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Departments of
* Microbiology-Immunology and
Pathology, and the Interdepartmental Immunobiology Center, Northwestern University Medical School, Chicago, IL 60611;
Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599; and
Department of Microbiology and Immunology, Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, TN 37232
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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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Activation of CD4+ T cells requires that their
TCR must recognize cognate Ags presented by class II MHC. The critical
importance of class II MHC Ag presentation in EAE initiation is
illustrated by the fact that treatment with anti-class II Abs
inhibits or ameliorates the disease (reviewed in Ref. 7).
A variety of proteins have important roles in the generation of the
functional peptide/class II MHC protein complex. Invariant chain (Ii)
and H-2M (DM) are critical components of the class II Ag processing
pathway. Newly synthesized class II 
- and 
-chains associate with
Ii in the endoplasmic reticulum (ER), forming a heterotrimeric complex,
which then forms a homotrimer, or trimer of trimers (8).
The Ii performs at least three critical functions in class II Ag
processing and presentation: it aids in the assembly of the class II
complex; it provides a signal sequence, targeting the class II/Ii
complex through the Golgi apparatus to the endocytic pathway; and a
portion of Ii, the class II-associated invariant chain peptide (CLIP)
occupies the class II peptide-binding groove, preventing the binding of
ER-resident peptides. Within endocytic and/or lysosomal compartments,
the Ii is proteolytically degraded in a stepwise fashion by cathepsins
(9), until only CLIP remains associated in the
peptide-binding groove of the class II heterodimer
(10). DM colocalizes with the class II/CLIP complexes in
the endocytic compartment, where it catalyzes the removal of the CLIP
peptide from the class II MHC peptide-binding groove and enables the
binding of antigenic peptides (11, 12, 13). DM also functions
as a peptide editor, removing peptides with high off-rates and
preferentially allowing the binding of high affinity peptides to class
II complexes (14). Upon binding antigenic peptides, the
class II/peptide complex traffics to the APC surface where
CD4+ T cells can recognize the complex and be
activated. Loss of expression of Ii or DM results in profound defects
in Ag processing and presentation, class II expression, and
CD4+ T cell development (12, 15, 16, 17). The expression of all of these proteins; class II, Ii,
and DM is regulated by the class II transcriptional activator (CIITA)
(18, 19, 20, 21). The CIITA is responsible for the constitutive
expression of class II Ag processing proteins in "professional"
APCs as well as the IFN-
-inducible expression of class II, Ii, and
DM in "nonprofessional" APCs. Mice deficient in CIITA have profound
defects in class II, Ii, and DM expression (20, 22, 23)
(although the effect on Ii expression is less severe) and substantial
decreases in CD4+ T cells due to the absence of
class II expression and defective thymic selection.
It is not clear which of a variety of candidate professional (e.g., macrophages, dendritic cells, and B cells) and nonprofessional (e.g., endothelial cells, microglia, and astrocytes) APCs present in the CNS during T cell-mediated demyelination are involved in presentation of myelin Ags to autoreactive CD4+ T cells active in disease initiation or relapses. In vivo studies using radiation bone marrow chimeras have demonstrated potential roles for both infiltrating macrophages, resident microglia, and CNS parenchymal cells in processing and presenting encephalitogenic Ags to CD4+ T cells (24, 25, 26, 27). Each of these cell populations express different levels of CIITA, class II, Ii, and DM, and the expression of these proteins can change with the activation of the different cell populations (28, 29). Thus, there is significant interest in the expression requirements of the class II Ag processing and presentation proteins and their impact on activation of encephalitogenic CD4+ T cell populations. Thus, we investigated the role of these proteins in Ag presentation and myelin-specific CD4+ T cell activation in the context of CNS disease in vivo, using mice deficient in the expression of Ii, DM, and, via the CIITA knockout (KO) mouse, deficient in Ii, DM, and class II. Mice deficient in CIITA, Ii, or DM are resistant to initiation of EAE by both active priming and adoptive transfer of wild-type (wt) myelin oligodendrocyte protein, MOG35–55-specific encephalitogenic T cells. Although both Ii- and DM-deficient APCs can present MOG peptide to CD4+ T cells, neither is capable of processing and presenting the encephalitogenic peptide of intact MOG protein. Remarkably, DM-deficient mice can prime a potent, peripheral Th1 response to MOG35–55, comparable to the response seen in wt mice, yet maintain resistance to EAE initiation and can adoptively transfer EAE to wt, but not DM-deficient, mice. Together, these data demonstrate that the inability to process antigenic peptide from intact myelin protein results in resistance to EAE and de novo processing and presentation of myelin Ags in the CNS is absolutely required for the initiation of autoimmune demyelinating disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 female mice, 5–6 wk old, were purchased from The Jackson Laboratory (Bar Harbor, ME). CIITA-, DM-, and Ii-deficient mice are described elsewhere (12, 16, 20). CIITA-deficient mice have been backcrossed onto the C57BL/6 background six generations. DM- and Ii-deficient mice have been backcrossed onto the C57BL/6 background 10 and 13 generations, respectively. Mutant mice were bred by homozygous brother-sister matings and all mice were housed in barrier conditions with the Center for Comparative Medicine at Northwestern University (Chicago, IL). Mice were maintained on standard laboratory food and water ad libitum. Paralyzed animals were afforded easier access to food and water.
Abs, peptides, and protein
Hybridomas producing the anti-class I and anti-class II Abs (M1/42 and M5/114, respectively) were purchased from American Type Culture Collection (Manassas, VA) and cultured in DMEM 10% FCS. Supernatants were tested for Ab, filtered, and sterilized before storage. MOG35–55 (MEVGWYRSPFSRVVHLYRNGK) was purchased from Genemed Synthesis (San Francisco, CA). PLP178–191 (NTWTTCQSIAFPSK) was purchased from Peptides International (Cleveland, OH). Amino acid composition was verified by mass spectrometry and purity (>98%) was assessed by HPLC. rMOG, consisting of the extracellular portion of MOG (aa 1–125) expressed in, and purified from Escherichia coli, was the generous gift of Dr. M. Gardinier (University of Iowa College of Medicine, Iowa City, IA).
Induction and clinical evaluation of peptide-induced EAE
For MOG35–55-induced EAE, 6- to 7-wk-old female mice were immunized s.c. with 200 µl of an emulsion containing 800 µg of Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI) and 200 µg MOG35–55 distributed over three spots on the flank. Each mouse additionally received 200 ng pertussis toxin (PTx) (List Biological Laboratories, Campbell, CA) in 200 µl PBS i.p. on days 0 and 2 postimmunization. Animals that were immunized twice received the same immunization 7 days after the first immunization, without PTx. For PLP178–191-induced EAE, 6- to 7-wk-old female mice were immunized s.c. with 200 µl of an emulsion containing 800 µg of M. tuberculosis H37Ra and 50 µg PLP178–191 distributed over three spots on the flank. Each mouse additionally received 200 ng PTx in 200 µl PBS i.p. on days 0 and 2 postimmunization. Individual animals were observed daily and clinical scores assessed in a blinded fashion on a 0–5 scale as follows: 0 = no abnormality, 1 = limp tail, 2 = limp tail and hind limb weakness (legs slip through cage top), 3 = hind limb paralysis, 4 = hind limb paralysis and forelimb weakness, and 5 = moribund. The data are reported as the mean daily clinical score ± SEM for all animals in a particular group and/or as the mean peak clinical score ± SEM, i.e. the mean clinical score for all animals at the peak of disease. Unless otherwise mentioned, all mice were age and sex-matched for all experiments.
Initiation of EAE by adoptive transfer
Female donor mice (6- to 10-wk-old) were immunized s.c. with 200 µl of an emulsion containing 800 µg of M. tuberculosis H37Ra and 200 µg MOG35–55 distributed over three spots on the flank. Draining lymph nodes (LN) were harvested from donor mice after 7–11 days for in vitro stimulation. LN cells were cultured (10 x 106 cells/ml) in DMEM containing 10% FBS, 1 mM glutamine, 1% penicillin-streptavidin, 1 mM nonessential amino acids and 5 x 10-5 M 2-ME (D-10; all products from Sigma-Aldrich, St. Louis, MO) with MOG35–55 peptide (30 µg/ml) and human rIL-12 (20 ng/ml; R&D Systems, Minneapolis, MN). After 72 h incubation, cells were counted, washed, and resuspended (2.5 x 107 T cell blasts/ml; 10–15 x 107 LN cells/ml) in buffered salt solution. T cell blasts were differentiated from other LN cells by size under microscopic observation. On day 0, 6- to 7-wk-old female B6 or KO mice were injected i.p. with 5 x 106 T cell blasts/mouse (in 200 µl). Recipient mice received 200 ng PTx in 200 µl PBS i.p. on days 0 and 2. Individual animals were observed daily and clinical scores were assessed as described above. For adoptive transfer of T cells from DM KO mice, donors and LN cells were prepared as described above, except that donors were primed with MOG35–55 on both days 0 and 7 and donors were sacrificed at day 14 postinitial priming.
Elicitation of delayed-type hypersensitivity (DTH) responses
DTH responses were measured using a 24-h ear-swelling assay. Prechallenge ear thickness was determined using a Mitutoyo model 7326 engineer’s micrometer (Schlesinger’s Tool, Brooklyn, NY). Immediately thereafter, mice were ear-challenged by injecting 10 µg of peptide (in 10 µl of saline) into the dorsal surface of the ear using a 100 µl syringe fitted with a 30-gauge needle. The increase in ear thickness was determined 24 h after ear challenge. Results are expressed in units of 10-4 inches ± SEM. Significance of ear swelling in experimental over naive mice was assessed by the Student’s t test.
Ag presentation assay
Spleens were collected from naive, C57BL/6 wt, or KO mice as indicated. RBCs were removed by hypotonic lysis and the remaining cells were used as APCs. The APCs were irradiated (3500 rad), washed, and cultured in 96-well microtiter plates at a density of 5 x 105 cells/well. Varying concentrations of MOG35–55 peptide or rMOG were added to the different APCs. A MOG35–55-specific T cell line (105 cells/well) was cocultured with the APCs and Ags in a total volume of 200 µl D-10. Cocultures were incubated for 96 h, being pulsed with 1 µCi/well [3H]TdR for the final 24 h of the 96-h incubation period. [3H]TdR uptake was detected using a Topcount microplate scintillation counter (Packard Instrument, Meriden, CT) and results are expressed as the mean of triplicate cultures ± SEM. The long-term, MOG35–55-specific T cell line was derived from a C57BL/6 mouse primed with MOG35–55/CFA as previously described (28). In brief, draining LN cells were isolated and restimulated in D-10 for 4 days with peptide, and rested in D10 plus human rIL-2 (2 U/ml; Roche, Indianapolis, IN) for a minimum of 2 wk. Peptide-specific restimulation and rest were repeated every 14–35 days.
In vitro proliferation assays
Draining LN were harvested from primed mice, counted, and cultured in 96-well microtiter plates at a density of 5 x 105 cells/well in a total volume of 200 µl HL-1 medium (BioWhittaker, Walkersville, MD; 1% penicillin/streptavidin, 1% glutamine). Cells were cultured with medium alone or different concentrations of peptide Ag for 72 h. Culture wells were pulsed with 1 µCi/well [3H]TdR for the final 20 h of the 72-h incubation period. [3H]TdR uptake was detected using a Topcount microplate scintillation counter and results are expressed as the mean of triplicate cultures ± SEM.
ELISPOT assays
Nitrocellulose-coated, 96-well flat-bottom microculture plates
(Whatman, Clifton NJ) were precoated overnight at 4°C with 100 µl
of anti-IFN-, anti-IL-4, or anti-IL-2 (R46A2 and 11B11
at 4 µg/well and JES6–1A12 at 2 µg/well, respectively) purchased
from BD PharMingen (San Diego, CA). Plates were washed four times with
sterile PBS and wells were blocked with 200 µl sterile DMEM 1% BSA
for 1 h at room temperature. LN cells (5 x
105) or 106 spleen cells
were cocultured with Ag at varying concentrations in HL-1 medium (1%
penicillin/streptavidin, 1% glutamine). Cultures were incubated at
37°C for 36 h. In mAb coculture experiments, Ab supernatants
were diluted 1/5 for a final volume of 200 µl/well. Plates
were subsequently washed three times with PBS and three times with
PBS/0.05% Tween20 (PBS/Tween). Biotinylated anti-IFN-,
anti-IL-2, or anti-IL-2 (XMG1.2, BVD6-24G2, and JES6-5H4,
respectively) at 2 µg/well diluted in PBS/Tween/1% BSA, were added
at 100 µl/well and incubated overnight at 4°C in a humidified
chamber. Plates were washed four times with PBS/Tween and incubated for
2 h at room temp with 100 µl/well anti-biotin alkaline
phosphatase (Vector Laboratories, Burlingame, CA) diluted 1/1000 in
PBS/Tween/1% BSA. Finally, plates were washed with PBS and developed
in nitroblue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate substrate
solution (Pierce, Rockford, IL). The developing reaction was quenched
after 30–45 min using distilled water. ELISPOTs were counted using the
ImmunoSpot series 1.0 analyzer (Resolution Technology, Cleveland, OH).
Samples were set up in triplicate and anti-CD3 mAb were included as
a positive control for each group.
Immunohistochemistry
Mice were anesthetized and perfused with 1x PBS on day 20
postimmunization or day 31 postadoptive transfer. Spinal cords were
removed by dissection, and 2- to 3-mm spinal cord blocks were
immediately frozen in OCT (Miles Laboratories; Elkhart, IN) in liquid
nitrogen. The blocks were stored at -80°C in plastic bags to prevent
dehydration. Six micrometer thick cross-sections from the lumbar region
(approximately L2-L3) were cut on a Reichert-Jung Cyocut CM1850
cryotome (Leica, Deerfield, IL), mounted on Superfrost Plus
electrostatically charged slides (Fisher, Pittsburgh, PA), air dried,
and stored at -80°C. Slides were stained using a Tyramide Signal
Amplification (TSA) Direct kit (NEN, Boston, MA) according to
manufacturer’s instructions. Lumbar sections from each group were
thawed, air-dried, fixed in 2% paraformaldehyde at room temperature,
and rehydrated in 1x PBS. Nonspecific staining was blocked using
anti-CD16/CD32, (FcIII/IIR, 2.4G2; BD PharMingen), and an
avidin/biotin blocking kit (Vector Laboratories) in addition to the
blocking reagent provided by the TSA kit. Tissues were stained with
biotin-conjugated Abs anti-mouse CD4 (H129.19) and anti-mouse
I-Ab (AF6-120.1) (BD PharMingen). Sections
were counterstained with 4',6'-diamidino-2-phenylindole (DAPI;
Sigma-Aldrich) and then coverslipped with Vectashield mounting medium
(Vector Laboratories). Slides were examined and images were acquired
via epifluorescence using the SPOT RT camera (Diagnostic Instruments,
Sterling Heights, MI) and Metamorph imaging software (Universal
Imaging, Downingtown, PA). Eight serial lumbar sections from each
sample per group were analyzed at x100 and x400 magnification.
Histologic evaluation
Mice were anesthetized and sacrificed by total body perfusion through the left ventricle using chilled 3% glutaraldehyde in PBS (pH 7.3). Spinal cords were dissected out and cut into 1 mm thick segments and postfixed in OsO4, dehydrated, and embedded in Epon. Toluidine blue stained sections from 10 segments/mouse were read and scored as follows: ± = mild inflammation without demyelination; 1+ = inflammation with focal demyelination; 2+ = inflammation with multiple foci of demyelination; 3+ = marked inflammation with bilateral, converging areas of demyelination; 4+ = extensive bilateral areas of demyelination and remyelination.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the role of class II in the priming of myelin-specific T
cells has been addressed using anti-MHC class II blocking Abs,
susceptibility to EAE had not been studied with mice genetically
deficient in class II expression. To investigate the possibility of
nonclass II-restricted mechanisms for initiation of EAE, we tested
CIITA KO mice for susceptibility to initiation of EAE by active priming
and adoptive transfer of encephalitogenic T cells. Age- and sex-matched
C57BL/6 wt and CIITA KO mice were primed with MOG
35–55/CFA and observed for clinical symptoms as
described in Materials and Methods. Although 100% of wt
mice were susceptible to disease, none of the CIITA KO mice showed any
clinical symptoms (Fig. 1
a).
In addition to defective class II expression, CIITA KO mice have a
radically altered CD4+ T cell repertoire
(20, 22, 23) and might not have CD4+
T cells capable of recognizing the MOG35–55
epitope. Surprisingly, when MOG35–55-primed mice
were tested for 24-h DTH responses, CIITA KO mice displayed
measurable Ag-specific responses (Fig. 1c), albeit
significantly reduced from those seen in wt mice. Analysis of the LN
responses from MOG35–55-primed wt and CIITA KO
mice 8 days postpriming by ELISPOT showed that the T cells produced the
Th1 cytokines IL-2 and IFN- (Fig. 2, a and b, respectively), although the frequency of
cytokine-producing LN cells from CIITA KO mice was lower and required
higher Ag doses for activation than LN cells from wt mice. The IL-2-
and IFN--producing cells from CIITA KO mice were not MOG-specific
CD8+ T cell responses as coculture with
anti-class I mAb (M1/42) did not block cytokine production, while
addition of anti-class II mAb (M5/114) completely abrogated Th1
cytokine production (Fig. 2, e and f). The lack
of cytokine production with the addition of M5/114 was not due to
nonspecific cytotoxicity, as cultures containing anti-CD3 mAb
were unaffected by the addition of the anti-class II mAb (data not
shown). Splenic Th1 responses were also reduced in CIITA KO mice
compared with wt mice (Fig. 2, c and d),
suggesting that residual class II MHC was present in both the spleen
and LN of CIITA KO mice. The lack of disease in CIITA KO mice
could potentially be due to a switch from IFN- production to IL-4
production by CD4+ T cells, resulting in an
inhibition of the inflammatory response. To address this possibility,
we compared IFN- and IL-4 responses from the LN and spleen of
MOG35–55-primed wt and CIITA KO mice. There was
little difference in the numbers of IFN-- and IL-4-producing cells
between wt and CIITA KO mice 13 days postpriming (Fig. 2, g
and h).
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b). Both wt and CIITA KO mice recipients showed significant
DTH responses (Fig. 1d). This recall response in the CIITA
KO mice eliminated the possibility that T cells primed by and
restricted to wt IAb were incapable of
recognizing MOG peptide presented by the residual class II present in
the CIITA KO mice. Priming of a peripheral Th1 response is insufficient for the initiation of EAE in Ii- and DM-deficient mice
CIITA mutant mice have dramatically reduced levels of class II,
Ii, and DM (20, 22, 23). Although the CIITA KO mice could
present antigenic peptide to MOG-specific T cells, the possibility
remained that due to the reduction of critical accessory molecules, the
APCs could not process protein Ags. Thus, mice deficient in Ii or DM
expression were tested for their susceptibility to initiation of EAE.
Age and sex-matched wt C57BL/6, Ii KO, and DM KO mice were primed with
MOG35–55/CFA and followed for disease as
described in Materials and Methods. Neither Ii-deficient nor
DM-deficient mice showed any clinical signs of disease (Fig. 3a), while 100% of wt mice
got sick. wt mice had a mean peak clinical score of 2.9 ± 0.5 and
histological analysis showed massive infiltration (Fig. 4a) and severe
demyelination (Fig. 3c, data not shown). When tested for in
vivo Th1 responses by DTH, Ii-deficient mice had no response, while,
similar to CIITA-deficient mice, DM KO mice had reduced, but
significant (p < 0.0002) MOG-specific ear
swelling (Fig. 5a).
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(Fig. 6, a and
c, respectively). In all cases, the frequency of Th1
cytokine-producing cells was less in the KO mice than in wt mice.
Strikingly, even though the Ii-deficient mice did not have a
MOG35–55-specific DTH response (Fig. 5a), their frequency of Th1 recall responses in vitro was
greater than that seen in DM-deficient mice (Fig. 6, a and
c).
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, b and c). Upon measure of Th1
responses, mice from every adoptively transferred group, including Ii
KO mice, elicited significant DTH responses (Fig. 5b),
although the responses in the DM KO and Ii KO mice were slightly and
dramatically reduced as compared with the wt response, respectively.
Surprisingly, when analyzed for their ability to drive Th1 recall
responses in vitro, LN cells from Ii-deficient animals showed no
responsiveness (Fig. 6, b and d). LN cells from
DM KO mice, in contrast, drove a high frequency of IL-2- and IFN--
(Fig. 6, b and d) producing T cells, although
reduced from the responses seen in wt mice. DM- and Ii-deficient mice have no CNS infiltration, inflammation, or demyelination after MOG35–55-priming or adoptive transfer of MOG-specific T cell blasts
DM-deficient mice can both prime and recall significant Th1
responses (Figs. 5 and 6), yet fail to show any overt symptoms of EAE.
Although adoptive transfer of wt encephalitogenic T cell blasts failed
to initiate disease in DM KO mice, the possibility remained that there
was a subclinical disease that exhibited no characteristic
motor defects. To eliminate this possibility,
MOG35–55-primed or adoptively transferred wt, Ii
KO, and DM KO mice were perfused and their spinal cords were analyzed
histologically for CNS infiltration and inflammation as described in
Materials and Methods. Although MOG-primed wt mice
showed severe signs of inflammation and demyelination (Fig. 3c), Ii KO and DM KO mice showed no clinical pathology.
Immunohistochemical analysis showed that spinal cords from wt mice
either MOG35–55-primed or adoptively transferred
with MOG35–55-specific T cell blasts showed
extensive infiltration of CD4+ T cells (Fig. 4, a and e), and widespread class II
(IAb) expression (Fig. 4, c and
g). F4/80 expression, indicative of macrophage infiltration
and/or microglial activation was also widespread in the wt tissues
(data not shown). In contrast, spinal cord sections from DM KO mice
showed no CD4+ T cell infiltration (Fig. 4, b and f), and no class II or F4/80 expression
(Fig. 4, d and h, and data not shown,
respectively). Similar to the DM KO mice, Ii-deficient mice, either
MOG35–55-primed or adoptively transferred,
showed no infiltration or CNS cell activation (data not shown),
confirming a lack of CNS inflammation in the absence of Ii or DM
expression.
DM- and Ii-deficient APCs cannot process and present the MOG35–55 epitope from intact MOG protein
The preponderance of data supported the hypothesis that DM KO
mice, and to a lesser extent, Ii KO mice do not have defective
MOG35–55-specific T cell responses. Both mutant
mice could prime Th1 responses and present peptide for recall
responses, yet failed to show any clinical signs of EAE. Indeed, the DM
KO mice could prime and elicit a significant peripheral Th1 recall
response to peptide in vivo. Thus, with the DM KO mice in particular,
the defect appeared to be at the level of Ag presentation in the target
organ. The Ii-deficient mice had a more profound defect in vivo;
failing to elicit peripheral T cell responses, suggesting a broader
defect than that seen in DM-deficient mice. Both Ii and DM
deficient mice have defects in the processing of intact protein Ags,
but the level of deficiency can be Ag-specific (35, 36, 37).
To assess the ability of the mutant APCs to present intact MOG,
splenocytes from wt, Ii KO, and DM KO mice were isolated and assessed
for their ability to present peptide vs recombinant protein to a
MOG35–55-specific T cell line. Although all of
the APCs could present the MOG35–55 peptide and
stimulate the line to proliferate (Fig. 7a), only the wt APCs could
process and present the rMOG protein (Fig. 7b). Thus,
resistance to EAE initiation in DM KO mice appeared to be at the level
of protein Ag processing and presentation.
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DM KO and Ii KO mice can differentially present peptide
Ags, in an Ag-dependent manner (35, 36, 37). Although
unlikely, it was possible that the defect in EAE initiation in the
mutant mice could be specific to CNS processing and presentation of the
MOG35–55 peptide. We thus tested the ability of
a peptide of proteolipid protein, PLP178–191, to
initiate disease in C57BL/6 mice. Similar to
MOG35–55-induced disease, wt mice developed
clinical disease with 100% incidence, while DM- and Ii-deficient mice
failed to show any signs of disease (Fig. 8a). Immunohistochemical
analysis of spinal cords from PLP178–191-primed
mice showed significant CD4+ T cell infiltration
and IAb and F4/80 expression in wt tissues, while
DM KO and Ii KO spinal cords showed little evidence of pathology (data
not shown). Interestingly, when measuring Th1 recall responses in vivo,
both DM- and Ii-deficient mice had significant
PLP178–191 reactivities (Fig. 8b),
although the response in Ii KO mice was not as significant as the
response elicited in DM KO mice (p < 0.02 and
p < 0.005, respectively). Ex vivo, LN cells from
PLP178–191-primed Ii KO and DM KO mice
proliferated to specific peptides in a dose-dependent fashion,
albeit at reduced levels compared with wt LN cells (Fig. 8c). Thus, the phenotype of peripheral Th1 responsiveness
upon myelin peptide immunization without initiation of clinical disease
in DM- and Ii-deficient mice does not appear to be an Ag-specific
phenomenon.
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DM KO mice displayed the ability to prime significant peripheral
MOG35–55-specific T cell responses without
initiating EAE. This data, in conjunction with their inability to
process rMOG protein and the failure to initiate disease following
adoptive transfer of wt encephalitogenic T cells, supported the
hypothesis that the failure to initiate EAE in the DM mutant is the
result of a failure to process CNS protein Ags. However, there remained
a slight possibility that the wt T cells transferred into the KO mice
were incapable of recognizing CNS Ags processed and presented by DM KO
APCs and that the peptide-primed T cell population in DM KO mice was
not potent enough to initiate clinical EAE. To address this concern, we
primed C57BL/6 wt and DM-deficient mice with
MOG35–55/CFA two times, 7 days apart, and
compared their Th1 responses in vivo and in vitro to mice primed only
once. Once again, DM KO mice were resistant to disease initiation,
whether primed one or two times, while all of the wt mice developed EAE
(Fig. 9a). Significantly, the
DTH response in DM KO mice primed twice was equivalent to the response
in wt mice primed once (Fig. 9b), suggesting that the
magnitude of Th1 responses in the two groups was identical. ELISPOT
analysis of LN cells from mice primed one or two times showed similar
results, with DM KO mice primed twice displaying similar frequencies of
IL-2- and IFN--producing T cells as seen in wt mice primed once
(data not shown). This suggested that unless there was a previously
undescribed defect in T cells from DM-deficient mice, these T cells
should have encephalitogenic potential. To test this hypothesis, we
used DM KO mice primed twice as adoptive transfer donors. All of the wt
recipients of MOG35–55-specific DM KO T cell
blasts showed significant disease, while none of the DM KO recipients
had any clinical signs (Fig. 9c), demonstrating that T cells
from DM KO mice were encephalitogenic.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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treatment, astrocytes from SJL/J mice up-regulate the
expression of the CIITA, class II, Ii, and DM, as well as a variety of
costimulatory molecules (28). Additionally, after IFN-
exposure, the SJL/J astrocytes could process the immunodominant PLP
epitope, PLP139–151, from intact protein and
present it to encephalitogenic CD4+ T cells
(28).
The CIITA has been described as the master switch for both constitutive
and IFN--induced class II expression. Findings from three distinct
CIITA-deficient mice have demonstrated a profound, but not total, loss
of class II (and accessory molecule) expression (20, 22, 23). Functional studies have shown that
CD4+ T cells from CIITA-deficient mice cannot
elicit a class II-restricted allogeneic response or prime an
anti-keyhole limpet hemocyanin T cell response
(22). Mora et al. (53) demonstrated that
while NOD mice deficient in CIITA expression did have pancreatic
infiltration of CD8+ T cells, B cells, and
macrophages, they did not develop diabetes, although an autoaggressive
CD8+ T cell clone could initiate a delayed
diabetes independent of CIITA function. To test for the requirement of
class II in EAE, we attempted to initiate disease in CIITA-deficient
mice. Not surprisingly, the CIITA KO mice were resistant to initiation
of EAE. This defect was not solely due to a failure in T cell
activation, as adoptively transferred syngeneic encephalitogenic T
cells could not initiate disease. Additionally, resistance to EAE was
not the result of a failure in MOG peptide presentation, as
CIITA-deficient mice could present peptide in vivo and in vitro.
Because the CIITA-deficient mice express little or no class II, there
was a possibility that the DTH and IFN- responses measured were
class I-restricted. Other disease models have provided evidence
supporting the role of CD8+ T cells in EAE, e.g.
Huseby et al. (54) recently described a role for myelin
basic protein (MBP)-specific CD8+
cytotoxic T cells in the initiation of EAE in C3H
(H-2k) mice. In a more similar model, Sun et al.
(55) described a potential role for
CD8+ T cells in the initiation of
MOG35–55-specific EAE in B6 mice. Thus, there
was a distinct possibility that in the CIITA mutant mice,
CD8+ T cells were mediating the MOG-specific
immune responses. To address this possibility, we preformed
immunohistochemistry on DTH lesions from
MOG35–55-primed mice. Immunohistochemical
analysis of ears following DTH showed significant
CD4+, but not CD8+, T cell
infiltrates in MOG35–55-challenged ears of both
wt and CIITA KO mice (data not shown), suggesting that the inflammatory
response was not mediated by CD8+ T cell
responses. Additionally, in ELISPOT analysis of
MOG35–55-primed wt and CIITA KO mice, the
inhibition of IL-2 and IFN- production by addition of anti-class
II mAb (Fig. 2, e and f) argues that trace levels
of class II were presenting MOG peptide to CD4+ T
cells, and that CD8+ T cells were not mediating
the inflammatory responses. Moreover, in vitro
MOG35–55-specific IFN- and IL-2 responses
were maintained using column purified CD4+ T
cells (data not shown). Lastly, both MOG35–55-
and PLP178–191-specific proliferative responses
from C57BL/6 mice were inhibited by coculture with anti-class II
mAb, while the addition of anti-class I mAb had no effect (data not
shown). These data show that while there are almost undetectable levels
of class II proteins expressed in CIITA KO mice, that level is
sufficient for the priming of some peptide-specific, peripheral
CD4+ T cell responses. This conclusion has
implications regarding the levels of class II required for the
initiation of peptide-induced immune responses and suggests that a
broader population of tissues may function as APCs as long as
prerequisite costimulatory molecules are present in cis or
in trans. This in turn may have important implications in
peptide vaccine design. Our data indicate that MHC class II levels are
sufficient for priming a Th1 peptide response in CIITA-deficient mice,
but the mice are not susceptible to EAE initiation perhaps due to the
lack of Ag-processing accessory molecules (e.g. Ii and DM) in the CNS
or the inability of nonprofessional APC to express functional
class II.
Ii and DM KO mice have significant defects in class II expression
and/or function. In the absence of Ii, class II - and -chains
fail to fold properly in the ER and the majority of the class II is
degraded. By associating with ER-resident peptides, a small but
significant population of class II dimers does assemble, and is
shunted directly to the cell surface by the default pathway (16, 17). Thus, Ii-deficient mice express low levels of class II that
are occupied with peptides easily exchanged for high-affinity peptides
at the cell surface. Loss of DM function, in contrast, does not
dramatically decrease class II expression. Class II is assembled and
transported to the endocytic compartment for peptide loading, but
without DM, CLIP is not removed form the class II peptide-binding
groove. The class II /CLIP complex is transported to the APC
surface where exogenous peptides could selectively exchange with CLIP
and be presented (12, 15, 34, 56). Thus in both cases,
there was the possibility of priming a myelin-specific T cell response
and it was of considerable interest whether the specific components of
the class II Ag-processing machinery were required for the activation
of encephalitogenic T cells in vivo.
Professional APCs are incapable of processing and presenting the MOG35–55 determinant from a rMOG protein in the absence of Ii or DM. Additionally, while the efficiency of peptide presentation by mutant APCs is decreased at lower Ag doses, both Ii- and DM-deficient mice can present both MOG35–55 and PLP178–191. Interestingly, the Ii-deficient mice had little response to MOG35–55 challenge in vivo, yet responded more vigorously to in vitro Ag restimulation than DM KO LN cells. This is likely due to the enhanced ability of mutant APCs to present peptide Ags in vitro, which is often more efficient in Ii KO APCs than peptide presentation by wt APCs (16). In vivo, we established that both Ii- and DM-deficient mice are resistant to initiation of EAE by active priming with either MOG35–55 or PLP178–191. In addition to defective in vivo Ag presentation, DM- and Ii-deficient mice have additional defects that could potentially contribute to disease resistance. Although both mutant mouse strains have altered CD4+ T cell development (12, 16), the inability to transfer disease to KO mice using wt MOG35–55-specific encephalitogenic T cell blasts, in conjunction with the demonstration of encephalitogenicity of MOG35–55-specific T cell blasts from DM-deficient mice when transferred into wt mice, argues against a T cell defect preventing disease. Indeed, the ability of DM KO mice to prime a T cell response that is encephalitogenic in wt mice indicates that, in this case, there is not a defect in the periphery of these mice. Ii KO mice, in contrast, have additional deficiencies; in particular, defective B cell maturation (57). However, it is unlikely that the B cell defect in Ii-deficient mice mediated resistance to active EAE, as Lyons et al. (58) demonstrated B cell deficient mice to be susceptible to MOG35–55-induced EAE. Moreover, Lyons and colleagues demonstrated that B cell-deficient mice had similar clinical symptoms and disease severity as compared with wt mice, suggesting that the loss of Ag presentation function in Ii-deficient mice, and not the absence of B cells, is responsible for the resistance to MOG35–55-induced EAE.
Activated T cells can cross the blood-brain barrier independent of Ag
specificity, and traffic through the CNS (59). If the T
cells encounter their cognate Ag (myelin peptide plus class II MHC),
they remain in the CNS and execute their effector function. Activated T
cells that enter the CNS express the necessary inflammatory mediators
to up-regulate the expression of class II and costimulatory molecules
(e.g. IFN-) in a variety of CNS cells. It is unclear whether the
initial source of peptide Ag is derived from free myelin peptides
present in the CNS, or if myelin proteins are endogenously processed
and presented by CNS APCs. In support of the former possibility,
Krogsgaard et al. (60) used a mAb specific for DR2
complexed to the encephalitogenic MBP85–99
peptide, demonstrating that the naive CNS tissues display low levels of
class II/myelin Ags. We have shown that myelin peptides can be
presented in vitro, to myelin specific CD4+ T
cells, independent of Ii or DM expression. Moreover, we have
demonstrated that DM- and Ii-deficient mice can prime and elicit a
myelin peptide-specific DTH response, which is indicative of a
Th1-mediated T cell response (61, 62). However, adoptively
transferred encephalitogenic T cells blasts, which can recognize
peptides presented by DM- and Ii-deficient APCs, and can infiltrate the
CNS (data not shown; Ref. (59)) fail to initiate EAE and
do not remain in the CNS (Fig. 4f). These data support the
hypothesis that free myelin peptides are not present in naive CNS
tissues of the various KO mice displaying defects in the ability to
process intact myelin proteins and that de novo endocytic processing of
myelin Ags and subsequent presentation by CNS APCs is required for the
reactivation of encephalitogenic T cells in the target organ, enabling
effector function and the initiation of EAE.
The observed absence of class I-restricted, CD8+
T cell responses in MOG35–55-primed wt and
mutant mice contrasts with the work of Sun et al.
(55). Our data suggest that potentially MOG-specific,
CD8+ T cells cannot initiate disease in C57BL/6
mice. Sun et al. (55) proposed that
CD4+ T cell help is required to elicit active
MOG35–55-specific CD8+ T
cell-mediated disease. We demonstrated that DM-deficient mice prime a
potent peripheral CD4+ T cell response, yet we
failed to observe any disease symptoms or CNS infiltration of
CD8+ T cells, and could not elicit any
MOG-specific CD8+ T cell responses. Moreover, Sun
et al. (55) suggested that as few as 5 x
105 previously activated
MOG35–55-specific CD8+ T
cells could adoptively transfer disease independent of
CD4+ T cell (and presumably class II) function.
Our findings in the B6 CIITA-, Ii-, and DM-KO mice contrast with these
results. In each adoptive transfer experiment, an average of 10 x
106 CD8+ T cells elicited
in wt mice and sensitized to MOG35–55 were
transferred along with the encephalitogenic CD4+
MOG-specific T cells into the various KO mice, all of which express
normal levels of MHC class I. In all experiments, there was never any
incidence of disease (Figs. 1b, 3b,
9c, and data not shown) or evidence of CNS infiltration of
CD8+ T cells. In support of our conclusions, a
recent publication using green fluorescent protein-expressing
cells to track adoptively transferred
MOG35–55-specific T cells in B6 mice
demonstrated that while a significant proportion of transferred cells
were CD8+, only CD4+ green
fluorescent protein-positive cells were detected in the CNS of
recipient mice (63). Although our results and those of
others downplay the role of CD8+ T cells in our
disease model, they do not conflict the work of Huseby et al.
(54), as the authors specifically primed the class I
Ag-processing pathway with a vaccinia virus expressing MBP to generate
the encephalitogenic T cells from C3H.shi (MBP-mutant),
H-2k mice. In a disease model distinct from
MOG-specific EAE in C57BL/6 mice, Huseby and colleagues demonstrate
that CD8+ T cells can mediate encephalomyelitis.
Their findings have important implications regarding factors
contributing to autoimmune disease and MS in particular, but do not
directly impact this study.
Note.
After submission of this manuscript for publication, Slavin et al. (64) published a paper entitled "Requirement for endocytic Ag processing and influence of invariant chain and H-2 M deficiencies in CNS autoimmunity." Similar to the results shown in this study, the authors use Ii- and DM-deficient mice to demonstrate the requirement of Ag processing in the CNS for the initiation of EAE in C57BL/6 mice by either active priming or adoptive transfer. We are very pleased by the similarities in results, as we strongly support each other’s conclusions.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stephen D. Miller, Department of Microbiology-Immunology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611. E-mail address: s-d-miller{at}northwestern.edu
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MS, multiple sclerosis; Ii, invariant chain; DM, H-2M; ER, endoplasmic reticulum; CLIP, class II-associated Ii peptide; CIITA, class II transcriptional activator; KO, knockout; wt, wild type; MOG, myelin oligodendrocyte protein; PTx, pertussis toxin; DTH, delayed-type hypersensitivity; PLP, proteolipid protein; MBP, myelin basic protein.
Received for publication October 9, 2001. Accepted for publication February 15, 2002.
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