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*
Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH 44106; and
University Hospital of Ulm, Section of Endocrinology, Ulm, Germany
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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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Most autoreactive T cells are thought to be deleted in the thymus. This is also the case for lymphocytes specific for the neuroantigen myelin basic protein (MBP) and proteolipid protein (PLP) (1, 2, 3). Low affinity cells, however, and T cells specific for determinants not expressed in the thymus escape this negative selection process. These lymphocytes persist as naive precursor cells, apparently ignorant of the endogenous autoantigen (4, 5, 6, 7), but upon priming they can give rise to autoimmune disease. Induction of EAE requires immunization with the neuroantigen itself or a cross-reactive foreign Ag. This supposedly mimics an infection that initiates the spontaneously developing autoimmunity in multiple sclerosis, for which EAE serves as a model. While the initial clonal expansion of the autoreactive precursors and their differentiation into effector cells occur in the dLN, little is known about their subsequent fate. Thus, it is not known whether the neuroantigen-specific T cells head directly for the target organ after becoming primed or whether they first randomly populate the immune periphery (8, 9). It is also not known what percentage of the autoimmune repertoire resides in the target organ as opposed to the immune periphery at various stages of the disease process. This information is of critical importance for various reasons. First, it is important to know whether there is a sizable peripheral reservoir of effector cells from which the autoimmune process in the target organ draws, as this would have to be included in immunotherapeutic considerations: T cells in the periphery should be more accessible to manipulation than those in the CNS. Second, it would be beneficial for immune-monitoring purposes to know how the numbers and functions of the neuroantigen-specific T cells in the periphery reflect those in the relevant target organ.
The population kinetics of these T cells are also unknown. While T cells that enter the CNS parenchyma are known to undergo apoptosis (10, 11), there is also evidence that during the inflammatory process in EAE, lymphoid neo-organogenesis is induced by lymphotoxin in the CNS (12). This may in turn give rise to ongoing stimulation of the neuroantigen-specific T cells and drive their clonal expansion. Moreover, these induced lymphoid tissues within the CNS may represent the site in which the amplification of the autoimmune process via determinant spreading occurs (13, 14, 15, 16). In this study, we address the organ distribution and fate of the first wave of peripherally primed effector cells, and the site of engagement of the second wave effector cells. Furthermore, we were interested in determining whether Th1/Th2 class-switching of the effector cells occurs in the course of EAE (17, 18, 19).
In this study, we set out to use a computer-assisted cytokine enzyme-linked immunospot analysis (ELISPOT) assay (20) to follow the PLP139–151 peptide-specific CD4 effector cells in the CNS and the periphery over the natural course of PLP139–151-induced EAE (21), measuring directly their frequency and cytokine signature at single cell resolution. Naive T cells do not secret cytokine, nor do memory cells in the absence of Ag (22). T cell differentiation into cytokine-producing effector cells depends on cycles of Ag-driven proliferation to open up the chromatin structure (23), requiring at least 3–4 days (20). In contrast, memory/effector T cells that underwent cytokine differentiation start producing the cytokine that they are precommitted to express within 24 h after Ag encounter. Therefore, the testing of freshly isolated T cells in ELISPOT assays of 24- to 48-h duration provides information on the frequency and the cytokine signature of the specific effector T cells in vivo. In previous work, we have shown that when freshly isolated LN or spleen cells were challenged with protein Ags or antigenic peptides, only in vivo primed CD4 memory cells produced cytokine in ELISPOT assays of such short duration (2, 20, 24, 25). Moreover, while we have established that accurate frequencies of the cytokine-producing T cells are measured in lymphoid tissues (20), we had to address whether this also holds true for T cells isolated from the CNS, where the Ag-presenting compartment fundamentally differs. While performing measurements of T cells isolated from the CNS, we had to account for conditions of optimal Ag presentation, including the contribution of the endogenously presented neuroantigen, and to address the concern of contamination with blood-borne T cells. After establishing the assay conditions for single cell CNS measurements, we studied the frequency and absolute numbers of PLP139–151-specific T cells after PLP139–151 immunization in the CNS and in the immune periphery. We provide evidence that the vast majority of effector cells reside in the periphery, not in the CNS, even at the peak of the disease. Furthermore, we show that the first wave of effector cells (the PLP139–151-specific T cells engaged by peripheral immunization) eventually exhausts without undergoing a Th2 switch. Finally, our data suggest that second wave immunity to the endogenous PLP178–191 peptide (15, 26) is engaged in the CNS itself.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SJL mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and kept at the animal facility of Case Western Reserve University under specific pathogen-free conditions. Female mice were used at 6–10 wk of age. PLP peptide 139–151 and PLP peptide 178–191 were purchased from Princeton Biomolecules (Langhorn, PA); OVA was purchased from Sigma (St. Louis, MO); and IFA was purchased from Life Technologies (Grand Island, NY). CFA was made by mixing Mycobacterium tuberculosis H37RA (Difco, Detroit, MI) at 2.5 mg/ml into IFA. A total of 0.5 mM of each peptide or of OVA was injected in CFA, s.c., 100 µl emulsion per mouse. Pertussis toxin (List Biological Laboratories, Campbell, CA) was injected on days 0 and 1, 0.2 ng at each time point. Starting from day 3 after immunization, mice were assessed for the development of paralytic symptoms, and the severity was recorded according to the standard scale: grade 1, floppy tail; grade 2, hind-leg weakness; grade 3, full hind-leg paralysis; grade 4, quadriplegia; grade 5, death. To ensure the nourishment of paralyzed mice, elongated water tubes were used and food was placed in the bedding.
Cell preparation from the various organs tested; flow cytometry
Cells from the CNS were prepared as follows. After sacrificing the animal, the spinal cord was removed from the entire vertebral column, placed into DMEM medium, and disrupted with the back of a syringe. The resulting cell suspension was filtered through a Falcon Cell Strainer 2350 (Becton Dickinson, San Jose, CA). The cells were washed twice with DMEM and subsequently counted. The cells were resuspended in HL-1 medium (BioWhittaker, Walkersville, MD) supplemented with 1% glutamine and plated typically at 5 x 104 to 5 x 105 cells/well, or at serial dilutions into ImmunoSpot M200 plates (Cellular Technology Limited, Cleveland, OH). Cells from the peritoneal cavity (PC) were obtained by lavage after injecting 7 ml of DMEM medium. Mouse blood was obtained by retroorbital bleeding, using heparin as an anticoagulant. The blood was diluted four times with sterile saline, and PBMCs were obtained by density-gradient centrifugation over a Ficoll gradient. Single cell suspensions from dLN and nondraining LN and the spleen were prepared as previously described (1). For flow cytometric studies of the cell populations obtained, we used directly labeled Abs specified, all from PharMingen (San Diego, CA). The stained cells were analyzed with a FACScan and Cell Quest software (Becton Dickinson). The cells isolated from spinal cords of mice with EAE contained 19–43% CD4 cells.
ELISPOT assays and ELISPOT image analysis
ImmunoSpot M200 plates (Cellular Technology Limited) were coated
overnight with the capture Abs in sterile PBS. R46A2 at 4 µg/ml
(purified from hybridoma in our laboratory) was used for capturing
IFN-
; JES6-1A12 at 3 µg/ml (PharMingen) for IL-2; 11B11 at 2
µg/ml (purified from hybridoma) for IL-4; TRFK5 at 5 µg/ml
(purified from hybridoma) was used for the capture of IL-5 and MP6-XT3
at 2.5 µg/ml for capture of TNF-
/
(PharMingen). The plates were
blocked for 1 h with 1% BSA in PBS and washed three times with
PBS. The freshly isolated cells from the various organs were plated in
HL-1/1% glutamine-supplemented medium in the numbers specified, with
or without Ag (7 µM, if not specified otherwise). In selected
experiments, cells and Ags were titrated. The plates were cultured at
37°C, 7% CO2 for 24 h (IFN-, IL-2, and
TNF-/ assays) or for 48 h (IL-4, IL-5 assays). The cells
were then discarded, the plates washed with PBS first, then with PBS
containing 0.025% Tween (PBST), and the detection Abs were added for
overnight incubation. XMG1.2 biotin (purified from hybridoma in our
laboratory) was used for IFN-; rat anti-mouse IL-4 biotin
(BVD6-24G2; PharMingen) was used for IL-4; rat anti-mouse IL-2
biotin (JES6-5H4; PharMingen) was used for IL-2; biotinylated TRFK4
(PharMingen) was used for IL-5; and biotinylated MP6-XT3 at 3 µg/ml
for the detection of TNF-/ (PharMingen). The plates were then
washed three times in PBST. Streptavidin-alkaline phosphatase
(Dako, Carpenteria, CA) was added at a 1/1000 dilution in PBST as a
third reagent for IL-2, IL-4, and IL-5, incubating for 2 h,
followed by three washes in PBS. The plates were developed using
nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate
(Kirkegaard & Perry Laboratories, Gaithersburg, MD) or HRP substrate
AEC (3-amino-9-ethylcarbazole) (Pierce, Rockford, IL) for 30 min. The
resulting spots were counted with an ImmunoSpot Series 1 Analyzer
(Cellular Technology Limited) specifically designed for the ELISPOT
assay. Digitized images were analyzed for the presence of areas in
which color density exceeds background by a factor set on the basis of
the difference between control wells (containing T cells and APC
without Ag) and experimental wells (containing Ag in addition). After
separating spots that touch or partially overlap, additional criteria
of spot size and circularity were applied to gate out speckles and
noise caused by spontaneous substrate precipitation, nonspecific Ab
binding, and ELISA effects. Objects that did not meet these criteria
were ignored, and areas that met them were recognized as spots,
counted, and highlighted.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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First we established the specificity of cytokine ELISPOT formation
for the Ags used in this study: PLP139–151,
PLP178–191, and OVA. SJL mice were injected with
these Ags in CFA, and the dLN were tested 9 days later (Table I
). In the presence of the immunizing Ag,
IFN-- and IL-2-producing cells were detected in the frequency range
20/106 to 150/106; IL-4 and
IL-5 were not detectable (<3/106). Notably, the
sensitivities of the IL-4 and IL-5 assays are equal to those of IFN-
and IL-2 (20, 24). Vigorous peptide-induced TNF-/
production (the Ab pair available does not distinguish between TNF-
and TNF-) was also seen (data not shown), further substantiating the
type 1 cytokine signature. The production of these cytokines was not
cross-reactively triggered by any of the three test Ags, and was also
not induced in naive mice (data not shown), suggesting specific
detection of memory cells. These immunizations with CFA therefore
induced highly polarized type 1 responses. Similar cytokine profiles
were seen in the spleen and PC, with the exception of an additional
IL-4 production in the spleen. This IL-4 was not produced by T cells,
however, but by mast cells. IL-3 secreted by the Ag-activated T cells
induced this bystander reaction (A. Y. K., manuscript in
preparation).
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and IL-2 spots detected (Fig. 1A). Similar results were
obtained when cells from spleens and the PC of
PLP139–151-primed mice were serially diluted
(data not shown). Confirming published work (25), we found
that the PLP139–151-reactive cells resided in
the CD4 cell fraction (data not shown). When freshly isolated bulk cell
populations from primed mice were studied at high cell numbers
(2.5 x
105/well-107/well),addition
of APC did not affect the spot count (data not shown).Overall, the
data suggest that Ag presentation in these compartments is
saturating, and that accurate frequency measurements of
PLP139–151-reactive type 1 CD4 memory cells are
made at single cell resolution in the absence of bystander cytokine
production.
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and of IL-2 was induced by
PLP139–151 peptide (Fig. 1
B);
PLP178–191 did not induce cytokine over
background at this early time point (see below). The signal-to-noise
performance of the CNS assays approximated those of the LN (Fig. 1)
with the exception of the first 2–3 days after the onset of clinical
EAE, when the medium background in CNS isolates tended to be somewhat
elevated compared with dLN. Image analysis showed that the size
distribution of the IFN- and IL-2 spots generated by CNS isolates
and dLN cells was comparable (data not shown). Also consistent with
single cell resolution of the CNS measurements, serial dilutions of CNS
cell isolates on a constant number of APC showed a linear relation for
IFN- and IL-2 spot formation (Fig. 1B). When the highest
numbers of CNS-derived cells (5 x 105 to
107 per well) were tested with or without added
APC, identical numbers of spots were seen (data not shown). These
data suggest that the APC compartment in the inflamed CNS is fully
competent in providing maximal stimulation/costimulation, revealing the
accurate frequencies of PLP139–151-specific
IFN-- and IL-2-producing memory cells in the isolate in the absence
of bystander reactions. The peptide-induced production of IFN- and
of IL-2 was invariably accompanied by vigorous TNF-/ spot
formation in the CNS isolates, but this occurred over a high medium
background, preventing accurate counting by image analysis. This
finding is consistent with reports that activated microglial cells and
infiltrating macrophages (27), rather than T cells, are
the primary source of TNF in the CNS of mice with EAE
(28). Also in accordance with this fact, and corroborating
another report (29), we did not detect significant
Ag-induced IL-4 or IL-5 production in such CNS recall assays at any
stage of the disease. Therefore, cognate production of these type 2
cytokines by T cells did not seem to contribute to the disease.
We were concerned that the detected responses might derive from
contamination by blood-borne CD4 cells as opposed to tissue-resident,
CNS-infiltrating cells. Arguing against this possibility is the fact
that PLP139–151 peptide reactivity was not
detected in CNS isolates before onset of EAE, although the chance for
blood cell contamination at these time points should be comparable
(Fig. 2, A vs B).
Furthermore, the frequency of
PLP139–151-specific cells in the blood was
20–60 per million for IFN-- or IL-2-producing cells (H.H.H.,
manuscript in preparation), >10-fold lower than that seen in the CNS
(Fig. 2B). If blood contamination contributed to the
results, the frequency of responses detected in the CNS isolates would
be expected to be lower than that in the blood. Finally, in the case of
blood contamination, the erythrocyte to CD4 cell ratio in the CNS
isolate should be similar to that in the blood. We found that the ratio
of CD4 cells to erythrocytes was 
1:2 in the CNS material, while it
was 1:3900 in the blood; hence, only 1 of every 2000 CD4 cells in
the CNS isolate could be attributed to blood contamination.
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We studied a total of 285 SJL mice with clinical disease at
various time points after immunization with
PLP139–151 peptide. The basic findings reported
in this work for this model were also reproduced in
MBP87–99-induced EAE of SJL mice and in myelin
oligodendrocyte glycoprotein:35–55-induced EAE of C57BL/6 mice. First,
we addressed whether, after priming, the neuroantigen-specific
precursors directly migrate to the CNS, or whether they disseminate
first in the immune periphery. Therefore, in addition to the dLN, we
also tested the spleen, as well as the PC, representing a nonlymphoid
compartment. The data for day 9 after immunization are shown in Fig. 2A. High frequency IFN--, IL-2 (and
TNF-/)-producing cells were present in the dLN, the spleen, and
the PC. At this time point, no cytokine-producing cells were detected
in the CNS. The PLP139–151-specific cells in the
PC became detectable on day 3 after immunization, and their numbers
peaked on day 9–12. From this point, their numbers gradually declined
(Figs. 2, B–D, and 3).
Therefore, in addition to the lymphoid tissues, a considerable
reservoir of effector cells resided in extralymphoid tissues, as
suggested by their high frequency in the PC; a considerable effector
cell mass builds up in the periphery before the neuroantigen-specific T
cells start to accumulate in detectable numbers in the CNS.
During acute EAE, neuroantigen-specific T cells are present in high frequencies in the CNS; the majority of the PLP139–151-specific cells, however, remains in the periphery
Clinical EAE developed at various time points after immunization,
typically between days 11 and 20. With the onset of paralysis,
PLP139–151-specific T cells also became
detectable in the CNS at high frequencies (Fig. 2B). When
the actual numbers of the neuropeptide-specific cells were calculated,
however, it was found that the vast majority continued to be present in
the periphery (Fig. 3). Their presence in the CNS vs the periphery did
not seem to reflect compartmentalization of subpopulations according to
pathogenicity (type A vs type B determinant specificity) (30, 31), because, reproducing data of others (7), we
also found that the cells from the periphery could readily mediate EAE
in adoptive transfers (data not shown). When the functional avidity of
the PLP139–151-specific cells was tested in the
CNS vs lymphoid tissues by measuring the peptide concentration that
induces 50% of the maximal spot numbers, no significant differences
were seen at any stage of EAE (A. Y. K., manuscript in
preparation). Therefore, neuroantigen-specific cells with higher
avidity are not selectively retained in the CNS, while those with lower
avidity continue to recirculate in the immune periphery. Moreover,
because the absolute number of
PLP139–151-reactive T cells in the periphery
continued to outnumber by far those in the CNS throughout the course of
EAE (Fig. 3), it appears that the disease-mediating effector cell pool
in the CNS draws from an extensive peripheral reservoir of effector
cells.
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The frequencies of PLP139–151-specific
cells in the periphery were highest before the onset of EAE and
gradually declined thereafter (Fig. 2). This decline was also reflected
in the absolute numbers of PLP139–151-reactive
cells in the periphery (Fig. 3). In the CNS, the frequencies and
absolute numbers of PLP139–151-specific cells
were highest at the onset of the first paralysis and declined with the
duration of the disease (Figs. 2 and 3). Invariably, for all mice
tested individually later than 70 days postimmunization
(n = 27), the reactivity to
PLP139–151 completely disappeared from their
CNS, while low numbers of peptide-reactive cells continued to be
present in the spleen (Figs. 2D and 3). This overall decline
in the numbers of the first wave effector cells, and their ultimate
disappearance from the CNS was a function of time: it occurred
irrespective of the number of relapses in this time frame, and was also
seen in the two other EAE models studied (data not shown). As stated
above, the cytokine signature of the
PLP139–151-specific T cells maintained its type
1 characteristic throughout the observation period, suggesting that the
eventual decline of type 1 activity is not a consequence of type 2
switching, but rather reflects the drop in absolute numbers of the
first wave T cells.
Priming of second wave T cells can be first detected in the CNS
The above data suggest that the
PLP139–151-specific effector cells become
exhausted in the course of EAE induced by immunization with this
peptide, yet the mice continued to exhibit chronic paralysis. Thus, we
were interested in testing whether determinant spreading had occurred,
which could explain why the disease progressed. We tested mice during
the first paralysis, and during the second episode of EAE for
reactivity to PLP178–191 (spreading from
PLP139–151 to 178–191 has been shown to be
essential for establishment of chronic EAE (15, 26)).
Reactivity to PLP178–191 was systematically
tested in the CNS, the spleen, the dLN (LN that drain the site of
immunization), and the superficial cervical LN (that might be dLN for
the brain). In four independent experiments,
PLP178–191-reactive cells were detected in the
CNS during the secondary paralysis, while the peripheral lymphoid
tissues did not show such responses (Table II). The data argue for determinant
spreading to occur in the inflamed target organ itself. Confirming
previous studies (15, 16), at later time points the
response to PLP178–191 emerged in the spleen
(day 72, data not shown). While the natural history of the second wave
response is beyond the scope of this study, these data support the
notion that determinant spreading provides a mechanism for the
persistence of the chronic disease after the first wave of effector
cells is exhausted.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There is renewed interest as to what extent neuroantigens expressed in
the thymus and the immune periphery contribute to the shaping of the
preimmune T cell repertoire (1, 2, 32, 33). In particular,
the PLP isoform that is expressed in the thymus of SJL mice does not
include the region that encodes the 139–151 peptide, and subsequently
precursor T cells specific for this peptide do not undergo negative
selection, but occur in relatively high frequencies in the immune
periphery (32, 33). Moreover, it was shown that in
unimmunized mice, these T cells are driven by the endogenous Ag into a
preactivated/memory state (33). The cytokine signature of
these precursor cells remained unresolved, however. The activation of
cytokine genes as required for T cell differentiation into an effector
cell requires extensive proliferation to open the chromatin structure
(23). We did not detect
PLP139–151-induced cytokine (IL-2, IL-4, IL-5,
or IFN-)-producing cells in unimmunized SJL mice. Therefore, the
PLP139–151-specific precursor cells do not seem
to have undergone cytokine differentiation in unimmunized hosts, and
hence do not qualify as effector cells.
The extent of engagement of cytokine-producing specific effector cells
after immunization was striking. By day 9, the frequency of
PLP139–151-specific IFN--producing CD4 cells
in dLN and spleen alone increased from <1/106
(Table I) to 200/106 in the dLN, and
600/106 in the spleen (Fig. 2A).
When corrected for absolute numbers, this translates into an increase
from <100 memory cells from the preimmune state to a total of
75,000 cells in dLN and the spleen. These numbers are still likely
to underrepresent the magnitude of clonal expansion, because the memory
cells are thought to disseminate early into extralymphoid tissues such
as the lung, the liver, the gut, the skin, and the bone marrow, these
being preferential sites for memory cell homing (8).
Indeed, studying the PC, we found high frequencies of peptide-specific
cells on day 9 after the immunization (Fig. 2A). Provided
the PC is representative of other extralymphoid sites from which
isolation of lymphocytes still awaits new techniques to be developed,
this finding suggests that a considerable effector cell mass resides in
extralymphoid tissues. While this clonal expansion of CD4 cells engaged
by immunization is impressive, it does not reach the dimensions of CD8
cell responses in viral infections, in which as many as 10–20% of all
T cells can be stained with the specific tetramer
(34, 35, 36, 37).
While the total number of PLP139–151-specific T
cells peaked in lymphoid tissues before the onset of clinical EAE (Fig. 3), and while high frequencies of
PLP139–151-specific cells were present in the
extralymphoid PC at these early time points, we could not detect these
cells in the CNS itself before the actual onset of the disease (Figs. 2A and 3). After becoming primed in the dLN, the
neuroantigen-specific T cells seem to disseminate
throughoutthe entire organism, as well as seeding into the PC. Being
T cell blasts, these lymphocytes should also be able to cross the
blood-brain barrier and to randomly seed in the CNS. Most of these
PLP139–151-specific T cell blasts end up in the
CNS parenchyma, where they, like T cell blasts of all specificities
that enter there, are prone to undergo apoptosis (11).
While the PLP139–151-reactive T cells,
therefore, are initially likely to randomly migrate to the brain and to
other sites of the body, apoptosis of these cells in the
immune-privileged CNS may explain why, at early time points, functional
T cells can be detected only in the immune periphery, and not the
CNS.
During EAE, T cells accumulate in the perivascular space, which is of
mesenchymal origin. As opposed to the parenchyma, this tissue does not
predispose T cells to apoptotic cell death (11). The
neuroantigen-specific T cells that end up in this compartment will be
selectively retained there as the consequence of autoantigen
recognition (38). The production of cytokines and
chemokines by these (initially few) T cells will lead to the initiation
of a local inflammatory reaction, promoting the further recruitment of
blood-borne lymphocytes and macrophages. Thus, it has been shown that
the development of the perivascular infiltrate in EAE depends on the
activation of microglia by IFN--producing T cells, resulting in
TNF- and chemokine production by the microglia (28, 39), and the induction of adhesion molecules (VCAM and ICAM)
that facilitate traffic of mononuclear cells across the endothelium.
The time required for the active development of this inflammatory
infiltrate in the CNS may explain why these neuroantigen-specific T
cells accumulate with a considerable delay in the target organ itself
relative to their presence in extralymphoid sites.
It has been a matter of controversy how many of the CNS-infiltrating T
cells are neuroantigen-specific vs bystander cells with different
specificity. For early parenchymal lesions, up to 50–80% specific
cells have been reported (40, 41). Other studies found the
specific cells to be a minor fraction of the CNS infiltrate
(42, 43, 44). These differences might be related to the stage
of EAE studied. As opposed to the requirement for T cells to be in the
blast stage for them to enter the noninflamed CNS, in the chronically
inflamed CNS, under the local influence of lymphotoxin, lymphoid
structures develop that facilitate the general recruitment of T cells
(12). Our data suggest that at any stage of active EAE,
functional neuroantigen-specific T cells are a minority (<1 in 1000
cells, Fig. 2B) within the infiltrate. Functional
measurements such as the one we performed, even if made at single cell
resolution, will miss cells that are anergized or undergo apoptosis.
There might be a higher number of
PLP139–151-specific cells present in the early
infiltrate, but in this case, these cells do not seem to be functional
effector cells.
Calculating the total numbers of
PLP139–151-specific T cells in the CNS vs those
in the peripheral lymphoid tissues, we found that, throughout EAE, less
than 20% of these cells resided in the CNS (Fig. 3). We propose that
the presence of PLP139–151-specific cells in
these two compartments reflects general migrational properties of
memory T cells rather than the selective compartmentalization of
subpopulations. We base this presumption on three observations. First,
the cells isolated from the immune periphery of mice with EAE can
adoptively transfer the disease. This provides evidence that they have
the migrational properties to enter the CNS, they have the specificity
to recognize the endogenous (type A) conformation of the peptide
(30, 31), and they secrete the pathogenic set of cytokines
(which could extend beyond those that we measured). Indeed (secondly),
when the cytokine signatures of the
PLP139–151-specific cells were directly
compared, they were identical for the CNS, the dLN, the peritoneal
lavage cells, and the spleen (the apparently peptide-specific IL-4
production in the spleen was found to be a cytokine-driven bystander
reaction). Third, the peptide dose that induced 50% maximal spot
numbers in the CNS isolates vs splenocytes was identical, suggesting
comparable functional avidity spectra for
PLP139–151-specific cells in both compartments.
This strongly argues against the retention of high affinity clones in
the CNS or their preferential expansion/exhaustion by the autoantigen
in the CNS in the course of the disease. The vast majority of effector
cells, therefore, seems to constitute a reservoir in the periphery,
from which the autoimmune process in the target organ draws. This
reservoir is largest before the onset of the disease, waning over 2–3
mo (Fig. 3). One explanation for this observation is that the effector
cells enter the CNS at the inflamed perivascular cuffs, from where they
migrate into the parenchyma. There they recognize Ag, possibly become
anergic, and undergo apoptosis (45, 46). This continuous
depletion of the effector cells in the CNS results in the drainage of
the peripheral effector cell pool until it eventually exhausts (Fig. 3). Because the PLP139–151-specific T cells
maintained the IFN-+,
IL-2+, TNF-/+
cytokine signature over the entire observation period without
converting to IL-4 and IL-5 production, we conclude that the loss in
numbers of cells producing the pathogenic type 1 cytokines does not
reflect type 2 switching.
The prevalence of type 1 cytokine activity in the CNS during the entire
course of EAE has also been observed by others (28, 29).
The data are consistent with studies on IL-4 knockout mice, suggesting
no critical role for this cytokine in the course of EAE (47, 48). Elevated IL-4 levels were measured in the CNS by PCR-based
methods, however (17, 49). PCR-based methods do not
provide information on frequencies of cytokine-producing cells, and do
not excel in providing quantitative results. Measuring actual
frequencies, we found that the cells that produce IFN-, IL-2, and
TNF-/ outnumbered at least 200-fold those producing IL-4 or IL-5.
Type 2 T cell activity by the first wave of effector cells, therefore,
does not prevail at any stage of EAE.
While our monitoring of the first wave effector cells showed that they
are exhausted within 2–3 mo, the mice continued to exhibit clinical
signs of EAE. It seemed conceivable, therefore, that a secondary wave
of effector T cells, engaged by determinant spreading, mediated the
later stages of the disease, as proposed by the "dynamic autoimmune
repertoire hypothesis" (14, 16). In the model we
studied, spreading to PLP178–191 has been shown
to occur, and is in fact required for disease progression (15, 26). Confirming these findings, we also observed spreading to
the PLP178–191 peptide in
PLP139–151-induced EAE. It has been a matter for
debate, however, where determinant spreading occurs. Our data seem to
suggest that second wave autoimmunity becomes engaged in the inflamed
target organ itself. We detected reactivity to
PLP178–191 in the CNS before this response
became detectable in the periphery (Table II). The development of the
aforementioned lymphoid structures in the inflamed perivascular
compartment of the CNS might be required for the spreading reaction to
occur and might also explain the delay with which it occurs. Cervical
LN are also candidates for the site of second wave priming. We tested
superficial cervical LN and we did not detect responses there. We did
not succeed in isolating deep cervical LN. From these (Fig. 2) and our
previous studies (20, 25) of the kinetics and topography
of the T cells primed in LN, we learned that responses in the spleen
follow those in the dLN with only 1–2 days of delay. Moreover, when
priming occurred in the periphery, the responses became detectable in
the periphery (including the spleen) before the CNS (Fig. 2).
Therefore, the fact that the second wave response to
PLP178–191 was detected in the CNS before it was
seen in the periphery (Table II) does not prove, but argues for
engagement of determinant spreading in the target organ itself. This
notion may explain why, frequently, cryptic determinants are targeted
(13). Involving different types of APC that are, in
addition, activated by the chronic local T cell-mediated inflammation,
the determinant hierarchy in the inflamed CNS is likely to
fundamentally differ from that displayed following Ag presentation by
the resting APC lineages present in lymphoid tissues or the blood
(14).
In summary, while semiquantitative T cell proliferation assays (which assess primarily IL-2-driven bystander cell proliferation (50)) suggested early on that autoimmune responses might be dynamic, involving exhaustion and spreading (13, 14, 16, 51), in this work we report the first direct monitoring of the autoreactive T cell pool at single cell resolution. These results provide direct evidence for a dynamic autoimmune response. Monitoring the frequency and cytokine signature of the first wave of effector cells in the periphery and the target organ itself during the course of EAE, we provide evidence for a large peripheral reservoir of memory/effector cells that outnumber by far those in the target organ. This reservoir seems to fuel the inflammatory process in the CNS, leading eventually to the exhaustion of the primary effector cell pool while engaging second wave autoimmunity in the CNS itself.
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Acknowledgments |
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Footnotes |
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2 O.S.T. and J.B. contributed equally.
3 Address correspondence and reprint requests to Dr. Paul V. Lehmann, Department of Pathology BRB 929, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106-4943.
4 Abbreviations used in this paper: EAE, experimental allergic encephalomyelitis; dLN, draining LN; ELISPOT, enzyme-linked immunospot analysis; LN, lymph node; MBP, myelin basic protein; PBST, PBS-Tween; PC, peritoneal cavity; PLP, proteolipid protein.
Received for publication August 23, 2000. Accepted for publication January 19, 2001.
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) and from the spleen plus the dLN (
). Legend to Fig. 2