The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Amend, B.
Right arrow Articles by Bischof, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Amend, B.
Right arrow Articles by Bischof, F.
The Journal of Immunology, 2006, 177: 4384-4390.
Copyright © 2006 by The American Association of Immunologists, Inc.

Induction of Autoimmunity by Expansion of Autoreactive CD4+CD62Llow Cells In Vivo1

Bastian Amend2,*, Hong Doster2,*, Christian Lange*, Evelyn Dubois*, Hubert Kalbacher{dagger}, Arthur Melms* and Felix Bischof3,*

* Hertie Institute for Clinical Brain Research, Department of General Neurology, University of Tübingen, Tübingen, Germany; and {dagger} Medical and Natural Sciences Research Center, University of Tübingen, Tübingen, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The prerequisites of peripheral activation of self-specific CD4+ T cells that determine the development of autoimmunity are incompletely understood. SJL mice immunized with myelin proteolipid protein (PLP) 139–151 developed experimental autoimmune encephalomyelitis (EAE) when pertussis toxin (PT) was injected at the time of immunization but not when injected 6 days later, indicating that PT-induced alterations of the peripheral immune response lead to the development of autoimmunity. Further analysis using IAs/PLP139–151 tetramers revealed that PT did not change effector T cell activation or regulatory T cell numbers but enhanced IFN-{gamma} production by self-specific CD4+ T cells. In addition, PT promoted the generation of CD4+CD62Llow effector T cells in vivo. Upon adoptive transfer, these cells were more potent than CD4+CD62Lhigh cells in inducing autoimmunity in recipient mice. The generation of this population was paralleled by higher expression of the costimulatory molecules CD80, CD86, and B7-DC, but not B7-RP, PD-1, and B7-H1 on CD11c+CD4+ dendritic cells whereas CD11c+CD8{alpha}+ dendritic cells were not altered. Collectively, these data demonstrate the induction of autoimmunity by specific in vivo expansion of CD4+CD62Llow cells and indicate that CD4+CD62Llow effector T cells and CD11c+CD4+ dendritic cells may be attractive targets for immune interventions to treat autoimmune diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The putative autoimmune disease multiple sclerosis (MS)4 and its animal model experimental autoimmune encephalomyelitis (EAE) are characterized by an inflammatory destruction of myelin and axons in the CNS (1, 2). CD4+ helper T cells specific for Ags in the CNS play a central role in orchestrating the effector processes of this self-directed immune response (3, 4). Autoimmune CNS inflammation in MS and EAE is preceded by activation of CD4+ T cells specific for CNS Ags in peripheral lymphoid organs (5, 6), but peripheral activation of CNS-specific T cells is not necessarily sufficient to induce autoimmunity in the CNS. In this study, we assessed the prerequisites of a peripheral immune response that lead to the development of CNS autoimmunity.

Pertussis toxin (PT), the major toxin produced by Bordetella pertussis, facilitates the development of autoimmunity in several experimental animal models including experimental autoimmune orchitis and EAE (7, 8). Within hours after i.v. injection, PT induces changes at the blood-brain barrier that have been believed to promote CNS autoimmunity by facilitating lymphocyte entry into the CNS (9, 10). In addition, in vitro incubation with PT enhances T cell proliferation, production of Th1- and Th2-type cytokines by T cells (11, 12, 13) and expression of MHC class II and costimulatory molecules on APCs (12, 13).

To determine the characteristics of the peripheral immune response that determine the development of autoimmunity in the CNS, we compared mice immunized with PLP139–151/CFA to mice that received, in addition, i.v. injections of PT. PLP139–151/CFA-immunized mice were completely free of clinical and histological signs of inflammation in the CNS whereas mice that received additional PT developed severe EAE. PT induced EAE in immunized mice only when given at the time of immunization but not when given directly before entry of immune cells into the CNS, indicating that its disease-inducing effect is due to changes of the peripheral immune response rather than alterations at the blood-brain barrier. More detailed analysis using I-As-PLP139–151 tetramers revealed that PT did not induce changes in the number of major cell types in secondary lymphoid organs and did not alter the kinetics of T cell activation or the generation of natural regulatory T cells. Instead, we observed a selective expansion of CD4+CD25lowCD62Llow effector T cells. This population was more potent than CD4+CD25lowCD62Lhigh cells in inducing a self-directed immune response as demonstrated by cytometric isolation and adoptive transfer experiments. The PT-induced expansion of this population of autoreactive T cells was accompanied by distinct alterations in APCs. Expression of CD80, CD86, and B7-DC but not B7-H1, B7-RP, and PD-1 on CD11c+CD4+ dendritic cells (DCs) was increased, whereas expression of these costimulatory molecules on CD11c+CD8{alpha}+ DCs was not altered. These data demonstrate a new mechanism for the induction of autoimmunity in the CNS and suggest that CD4+CD25lowCD62Llow effector T cells and CD11c+CD4+ may be attractive targets for immune interventions to treat autoimmune diseases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice, Ags, and Abs

SJL mice were purchased from Harlan Winkelmann and maintained in the animal facility at the Hertie Institute for Clinical Brain Research (Tübingen, Germany). In all experiments, 6- to 12-wk old female mice were used according to approved protocols. The peptides myelin basic protein (MBP) 84–96 (VVHFFKNIVTPRTP) and PLP139–151 (HCLGKWLGHPDKF) were synthesized using standard 9-fluorenylmethoxycarbonyl (FMOC) chemistry and purified by HPLC. All Abs were purchased from BD Pharmingen (except for mAbs against B7-DC, B7-H1, B7-RP, and PD-1 which were purchased from eBioscience).

Production of MHC II tetramers

Recombinant MHC class II tetramers were produced essentially as described (6). The proteins were expressed in suspension cultures of Sf9 cells in protein-free insect medium (BD Pharmingen). Recombinant monomers were purified under native conditions from the supernatant using Ni+ chromatography (Qiagen) and biotinylated as described. Tetramers were formed by incubation with PE- or APC- labeled streptavidin (Molecular Probes, MoBiTec).

Immunization and EAE induction

For induction of EAE, 50 nmol of the peptide PLP139–151 in PBS emulsified with an equal amount of CFA containing 200 µg of mycobacterium tuberculosis H37RA (Difco) was injected s.c. on the back of the foot. When indicated, mice received in addition a single i.v. injection of 200 ng of pertussis toxin (List Biological Laboratories) or pertussis toxin B-oligomer (Sigma-Aldrich) in PBS. Clinical signs of EAE were assessed according to the following score: 0, no apparent abnormalities; 1, tail or hind limb weakness; 2, limp tail and hind limb weakness; 3, severe hind limb paresis; 4, complete hind limb paralysis and front limb weakness; 5, dead (dead mice were scored 5 if they had previously shown signs of progressive disease).

Flow cytometry

Single-cell suspensions were prepared from draining lymph nodes (DLN) and spleen as described (6). Cells were incubated with 20 µg/ml tetramer in 20 µl of PBS containing 1% serum albumin and 0.02% NaN3 at 4°C overnight, stained on ice with additional Abs for 20 min and directly before analysis with 1 µg/ml propidium iodide. Flow cytometric analysis was performed on life cells using a FACSCalibur System (BD Biosciences) or a Cyan Cytometer (DakoCytomation) and analyzed using Summit software (Dakocytomation).

Tetramer staining and intracellular cytokine staining

A total of 500,000 cells was incubated with 20 µg/ml tetramer at 4°C in 20 µl of PBS containing 1% serum albumin and 0.02% NaN3 overnight and then stained with anti-CD4 FITC for 20 min on ice. Cells were then fixed and permeabilized with Cytofix/Cytoperm (BD Pharmingen) for 20 min, washed twice with Wash/Perm (BD Pharmingen), and incubated with anti-cytokine Abs or anti-active caspase-3 Abs (anti-IFN{gamma}-PE (1/300), anti-IL-4-PE (1/300), anti-active caspase-3-PE (4 µl), all from BD Pharmingen) in 20 µl of Wash/Perm for 30 min on ice. Then cells were washed and analyzed by flow cytometry.

Histology

CNS samples including brain and spinal cord were obtained from CO2-sacrificed mice that had been extensively transcardially perfused with PBS. Brain and spinal cord were snap-frozen in liquid nitrogen and stored at –70°C. Cryostat sections (7–8 µm) were air-dried and stained with H&E. For immunofluorescence microscopy, cryostat sections were fixed with acetone on cover slips and air-dried. Sections were incubated with supernatant of MOMA-1 mAb (kindly provided by G. Kraal, Free University of Amsterdam, The Netherlands) (14) and anti-rabbit Fab-Cy3 (Molecular Probes), FITC-labeled anti-CD4 (BD Pharmingen) and 4',6'-diamidino-2-phenylindole (DAPI) (Molecular Probes).

Isolation and adoptive transfer of purified cell populations

Single-cell suspensions were prepared from spleen 10 days after immunization with PLP139–151 peptide/CFA. CD4+CD62Llow and CD4+CD62Lhigh cell populations were isolated by magnetic separation using the MACS CD4CD62L T cell isolation kit (Miltenyi Biotec). The purity of the isolated cell populations was assessed by flow cytometry. More than 90% of CD4+ T cells was CD62Lhigh in the CD4+CD62Lhigh population and >90% of CD4+ cells was CD62Llow in the CD4+CD62Llow population. Ten million CD4+CD62Llow or CD4+CD62Lhigh cells were adoptively transferred into naive SJL mice 1 day before immunization with 50 nmol of PLP139–151 peptide in CFA.

Data analysis

Statistical significance was tested by Student’s t test with an {alpha} level of 0.05. Error bars indicate SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
PT induces EAE in neuroantigen/CFA immunized mice when given at the time of immunization but not when given at day 6 after immunization

EAE is classically induced in rodents by s.c. injection of Ag emulsified in CFA plus i.v. injection of PT (8). Although PT is not an absolute requirement for EAE induction, it greatly facilitates the development of the disease. Mice immunized with PLP139–151/CFA which received in addition PT on the day of immunization developed severe clinical disease with a relapsing remitting disease course (Fig. 1A) and perivascular inflammatory infiltrations in the CNS (Fig. 1B). In contrast, control mice immunized with PLP139–151/CFA that received i.v. injections of PBS did not develop clinical or histological signs of EAE (Fig. 1).


Figure 1
View larger version (93K):
[in this window]
[in a new window]
  		FIGURE 1. PT induces EAE in neuroantigen/CFA-immunized mice when given at the time of immunization but not when given directly before entry of autoreactive T cells into the CNS. A, Mice were immunized with 50 nmol of PLP139–151 peptide in CFA and received in addition i.v. injections of PBS on day 0 or PT on day 0 or day 6 after immunization as indicated (n = 4 per group; error bars indicate SEM). Mice immunized with PLP139–151/CFA that received in addition 200 ng of PT oligomer B as additional control did not develop clinical or histological signs of EAE (data not shown). B, Representative CNS sections 14 days after immunization with PLP/CFA plus i.v. injection of PT or PBS as indicated. C, CD4+ T cells have access to the T cell areas of the spleen. Mice were immunized with PLP139–151/CFA. One group received in addition 200 ng of PT i.v. After 10 days, spleen sections were prepared and stained with an Ab against marginal zone macrophages (MOMA-1, red) and anti-CD4 (green) and examined by fluorescence microscopy.

 
In PLP139–151-induced EAE in SJL mice, autoreactive T lymphocytes start to infiltrate the CNS at day 8 after immunization (6). To assess whether the disease-enhancing effect of PT is due to its effects on the peripheral activation of the immune response or by alterations at the blood-brain barrier, PLP139–151/CFA immunized mice received PT on day 6 after immunization, 2 days before entry of self-reactive lymphocytes into the CNS. These mice developed only faint disease symptoms starting at day 28 post immunization (Fig. 1A) indicating that PT enhances the development of CNS autoimmunity by interfering with the generation of the immune response in the periphery rather than by directly enhancing the permeability of the blood-brain barrier.

The frequency of lymphocytes, NK cells and DCs in DLN and spleen is not significantly altered by PT

To characterize the PT-induced changes of the peripheral immune response, we first assessed the cellular composition of DLN and spleen in immunized mice. Flow cytometric analysis of cells isolated from DLN and spleen 10 days after immunization with neuroantigen/CFA revealed that the frequency of T cells, B cells, NK cells, and different DC populations (CD11c+CD4+ DCs, CD11c+CD8{alpha}+ DCs, and CD11c+B220+Gr-1+ plasmacytoid DCs) was not significantly altered by PT (Table I).


View this table:
[in this window]
[in a new window]
  		Table I. PT does not significantly alter the cellular composition of DLN and spleen in immunized micea

 
CD4+ T cells have access to the T cell areas of the spleen in PT-treated mice

The requirement of peripheral lymphoid organs for lymphocyte entry into the CNS is a matter of ongoing debate. After adoptive transfer, CNS-specific lymphocytes first migrate to peripheral lymph nodes and spleen, where they undergo profound phenotypic changes before they enter the CNS (15), whereas after EAE induction in mice that genetically lack lymph nodes and display a disturbed splenic architecture, lymphocytes show normal infiltration into the CNS (16). Earlier reports indicated that PT might inhibit migration of T and B cells into the T cell areas of the spleen (17). This led to the hypothesis that PT enhances EAE by inhibiting access of lymphocytes to the sites of peripheral tolerance induction in secondary lymphoid organs (17). To directly test this hypothesis, spleen sections of mice 10 days after EAE induction were stained with anti-CD4 and MOMA-1, an Ab against marginal zone macrophages, and examined by immunofluorescence microscopy. CD4+ T cells were present in the T cell areas of the spleen in immunized mice with or without additional administration of PT (Fig. 1C) and thus have access to the sites of peripheral tolerance induction.

PT promotes Th1 differentiation of autoreactive T cells in vivo but does not alter T cell activation

Autoreactive, CNS-specific Th1 cells play a central role in the pathogenesis of EAE (4). We next asked whether the enhanced autoreactivity in PT-injected mice is related to increased production of Th1-type cytokines. Lymphocytes isolated from DLN 8 days after immunization with PLP139–151/CFA were analyzed after simultaneous staining with anti-CD4, I-As-PLP139–151 tetramers (6) and Abs against intracellular IFN-{gamma} or IL-4. Autoreactive Th cells of mice that received PT at the time of immunization expressed higher levels of intracellular IFN-{gamma} than mice that did not receive PT although the levels of intracellular IL-4 were similar (Fig. 2A). In addition, the kinetics of in vivo activation of autoreactive T cells following immunization was not altered by PT as assessed by expression of the activation marker CD44 (Fig. 2B). PT thus promotes the production of Th1-type cytokines in immunized mice but does not significantly alter T cell activation.


Figure 2
View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 2. PT increases IFN-{gamma} production by autoreactive CD4 T cells but does not alter T cell activation. A, Cells isolated from DLN 8 days after immunization with PLP139–151/CFA and additional i.v. injection of PT or PBS were stained with anti-CD4, I-As-PLP139–151 tetramers and Abs against intracellular IFN-{gamma} and IL-4 as indicated and analyzed by flow cytometry. One representative of three independent experiments. B, Cells were isolated from DLN at the indicated time points after EAE induction and analyzed by flow cytometry after staining with anti-CD4, I-As-PLP139–151 tetramers and anti-CD44 ({blacktriangleup}: CD4+/I-As-PLP139–151+, {square}amp;: CD4+/I-As-PLP139–151– cells; error bars indicate SEM).

 
The numbers of CD4+CD25+ regulatory T cells are not altered by PT

Natural regulatory T cells are central to the regulation of peripheral tolerance (18). Intravenous injection of PT into mice immunized with neuroantigen/CFA did not induce changes in the frequency of CD4+CD25+CD69– natural regulatory T cells in DLN (Fig. 3), indicating that regulatory T cells do not play a significant role in the induction of CNS autoimmunity by PT.


Figure 3
View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 3. PT does not significantly alter the frequency of CD4+CD25+CD69– regulatory T cells. Cells isolated from DLN 8 days after immunization with PLP139–151/CFA and additional i.v. injection of PT or PBS were stained with Abs against CD4, CD25, and CD69 and analyzed by flow cytometry. A, Dot plots are gated on CD4+ cells. B, The frequency of CD25+CD69– cells of all CD4+ cells is not altered by PT (*, p = 0.4; error bars indicate SEM; one representative of three independent experiments).

 
PT induces the generation of CD4+CD62Llow effector T cells

The lymph node homing receptor CD62L (L-selectin) is highly expressed on naive T cells and down-regulated after T cell activation (19). We asked whether the induction of autoimmunity by PT correlates to differential expression of CD62L by autoreactive CD4+ T cells. First, the kinetics of CD62L expression during the induction phase of EAE was assessed. In unimmunized mice, 80% of CD4 T cells in the DLN and 30% of CD4 T cells in the spleen expressed CD62L (Fig. 4A). Following EAE induction by s.c. injection of PLP139–151/CFA and i.v. injection of PT, the proportion of CD62L-expressing CD4 T cells in DLN declined to 30% at day 8 after immunization whereas the frequency of CD62Lhigh cells in the spleen remained constant at 20–30% (Fig. 4A). The influence of PT on the proportion of CD62Lhigh and CD62Llow expressing CD4+CD25– effector T cells was further analyzed. PT induced a marked increase in the population of CD4+CD25–CD62Llow effector T cells (Fig. 4B) whereas the frequency of CD62L expressing CD4+CD25+ regulatory T cells was not significantly altered (Fig. 4B). The induction of CNS autoimmunity by PT thus correlates to in vivo expansion of CD4+CD25–CD62Llow effector T cells.


Figure 4
View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 4. PT induced expansion of CD4+CD25–CD62Llow cells. A, EAE was induced by s.c. immunization with PLP139–151/CFA and i.v. injection of PT. Mice were sacrificed at different time points after disease induction, and cells isolated from DLN and spleen were analyzed by flow cytometry after simultaneous staining with I-As-PLP139–151 tetramers and Abs against CD4 and CD62L (n = 4 per time point; error bars indicate SEM). B, Mice were immunized s.c. with PLP139–151/CFA and received in addition i.v. injections of PT or PBS. Ten days later, flow cytometry of spleen cells was performed after simultaneous staining with Abs against CD4 and CD62L. Representative dot plots are gated on CD4+ T cells (n = 6 per group in three independent experiments; error bars indicate SEM).

 
CD4+CD62Llow effector T cells mediate autoimmunity upon adoptive transfer

To assess the potency of the expanded population of CD4+CD62Llow effector T cells to induce EAE, CD4+CD62Llow, and CD4+CD62Lhigh cells were adoptively transferred to naive SJL mice. Recipient mice were immunized with PLP139–151/CFA without additional injection of PT. Adoptive transfer of both cell populations induced EAE in immunized mice. EAE was more severe and developed earlier in mice that received CD4+CD62Llow cells compared with mice that received CD4+CD62Lhigh cells (Fig. 5).


Figure 5
View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 5. CD4+CD62Llow cells are more potent in inducing EAE than CD4+CD62Lhigh cells. CD4+CD62Llow or CD4+CD62Lhigh cells were isolated from the spleen of mice immunized with PLP139–151/CFA by magnetic separation. More than 90% of CD4+ T cells were CD62Llow in the CD4+CD62Llow population and >90% of CD4+ cells were CD62Lhigh in the CD4+CD62Lhigh population as determined by flow cytometry. Ten million cells were adoptively transferred into naive SJL mice and control mice received i.v. injections of PBS as indicated. Recipient mice of all groups were immunized 1 day later with 50 nmol of PLP139–151 peptide in CFA (n = 4 per group; error bars indicate SEM; mean disease onset at day 14 and maximal mean disease score of 2.0 in mice that received CD4+CD62Lhigh cells and mean disease onset at day 7 and maximal mean disease score of 4.5 in mice that received CD4+CD62Llow cells).

 
PT induces expression of CD80, CD86, and B7-DC on CD11c+CD4+ DC

We next sought to determine whether the established alterations in the CD4+ T cell compartment correlate to distinct changes in APCs. Because the number of the different DC subsets in secondary lymphoid organs was not altered (Table I), we asked whether PT might influence costimulatory molecule expression by APCs. The expression of CD80, CD86, and B7-DC on CD11c+CD4+ DC in the spleen was increased by PT (Fig. 6), while expression of B7-H1, B7-RP, and PD-1 on CD11c+CD4+ cells and costimulatory molecule expression (CD80, CD86, B7-DC, B7-H1, B7-RP, and PD-1) on CD11c+CD8{alpha}+ cells was not significantly altered (data not shown).


Figure 6
View larger version (34K):
[in this window]
[in a new window]
  		FIGURE 6. PT induces CD80, CD86, and B7-DC on CD11c+CD4+ DC. Mice were immunized subcutaneously with PLP139–151/CFA and received in addition i.v. injections of PT or PBS, respectively. Ten days later, flow cytometry of spleen cells was performed after simultaneous staining with Abs against CD4, CD11c, and either CD80, CD86, or B7-DC as indicated (error bars indicate SEM; *, p < 0.03, t test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study assesses the cellular mechanisms that underlie the induction of autoimmunity in the CNS. It has long been recognized that the development of autoimmunity in the CNS is preceded by activation of self-specific lymphocytes in peripheral lymphoid organs (5, 6), but the characteristics of the peripheral immune response that determine the development CNS autoimmunity have not yet been resolved.

Previous studies established that PT causes changes in peripheral lymphoid organs as well as alterations at the blood-brain barrier that take place within hours of injection (9, 10). We demonstrated that the development of EAE is due to the effects of PT on the peripheral immune response and not to changes at the blood-brain barrier. Induction of EAE by PT required administration of PT before peripheral activation of lymphocytes whereas injection of PT after lymphocyte activation (6) and before entry of lymphocytes into the CNS (6) did not have any disease enhancing effect. This is perfectly in line with the fact that PT is not required for EAE induction by adoptive transfer of in vitro activated CNS-specific CD4 T cells (20). In addition, PT-induced alterations at the blood-brain barrier are unlikely to contribute to autoimmunity in the CNS because migration of lymphocytes through endothelial barriers is controlled by chemokines and chemokine receptors. Chemokines and their receptors signal through G proteins, and G proteins are blocked by PT. For this reason, PT inhibits rather than facilitates migration of lymphocytes across endothelial walls, and it has been shown that this is indeed the case (21, 22, 23).

PT did not induce changes in the composition of major cellular components of lymph nodes and spleen. The frequencies of T cells, NK cells, and different types of DC were not significantly altered and PT did not influence number and activation of self-specific CD4 T cells. These findings were surprising, because the clinical and histological differences in both experimental groups were dramatic, and we thus expected to find more pronounced effects on major cellular subtypes in peripheral lymphoid organs. In addition, PT did not induce changes in the numbers of natural regulatory T cells indicating that regulatory T cells do not play a major role in the induction of CNS autoimmunity by PT. This was also unexpected because in a number of experimental systems, natural regulatory T cells have been shown to be central to the regulation of immunological tolerance toward CNS Ags (24, 25, 26).

Importantly, the induction of EAE in neuroantigen/CFA immunized mice by PT was related to expansion of CD4+CD25–CD62Llow effector T cells. Down-regulation of CD62L on CD4+ T cells was not simply due to increased T cell activation, because expression of other T cell activation markers including CD44, CD25, and CD69 was not significantly altered (Figs. 2 and 4A and data not shown). These data demonstrate differential regulation of CD25, CD69, CD44, and CD62L, which are often used as general markers for T cell activation. In contrast, >90% of CNS-specific cells isolated from the CNS of mice with severe EAE are CD62Llow and display also other activation markers including CD44, CD69, and CD25 (6) (data not shown).

The population of CD4+CD25–CD62Llow cells was more potent in inducing EAE after adoptive transfer than CD4+CD25–CD62Lhigh cells. The higher capacity of CD62Llow helper T cells to mediate a self-directed immune response could be explained by several possible mechanisms. CD62Llow cells lack the ability to home to peripheral lymphoid organs (21) while CD62L is not required for lymphocyte migration into the CNS (27). A higher number of CNS-specific CD4 T cells may thus be available for CNS entry. This notion is consistent with the absence of detectable lymphocyte infiltration in the CNS of immunized mice that did not receive PT (Fig. 1).

Moreover, the higher autoreactivity of CD62Llow cells could be related to properties of these cells that are distinct from CD62L expression. CD62Llow cells may produce higher amounts of Th1-type cytokines that critically determine the magnitude of the inflammatory response (4). This is supported by increased production of IFN-{gamma} by self-specific CD4 T cells after PT injection (Fig. 2). In line with this, a higher production of IFN-{gamma} in CD62Llow compared with CD62Lhigh cells has been reported in CD8+ CTLs and memory CD4+ T cells (28, 29). In addition, CD62Llow cells may be more susceptible to peripheral tolerance mechanisms (17).

The role of CD62L in the development of autoimmune diseases is controversial. CD62L-deficient mice show a reduced capacity to generate primary T cell responses (30) and to develop EAE (27). Treatment with a blocking Ab against CD62L reduces the spontaneous development of insulitis in NOD mice (31) but has been reported not to significantly alter EAE (20). However, CD62Llow cells still express detectable levels of CD62L and may thus be functionally different from CD62L-deficient cells. This notion is supported by the fact that CNS-specific CD4 T cells in CD62L-deficient mice enter the CNS but do not induce myelin destruction or EAE symptoms (27), although in the experiments reported here, CD62Llow cells induced severe lymphocyte infiltration, inflammation, and clinical disease (Fig. 1).

The conflicting results on the role of CD62L in peripheral tolerance may be due to the broad tissue distribution of CD62L. CD62L is expressed on CD4+ T cells, CD8+ T cells, B cells, neutrophils, monocytes, eosinophils (32), and NK cells (33), and expression of CD62L on these different cell types is likely to have distinct consequences in vivo. Our data demonstrate that low expression of CD62L specifically on CD4+ T cells leads to the breakdown of peripheral tolerance and the development of autoimmunity.

A role for CD62L in discriminating different subtypes of helper T cells has previously been established for Ag-experienced cells. Memory CD4 T cells, which express high levels of CD62L (central memory T cells), migrate through peripheral lymph nodes whereas CD4+CD62Llow memory T cells (effector memory T cells) produce higher amounts of IFN-{gamma} and mediate strong effector functions during recall responses (34). In the CD8+ T cell compartment, CD62Llow cells generate stronger proliferative responses and secrete higher amounts of IFN-{gamma} after viral infection compared with CD62Lhigh cells (29). In addition, CD8+CD62Llow cells preferentially secrete Th1-type cytokines after viral challenge (29). These data are consistent with our results, which demonstrate that the propagation of CD4+CD62Llow cells correlates to increased production of IFN-{gamma} and the development of CNS autoimmunity.

PT induced expression of the costimulatory molecules CD80, CD86, and B7-DC but not B7-H1, B7-RP, and PD-1 on CD4+ DC although costimulatory molecule expression on CD8{alpha}+ DC was not altered. These data thus demonstrate distinct changes in peripheral CD4+ DC that are related to the propagation of CD4+CD62Llow T cells and the development of autoimmunity in the CNS. CD4+ DC are a major subgroup of DC which take up and process pathogen-derived Ags and stimulate specific CD4 T cells in the T cell areas of the spleen. CD8{alpha}+ DC preferentially capture apoptotic cells and present Ags of these cells to CD4+ and CD8+ T cells in a tolerogenic manner (35). Our results thus indicate that the induction of CNS autoimmunity by PT is related to enhanced T cell activation via CD4+ DC and not via alterations in tolerogenic CD8{alpha}+ DC.

B7-DC is a member of the B7 family of costimulatory molecules which is mainly expressed by DC and closely homologous to its broadly expressed relative B7-H1. B7-DC and B7-H1 both interact with PD-1. The precise function of B7-DC in vivo is presently unclear (36). Some reports indicate that B7-DC inhibits T cell responses via PD-1 while it enhances T cell activation by interacting with an as yet unidentified receptor (36, 37). Our results show that increased expression of B7-DC on CD4+ DC is related to the induction of CNS autoimmunity and further demonstrate differential in vivo regulation of B7-DC and its closest relative B7-H1.

In conclusion, the data presented in this report demonstrate a new mechanism that leads to the development of organ-specific autoimmunity. PT induced EAE by specifically increasing the number of CD4+CD62Llow cells, and these cells were more potent than CD4+CD62Lhigh cells in triggering CNS autoimmunity. These data further indicate a role for CD62L on CD4+ T cells and CD80, CD86, and B7-DC on CD4+ DC in the regulation of peripheral tolerance. CD4+CD62Llow cells and CD4+ DC may thus be attractive targets for therapeutic immune interventions to treat autoimmune diseases.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by The Gemeinnützige Hertie-Stiftung (1.01.1/04/006 to F.B. and A.M.), the Deutsche Forschungsgemeinschaft (BI 603/5-1 to F.B.), the Interdisciplinary Clinical Research Center (IZKF) Tübingen, Germany (to F.B.) and the Boehringer Ingelheim Fonds (to C.L.). Back

2 B.A. and H.D. contributed equally to this work. Back

3 Address correspondence and reprint request to Dr. Felix Bischof, Hertie Institute for Clinical Brain Research, Department of General Neurology, University of Tübingen, Hoppe-Seyler-Strasse 3, 72076 Tübingen, Germany. E-mail address: Felix.Bischof{at}uni-tuebingen.de Back

4 Abbreviations used in this paper: MS, multiple sclerosis; DC, dendritic cell; DLN, draining lymph node; EAE, experimental autoimmune encephalomyelitis; PLP, proteolipid protein; PT, pertussis toxin. Back

Received for publication March 9, 2006. Accepted for publication July 17, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Frohman, E. M., M. K. Racke, C. S. Raine. 2006. Multiple sclerosis–the plaque and its pathogenesis. N. Engl. J. Med. 354: 942-955. [Free Full Text]
  2. Noseworthy, J. H., C. Lucchinetti, M. Rodriguez, B. G. Weinshenker. 2000. Multiple sclerosis. N. Engl. J. Med. 343: 938-952. [Free Full Text]
  3. Brosnan, C. F., C. S. Raine. 1996. Mechanisms of immune injury in multiple sclerosis. Brain Pathol 6: 243-257. [Medline]
  4. Kuchroo, V. K., A. C. Anderson, H. Waldner, M. Munder, E. Bettelli, L. B. Nicholson. 2002. T cell response in experimental autoimmune encephalomyelitis (EAE): role of self and cross-reactive antigens in shaping, tuning, and regulating the autopathogenic T cell repertoire. Annu. Rev. Immunol. 20: 101-123. [Medline]
  5. Steinman, L.. 1996. Multiple sclerosis: a coordinated immunological attack against myelin in the central nervous system. Cell 85: 299-302. [Medline]
  6. Bischof, F., M. Hofmann, T. N. M. Schumacher, F. A. Vyth-Dreese, R. Weissert, H. Schild, A. M. Kruisbeek, A. Melms. 2004. Analysis of autoreactive CD4 T cells in experimental autoimmune encephalomyelitis after primary and secondary challenge using MHC class II tetramers. J. Immunol. 172: 2878-2884. [Abstract/Free Full Text]
  7. Mochizuki, M., J. Charley, T. Kuwabara, R. B. Nussenblatt, I. Gery. 1983. Involvement of the pineal gland in rats with experimental autoimmune uveitis. Invest. Ophthalmol. Vis. Sci. 24: 1333-1338. [Abstract/Free Full Text]
  8. Lee, J. M., P. K. Olitsky. 1955. Simple method for enhancing development of acute disseminated encephalomyelitis in mice. Proc. Soc. Exp. Biol. Med. 89: 263-266. [Medline]
  9. Amiel, S. A.. 1976. The effects of Bordetella pertussis vaccine on cerebral vascular permeability. Br. J. Exp. Pathol. 57: 653-662. [Medline]
  10. Tonra, J. R., B. S. Reiseter, R. Kolbeck, K. Nagashima, R. Robertson, B. Keyt, R. M. Lindsay. 2001. Comparison of the timing of acute blood-brain barrier breakdown to rabbit immunoglobulin G in the cerebellum and spinal cord of mice with experimental autoimmune encephalomyelitis. J. Comp. Neurol. 430: 131-144. [Medline]
  11. Kong, A. S., S. I. Morse. 1977. The in vitro effects of Bordetella pertussis lymphocytosis-promoting factor on murine lymphocytes. I. Proliferative response. J. Exp. Med. 145: 151-162. [Abstract/Free Full Text]
  12. Ryan, M., L. McCarthy, R. Rappuoli, B. P. Mahon, K. H. Mills. 1998. Pertussis toxin potentiates Th1 and Th2 responses to co-injected antigen: adjuvant action is associated with enhanced regulatory cytokine production and expression of the co-stimulatory molecules B7-1, B7-2 and CD28. Int. Immunol. 10: 651-662. [Abstract/Free Full Text]
  13. Hofstetter, H. H., C. L. Shive, T. G. Forsthuber. 2002. Pertussis toxin modulates the immune response to neuroantigens injected in incomplete Freund’s adjuvant: induction of Th1 cells and experimental autoimmune encephalomyelitis in the presence of high frequencies of Th2 cells. J. Immunol. 169: 117-125. [Abstract/Free Full Text]
  14. Kraal, G.. 1992. Cells in the marginal zone of the spleen. Int. Rev. Cytol. 132: 31-74. [Medline]
  15. Flugel, A., T. Berkowicz, T. Ritter, M. Labeur, D. E. Jenne, Z. Li, J. W. Ellwart, M. Willem, H. Lassmann, H. Wekerle. 2001. Migratory activity and functional changes of green fluorescent effector cells before and during experimental autoimmune encephalomyelitis. Immunity 14: 547-560. [Medline]
  16. Greter, M., F. L. Heppner, M. P. Lemos, B. M. Odermatt, N. Goebels, T. Laufer, R. J. Noelle, B. Becher. 2005. Dendritic cells permit immune invasion of the CNS in an animal model of multiple sclerosis. Nat. Med. 11: 328-334. [Medline]
  17. Cyster, J. G., C. C. Goodnow. 1995. Pertussis toxin inhibits migration of B and T lymphocytes into splenic white pulp cords. J. Exp. Med. 182: 581-586. [Abstract/Free Full Text]
  18. Sakaguchi, S.. 2005. Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat. Immunol. 6: 345-352. [Medline]
  19. Butcher, E. C., L. J. Picker. 1996. Lymphocyte homing and homeostasis. Science 272: 60-66. [Abstract]
  20. Brocke, S., C. Piercy, L. Steinman, I. L. Weissman, T. Veromaa. 1999. Antibodies to CD44 and integrin {alpha}4, but not L-selectin, prevent central nervous system inflammation and experimental encephalomyelitis by blocking secondary leukocyte recruitment. Proc. Natl. Acad. Sci. USA 96: 6896-6901. [Abstract/Free Full Text]
  21. Warnock, R. A., S. Askari, E. C. Butcher, U. H. von Andrian. 1998. Molecular mechanisms of lymphocyte homing to peripheral lymph nodes. J. Exp. Med. 187: 205-216. [Abstract/Free Full Text]
  22. Alt, C., M. Laschinger, B. Engelhardt. 2002. Functional expression of the lymphoid chemokines CCL19 (ELC) and CCL 21 (SLC) at the blood-brain barrier suggests their involvement in G-protein-dependent lymphocyte recruitment into the central nervous system during experimental autoimmune encephalomyelitis. Eur. J. Immunol. 32: 2133-2144. [Medline]
  23. Adamson, P., B. Wilbourn, S. Etienne-Manneville, V. Calder, E. Beraud, G. Milligan, P. O. Couraud, J. Greenwood. 2002. Lymphocyte trafficking through the blood-brain barrier is dependent on endothelial cell heterotrimeric G-protein signaling. FASEB J. 16: 1185-1194. [Abstract/Free Full Text]
  24. Chorny, A., E. Gonzalez-Rey, A. Fernandez-Martin, D. Pozo, D. Ganea, M. Delgado. 2005. Vasoactive intestinal peptide induces regulatory dendritic cells with therapeutic effects on autoimmune disorders. Proc. Natl. Acad. Sci. USA 102: 13562-13567. [Abstract/Free Full Text]
  25. McGeachy, M. J., L. A. Stephens, S. M. Anderton. 2005. Natural recovery and protection from autoimmune encephalomyelitis: contribution of CD4+CD25+ regulatory cells within the central nervous system. J. Immunol. 175: 3025-3032. [Abstract/Free Full Text]
  26. Reddy, J., H. Waldner, X. Zhang, Z. Illes, K. W. Wucherpfennig, R. A. Sobel, V. K. Kuchroo. 2005. Cutting Edge: CD4+CD25+ regulatory T cells contribute to gender differences in susceptibility to experimental autoimmune encephalomyelitis. J. Immunol. 175: 5591-5595. [Abstract/Free Full Text]
  27. Grewal, I. S., H. G. Foellmer, K. D. Grewal, H. Wang, W. P. Lee, D. Tumas, C. A. Janeway, Jr, R. A. Flavell. 2001. CD62L is required on effector cells for local interactions in the CNS to cause myelin damage in experimental allergic encephalomyelitis. Immunity 14: 291-302. [Medline]
  28. Seder, R. A., R. Ahmed. 2003. Similarities and differences in CD4+ and CD8+ effector and memory T cell generation. Nat. Immunol. 4: 835-842. [Medline]
  29. Roberts, A. D., K. H. Ely, D. L. Woodland. 2005. Differential contributions of central and effector memory T cells to recall responses. J. Exp. Med. 202: 123-133. [Abstract/Free Full Text]
  30. Xu, J., I. S. Grewal, G. P. Geba, R. A. Flavell. 1996. Impaired primary T cell responses in L-selectin-deficient mice. J. Exp. Med. 183: 589-598. [Abstract/Free Full Text]
  31. Yang, X. D., N. Karin, R. Tisch, L. Steinman, H. O. McDevitt. 1993. Inhibition of insulitis and prevention of diabetes in nonobese diabetic mice by blocking L-selectin and very late antigen 4 adhesion receptors. Proc. Natl. Acad. Sci. USA 90: 10494-10498. [Abstract/Free Full Text]
  32. Iwabuchi, K., J. Ohgama, K. Ogasawara, C. Iwabuchi, I. Negishi, R. A. Good, K. Onoe. 1991. Distribution of MEL-14+ cells in various lymphoid tissues. Immunobiology 182: 161-173. [Medline]
  33. Chen, S., H. Kawashima, J. B. Lowe, L. L. Lanier, M. Fukuda. 2005. Suppression of tumor formation in lymph nodes by L-selectin-mediated natural killer cell recruitment. J. Exp. Med. 202: 1679-1689. [Abstract/Free Full Text]
  34. Zaph, C., J. Uzonna, S. M. Beverley, P. Scott. 2004. Central memory T cells mediate long-term immunity to Leishmania major in the absence of persistent parasites. Nat. Med. 10: 1104-1110. [Medline]
  35. Chung, Y., J. H. Chang, M. N. Kweon, P. D. Rennert, C. Y. Kang. 2005. CD8{alpha}-11b+ dendritic cells but not CD8{alpha}+ dendritic cells mediate cross-tolerance toward intestinal antigens. Blood 106: 201-206. [Abstract/Free Full Text]
  36. Shin, T., K. Yoshimura, E. B. Crafton, H. Tsuchiya, F. Housseau, H. Koseki, R. D. Schulick, L. Chen, D. M. Pardoll. 2005. In vivo costimulatory role of B7-DC in tuning T helper cell 1 and cytotoxic T lymphocyte responses. J. Exp. Med. 201: 1531-1541. [Abstract/Free Full Text]
  37. Radhakrishnan, S., E. Celis, L. R. Pease. 2005. B7-DC cross-linking restores antigen uptake and augments antigen-presenting cell function by matured dendritic cells. Proc. Natl. Acad. Sci. USA 102: 11438-11443. [Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Amend, B.
Right arrow Articles by Bischof, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Amend, B.
Right arrow Articles by Bischof, F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS