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Section of Immunobiology, Yale University School of Medicine, and Howard Hughes Medical Institute, New Haven, CT 06510
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been hypothesized that T cell-mediated autoimmune diseases are the result of inappropriate Ag presentation of either a self-Ag or an Ag with the capacity to mimic a self-Ag in the peripheral lymphoid tissues. An activated or memory T cell specific for a self peptide then has the potential to meet the self Ag where it is naturally expressed. In the case of experimental autoimmune encephalomyelitis (EAE), autoreactive T cells would re-encounter their specific self-Ag in the CNS. Immunization of mice with several different protein components of myelin, including myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein, are capable of eliciting an immune response resulting in the quintessential symptoms of EAE: ascending paralysis involving the tail and then the limbs (9, 10). EAE is a demyelinating disease in rodents and is used as a model of the human disease multiple sclerosis (9, 10, 11). EAE is characterized by focal areas of inflammation and demyelination throughout the CNS and can be actively induced in genetically susceptible animals by the injection of myelin Ags in CFA or passively by the adoptive transfer of activated CD4 T cells producing type 1 cytokines specific for myelin Ags, provided they express the adhesion molecule VLA-4 (9, 10, 12). In I-Au mice, the primary immunogenic peptide of MBP is the acetylated N-terminal peptide from residues 1 to 11 (Ac1–11) (9).
In this study, we show that DC capable of processing and presenting the self Ag MBP migrate to the lymph nodes via the afferent lymphatics, and interact with naive Ac1–11-specific T cells leading to the induction of EAE. In addition, Ac1–11-specific T cells were observed in the brains and spinal cords of mice 20 days following this unique means of EAE induction. These data demonstrate that presentation of a self Ag by DC in the periphery results in the activation of MBP-specific T cells and can lead to an overt autoimmune disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B10.PL mice (I-Au) were purchased from The Jackson Laboratory (Bar Harbor, ME). MBP-TCR transgenic (tg) mice were generated as previously described (13) and reared in our colony at Yale University. All mice used were between 5 and 10 wk of age.
Peptides and Abs
The MBP Ac1–11 (Ac-ASQKRPSQRSK) and the MBP121–140 (GFGYGGRASDYKSAHKGFKG) peptide were synthesized and HPLC purified by the W. M. Keck Biotechnology Resource Laboratory (Yale University). GK1.5 (rat anti-mouse CD4), HO-2.2 (mouse anti-mouse CD8), and RA3-3A1/6.1 (rat anti-mouse B220) were purchased from the American Type Culture Collection (Manassas, VA) and maintained in our laboratory. A clonotypic mAb (19G) specific for the MBP-TCR (12), Y3JP (mouse anti-mouse I-A), and Y19 (rat anti-mouse Thy1) were produced in our laboratory using standard hybridoma generation techniques. Anti-mouse CD4-biotin and anti-mouse Vß8.1,8.2-biotin were purchased from PharMingen (San Diego, CA).
Cells
DC were isolated as previously described (14).
Briefly, BM was flushed from the femurs and tibias of B10.PL mice. BM
cells were depleted of B cells, T cells, and I-A+ cells
using Abs and complement. The mAb used were GK1.5, HO-2.2, Y19,
RA3-3A1/6.1, and Y3JP. The remaining mononuclear cells were plated in
12-well plates at 0.5–1 x 106 cells/ml in RPMI 1640
medium supplemented with 5% FCS, 1 mM L-glutamine, 20
µg/ml gentamicin, 50 µM 2 ME, and 1% culture supernatant from
J558L cells transfected with a GM-CSF construct (kindly provided by
Fritz Melchers, Basel Institute of Immunology, Basel, Switzerland).
After 2 days, the nonadherent cells were gently removed and discarded,
and fresh medium was added every 2 days to the remaining adherent
cells. On day 6, the nonadherent and loosely adherent cells were
removed and replated into 100-mm culture tissues in the above medium.
Following overnight incubation, the nonadherent cells were collected
and pulsed with medium alone or Ac1–11 (100 µg/ml) for
up to 6 h in 100-mm culture dishes and washed twice in PBS before
transfer into mice. Purity of the DC was >60% as determined by flow
cytometry examining expression of B7-2 and high levels of MHC class II
(Y3JP). MBP-TCR CD4 T cells were isolated from the spleen of MBP-TCR tg
mice. Briefly, spleens were minced and RBC were removed followed by
depletion of cells expressing CD8, B220, and I-A using mAb and
complement. The mAb used were HO-2.2, RA3-3A1/6.1, and Y3JP. Purity of
the T cells was assessed using mAb specific for mouse CD4, CD8, B220,
ß TCR, Vß8.1,8.2, and the MBP-TCR clonotypic mAb 19G. The
MBP.TCR 31 clone was isolated from the spleen of a MBP tg mouse we
prepared (13) that expresses a TCR specific for
Ac1–11 and restricted to I-Au. The MBP.TCR 31
clone was obtained as previously described (15) using a
modified procedure of Srendi et al (16). The MBP.TCR 31
clone was maintained by restimulation in Click’s Eagle Hank’s amino
acid (EHAA) medium containing 10% FCS, 2 U/ml IL-2, and 5 µg/ml
Ac1–11 in the presence of inactivated spleen cells from
B10.PL mice (I-Au) every 3–6 wk and allowed to rest at
least 10 days before use. The MBP.TCR 31 clone is CD4+ and
expresses the MBP-TCR as assessed by staining with mAb 19G.
T cell activation assay
MBP.TCR 31 cloned cells (1 x 104) or splenic CD4+ MBP-TCR CD4 T cells (1 x 104) were incubated with 1 x 104 BM-derived DC isolated as described above in the presence or absence of 1:10 dilutions of the peptides Ac1–11 or MBP121–140 from 0.001 to 1 µg/ml in Click’s EHAA medium containing 5% FCS. In addition, 1 x 104 cloned T cells were incubated with increasing numbers of DC that had been pulsed with medium alone, 100 µg/ml Ac1–11, or 100 µg/ml whole mouse MBP (mMBP). CD4+ T cells (2 x 105), isolated from the spleen of a mouse 30 days posttransfer of Ac1–11-pulsed DC and MBP-TCR CD4 T cells (as described below), were cocultured with 2 x 105 inactivated H-2u splenocytes in the presence of increasing concentrations of Ac1–11. Proliferation was detected at 72 h by the addition of 1 µCi [3H]TdR to each well for the last 15–18 h of culture. Individual data points were set up in duplicate.
Cytokine secretion
IL-2 production from splenic CD4+ MBP-TCR CD4 T
cells was detected using CTLL-2 responder cells. Culture supernatants
were collected 24 h following coculture of 1 x
104 MBP-TCR CD4 T cells and 1 x 105
inactivated H-2u splenocytes in the presence of increasing
concentrations of Ac1–11. Prior to the addition of 5000
CTLL-2 cells, the culture supernatants were frozen at -70°C to kill
any viable cells. Following culture for 18 h, the cells were
pulsed with 1 µCi [3H]TdR and harvested after 4 h.
IFN-
was detected from culture supernatants collected 24 h
following coculture of 2 x 105 CD4+
splenic T cells, isolated from a mouse injected 30 days prior with
Ac1–11-pulsed DC and splenic MBP-TCR tg T cells, and
2 x 105 inactivated H-2u splenocytes in
the presence of increasing concentrations of Ac1–11.
IFN- was detected by ELISA as previously described
(15).
EAE induction
Irradiated (600 rads) female B10.PL mice were s.c. injected in the footpads (0.5 x 106 cells) and in each internal flank (1 x 106 cells) with BM-derived DC in PBS that had been pulsed with medium alone or Ac1–11. Twenty-four hours later, 10 x 106 MBP-TCR CD4 splenic T cells in PBS, isolated as described above, were injected i.v. into each animal. Individual animals were assessed daily starting at day 6 for symptoms of EAE and scored using a scale from 1 to 5 as follows: 0, no disease; 1, limp tail and/or wobbly walk; 2, hind limb paresis; 3, hind limb paralysis; 4, hind and fore limb paralysis; 5, death.
Labeling and in vivo detection of T cells and DC
DC and MBP-TCR tg T cells isolated as described above were labeled with the fluorescent dyes 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) (6 mg/ml) and 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanine perchlorate (DiI; 2.5 mg/ml) (Molecular Probes, Eugene, OR), respectively, for 30 min at 37°C after which they were washed three times in PBS. DC (0.5 x 106) pulsed with medium alone or with 100 µg/ml Ac1–11 were injected s.c. into each footpad followed by i.v. injection of 10 x 106 MBP-TCR tg CD4 T cells. After 24 h, the popliteal lymph nodes were harvested and fixed in paraformaldehyde-lysine-periodate overnight. The lymph nodes were sucrose infused prior to embedding in Tissue Tek OCT (Miles, Elkhart, IL) and frozen in isopentane. Frozen sections 7 µM thick were generated and visualized on a fluorescent microscope.
Immunohistopathological analysis
Brains and spinal cords were removed from perfused mice and fixed in paraformaldehyde-lysine-periodate. After 24 h, the tissues were sucrose infused, embedded in Tissue Tek OCT (Miles), and frozen in isopentane. For immunohistochemistry, frozen sections 7 µM thick were stained with biotinylated anti-CD4, biotinylated anti-Vß8.1,8.2, or biotinylated 19G. The color was developed using HistoMark Red Phosphatase System (Kiregaard & Perry Laboratories, Gaithersburg, MD) and counterstained with hematoxylin.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine the presentation of a MBP self peptide by DC, we
derived DC from the BM of B10.PL mice (H-2u), a mouse
strain that is genetically susceptible to the CNS autoimmune disease
EAE. To detect Ag presentation by the DC, we used a CD4+ T
cell clone (MBP.TCR 31) isolated from a mouse tg for the rearranged TCR
- and ß-chains of a cloned T cell line called clone 19 that is
restricted to I-Au and specific for Ac1–11
(12). As shown in Fig. 1
A, proliferation of the
MBP.TCR 31 clone occurred following presentation of the
Ac1–11 peptide by BM-derived DC in a dose-dependent
manner. The specificity of the MBP.TCR 31 clone to Ac1–11
is demonstrated by the lack of proliferation to the MBP peptide
composed of residues 121–140, which binds to I-Au and is
encephalitogenic in (B10.PL x SJL/J)F1 mice (B.
N. Dittel and C. A. Janeway, unpublished observations). Because
the goal of this study was to use Ac1–11-pulsed DC in vivo
to generate an immune response culminating in EAE, we examined the
proliferative response of the MBP.TCR 31 clone to
Ac1–11-pulsed DC. As shown in Fig. 1B,
proliferation of the MBP.TCR 31 clone was enhanced with the addition of
increasing numbers of Ac1–11-pulsed DC. In contrast, no
proliferation was observed when the DC were pulsed with medium alone.
Because Ag uptake and processing have been shown to change with DC
maturation (17), we tested whether the DC used in this
study were capable of protein Ag uptake leading to processing and
presentation of the Ac1–11 peptide. This is demonstrated
in Fig. 1C by the proliferative response of the MBP.TCR 31
to DC pulsed with whole mMBP.
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DC have been shown to form clusters with Ag-specific T cells in
vitro (18) and in vivo (8). Because this
clustering is believed to facilitate Ag presentation, we examined
whether DC would preferentially form interactions and clusters with
MBP-TCR bearing naive CD4 T cells in the lymph node of B10.PL mice when
pulsed with the self Ag Ac1–11 as compared to DC pulsed
with medium alone. BM-derived DC were labeled with the lipophilic dye
DiO (green fluorescence) and injected s.c. in the footpads of B10.PL
mice. Preliminary studies showed that DC could be detected in the T
cell areas of the popliteal lymph nodes 24 h after adoptive
transfer. The kinetics of DC detection that we observed in the lymph
node was consistent with that previously described by Jenkins and
colleagues (8). As shown in Fig. 2, we also were able to detect the
presence of DiI-labeled (red fluorescence) MBP-TCR tg splenic CD4 T
cells in the lymph nodes 24 h following adoptive transfer by i.v.
injection. The simultaneous adoptive transfer of MBP-TCR CD4 T cells
and Ac1–11-pulsed DC resulted in the detection of clusters
of cells composed of green fluorescing DiO-labeled DC interacting with
and surrounded by red fluorescing DiI-labeled T cells in the lymph node
24 h following transfer (Fig. 2D). A lower power
magnification field is shown in Fig. 2B, with the yellow
fluorescence indicating areas of direct interaction between the
DiI-labeled T cells and the DiO-labeled DC. When DC pulsed with medium
alone were transferred with MBP-TCR CD4 T cells, few cell-cell
interactions and little T cell-DC clustering was observed as shown by
high power (Fig. 2C) and by low power (Fig. 2A)
magnifications. These data show that the interaction between DC
presenting the Ac1–11 peptide and the MBP-TCR CD4 T cells
are specific for self peptide.
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We chose to use CD4 T cells isolated from MBP-TCR tg mice in our
experiment in Fig. 2
because DC have been shown preferentially to
interact with and activate naive T cells. CD4+ T cells
isolated from the spleen of a MBP-TCR tg mouse are 95%
Vß8.2+ (data not shown), the ß-chain expressed by the
MBP TCR. We found that of the Vß8.2+ T cells, >98% were
of the naive phenotype as determined by the expression of low levels of
CD44 and high levels of CD45RB. Thus, only 1.2–1.5% of the
Vß8.2+ T cells expressed the memory phenotype,
CD44high and CD45RBlow (data not shown)
(19). The specificity of the MBP-TCR CD4 T cells is shown
in Fig. 3A by the
dose-dependent proliferation and IL-2 production following stimulation
with Ac1–11. Because we detected a small population of
memory cells that are known to produce abundant quantities of either
Th1 or Th2 cytokines upon stimulation (19), we examined
the MBP-TCR CD4 T cells for the production of IL-4 and IFN- (Fig. 3A). We did not detect either IL-4 or IFN- by ELISA (data
not shown). The production of IL-2 in the absence of IL-4 and IFN-
further suggests a naive phenotype of the tg T cells. To examine
whether we could prime for a Th1 or Th2 response using DC, we isolated
CD4+ splenocytes from mice 30 days following transfer of
Ac1–11-pulsed DC and MBP-TCR CD4 T cells and tested for
proliferation and cytokine production. The successful priming of
MBP-TCR T cells is shown in Fig. 3B by the dose-dependent
proliferation and IFN- production in response to stimulation with
Ac1–11. No IL-4 production was detected (data not shown),
demonstrating priming of a Th1, but not Th2, response.
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Because we were able to demonstrate an Ag-specific interaction
between DC and MBP-TCR CD4 T cells in vivo in lymph nodes leading to a
Th1 response, we asked whether this interaction could lead to the
induction of EAE. As shown in Fig. 4A, symptoms of EAE occurred
as early as day 8 following adoptive transfer of naive MBP-TCR CD4 T
cells and Ac1–11-pulsed DC. As a comparison, animals that
were transferred with MBP-TCR CD4 T cells and medium-pulsed DC
exhibited no signs of EAE (Fig. 4A). All animals that
received peptide-loaded DC exhibited signs of EAE by day 11, and peak
disease was reached on day 20. The animals were sacrificed on day 20 at
the peak of disease for the analysis of CNS cell infiltration (Fig. 5). In an extended experiment in which
the animals continued being examined for EAE after the peak day of
disease, the mice were able to resolve most symptoms of disease and
recover (Fig. 4B). These data show that DC can present the
Ac1–11 self peptide to MBP-TCR T cells resulting in
autoimmunity. The induction of EAE is dependent upon the transfer of
live DC, as i.p. injection of Ac1–11 (100 µg) or
transfer of paraformaldehyde-fixed DC pulsed with Ac1–11
in combination with transferred MBP-TCR T cells did not result in EAE.
In the experiments shown in Fig. 4, the DC were injected 24 h
prior to the adoptive transfer of MBP-TCR T cells. This time delay is
not required, as EAE also occurs when the DC and T cells are
transferred on the same day (data not shown).
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Because EAE is a disease of the CNS associated with the
destruction of surrounding oligodendrocytes of the myelin sheath,
disease symptoms should be accompanied by infiltration of
encephalitogenic T cells into the brain and spinal cord. Using the
animals shown in Fig. 4A, we examined brain and spinal cord
for the presence of MBP-TCR CD4 T cells. Because the MBP-TCR contains a
Vß8.2+ ß-chain, we used a mAb specific for Vß8.1/8.2
to detect the presence of transgenic T cells. We were able to visualize
the presence of both CD4+ (Fig. 5A) and
Vß8.2+ (Fig. 5B) cells in the brains and
spinal cords (data not shown) of mice with active EAE. To confirm that
the T cell infiltration observed contained MBP-TCR CD4 T cells, we
stained the tissue sections with a clonotypic mAb specific for the
MBP-TCR (Fig. 5C). Mice from the same experiment which
received medium-pulsed DC and did not exhibit signs of EAE showed
little to no detectable infiltration of CD4+,
Vß8.2+, or MBP-TCR+ (Fig. 5 D,
E, and F, respectively) cells in the brain.
We have previously shown that in vitro-activated
Ac1–11-specific cloned T cells induced EAE upon
adoptive transfer and were detected in the CNS of sick animals
(12). Our ability to reproduce this result using our DC
induction model is important for future studies on the dynamics of cell
migration into the brain and on demyelination.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we describe a novel method of EAE induction using
BM-derived DC pulsed with the Ac1–11 MBP peptide capable
of interacting with and activating naive Ac1–11-specific T
cells in vivo leading to EAE. We sought to develop a single model of
EAE facilitating the study of the autoimmune process including
initiation, effector, and recovery phases in one disease process.
Building upon our previous work using B10.PL mice (20) and
utilizing our Ac1–11-specific TCR tg mouse
(13), we have successfully used DC as adjuvants to induce
EAE. In our model, BM-derived DC were pulsed with Ac1–11
prior to s.c. injection into irradiated recipient mice, followed by
i.v. injection of splenic naive CD4+ tg T cells. In
concordance with our previous studies using B10.PL mice
(20), we observed an acute monophasic disease course (Fig. 4B) followed by infiltration of MBP-TCR tg T cells into the
brain parenchyma at a level we have previously observed only in passive
EAE induction (Fig. 5) (12).
One of the major drawbacks to the study of EAE using active induction models is the necessity to use CFA as an adjuvant. CFA has been shown to preferentially induce Th1 immune responses over Th2 responses (23). This immune skewing can be beneficial, since a variety of studies have shown that the pathogenic T cell is typically of the Th1 phenotype (10), but has limitations in determining a role for Th2 cells. Th2 cells have been shown to be encephalitogenic under specific conditions (24) and are thought to be important in protection from EAE (10, 25, 26). Thus, the absence of CFA in our EAE model will allow a more precise study of the dynamics of cytokine profiles in EAE that have been suggested to switch from a Th1 pattern during the effector phase to a Th2 pattern during the recovery phase (10). In addition, we have eliminated the need for pertussis toxin, which inhibits G-protein function and has been shown to be immunomodulatory (27, 28).
In the mouse, the induction of EAE following the adoptive transfer of
activated encephalitogenic T cells typically requires sublethal
irradiation of the host animal. The irradiation is believed to
facilitate the migration of T cells into the CNS, perhaps by
up-regulating cytokines and adhesion molecules, leading to disease.
Although irradiated animals were used in the experiments in Fig. 4, we
were able to induce EAE in nonirradiated mice using our DC-based model
of EAE induction (data not shown). Thus, DC alone in the absence of a
stimulus provided by the mycobacteria in CFA, and in the absence of
inflammatory signals provided by irradiation, can present self Ags
resulting in the activation of self-reactive T cells leading to
autoimmunity.
DC pulsed with tumor Ags have been used successfully in the treatment
of a variety of animal tumor models (29). Although these
studies have confirmed the efficient immune stimulatory activity of the
adaptive immune response by DC, there remains much to learn about DC
cell physiology. DC of BM origin are now known to reside in most
tissues of the body. These DC are thought to be immature with the
capacity for efficient and rapid Ag uptake leading to processing and
presentation of Ags. In the case of foreign microbial Ags that activate
DC, the DC are triggered to migrate to the draining lymph nodes via the
afferent lymphatics. DC activation also initiates a rapid maturation
process culminating with a mature DC with limited ability to take up
and process Ag, high cell surface levels of MHC class II and B7-2, and
a striking change in phenotype to a cell with numerous extended
processes. This mature DC, now residing in the T cell zone, has a very
slow turnover of peptide-bound MHC class II molecules on the cell
surface (30). This DC maturation process was dramatically
illustrated in vitro using BM-derived DC by tracking the location of
MHC class II molecules from intracellular vesicles to the cell surface
in combination with changes in morphology (17). Thus, the
use of DC therapeutically requires the DC to be immature upon Ag
pulsing. This is particularly important with protein Ags where peptide
exchange on the cell surface is not a feasible mechanism
(31). Although DC at various stages of maturation can
present peptide Ags to T cell clones (32), only immature
and not fully mature DC were shown to stimulate T cell clones when
whole protein Ag was used (18). In this study, we used
BM-derived DC that were enriched by replating overnight to allow cells
of the monocyte lineage to strongly adhere. Although replating induces
DC maturation, proliferation of the Ac1–11-specific
MBP.TCR 31 clone occurred when the stimulating Ag was whole mMBP
protein (Fig. 1C). This result demonstrates that the DC used
in this study were capable of protein Ag uptake leading to processing
and presentation.
Our use of T cells that are activated in vivo on peptide-loaded DC more
closely mimics the actual series of events that occur between T cell
priming and EAE induction in vivo, as compared with peptide
immunization or the use of in vitro-activated T cell clones. In
previous experiments, we have successfully induced EAE using a T cell
clone expressing the Ac1–11-specific TCR from which the
MBP tg mouse was generated (12). Although these
experiments were successful, the use of T cell clones has limitations.
The most problematic is the loss of encephalitogenic potential with
continuous passage in culture as the result of phenotypic changes.
Although phenotypic variations is not a concern in our model, a
potential drawback to the use of CD4+ T cells from the
MBP-TCR tg mouse is the presence of Ac1–11-specific Th1
memory cells that could rapidly expand and produce Th1 cytokines
leading to EAE. In our model, this seems not to be the case since
95% of the CD4+ T cells bear the Vß8.2 TCR chain with
only 1.2–1.5% of the starting tg T cell population of the memory
phenotype (data not shown). This is consistent with a report by Linton,
et al. (33), showing that CD4+ tg T cells in
the AND TCR tg mouse are of a naive phenotype, even upon aging. In
contrast, the memory cells detected showed little to no expression of
the TCR tg. In addition, we were able to induce severe EAE with hind
limb paralysis using Ac1–11-pulsed DC and transfer of only
4 x 106 Vß8.2+ MBP-TCR tg T cells, of
which only 6 x 104 would be memory cells (data not
shown). Although EAE has been shown to be induced following transfer of
1 x 105 activated encephalitogenic T cells, EAE onset
was delayed until approximately day 28 and required both irradiation
and injection of Bordetella pertussis (34). In
addition, we have been able to induce EAE following the elimination of
memory cells by cell sorting (data not shown).
A further expansion of our DC-based EAE induction model would be to induce EAE with Ac1–11-pulsed DC in the absence of transferred MBP-TCR T cells. To date, we have not been successful in inducing EAE in B10.PL mice without the transfer of MBP-TCR tg T cells (data not shown). We feel that a low precursor frequency of autoreactive T cells in the B10.PL mouse in combination with the reported low affinity of the Ac1–11 peptide for the I-Au MHC class II molecule (35) is not sufficient for the development of an immune response leading to autoimmunity. Another contributing factor is the transient presence of the peptide-pulsed DC in the lymph nodes, which decline in number 24 h following transfer (8). The use of CFA allows the slow constant release of Ag over a time frame of weeks. We feel that the transferred DC, although competent APC able to activate naive T cells, do not remain in the lymph nodes for a sufficient time to induce an autoimmune response. We are currently performing experiments using a modified Ac1–11 peptide with increased affinity for the I-Au MHC class II molecule combined with twice weekly transfers of DC to test our hypothesis.
A common strategy employed by a variety of investigators for preventing or diminishing the clinical symptoms of EAE is tolerance induction. Various mechanisms of tolerance induction have been utilized including the use of peptides (36, 37), thymic dendritic cells (38), and blocking of costimultory molecules (39, 40). A likely common mechanism of these tolerogenic models is the prevention of the primary immune response. However, in multiple sclerosis patients, T cells with specificity for myelin Ags existing in the peripheral blood have an activated phenotype when compared to T cells from normal controls with comparable specificities (41, 42, 43). The long-term persistence of MBP-specific T cell clones in a multiple sclerosis patient suggests in vivo activation of the self reactive T cells (44). Thus, a mechanism of tolerance induction capable of rendering the myelin Ag-specific T cells unresponsive would be beneficial. We are currently using our DC EAE induction model to examine whether DC can deliver a tolerogenic signal to Ac1–11-specific naive T cells present prior to primary immunization and, more importantly, to primed CD4+ T cells after immunization. It is our hope that these and future studies using our DC EAE induction model will lead to therapies allowing the prevention or amelioration of multiple sclerosis.
Thus, our DC induction model of EAE allows the delivery of the encephalitogenic peptide Ac1–11 directly to the lymph node via migration of DC through the afferent lymphatics without the need for conventional adjuvants or in vitro T cell activation. Here, the DC encounter naive recirculating CD4 T cells and arrest their migration, allowing Ag-specific activation of MBP-specific T cells into capable EAE effector cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bonnie N. Dittel, Section of Immunobiology, Yale University School of Medicine, P.O. Box 208011, 310 Cedar Street, FMB 402, New Haven, CT 06520-8011. E-mail address:
3 Abbreviations used in this manuscript: DC, dendritic cells; BM, bone marrow; MBP, myelin basic protein; mMBP, mouse MBP; EAE, experimental autoimmune encephalomyelitis; PLP, proteolipid protein; tg, transgenic; DiI, 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanine perchlorate; DiO, 3,3'-dioctadecyloxacarbocyanine perchlorate.
Received for publication January 11, 1999. Accepted for publication April 22, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4 integrin by CD4 T cells is required for their entry into brain parenchyma. J. Exp. Med. 177:56.
in T helper 1, T helper 2, and T helper 0 clones bearing the same TCR. J. Immunol. 158:4065.[Abstract]
and tumor necrosis factor production. J. Exp. Med. 180:2227.This article has been cited by other articles:
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 R. L. Lakey, T. G. Morgan, A. D. Rowan, J. D. Isaacs, T. E. Cawston, and C. M. U. Hilkens A novel paradigm for dendritic cells as effectors of cartilage destruction Rheumatology, May 1, 2009; 48(5): 502 - 507. [Abstract] [Full Text] [PDF]
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C. Capini, M. Jaturanpinyo, H.-I Chang, S. Mutalik, A. McNally, S. Street, R. Steptoe, B. O'Sullivan, N. Davies, and R. Thomas Antigen-Specific Suppression of Inflammatory Arthritis Using Liposomes J. Immunol., March 15, 2009; 182(6): 3556 - 3565. [Abstract] [Full Text] [PDF]
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A. Liston, R. E. Kohler, S. Townley, S. Haylock-Jacobs, I. Comerford, A. C. Caon, J. Webster, J. M. Harrison, J. Swann, I. Clark-Lewis, et al. Inhibition of CCR6 Function Reduces the Severity of Experimental Autoimmune Encephalomyelitis via Effects on the Priming Phase of the Immune Response J. Immunol., March 1, 2009; 182(5): 3121 - 3130. [Abstract] [Full Text] [PDF]
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 A. L. Zozulya, S. Ortler, J. Lee, C. Weidenfeller, M. Sandor, H. Wiendl, and Z. Fabry Intracerebral Dendritic Cells Critically Modulate Encephalitogenic versus Regulatory Immune Responses in the CNS J. Neurosci., January 7, 2009; 29(1): 140 - 152. [Abstract] [Full Text] [PDF]
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C. Ling, Y. I. Verbny, M. I. Banks, M. Sandor, and Z. Fabry In Situ Activation of Antigen-Specific CD8+ T Cells in the Presence of Antigen in Organotypic Brain Slices J. Immunol., June 15, 2008; 180(12): 8393 - 8399. [Abstract] [Full Text] [PDF]
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J. Tang, W. Zhu, P. B. Silver, S.-B. Su, C.-C. Chan, and R. R. Caspi Autoimmune Uveitis Elicited with Antigen-Pulsed Dendritic Cells Has a Distinct Clinical Signature and Is Driven by Unique Effector Mechanisms: Initial Encounter with Autoantigen Defines Disease Phenotype J. Immunol., May 1, 2007; 178(9): 5578 - 5587. [Abstract] [Full Text] [PDF]
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 X. Liu, C. Ciumas, Y.-M. Huang, K. R Steffensen, H. Lian, H. Link, and B.-G. Xiao Autoantigen-pulsed dendritic cells constitute a beneficial cytokine and growth factor network in ameliorating experimental allergic encephalomyelitis Multiple Sclerosis, August 1, 2005; 11(4): 381 - 389. [Abstract] [PDF]
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 C. Weir, C. C. A. Bernard, and B. T. Backstrom IL-5-deficient mice are susceptible to experimental autoimmune encephalomyelitis Int. Immunol., November 1, 2003; 15(11): 1283 - 1289. [Abstract] [Full Text] [PDF]
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E. Hauben, A. Gothilf, A. Cohen, O. Butovsky, U. Nevo, I. Smirnov, E. Yoles, S. Akselrod, and M. Schwartz Vaccination with Dendritic Cells Pulsed with Peptides of Myelin Basic Protein Promotes Functional Recovery from Spinal Cord Injury J. Neurosci., September 24, 2003; 23(25): 8808 - 8819. [Abstract] [Full Text] [PDF]
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W. Hou, Y. Wu, S. Sun, M. Shi, Y. Sun, C. Yang, G. Pei, Y. Gu, C. Zhong, and B. Sun Pertussis Toxin Enhances Th1 Responses by Stimulation of Dendritic Cells J. Immunol., February 15, 2003; 170(4): 1728 - 1736. [Abstract] [Full Text] [PDF]
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B. P. Leung, M. Conacher, D. Hunter, I. B. McInnes, F. Y. Liew, and J. M. Brewer A Novel Dendritic Cell-Induced Model of Erosive Inflammatory Arthritis: Distinct Roles for Dendritic Cells in T Cell Activation and Induction of Local Inflammation J. Immunol., December 15, 2002; 169(12): 7071 - 7077. [Abstract] [Full Text] [PDF]
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G-X Zhang, M Kishi, H Xu, and A Rostami Mature bone marrow-derived dendritic cells polarize Th2 response and suppress experimental autoimmune encephalomyelitis Multiple Sclerosis, December 1, 2002; 8(6): 463 - 468. [Abstract] [PDF]
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 J. W. Greiner, H. Zeytin, M. R. Anver, and J. Schlom Vaccine-based Therapy Directed against Carcinoembryonic Antigen Demonstrates Antitumor Activity on Spontaneous Intestinal Tumors in the Absence of Autoimmunity Cancer Res., December 1, 2002; 62(23): 6944 - 6951. [Abstract] [Full Text] [PDF]
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H. Holcombe, I. Mellman, C. A. Janeway Jr., K. Bottomly, and B. N. Dittel The Immunosuppressive Agent 15-Deoxyspergualin Functions by Inhibiting Cell Cycle Progression and Cytokine Production Following Naive T Cell Activation J. Immunol., November 1, 2002; 169(9): 4982 - 4989. [Abstract] [Full Text] [PDF]
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 C. Scheinecker, R. McHugh, E. M. Shevach, and R. N. Germain Constitutive Presentation of a Natural Tissue Autoantigen Exclusively by Dendritic Cells in the Draining Lymph Node J. Exp. Med., October 21, 2002; 196(8): 1079 - 1090. [Abstract] [Full Text] [PDF]
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K. L. Legge, R. K. Gregg, R. Maldonado-Lopez, L. Li, J. C. Caprio, M. Moser, and H. Zaghouani On the Role of Dendritic Cells in Peripheral T Cell Tolerance and Modulation of Autoimmunity J. Exp. Med., July 15, 2002; 196(2): 217 - 227. [Abstract] [Full Text] [PDF]
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K. Kawahata, Y. Misaki, M. Yamauchi, S. Tsunekawa, K. Setoguchi, J.-i. Miyazaki, and K. Yamamoto Peripheral Tolerance to a Nuclear Autoantigen: Dendritic Cells Expressing a Nuclear Autoantigen Lead to Persistent Anergic State of CD4+ Autoreactive T Cells After Proliferation J. Immunol., February 1, 2002; 168(3): 1103 - 1112. [Abstract] [Full Text] [PDF]
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M. Kobayashi, E. Azuma, M. Ido, M. Hirayama, Q. Jiang, S. Iwamoto, T. Kumamoto, H. Yamamoto, M. Sakurai, and Y. Komada A Pivotal Role of Rho GTPase in the Regulation of Morphology and Function of Dendritic Cells J. Immunol., October 1, 2001; 167(7): 3585 - 3591. [Abstract] [Full Text] [PDF]
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Y.-M. Huang, M. Kouwenhoven, Y.-P. Jin, R. Press, W.-X. Huang, and H. Link Dendritic cells derived from patients with multiple sclerosis show high CD1a and low CD86 expression Multiple Sclerosis, April 1, 2001; 7(2): 95 - 99. [Abstract] [PDF]
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 M. Pashenkov, Y.-M. Huang, V. Kostulas, M. Haglund, M. Soderstrom, and H. Link Two subsets of dendritic cells are present in human cerebrospinal fluid Brain, March 1, 2001; 124(3): 480 - 492. [Abstract] [Full Text] [PDF]
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 B. Serafini, S. Columba-Cabezas, F. Di Rosa, and F. Aloisi Intracerebral Recruitment and Maturation of Dendritic Cells in the Onset and Progression of Experimental Autoimmune Encephalomyelitis Am. J. Pathol., December 1, 2000; 157(6): 1991 - 2002. [Abstract] [Full Text] [PDF]
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Y. Wan, J. Bramson, A. Pilon, Q. Zhu, and J. Gauldie Genetically Modified Dendritic Cells Prime Autoreactive T Cells through a Pathway Independent of CD40L and Interleukin 12: Implications for Cancer Vaccines Cancer Res., June 1, 2000; 60(12): 3247 - 3253. [Abstract] [Full Text]
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G. P. Linette, S. Shankara, S. Longerich, S. Yang, R. Doll, C. Nicolette, F. I. Preffer, B. L. Roberts, and F. G. Haluska In Vitro Priming with Adenovirus/gp100 Antigen-Transduced Dendritic Cells Reveals the Epitope Specificity of HLA-A*0201-Restricted CD8+ T Cells in Patients with Melanoma J. Immunol., March 15, 2000; 164(6): 3402 - 3412. [Abstract] [Full Text] [PDF]
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 R. M. Steinman and M. C. Nussenzweig Inaugural Article: Avoiding horror autotoxicus: The importance of dendritic cells in peripheral T cell tolerance PNAS, January 8, 2002; 99(1): 351 - 358. [Abstract] [Full Text] [PDF]
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), with Ac1–11
(100 µg/ml) (B, 
), or with mMBP (100 µg/ml)
(C, 
) as U/ml. IFN-
