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Expanded T cell receptor V restricted T cells from patients with sporadic inclusion body myositis are proinflammatory and cytotoxic CD28null T cells.

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ARTHRITIS & RHEUMATISM
Vol. 62, No. 11, November 2010, pp 3457–3466
DOI 10.1002/art.27665
© 2010, American College of Rheumatology
Expanded T Cell Receptor V␤–Restricted T Cells From
Patients With Sporadic Inclusion Body Myositis Are
Proinflammatory and Cytotoxic CD28null T Cells
Jayesh M. Pandya,1 Andreas E. R. Fasth,1 Mei Zong,1 Snjolaug Arnardottir,2 Lara Dani,1
Eva Lindroos,1 Vivianne Malmström,1 and Ingrid E. Lundberg1
Objective. Sporadic inclusion body myositis
(IBM) is characterized by T cell infiltrates in muscle
tissue, but their functional role is unclear. Systemic
signs of inflammation are lacking, and the absence of
beneficial effects following immunosuppression has
challenged the notion of a role for the immune system.
This study was undertaken to investigate the phenotype
and functionality of T cells, specifically a subset of
proinflammatory, cytotoxic, and apoptosis-resistant T
cells defined as CD28null T cells, in the pathogenesis of
sporadic IBM.
Methods. A cohort of 27 patients with sporadic
IBM was analyzed for the frequency of circulating and
muscle-infiltrating CD28null T cells. The T cell receptor
(TCR) V␤ usage was determined using flow cytometry
and immunohistochemistry. Anti-CD3–stimulated peripheral blood mononuclear cells were analyzed for intracellular interferon-␥ and cytotoxic potential by flow
cytometry.
Results. We found striking accumulations of both
CD8ⴙCD28null and CD4ⴙCD28null T cells, which represented the TCR V␤–expanded T cells in sporadic IBM.
Such CD28null T cells were abundant both in the inflamed muscle tissue and in the circulation. Although
the specific TCR V␤ expansions varied between patients,
both CD8ⴙCD28null and CD4ⴙCD28null T cells consistently displayed a highly proinflammatory and cytotoxic
potential.
Conclusion. Our results suggest that CD28null T
cell expansions represent the previously described expanded T cell subsets in sporadic IBM, and their proinflammatory capacity and presence in both muscle tissue
and the circulation may imply a role of immune activation
in sporadic IBM. In addition, CD4ⴙCD28null T cells may
exert cytotoxic effects directly on muscle fibers due to a
cytotoxic potential similar to that in CD8ⴙ T cells.
Sporadic inclusion body myositis (IBM) is a
chronic progressive myopathy that leads to severe disability, and presently, there is no successful long-term
treatment. Sporadic IBM is characterized clinically by
skeletal muscle weakness and muscle atrophy, and histologically by T cell infiltrates, vacuolar degeneration,
and accumulation of amyloid-related proteins in muscle
fibers (1,2). Although the cause of sporadic IBM is
unclear, 2 main mechanisms have been suggested: muscle degeneration and/or autoinflammation. A role for an
autoimmune component in this puzzling disease is supported by accumulating evidence, such as the strong
association with specific HLA alleles (e.g., DRB1*0301,
DRB3*0101, and DQB1*0201) (3); association with
immune-mediated conditions and the presence of autoantibodies in some patients (4); and the presence of
clonally expanded T cells (5–7), plasma cells (8), and
antigen-presenting cells in muscle tissue (9).
In particular, cytotoxic CD8⫹ T cells invading
class I major histocompatibility complex (MHC)–
expressing muscle fiber have been implicated in the
pathogenesis of sporadic IBM (10,11). These autoinvasive CD8⫹ T cells have been demonstrated to be
clonally expanded in the muscle tissue of patients with
Supported by grants from the Swedish Research Council, the
Swedish Rheumatism Association, King Gustaf V’s 80-Year Foundation, Karolinska Institutet, the European Union Sixth Framework
Programme (project AutoCure; LSH-018661), and by the Stockholm
County Council and Karolinska Institutet through a regional agreement on medical training and clinical research (ALF).
1
Jayesh M. Pandya, MSc, Andreas E. R. Fasth, PhD, Mei
Zong, MD, Lara Dani, MD, Eva Lindroos, MLT, Vivianne Malmström, PhD, Ingrid E. Lundberg, MD, PhD: Karolinska University
Hospital, Solna, and Karolinska Institutet, Stockholm, Sweden; 2Snjolaug Arnardottir, MD, PhD: Karolinska Institutet, Stockholm, Sweden.
Address correspondence and reprint requests to Jayesh
Pandya, MSc, Rheumatology Unit, CMM L8:04, Karolinska Universit y Hospital, SE-171 76 Stockholm, Sweden. E-mail:
Jayesh.Pandya@ki.se.
Submitted for publication March 18, 2010; accepted in revised
form July 13, 2010.
3457
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PANDYA ET AL
sporadic IBM, with a restricted variation in the third
complementarity-determining region of the T cell receptor (TCR), which persists over several years (7,12). In
addition to muscle, clonal expansions of CD8⫹ T cells
have been found in the peripheral blood of patients with
sporadic IBM (13), and peripheral blood and muscle
tissue have been shown to share T cell expansions (14).
However, an enigma is the observation that sporadic IBM is mostly resistant to conventional immunosuppressive treatment, including high doses of glucocorticoids in combination with other disease-modifying
agents, which challenges the notion that T cells and
immune components play a role in this disease (15,16).
In this context, the proinflammatory CD28null T cells are
of particular interest, since they are long-lived and
resistant to apoptosis (17–19). These cells are terminally
differentiated T cells lacking CD28, are often clonally
expanded, and have acquired new effector functions and
up-regulated a set of activating receptors mostly
associated with natural killer (NK) cells (20,21).
CD8⫹CD28null T cells emerge from repeated antigen
stimulation and are increased in the peripheral blood of
individuals with chronic viral infections, such as EpsteinBarr virus, human cytomegalovirus (CMV), and human
immunodeficiency virus (22,23). CD4⫹CD28null T cells
are less frequently found in the general population, but
are overrepresented in the peripheral blood of patients
with inflammatory and autoimmune diseases, such as
multiple sclerosis, cardiovascular diseases, rheumatoid
arthritis, and Crohn’s disease (24–26). Recently, we
demonstrated the presence of CD28null T cells of both
the CD4⫹ and CD8⫹ subtypes in the muscle tissue of
patients with dermatomyositis (DM) and polymyositis
(PM) and showed that these cells correlated with disease
variables (27).
Despite a number of reports indicating a role of
T cells in the pathogenesis of sporadic IBM, the specific
phenotype and functionality of T cells in this condition is
poorly understood. In this study, we provide evidence
that the TCR V␤–restricted T cells in patients with
sporadic IBM are proinflammatory and cytotoxic
CD28null T cells, belonging to both the CD8⫹ and the
CD4⫹ T cell compartments, and that these cells dominate the T cell infiltrates within muscle tissue.
PATIENTS AND METHODS
Patients and healthy subjects. Twenty-seven consecutive patients (16 men and 11 women) who had sporadic IBM
according to published criteria (28) and regularly visited the
rheumatology clinic (n ⫽ 20 patients) or the neurology clinic
(n ⫽ 7 patients) at Karolinska University Hospital were
enrolled in the study. At the time of blood sampling, their
median age was 76 years (range 46–85 years). The median age
of the men was 76 years (range 46–84 years), and the median
age of the women was 70 years (range 63–85 years). The
median disease duration was 2.5 years (range 0–12 years) for
the entire group, 2.5 years (range 0.1–12 years) for the men,
and 2.5 years (range ⫺0.04–8.9 years) for the women. At the
time of blood sampling, 13 patients were untreated, 9 were
receiving prednisolone (either alone [n ⫽ 1] or in combination
with methotrexate [n ⫽ 5], azathioprine [n ⫽ 2], or intravenous
immunoglobulin [n ⫽ 1]), 2 were receiving methotrexate, and
3 were receiving anakinra. Immunoregulatory treatment in the
3 months before blood sampling was considered to be ongoing
treatment. Patient records were scrutinized for any infection in
the 6 months preceding blood sampling. Information was
available for 25 patients. Muscle biopsy specimens obtained at
the same time as blood sampling were available for 13 patients,
and were included in this study. Peripheral blood was analyzed
from 29 healthy subjects with a median age of 54 years (range
46–82 years). The study was approved by the local Ethics
Committee of the Karolinska University Hospital, and both
patients and healthy subjects gave informed consent.
Flow cytometric analysis. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll separation
(Ficoll-Paque Plus; GE Healthcare Biosciences) and screened
for CD8⫹CD28null and CD4⫹CD28null phenotypes by flow
cytometry. Antibodies used were CD3 Pacific Blue (clone
UCHT1), phycoerythrin–Cy7 (PE-Cy7)–conjugated CD4
(SK3), allophycocyanin (APC)–conjugated CD28 (CD28.2;
BD PharMingen), and peridinin chlorophyll A protein
(PerCP)–conjugated CD8 (SK1; Becton Dickinson). Cells
were acquired by flow cytometry on a CyAn ADP Analyzer
(Beckman Coulter) and analyzed with Flow Jo software
(Tree Star). The percentages of CD8⫹CD28 null and
CD4⫹CD28null T cells were calculated as the frequencies of
CD28-negative cells in the gated CD3⫹CD8⫹ or CD3⫹CD4⫹
populations, respectively.
TCR V␤ analysis. The TCR V␤ usage was determined
by the IOTest1 ␤ Mark kit (Beckman Coulter) for 8 selected
patients with a high frequency (exceeding the median) of
CD8⫹CD28null T cells and/or CD4⫹CD28null T cells (Table
1). The TCR V␤ stainings were combined with CD3 PB,
PerCP-conjugated CD8, PE-Cy7–conjugated CD4, APC–
conjugated CD28, APC-Cy7–conjugated CD14 (MphiP9), and
APC-Cy7–conjugated CD19 (SJ25C1; BD PharMingen)
fluorochrome-conjugated antibodies. CD14 and CD19 antibodies were used to exclude monocytes and B cells in order to
increase the purity of the CD3⫹ population. The cells were
analyzed by 7-color flow cytometry, using a CyAn ADP
Analyzer and analyzed with Flow Jo software. A frequency of
⬎2 SD from the frequency found in healthy subjects was
considered to be a significant TCR V␤ restriction.
Functional assays. PBMCs from 4 patients analyzed
for TCR V␤ were cultured for 6 hours of 72 hours at a density
of 200,000 cells/well in 96-well plates (Sarstedt) coated with
anti-CD3 antibodies (OKT3) at a 2.5 ␮g/ml concentration in
RPMI with 5% heat-inactivated pooled male human AB serum
from the Karolinska University Hospital Blood Center. APCconjugated antibody to CD107a (Becton Dickinson) was added
at time point 0 for 6-hour stimulation cultures and during the
last 5–6 hours for 72-hour stimulation cultures. BD GolgiStop
(containing monensin) and brefeldin A (Sigma-Aldrich) were
added for the last 4 hours of all of the stimulations at a final
CD28null T CELLS IN SPORADIC INCLUSION BODY MYOSITIS
3459
Table 1. TCR V␤ expansions and demographic characteristics of the patients with sporadic inclusion body myositis who were screened for TCR
V␤ repertoire in peripheral blood*
CD8⫹
CD28null
cells, %
CD4⫹
CD28null
cells, %
TCR V␤ expansions
in CD8⫹ T cells†
TCR V␤
expansions in
CD4⫹ T
cells†
TCR V␤
analysis in
muscle
biopsy
specimen
Patient/
age/sex
Disease
duration,
months
1/80/M
2/66/M
16
73
93
31
72
17.5
V␤14, V␤22
V␤7.2
V␤13.6, V␤5.1
V␤5.1
Yes
Yes
3/81/M
35
85
70
V␤2, V␤22
V␤7.1, V␤5.1,
V␤16
NA
4/62/F
107
40
13.8
V␤1, V␤22
V␤21.3
Yes
5/83/F
6/84/F‡
⫺0.5
3
40
88
15
37
V␤20, V␤21.3
V␤21.3, V␤13.2
No
Yes
7/76/M§
8
70.5
V␤17
V␤22, V␤5.1,
V␤21.3,
V␤7.1
None
Yes
8/78/F¶
7
75
V␤20
Yes
1.6
18
V␤2
V␤21.3, V␤13.1, V␤1,
V␤13.2, V␤13.6
Treatment at the
time of sampling
Infections in the
6 months prior
to sampling
No treatment
Prednisolone 2.5
mg/day
Prednisolone 10
mg/day, MTX
20 mg/week
Prednisolone
1.25 mg/day;
MTX 15 mg/
week, IVIG
No treatment
Prednisolone 7.5
mg/day
Upper airways
None
Prednisolone 2.5
mg/day; AZA
100 mg/day;
anakinra 100
mg/day
Prednisolone 20
mg/day
Herpes zoster
None
None
Pneumonia
Urinary tract
Urinary tract
* NA ⫽ not available; MTX ⫽ methotrexate; IVIG ⫽ intravenous immunoglobulin; AZA ⫽ azathioprine.
† Expansions are shown in sequence, from the most restricted T cell receptor (TCR) V␤ to the least restricted TCR V␤ in peripheral blood.
‡ Also diagnosed as having systemic lupus erythematosus.
§ Also diagnosed as having Bechterew’s disease.
¶ Also diagnosed as having Sjögren’s syndrome.
concentration of 6 ␮g/ml and 10 ␮g/ml, respectively. Cells were
fixed and permeabilized using a BD Cytofix/Cytoperm
Fixation/Permeabilization kit and stained for intracellular
interferon-␥ (IFN␥) using PE-Cy7–conjugated IFN␥ antibody
(B27; Becton Dickinson). The stainings were combined with
fluorochrome-conjugated antibodies to CD3, CD8, CD4,
CD28, CD14, CD19 (as described above), TCR V␤, and
PE-conjugated CD244 (2B4) (clone C1.7; Immunotech). In
addition, aqua fluorescent reactive dye from Live/Dead Fixable Dead Cell Stain kit (Invitrogen) was added to all of the
stainings in order to exclude dead cells from the analysis. Cells
were acquired by a CyAn ADP Analyzer and analyzed by Flow
Jo.
Muscle biopsy specimens and immunostaining. Biopsy
specimens were obtained from the vastus lateralis or anterior
tibialis muscle by a “semi-open” technique under local anesthesia (29). The biopsy specimens were immediately frozen in
isopentane chilled by liquid nitrogen and stored at ⫺80°C. The
presence of CD28null T cells in muscle tissue should be
investigated by expression of a positive marker to allow direct
quantification and reduce the risk of inclusion of recently
activated T cells temporarily down-regulating CD28. We have
previously demonstrated the use of CD244 as a surrogate
marker for CD28null T cells in the muscle tissue of DM and PM
patients (27). A similar immunohistochemistry protocol was
used in the present study, combining CD3 and TCR V␤ with
CD244 (27). An extra blocking step was introduced to block
endogenous biotin or biotin-binding activity in tissue sections
using an Avidin/Biotin Blocking kit (Vector).
Primary antibodies included goat anti-human CD244
(R&D Systems), mouse monoclonal anti-CD3 (clone SK7;
Becton Dickinson), and mouse and rat monoclonal antibodies
against human TCR V␤ (all from Immunotech/Beckman
Coulter). Respective isotype control antibodies were irrelevant
mouse IgG1 (Dako), mouse IgG2a (Sigma), mouse IgG2b
(BD Biosciences), and rat IgG1 (R&D Systems). Biotinylated
secondary antibodies included horse anti-mouse IgG (Vector),
goat anti-rat IgG (Vector), or donkey anti-sheep/goat IgG
(The Binding Site). After the secondary antibody step, sections
were washed, incubated with peroxidase-conjugated ExtrAvidin (1:2500; Sigma-Aldrich), developed using a Peroxidase
Substrate kit (Vector) containing diaminobenzidine, and counterstained with hematoxylin.
Stained tissue sections were examined with a Leica
DM RXA2 microscope (Leica Microsystems) equipped with a
Leica DC digital color video camera 300F (Leica
Microsystems) connected to a PC computer. The number of
cells expressing CD244 and CD3 per unit area was assessed
quantitatively using a computer-assisted Quantimet 600 image
analyzer (Leica). Sections containing ⬎50 CD3⫹ T cells/mm2
were categorized as having a large T cell infiltrate. Sections
containing ⬍50 CD3⫹ T cells/mm2 positive for CD3 were
categorized as having small T cell infiltrates.
Statistical analysis. The Mann-Whitney 2-tailed test
was used to compare the frequencies of CD4⫹CD28null and
CD8⫹CD28null T cells in peripheral blood between healthy
subjects and patients with sporadic IBM, and to compare the
frequencies of CD28null T cells between peripheral blood and
3460
PANDYA ET AL
muscle tissue. The correlations between muscle-infiltrating
CD3⫹ and CD244⫹ cells per area were investigated by
Spearman’s 2-tailed correlation test. The computer software
program GraphPad Prism, version 5.02 was used for all
graphing and curve fitting.
RESULTS
ber of CD3 and CD244 positively stained cells per area
(Spearman’s r ⫽ 0.89, P ⫽ 0.012; n ⫽ 7) (Figure 1D).
The ratio between the number of CD244⫹ cells/mm2
and CD3⫹ cells/mm2 was considered to be the frequency of CD28null T cells in muscle tissue (median
72%, range 45.5–96%). Comparing the frequencies of
circulating and muscle-infiltrating CD28null T cells in
High frequencies of CD28null T cells in the peripheral blood of patients with sporadic IBM. Patients
with sporadic IBM displayed significantly higher median
frequencies of circulating CD8⫹CD28null T cells than
did healthy subjects (67% in patients with IBM versus
32% in healthy subjects; P ⬍ 0.01) (Figure 1A). The
frequency of circulating CD4⫹CD28null T cells was also
significantly higher in patients with sporadic IBM (median 11.5% in patients versus 2% in healthy subjects;
P ⫽ 0.0001) (Figure 1B). There was a significant correlation between the size of the circulating CD4⫹CD28null
and CD8⫹CD28null T cell populations (Spearman’s r ⫽
0.64, P ⫽ 0.001). The sizes of both CD28null T cell
populations were stable over a followup period of up to
2 years in repeated blood samples from 6 patients (data
not shown). Clonal T cell expansions could be due to
recent infections; however, we did not observe any
significant difference between the frequencies of
CD8⫹CD28null T cells in the group of patients with a
preceding infection (median 72.2%; n ⫽ 10) and the
group of patients with no infections (median 56.2%; n ⫽
15) (P ⫽ 0.211). Similarly, no significant difference was
observed between the frequencies of CD4⫹CD28null T
cells in the group of patients with infections (median
8.1%) and the group of patients with no infections
(median 12.0) (P ⫽ 0.867).
Higher frequency of CD28null T cells in inflamed
muscle compared with peripheral blood. CD244, in
combination with CD3, is a surrogate marker for
CD28null T cells in muscle tissue. We used these markers
in immunohistochemistry to analyze the muscle tissue of
patients with sporadic IBM for the presence and frequency of CD28null T cells. Seven of 13 muscle biopsy
specimens obtained from patients contained large
CD3⫹ T cell infiltrates with ⬎50 CD3⫹ cells/mm2, and
these were used for detailed phenotyping of the CD3⫹
T cells. In consecutive sections of muscle biopsy specimens, we found that most of the areas that were positive
for CD3 were also positive for the CD244 marker. A
representative immunohistochemistry staining depicting
the same area as positive for CD3 and CD244 in 2
consecutive sections of a muscle biopsy specimen from a
patient with sporadic IBM is shown in Figure 1C. We
found a significant linear correlation between the num-
Figure 1. Increased frequency of CD28null T cells in the peripheral blood
and muscle tissue of patients with sporadic inclusion body myositis (IBM).
A and B, Significantly higher frequencies of CD28null T cells in the CD8⫹
(A) and CD4⫹ (B) T cell populations in peripheral blood mononuclear
cells (PBMCs) from patients with sporadic IBM than in PBMCs from
healthy controls (HC). Each data point represents a single subject;
horizontal lines show the median. C, Staining of a muscle biopsy sample
from a representative patient with IBM. Muscle biopsy specimens from
patients with sporadic IBM (n ⫽ 13) were subjected to immunohistochemical analysis for the presence of CD3 and CD244. Most cells that
were positive for CD3 were also positive for CD244 in 7-␮m–thick
consecutive sections from specimens with mononuclear infiltrates (original magnification ⫻ 320). D, Significant correlation between the number
of CD3⫹ and the number of CD244⫹ cells per unit area within the
muscle tissue of patients with sporadic IBM, as determined by Spearman’s
correlation test. E, Significantly higher frequency of CD28null T cells in
muscle tissue (ratio of CD244⫹ cells per area to CD3⫹ cells per area on
consecutive muscle sections) than of CD28null T cells in peripheral blood.
P values were determined by the Mann-Whitney test.
CD28null T CELLS IN SPORADIC INCLUSION BODY MYOSITIS
Figure 2. Higher proinflammatory and cytotoxic functions in CD28null T
cells than in CD28⫹ subsets, in both the CD4⫹ and CD8⫹ compartments. For functional analysis, peripheral blood mononuclear cells from
patients with sporadic inclusion body myositis (IBM; n ⫽ 4) were
stimulated with plate-bound anti-CD3 for 6 hours, and the frequencies of
intracellular interferon-␥ (IFN␥)– and extracellular CD107a–expressing T
cells were determined by multicolor flow cytometry. A, Frequency of
IFN␥-containing cells in the CD28null subset (upper panel), and surface
expression of CD107a⫹ on CD28null T cells (lower panel), compared with
CD28⫹ counterparts within the CD8⫹ T cell compartment. While the
frequency of IFN␥-containing cells in the CD28null subset was significantly
higher, the surface expression of CD107a⫹ was not always found to be
increased compared with the CD8⫹CD28⫹ T cell subset. B, Higher
frequencies of both intracellular IFN␥-containing and CD107a⫹ cells in
CD4⫹CD28null T cell subsets compared with their CD28⫹ counterparts
within the CD4⫹ T cell compartment for all patients. C and D, Flow
cytometry plots of cells from a representative patient for CD8⫹ (C) and
CD4⫹ (D) T cell compartments. The percentages displayed on the plots
are the percentages of CD28null or CD28⫹ T cells that were IFN␥⫹ or
CD107a⫹ cells, after the frequency of positive cells in unstimulated
samples was deducted.
patients with sporadic IBM, we found a significantly
higher ratio of CD28null T cells in muscle tissue (median
72%) than in peripheral blood (median 35%; P ⬍ 0.05)
(n ⫽ 7) (Figure 1E).
Taken together, these data demonstrate that the
3461
higher frequencies of CD28null T cell subsets in the
peripheral blood of patients with sporadic IBM are
higher than those in healthy individuals and that the
majority of muscle-infiltrating T cells in these patients
are of the CD28null phenotype.
Higher IFN␥ secretion and degranulation potential in circulating CD8ⴙCD28null and CD4ⴙCD28null T
cell subsets compared with the CD28ⴙ T cell subset.
Next, we investigated whether the CD28null T cells were
proinflammatory in function. PBMCs from 4 patients
with sporadic IBM with a high frequency (exceeding
the median) of CD8⫹CD28 null T cells and/or
CD4⫹CD28null T cells were stimulated with anti-CD3
and stained for intracellular IFN␥ and CD107a surface
expression. The latter is an indirect measure of cytotoxicity and degranulation, i.e., release of perforin and
granzyme B (30,31). Following 6 hours of stimulation,
the CD8⫹CD28null T cell populations displayed higher
frequencies of intracellular IFN␥-containing cells (median 5.6%) compared with their CD28⫹ counterparts
(median 1.4%; P ⬍ 0.05) (Figure 2A, upper panel). The
frequency of CD107a⫹CD8⫹CD28null T cells (median
2.6%) was similar to that of the CD8⫹CD28⫹ T cell
subset (median 1.4%) (Figure 2A, lower panel).
Within the CD4⫹ compartment, CD4⫹ CD28null
T cell populations displayed much higher frequencies of
both intracellular IFN␥-containing cells (median 8.8%;
P ⬍ 0.05) and CD107a⫹ cells (median 1.9%; P ⬍ 0.05)
compared with their CD28⫹ counterparts (median 0.7%
for IFN␥ and 0.2% for CD107a⫹) (Figure 2B). Flow
cytometry plots from the 6-hour anti-CD3 stimulation
experiments with specimens obtained from a representative patient are shown in Figures 2C and D. The data
from the 72-hour stimulation experiments were similar
with respect to the relative differences between CD28null
and CD28⫹ subsets, although there were magnitude
differences compared with the results from the 6-hour
cultures (data not shown). Overall, these results demonstrate that both the CD8⫹CD28null and CD4⫹CD28null
T cell subsets are true effector cells with a capacity to act
both by cytokine release and by direct cytotoxicity.
Pronounced TCR V␤ restriction of CD28null T cells
in both peripheral blood and muscle infiltrates. Previous
studies have shown clonal expansions in the CD8⫹ T cell
populations of patients with sporadic IBM (12,14), a feature shared with CD28null T cells (19). To investigate
whether the CD28null T cell populations in our patient
cohort were restricted in their TCR V␤ usage, we analyzed
the TCR V␤ repertoire in 8 patients with high frequencies
of CD8⫹CD28null T cells and/or CD4⫹CD28null T cells in
the blood (Table 1). Not only CD8⫹, but also CD4⫹ T cell
3462
PANDYA ET AL
Figure 3. Skewed T cell receptor (TCR) V␤ repertoire in patients with sporadic inclusion body myositis (IBM), due to TCR V␤ restriction in the
CD28null population. Patients with sporadic IBM (n ⫽ 8) with high frequencies of CD28null T cells were screened for TCR V␤ restriction. All patients
with sporadic IBM were found to display a skewed TCR V␤ repertoire within CD8⫹ and CD4⫹ T cells compared with mean frequencies in healthy
controls (HC). A and C, TCR V␤ that were found to be significantly increased in CD8⫹ T cells (A) and CD4⫹ T cells (C) in any of the patients
screened are displayed. Shaded bars show expanded TCR V␤; open bars show nonexpanded TCR V␤. B and D, Comparison of the CD28null versus
CD28⫹ T cells within CD8⫹ T cell compartments (B) and CD4⫹ T cell compartments (D). The expansions were mainly observed within the
CD28null T cell populations, leading to an overall expansion in the CD8⫹ or CD4⫹ T cell population. Results from 2 representative patients (patient
7 and patient 2) are shown. E and F, Immunohistochemistry staining of specimens from 2 representative patients, displaying the presence of the same
TCR V␤–containing cells in muscle tissue as were dominant in circulating CD8⫹ (E) or CD4⫹ (F) T cell populations. Immunohistochemistry
staining for CD3, isotype control for TCR V␤ antibodies and nondominant TCR V␤ on consecutive muscle biopsy sections is shown along with
dominant TCR V␤ staining (original magnification ⫻ 250).
populations from screened patients with sporadic IBM
were found to have a skewed TCR repertoire.
Figure 3 summarizes the TCR V␤ restriction
patterns in CD8⫹ and CD4⫹ T cells in peripheral
blood. In total, 10 and 8 expansions were found in the
CD8⫹ and CD4⫹ T cells, respectively, for all patients.
Different patients were found to be restricted in different TCR V␤ (Table 1). Also, CD8⫹ and CD4⫹ T cell
populations displayed different TCR V␤ restrictions within
the same patient. Moreover, the clonal expansions were
found to be consistent over a period of up to 2 years (n ⫽
3) (data not shown). Importantly, when we compared the
CD28null versus CD28⫹ T cells within the CD8⫹ and
CD4⫹ T cell compartments, the expansions were confined
within CD28null T cell populations (Figure 3). In fact, we
found that the TCR V␤ restrictions observed in the total
CD8⫹ and CD4⫹ T cell populations were mainly due to
clonally expanded CD28null T cells. This was consistent for
all patients investigated.
To investigate whether the clonally expanded,
dominant TCR V␤ in peripheral blood were also present
in inflamed muscle tissue, immunohistochemistry analyses of muscle biopsy specimens were performed in 6
patients with known TCR V␤ profiles in peripheral
blood and where muscle biopsy samples were available.
The dominant TCR V␤ from peripheral blood CD8⫹
and CD4⫹ T cells were also dominant in muscle tissue.
Immunohistochemistry stainings of samples from representative patients are displayed in Figures 3E and F for
CD8⫹ and CD4⫹ T cells, respectively. As a control
experiment, nondominant TCR V␤ in peripheral blood
were analyzed in muscle tissue, and none of these were
found in significant numbers in the muscle biopsy specimens (Figures 3E and F).
Taken together, these results demonstrate that
CD28null T cells display a limited TCR V␤ repertoire in
blood and that the same T cell populations are also
CD28null T CELLS IN SPORADIC INCLUSION BODY MYOSITIS
found in the inflamed muscles of patients with sporadic
IBM. These expanded TCR V␤ subsets are indicative of
persistent antigen-specific activation.
Proinflammatory and cytotoxic functions of
CD28null T cells expressing dominant TCR V␤. Persistent antigen-specific activation and clonal expansion
could also indicate functional senescence of T cells.
Therefore, we next investigated whether the TCR V␤–
dominant CD28null T cells were senescent cells, exhausted in their effector functions due to repeated
division, or if they were still capable of exerting proinflammatory and cytotoxic functions. To this end, functional analyses were performed in anti-CD3–stimulated
Figure 4. Dominant T cell receptor (TCR) V␤–expressing CD28null T
cells are not functionally senescent. In multicolor flow cytometry–based
functional analysis experiments, peripheral blood mononuclear cells from
3 patients with sporadic inclusion body myositis (IBM) were stained for
circulating dominant TCR V␤. A and B, Similar interferon-␥ (IFN␥)
secretion and degranulation capacity in CD28null T cells with dominant
TCR V␤ and CD28null cells with nondominant TCR V␤ in both
CD8⫹CD28null (A) and CD4⫹CD28null (B) T cell compartments. C and
D, Flow cytometry plots of CD8⫹CD28null (C) and CD4⫹CD28null (D) T
cells from a representative patient. The percentages displayed on the plots
are the percentages of dominant V␤⫹ or dominant V␤⫺ CD28null T cells
that were IFN-␥⫹ or CD107a⫹ cells, after the frequency of positive cells
in unstimulated samples was deducted.
3463
PBMCs from 3 patients with sporadic IBM where the
dominant TCR V␤ were stained in parallel. Interestingly, CD28null T cells with dominant TCR V␤ were
found to display equal capacity with regard to IFN␥
secretion and degranulation as nondominant CD28null T
cells in both the CD8⫹ and CD4⫹ T cell compartments
(Figures 4A and B). Flow cytometry plots of cells from a
representative patient are displayed in Figures 4C and D.
The results indicate that CD28null T cells expressing dominant TCR V␤ are proinflammatory and cytotoxic effector
cells and are not driven to functional senescence.
DISCUSSION
It is still debated whether T cells play a role in the
pathogenesis of sporadic IBM or whether sporadic IBM
is merely a degenerative muscle disease. In this study, we
demonstrated that proinflammatory CD28null T cells are
the dominant T cell subset in peripheral blood and in the
inflamed muscles of patients with sporadic IBM. The
CD28null expansions were not confined to CD8⫹ only,
but were also prominent in the CD4⫹ T cell populations. These cells displayed a strong proinflammatory
and cytotoxic phenotype, more so than the corresponding CD28⫹ subsets. Additionally, both the
CD8⫹CD28null and the CD4⫹CD28null T cell populations were highly TCR V␤–restricted in the peripheral
blood, indicative of persistent stimulation by a limited
number of antigens. Strikingly, these cells were not
functionally senescent but retained a highly proinflammatory and cytotoxic function, and the same TCR
V␤–expressing subsets were also abundant in inflamed
muscle tissue.
Previous reports have described the clonal
expansions of CD8⫹ T cells in both the muscle and
blood of patients with sporadic IBM (13,14) and the
persistent nature of expanded CD8⫹ T clones in the
muscle tissue of patients with sporadic IBM (7,12). In
the present study, we demonstrated that the observed
clonal T cell expansions in patients with sporadic IBM
are due to expansions in the CD28null T cell populations.
Importantly, this feature is not limited to CD8⫹ T cells,
but is also found in the CD4⫹ compartment. Expanded
T cells with the same TCR V␤ expression were found in
the peripheral blood and muscle. Furthermore, in muscle tissue, many of these cells were surrounding muscle
fibers and were localized close to the muscle fiber
membrane, which could indicate a role of TCR V␤–
dominant CD8⫹CD28null and CD4⫹CD28null T cells in
muscle fiber destruction in sporadic IBM. In addition,
our findings support the notion of an exchange between
3464
blood and muscle and/or specific recruitment of
CD28null T cells to muscle tissue.
An accumulation of CD4⫹CD28null T cells in
tissue has previously been demonstrated in the vessel
walls in inflammatory cardiovascular disorders and
shown to correlate with clinical disease variables (32,33).
Recent results from our group demonstrated that
CD28null of both the CD4⫹ and CD8⫹ T cells dominate
the T cell infiltrates within the muscle tissue of patients
with DM and PM (27). Similarly, we showed in the
present study that in patients with sporadic IBM, the T
cell infiltrates in inflamed muscle tissue are dominated
by CD28null populations. Using a surrogate marker, such
as CD244, requires caution in interpretation since other
cells, such as NK cells, may also express CD244. However, the colocalization and strong correlation between
CD3 and CD244 expression in the muscle tissue strongly
indicate that CD244⫹ cells in this compartment are
CD3⫹ T cells. This was also shown in DM and PM,
where CD244 was mainly expressed by cells coexpressing CD3 (27). Of note, CD244 expression on T
cells is not a general phenomenon at the site of inflammation, since only occasional CD3⫹ T cells in the
synovial membrane of RA patients express CD244 (34).
The factors responsible for the observed
CD8⫹CD28null and CD4⫹CD28null T cell expansions
may be common to both subsets or may differ. Possible
common factors include chronic viral infections. Indeed,
the results of a number of recent studies indicate a link
between retroviral infection and the pathogenesis of
sporadic IBM (35,36). However, we could not explain
the CD28null T cell expansions by the presence of
previous infections in our patient cohort. The cooccurrence of both CD8⫹CD28null and CD4⫹CD28null
T cells could also indicate a common cause, such as
accelerated telomere erosion in an IFN␣-rich environment (37,38). Interestingly, plasmacytoid dendritic cells
are known to produce high levels of IFN␣ in response to
human CMV, and IFN␣ has been shown to accelerate
phenotypic differentiation and telomere erosion in
CD4⫹ T cells (39). Strikingly, recent studies have demonstrated a link between the long-term use of IFN␣,
particularly for the treatment of chronic hepatitis C virus
infection, and the development or exacerbation of numerous autoimmune phenomena and a spectrum of
myopathies, including IBM (40,41). The factors described above are possible mechanisms of CD28null T
cell expansions in patients with sporadic IBM.
There are several possible mechanisms by which
CD8⫹CD28null and CD4⫹CD28null T cells could harm
the muscle fibers in sporadic IBM. In patients with
sporadic IBM, the endomysial inflammatory cells have
PANDYA ET AL
been demonstrated to be predominated by activated
CD8⫹ T cells (10); therefore, the autoimmune pathogenesis in sporadic IBM is believed to be mediated
mainly by class I MHC–restricted CD8⫹ T cells invading
non-necrotic muscle fibers, a typical histopathologic
feature of sporadic IBM (10,11). Although CD4⫹ T cells
have also been detected in endomysial infiltrate in
previous studies, they were considered less important in
the pathogenesis of sporadic IBM, since CD4⫹ T cells
are not generally cytotoxic. However, our data demonstrate that a vast number of the CD4⫹ T cells in the
muscle tissue of patients with sporadic IBM are of the
CD4⫹CD28null phenotype and have cytotoxic potential
similar to that of CD8⫹ T cells. Therefore, the
CD4⫹CD28null T cells may also exert cytotoxic effects
directed to muscle fibers.
We also demonstrated that CD28null T cells are
potent producers of IFN␥, which alone or in combination with other cytokines can induce a chain of inflammatory events: up-regulation of class I or class II MHC
on muscle fibers and Fas-mediated apoptosis of muscle
cells, leading to myopathic conditions (42,43). Expression of MHC molecules on muscle fibers could allow T
cell–muscle cell interaction. However, based on the
results of previous studies, this has been regarded as
unlikely because the costimulatory molecules CD80/86,
which are normally required for functional interaction,
are not expressed by inflamed muscle fibers (44,45).
Nonetheless, CD28null T cells, which are independent of
costimulation by CD80/86, could be activated following
interaction with MHC-expressing muscle fibers and
cause muscle fiber damage. The function of CD28null T
cells could be potentiated by unconventional costimulatory molecules, such as CD244. We have recently demonstrated that de novo expressed NK receptors on
CD28null T cells in an additive pattern compensate for
suboptimal TCR stimulation (21). In addition, decreased susceptibility to immunoregulation by Treg cells
can potentially further strengthen the pathogenic role of
CD28null T cells (46).
The present study has some limitations. The
absence of large T cell infiltrates despite myopathic
features in some muscle biopsy specimens from patients
with sporadic IBM strengthens the general belief that
the role of T cells may be more important in certain
stages of the disease. Nevertheless, we cannot rule out
the possibility that we have missed infiltrates by chance
due to the patchy nature of infiltrates. The age ranges of
patients and healthy controls were similar, but the
median age of the patients was higher than for the
controls. However, we did not observe a significant
correlation between CD28null T cell frequencies and age
CD28null T CELLS IN SPORADIC INCLUSION BODY MYOSITIS
in healthy subjects or in patients; therefore, it is not
likely that the difference in the frequencies of CD28null
T cells between patients and controls could be explained
by the age difference at the beginning of the study.
A pathogenic role of CD28null T cells may not
be a general feature of sporadic IBM in all patients,
since we observed variable frequencies of CD28null T
cells. Instead, we suggest that CD28null T cell expansions
in patients with sporadic IBM could serve as a biomarker
for the presence of proinflammatory T cells and might
support an indication to choose therapies targeting
CD28null T cells for a subgroup of patients with sporadic
IBM. A recent proof-of-principle study concluded that
in patients with sporadic IBM one series of alemtuzumab infusions (a humanized monoclonal antibody to
CD52 that causes depletion of or a severe reduction in
peripheral blood lymphocytes and certain leukocyte
subsets) could slow down disease progression for up to 6
months, improve the strength of some patients, and
reduce endomysial inflammation and stressor molecules
in muscle tissue (47). These results strengthen the
notion of a role of inflammation and T cells in patients
with sporadic IBM. At the same time, we do not rule out
the possibility that other suggested mechanisms contribute to disease pathogenesis, especially in patients with
sporadic IBM with low CD28null T cell frequencies (48).
In conclusion, we identified CD28null T cells to be
the TCR V␤–expanded T cells in muscle and peripheral
blood in patients with sporadic IBM. CD28null T cells
constituted a majority of the T cells in muscle cell
infiltrates. Since these cells are proinflammatory and
have NK cell properties, our findings support the notion
that immune mechanisms play a role in the pathogenesis
of sporadic IBM and indicate that selective targeting of
CD28null T cells may provide effective therapy for patients with sporadic IBM.
ACKNOWLEDGMENTS
We are grateful to Ms Adina Nisell for contributing to
the collection of patient demographic data, Mrs. Annika van
Vollenhoven for outstanding technical help with the flow
cytometry, and Mrs. Eva Jemseby and Mrs. Gull-britt Almgren
for organizing blood sampling and the biobank.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Pandya had full access to all of
the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis.
Study conception and design. Fasth, Malmström, Lundberg.
Acquisition of data. Pandya, Zong, Arnardottir, Dani, Lindroos,
Lundberg.
3465
Analysis and interpretation of data. Pandya, Fasth, Malmström,
Lundberg.
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