Protein Kinase Cθ Controls Th1 Cells in
Experimental Autoimmune
Encephalomyelitis
This information is current as
of June 17, 2017.
Shahram Salek-Ardakani, Takanori So, Beth S. Halteman,
Amnon Altman and Michael Croft
J Immunol 2005; 175:7635-7641; ;
doi: 10.4049/jimmunol.175.11.7635
http://www.jimmunol.org/content/175/11/7635
Subscription
Permissions
Email Alerts
This article cites 23 articles, 8 of which you can access for free at:
http://www.jimmunol.org/content/175/11/7635.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2005 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
References
The Journal of Immunology
Protein Kinase C␪ Controls Th1 Cells in Experimental
Autoimmune Encephalomyelitis1
Shahram Salek-Ardakani,* Takanori So,* Beth S. Halteman,* Amnon Altman,† and
Michael Croft2*
M
ultiple sclerosis (MS)3 is the most common demyelinating disease that affects the CNS (1). The best-characterized model for MS is experimental allergic encephalomyelitis (EAE) (1, 2). The pathology and clinical
presentation of EAE are typically characterized by lymphocytic
and mononuclear cell infiltration of the CNS, an increase in bloodbrain barrier permeability, astrocytic hypertrophy, demyelination,
and acute, chronic, or chronic relapsing paralysis (1, 3). There is
strong clinical and experimental evidence indicating that activation
and differentiation of myelin-specific precursor CD4 T cells into
encephalitogenic Th1-type cells are essential for initiation of the
disease (2, 4, 5). Accordingly, T cell lines and clones that transfer
EAE in the rat and mouse produce the Th1 cytokines IL-2, IFN-␥,
and TNF-␣, and these cytokines are present in the CNS of animals
with active EAE or that of MS patients (5).
Many efforts are presently directed toward inactivating or eliminating encephalitogenic CD4 T cells or inducing immune deviation away from the Th1 phenotype ((2, 6, 7). For example, blocking activation of CD4 T cells with Abs to CD4, MHC class II,
costimulatory molecules, and adhesion molecules, can strongly inhibit the induction and progression of EAE (2, 6, 7). However,
these approaches lack Ag specificity and might also result in generalized immune suppression (6). A more useful and clinically
applicable approach would be to target molecules that alter the
encephalitogenic potential of the autoreactive CD4⫹ T cell popu-
*Division of Molecular Immunology and †Division of Cell Biology, La Jolla Institute
for Allergy and Immunology, San Diego, CA 92121
Received for publication July 6, 2005. Accepted for publication September 27, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants AI50498 and
CA91837 (to M.C.). This is publication 710 from the La Jolla Institute for Allergy and
Immunology.
2
Address correspondence and reprint requests to Dr. Michael Croft, Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science
Center Drive, San Diego, CA 92121. E-mail address: [email protected]
3
Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental allergic
encephalomyelitis; HPRT, hypoxanthine phosphoribosyltransferase; LN, lymph node;
MOG, myelin oligodendrocyte glycoprotein; wt, wild type.
Copyright © 2005 by The American Association of Immunologists, Inc.
lation while at the same time minimizing the effects on other immune responses.
T cell activation requires TCR interaction with MHC-peptide
complexes in parallel with engagement of costimulatory molecules
such as CD28. Emerging data have suggested that protein kinase
C␪ (PKC␪) may be central to TCR- and CD28-specific signals that
lead to T cell activation, proliferation, and cytokine production.
Considerable in vitro data have suggested that PKC␪ is essential to
many events involved in signaling T cells to become activated (8,
9). Based on these studies, it was widely assumed that T cell activation and differentiation are absolutely dependent on PKC␪.
However, more recent in vivo data have challenged this idea. Using gene-deficient mice, CTL responses to lymphocytic choriomeningitis virus infection and CD4-dependent Ab responses to
vesicular stomatitis virus infection were normal in the absence of
PKC␪ (10). However, strongly impaired CD8 responses and a
form of CTL anergy were observed when soluble peptide Ag was
administered in vivo, implying that the requirement for PKC␪
could be dictated by either the subset of T cells responding or the
inflammatory milieu that accompanies T cell priming. In support
of this, two recent reports showed that PKC␪ was crucially involved in several Th2-type responses, such as that to Nippostrongylus brasiliensis and during asthmatic lung inflammation, but
it was dispensable for the protective Th1 response to Leishmania
major infection and for Th1-directed lung inflammation (11, 12).
This suggested a lesser role for PKC␪ in Th1 immune responses.
In the present study we show, in contrast to previous reports of
Th1-dominated responses, that without PKC␪, the peripheral T cell
response to myelin oligodendrocyte glycoprotein (MOG), T cell
entry into the CNS, and Th1 cytokine production were strongly
impaired, resulting in minimal clinical signs of EAE. These results
highlight further complexities in PKC␪ biology and have implications for how MS is controlled.
Materials and Methods
Mice
The studies reported in this paper conform to the principles outlined by the
Animal Welfare Act and the National Institutes of Health guidelines for the
care and use of animals in biomedical research. PKC␪-deficient mice
(PKC⫺/⫺; originally on a 129-C57BL/6J background) were a gift from Dr.
D. Littman (New York University, School of Medicine, New York, NY)
0022-1767/05/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Molecules that regulate encephalitogenic T cells are of interest for multiple sclerosis. In this study we show that protein kinase C␪
(PKC␪) is critical for the development of Ag-specific Th1 cells in experimental allergic encephalomyelitis (EAE), a model of
multiple sclerosis. PKC␪-deficient mice immunized with myelin oligodendrocyte glycoprotein failed to develop cell infiltrates and
Th1 cytokines in the CNS and were resistant to the development of clinical EAE. CD4 T cells became primed and accumulated
in secondary lymphoid organs in the absence of PKC␪, but had severely diminished IFN-␥, TNF, and IL-17 production. Increasing
Ag exposure and inflammatory conditions failed to induce EAE in PKC␪-deficient mice, showing a profound defect in the myelin
oligodendrocyte glycoprotein-reactive T cell population. These data provide evidence of a pivotal role for PKC␪ in the generation
and effector function of autoimmune Th1 cells. The Journal of Immunology, 2005, 175: 7635–7641.
7636
(13) and were backcrossed for more than five generations onto the
C57BL/6J background. These mice have no defect in thymic selection and
bear normal numbers of peripheral T cells. Wild-type (wt; PKC⫹/⫹)
C57BL/6J littermates were used as controls and were bred as previously
described (12).
Induction of EAE
Assay of T cell function ex vivo
Spleen and draining lymph nodes (LN; lumbar and inguinal LN) were
collected and after lysing RBC with ACK lysis buffer, cells were resuspended in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% FCS (Omega Scientific), 1% L-glutamine (Invitrogen
Life Technologies), 100 ␮g/ml streptomycin, 100 U/ml penicillin, and 50
␮M 2-ME (Sigma-Aldrich). Cells were plated at 2 ⫻ 105/well in roundbottom, 96-well microtiter plates in 200 ␮l with increasing concentrations
of MOG33–55 peptide (0.1–200 ␮g/ml) for 72 h at 37°C. After 66 h, 1 ␮Ci
of [3H]thymidine (ICN Biomedicals) was added, and thymidine incorporation was measured using a Betaplate scintillation counter (Tomtec). Each
in vitro stimulation assay was performed in triplicate. Supernatants were
harvested after 72 h of in vitro restimulation for cytokine analysis.
Histological assessment for EAE
Mice were anesthetized with sodium pentobarbital and intracardially perfused through the left ventricle with ice-cold PBS. Spinal cords and brains
from EAE-induced mice were dissected, fixed in 10% zinc-buffered formalin (Protocal; Fisher Diagnostics), and paraffin embedded, and sections
(5 ␮m) were stained with H&E. To ensure comparable analyses between
different groups, six to eight randomly selected sections were analyzed per
animal.
Phenotypic and intracellular cytokine staining of T cells
After blocking of FcRs with excess anti-Fc (24.G2), cells were incubated
with fluorescently conjugated Abs against CD4 (FITC or allophycocyanin),
CD3 (PE), CD8 (FITC), OX40 (PE), CD25 (PE), and CD69 (PerCP). Flow
cytometric measurement of cytokine production in T cells was performed
after lysing RBCs and LN or splenocytes were plated in round-bottom,
96-well microtiter plates in 200 ␮l with MOG peptide (100 ␮g/ml) for 72 h
at 37°C. GolgiPlug (BD Biosciences) was added, and the incubation was
continued for 8 h. Washed cells were stained with anti-CD4, fixed with
Cytofix/Cytoperm (BD Biosciences), and then stained with anti-IL-2 (FITC
and PE), anti-IFN-␥ (FITC and allophycocyanin), and anti-TNF-␣ (FITC
or allophycocyanin; all from BD Pharmingen). Samples were analyzed
after gating on CD4⫹ T cells on a FACSCalibur flow cytometer using
CellQuest (BD Biosciences) and FlowJo software (TreeStar).
5⬘-TGGGAGTAGACA AGGTACAACCC-3⬘), IL-17 (forward primer, 5⬘GCAAGAGATCCGGTCCTGA-3⬘; reverse primer, 5⬘-AGCATCTTCTC
GACCCTGAA-3⬘), CD3-⑀ (forward primer, 5⬘-TCTCGGAAGTCGAG
GACAGT-3⬘; reverse primer, 5⬘-TTGAGGCTGGTGTGTAGCAG-3⬘),
CD4 (forward primer, 5⬘-TCAAGATACCCCAGGTCTCG-3⬘; reverse
primer, 5⬘-CACCACCAGGTTCACTTCCT-3⬘), CD8⫺␣ (forward primer,
5⬘-ACTGCAAGGAAGCAAGTGGT-3⬘; reverse primer, 5⬘-CACCGCTA
AAGGCAGTTCTC-3⬘), F4/80 (forward primer, 5⬘-GCTGTGAGATTGT
GGAAGCA-3⬘; reverse primer, 5⬘-AGTTTGCCATCCGGTTACAG-3⬘),
and HPRT (forward primer, 5⬘-CTGGTGAAAAGGACCTCTCG-3⬘;
reverse
primer,
5⬘-TGAAGTACTCATTATAGTCAAGGGCA-3⬘)
were used.
Statistics
Statistical significance was analyzed by Student’s t test. Unless otherwise
indicated, data represent the mean ⫾ SD, with p ⬍ 0.05 considered statistically significant.
Results
PKC␪-deficient mice are resistant to EAE
To examine whether PKC␪ is involved in the development of autoimmune Th1 responses in vivo, groups of wt (PKC␪⫹/⫹) and
PKC␪⫺/⫺ mice were immunized with MOG peptide in CFA plus
pertussis toxin. The wt mice developed reproducible acute EAE
(Fig. 1 and Table I). On the average, clinical signs of disease were
evident on day 12 and peaked between days 14 and 18. The acute
phase was followed by remission that took place between days 25
and 40. In contrast, PKC␪⫺/⫺ mice displayed minimal clinical
signs of disease (Fig. 1 and Table I). The disease incidence (52 vs
94%), day of onset (19.83 vs 11.76), and mean maximal disease
score (0.91 vs 2.71) were all significantly lower in mice lacking
PKC␪ (Table I). These results provide clear evidence that PKC␪ is
required for the initiation of clinical EAE.
PKC␪-deficient mice show minimal CNS inflammation and Th1
cytokines after EAE induction
Immunization with MOG leads to the generation of encephalitogenic Th1 cells in the local draining LNs, followed by migration of
T cells into the CNS. Ag-specific Th1 cells produce cytokines,
chemokines, and proinflammatory mediators that initiate inflammation and demyelination, ultimately leading to EAE. To initially
try to understand why PKC␪⫺/⫺ mice were resistant to EAE, spinal cords and brains were examined (Fig. 2). Before the onset of
disease (day 9), histological examination of either wt or PKC␪⫺/⫺
tissue revealed no visible signs of inflammatory lesions or cell
RT-PCR
Spinal cords were extruded by flushing the vertebral canal with cold PBS.
Quantitative RT-PCR was performed with SYBR Green I dye using an
ABI GeneAmp 5700 Sequence BioDetector (PE Biosystems). Total RNA
was extracted using TRIzol (Invitrogen Life Technologies) and reverse
transcribed using oligo(dT)12–18 (SuperScript II; Invitrogen Life Technologies). Each transcript was analyzed concurrently on the same plate with
hypoxanthine phosphoribosyltransferase (HPRT), and levels of mRNA
were normalized to HPRT abundance. Sequence-specific primers for murine IFN-␥ (forward primer, 5⬘-GGATGCATTCATGAGTATTGC-3⬘; reverse primer, 5⬘-CCTTTTCCGCTTCCTGAGG-3⬘), TNF-␣ (forward
primer, 5⬘-CATCTTCTC AAAATTCGAGTGACAA-3⬘; reverse primer,
FIGURE 1. PKC␪-deficient mice display minimal clinical signs of
EAE. Groups of 12 PKC␪-deficient (PKC⫺/⫺; E) and wt (PKC⫹/⫹; F)
mice were immunized s.c. at the base of the tail with MOG in CFA. Pertussis toxin was given i.p. at the time of immunization and 48 h later. Mice
were monitored for clinical signs of EAE for 40 days and were scored on
an arbitrary scale of 0 –5 as described in Materials and Methods. Results
are the mean ⫾ SEM from 12 mice/group.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
MOG (35-MEVGWYRSPFSRVVHLY RNGK-55) was synthesized by
A&A Laboratories. Six- to 9-wk-old female and male mice were immunized by s.c. injection at the base of the tail with 150 ␮g of MOG33–55
peptide in CFA. The mice received injections i.p. with 300 ng of pertussis
toxin on days 0 and 2 after immunization. Where indicated, experimental
animals received the exact same immunization again 7 or 40 days after the
first immunization. Individual animals were scored daily for clinical signs
of EAE using the following criteria: 0, no detectable signs of disease; 0.5,
distal limp tail; 1, complete limp tail; 1.5, limp tail and hind limb weakness;
2, unilateral partial hind paralysis; 2.5, bilateral partial hind limb paralysis;
3, complete bilateral hind limb paralysis; 3.5, complete bilateral hind limb
paralysis and unilateral forelimb; 4, total paralysis of fore and hind limbs;
and 5, death. The average day of EAE onset was calculated by adding the
first day of clinical signs for individual mice and dividing by the number
of mice in the group. The average maximum disease score was calculated
by averaging the highest clinical score for each mouse. For those animals
that showed no clinical signs of disease, the onset was arbitrarily calculated
as 1 d after the experiment was terminated.
PKC␪ IS CRITICAL FOR THE DEVELOPMENT OF EAE
The Journal of Immunology
7637
Table I. Clinical parameters of MOG-induced EAE in wt and PKC␪-deficient micea
a
Mouse Strain
No. of Mice/Group
EAE Incidence
(%)
% Mortality
Mean EAE Onset
(range)
Mean Maximal EAE Score
(range)
C57BL/6
(PKC⫹/⫹)
C57BL/6
(PKC⫺/⫺)
Female ⫽ 22
Male ⫽ 12
Female ⫽ 23
Male ⫽ 11
32/34
(94%)
18/34ⴱ
(52%)
4/34
(11.76%)
0/34
(0%)
11.76
(8 –16)
19.83ⴱⴱ
(10 – 41)
2.71
(1–5)
0.91ⴱⴱⴱ
(1–35)
ⴱ, p ⬍ 0.000002; ⴱⴱ, p ⬍ 0.0000002; ⴱⴱⴱ, p ⬍ 0.000000002.
FIGURE 2. PKC␪-deficient mice display minimal
CNS inflammation and Th1 cytokine production after
active EAE induction. Groups of four PKC␪-deficient
(䡺) and wt (f) mice were immunized with MOG as
described in Fig. 1. a, CNS histopathology. Representative fields from the anterior lumbar spinal cords of
mice during the acute (day 14) phase of EAE. Sections
are stained with H&E. Magnification, ⫻100. mRNA
levels of CD3, CD4, CD8, and F4/80 (b) and mRNA
levels of IFN-␥, TNF, IL-2, and IL-17 (c) were determined by RT-PCR in the spinal cord at the indicated
time points after immunization with MOG. Values are
calculated as the fold increase vs untreated normal controls. Shown are mean ⫾ SEM for each group (n ⫽ 4
mice/group) from one of two experiments. ⴱ, p ⬍ 0.05
(wt vs knockout) as determined by Student’s t test.
CD4, CD8, and F4/80⫹ mRNA transcript levels at the peak of the
disease (Fig. 2b). Again, this analysis correlated well with the general lack of inflammation seen in H&E-stained spinal cord tissue
sections (Fig. 2a). Similar results were obtained when brains were
isolated from each experimental group (data not shown).
Proinflammatory cytokines derived from Th1 cells and macrophages in the CNS play an important role in the pathogenesis of
MS and the development of EAE. Consistent with lesser CNS
inflammation, mRNA for all the major cytokines implicated in
EAE, namely, IL-2, TNF, IFN-␥, and IL-17, was significantly reduced in the spinal cords (Fig. 2c) and brains (data not shown) of
PKC␪⫺/⫺ mice compared with wt littermates. These results indicated that PKC␪ affected the amount of mononuclear cell infiltration and Th1 cytokine production in the CNS throughout the
course of EAE, implying that this was responsible for the limited
disease in PKC␪⫺/⫺ mice.
Normal, but delayed, accumulation of activated CD4 T cells in
the absence of PKC␪
To quantify the extent of T cell priming after EAE induction, we
performed kinetic evaluation of the number and activation state of
CD4 T cells in the draining LN of wt and PKC␪⫺/⫺ animals. In the
absence of a specific method to track MOG-reactive T cells, cell
surface expression of CD44 and OX40, molecules associated with
T cell priming and activation, were assessed. The percentages of
CD3⫹CD4⫹ T cells in the draining LN of PKC␪⫺/⫺ mice were
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
infiltration (data not shown). Between day 9 and the peak of the
disease on day 14, the numbers of inflammatory cells and lesions
increased in all wt mice that had clinical signs of EAE. In striking
contrast and consistent with the lack of physical signs of EAE, the
numbers and sizes of the inflammatory lesions were dramatically
reduced in PKC␪⫺/⫺ mice (Fig. 2a).
Previous studies have demonstrated that T cells and F4/80⫹
macrophages (14, 15) are necessary for the induction of EAE in
C57BL/6 mice. To determine whether a lack of PKC␪ resulted in
fewer leukocytes in the CNS, we examined bulk infiltration at the
preclinical (day 9) and acute (day 14) phases of the disease. At
each time point, samples from four different spinal cords (Fig. 2b)
and brains (data not shown) were subjected to real-time PCR using
specific oligonucleotide primers for CD3, CD4, CD8, and F4/80⫹.
All amplifications were calibrated to HRPT, which enabled quantitative analysis of the dynamics of mRNA transcription of CD3,
CD4, CD8, and F480⫹ at the site of inflammation. Fig. 2b shows
a representative result from each time point of the experiment.
Consistent with the histological data, CD3, CD4, CD8, and F4/80⫹
mRNA transcript levels were scarcely detectable during the preclinical phase of the disease on day 9, but increased significantly
in the spinal cords isolated from wt mice at the acute phase (day
14) of EAE (Fig. 2b). This increase in the transcription of CD3,
CD4, CD8, and F4/80⫹ mRNA correlated well with the rapid onset
of clinical disease (compare Fig. 2b and Fig. 1). In sharp contrast,
the spinal cords from PKC␪⫺/⫺ mice had significantly lower CD3,
7638
PKC␪ deficiency results in Th1 cell hyporesponsiveness
We next considered the possibility that the PKC␪ deficiency resulted in a state of hyporesponsiveness in the responding CD4 T
cells and an inability to produce Th1 proinflammatory cytokines.
At various time points after immunization, draining LN cells were
stimulated with MOG (Fig. 4), and IL-2, IFN-␥, and TNF production and the ability to proliferate were measured. LN cells isolated
from wt mice 3 or 14 days after immunization showed enhanced
IL-2 production (Fig. 4a) and concentration-dependent proliferation (Fig. 4b). However, in the absence of PKC␪, both IL-2 pro-
duction and proliferation were reduced. As additional proof of hyporesponsiveness, we assessed the production of the effector Th1
cytokines, IFN-␥ and TNF, during the stages of disease (Fig. 4c).
In contrast to CD4 cells from wt mice, those from PKC␪⫺/⫺ mice
exhibited markedly impaired IFN-␥ and TNF production in response to MOG at all stages of disease. This was substantiated
when cytokine production was assayed in supernatants from these
cultures (data not shown). This demonstrates that the failure to
induce EAE in PKC␪⫺/⫺ mice was probably due to the inability to
generate functionally competent, MOG-specific effector Th1 cells.
Increasing Ag and inflammation fails to induce EAE in
PKC␪-deficient mice
Lastly, we examined whether the requirement for PKC␪ in the
development of EAE could be overcome by increasing Ag signal
strength and/or inflammatory conditions. Mice were immunized
with MOG in CFA on day 7 as well as day 0, with pertussis toxin
given on days 0, 2, 7, and 9. With this protocol all wt mice developed clinical signs of disease, with two mortalities on days 12
and 24 (Fig. 5a). The 50% mortality rate observed with this protocol is significantly higher than that seen in wt mice (11.76%; see
Table I) that were immunized once. Under these stringent inflammatory conditions, all PKC␪⫺/⫺ mice remained refractory to EAE
induction.
We next waited until wt and PKC␪⫺/⫺ mice showed remission
of disease after the first acute episode, and on day 40 after initial
immunization reinjected MOG in CFA/pertussis and monitored for
signs of EAE for an additional 40 days. As shown in Fig. 5b, in
addition to an earlier onset of disease (day 2 compared with day
12), all wt mice developed a chronic-progressive form of EAE
(clinical score of 2–2.5) with no signs of remission. In contrast,
recall responses in PKC␪⫺/⫺ mice were profoundly reduced, as
shown by significantly milder signs of EAE.
FIGURE 3. PKC␪ is transiently required for accumulation of activated CD4 T cells during priming with MOG. Groups of four PKC␪-deficient (PKC⫺/⫺)
and wt (PKC⫹/⫹) mice were immunized with MOG. Naive mice were used as controls. On day 3 (a), day 8 (b), and day 14 (c), draining LNs (inguinal
and lumbar) were collected, and total cell numbers were enumerated. Top panel, Percentage (left) and absolute numbers (right) of CD3⫹CD4⫹ T cells.
Middle panel, Percentage (left) and absolute numbers (right) of CD44high-expressing CD3⫹CD4⫹ T cells. Gray histograms indicate isotype control mAb.
Bottom panel, Percentage (left) and absolute numbers (right) of OX40⫹ cells after gating on CD4⫹CD44high T cells. Quadrant settings, distinguishing
positive from background fluorescence, were determined by staining with isotype-matched control mAbs (data not shown). ⴱ, p ⬍ 0.0001 (vs wt mice
immunized with MOG). Similar results were obtained in two independent experiments.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
comparable to those in wt mice at all time points examined (Fig.
3, a, b, and c, top panel). However, when converted into total CD4
T cell numbers, significantly fewer CD4 T cells were detected in
the LN of PKC␪⫺/⫺ mice on day 3 after immunization, although
comparable numbers were detected on days 8, 14 (Fig. 3, b and c),
and 25 (data not shown). Consistent with this, analysis of
CD44high-expressing CD4 T cells as marker of activation revealed
significantly greater numbers in wt mice on day 3, but not at later
time points from day 8 onward. OX40 is selectively expressed on
encephalitogenic or Ag-experienced CD4 T cells (16, 17). Analysis of OX40-expressing cells within the CD4⫹CD44high population also showed a significant reduction in both percentages and
absolute numbers induced in PKC␪⫺/⫺ mice during the first 3 days
after Ag priming, but not thereafter. Comparable results were obtained when CD25- or CD69-expressing cells were analyzed
within the CD4⫹CD44high population (data not shown). Collectively, these data imply that early during Ag encounter, PKC␪
affected T cell priming, but that activated, possibly MOG-specific,
T cells were generated with increasing time. Although there are
caveats to these phenotypic analyses, this suggests that the lack of
clinical signs of EAE and the reduced CNS inflammation seen in
PKC␪⫺/⫺ mice were not due to an inability of PKC␪-deficient
CD4 T cells to respond in the secondary lymphoid organs.
PKC␪ IS CRITICAL FOR THE DEVELOPMENT OF EAE
The Journal of Immunology
7639
The development of functionally competent, MOG-specific Th1
cells requires costimulatory signals, such as that from OX40 (18 –
21). Therefore, we considered the possibility that signaling
through OX40 may overcome the Ag-specific T cell hyporesponsiveness seen in PKC␪⫺/⫺ mice and administered agonistic antiOX40 on day 2 (Fig. 5c) or day 7 (Fig. 5d). The onset of EAE in
wt mice that received anti-OX40 was almost identical with that in
control mice, but disease progression was accelerated and resulted
in significantly more severe clinical signs of disease and increased
mortality. In contrast, PKC␪⫺/⫺ mice remained largely resistant to
EAE induction. Collectively, the data suggest that PKC␪ is absolutely required for the development of functionally competent,
MOG-specific Th1 cells.
Discussion
In the present study we provide evidence to show that PKC␪ is
essential for the development and persistence of Ag-specific Th1
cells in EAE. We show that a PKC␪ deficiency significantly affects
the peripheral T cell responses of mice to MOG, and the result is
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 4. PKC␪ is required for MOG-specific T
cell proliferation and Th1 cytokine production in secondary lymphoid organs. Groups of four PKC␪-deficient (PKC⫺/⫺; E) and wt (PKC⫹/⫹; F) mice were immunized with MOG as described in Fig. 1. a,
Intracellular IL-2 production from draining LN CD4
cells after restimulation in vitro with 100 ␮g/ml MOG
for 72 h. Numbers represent the mean percentage of
positive cells from four individual mice. b, Proliferation
from draining LN cells in vitro after 72 h of stimulation
with increasing concentrations of MOG on day 14. Results are the average cpm from triplicate cultures using
cells from four individual mice per group. c, Kinetics of
intracellular IFN-␥ and TNF production after restimulating LN CD4 cells with MOG peptide (100 ␮g/ml).
Numbers represent the mean percentage of IFN-␥-positive CD4 cells from four mice. Quadrant settings, distinguishing positive from background fluorescence,
were determined by staining with isotype-matched control mAbs (data not shown). Similar results were obtained in two independent experiments.
diminished inflammatory cells in CNS tissue and lower Th1 cytokine production, resulting in delayed EAE onset and minimal
clinical signs of disease.
Analysis of spinal cords and brains revealed that PKC␪ deficiency significantly affected mononuclear cell (CD4⫹, CD8⫹, and
F4/80⫹) accumulation in CNS tissue as well as Th1 (IL-2, IFN-␥,
TNF, and IL-17) cytokine production. Detailed analysis of CD4⫹
T cells isolated from the peripheral lymphoid organs of PKC␪deficient mice suggested normal, but delayed, accumulation of activated T cells in the absence of PKC␪. When functionality was
examined ex vivo in response to MOG, PKC␪⫺/⫺ CD4 T cells
were impaired in their ability to produce IL-2, to proliferate, and to
produce the effector Th1 cytokines, IFN-␥ and TNF, regardless of
the time after immunization. These results show that PKC␪ deficiency results in impaired maturation of MOG-reactive CD4 T
cells into functionally competent effector Th1 cells. Resistance to
EAE is then most likely not simply due to blocking T cells from
entering the CNS, although the latter associated with this hyporesponsiveness could also contribute to the phenotype observed.
7640
PKC␪ IS CRITICAL FOR THE DEVELOPMENT OF EAE
An important observation from our studies is the demonstration
that increasing Ag dose and inflammatory conditions did not overcome the PKC␪ dependence for EAE development. PKC␪⫺/⫺
mice, which were resistant to the initial episode of EAE, remained
unresponsive to EAE reinduction several months after initial priming, indicating that PKC␪ deficiency results in a long-lasting deficiency in the MOG-specific CD4 T cell population. The phenotype of CD28-deficient mice after single immunization with MOG
is very similar to that reported in this paper with PKC␪-deficient
mice (21–23), potentially correlating with the idea put forward in
the literature that PKC␪ is central to CD28 signaling. However, in
contrast with our data, CD28-deficient mice were shown to develop EAE with severe Th1 infiltration and demyelination after
repeated immunization with MOG (21, 22). Based on the latter
results, it was proposed that CD28 costimulation does not regulate
immunological anergy in EAE, but, rather, adjusts the threshold
for activation of autoreactive Th1 cells. Interestingly, blockade of
OX40-OX40L (21) or CD40-CD40L (22) interactions inhibits the
development of EAE in repeatedly immunized, CD28-deficient
mice, indicating that additional Ag exposure results in up-regulation of alternate costimulatory signaling pathways that can compensate for the lack of CD28. In this regard, we show that coadministration of an agonistic anti-OX40 resulted in the development
of a more severe EAE in wt mice, whereas PKC␪⫺/⫺ mice remained largely resistant to EAE induction. Thus, the hyporesponsive phenotype observed in PKC␪⫺/⫺ CD4 T cells, but not in
CD28⫺/⫺ CD4 T cells, suggests that in addition to CD28, PKC␪
may facilitate the development of encephalitogenic Th1 cells via
alternative costimulatory pathways, such as through OX40.
The role of PKC␪ in experimental models of inflammatory diseases has been recently studied in several model systems. PKC␪
was shown to be critical for the development of asthmatic lung
inflammatory responses controlled by Th2 cells and also for the
Th2 response to the parasite N. brasiliensis (11, 12). Interestingly,
PKC␪-deficient mice on both the C57BL/6 background and the
susceptible BALB/c background were able to mount a normal protective Th1 response against L. major infection, implying that
PKC␪ might be preferentially involved in the development and
effector function of Th2, but not Th1, cells (11). In additional
support of this, memory Th1 responses in the lung were normal in
PKC␪⫺/⫺ mice that were immunized and subsequently challenged
via the airways with OVA (12). In the latter study, however, the
PKC␪ deficiency was shown to significantly affect early in vivo
priming of both Th1 and Th2 cells after immunization. This implied that the requirement for PKC␪ in the generation of Th1 cells
might be overcome with time and/or by the overall level of costimulatory and inflammatory signals that accompany Th1 priming. The data presented in this paper extend these studies by showing that PKC␪ is certainly not selective for Th2 inflammation, but
can also be critically involved in inflammation driven by some, but
obviously not all, Th1 populations.
An interesting question, then, is why PKC␪⫺/⫺ mice failed to
generate efficient Th1 responses to MOG in the model of EAE, but
were largely normal in generating Th1 immunity to Leishmania
Ags (11) and to OVA when given via the airways (12). At present
we cannot answer this, and the exact parameters that govern the
dependence on PKC␪ of any one given T cell response remain to
be determined. It is possible that the nature of the Ag plays a key
role, whether it is foreign or self, or peptide or protein. Alternatively, the target organ might be crucial to how critical PKC␪ is for
the overall response. This could encompass not only the nature of
the T cell populations and their affinity for Ag, but also the range
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 5. Increasing Ag exposure or costimulation
fails to induce EAE in PKC␪-deficient mice. Groups of
four PKC␪-deficient (PKC⫺/⫺; E and 〫) and wt
(PKC⫹/⫹; F and ⽧) mice were immunized with MOG
as described in Fig. 1. Seven (a) or 40 (b) days later,
mice were reimmunized with MOG and pertussis and
monitored for clinical signs of EAE. Two (c) or 7 days
(d) later, mice received injections i.p. with either an
OX40 agonistic Ab (OX86; ⽧ and 〫) or an isotypematched control IgG (F and E), and monitored for clinical signs of EAE. Shown are the mean clinical scores
per group. Individual mouse death is indicated by a
cross. Results are the mean ⫾ SEM from four mice per
group.
The Journal of Immunology
of molecules required for trafficking to the organ, and the availability of Ag at the site of T cell priming or at the site of inflammation. All these parameters differ in the three Th1 models that
have now been analyzed, so it will be informative to test these and
other possibilities in the future. In particular, for those who are
interested in therapeutic targeting of autoimmune diseases, it will
be particularly important to know whether PKC␪ is essential for
the induction of other T cell-mediated autoimmune diseases.
In conclusion, our results indicate that PKC␪ plays a crucial role
in the development of the Th1 autoimmune inflammatory process
that leads to CNS inflammation and clinical EAE. These findings
have important ramifications not only for our understanding of the
basic mechanisms of autoimmunity, but also might offer the hope
of designing more efficient and highly specific therapeutic strategies for the treatment of autoimmune disorders.
Acknowledgments
We thank Dr. Dan Littman for making available the PKC␪-deficient mice.
The authors have no financial conflict of interest.
References
1. Steinman, L. 2001. Multiple sclerosis: a two-stage disease. Nat. Immunol. 2:
762–764.
2. Martin, R., H. F. McFarland, and D. E. McFarlin. 1992. Immunological aspects
of demyelinating diseases. Annu. Rev. Immunol. 10: 153–187.
3. Ransohoff, R. M., P. Kivisakk, and G. Kidd. 2003. Three or more routes for
leukocyte migration into the central nervous system. Nat. Rev. Immunol. 3:
569 –581.
4. Zamvil, S. S., and L. Steinman. 1990. The T lymphocyte in experimental allergic
encephalomyelitis. Annu. Rev. Immunol. 8: 579 – 621.
5. Kuchroo, V. K., A. C. Anderson, H. Waldner, M. Munder, E. Bettelli, and
L. B. Nicholson. 2002. T cell response in experimental autoimmune encephalomyelitis (EAE): role of self and cross-reactive antigens in shaping, tuning, and
regulating the autopathogenic T cell repertoire. Annu. Rev. Immunol. 20:
101–123.
6. Martin, R., C. S. Sturzebecher, and H. F. McFarland. 2001. Immunotherapy of
multiple sclerosis: where are we? Where should we go? Nat. Immunol. 2:
785–788.
7. Chernajovsky, Y., D. J. Gould, and O. L. Podhajcer. 2004. Gene therapy for
autoimmune diseases: quo vadis? Nat. Rev. Immunol. 4: 800 – 811.
8. Altman, A., N. Isakov, and G. Baier. 2000. Protein kinase C␪: a new essential
superstar on the T-cell stage. Immunol. Today 21: 567–573.
9. Altman, A., and M. Villalba. 2003. Protein kinase C-␪ (PKC␪): it’s all about
location, location, location. Immunol. Rev. 192: 53– 63.
10. Berg-Brown, N. N., M. A. Gronski, R. G. Jones, A. R. Elford, E. K. Deenick,
B. Odermatt, D. R. Littman, and P. S. Ohashi. 2004. PKC␪ signals activation
versus tolerance in vivo. J. Exp. Med. 199: 743–752.
11. Marsland, B. J., T. J. Soos, G. Spath, D. R. Littman, and M. Kopf. 2004. Protein
kinase C␪ is critical for the development of in vivo T helper (Th) 2 cell but not
Th1 cell responses. J. Exp. Med. 200: 181–189.
12. Salek-Ardakani, S., T. So, B. S. Halteman, A. Altman, and M. Croft. 2004.
Differential regulation of Th2 and Th1 lung inflammatory responses by protein
kinase C␪. J. Immunol. 173: 6440 – 6447.
13. Sun, Z., C. W. Arendt, W. Ellmeier, E. M. Schaeffer, M. J. Sunshine, L. Gandhi,
J. Annes, D. Petrzilka, A. Kupfer, P. L. Schwartzberg, et al. 2000. PKC-␪ is
required for TCR-induced NF-␬B activation in mature but not immature T lymphocytes. Nature 404: 402– 407.
14. Austyn, J. M., and S. Gordon. 1981. F4/80, a monoclonal antibody directed
specifically against the mouse macrophage. Eur. J. Immunol. 11: 805– 815.
15. Perry, V. H., and S. Gordon. 1988. Macrophages and microglia in the nervous
system. Trends Neurosci. 11: 273–277.
16. Weinberg, A. D., J. J. Wallin, R. E. Jones, T. J. Sullivan, D. N. Bourdette,
A. A. Vandenbark, and H. Offner. 1994. Target organ-specific up-regulation of
the MRC OX-40 marker and selective production of Th1 lymphokine mRNA by
encephalitogenic T helper cells isolated from the spinal cord of rats with experimental autoimmune encephalomyelitis. J. Immunol. 152: 4712– 4721.
17. Buenafe, A. C., A. D. Weinberg, N. E. Culbertson, A. A. Vandenbark, and
H. Offner. 1996. V␤ CDR3 motifs associated with BP recognition are enriched in
OX-40⫹ spinal cord T cells of Lewis rats with EAE. J. Neurosci. Res. 44:
562–567.
18. Weinberg, A. D., K. W. Wegmann, C. Funatake, and R. H. Whitham. 1999.
Blocking OX-40/OX-40 ligand interaction in vitro and in vivo leads to decreased
T cell function and amelioration of experimental allergic encephalomyelitis.
J. Immunol. 162: 1818 –1826.
19. Ndhlovu, L. C., N. Ishii, K. Murata, T. Sato, and K. Sugamura. 2001. Critical
involvement of OX40 ligand signals in the T cell priming events during experimental autoimmune encephalomyelitis. J. Immunol. 167: 2991–2999.
20. Nohara, C., H. Akiba, A. Nakajima, A. Inoue, C. S. Koh, H. Ohshima, H. Yagita,
Y. Mizuno, and K. Okumura. 2001. Amelioration of experimental autoimmune
encephalomyelitis with anti-OX40 ligand monoclonal antibody: a critical role for
OX40 ligand in migration, but not development, of pathogenic T cells. J. Immunol. 166: 2108 –2115.
21. Chitnis, T., N. Najafian, K. A. Abdallah, V. Dong, H. Yagita, M. H. Sayegh, and
S. J. Khoury. 2001. CD28-independent induction of experimental autoimmune
encephalomyelitis. J. Clin. Invest. 107: 575–583.
22. Girvin, A. M., M. C. Dal Canto, and S. D. Miller. 2002. CD40/CD40L interaction
is essential for the induction of EAE in the absence of CD28-mediated co-stimulation. J. Autoimmun. 18: 83–94.
23. Chang, T. T., C. Jabs, R. A. Sobel, V. K. Kuchroo, and A. H. Sharpe. 1999.
Studies in B7-deficient mice reveal a critical role for B7 costimulation in both
induction and effector phases of experimental autoimmune encephalomyelitis.
J. Exp. Med. 190: 733–740.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Disclosures
7641