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α4-integrins control viral meningoencephalitis through differential recruitment of T helper cell subsets

α4-integrins control viral meningoencephalitis through differential recruitment of T helper cell... Introduction: Natalizumab blocks α4-integrins and is a prototypic agent for a series of anti-inflammatory drugs that impair trafficking of immune cells into the CNS. However, modulation of the access of immune cells to the CNS is associated with impaired immune surveillance and detrimental viral infections of the CNS. Here, we explored the potency of cellular immune responses within the CNS to protect against viral encephalitis in mice with T cell conditional disruption of VLA-4 integrin (α4β1) expression. Results: While VLA-4 expression in virus specific Th1 cells is non-redundant for their ability to access the CNS, α4-integrin deficient Th17 cells enter the CNS compartment and generate an inflammatory milieu upon intrathecal vaccinia virus (VV) infection. However, in contrast to Th1 cells that can adopt direct cytotoxic properties, Th17 cells fail to clear the virus due to insufficient Eomes induced perforin-1 expression. Conclusion: The quality of the intrathecal cellular antiviral response under conditions of impaired VLA-4 function jeopardizes host protection. Our functional in vivo data extend our mechanistic understanding of anti-viral immunity in the CNS and help to estimate the risk potential of upcoming therapeutic agents that target the trafficking of immune cells into distinct anatomical compartments. Introduction While Th1 cells maintain high amounts of VLA-4 expres- Autoimmune inflammation of the CNS in multiple scler- sion, Th17 cells are low in VLA-4. As a consequence, osis (MS) and its animal model, experimental autoimmune blockade of VLA-4 is more efficient in preventing the encephalomyelitis (EAE), is mediated by antigen specific recruitment of Th1 cells than of Th17 cells into the Th1 and Th17 cells [1]. For many years integrin targeted CNS parenchyma. blocking of T helper cell trafficking into the CNS has Although considered as an “immune privileged” organ, appeared to be an attractive approach to treat immuno- the CNS is still patrolled by T cells as a means of immune + + pathologyinMS[2].Inparticular, monoclonal anti- surveillance [5]. The contribution of CD4 vs CD8 bodies (natalizumab) to the α4 subunit of the integrin effector memory T cells in the migratory and resident VLA-4 (α4β1 heterodimer) were successfully used to pools of lymphocytes specific to a given pathogen has prevent the influx of immune cells into the CNS and to been investigated in skin infection but is unclear in the treat CNS autoimmunity [3]. However, in experimental CNS [6]. In the treatment of organ specific autoimmunity models it has been shown that distinct encephalitogenic and chronic inflammation, efforts are increasing to market T cell subsets vary in their equipment with VLA-4 [4]. compounds that either inhibit immune cell trafficking [7-10] or cytokine networks that affect distinct T helper cell subsets in a differential manner (anti-IL-23p19, anti- * Correspondence: korn@lrz.tum.de † IL-17A [11,12], anti-GM-CSF (NCT01517282), anti-IL-6R Equal contributors [13]). However, preclinical models to investigate niche Klinikum rechts der Isar, Department of Neurology, Technische Universität München, Ismaninger Str. 22, 81675 Munich, Germany specific immune surveillance and host defense in the CNS Munich Cluster for Systems Neurology (SyNergy), Munich, Germany are rare. Indeed, efalizumab, a blocking antibody to the Full list of author information is available at the end of the article © 2014 Rothhammer et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 2 of 16 http://www.actaneurocomms.org/content/2/1/27 integrin αL was withdrawn from the market in 2009 cisterna magna of mice in deep anaesthesia by means of because of viral meningitis and cases of JC virus induced transcutaneous suboccipital puncture. Clinical signs of progressive multifocal leukencephalopathy (PML) [14]. disease as well as weight loss in percent of initial weight Here, we established a CNS specific viral infection (means + SEM) were monitored daily. model that allowed us to analyze the contribution of dis- For adoptive transfer experiments, naïve T cells were + + tinct T helper cell subsets to host protection. We chose isolated by magnetic sorting (CD4 CD62L ; T cell isola- vaccinia virus (VV) infection where the importance of tion kit II, mouse; Miltenyi Biotec, Germany) from virus specific T helper cell responses has been analyzed CD45.1 OT-II mice and differentiated in vitro into Th1 previously [15,16]. Vaccinated mice were found to be or Th17 cells. The differentiation status was checked on protected from intrathecal (i.th.) infection with VV due day 4 by intracellular cytokine staining and 2 × 10 cyto- + -/- to cellular immunity. In the absence of CD8 T cells, kine positive T cells were injected i.v. into Rag1 recipi- Th1 like cells were sufficient to protect mice from intra- ent mice, which had been infected with VV-Ova one day thecal VV infection. Access of Th1 cells into the infected prior to T cell transfer. CNS compartment was dependent on VLA-4 expression. Animals were kept in a specific pathogen-free facility Although virus specific Th17 cells were able to migrate at the Technische Universität München. All experimen- into the CNS in the absence of VLA-4, CNS recruited tal protocols were approved by the standing committee and infected macrophages were not cleared by Th17 for experimentation with laboratory animals of the cells since Th17 cells – in contrast to Th1 cells – were Bavarian state authorities (“Governmental Department deficient in perforin-1 expression. These data highlight of Upper Bavaria, Approved animal experimental pro- a dominant role of Th1 cells in antiviral tissue-specific posals No 55.2-1-54-2531-88-08 and No 55.2.1-54-2532- immunity. Our data further suggest that as in auto- 29-13 according to §8.1, German law for experimentation immune inflammation of the CNS, virus specific Th1 with laboratory animals”) and carried out in accordance cells are dependent on VLA-4 to enter into the CNS with the corresponding guidelines. and virus infection does not overcome the requirement for Th1 cells to express VLA-4. Thus, integrin targeted Virus strains therapeutic interventions in autoimmunity and chronic Replication competent VV Western Reserve strain (VV) inflammation need to be refined in order to not was provided by B Moss (National Institutes of Health, jeopardize organ specific immune surveillance and Bethesda, MD). Recombinant viruses encoding en- host protection. hanced green fluorescent protein (VV eGFP) or full- length ovalbumin (VV-Ova) based on the Western Materials and methods Reserve strain were provided by JW Jewdell and JR Ben- Animals, immunization, and infection nink (National Institutes of Health, Bethesda, MD). VV flox/flox Foxp3gfp.KI mice [17,18] and Itga4 mice [19] have and replication deficient Modified Vaccinia Virus -/- been described previously. CD4 Cre mice, Ifng mice, Ankara strain (MVA, cloned isolate IInew) used in this -/- -/- Rag1 mice, Prf1 mice, and wild type C57BL/6 mice were studywerepropagatedand titeredaccording to stand- obtained from Jackson Laboratories. CD45.1 OT-II mice ard methodology [21]. were kindly provided by DH Busch (Institute for Medical Microbiology, Immunology, and Hygiene, Technische T cell differentiation + high - Universität München). All mouse strains were on pure Naïve T cells (CD4 CD62L CD25 ) were isolated from C57BL/6 background. lymph nodes and spleen by magnetic sorting (T cell iso- Mice were immunized by subcutaneous injection of lation kit II, mouse; Miltenyi Biotec, Germany). Purity 100 μl of an emulsion of 1 × 10 IU MVA or PBS in was in general higher than 95% as controlled by FACS complete Freund’s adjuvant (CFA). For in vivo blockade staining. Naïve T cells were stimulated for 3 to 5 days of IFN-γ, mice were treated with every other day i.p. in- with plate-bound antibody to CD3 (145-2C11, 4 μg/ml) jections of a neutralizing antibody to IFN-γ (R4-6A2, and antibody to CD28 (PV-1, 2 μg/ml). Recombinant BioXCell, West Lebanon, USA; 200 μg) or isotype control cytokines were added to the differentiation cultures as starting on day 9 after immunization. In a similar regimen, indicated: human TGF-β1 (2 ng/ml) and mouse IL-6 blocking antibodies to integrin α4 (PS/2, BioXCell, West (50 ng/ml) for Th17, mouse IL-12 (10 ng/ml) and anti- Lebanon, USA; 200 μg), depleting antibodies to CD8 IL-4 (10 μg/ml) for Th1, all R&D Systems. (YTS169.4, BioXcell; 200 μg) or CD4 (GK1.5, BioXcell; 200 μg) were administered every other day from day 9 or Preparation of splenic and CNS mononuclear cells and day 10 after immunization, respectively. antibody staining Intrathecal infection was performed as previously Mononuclear cells were isolated from either spleen or described [20]. In brief, VV was inoculated into the CNS at the peak of disease (d4-d5 after intrathecal Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 3 of 16 http://www.actaneurocomms.org/content/2/1/27 challenge). After perfusion through the left cardiac ven- Quantitative PCR analysis tricle with cold PBS, the brain including cerebellum was For quantitative PCR, RNA was extracted from magnetic dissected and the spinal cord flushed out with PBS by bead-purified or flow cytometry-sorted cells ex vivo or hydrostatic pressure. CNS tissue was digested with colla- after in vitro differentiation using RNeasy columns (Qiagen, genase D (2.5 mg/ml, Roche Diagnostics, Indianapolis Valencia, CA). Complementary DNA was transcribed as IN) and DNAseI (1 mg/ml, Sigma, Saint Louis, MO) at recommended (Applied Biosystems, Foster City, CA) and 37°C for 45 min. Mononuclear cells were isolated by used as template for quantitative PCR. Primer plus probe passing the tissue through a cell strainer (70 μm) and mixtures were obtained from Applied Biosystems. The percoll gradient (37% over 70%) centrifugation. Mono- Taqman analysis was performed on a StepOne system nuclear cells were removed from the interphase, washed from Applied Biosystems. The gene expression was and resuspended in culture medium for further analysis. normalized to the expression of β-actin. For isolation of mononuclear cells from spleen, spleens were mashed through a cell strainer (70 μm) and red Western blotting blood cells were removed using BD Pharm Lyse (BD T cells were lysed and denatured using RiPA buffer Biosciences). Surface staining of T cells was carried out (Sigma-Aldrich). The protein lysates were separated by with antibodies to CD3 (14-2C11), CD4 (RM4-5), CD8 SDS-PAGE in 4–12% NuPAGE Bis-Tris Mini gels and (53-6.7), CD11b (M1/70), CD25 (PC61 or 7D4), CD44 transferred to nitrocellulose membranes (Invitrogen). (IM7), CD45 (30-F11) and Nk1.1 (PK136). All antibodies After blocking with 5% low-fat dry milk in TBS-T, were purchased from BD Biosciences. Fluorescence- membranes were incubated with primary antibodies to labeled MHC class I H-2K /B8R (TSYKFESV) mul- Prf-1 (ab7203, Abcam) and β-actin (Abcam) in blocking 20-27 timers were provided by DH Busch. solution overnight at 4°C. Primary antibody binding was detected with HRP-conjugated secondary antibodies Intracellular cytokine staining (Dianova). The signal was visualized by enhanced chemi- Cells were stimulated in culture medium containing luminescence (Novex ECL, Invitrogen). phorbol 12-myristate 13-acetate (PMA, 50 ng/ml, Sigma), ionomycin (1 μg/ml, Sigma), and monensin (GolgiStop Antigen specific proliferative and cytokine responses 1 μl/ml, BD Biosciences) at 37°C and 10% CO for For CD154 (CD40L) staining, spleens from MVA immu- - flox/flox + flox/flox 4 hours. After staining of surface markers, cells were nized CD4 Cre × Itga4 or CD4 Cre × Itga4 fixedand permeabilized(Cytofix/Cytoperm andPerm/ mice were dissected on day 10 after immunization. Sin- Wash buffer, BD Biosciences) followed by staining with gle cell suspensions were prepared and cells were seeded monoclonal antibodies to mouse IL-2, IL-17, or IFN-γ on a 12 well flat-bottom plate at a density of 2 × 10 (BD Biosciences) and flow cytometric analysis (CYAN, cells/well. Single cells were restimulated with a mixture Beckmann/Coulter). of I-A -restricted VV peptides (A33R, B2R, B5R, L4R; 30 μg/ml each) for 6 hours in the presence of brefeldin Histologic analysis A(5 μg/ml) during the last 3 hours of incubation followed For detection of VV-infected cells or macrophages, para- by surface and intracellular staining for CD40L (CD154 formaldehyde (PFA) (4%) fixed and paraffin embedded (MR1), eBioscience) and cytokines as indicated. A33R 116-130 CNS tissue sections were incubated with Bond Primary (YQLFSDAKANCTAES), B2R (VKDKYMWCYSQ 46-60 Antibody Diluent (Leica) containing either polyclonal VNKR), B5R (FTCDQGYHSSDPNAV) and L4R 46-60 176-190 rabbit anti-VV serum (1:1000; Quartett Immunodiagnos- (ISKYAGINILNVYSP) were obtained from Biosyntan, tika & Vertriebs-GmbH, Berlin) or monoclonal anti- Berlin. bodies against Mac-3 (1:750; M3/84) purchased from BD Pharmingen. IHC staining was performed on an auto- Retroviral transduction of T cells mated Leica BOND-MAX instrument using Bond Polymer pMIG Eomes (GFP) and control pMIG (GFP) retroviral Refine Detection Solution for DAB. For detection of GFAP, constructs were a kind gift from SL Reiner (University of PFA-fixed and paraffin-embedded CNS sections were Pennsylvania, Philadelphia, PA) and FJ Quintana (Harvard incubated with Dako polyclonal rabbit anti-GFAP anti- Medical School, Boston, MA). Phoenix-Eco cells (PMID: bodies (Z0034; 1:13000) in Ventana buffer and staining 18432682, a kind gift of H-M Jaeck, Erlangen) were transi- was performed on a Ventana NexES IHC Slide Stainer ently transfected with plasmids by calcium phosphate pre- using iVIEW DAB Detection Kit (Ventana). Images cipitation in the presence of 25 μM chloroquine (Sigma were taken using the Leica SCN400 slide scanner ana- Aldrich). Retroviral supernatants were collected two days lysis software or were acquired on an Olympus BX53 post transfection. T cells were transduced 24 hours post Microscope (DP72 camera) using the cellSens 1.8 polyclonal in vitro activation of naïve sorted CD4 Tcells digital imaging software (Olympus). with TGF-β and IL-6 (Th17). Retroviral supernatant and Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 4 of 16 http://www.actaneurocomms.org/content/2/1/27 4 μg/ml polybrene (Merck Millipore) were added to the CNS and ovaries of mice injected i.th. with 1.000 PFUs pre-committed Th17 cells and a spin transduction was of VV. While high titers of VV were measured in the performed (2000 rpm, RT, 1 hour). Cells were further CNS, virus was not detected in the ovaries of intra- cultured for 3 days in Th17 polarizing conditions before thecally infected mice (Figure 1B) indicating that intra- GFP expressing cells were purified by FACS sorting and thecal injection of VV led to a compartmentalized subjected to quantitative PCR analysis. infection within the CNS without systemic dissemin- ation. Within the CNS compartment, VV antigen was Plaque assay detected in ventricular lining cells and plexus epithe- CNS tissue was digested as described and single cell sus- lium cells as well as in astrocytes of the glia limitans. pensions were obtained by passing digested CNS tissue Some innate immune cells of monocytic origin that through a 70-μm nylon filter. Pelleted cells were sub- were recruited to the infected CNS compartment were jected to three freeze-thaw cycles (-80°C and 37°C) and also positive for VV antigen (Figure 1C). sonicated three times for 1 min. Serial dilutions in In order to determine immune cell targets of VV RPMI-1640 medium containing 10% FCS were added in within the CNS, we infected wild type mice with recom- duplicates to 90% confluent RK-13 cells seeded in 6-well binant VV expressing enhanced green fluorescent pro- plates and incubated for 24 hours at 37°C. Plaques were tein (VV eGFP). When analyzing eGFP expression in counted after crystal violet staining. distinct cell populations of the CNS on day 4 after infec- tion, we found a sizable fraction of eGFP expressing cells high high Neutralization assay only within the CD11b CD45 macrophage com- Mouse serum samples were collected at day 10 post partment (Figure 2). This is in line with a recent study + + immunization (MVA/CFA or PBS/CFA). All serum sam- which identified infiltrating CD11b CD45 inflammatory ples were heat-inactivated at 56°C for 30 min prior to monocytes as the predominant VV infected leukocyte testing. Serum dilutions were incubated with 10,000 population during VV skin infection [22]. Microglial + low PFU of sucrose gradient purified VV eGFP for 1 hour at cells (CD11b CD45 ), T cells and B cells did not show 37°C and 5% CO in a 96-well plate. RK-13 cells were eGFP expression. In addition, we were unable to detect added and infection (MOI = 0.1) was carried out over eGFP expressing cells in the spleens of intrathecally night at 37°C and 5% CO . Cells were harvested, washed injected mice, further demonstrating that VV infection and fixed in 1% paraformaldehyde. Percentage of was restricted to the CNS compartment. Importantly, + high VV-infected cells was determined by measuring eGFP ex- infected CD11b CD45 macrophages expressed MHC pression in flow cytometric analyses (CYAN, Beckmann/ class II (Figure 2B). Thus, phagocytic cells were re- Coulter). The percentage of virus neutralization was cruited from the systemic compartment in response to defined as (1-[percentage of GFP-expressing cells]/ i.th. VV infection and became targets of virus replication [percentage of GFP-expressing cells in controls with- but were unable to control the infection (see Figure 1A). out serum]) × 100. Immunization with modified vaccinia virus (MVA) protects Statistical analysis against VV encephalitis Statistical evaluations of cell frequency measurements As macrophages became infected during VV encephalitis and gene expression levels were performed with the but failed to eliminate the virus, we hypothesized that unpaired Student’s t test. Two-tailed p values < 0.05 adaptive cellular immune responses were required for were considered significant. Weight scores are given as virus control. In order to investigate the contribution of means ± SEM. Multiple comparisons were performed antigen specific effector T helper cells to host defense by two-way-ANOVA and Bonferroni post-testing. against i.th. VV infection, we immunized wild type mice with a replication deficient strain of VV (MVA) emulsi- Results fied in complete Freund’s adjuvant (CFA) 10 days prior Intrathecal vaccinia virus (VV) infection causes to i.th. challenge with VV. CFA induces both Th1 and lethal encephalitis Th17 responses. Here, we were interested in investigat- To establish a model of viral encephalitis, female C57BL/6 ing the role of these T helper cell subsets in the trade-off mice were injected intrathecally with VV by suboccipital between host protection and immunopathology during puncture of the cisterna magna. By titrating the dose of infectious encephalitis. While sham-immunized mice the inoculum, the half lethal dose (LD50) was determined died at day 6 after infection and largely failed to recruit to be approximately 500 plaque forming units (PFU) either CD4 T helper cells or VV-specific CTLs into the (Figure1A).Insystemicinfection, VVisknown to pref- CNS compartment (Figure 3A,B), MVA immunized erentially replicate in the ovaries upon hematogenous animals harbored both antigen specific CTLs and CD4 T dissemination. We therefore measured viral loads in cells in the CNS and recovered from VV encephalitis Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 5 of 16 http://www.actaneurocomms.org/content/2/1/27 Figure 1 Intrathecal infection with VV causes lethal encephalitis. (A) C57BL/6 wild type mice were injected intrathecally by suboccipital puncture of the cisterna magna with various doses of VV. Percentage of surviving mice are depicted in Kaplan-Meier curves (n = 5 per group). (B) Virus loads in CNS and ovaries of wild type mice infected intrathecally with VV were measured on day 5 after infection (Student’st test, n ≥ 5). (C) Wild type mice injected with VV were subjected to histologic analyses on day 5 after infection. Unless otherwise indicated, scale bars represent 200 μm in the low magnification and 20 μm in the higher magnification photomicrographs. (Figure 3A-C). While CTLs re-isolated from the CNS Th17 cells into the CNS compartment. In the EAE largely produced IFN-γ, the intrathecal CD4 effector T cell model, Th1 cells depend on α4-integrins in order to compartment comprised IFN-γ producers, IL-17 producers, enter into the CNS during autoimmune inflammation. and IFN-γ/IL-17 double producers (Figure 3C). In sum- In contrast, Th17 cells are able to access the CNS com- mary, protective immunity against intrathecal VV infec- partment independently of α4-integrins using an LFA-1 tion was associated with the mobilization of both CTLs dependent mechanism [4]. Here, we treated our MVA and CD4 effector T helper cells to the CNS. immunized mice with blocking antibodies to integrin α4 (PS/2) prior to i.th. VV challenge (Figure 4A). Notably, Antibody mediated blockade of α4-integrins leads to MVA immune mice survived i.th. VV infection in spite relative enrichment of Th17 cells in the CNS of integrin α4 blockade (Figure 4A). As expected, we We next wished to dissect the specific contribution of found a relative abundance of IL-17 producing T cells as different T cell subsets to the protective effects of compared with IFN-γ positive T helper cells (Figure 4B). vaccination. Here, we took advantage of the differential This finding supported the idea that – similar to T integrin requirement for the recruitment of Th1 cells vs cell recruitment in autoimmune inflammation of the Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 6 of 16 http://www.actaneurocomms.org/content/2/1/27 SPL CNS 4 4 10 10 3 3 10 10 1 3 2 2 10 10 1 1 10 10 0 0 10 10 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 CD11b SPL CNS 4 4 10 10 38.6 0 34.7 0.10 3 3 10 10 2 2 10 10 1 1 10 10 0 0.25 0 0 10 10 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 4 4 10 10 29.4 0.04 80.7 1.31 3 3 10 10 2 2 10 10 1 1 10 10 0 0.07 0 0 10 10 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 4 4 10 10 62 0 42.5 6.81 3 3 10 10 2 2 10 10 1 1 10 10 0 0.81 0 0 10 10 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 VV (eGFP) Figure 2 MHC class II positive macrophages are targets of VV infection in the CNS. Wild type mice were infected intrathecally with 1.000 PFUs of VV (eGFP), a replication competent recombinant VV expressing enhanced green fluorescent protein (eGFP) in infected cells. On day 4 after infection, mice were sacrificed, and spleen and CNS mononuclear cells were isolated and analyzed by flow cytometry. In the live cell gate, low high the following populations were defined based on their expression of CD11b and CD45: (A) spleen (SPL), left panel: (1) CD11b CD45 int high high high low high lymphocytes, (2) CD11b CD45 dendritic cells, (3) CD11b CD45 macrophages. (A) CNS, right panel: (1) CD11b CD45 lymphocytes, (2) int int high high CD11b CD45 microglia, (3) CD11b CD45 macrophages. (B) These populations were analyzed as to their expression of eGFP and MHC class II by flow cytometry. Numbers indicate percentages of positive cells in each quadrant or gate (representative out of 3 independent experiments). MHC II CD45 Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 7 of 16 http://www.actaneurocomms.org/content/2/1/27 CNS – Th1 cells and Th17 cells exhibited distinct deficient T helper cells re-isolated from the CNS of integrin requirements for their entry into CNS in VV challenged mice exhibited a Th17 like phenotype the context of a local virus infection. Interestingly, while the number of Th1 like cells was significantly while the absolute number of CD4 T cells was reduced in reduced in α4 CKO animals as compared with controls the CNS, which was largely due to the reduction of IFN-γ (Figure 5D, E). Consistent with their cytokine produc- producing CD4 effector T cells, the number of CTLs tion upon ex vivo stimulation, CNS derived α4-integrin recruited to the CNS compartment was not significantly deficient T helper cells expressed higher amounts of reduced in anti-integrin α4 treated mice (Figure 4C). Th17 signature markers such as Rorc, Il17, Il22, Il6, Thus, integrin α4 mediated mechanisms appeared to be Ccr6, or Il1r1 whereas Th1 associated genes like Cxcr3, redundant for the recruitment of effector CD8 Tcells Ccr5, or Ccr2 prevailed in wild type T helper cells into the infected CNS. (Figure 5F). In conclusion, while Th1 cells appeared to be sufficient to provide host protection in intrathecal Th17 cells fail to clear cerebral VV infection VV infection, Th17 cells failed to control cerebral virus Similar to the murine system, neutralization of α4- replication in the absence of CD8 T cells resulting in integrins by natalizumab in humans mainly blocks CD4 T lethal encephalitis. cells from entering into the CNS compartment but To corroborate that Th1 immunity was sufficient to spares CD8 T cells [23,24]. Yet, intracerebral reconsti- improve the outcome of intrathecal viral infection, we tution of CD4 effector T helper cell responses has established a model of adoptively transferred host pro- been proposed to be necessary for efficient clearance tection using OT-II T cells, which carry a transgenic T of various viruses from the CNS including JC virus cell receptor specific to ovalbumin, in combination with [25]. We wanted to dissect the essential features of an a recombinant VV expressing ovalbumin in infected cells intracerebral T helper cell response that would be able (VV-Ova). Naive OT-II cells were differentiated in vitro to control viral infection. Thus, we combined a system into Th1 cells or Th17 cells followed by transfer into -/- of T cell conditional ablation of α4-integrin expression Rag1 mice, which had been infected intrathecally with + flox/flox (CD4 Cre xItga4 mice, α4 CKO mice) with VV-Ova one day prior to adoptive transfer (Figure 6). antibody mediated depletion of CD8 T cells in order While non-transferred Rag1 deficient recipients and recip- to investigate the differential contribution of Th1 cells ients of Th17 cells rapidly died, Th1 recipients survived vs Th17 cells to host protection in CNS infection in significantly longer than their counterparts (Figure 6A). the absence of CTLs. To exclude possible alterations Despite a significant difference in clinical outcome, equal in priming of antigen specific T helper cell responses amounts of transferred Th1 cells and Th17 cells were re- in the peripheral immune compartment of α4CKO isolated from the CNS of infected mice (Figure 6B). Thus, mice, we compared the fractions of antigen specific T on a per cell basis, antigen specific Th1 cells appeared to cells on day 10 after subcutaneous immunization with be more potent in alleviating i.th. virus infection than - flox/flox MVA/CFA in the spleens of CD4 Cre xItga4 antigen specific Th17 cells. + flox/flox (wild type control) versus CD4 Cre xItga4 (α4 CKO) mice (Additional file 1: Figure S1). Upon ex vivo Th17 cells fail to clear i.th. VV infection due to lack of restimulation with I-A restricted VV epitopes, the perforin-1 expression + + fractions of antigen specific (CD40L )CD4 T cells It was possible that the reduced capacity of Th17 cells to and their cytokine profile were similar in wild type vs provide host protection in intrathecal VV infection was α4 CKO mice. Moreover, the anti-VV neutralizing serum simply due to reduced availability of IFN-γ within the response was equally effective in both groups on day 10 CNS compartment. In order to test this possibility, we after immunization (Additional file 1: Figure S1). Thus, neutralized IFN-γ by monoclonal antibodies in MVA sensitization for adaptive cellular immune responses immune and CD8 T cell depleted wild type mice against VV in draining lymph nodes and spleen was not (Additional file 2: Figure S2). In this purely T helper cell impaired by the lack of α4-integrins on T cells. dependent scenario, VV specific Th1 and Th17 cells Next, we challenged MVA immune and CD8 depleted were generated and had access to the VV challenged wild type and α4 CKO mice with i.th. VV. In contrast to CNS. Notably, the mice recovered from VV encephalitis control littermates, α4 CKO mice rapidly lost weight despite ablation of IFN-γ. Efficiency of IFN-γ blockade and succumbed to infection (Figure 5A). Even in the was documented by reduced levels of MHC class II + + complete absence of CD8 T cells (Figure 5B), CD4 ef- expression on microglial cells in the CNS compartment fector T helper cells were protective in wild type mice (Additional file 2: Figure S2). These data suggested that but failed to control virus replication in the CNS of α4 IFN-γ was redundant as an effector molecule in the CKO mice (Figure 5C). Recapitulating our observations clearance of intrathecal VV infection and refuted the with anti-α4 integrin (PS/2) administration, α4-integrin idea that diminished IFN-γ expression by Th17 cells Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 8 of 16 http://www.actaneurocomms.org/content/2/1/27 [d] -10 0 4 Immunization Challenge Analysis PBS/CFA MVA/CFA Time after challenge (days) p=0.0065 p=0.0293 150,000 600,000 100,000 400,000 50,000 200,000 0 0 PBS/CFA MVA/CFA 0.87 61.1 CD8+ T cells 0.93 0.23 3.4 3.67 CD4+ T cells 53.9 78.3 0 0 0 0.06 CD8+ T cells 95.3 IFN-γ Figure 3 Immunization with modified VV Ankara (MVA) confers protective immunity against viral encephalitis. Wild type mice were immunized subcutaneously with a replication deficient strain of VV (Modified Vaccinia Virus Ankara, MVA) or PBS emulsified in complete Freund’s Adjuvant (CFA). On day 10 after immunization, mice were challenged intrathecally with VV, and weight courses were monitored daily as percentage of initial body weight (A, n = 5 mice per group). On day 4 after infection, CNS infiltrating mononuclear cells were isolated. (B) Absolute numbers of CNS + + infiltrating CD4 and CD8 T cells. Horizontal bars indicate means (Student’s t test; n ≥ 5 per group). (C) CNS derived T cells were analyzed as to their specificity to the VV MHC class I restricted epitope B8R by multimer staining and to their IL-17 and IFN-γ cytokine status by intracellular + + + + cytokine staining in the CD3 CD8 CTL and CD3 CD4 T helper cell compartment, respectively. Numbers indicate percentages of cells in the depicted gates or quadrants; representative out of more than 5 independent experiments). PBS/CFA MVA/CFA PBS/CFA MVA/CFA CD4+ T cells (absolute number, CNS) Fraction of original body weight (%) IL-17 K /B8R CD8+ T cells (absolute numbers, CNS) Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 9 of 16 http://www.actaneurocomms.org/content/2/1/27 -/- was responsible for the failure to clear intracerebral not impaired in Prf1 mice. Moreover, wild type mice VV infection. that were vaccinated with MVA and then depleted of + + In order to define potential molecular mechanisms of CD4 T cells in addition to CD8 T cells succumbed to T helper cell mediated host protection in intracerebral intrathecal challenge with VV although they had similar viral infection, we screened the expression profile of fractions of NK cells and NK T cells in the CNS as + + highly purified CD4 effector T cells isolated from the their CD4 T cell replete counterparts (Additional file 3: CNS of VV challenged wild type vs α4 CKO mice for Figure S3) suggesting that alternative sources of Prf1 molecules directly involved in virus defense (Figure 7A). other than CD4 T cells were insufficient to provide CD4 effector T cells expressed Tnf, Grzmb, and Fasl protection in this model. Taken together, these data irrespective of whether they were derived from the Th1 demonstrated that Prf1 expression was indispensable biased inflammatory infiltrate of wild type mice or from for effector T helper cell mediated control of intra- the Th17 biased inflammatory milieu of α4 CKO mice. thecal VV infection. In contrast, the expression of perforin-1 (Prf1)was markedly reduced in α4-integrin deficient as compared Discussion with wild type T helper cells. In order to correlate lack In this study, we tested the concept that host defense of Prf1 expression with the Th17 transcriptional pro- against viral infections of the CNS requires distinct T + + - - gram, we purified CD3 CD4 CD44 Foxp3 naïve T cells helper cell subsets. We show that access inhibition to from Foxp3gfp.KI mice and stimulated them without the CNS of selected T helper cell subsets by integrin exogenous cytokines (Th0) or differentiated them into targeted interventions results in the failure of host Th1 cells or Th17 cells (Figure 7B). Consistent with our defense. Intrathecal infection with vaccinia virus (VV) + + in vivo data, we found a significant reduction in Prf1 was controlled in vaccinated mice when CD4 and CD8 T mRNA and protein in Th17 as compared with Th1 and cells had access to the CNS compartment. Blockade of Th0 cells. Notably, protein expression of Prf1 was only α4-integrins by neutralizing antibodies or T cell condi- seen at late time points (Figure 7B). Since expression of tional disruption of α4-integrin expression did not prevent Prf1 has been reported to depend on the expression of CTLs and Th17 cells from entering the CNS parenchyma the transcription factor eomesodermin (Eomes) [26], we while Th1 cells were blocked from migrating into the + + measured RNA levels of Eomes in CD4 T cells isolated CNS. When CD8 CTLs were depleted, host defense from brains of VV infected wild type and α4 CKO mice. against intrathecal VV infection was still maintained as Consistent with the low expression of Prf1 in Th17 cells, long as efficient Th1 like responses were operational Eomes mRNA levels were reduced in CD4 T cells iso- within the CNS. In contrast, Th17 cells alone failed to lated from α4 CKO mice as compared with wild type rescue individuals from CNS infection despite a strong in- controls (Figure 7C). To formally validate whether flammatory response. Perforin-1 expression by T helper reduced Eomes expression in Th17 cells accounted for cells was required for clearance of intrathecal VV infection diminished Prf1 expression as compared with Th0 and and Th17 cells failed to eliminate VV in the CNS due to Th1 cells, we polarized Th17 cells in vitro and overex- lack of Eomes dependent Prf1 expression. pressed Eomes by retroviral transduction (Figure 7D). Adaptive cellular immune responses are necessary for + + FACS sorted transduced (GFP ) Th17 cells expressed the clearance of systemic VV infection. CD8 T cells abundant levels of Eomes as compared with control recognize epitopes from early antigens of VV but robust vector proving effective transduction; concomitantly, Prf1 CD4 T cell responses are also required for direct and RNA was significantly increased in Eomes transduced indirect antiviral effects [27-30]. Niche restricted or Th17 cells. These data suggested that the failure of Th17 organ specific infection may afford specific effector func- cells to express Prf1 was due to reduced Eomes expression. tions. For example, VV skin infection is contained and In order to explore the importance of CD4 T cell de- cleared by a temporally and spatially organized inter- + + rived Prf1 for successful host defense in CNS virus infec- action of Ly6G innate immune cells and CD8 CTLs -/- tion in vivo, we immunized Prf1 deficient mice (Prf1 ) [22]. In the ovaries, clearance of VV correlates with the with MVA/CFA and established viral encephalitis in number of effector memory CTLs that are present in the CD8 T cell depleted animals by intrathecal infection ovaries of VV immunized mice before challenge with the with VV (Figure 8). While wild type mice recovered virus [31]. Here, we established an intrathecal infection from i.th. VV challenge, Prf1 deficient mice succumbed model with VV and used this system as an in vivo surro- to viral encephalitis. Numbers and fractions of CNS in- gate to correlate the access of distinct T helper cell sub- filtrating immune cells were comparable between groups sets with successful host defense. Intrathecal infection of (data not shown) indicating that antigen specific priming naive wild type C57BL/6 mice with VV was lethal be- in the peripheral immune compartment and establish- cause adaptive antiviral immune responses did not de- ment of inflammatory infiltrates within the CNS were velop upon i.th. infection alone although innate immune Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 10 of 16 http://www.actaneurocomms.org/content/2/1/27 PBS/CFA + rIgG MVA/CFA + rIgG rIgG / PS/2 MVA/CFA + PS/2 [d] -10 -1 0 1 3 5 Immunization Challenge Time after challenge (days) PBS/CFA + rIgG MVA/CFA + rIgG MVA/CFA + PS/2 CD4+ T cells 3.85 0.58 11.4 11.7 16.8 11.6 12.7 59.4 48.8 IFN-γ p=0.026 ns 80,000 20,000 60,000 15,000 40,000 10,000 20,000 5,000 0 0 p= 0.049 ns p=0.003 p=0.042 150,000 1,500 100,000 1,000 50,000 500 0 0 Figure 4 Antibody mediated blockade of integrin α4 prevents entry of Th1, but not Th17 cells or CD8 T cells into the CNS. Wild type mice were sham immunized (PBS/CFA) or immunized with MVA/CFA followed by intrathecal VV challenge on day 10 after immunization. Control IgG or antibodies to integrin α4 (PS/2) were administered every other day starting one day prior to VV challenge. (A) Weight courses of VV challenged animals (n = 5 per group). (B, C) On day 4 after infection, CNS infiltrating mononuclear cells were isolated and the absolute number of + + + CD4 Thelper cells and CD8 CTLs as well as the cytokine phenotype of CD4 T helper cells from the CNS were assessed based on intracellular staining for IFN-γ and IL-17. (B; numbers indicate percentages of CD4 T cells in each quadrant; representative out of 5 independent experiments. The absolute numbers of T cells were compared by Student’sttest, n ≥ 3). + high cells and in particular CD11b CD45 monocytic cells Migration of effector cells into the CNS is mediated by were recruited to the infected CNS. Active immunization chemokine receptors and integrins. Several cytolytic and of mice with MVA in CFA prior to i.th. VV challenge non-cytolytic viral infection models of the CNS were protected the animals from lethal encephalitis. Protec- used to investigate principles of immune cell recruit- tion was independent of neutralizing antibodies. How- ment into the CNS. Depending on the viral agent, ever, access of VV specific Th1 cells into the CNS immune cell recruitment into the CNS cleared the compartment was necessary and sufficient for host pathogen or in addition, caused immunopathogenic tis- protection. sue damage. CCR5 and CXCR3 were identified to be PBS/CFA + PBS MVA/CFA + rIgG MVA/CFA + PS/2 PBS/CFA + PBS MVA/CFA + rIgG PBS/CFA + PBS MVA/CFA + PS/2 MVA/CFA + rIgG MVA/CFA + PS/2 PBS/CFA + PBS MVA/CFA + rIgG MVA/CFA + PS/2 CD4+ T cells IFN-γ+ IL-17- CD4+ T cells (absolute number, CNS) (absolute number, CNS) IL-17 Fraction of original body weight (%) CD8+ T cells IL-17+ IFN-γ- CD4+ T cells -3 (absolute number x 10 , CNS) (absolute number, CNS) Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 11 of 16 http://www.actaneurocomms.org/content/2/1/27 A anti-CD8 B rIgG anti-CD8 [d] -10 0 2 46 8.9 0 Immunization Challenge WT 72 86 WT MVA/CFA + anti-CD8 α4 CKO MVA/CFA + anti-CD8 5.3 0 80 α4 CKO 81.9 87.4 CD4 02468 10 Time after challenge (days) p=0.0092 p=0.021 2.5 500,000 2.0 400,000 300,000 1.5 200,000 1.0 100,000 5.0 0 0 E F Tbx21 Rorc Ifng p=0.008 40,000 ns Il17 Il22 300,000 Il6 30,000 Cxcr3 200,000 Ccr5 20,000 Ccr2 Ccr6 100,000 10,000 Il23r Il1r1 0 0 -2 0 2 Fold change in relative expression (α4 CKO/W T CNS, log , n=4) Figure 5 VV specific Th17 cells are relatively enriched in the CNS compartment of α4 CKO mice but do not control i.th. VV infection. - flox/flox + CD4 Cre x Itga4 mice (controls, WT) and α4 CKO mice were immunized with MVA/CFA, depleted of CD8 T cells, and challenged i.th. with VV. (A) After intrathecal infection with VV, the clinical course was monitored and weight loss was calculated as percentage of initial body weight (n = 5 per group). On day 5 after infection, mice of each group were sacrificed. (B) The efficacy of CD8 T cell depletion was analyzed by flow + - cytometry in CNS mononuclear cells (gate on CD3 CD19 cells). (C) The viral load in the CNS was measured by means of plaque assay. CNS + + infiltrating CD4 T cells (D) and absolute numbers of cytokine positive CD4 T cells (E) were calculated by surface staining and intracellular cytokine analysis (Student’s t test, n ≥ 4 per group, representative out of n ≥ 4 independent experiments). (F) CD4 T cells were isolated by flow cytometric purification from the CNS compartment of MVA immunized wild type vs α4 CKO mice 5 days after i.th. VV challenge. The expression profile of the indicated genes was determined by quantitative PCR analysis, n = 4. important for immune cell recruitment into the CNS in cells for the transmigration of immune cells across the west nile or MHV and LCMV infection, respectively blood brain barrier [36]. Early work suggested that accu- [32-35]. Chemotaxis of immune cells must be comple- mulation of antigen specific T cells in the CNS compart- mented by integrin mediated interaction with endothelial ment of sindbis virus infected animals is mediated by WT anti-CD8 α4 CKO anti-CD8 WT + anti-CD8 WT anti-CD8 α4 CKO+ anti-CD8 α4 CKO anti-CD8 WT + anti-CD8 α4 CKO + anti-CD8 IFN-γ+ IL-17- CD4+ T cells (absolute number, CNS) -6 Fraction of original PFU x 10 / g CNS tissue body weight (%) CD4+ T cells IL-17+ IFN-γ- CD4+ T cells (absolute number, CNS) (absolute number, CNS CD8 Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 12 of 16 http://www.actaneurocomms.org/content/2/1/27 Infection of Adoptive transfer of -/- Rag1 mice Th1 or Th17 cells [d] -1 0 ns p=0.0006 p=0.0165 100 No transfer Th1 80 8 Th17 40 6 20 5 No transfer Th1 Th17 Time after infection (days) 30,000 6,000 20,000 4,000 10,000 2,000 No transfer Th1 Th17 No transfer Th1 Th17 + - Figure 6 Adoptive transfer of antigen specific Th1 cells but not Th17 cells promotes survival after intrathecal virus infection. Naïve CD4 CD25 CD44 Tcells from OT-II Ova specific TCR transgenic mice were polarized under Th1 or Th17 conditions in vitro.Onday four of 323-339 -/- differentiation, activated T cells were transferred into Rag1 mice, which had been infected with a recombinant VV expressing ovalbumin (VV-Ova) one day earlier. (A) Kaplan-Meier curves of survival (left) and statistical evaluation of time to death (right) are shown. Horizontal bars in the right plot indicate mean survival time in days (ANOVA, n ≥ 5per group). (B) Mice were sacrificed on day 6 after transfer and absolute numbers of adoptively + + transferred CD3 CD4 T cells in spleens and CNS were assessed (means are indicated by horizontal bars, n = 3 per group). engagement of LFA-1 but not VLA-4 [37]. Using gen- systemic lymphoid compartment prior to intracerebral etic tools, we have recently shown that distinct T helper challenge [42]. In contrast to Th17 cells, VLA-4 expres- cell subsets use distinct integrins to access the CNS sion is indispensable and non-redundant for the capacity compartment in autoimmune inflammation. The entry of antigen experienced Th1 cells to infiltrate into the of Th17 cells into the subarachnoid space most likely subarachnoid space and the CNS compartment. Here, requires CCR6 and LFA-1 but is independent of VLA-4 we provideevidencethatMHC classIIpositiveinfected [4,38]. In fact, Th17 cells express low levels of α4- phagocytic cells in the CNS need to be cleared by Th1 integrins since TGF-β, which is required for Th17 cell cells in order to rescue mice from lethal encephalitis. commitment [17,39,40], inhibits the expression of α4- Thus, MVA-immune T cell conditional α4 KO mice integrins [41]. Interestingly, antigen specific CD8 succumbed to intrathecal VV challenge because they CTLs also migrated into the CNS independently of α4- failed to recruit Th1 cells into the CNS despite regular integrins (both in α4-integrin antibody blockade and T Th17 cell accumulation. cell conditional genetic disruption of Itga4). This is Antigen specific CD4 T cells communicated with consistent with the observation that CD4 Tcell are virus infected macrophages within the CNS parenchyma preferentially reduced in CSF of natalizumab treated and only eliminated the infected cells when they were patients as compared with CD8 T cells resulting in the sufficient in perforin-1 expression. In ectromelia infec- + + reversion of the CSF CD4 /CD8 ratio [23]. Although tion, a natural mouse pox infection, perforin-1 expres- + + one report suggested that CD8 T cell influx into the CNS sion by CD4 T cells is required to clear the pathogen, was prevented by a blocking antibody to α4-integrins in and CD4 T cell mediated killing of hematopoietic MHC an intracerebral corona virus infection model, it is unlikely class II expressing infected target cells occurs in the that the recruitment of antigen specific CTLs was assessed draining lymph nodes, the liver, and the spleen [28]. Dir- by Ifergan et al. since their model did not allow for active ect cytotoxic effects of antigen specific CD4 T cells priming and activation of adaptive T cell responses in the were also reported in systemic VV infection [43]. In our OT-II T cells (absolute number, spleen) Percent survival OT-II T cells (absolute number, CNS) Survival time (days) Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 13 of 16 http://www.actaneurocomms.org/content/2/1/27 Tnf Prf1 Grzmb Fasl -2 -1 0 1 Fold change in relative expression (α4 CKO/W T CNS, log , n=4) d3 d4 d5 B MW (kD) Th0 Th1 Th17 Th0 Th1 Th17 Th0 Th1 Th17 p<0.0001 0.5 0.4 Prf1 0.3 0.2 0.1 0.0 38 Th0 Th1 Th17 Actin C D p=0.0193 p=0.0006 7.2 p=0.0011 1.5 0.8 pMIG 0.6 1.0 0.4 0.20 0.5 0.2 0.10 5.9 0.0 0.00 0.0 pMIG Eomes GFP + + Figure 7 Th17 cells fail to control VV encephalitis due to lack of Prf1 expression. (A) CD3 CD4 T cells were highly purified by FACS sorting from the CNS of MVA/CFA immunized wild type or α4 CKO mice on day 4 after intrathecal infection. Fold change in relative expression of the indicated genes in α4 deficient vs. wild type control CD4 T cells (log scale, means + SD, n = 4). (B) Flow cytometrically purified naïve + + - - CD3 CD4 CD44 Foxp3 T cells were stimulated without exogenous cytokines (Th0) or differentiated into Th1 cells (IL-12 + anti-IL-4), or Th17 cells (TGF-β + IL-6). Levels of Prf1 RNA expression were measured by quantitative RT-PCR (left in B, means + SD, Student’st test,n = 4). Time course of Prf1 protein expression was determined by Western Blot in Th0, Th1, and Th17 cells from day 3 to day 5 of culture (right in B). (C) RNA levels of Eomes were measured in wild type and α4 integrin deficient T cells isolated from the CNS of VV infected mice (means + SD, n = 4). (D) Naïve T cells were differentiated into Th17 cells in vitro and transduced retrovirally with a control vector (pMIG) or an Eomes GFP vector (pMIG Eomes). GFP cells were purified by flow cytometry and RNA levels of Eomes and Prf1 were measured in control transduced Th17 cells vs Th17 cells that ectopically expressed Eomes (means + SD, n = 4). anti-CD8 WT (+anti-CD8) Prf1-/- (+anti-CD8) [d] -10 0 2 46 Immunization Challenge Time after challenge (days) -/- -/- Figure 8 Prf1 mice succumb to VV encephalitis. Wild type and Prf1 mice were immunized with MVA/CFA and continuously depleted of CD8 T cells after intrathecal VV challenge. Weight courses of infected animals (n = 5 per group). WT α4 CKO pMIG (GFP) pMIG Eomes (GFP) pMIG (GFP) pMIG Eomes (GFP) Eomes (rel. expression) in CNS CD4+ T cells Prf1 (rel. expression) Fraction of original body weight (%) Eomes (rel. expression) Prf1 (rel. expression) Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 14 of 16 http://www.actaneurocomms.org/content/2/1/27 model, T cell conditional α4-integrin deficient mice be- a latent virus infection of oligodendrocytes. However, the haved like perforin-1 deficient animals and succumbed intracerebral anti-JC virus CD4 T cell response contributes to infection because perforin-1 expressing Th1 cells did substantially to JCV control because efficient clearance of not reach the CNS compartment in the absence of VLA- JC virus from the CNS after immune reconstitution by 4. Like in EAE, the cytokine phenotype of T helper cells wash-out of natalizumab in MS patients with PML is asso- that entered into the CNS compartment in the absence ciated with the re-occurrence of Th1 cells in the CNS [53]. of α4-integrin showed a Th17 profile [4]. Th17 cells did not express perforin-1. Initial Th17 cell commitment is Conclusions sensitive to IL-2 and high IL-2 concentrations inhibit Our experimental model provides a mechanistic explanation Th17 differentiation [44]. In contrast, IL-2 promotes the for insufficient virus control under conditions of altered T expression of granzyme B and perforin-1 and boosts cell migration. Our data raise the concern that treatment of cytotoxic capacity of CD4 T cells [45]. While the tran- humans with compounds like natalizumab that interfere scriptional control of granzyme B and perforin-1 has in with T cell trafficking not only by quantitatively reducing the part been investigated in CD8 T cells [46], we found migration of T cells into the CNS but also by selectively tar- that perforin-1 expression in Th1 cells is associated with geting distinct T cell subsets may evoke serious deficiencies Eomes and overexpression of Eomes in Th17 cells par- in cellular immune responses at privileged anatomical sites tially reconstituted perforin-1 expression. Similar obser- like the CNS. As a result, primary infections or unexpected vations have been reported for Th2 cells that per se also reactivations of latent viruses may be facilitated. lack perforin-1 [26], which correlates with their low cytotoxic efficacy. However, lack of perforin-1 expression Supporting data in Th17 cells may not be the only mechanism to impair The data sets supporting the results of this article are their antiviral capacity. For example, Th17 cells are able included within the article and its additional files. to mount a protective response in primary lung infection with influenza in IL-10 KO mice suggesting that Th17 Additional files cells are particularly susceptible to IL-10 mediated sup- Additional file 1: Figure S1. α4 integrin deficiency in T cells does not pression [47]. In addition, induction of prosurvival fac- alter priming or humoral responses against VV after immunization. (A) CD4 tors by Th17 cells may contribute to viral persistence in - flox/flox + flox/flox Cre x Itga4 mice (wild type control (WT)) or CD4 Cre xItga4 the CNS in Theiler’s virus infection [48]. Conversely, it mice (α4CKO)wereimmunizedwith MVA/CFA.Onday 10 after immunization, splenocytes were isolated and restimulated with an I-Ab is possible that in some anatomical niches, Th17 cells restricted peptide epitope mix of VV. Fractions of antigen specific cytokine may contribute to protective antiviral immune responses, producing CD4 T cells were determined by intracellular CD40L (CD154) e.g. by effective IL-17 mediated recruitment of neutrophils and cytokine staining. Numbers in the gates represent means ± SD, n = 3. (B) Specific serum neutralization capacity of sera collected on day 10 in lung infection [49]. Also, IL-21, which is produced by after immunization of wild type or α4 CKO mice vaccinated with PBS/CFA Th17 cells, might have a role in the modulation of primary or MVA/CFA (specific neutralization capacity in percent of maximum, and recall CTL responses by increasing their proliferation means + SD, n = 5). and reducing their sensitivity to exhaustion [50]. However, Additional file 2: Figure S2. IFN-γ expression in CD4 T cells is not required for virus control in the CNS. (A) Wild type mice were immunized overall it is a more common scenario that antiviral Th17 with MVA/CFA and challenged intrathecally with VV on day 10. Starting responses fail to clear viral pathogens but rather drive one day before VV challenge, neutralizing antibodies to IFN-γ (or control deleterious immunopathology in the skin [51], in the IgG) and depleting antibodies to CD8 were applied alternatingly every other day. Weight courses of infected animals were measured every day cornea [52], and also in the CNS. after infection and depicted relative to initial body weight (n = 5 per We have shown that blockade or genetic lack of α4- group). (B) Expression of MHC class II on CNS microglial cells (gating on int int integrins on T cells selectively modulated the access of T CD11b CD45 , upper panel) was measured on day 6 after infection using surface staining and flow cytometric analysis. Numbers indicate helper cell subsets to the CNS. Preferential limitation of percentages. Histogram: black line: MHC class II expression on migroglia Th1 trafficking jeopardized host defense against intrathecal from control IgG treated mice, grey/shaded line: MHC class II expression VV infection. Nevertheless, our experimental system has on microglia from anti-IFN-γ treated mice. The numbers in the histogram represent mean fluorescence intensities. Representative several limitations: First, i.th. inoculation of VV does not out of 3 independent experiments. represent a natural route of CNS infection. Yet, Additional file 3: Figure S3. CD4 T cells mediate protection against immunization with virus antigens in CFA provides an VV encephalitis in the absence of CD8 T cells. Wild type mice efficient way to generate both an antigen specific Th1 and immunized with MVA/CFA were challenged i.th. with VV and depleted of CD8 T cells. Concomitantly, animals were treated with isotype control or Th17 response in the secondary lymphoid tissue and to depleting antibodies to CD4 every other day. (A) Percentage of surviving study their trafficking and effector functions upon i.th. chal- mice are depicted in Kaplan-Meier curves of survival (n = 6 per group). lenge with a well characterized pathogen. Second, i.th. VV (B) CNS infiltrating mononuclear cells were analyzed by flow cytometry. Upper panel: Staining for CD4 and CD8 in the CD3 T cell gate. Lower infection results in an acute encephalomyelitis, which panel: Staining for CD3 and Nk1.1 in the CD4 gate (representative out of causes rapid decay and death of infected animals; in con- 4 independent experiments). trast, JC virus induced PML is due to insufficient control of Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 15 of 16 http://www.actaneurocomms.org/content/2/1/27 Competing interests 10. Miller DH, Weber T, Grove R, Wardell C, Horrigan J, Graff O, Atkinson G, Dua P, The authors declare that they have no competing interests. Yousry T, Macmanus D, Montalban X: Firategrast for relapsing remitting multiple sclerosis: a phase 2, randomised, double-blind, placebo-controlled trial. Lancet Neurol 2012, 11:131–139. doi:10.1016/S1474-4422(11)70299-X. Authors’ contributions 11. Hueber W, Patel DD, Dryja T, Wright AM, Koroleva I, Bruin G, Antoni C, TK, VR and AM designed the study and wrote the manuscript. VR and AM Draelos Z, Gold MH, Psoriasis Study Group, Durez P, Tak PP, Gomez-Reino JJ, contributed equally, carried out all the experiments and where herein Rheumatoid Arthritis Study Group, Foster CS, Kim RY, Samson CM, Falk NS, supported by GG, FP, and SH. DHB provided fluorescence-labeled MHC class I Chu DS, Callanan D, Nguyen QD, Uveitis Study Group, Rose K, Haider A, Di H-2K /B8R (TSYKFESV) multimers. Histological analyses were carried out with 20-27 Padova F: Effects of AIN457, a fully human antibody to interleukin-17A, the support of MH’s group. GG, DHB, MH, BH and ID gave conceptional input on psoriasis, rheumatoid arthritis, and uveitis. Sci Transl Med 2010, and revised the manuscript. All authors read and approved the final manuscript. 2:52ra72. doi:10.1126/scitranslmed.3001107. 12. Leonardi C, Matheson R, Zachariae C, Cameron G, Li L, Edson-Heredia E, Acknowledgments Braun D, Banerjee S: Anti-interleukin-17 monoclonal antibody ixekizumab TK is recipient of a Heisenberg award by the Deutsche in chronic plaque psoriasis. N Engl J Med 2012, 366:1190–1199. Forschungsgemeinschaft (DFG, KO2964/3-2). This work was supported by doi:10.1056/NEJMoa1109997. SyNergy (Munich Cluster of Systems Neurology) and by other grants from 13. Yazici Y, Curtis JR, Ince A, Baraf H, Malamet RL, Teng LL, Kavanaugh A: the DFG (SFB1054/B06, TR128/A06, A07) as well as by intramural funding Efficacy of tocilizumab in patients with moderate to severe active of the Klinikum rechts der Isar (KKF B11-11). MH was supported by the rheumatoid arthritis and a previous inadequate response to Helmholtz foundation. disease-modifying antirheumatic drugs: the ROSE study. Ann Rheum Dis 2012, 71:198–205. doi:10.1136/ard.2010.148700. Author details 14. 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α4-integrins control viral meningoencephalitis through differential recruitment of T helper cell subsets

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Springer Journals
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Copyright © 2014 by Rothhammer et al.; licensee BioMed Central Ltd.
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Biomedicine; Neurosciences; Pathology; Neurology
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2051-5960
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10.1186/2051-5960-2-27
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24606807
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Abstract

Introduction: Natalizumab blocks α4-integrins and is a prototypic agent for a series of anti-inflammatory drugs that impair trafficking of immune cells into the CNS. However, modulation of the access of immune cells to the CNS is associated with impaired immune surveillance and detrimental viral infections of the CNS. Here, we explored the potency of cellular immune responses within the CNS to protect against viral encephalitis in mice with T cell conditional disruption of VLA-4 integrin (α4β1) expression. Results: While VLA-4 expression in virus specific Th1 cells is non-redundant for their ability to access the CNS, α4-integrin deficient Th17 cells enter the CNS compartment and generate an inflammatory milieu upon intrathecal vaccinia virus (VV) infection. However, in contrast to Th1 cells that can adopt direct cytotoxic properties, Th17 cells fail to clear the virus due to insufficient Eomes induced perforin-1 expression. Conclusion: The quality of the intrathecal cellular antiviral response under conditions of impaired VLA-4 function jeopardizes host protection. Our functional in vivo data extend our mechanistic understanding of anti-viral immunity in the CNS and help to estimate the risk potential of upcoming therapeutic agents that target the trafficking of immune cells into distinct anatomical compartments. Introduction While Th1 cells maintain high amounts of VLA-4 expres- Autoimmune inflammation of the CNS in multiple scler- sion, Th17 cells are low in VLA-4. As a consequence, osis (MS) and its animal model, experimental autoimmune blockade of VLA-4 is more efficient in preventing the encephalomyelitis (EAE), is mediated by antigen specific recruitment of Th1 cells than of Th17 cells into the Th1 and Th17 cells [1]. For many years integrin targeted CNS parenchyma. blocking of T helper cell trafficking into the CNS has Although considered as an “immune privileged” organ, appeared to be an attractive approach to treat immuno- the CNS is still patrolled by T cells as a means of immune + + pathologyinMS[2].Inparticular, monoclonal anti- surveillance [5]. The contribution of CD4 vs CD8 bodies (natalizumab) to the α4 subunit of the integrin effector memory T cells in the migratory and resident VLA-4 (α4β1 heterodimer) were successfully used to pools of lymphocytes specific to a given pathogen has prevent the influx of immune cells into the CNS and to been investigated in skin infection but is unclear in the treat CNS autoimmunity [3]. However, in experimental CNS [6]. In the treatment of organ specific autoimmunity models it has been shown that distinct encephalitogenic and chronic inflammation, efforts are increasing to market T cell subsets vary in their equipment with VLA-4 [4]. compounds that either inhibit immune cell trafficking [7-10] or cytokine networks that affect distinct T helper cell subsets in a differential manner (anti-IL-23p19, anti- * Correspondence: korn@lrz.tum.de † IL-17A [11,12], anti-GM-CSF (NCT01517282), anti-IL-6R Equal contributors [13]). However, preclinical models to investigate niche Klinikum rechts der Isar, Department of Neurology, Technische Universität München, Ismaninger Str. 22, 81675 Munich, Germany specific immune surveillance and host defense in the CNS Munich Cluster for Systems Neurology (SyNergy), Munich, Germany are rare. Indeed, efalizumab, a blocking antibody to the Full list of author information is available at the end of the article © 2014 Rothhammer et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 2 of 16 http://www.actaneurocomms.org/content/2/1/27 integrin αL was withdrawn from the market in 2009 cisterna magna of mice in deep anaesthesia by means of because of viral meningitis and cases of JC virus induced transcutaneous suboccipital puncture. Clinical signs of progressive multifocal leukencephalopathy (PML) [14]. disease as well as weight loss in percent of initial weight Here, we established a CNS specific viral infection (means + SEM) were monitored daily. model that allowed us to analyze the contribution of dis- For adoptive transfer experiments, naïve T cells were + + tinct T helper cell subsets to host protection. We chose isolated by magnetic sorting (CD4 CD62L ; T cell isola- vaccinia virus (VV) infection where the importance of tion kit II, mouse; Miltenyi Biotec, Germany) from virus specific T helper cell responses has been analyzed CD45.1 OT-II mice and differentiated in vitro into Th1 previously [15,16]. Vaccinated mice were found to be or Th17 cells. The differentiation status was checked on protected from intrathecal (i.th.) infection with VV due day 4 by intracellular cytokine staining and 2 × 10 cyto- + -/- to cellular immunity. In the absence of CD8 T cells, kine positive T cells were injected i.v. into Rag1 recipi- Th1 like cells were sufficient to protect mice from intra- ent mice, which had been infected with VV-Ova one day thecal VV infection. Access of Th1 cells into the infected prior to T cell transfer. CNS compartment was dependent on VLA-4 expression. Animals were kept in a specific pathogen-free facility Although virus specific Th17 cells were able to migrate at the Technische Universität München. All experimen- into the CNS in the absence of VLA-4, CNS recruited tal protocols were approved by the standing committee and infected macrophages were not cleared by Th17 for experimentation with laboratory animals of the cells since Th17 cells – in contrast to Th1 cells – were Bavarian state authorities (“Governmental Department deficient in perforin-1 expression. These data highlight of Upper Bavaria, Approved animal experimental pro- a dominant role of Th1 cells in antiviral tissue-specific posals No 55.2-1-54-2531-88-08 and No 55.2.1-54-2532- immunity. Our data further suggest that as in auto- 29-13 according to §8.1, German law for experimentation immune inflammation of the CNS, virus specific Th1 with laboratory animals”) and carried out in accordance cells are dependent on VLA-4 to enter into the CNS with the corresponding guidelines. and virus infection does not overcome the requirement for Th1 cells to express VLA-4. Thus, integrin targeted Virus strains therapeutic interventions in autoimmunity and chronic Replication competent VV Western Reserve strain (VV) inflammation need to be refined in order to not was provided by B Moss (National Institutes of Health, jeopardize organ specific immune surveillance and Bethesda, MD). Recombinant viruses encoding en- host protection. hanced green fluorescent protein (VV eGFP) or full- length ovalbumin (VV-Ova) based on the Western Materials and methods Reserve strain were provided by JW Jewdell and JR Ben- Animals, immunization, and infection nink (National Institutes of Health, Bethesda, MD). VV flox/flox Foxp3gfp.KI mice [17,18] and Itga4 mice [19] have and replication deficient Modified Vaccinia Virus -/- been described previously. CD4 Cre mice, Ifng mice, Ankara strain (MVA, cloned isolate IInew) used in this -/- -/- Rag1 mice, Prf1 mice, and wild type C57BL/6 mice were studywerepropagatedand titeredaccording to stand- obtained from Jackson Laboratories. CD45.1 OT-II mice ard methodology [21]. were kindly provided by DH Busch (Institute for Medical Microbiology, Immunology, and Hygiene, Technische T cell differentiation + high - Universität München). All mouse strains were on pure Naïve T cells (CD4 CD62L CD25 ) were isolated from C57BL/6 background. lymph nodes and spleen by magnetic sorting (T cell iso- Mice were immunized by subcutaneous injection of lation kit II, mouse; Miltenyi Biotec, Germany). Purity 100 μl of an emulsion of 1 × 10 IU MVA or PBS in was in general higher than 95% as controlled by FACS complete Freund’s adjuvant (CFA). For in vivo blockade staining. Naïve T cells were stimulated for 3 to 5 days of IFN-γ, mice were treated with every other day i.p. in- with plate-bound antibody to CD3 (145-2C11, 4 μg/ml) jections of a neutralizing antibody to IFN-γ (R4-6A2, and antibody to CD28 (PV-1, 2 μg/ml). Recombinant BioXCell, West Lebanon, USA; 200 μg) or isotype control cytokines were added to the differentiation cultures as starting on day 9 after immunization. In a similar regimen, indicated: human TGF-β1 (2 ng/ml) and mouse IL-6 blocking antibodies to integrin α4 (PS/2, BioXCell, West (50 ng/ml) for Th17, mouse IL-12 (10 ng/ml) and anti- Lebanon, USA; 200 μg), depleting antibodies to CD8 IL-4 (10 μg/ml) for Th1, all R&D Systems. (YTS169.4, BioXcell; 200 μg) or CD4 (GK1.5, BioXcell; 200 μg) were administered every other day from day 9 or Preparation of splenic and CNS mononuclear cells and day 10 after immunization, respectively. antibody staining Intrathecal infection was performed as previously Mononuclear cells were isolated from either spleen or described [20]. In brief, VV was inoculated into the CNS at the peak of disease (d4-d5 after intrathecal Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 3 of 16 http://www.actaneurocomms.org/content/2/1/27 challenge). After perfusion through the left cardiac ven- Quantitative PCR analysis tricle with cold PBS, the brain including cerebellum was For quantitative PCR, RNA was extracted from magnetic dissected and the spinal cord flushed out with PBS by bead-purified or flow cytometry-sorted cells ex vivo or hydrostatic pressure. CNS tissue was digested with colla- after in vitro differentiation using RNeasy columns (Qiagen, genase D (2.5 mg/ml, Roche Diagnostics, Indianapolis Valencia, CA). Complementary DNA was transcribed as IN) and DNAseI (1 mg/ml, Sigma, Saint Louis, MO) at recommended (Applied Biosystems, Foster City, CA) and 37°C for 45 min. Mononuclear cells were isolated by used as template for quantitative PCR. Primer plus probe passing the tissue through a cell strainer (70 μm) and mixtures were obtained from Applied Biosystems. The percoll gradient (37% over 70%) centrifugation. Mono- Taqman analysis was performed on a StepOne system nuclear cells were removed from the interphase, washed from Applied Biosystems. The gene expression was and resuspended in culture medium for further analysis. normalized to the expression of β-actin. For isolation of mononuclear cells from spleen, spleens were mashed through a cell strainer (70 μm) and red Western blotting blood cells were removed using BD Pharm Lyse (BD T cells were lysed and denatured using RiPA buffer Biosciences). Surface staining of T cells was carried out (Sigma-Aldrich). The protein lysates were separated by with antibodies to CD3 (14-2C11), CD4 (RM4-5), CD8 SDS-PAGE in 4–12% NuPAGE Bis-Tris Mini gels and (53-6.7), CD11b (M1/70), CD25 (PC61 or 7D4), CD44 transferred to nitrocellulose membranes (Invitrogen). (IM7), CD45 (30-F11) and Nk1.1 (PK136). All antibodies After blocking with 5% low-fat dry milk in TBS-T, were purchased from BD Biosciences. Fluorescence- membranes were incubated with primary antibodies to labeled MHC class I H-2K /B8R (TSYKFESV) mul- Prf-1 (ab7203, Abcam) and β-actin (Abcam) in blocking 20-27 timers were provided by DH Busch. solution overnight at 4°C. Primary antibody binding was detected with HRP-conjugated secondary antibodies Intracellular cytokine staining (Dianova). The signal was visualized by enhanced chemi- Cells were stimulated in culture medium containing luminescence (Novex ECL, Invitrogen). phorbol 12-myristate 13-acetate (PMA, 50 ng/ml, Sigma), ionomycin (1 μg/ml, Sigma), and monensin (GolgiStop Antigen specific proliferative and cytokine responses 1 μl/ml, BD Biosciences) at 37°C and 10% CO for For CD154 (CD40L) staining, spleens from MVA immu- - flox/flox + flox/flox 4 hours. After staining of surface markers, cells were nized CD4 Cre × Itga4 or CD4 Cre × Itga4 fixedand permeabilized(Cytofix/Cytoperm andPerm/ mice were dissected on day 10 after immunization. Sin- Wash buffer, BD Biosciences) followed by staining with gle cell suspensions were prepared and cells were seeded monoclonal antibodies to mouse IL-2, IL-17, or IFN-γ on a 12 well flat-bottom plate at a density of 2 × 10 (BD Biosciences) and flow cytometric analysis (CYAN, cells/well. Single cells were restimulated with a mixture Beckmann/Coulter). of I-A -restricted VV peptides (A33R, B2R, B5R, L4R; 30 μg/ml each) for 6 hours in the presence of brefeldin Histologic analysis A(5 μg/ml) during the last 3 hours of incubation followed For detection of VV-infected cells or macrophages, para- by surface and intracellular staining for CD40L (CD154 formaldehyde (PFA) (4%) fixed and paraffin embedded (MR1), eBioscience) and cytokines as indicated. A33R 116-130 CNS tissue sections were incubated with Bond Primary (YQLFSDAKANCTAES), B2R (VKDKYMWCYSQ 46-60 Antibody Diluent (Leica) containing either polyclonal VNKR), B5R (FTCDQGYHSSDPNAV) and L4R 46-60 176-190 rabbit anti-VV serum (1:1000; Quartett Immunodiagnos- (ISKYAGINILNVYSP) were obtained from Biosyntan, tika & Vertriebs-GmbH, Berlin) or monoclonal anti- Berlin. bodies against Mac-3 (1:750; M3/84) purchased from BD Pharmingen. IHC staining was performed on an auto- Retroviral transduction of T cells mated Leica BOND-MAX instrument using Bond Polymer pMIG Eomes (GFP) and control pMIG (GFP) retroviral Refine Detection Solution for DAB. For detection of GFAP, constructs were a kind gift from SL Reiner (University of PFA-fixed and paraffin-embedded CNS sections were Pennsylvania, Philadelphia, PA) and FJ Quintana (Harvard incubated with Dako polyclonal rabbit anti-GFAP anti- Medical School, Boston, MA). Phoenix-Eco cells (PMID: bodies (Z0034; 1:13000) in Ventana buffer and staining 18432682, a kind gift of H-M Jaeck, Erlangen) were transi- was performed on a Ventana NexES IHC Slide Stainer ently transfected with plasmids by calcium phosphate pre- using iVIEW DAB Detection Kit (Ventana). Images cipitation in the presence of 25 μM chloroquine (Sigma were taken using the Leica SCN400 slide scanner ana- Aldrich). Retroviral supernatants were collected two days lysis software or were acquired on an Olympus BX53 post transfection. T cells were transduced 24 hours post Microscope (DP72 camera) using the cellSens 1.8 polyclonal in vitro activation of naïve sorted CD4 Tcells digital imaging software (Olympus). with TGF-β and IL-6 (Th17). Retroviral supernatant and Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 4 of 16 http://www.actaneurocomms.org/content/2/1/27 4 μg/ml polybrene (Merck Millipore) were added to the CNS and ovaries of mice injected i.th. with 1.000 PFUs pre-committed Th17 cells and a spin transduction was of VV. While high titers of VV were measured in the performed (2000 rpm, RT, 1 hour). Cells were further CNS, virus was not detected in the ovaries of intra- cultured for 3 days in Th17 polarizing conditions before thecally infected mice (Figure 1B) indicating that intra- GFP expressing cells were purified by FACS sorting and thecal injection of VV led to a compartmentalized subjected to quantitative PCR analysis. infection within the CNS without systemic dissemin- ation. Within the CNS compartment, VV antigen was Plaque assay detected in ventricular lining cells and plexus epithe- CNS tissue was digested as described and single cell sus- lium cells as well as in astrocytes of the glia limitans. pensions were obtained by passing digested CNS tissue Some innate immune cells of monocytic origin that through a 70-μm nylon filter. Pelleted cells were sub- were recruited to the infected CNS compartment were jected to three freeze-thaw cycles (-80°C and 37°C) and also positive for VV antigen (Figure 1C). sonicated three times for 1 min. Serial dilutions in In order to determine immune cell targets of VV RPMI-1640 medium containing 10% FCS were added in within the CNS, we infected wild type mice with recom- duplicates to 90% confluent RK-13 cells seeded in 6-well binant VV expressing enhanced green fluorescent pro- plates and incubated for 24 hours at 37°C. Plaques were tein (VV eGFP). When analyzing eGFP expression in counted after crystal violet staining. distinct cell populations of the CNS on day 4 after infec- tion, we found a sizable fraction of eGFP expressing cells high high Neutralization assay only within the CD11b CD45 macrophage com- Mouse serum samples were collected at day 10 post partment (Figure 2). This is in line with a recent study + + immunization (MVA/CFA or PBS/CFA). All serum sam- which identified infiltrating CD11b CD45 inflammatory ples were heat-inactivated at 56°C for 30 min prior to monocytes as the predominant VV infected leukocyte testing. Serum dilutions were incubated with 10,000 population during VV skin infection [22]. Microglial + low PFU of sucrose gradient purified VV eGFP for 1 hour at cells (CD11b CD45 ), T cells and B cells did not show 37°C and 5% CO in a 96-well plate. RK-13 cells were eGFP expression. In addition, we were unable to detect added and infection (MOI = 0.1) was carried out over eGFP expressing cells in the spleens of intrathecally night at 37°C and 5% CO . Cells were harvested, washed injected mice, further demonstrating that VV infection and fixed in 1% paraformaldehyde. Percentage of was restricted to the CNS compartment. Importantly, + high VV-infected cells was determined by measuring eGFP ex- infected CD11b CD45 macrophages expressed MHC pression in flow cytometric analyses (CYAN, Beckmann/ class II (Figure 2B). Thus, phagocytic cells were re- Coulter). The percentage of virus neutralization was cruited from the systemic compartment in response to defined as (1-[percentage of GFP-expressing cells]/ i.th. VV infection and became targets of virus replication [percentage of GFP-expressing cells in controls with- but were unable to control the infection (see Figure 1A). out serum]) × 100. Immunization with modified vaccinia virus (MVA) protects Statistical analysis against VV encephalitis Statistical evaluations of cell frequency measurements As macrophages became infected during VV encephalitis and gene expression levels were performed with the but failed to eliminate the virus, we hypothesized that unpaired Student’s t test. Two-tailed p values < 0.05 adaptive cellular immune responses were required for were considered significant. Weight scores are given as virus control. In order to investigate the contribution of means ± SEM. Multiple comparisons were performed antigen specific effector T helper cells to host defense by two-way-ANOVA and Bonferroni post-testing. against i.th. VV infection, we immunized wild type mice with a replication deficient strain of VV (MVA) emulsi- Results fied in complete Freund’s adjuvant (CFA) 10 days prior Intrathecal vaccinia virus (VV) infection causes to i.th. challenge with VV. CFA induces both Th1 and lethal encephalitis Th17 responses. Here, we were interested in investigat- To establish a model of viral encephalitis, female C57BL/6 ing the role of these T helper cell subsets in the trade-off mice were injected intrathecally with VV by suboccipital between host protection and immunopathology during puncture of the cisterna magna. By titrating the dose of infectious encephalitis. While sham-immunized mice the inoculum, the half lethal dose (LD50) was determined died at day 6 after infection and largely failed to recruit to be approximately 500 plaque forming units (PFU) either CD4 T helper cells or VV-specific CTLs into the (Figure1A).Insystemicinfection, VVisknown to pref- CNS compartment (Figure 3A,B), MVA immunized erentially replicate in the ovaries upon hematogenous animals harbored both antigen specific CTLs and CD4 T dissemination. We therefore measured viral loads in cells in the CNS and recovered from VV encephalitis Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 5 of 16 http://www.actaneurocomms.org/content/2/1/27 Figure 1 Intrathecal infection with VV causes lethal encephalitis. (A) C57BL/6 wild type mice were injected intrathecally by suboccipital puncture of the cisterna magna with various doses of VV. Percentage of surviving mice are depicted in Kaplan-Meier curves (n = 5 per group). (B) Virus loads in CNS and ovaries of wild type mice infected intrathecally with VV were measured on day 5 after infection (Student’st test, n ≥ 5). (C) Wild type mice injected with VV were subjected to histologic analyses on day 5 after infection. Unless otherwise indicated, scale bars represent 200 μm in the low magnification and 20 μm in the higher magnification photomicrographs. (Figure 3A-C). While CTLs re-isolated from the CNS Th17 cells into the CNS compartment. In the EAE largely produced IFN-γ, the intrathecal CD4 effector T cell model, Th1 cells depend on α4-integrins in order to compartment comprised IFN-γ producers, IL-17 producers, enter into the CNS during autoimmune inflammation. and IFN-γ/IL-17 double producers (Figure 3C). In sum- In contrast, Th17 cells are able to access the CNS com- mary, protective immunity against intrathecal VV infec- partment independently of α4-integrins using an LFA-1 tion was associated with the mobilization of both CTLs dependent mechanism [4]. Here, we treated our MVA and CD4 effector T helper cells to the CNS. immunized mice with blocking antibodies to integrin α4 (PS/2) prior to i.th. VV challenge (Figure 4A). Notably, Antibody mediated blockade of α4-integrins leads to MVA immune mice survived i.th. VV infection in spite relative enrichment of Th17 cells in the CNS of integrin α4 blockade (Figure 4A). As expected, we We next wished to dissect the specific contribution of found a relative abundance of IL-17 producing T cells as different T cell subsets to the protective effects of compared with IFN-γ positive T helper cells (Figure 4B). vaccination. Here, we took advantage of the differential This finding supported the idea that – similar to T integrin requirement for the recruitment of Th1 cells vs cell recruitment in autoimmune inflammation of the Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 6 of 16 http://www.actaneurocomms.org/content/2/1/27 SPL CNS 4 4 10 10 3 3 10 10 1 3 2 2 10 10 1 1 10 10 0 0 10 10 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 CD11b SPL CNS 4 4 10 10 38.6 0 34.7 0.10 3 3 10 10 2 2 10 10 1 1 10 10 0 0.25 0 0 10 10 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 4 4 10 10 29.4 0.04 80.7 1.31 3 3 10 10 2 2 10 10 1 1 10 10 0 0.07 0 0 10 10 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 4 4 10 10 62 0 42.5 6.81 3 3 10 10 2 2 10 10 1 1 10 10 0 0.81 0 0 10 10 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 VV (eGFP) Figure 2 MHC class II positive macrophages are targets of VV infection in the CNS. Wild type mice were infected intrathecally with 1.000 PFUs of VV (eGFP), a replication competent recombinant VV expressing enhanced green fluorescent protein (eGFP) in infected cells. On day 4 after infection, mice were sacrificed, and spleen and CNS mononuclear cells were isolated and analyzed by flow cytometry. In the live cell gate, low high the following populations were defined based on their expression of CD11b and CD45: (A) spleen (SPL), left panel: (1) CD11b CD45 int high high high low high lymphocytes, (2) CD11b CD45 dendritic cells, (3) CD11b CD45 macrophages. (A) CNS, right panel: (1) CD11b CD45 lymphocytes, (2) int int high high CD11b CD45 microglia, (3) CD11b CD45 macrophages. (B) These populations were analyzed as to their expression of eGFP and MHC class II by flow cytometry. Numbers indicate percentages of positive cells in each quadrant or gate (representative out of 3 independent experiments). MHC II CD45 Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 7 of 16 http://www.actaneurocomms.org/content/2/1/27 CNS – Th1 cells and Th17 cells exhibited distinct deficient T helper cells re-isolated from the CNS of integrin requirements for their entry into CNS in VV challenged mice exhibited a Th17 like phenotype the context of a local virus infection. Interestingly, while the number of Th1 like cells was significantly while the absolute number of CD4 T cells was reduced in reduced in α4 CKO animals as compared with controls the CNS, which was largely due to the reduction of IFN-γ (Figure 5D, E). Consistent with their cytokine produc- producing CD4 effector T cells, the number of CTLs tion upon ex vivo stimulation, CNS derived α4-integrin recruited to the CNS compartment was not significantly deficient T helper cells expressed higher amounts of reduced in anti-integrin α4 treated mice (Figure 4C). Th17 signature markers such as Rorc, Il17, Il22, Il6, Thus, integrin α4 mediated mechanisms appeared to be Ccr6, or Il1r1 whereas Th1 associated genes like Cxcr3, redundant for the recruitment of effector CD8 Tcells Ccr5, or Ccr2 prevailed in wild type T helper cells into the infected CNS. (Figure 5F). In conclusion, while Th1 cells appeared to be sufficient to provide host protection in intrathecal Th17 cells fail to clear cerebral VV infection VV infection, Th17 cells failed to control cerebral virus Similar to the murine system, neutralization of α4- replication in the absence of CD8 T cells resulting in integrins by natalizumab in humans mainly blocks CD4 T lethal encephalitis. cells from entering into the CNS compartment but To corroborate that Th1 immunity was sufficient to spares CD8 T cells [23,24]. Yet, intracerebral reconsti- improve the outcome of intrathecal viral infection, we tution of CD4 effector T helper cell responses has established a model of adoptively transferred host pro- been proposed to be necessary for efficient clearance tection using OT-II T cells, which carry a transgenic T of various viruses from the CNS including JC virus cell receptor specific to ovalbumin, in combination with [25]. We wanted to dissect the essential features of an a recombinant VV expressing ovalbumin in infected cells intracerebral T helper cell response that would be able (VV-Ova). Naive OT-II cells were differentiated in vitro to control viral infection. Thus, we combined a system into Th1 cells or Th17 cells followed by transfer into -/- of T cell conditional ablation of α4-integrin expression Rag1 mice, which had been infected intrathecally with + flox/flox (CD4 Cre xItga4 mice, α4 CKO mice) with VV-Ova one day prior to adoptive transfer (Figure 6). antibody mediated depletion of CD8 T cells in order While non-transferred Rag1 deficient recipients and recip- to investigate the differential contribution of Th1 cells ients of Th17 cells rapidly died, Th1 recipients survived vs Th17 cells to host protection in CNS infection in significantly longer than their counterparts (Figure 6A). the absence of CTLs. To exclude possible alterations Despite a significant difference in clinical outcome, equal in priming of antigen specific T helper cell responses amounts of transferred Th1 cells and Th17 cells were re- in the peripheral immune compartment of α4CKO isolated from the CNS of infected mice (Figure 6B). Thus, mice, we compared the fractions of antigen specific T on a per cell basis, antigen specific Th1 cells appeared to cells on day 10 after subcutaneous immunization with be more potent in alleviating i.th. virus infection than - flox/flox MVA/CFA in the spleens of CD4 Cre xItga4 antigen specific Th17 cells. + flox/flox (wild type control) versus CD4 Cre xItga4 (α4 CKO) mice (Additional file 1: Figure S1). Upon ex vivo Th17 cells fail to clear i.th. VV infection due to lack of restimulation with I-A restricted VV epitopes, the perforin-1 expression + + fractions of antigen specific (CD40L )CD4 T cells It was possible that the reduced capacity of Th17 cells to and their cytokine profile were similar in wild type vs provide host protection in intrathecal VV infection was α4 CKO mice. Moreover, the anti-VV neutralizing serum simply due to reduced availability of IFN-γ within the response was equally effective in both groups on day 10 CNS compartment. In order to test this possibility, we after immunization (Additional file 1: Figure S1). Thus, neutralized IFN-γ by monoclonal antibodies in MVA sensitization for adaptive cellular immune responses immune and CD8 T cell depleted wild type mice against VV in draining lymph nodes and spleen was not (Additional file 2: Figure S2). In this purely T helper cell impaired by the lack of α4-integrins on T cells. dependent scenario, VV specific Th1 and Th17 cells Next, we challenged MVA immune and CD8 depleted were generated and had access to the VV challenged wild type and α4 CKO mice with i.th. VV. In contrast to CNS. Notably, the mice recovered from VV encephalitis control littermates, α4 CKO mice rapidly lost weight despite ablation of IFN-γ. Efficiency of IFN-γ blockade and succumbed to infection (Figure 5A). Even in the was documented by reduced levels of MHC class II + + complete absence of CD8 T cells (Figure 5B), CD4 ef- expression on microglial cells in the CNS compartment fector T helper cells were protective in wild type mice (Additional file 2: Figure S2). These data suggested that but failed to control virus replication in the CNS of α4 IFN-γ was redundant as an effector molecule in the CKO mice (Figure 5C). Recapitulating our observations clearance of intrathecal VV infection and refuted the with anti-α4 integrin (PS/2) administration, α4-integrin idea that diminished IFN-γ expression by Th17 cells Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 8 of 16 http://www.actaneurocomms.org/content/2/1/27 [d] -10 0 4 Immunization Challenge Analysis PBS/CFA MVA/CFA Time after challenge (days) p=0.0065 p=0.0293 150,000 600,000 100,000 400,000 50,000 200,000 0 0 PBS/CFA MVA/CFA 0.87 61.1 CD8+ T cells 0.93 0.23 3.4 3.67 CD4+ T cells 53.9 78.3 0 0 0 0.06 CD8+ T cells 95.3 IFN-γ Figure 3 Immunization with modified VV Ankara (MVA) confers protective immunity against viral encephalitis. Wild type mice were immunized subcutaneously with a replication deficient strain of VV (Modified Vaccinia Virus Ankara, MVA) or PBS emulsified in complete Freund’s Adjuvant (CFA). On day 10 after immunization, mice were challenged intrathecally with VV, and weight courses were monitored daily as percentage of initial body weight (A, n = 5 mice per group). On day 4 after infection, CNS infiltrating mononuclear cells were isolated. (B) Absolute numbers of CNS + + infiltrating CD4 and CD8 T cells. Horizontal bars indicate means (Student’s t test; n ≥ 5 per group). (C) CNS derived T cells were analyzed as to their specificity to the VV MHC class I restricted epitope B8R by multimer staining and to their IL-17 and IFN-γ cytokine status by intracellular + + + + cytokine staining in the CD3 CD8 CTL and CD3 CD4 T helper cell compartment, respectively. Numbers indicate percentages of cells in the depicted gates or quadrants; representative out of more than 5 independent experiments). PBS/CFA MVA/CFA PBS/CFA MVA/CFA CD4+ T cells (absolute number, CNS) Fraction of original body weight (%) IL-17 K /B8R CD8+ T cells (absolute numbers, CNS) Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 9 of 16 http://www.actaneurocomms.org/content/2/1/27 -/- was responsible for the failure to clear intracerebral not impaired in Prf1 mice. Moreover, wild type mice VV infection. that were vaccinated with MVA and then depleted of + + In order to define potential molecular mechanisms of CD4 T cells in addition to CD8 T cells succumbed to T helper cell mediated host protection in intracerebral intrathecal challenge with VV although they had similar viral infection, we screened the expression profile of fractions of NK cells and NK T cells in the CNS as + + highly purified CD4 effector T cells isolated from the their CD4 T cell replete counterparts (Additional file 3: CNS of VV challenged wild type vs α4 CKO mice for Figure S3) suggesting that alternative sources of Prf1 molecules directly involved in virus defense (Figure 7A). other than CD4 T cells were insufficient to provide CD4 effector T cells expressed Tnf, Grzmb, and Fasl protection in this model. Taken together, these data irrespective of whether they were derived from the Th1 demonstrated that Prf1 expression was indispensable biased inflammatory infiltrate of wild type mice or from for effector T helper cell mediated control of intra- the Th17 biased inflammatory milieu of α4 CKO mice. thecal VV infection. In contrast, the expression of perforin-1 (Prf1)was markedly reduced in α4-integrin deficient as compared Discussion with wild type T helper cells. In order to correlate lack In this study, we tested the concept that host defense of Prf1 expression with the Th17 transcriptional pro- against viral infections of the CNS requires distinct T + + - - gram, we purified CD3 CD4 CD44 Foxp3 naïve T cells helper cell subsets. We show that access inhibition to from Foxp3gfp.KI mice and stimulated them without the CNS of selected T helper cell subsets by integrin exogenous cytokines (Th0) or differentiated them into targeted interventions results in the failure of host Th1 cells or Th17 cells (Figure 7B). Consistent with our defense. Intrathecal infection with vaccinia virus (VV) + + in vivo data, we found a significant reduction in Prf1 was controlled in vaccinated mice when CD4 and CD8 T mRNA and protein in Th17 as compared with Th1 and cells had access to the CNS compartment. Blockade of Th0 cells. Notably, protein expression of Prf1 was only α4-integrins by neutralizing antibodies or T cell condi- seen at late time points (Figure 7B). Since expression of tional disruption of α4-integrin expression did not prevent Prf1 has been reported to depend on the expression of CTLs and Th17 cells from entering the CNS parenchyma the transcription factor eomesodermin (Eomes) [26], we while Th1 cells were blocked from migrating into the + + measured RNA levels of Eomes in CD4 T cells isolated CNS. When CD8 CTLs were depleted, host defense from brains of VV infected wild type and α4 CKO mice. against intrathecal VV infection was still maintained as Consistent with the low expression of Prf1 in Th17 cells, long as efficient Th1 like responses were operational Eomes mRNA levels were reduced in CD4 T cells iso- within the CNS. In contrast, Th17 cells alone failed to lated from α4 CKO mice as compared with wild type rescue individuals from CNS infection despite a strong in- controls (Figure 7C). To formally validate whether flammatory response. Perforin-1 expression by T helper reduced Eomes expression in Th17 cells accounted for cells was required for clearance of intrathecal VV infection diminished Prf1 expression as compared with Th0 and and Th17 cells failed to eliminate VV in the CNS due to Th1 cells, we polarized Th17 cells in vitro and overex- lack of Eomes dependent Prf1 expression. pressed Eomes by retroviral transduction (Figure 7D). Adaptive cellular immune responses are necessary for + + FACS sorted transduced (GFP ) Th17 cells expressed the clearance of systemic VV infection. CD8 T cells abundant levels of Eomes as compared with control recognize epitopes from early antigens of VV but robust vector proving effective transduction; concomitantly, Prf1 CD4 T cell responses are also required for direct and RNA was significantly increased in Eomes transduced indirect antiviral effects [27-30]. Niche restricted or Th17 cells. These data suggested that the failure of Th17 organ specific infection may afford specific effector func- cells to express Prf1 was due to reduced Eomes expression. tions. For example, VV skin infection is contained and In order to explore the importance of CD4 T cell de- cleared by a temporally and spatially organized inter- + + rived Prf1 for successful host defense in CNS virus infec- action of Ly6G innate immune cells and CD8 CTLs -/- tion in vivo, we immunized Prf1 deficient mice (Prf1 ) [22]. In the ovaries, clearance of VV correlates with the with MVA/CFA and established viral encephalitis in number of effector memory CTLs that are present in the CD8 T cell depleted animals by intrathecal infection ovaries of VV immunized mice before challenge with the with VV (Figure 8). While wild type mice recovered virus [31]. Here, we established an intrathecal infection from i.th. VV challenge, Prf1 deficient mice succumbed model with VV and used this system as an in vivo surro- to viral encephalitis. Numbers and fractions of CNS in- gate to correlate the access of distinct T helper cell sub- filtrating immune cells were comparable between groups sets with successful host defense. Intrathecal infection of (data not shown) indicating that antigen specific priming naive wild type C57BL/6 mice with VV was lethal be- in the peripheral immune compartment and establish- cause adaptive antiviral immune responses did not de- ment of inflammatory infiltrates within the CNS were velop upon i.th. infection alone although innate immune Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 10 of 16 http://www.actaneurocomms.org/content/2/1/27 PBS/CFA + rIgG MVA/CFA + rIgG rIgG / PS/2 MVA/CFA + PS/2 [d] -10 -1 0 1 3 5 Immunization Challenge Time after challenge (days) PBS/CFA + rIgG MVA/CFA + rIgG MVA/CFA + PS/2 CD4+ T cells 3.85 0.58 11.4 11.7 16.8 11.6 12.7 59.4 48.8 IFN-γ p=0.026 ns 80,000 20,000 60,000 15,000 40,000 10,000 20,000 5,000 0 0 p= 0.049 ns p=0.003 p=0.042 150,000 1,500 100,000 1,000 50,000 500 0 0 Figure 4 Antibody mediated blockade of integrin α4 prevents entry of Th1, but not Th17 cells or CD8 T cells into the CNS. Wild type mice were sham immunized (PBS/CFA) or immunized with MVA/CFA followed by intrathecal VV challenge on day 10 after immunization. Control IgG or antibodies to integrin α4 (PS/2) were administered every other day starting one day prior to VV challenge. (A) Weight courses of VV challenged animals (n = 5 per group). (B, C) On day 4 after infection, CNS infiltrating mononuclear cells were isolated and the absolute number of + + + CD4 Thelper cells and CD8 CTLs as well as the cytokine phenotype of CD4 T helper cells from the CNS were assessed based on intracellular staining for IFN-γ and IL-17. (B; numbers indicate percentages of CD4 T cells in each quadrant; representative out of 5 independent experiments. The absolute numbers of T cells were compared by Student’sttest, n ≥ 3). + high cells and in particular CD11b CD45 monocytic cells Migration of effector cells into the CNS is mediated by were recruited to the infected CNS. Active immunization chemokine receptors and integrins. Several cytolytic and of mice with MVA in CFA prior to i.th. VV challenge non-cytolytic viral infection models of the CNS were protected the animals from lethal encephalitis. Protec- used to investigate principles of immune cell recruit- tion was independent of neutralizing antibodies. How- ment into the CNS. Depending on the viral agent, ever, access of VV specific Th1 cells into the CNS immune cell recruitment into the CNS cleared the compartment was necessary and sufficient for host pathogen or in addition, caused immunopathogenic tis- protection. sue damage. CCR5 and CXCR3 were identified to be PBS/CFA + PBS MVA/CFA + rIgG MVA/CFA + PS/2 PBS/CFA + PBS MVA/CFA + rIgG PBS/CFA + PBS MVA/CFA + PS/2 MVA/CFA + rIgG MVA/CFA + PS/2 PBS/CFA + PBS MVA/CFA + rIgG MVA/CFA + PS/2 CD4+ T cells IFN-γ+ IL-17- CD4+ T cells (absolute number, CNS) (absolute number, CNS) IL-17 Fraction of original body weight (%) CD8+ T cells IL-17+ IFN-γ- CD4+ T cells -3 (absolute number x 10 , CNS) (absolute number, CNS) Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 11 of 16 http://www.actaneurocomms.org/content/2/1/27 A anti-CD8 B rIgG anti-CD8 [d] -10 0 2 46 8.9 0 Immunization Challenge WT 72 86 WT MVA/CFA + anti-CD8 α4 CKO MVA/CFA + anti-CD8 5.3 0 80 α4 CKO 81.9 87.4 CD4 02468 10 Time after challenge (days) p=0.0092 p=0.021 2.5 500,000 2.0 400,000 300,000 1.5 200,000 1.0 100,000 5.0 0 0 E F Tbx21 Rorc Ifng p=0.008 40,000 ns Il17 Il22 300,000 Il6 30,000 Cxcr3 200,000 Ccr5 20,000 Ccr2 Ccr6 100,000 10,000 Il23r Il1r1 0 0 -2 0 2 Fold change in relative expression (α4 CKO/W T CNS, log , n=4) Figure 5 VV specific Th17 cells are relatively enriched in the CNS compartment of α4 CKO mice but do not control i.th. VV infection. - flox/flox + CD4 Cre x Itga4 mice (controls, WT) and α4 CKO mice were immunized with MVA/CFA, depleted of CD8 T cells, and challenged i.th. with VV. (A) After intrathecal infection with VV, the clinical course was monitored and weight loss was calculated as percentage of initial body weight (n = 5 per group). On day 5 after infection, mice of each group were sacrificed. (B) The efficacy of CD8 T cell depletion was analyzed by flow + - cytometry in CNS mononuclear cells (gate on CD3 CD19 cells). (C) The viral load in the CNS was measured by means of plaque assay. CNS + + infiltrating CD4 T cells (D) and absolute numbers of cytokine positive CD4 T cells (E) were calculated by surface staining and intracellular cytokine analysis (Student’s t test, n ≥ 4 per group, representative out of n ≥ 4 independent experiments). (F) CD4 T cells were isolated by flow cytometric purification from the CNS compartment of MVA immunized wild type vs α4 CKO mice 5 days after i.th. VV challenge. The expression profile of the indicated genes was determined by quantitative PCR analysis, n = 4. important for immune cell recruitment into the CNS in cells for the transmigration of immune cells across the west nile or MHV and LCMV infection, respectively blood brain barrier [36]. Early work suggested that accu- [32-35]. Chemotaxis of immune cells must be comple- mulation of antigen specific T cells in the CNS compart- mented by integrin mediated interaction with endothelial ment of sindbis virus infected animals is mediated by WT anti-CD8 α4 CKO anti-CD8 WT + anti-CD8 WT anti-CD8 α4 CKO+ anti-CD8 α4 CKO anti-CD8 WT + anti-CD8 α4 CKO + anti-CD8 IFN-γ+ IL-17- CD4+ T cells (absolute number, CNS) -6 Fraction of original PFU x 10 / g CNS tissue body weight (%) CD4+ T cells IL-17+ IFN-γ- CD4+ T cells (absolute number, CNS) (absolute number, CNS CD8 Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 12 of 16 http://www.actaneurocomms.org/content/2/1/27 Infection of Adoptive transfer of -/- Rag1 mice Th1 or Th17 cells [d] -1 0 ns p=0.0006 p=0.0165 100 No transfer Th1 80 8 Th17 40 6 20 5 No transfer Th1 Th17 Time after infection (days) 30,000 6,000 20,000 4,000 10,000 2,000 No transfer Th1 Th17 No transfer Th1 Th17 + - Figure 6 Adoptive transfer of antigen specific Th1 cells but not Th17 cells promotes survival after intrathecal virus infection. Naïve CD4 CD25 CD44 Tcells from OT-II Ova specific TCR transgenic mice were polarized under Th1 or Th17 conditions in vitro.Onday four of 323-339 -/- differentiation, activated T cells were transferred into Rag1 mice, which had been infected with a recombinant VV expressing ovalbumin (VV-Ova) one day earlier. (A) Kaplan-Meier curves of survival (left) and statistical evaluation of time to death (right) are shown. Horizontal bars in the right plot indicate mean survival time in days (ANOVA, n ≥ 5per group). (B) Mice were sacrificed on day 6 after transfer and absolute numbers of adoptively + + transferred CD3 CD4 T cells in spleens and CNS were assessed (means are indicated by horizontal bars, n = 3 per group). engagement of LFA-1 but not VLA-4 [37]. Using gen- systemic lymphoid compartment prior to intracerebral etic tools, we have recently shown that distinct T helper challenge [42]. In contrast to Th17 cells, VLA-4 expres- cell subsets use distinct integrins to access the CNS sion is indispensable and non-redundant for the capacity compartment in autoimmune inflammation. The entry of antigen experienced Th1 cells to infiltrate into the of Th17 cells into the subarachnoid space most likely subarachnoid space and the CNS compartment. Here, requires CCR6 and LFA-1 but is independent of VLA-4 we provideevidencethatMHC classIIpositiveinfected [4,38]. In fact, Th17 cells express low levels of α4- phagocytic cells in the CNS need to be cleared by Th1 integrins since TGF-β, which is required for Th17 cell cells in order to rescue mice from lethal encephalitis. commitment [17,39,40], inhibits the expression of α4- Thus, MVA-immune T cell conditional α4 KO mice integrins [41]. Interestingly, antigen specific CD8 succumbed to intrathecal VV challenge because they CTLs also migrated into the CNS independently of α4- failed to recruit Th1 cells into the CNS despite regular integrins (both in α4-integrin antibody blockade and T Th17 cell accumulation. cell conditional genetic disruption of Itga4). This is Antigen specific CD4 T cells communicated with consistent with the observation that CD4 Tcell are virus infected macrophages within the CNS parenchyma preferentially reduced in CSF of natalizumab treated and only eliminated the infected cells when they were patients as compared with CD8 T cells resulting in the sufficient in perforin-1 expression. In ectromelia infec- + + reversion of the CSF CD4 /CD8 ratio [23]. Although tion, a natural mouse pox infection, perforin-1 expres- + + one report suggested that CD8 T cell influx into the CNS sion by CD4 T cells is required to clear the pathogen, was prevented by a blocking antibody to α4-integrins in and CD4 T cell mediated killing of hematopoietic MHC an intracerebral corona virus infection model, it is unlikely class II expressing infected target cells occurs in the that the recruitment of antigen specific CTLs was assessed draining lymph nodes, the liver, and the spleen [28]. Dir- by Ifergan et al. since their model did not allow for active ect cytotoxic effects of antigen specific CD4 T cells priming and activation of adaptive T cell responses in the were also reported in systemic VV infection [43]. In our OT-II T cells (absolute number, spleen) Percent survival OT-II T cells (absolute number, CNS) Survival time (days) Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 13 of 16 http://www.actaneurocomms.org/content/2/1/27 Tnf Prf1 Grzmb Fasl -2 -1 0 1 Fold change in relative expression (α4 CKO/W T CNS, log , n=4) d3 d4 d5 B MW (kD) Th0 Th1 Th17 Th0 Th1 Th17 Th0 Th1 Th17 p<0.0001 0.5 0.4 Prf1 0.3 0.2 0.1 0.0 38 Th0 Th1 Th17 Actin C D p=0.0193 p=0.0006 7.2 p=0.0011 1.5 0.8 pMIG 0.6 1.0 0.4 0.20 0.5 0.2 0.10 5.9 0.0 0.00 0.0 pMIG Eomes GFP + + Figure 7 Th17 cells fail to control VV encephalitis due to lack of Prf1 expression. (A) CD3 CD4 T cells were highly purified by FACS sorting from the CNS of MVA/CFA immunized wild type or α4 CKO mice on day 4 after intrathecal infection. Fold change in relative expression of the indicated genes in α4 deficient vs. wild type control CD4 T cells (log scale, means + SD, n = 4). (B) Flow cytometrically purified naïve + + - - CD3 CD4 CD44 Foxp3 T cells were stimulated without exogenous cytokines (Th0) or differentiated into Th1 cells (IL-12 + anti-IL-4), or Th17 cells (TGF-β + IL-6). Levels of Prf1 RNA expression were measured by quantitative RT-PCR (left in B, means + SD, Student’st test,n = 4). Time course of Prf1 protein expression was determined by Western Blot in Th0, Th1, and Th17 cells from day 3 to day 5 of culture (right in B). (C) RNA levels of Eomes were measured in wild type and α4 integrin deficient T cells isolated from the CNS of VV infected mice (means + SD, n = 4). (D) Naïve T cells were differentiated into Th17 cells in vitro and transduced retrovirally with a control vector (pMIG) or an Eomes GFP vector (pMIG Eomes). GFP cells were purified by flow cytometry and RNA levels of Eomes and Prf1 were measured in control transduced Th17 cells vs Th17 cells that ectopically expressed Eomes (means + SD, n = 4). anti-CD8 WT (+anti-CD8) Prf1-/- (+anti-CD8) [d] -10 0 2 46 Immunization Challenge Time after challenge (days) -/- -/- Figure 8 Prf1 mice succumb to VV encephalitis. Wild type and Prf1 mice were immunized with MVA/CFA and continuously depleted of CD8 T cells after intrathecal VV challenge. Weight courses of infected animals (n = 5 per group). WT α4 CKO pMIG (GFP) pMIG Eomes (GFP) pMIG (GFP) pMIG Eomes (GFP) Eomes (rel. expression) in CNS CD4+ T cells Prf1 (rel. expression) Fraction of original body weight (%) Eomes (rel. expression) Prf1 (rel. expression) Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 14 of 16 http://www.actaneurocomms.org/content/2/1/27 model, T cell conditional α4-integrin deficient mice be- a latent virus infection of oligodendrocytes. However, the haved like perforin-1 deficient animals and succumbed intracerebral anti-JC virus CD4 T cell response contributes to infection because perforin-1 expressing Th1 cells did substantially to JCV control because efficient clearance of not reach the CNS compartment in the absence of VLA- JC virus from the CNS after immune reconstitution by 4. Like in EAE, the cytokine phenotype of T helper cells wash-out of natalizumab in MS patients with PML is asso- that entered into the CNS compartment in the absence ciated with the re-occurrence of Th1 cells in the CNS [53]. of α4-integrin showed a Th17 profile [4]. Th17 cells did not express perforin-1. Initial Th17 cell commitment is Conclusions sensitive to IL-2 and high IL-2 concentrations inhibit Our experimental model provides a mechanistic explanation Th17 differentiation [44]. In contrast, IL-2 promotes the for insufficient virus control under conditions of altered T expression of granzyme B and perforin-1 and boosts cell migration. Our data raise the concern that treatment of cytotoxic capacity of CD4 T cells [45]. While the tran- humans with compounds like natalizumab that interfere scriptional control of granzyme B and perforin-1 has in with T cell trafficking not only by quantitatively reducing the part been investigated in CD8 T cells [46], we found migration of T cells into the CNS but also by selectively tar- that perforin-1 expression in Th1 cells is associated with geting distinct T cell subsets may evoke serious deficiencies Eomes and overexpression of Eomes in Th17 cells par- in cellular immune responses at privileged anatomical sites tially reconstituted perforin-1 expression. Similar obser- like the CNS. As a result, primary infections or unexpected vations have been reported for Th2 cells that per se also reactivations of latent viruses may be facilitated. lack perforin-1 [26], which correlates with their low cytotoxic efficacy. However, lack of perforin-1 expression Supporting data in Th17 cells may not be the only mechanism to impair The data sets supporting the results of this article are their antiviral capacity. For example, Th17 cells are able included within the article and its additional files. to mount a protective response in primary lung infection with influenza in IL-10 KO mice suggesting that Th17 Additional files cells are particularly susceptible to IL-10 mediated sup- Additional file 1: Figure S1. α4 integrin deficiency in T cells does not pression [47]. In addition, induction of prosurvival fac- alter priming or humoral responses against VV after immunization. (A) CD4 tors by Th17 cells may contribute to viral persistence in - flox/flox + flox/flox Cre x Itga4 mice (wild type control (WT)) or CD4 Cre xItga4 the CNS in Theiler’s virus infection [48]. Conversely, it mice (α4CKO)wereimmunizedwith MVA/CFA.Onday 10 after immunization, splenocytes were isolated and restimulated with an I-Ab is possible that in some anatomical niches, Th17 cells restricted peptide epitope mix of VV. Fractions of antigen specific cytokine may contribute to protective antiviral immune responses, producing CD4 T cells were determined by intracellular CD40L (CD154) e.g. by effective IL-17 mediated recruitment of neutrophils and cytokine staining. Numbers in the gates represent means ± SD, n = 3. (B) Specific serum neutralization capacity of sera collected on day 10 in lung infection [49]. Also, IL-21, which is produced by after immunization of wild type or α4 CKO mice vaccinated with PBS/CFA Th17 cells, might have a role in the modulation of primary or MVA/CFA (specific neutralization capacity in percent of maximum, and recall CTL responses by increasing their proliferation means + SD, n = 5). and reducing their sensitivity to exhaustion [50]. However, Additional file 2: Figure S2. IFN-γ expression in CD4 T cells is not required for virus control in the CNS. (A) Wild type mice were immunized overall it is a more common scenario that antiviral Th17 with MVA/CFA and challenged intrathecally with VV on day 10. Starting responses fail to clear viral pathogens but rather drive one day before VV challenge, neutralizing antibodies to IFN-γ (or control deleterious immunopathology in the skin [51], in the IgG) and depleting antibodies to CD8 were applied alternatingly every other day. Weight courses of infected animals were measured every day cornea [52], and also in the CNS. after infection and depicted relative to initial body weight (n = 5 per We have shown that blockade or genetic lack of α4- group). (B) Expression of MHC class II on CNS microglial cells (gating on int int integrins on T cells selectively modulated the access of T CD11b CD45 , upper panel) was measured on day 6 after infection using surface staining and flow cytometric analysis. Numbers indicate helper cell subsets to the CNS. Preferential limitation of percentages. Histogram: black line: MHC class II expression on migroglia Th1 trafficking jeopardized host defense against intrathecal from control IgG treated mice, grey/shaded line: MHC class II expression VV infection. Nevertheless, our experimental system has on microglia from anti-IFN-γ treated mice. The numbers in the histogram represent mean fluorescence intensities. Representative several limitations: First, i.th. inoculation of VV does not out of 3 independent experiments. represent a natural route of CNS infection. Yet, Additional file 3: Figure S3. CD4 T cells mediate protection against immunization with virus antigens in CFA provides an VV encephalitis in the absence of CD8 T cells. Wild type mice efficient way to generate both an antigen specific Th1 and immunized with MVA/CFA were challenged i.th. with VV and depleted of CD8 T cells. Concomitantly, animals were treated with isotype control or Th17 response in the secondary lymphoid tissue and to depleting antibodies to CD4 every other day. (A) Percentage of surviving study their trafficking and effector functions upon i.th. chal- mice are depicted in Kaplan-Meier curves of survival (n = 6 per group). lenge with a well characterized pathogen. Second, i.th. VV (B) CNS infiltrating mononuclear cells were analyzed by flow cytometry. Upper panel: Staining for CD4 and CD8 in the CD3 T cell gate. Lower infection results in an acute encephalomyelitis, which panel: Staining for CD3 and Nk1.1 in the CD4 gate (representative out of causes rapid decay and death of infected animals; in con- 4 independent experiments). trast, JC virus induced PML is due to insufficient control of Rothhammer et al. Acta Neuropathologica Communications 2014, 2:27 Page 15 of 16 http://www.actaneurocomms.org/content/2/1/27 Competing interests 10. Miller DH, Weber T, Grove R, Wardell C, Horrigan J, Graff O, Atkinson G, Dua P, The authors declare that they have no competing interests. Yousry T, Macmanus D, Montalban X: Firategrast for relapsing remitting multiple sclerosis: a phase 2, randomised, double-blind, placebo-controlled trial. Lancet Neurol 2012, 11:131–139. doi:10.1016/S1474-4422(11)70299-X. Authors’ contributions 11. Hueber W, Patel DD, Dryja T, Wright AM, Koroleva I, Bruin G, Antoni C, TK, VR and AM designed the study and wrote the manuscript. VR and AM Draelos Z, Gold MH, Psoriasis Study Group, Durez P, Tak PP, Gomez-Reino JJ, contributed equally, carried out all the experiments and where herein Rheumatoid Arthritis Study Group, Foster CS, Kim RY, Samson CM, Falk NS, supported by GG, FP, and SH. DHB provided fluorescence-labeled MHC class I Chu DS, Callanan D, Nguyen QD, Uveitis Study Group, Rose K, Haider A, Di H-2K /B8R (TSYKFESV) multimers. Histological analyses were carried out with 20-27 Padova F: Effects of AIN457, a fully human antibody to interleukin-17A, the support of MH’s group. GG, DHB, MH, BH and ID gave conceptional input on psoriasis, rheumatoid arthritis, and uveitis. Sci Transl Med 2010, and revised the manuscript. All authors read and approved the final manuscript. 2:52ra72. doi:10.1126/scitranslmed.3001107. 12. 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