1. Engelmann S, Togni M, Thielitz A, Reichardt P, Kliche S, Reinhold D, Schraven B, Reinhold A. T Cell-Independent Modulation of Experimental Autoimmune
Encephalomyelitis in ADAP-Deficient Mice. J Immunol. 2013 Oct 7. [Epub ahead of print]
2. Etemire E, Krull M, Hasenberg M, P Reichardt P#+ and M Gunzer#+. Transiently reduced PI3K/Akt activity drives the development of regulatory function in antigen-stimulated naïve T-cells. PLOS One. 2013: 8(7):e68378.
+Corresponding Author, #Joint Senior Authors
3. Teles A, Schumacher A, Kühnle MC, Linzke N, Thuere C, Reichardt P, Tadokoro CE, Hämmerling GJ, Zenclussen AC. Control of uterine microenvironment by foxp3(+) cells facilitates embryo implantation. Front Immunol. 2013:4:158.
4. Philipsen L, Engels T, Schilling K, Gurbiel S, Fischer KD, Tedford K, Schraven B#, Gunzer M#, Reichardt P#+. Multi-molecular analysis of stable immunological synapses reveals sustained recruitment and sequential assembly of signaling clusters.
Mol Cell Proteomics. 2013;12(9):2551-67. +Corresponding Author, #Joint Senior Authors
5. Reichardt P.*, Patzak I.*, Jones K., Etemire E., Gunzer M., Hogg N. A role for LFA-1 in delaying T lymphocyte egress from lymph nodes. EMBO J. 2013;32(6):829-43.
*Joint First authors
6. Stirnweiss A, Hartig R, Gieseler S, Lindquist JA, Reichardt P, Philipsen L, Simeoni L, Poltorak M, Merten C, Zuschratter W, Prokazov Y, Paster W, Stockinger H, Harder T, Gunzer M, Schraven B. T cell activation results in conformational changes in the Src family kinase Lck to induce its activation. Sci Signal. 2013; 6(263):ra13.
7. Arndt B, M Poltorak, BS. Kowtharapu, Reichardt P, L Philipsen, JA. Lindquist, B Schraven and L Simeoni. Analysis of TCR activation kinetics in primary human T cells upon focal or soluble stimulation. J Immunol Meth 2013; 387:276-83
8. Schmidt A, N Oberle, EM Weiß, D Vobis, S Frischbutter, R Baumgrass, CS Falk, M Haag, B Brügger, H Lin, GW Mayr, Reichardt P, M Gunzer, E Suri-Payer and PH Krammer.
Human regulatory T cells rapidly suppress T cel receptor-induced Ca2+s, NF-κB and NFAT signaling in conventional T cells. Sci Sign 2011;4(204):ra90.
9. Ocaña-Morgner C, Reichardt P, Chopin M, Braungart S, Wahren C, Gunzer M, Jessberger R. Sphingosine 1-Phosphate-Induced Motility and Endocytosis of Dendritic Cells Is Regulated by SWAP-70 through RhoA. J Immunol. 2011;186(9):5345-55.
10.Herroeder S, Reichardt P, Sassmann A, Zimmermann B, Hogg N, Hollmann MW, Fischer KD, Vogt S, Grosse R, Gunzer M, Offermanns S, Wettschureck N. G-proteins of the G12/G13 family shape immune functions by controlling CD4+ T-cell adhesiveness.
Immunity 2009;30:708-20.
11. Horn J, Wang X, Reichardt P, Stradal TE, Warnecke N, Simeoni L, Gunzer M, Yablonski D, Schraven B, Kliche S. Src homology 2-domain containing leukocyte-specific phosphoprotein of 76 kDa is mandatory for TCR-mediated inside-out signaling, but dispensable for CXCR4-mediated LFA-1 activation, adhesion, and migration of T cells.
12. Jones DS, Reichardt P, Ford ML, Edwards LJ, Evavold BD. TCR antagonism by peptide requires high TCR expression.
J Immunol. 2008;181:1760-6.
13.Waibler Z, Sender LY, Merten C, Hartig R, Kliche S, Gunzer M, Reichardt P, Kalinke U, Schraven B. Signaling signatures and functional properties of anti-human CD28 superagonistic antibodies. PLoS ONE. 2008;3:e1708.
14.Reichardt P, Dornbach B, Rong S, Beissert S, Gueler F, Loser K, Gunzer M.
Naive B-cells generate regulatory T-cells in the presence of a mature immunologic synapse. Blood. 2007;110:1519-1529.
15. Reichardt P+, Müller D, Posselt U, Vorberg B, Diez U, Schlink U, Reuter W, Borte M.
Fatty acids in colostrum from mothers of children at high risk of atopy in relation to clinical and laboratory signs of allergy in the first year of life. Allergy.2004;59(4):394-400.
+Corresponding author
16. Reichardt P, Schreiber A, Wichmann G, Metzner G, Efer J, Raabe F. Identification and quantification of in vitro adduct formation between protein reactive xenobiotics and a lysine-containing model peptide. Environ Toxicol.2003;18:29-36.
17.Reichardt P+, Lehmann I, Sierig G, Borte M. Analysis of T-cell receptor V-beta 2 in peripheral blood lymphocytes as a diagnostic marker for Kawasaki disease.
Infection.2002;30:360-4.
18. Reichardt, P+; Dähnert, I.; Tiller, G.; Häusler, H.-J. Possible activation of an intra-myocardial inflammatory process (S. aureus) after treatment with infliximab in a boy with Crohn’s disease. European Journal of Pediatrics.2002;161:281-3.
+Corresponding author
19.Reichardt P+, Apel TW, Domula M, Tröbs RB, Krause I, Bierbach U, Neumann HP, Kiess W. J Recurrent polytopic chromaffin paragangliomas in a 9-year-old boy resulting from a novel germline mutation in the von Hippel-Lindau gene. J Pediatr Hematol Oncol. 2002: 24:145-8.
+Corresponding author
20. Reichardt, P+, W. Handrick, A. Linke, R. Schille, W. Kiess. Leukocytopenia, thrombocytopenia and fever related to piperacillin/tazobactam treatment - a retrospective analysis in 38 children with cystic fibrosis. Infection.1999;27:355-6 +Corresponding author
- - -
Appendix 01
Engelmann S, Togni M, Thielitz A, Reichardt P, Kliche S, Reinhold D, Schraven B, Reinhold A.
T Cell-Independent Modulation of Experimental Autoimmune Encephalomyelitis in ADAP-Deficient Mice.
J Immunol. 2013 Oct 7. [Epub ahead of print]
IF: 5.5
The Journal of Immunology
T Cell–Independent Modulation of Experimental
Autoimmune Encephalomyelitis in ADAP-Deficient Mice
Swen Engelmann,*,1 Mauro Togni,*,1 Anja Thielitz,† Peter Reichardt,* Stefanie Kliche,*
Dirk Reinhold,* Burkhart Schraven,*,‡ and Annegret Reinhold*
The adhesion- and degranulation-promoting adaptor protein (ADAP), expressed in T cells, myeloid cells, and platelets, is known to regulate receptor-mediated inside-out signaling leading to integrin activation and adhesion. In this study, we demonstrate that, upon induction of active experimental autoimmune encephalomyelitis (EAE) by immunization with the myelin oligodendrocyte glyco-protein35–55 peptide, ADAP-deficient mice developed a significantly milder clinical course of EAE and showed markedly less inflammatory infiltrates in the CNS than wild-type mice. Moreover, ADAP-deficient recipients failed to induce EAE after adoptive transfer of myelin oligodendrocyte glycoprotein–specific TCR-transgenic T cells (2D2 T cells). In addition, ex vivo fully activated 2D2 T cells induced significantly less severe EAE in ADAP-deficient recipients. The ameliorated disease in the absence of ADAP was not due to expansion or deletion of a particular T cell subset but rather because of a strong reduction of all inflammatory leukocyte populations invading the CNS. Monitoring the adoptively transferred 2D2 T cells over time demonstrated that they accumulated within the lymph nodes of ADAP-deficient hosts. Importantly, transfer of complete wild-type bone marrow or even bone marrow of 2D2 TCR–transgenic mice was unable to reconstitute EAE in the ADAP-deficient animals, indicating that the milder EAE was dependent on (a) radio-resistant nonhematopoietic cell population(s). Two-photon microscopy of lymph node explants revealed that adoptively transferred lymphocytes accumulated at lymphatic vessels in the lymph nodes of ADAP-deficient mice. Thus, our data identify a T cell–independent mechanism of EAE modulation in ADAP-deficient mice. The Journal of Immunology, 2013, 191: 000–000.
A
daptor proteins play crucial roles in organizing mo-lecular signaling complexes called signalosomes. They are subdivided in transmembrane adapter proteins and cytosolic adaptor proteins (1). The cytosolic adapter protein, adhesion- and degranulation-promoting adapter protein (ADAP), was originally identified on the basis of its association with SLP-76 (SLP-76–associated protein of 130 kDa also known as SLAP-130) (2) and with the Src family kinase Fyn (Fyn-binding protein called Fyb) (3). ADAP is expressed in T cells, NK cells, and myeloid cells but not in mature B cells (4). ADAP possesses a number of protein–protein interaction domains. These include a proline-rich region, tyrosine-based signaling motifs, two helical SH3 domains, two putative nuclear localization sites, and an Ena-Vasp homology (EVH1) domain binding site (5).It is well established that ADAP couples TCR and chemokine receptor stimulation to the activation of integrins via a process called inside-out signaling. ADAP-deficient T cells display altered TCR-mediated adhesion, diminished LFA-1 activation (6, 7), and impaired conjugate formation with APCs (8). In addition to its role in TCR-mediated adhesion and T cell interaction with APCs, ADAP is also involved in chemokine receptor CCR7-mediated LFA-1 affinity/avidity regulation, adhesion, homing, as well as T cell motility within in the lymph nodes (9).
ADAP is also required for NF-kB activation in T cells (10). In this study, ADAP is critical for the assembly of the Carma1-Bcl10-Malt1-complex (11). Furthermore, ADAP associates with the adap-tor molecule Nck (12). It has been shown that the functional coop-eration between ADAP and Nck stabilizes the interaction of SLP-76 and the Wiskott–Aldrich syndrome protein (13). Thus, ADAP is also involved in the regulation of actin cytoskeleton rearrangement after TCR stimulation.
Recent work has established that ADAP may also play a role in outside-in signaling from integrins in T cells. Following LFA-1 stimulation of T cells, the ADAP-dependent formation of a ring-shaped actin reorganization called actin cloud was discovered (14). This LFA-1–mediated costimulation enhances IL-2 produc-tion, F-actin clustering, T cell polarizaproduc-tion, and T cell motility (15).
Besides its regulation of outside-in signaling in T cells, ADAP is also required for optimal CD11c integrin–mediated outside-in sig-naling in dendritic cells (DCs) (16).
ADAP-deficient mice have been studied in a limited number of disease models. In transplantation models, ADAP-deficient mice showed prolonged heart graft survival and ameliorated rejec-tion of intestinal allografts (17, 18). In contrast, ADAP-deficient TCR-transgenic mice revealed an increased incidence of autoim-mune diabetes (19). In platelets, loss of ADAP results in impaired
*Institute of Molecular and Clinical Immunology, Otto von Guericke University, 39120 Magdeburg, Germany;†Clinic of Dermatology and Venereology, Otto von Guericke University, 39120 Magdeburg, Germany; and‡Department of Immune Control, Helmholtz Center for Infection Research, Braunschweig, Germany
1S.E. and M.T. equally contributed.
Received for publication December 5, 2012. Accepted for publication September 9, 2013.
The work was supported by Deutsche Forschungsgemeinschaft Grants RE 2907/2-1 (to A.R.) and SFB854 TP12 (to B.S.).
Address correspondence and reprint requests to Prof. Burkhart Schraven, Institute of Molecular and Clinical Immunology, Medical Faculty, Otto von Guericke University, Leipziger Strasse 44, 39120 Magdeburg, Germany. E-mail address: schraven@med.
ovgu.de
The online version of this article contains supplemental material.
Abbreviations used in this article: ADAP, adhesion- and degranulation-promoting adaptor protein; DC, dendritic cell; EAE, experimental autoimmune encephalomy-elitis; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; S1P, sphingosine 1-phosphate.
Published October 7, 2013, doi:10.4049/jimmunol.1203340
formation after carotid artery injury in vivo (21). Recently, it was shown that loss of ADAP attenuated neutrophil recruitment in an ischemia–reperfusion-induced acute kidney injury model (22).
Experimental autoimmune encephalomyelitis (EAE) is a proin-flammatory autoimmune disorder that targets the CNS and serves as an animal model for the human disease multiple sclerosis (MS).
EAE can be induced in C57BL/6 mice by immunization with the encephalitogenic peptide myelin oligodendrocyte glycoprotein (MOG)35–55. Historically, the disease is considered to be mediated by Thl cells because the adoptive transfer of activated brain-specific CD4+cells is sufficient to induce EAE in recipient ani-mals. Recent evidence suggests that a proinflammatory cascade of Th17 cells, IL-6 and TGF-b1, in the CNS plays a critical role in the pathogenesis of EAE and MS (23, 24). The role of B cells and myelin-specific Abs is still controversial, because depending on the model, the presence of B cells is necessary or dispensable for the development of the disease (25, 26). However, it is clear that B cells are important in the relapsing phase by their production of the anti-inflammatory cytokine IL-10 (27).
During the past few years, an important immunological role of APCs, especially DCs and CNS-resident microglia, is emerging.
In the normal CNS, microglia cells express low levels of MHC molecules. During EAE, the expression of costimulatory and MHC molecules on microglia increases, and this may play an important role in the reactivation of T cells that infiltrate the CNS (28). Bone marrow (BM)–derived DCs are the most potent APC population at priming naive T cells in secondary lymphoid organs. Indeed, it was shown that peripherally derived infiltrating DCs rather than radio-resistant CNS-resident microglia cells are involved in the pathogenesis of EAE (29).
In this paper, we report that ADAP-deficient mice undergo milder EAE. This is shown for active EAE as well as in a passive transfer EAE model. We further demonstrate that the ameliorated course of disease in the absence of ADAP is not primarily because of an intrinsic activation defect of T cells. Radiation-induced BM chimeras surprisingly reveal that the attenuated course of EAE is dependent on a radio-resistant nonhematopoietic compartment. We provide evidence that in ADAP-deficient hosts adoptively trans-ferred T cells accumulate at LYVE-1+lymphatic vessels in the lymph nodes. Thus, it appears that trapping of T cells in the lymph nodes results in reduced infiltration of inflammatory cells into the CNS and consequently in strongly attenuated EAE.
Materials and Methods
Mice
ADAP-deficient mice (7) were backcrossed to C57BL/6JBom for at least 10 generations. Lck-deficient mice were provided by Dr. T. Mak (Uni-versity of Toronto, Toronto, ON, Canada) (30). MOG35–55-specific TCR trans-genic mice (2D2 mice) were provided by Dr. V. Kuchroo (Harvard Medical School, Boston, MA) (31). Congenic C57/BL6-Ly 5.1 mice (B6.SJL) were purchased from Charles River Laboratories. The mice were bred and maintained under specific pathogen-free conditions in the central animal facility of the medical faculty of the University of Magdeburg. In all experiments, 8- to 12-wk-old littermate mice were used. All procedures were conducted according to protocols approved by the local authorities.
EAE induction
Induction of EAE was performed as described earlier (32). Briefly, active EAE was induced by immunization with 200mg MOG p35–55 emulsified in CFA (Sigma-Aldrich) containing 800mg heat-killedMycobacterium
as the average of all individual disease scores of each group, including mice not developing clinical signs of EAE.
For the adoptive transfer experiments, splenic T cells were isolated using aPanT cell isolation kit and AutoMACS (Miltenyi Biotec). The indicated number of purified T cells in 100ml PBS was injected into the tail vein.
One week later, EAE was induced by immunization with MOG in CFA.
Passive induction of EAE by adoptive transfer of polarized MOG-specific Th1 cells was performed as described by Yang et al. (34). Briefly, splenocytes from 2D2 mice were cultured and stimulated with MOG35–55(20mg/ml) in the presence of 5mg/ml IL-2/IL-7 (Miltenyi Biotec) for 2 d. At the end of this incubation period, cells were expanded with IL-2 and IL-7 for another 4 d. Subsequently, cells were reactivated for 24 h with plate-bound anti-CD3 and anti-CD28 (1mg/ml) in the presence of IL-12 (20 ng/ml; R & D Sys-tems) and IL-18 (20 ng/ml; Biozol). Activated T cells were collected and washed, and 2–53106cells were transferred i.p. into recipient mice.
Ab-mediated depletion of host T cells was performed using anti-Thy1.2 Ab. Recipient mice were repeatedly injected i.p. with 500mg anti-Thy1.2 mAb (clone 30H12; BioXCell) 2 d before transfer and on days 2, 6, and 10 after adoptive transfer of activated 2D2 T cells. Blood samples were taken on days 0, 2, 6, and 10 to control depletion of host T cells.
Immunohistological analysis
Mice were killed and cardially perfused with NaCl. Spinal cords were removed and fixed in 4% formaldehyde. Paraffin-embedded spinal cord longitudinal sections were stained with H&E for visualization of inflam-matory infiltrates and evaluated in a blinded manner for the amount of inflammation as described previously (35).
ELISA
For the determination of specific anti-MOG Abs, plates were coated with MOG35–55(10mg/ml) in bicarbonate buffer overnight at 37˚C. After blocking with 1% BSA, the plates were incubated with serial dilutions of mouse serum overnight at 4˚C. Specific binding was detected using alkaline phosphatase–
labeled goat anti-mouse IgG (subclasses 1+2a+2b+3, Fcg-fragment specific;
Dianova, Hamburg, Germany).
Cytokine concentration
Levels of cytokines in plasma were determined using the Th1/Th2/Th17 cytometric bead array kit (BD Biosciences).
Isolation of leukocytes from spinal cord
Mice were killed with CO2and cardially perfused through the left ventricle using NaCl. Spinal cords were extruded by flushing the vertebral canal with PBS. CNS tissue was cut into pieces and treated with collagenase (2.5 mg/ml; Roche Diagnostics) and DNAse I (1 mg/ml; Sigma-Aldrich) for 45 min at 37˚C. Tissue was ground through a cell strainer (70mm), washed, resuspended in 37% Percoll, and layered onto 70% Percoll. After centri-fugation (2000 rpm, 25 min), cells were removed from the interphase, washed, and stained for FACS analysis.
Flow cytometry
Flow cytometric analysis was performed on BD LSRFortessa using BD FACSDiva software (BD Biosciences). The following Abs were used for staining: CD3, CD4, CD8, Va3.2, CD11b, anti-CD11c, anti CD45, anti-CD45.1 and anti-CD45.2, anti-podoplanin (Gp38), and anti-CD31 (all BD Biosciences).
For staining of stromal cells, pooled lymph nodes were cut into pieces and digested with collagenase IV (final concentration, 4 mg/ml; Invitrogen) and DNAse (final concentration, 100mg/ml; Sigma-Aldrich) for 25 min at 37˚C. Cells were stained for podoplanin (Gp38) and CD31. For flow cytometric analysis, the CD45-negative cells were gated and analyzed for the expression of podoplanin, CD31, and ADAP. The polyclonal sheep anti-ADAP antiserum and the respective preimmune serum were provided by G. Koretzky (University of Pennsylvania, Philadelphia, PA) and were used for intracellular staining as described previously (4). The monoclonal anti-Fyb Ab was purchased from BD Biosciences.
Radiation-induced BM chimeric mice
BM donor mice were killed using CO2, and BM cells were isolated by
2 EAE IN ADAP-DEFICIENT MICE
1 wk after transplantation. Engraftment took place over 6–8 wk of re-covery. Successful reconstitution was checked by flow cytometry using congenic markers CD45.1 and CD45.2.
Microglia function
Primary microglia cultures were prepared from wild-type and ADAP-deficient 1- to 3-d-old pups. The cerebral cortices and meninges were removed, and the brains of up to 10 mice were pooled. A single-cell suspension was generated by several rounds of enzymatic and mechanical digestion with the neural dissociation kit (Miltenyi Biotec). Subsequently, microglia cells were isolated using CD11b microbeads (Miltenyi Biotec) as described in the manufactures protocol (purity.90%). Cells (53104) were cultured in 96-well plates in DMEM (Life Technologies) supplemented with 10% FCS (Life Technolo-gies), 1% gentamicin, 1% penicillin/streptomycin, and 0.5% glutamine. After resting for 24 h, microglia cells were stimulated with LPS fromSalmonella minnesota(1mg/ml; Sigma-Aldrich). Following stimulation for 24 h, super-natants were removed and stored for NO and cytokine measurement at 220˚C. Concentration of TNF-awas determined using Mouse CBA Kit (BD Biosciences). NO production was quantified using Griess Reagent System (Promega) following the manufacturer’s protocol.
Two-photon microscopy of inguinal lymph node explants Splenic T cells and B cells from congenic B6.SJL mice were isolated by magnetic depletion (Miltenyi Biotec). Purified B cells were labeled with CFSE (5mM), and purified T cells were labeled with Cell Tracker Orange (5mM; both Molecular Probes). Approximately 103106cells (ratio 1:1) were administered i.v. by retro-orbital injection into each recipient mouse.
Lymphatic vessels were visualized by Pacific Blue–labeled anti–LYVE-1 Ab (10mg in 30ml PBS) applied s.c. into the right flank 24 h before im-aging. The left lymph node was left unstained and was used as control for FACS analysis. The inguinal lymph nodes of wild-type and ADAP-deficient recipients were imaged 48 h after adoptive transfer. Two-photon micros-copy of explanted lymph nodes was performed as reported previously (36).
The inguinal lymph node was surgically removed, immediately immersed in PBS, and placed with the medullary (hilar side) or the follicular (convex) side up, respectively, onto a glass slide. Imaging was performed at a 50 and 150mm depth. Microscopy was performed using a ZeissLSM710 (Carl Zeiss, Jena, Germany) equipped with a MaiTai DeepSee Femtosecond-Laser (Spectra-Physics, Darmstadt, Germany) tuned to 850 nm on an AxioExaminer upright stage with a320, numerical aperture 1.0 (Carl Zeiss) water dipping lens. Image detection was done with three non-descanned detectors, typically equipped with emission detection filters of 565–610 nm (red), 500–500 nm (green), and ShortPass485 nm (blue).
Individual red, green, blue, z-stacks of 606 3 606 mm images were recorded and tiled to combine up to 634 of such fields. Rendering of images was performed using Volocity software (version 4.3; Improvision, Waltham, MA). The number of LYVE-1–associated T cells and B cells was quantified in at least 20 defined areas of two-photon microscopic images.
Statistical analysis
Results are expressed as mean6SEM. Student unpairedttest was used to assess the statistical significance of the differences. Statistical comparison of EAE disease severity between different two groups of animals was accomplished by performing nonparametric Wilcoxon matched pairs test using GraphPad Prism software (37).p,0.05 was considered significant.
Results
ADAP-deficient mice developed milder EAE
EAE (the mouse model of MS) can be induced in C57BL/6 mice by s.c. immunization with MOG35–55peptide in mycobacteria-containing CFA. To assess the consequences of ADAP deficiency in this disease model, we induced EAE in ADAP-deficient mice and wild-type mice. Fig. 1A shows that ADAP-deficient mice un-dergo a significantly milder disease compared with wild-type mice.
FIGURE 1. Milder EAE in ADAP-deficient mice. (A) EAE was induced following immunization with MOG35–55peptide in CFA. The clinical score of EAE was assessed for 35 d after immunization (mean6SD;n= 5;p,0.05 wild-type [WT] versus ADAP). (B) Representative histology of spinal cord longitudinal sections. Inflammatory infiltration was visualized by H&E staining. The arrowhead shows a typical heavy inflammatory cellular infiltration in
The Journal of Immunology 3
The milder clinical score of ADAP-deficient mice was further re-flected by significantly reduced numbers of inflammatory foci in the spinal cord (Fig. 1B, 1C) where total inflammatory foci as well those in the meninges and the parenchyma were reduced. In addi-tion, the levels of MOG-specific IgG Abs in the serum were sig-nificantly lower in ADAP-deficient-mice compared with wild-type mice (Fig. 1D). Taken together, these results suggest that loss of ADAP leads to a strongly attenuated clinical course of EAE, which is accompanied by lower inflammatory infiltrates in the CNS and reduced concentrations of anti-MOG Abs in the serum.
It has been reported previously that ADAP-deficient T cells show defective proliferation, IL-2 production, as well as impaired TCR-mediated LFA-1 clustering and adhesion (6, 7). To assess whether the observed amelioration of EAE in the absence of ADAP was due to impaired function of the ADAP-deficient T cell, we
per-formed an adoptive transfer experiment in which wild-type T cells or ADAP-deficient T cells were transferred into Lck-deficient ani-mals, which completely lack mature T cells. After 7 d, EAE was induced by immunization with MOG35–55peptide. The onset of EAE was delayed, although not statistically significant, when ADAP-deficient T cells were adoptively transferred into the Lck-deficient hosts compared with transferred wild-type T cells (Fig.
1E). This is possibly due to the well established defect in inside-out signaling of ADAP-deficient T cells (6, 7). However, both wild-type and ADAP-deficient T cells produced the same clinical score of disease at later time points of disease. These data indicate 1) that ADAP-deficient T cells are principally capable to induce EAE in T cell–deficient hosts and 2) that non-T cell–specific functions of ADAP are likely responsible for the blunted course of EAE in the absence of ADAP.
FIGURE 2. Reduced EAE severity in ADAP-deficient mice after adoptive transfer of 2D2-transgenic T cells. (A) Wild-type (WT) and ADAP-deficient mice were adoptively transferred with MOG-specific transgenic T cells (2D2). One week later, EAE was induced by immunization with MOG35–55peptide in CFA. The severity of EAE is presented as clinical score (mean6SD;n= 5;p,0.05 WT + 2D2 T cells versus ADAP + 2D2 T cells). (B) Splenic T cells of 2D2 mice were stimulated in vitro with MOG35–55in the presence of IL-2 and IL-7 and were reactivated with plate-bound anti-CD3 and anti-CD28 in the presence of IL-12 and IL-18. Fully activated transgenic *2D2 T cells were adoptively transferred into WT or ADAP-deficient recipients, and EAE course was monitored. Data are shown as mean6SD of one representative experiment (n= 13 for WT andn= 10 for ADAP, respectively;p,0.05 WT + *2D2
4 EAE IN ADAP-DEFICIENT MICE
2D2 TCR–transgenic T cells do not reconstitute EAE in ADAP-deficient mice
To further substantiate this assumption we performed an additional adoptive transfer experiment where we transferred 2D2 TCR–
transgenic T cells (the 2D2 T cells express a TCR that specifically recognizes the MOG33–55peptide) into either wild-type or ADAP-deficient recipient mice. Subsequently, we induced EAE in the two cohorts by immunization with MOG35–55peptide. As shown in Fig.
2A, even under these experimental conditions, ADAP-deficient mice display a significantly milder disease score compared with wild-type mice, thus corroborating that T cell–extrinsic functions of ADAP are responsible for the ameliorated disease in ADAP-deficient animals.
The blunted course of EAE even after transfer of MOG-specific 2D2 cells could be due to an impaired priming phase of the T cell response or due to an altered effector phase (or both). To distinguish between these possibilities, we by-passed the induction phase of the immune response in the peripheral lymphoid organs by transferring ex vivo–activated and polarized 2D2-transgenic T cells. To this end primary non-activated 2D2 TCR transgenic T cells were isolated and ex vivo activated for 2 rounds using MOG35–55peptide fol-lowed by one round of in vitro stimulation using anti-CD3/anti-CD28 Abs. This procedure resulted in the generation of fully ac-tivated *2D2 T cells. The *2D2 T cells were then transferred into either wild-type or ADAP-deficient hosts and the clinical score of the diseased animals was monitored over time. This experiment showed that even the ex vivo–activated *2D2 T cells induced
significantly less severe EAE in ADAP-deficient recipients com-pared with wild-type recipients (Fig. 2B). Histological analysis of the spinal cords confirmed the clinical data by showing less cellular infiltration and significantly reduced numbers of meningeal, pa-renchymal, and total inflammatory foci in the ADAP-deficient recipients (Fig. 2C, 2D). Similarly, plasma concentration of the proinflammatory cytokine IFN-gthat plays a major pathophysio-logical role in EAE was significantly lower in ADAP-deficient mice. In contrast, no differences in the serum levels of IL-17A and TNF-awere observed between the two cohorts (Fig. 2E).
To exclude the possibility that endogenous ADAP-deficient T cells contribute to the milder EAE in ADAP-deficient mice, we performed Ab-mediated depletion of host T cells. Therefore, recipient mice were repeatedly injected with an anti-Thy1.2 Ab 2 d before adoptive transfer and on days 2, 6, and 10 after transfer.
This Ab-mediated depletion of host T cells does not alter EAE severity in ADAP-deficient and wild-type recipient mice after adoptive transfer of fully activated 2D2-transgenic T cells (Fig. 3).
In summary, the data shown in Figs. 2 and 3 clearly demonstrate that T cell–extrinsic functions of ADAP impair the inflammatory response in the ADAP-deficient animals.
ADAP-deficient mice showed reduced leukocyte infiltration in the spinal cord
We next attempted to characterize the immune cell populations that infiltrate the CNS during EAE in ADAP-deficient animals following transfer of *2D2 TCR–transgenic cells. Therefore, we FIGURE 3. Reduced EAE severity in
ADAP-defi-cient mice after adoptive transfer of 2D2-transgenic T cells and depletion of host T cells. (A) To deplete host T cells, recipient mice were repeatedly injected i.p. with 500mg anti-Thy1.2 mAb 2 d before adoptive transfer and on days 2, 6, and 10 after transfer. Starting on day 5, the disease progression was monitored daily.
Data are shown as mean6SD of one representative out of two independent experiment (n= 6 mice/group;
p,0.05 wild-type [WT] + *2D2 T cells + anti-Thy1.2 versus ADAP + *2D2 T cells + anti-Thy1.2). (B) FACS plots demonstrate the depletion of endogenous T cells in peripheral blood taken on day 6 after transfer.
FIGURE 4. Less inflammatory cells in the spinal cord in ADAP-deficient mice. EAE was induced by adoptive transfer of fully activated MOG-specific *2D2-transgenic T cells. At day 14 after transfer, leukocytes were isolated from the spinal cord by Percoll gradient.
(A) Cells were stained with Abs to CD45 and CD11b and analyzed by flow cytometry: R2, CNS-resident microglia; R4, invading macrophages and granulocytes;
and R6, invading lymphocytes. (B) Absolute cell numbers of these CNS subpopulations are depicted. Transgenic
*2D2 T cells were identified by the presence of the
The Journal of Immunology 5
isolated the leukocytes from the spinal cord at the clinical peak of disease and assessed their phenotypes by flow cytometry. Ex-pression of CD45 was used to distinguish CNS-resident microglia (CD45 low) from invading leukocytes (CD45 high, R2; Fig. 4A).
The CNS invading cell populations were further distinguished by the presence (macrophages and granulocytes) or absence (lympho-cytes) of CD11b. This analysis revealed that ADAP-deficient mice showed consistently fewer invading leukocytes than their wild-type counterparts (regions R4 and R6; Fig. 4A). A deeper analysis of the leukocytes showed that significantly less lymphocytes, including TCR-transgenic Va3.2 T cells, macrophages and DCs invaded the CNS of ADAP-deficient mice. In contrast, no difference in the number of resident microglia was observed (Fig. 4B). Further sub-typing of the infiltrating CD4+T cells showed similar frequencies of FoxP3 regulatory T cells, IL-17+Th17 cells, IFN-g+Th1 cells and IL-10–producing cells (data not shown). Thus, loss of ADAP leads to a globally impaired infiltration of leukocytes into the CNS without affecting a particular T cell or leukocyte subpopulation.
*2D2 T cells accumulate in the lymph nodes in ADAP-deficient mice
After induction of passive EAE, activated encephalitogenic T cells home through the secondary lymphoid organs before they enter the CNS (38). To localize the ex vivo–activated inflammatory *2D2 cells over time, we isolated lymph nodes, spleen, blood, and spinal cord at days 7, 10, and 14 after adoptive transfer and quantified the transgenic T cells within the individual organs by flow cytometry.
This approach revealed no significant differences between wild-type and ADAP-deficient recipients in the absolute number of
*2D2 T cells in the spleen and the percentage of *2D2 T cells in the blood (Fig. 5A). However, at the same time, we recovered significantly more *2D2 T cells from the lymph nodes and sig-nificantly less *2D2 T cells from the spinal cord of ADAP-deficient recipients. This indicates that in the absence of ADAP the ac-tivated *2D2-transgenic T cells are retained within the peripheral lymph nodes.
Next, we investigated whether the accumulation of T cells within the lymph nodes was a specific property of the ex vivo–activated
*2D2 T cells. Therefore, we repeated the adoptive transfer experi-ments this time using nonactivated 2D2 T cells (Fig. 5B). Also under these experimental conditions, we found an increased percentage of nonactivated 2D2 T cells in the lymph nodes of ADAP-deficient recipients. At the same time, similar numbers of nonactivated 2D2 T cells were recovered from the blood and the spleen of both ADAP-deficient and wild-type mice. Collectively, these results demonstrate that in ADAP-deficient recipients, T cells accumulate in the peripheral lymph nodes independent of their activation state.
Nonhematopoietic radio-resistant cells partially mediate the milder course of EAE in ADAP-deficient mice
To dissect whether the accumulation of T cells in the lymph nodes of ADAP-deficient mice was mediated by cells of the hematopoietic system or by nonhematopoietic cells (e.g., stromal cells of the lymph node), we generated radiation BM chimeras and recon-stituted the hematopoiesis of ADAP-deficient recipients with wild-type BM. To track the reconstituted cells, we used the B6.SJL congenic mouse strain carrying the CD45.1 (Ly-5.1) allele. Eight weeks after reconstitution, EAE was induced by immunization with MOG35–55peptide in CFA. As shown in Fig. 6A, even after transfer
mice. After reconstitution, EAE was induced by two injections of pertussis toxin. As shown in Fig. 6B, wild-type recipients devel-oped severe disease whereas ADAP-deficient recipients did not.
These data show that the attenuated EAE in ADAP-deficient mice is mediated via a radio-resistant nonhematopoietic mechanism.
Microglia is the CNS-resident radio-resistant macrophage, critical for the development of EAE by the release of cytokines as well as reactive oxygen species (39). To assess whether ADAP deficiency might result in diminished function of radio-resistant microglia cells, we investigated functional activity of wild-type and ADAP-deficient primary microglia. For this purpose, NO production and
FIGURE 5. Accumulation of adoptively transferred T cells in the lymph nodes of ADAP-deficient mice. (A) Wild-type (WT) and ADAP-deficient recipients were adoptively transferred with fully activated *2D2-transgenic T cells. At days 7, 10, and 14 after transfer, the number of activated *2D2 T cells (gated on CD4+Va3.2+) in spleen, lymph nodes, spinal cord, and
6 EAE IN ADAP-DEFICIENT MICE