cell responses generated by dendritic cells expressing the early HIV-1 36. Davoust, N., C. Vuaillat, G. Androdias, and S. Nataf. 2008. From bone marrow proteins Tat, Rev and Nef. Vaccine 26: 3735–3741. to microglia: barriers and avenues. Trends Immunol. 29: 227–234. 15. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of 37. Li, H. Y., E. H. Say, and X. F. Zhou. 2007. Isolation and characterization of

immunity. Nature 392: 245–252. http://www.jimmunol.org/ 16. Colonna, M., G. Trinchieri, and Y. J. Liu. 2004. Plasmacytoid dendritic cells in progenitors from adult dorsal root ganglia. Stem. Cells 25: immunity. Nat. Immunol. 5: 1219–1226. 2053–2065. 17. Prechtel, A. T., and A. Steinkasserer. 2007. CD83: an update on functions and 38. Wakim, L. M., J. Waithman, N. van Rooijen, W. R. Heath, and F. R. Carbone. prospects of the maturation marker of dendritic cells. Arch. Dermatol. Res. 299: 2008. Dendritic cell-induced memory T cell activation in nonlymphoid tissues. 59–69. Science 319: 198–202. 18. Crow, M. K., and H. G. Kunkel. 1982. Human dendritic cells: major stimulators 39. Pereira, R. A., D. C. Tscharke, and A. Simmons. 1994. Upregulation of class I of the autologous and allogeneic mixed leucocyte reactions. Clin. Exp. Immunol. major histocompatibility complex gene expression in primary sensory neurons, 49: 338–346. satellite cells, and Schwann cells of mice in response to acute but not latent 19. Suvas, S., A. K. Azkur, and B. T. Rouse. 2006. Qa-1b and CD94-NKG2a inter- herpes simplex virus infection in vivo. J. Exp. Med. 180: 841–850. action regulate cytolytic activity of herpes simplex virus-specific memory CD8ϩ 40. Liu, T., Q. Tang, and R. L. Hendricks. 1996. Inflammatory infiltration of the T cells in the latently infected trigeminal ganglia. J. Immunol. 176: 1703–1711. after herpes simplex virus type 1 corneal infection. J. Virol.

20. Kaiser, B. K., F. Barahmand-Pour, W. Paulsene, S. Medley, D. E. Geraghty, and 70: 264–271. by guest on September 28, 2021 R. K. Strong. 2005. Interactions between NKG2x immunoreceptors and HLA-E 41. Dilger, R. N., and R. W. Johnson. 2008. Aging, microglial cell priming, and the ligands display overlapping affinities and thermodynamics. J. Immunol. 174: discordant central inflammatory response to signals from the peripheral immune 2878–2884. system. J. Leukocyte Biol. 84: 932–939. 21. Blank, C., and A. Mackensen. 2007. Contribution of the PD-L1/PD-1 pathway to 42. Perry, V. H., M. K. Matyszak, and S. Fearn. 1993. Altered antigen expression of T-cell exhaustion: an update on implications for chronic infections and tumor microglia in the aged rodent CNS. Glia 7: 60–67. evasion. Cancer Immunol. Immunother. 56: 739–745. 43. Knickelbein, J. E., K. M. Khanna, M. B. Yee, C. J. Baty, P. R. Kinchington, and ϩ 22. Sharpe, A. H., E. J. Wherry, R. Ahmed, and G. J. Freeman. 2007. The function R. L. Hendricks. 2008. Noncytotoxic lytic granule-mediated CD8 T cell inhi- of programmed cell death 1 and its ligands in regulating autoimmunity and in- bition of HSV-1 reactivation from neuronal latency. Science 322: 268–271. fection. Nat. Immunol. 8: 239–245. 44. Liu, T., K. M. Khanna, B. N. Carriere, and R. L. Hendricks. 2001. Gamma 23. Barber, D. L., E. J. Wherry, D. Masopust, B. Zhu, J. P. Allison, A. H. Sharpe, interferon can prevent herpes simplex virus type 1 reactivation from latency in G. J. Freeman, and R. Ahmed. 2006. Restoring function in exhausted CD8 T cells sensory neurons. J. Virol. 75: 11178–11184. during chronic viral infection. Nature 439: 682–687. 45. Muhlbauer, M., M. Fleck, C. Schutz, T. Weiss, M. Froh, C. Blank, 24. Maier, H., M. Isogawa, G. J. Freeman, and F. V. Chisari. 2007. PD-1:PD-L1 J. Scholmerich, and C. Hellerbrand. 2006. PD-L1 is induced in hepatocytes by interactions contribute to the functional suppression of virus-specific CD8ϩ T viral infection and by interferon-␣ and -␥ and mediates T cell apoptosis. J. Hepa- lymphocytes in the liver. J. Immunol. 178: 2714–2720. tol. 45: 520–528.