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IMMUNOBIOLOGY
Anti-cytokine autoantibodies are associated with opportunistic infection in
patients with thymic neoplasia
*Peter D. Burbelo,1 *Sarah K. Browne,2 Elizabeth P. Sampaio,2,3 Giuseppe Giaccone,4 Rifat Zaman,2 Ervand Kristosturyan,2
Arun Rajan,4 Li Ding,2 Kathryn H. Ching,1 Arlene Berman,4 Joao B. Oliveira,5 Amy P. Hsu,1 Caitlin M. Klimavicz,1
Michael J. Iadarola,1 and Steven M. Holland2
1Neurobiology and Pain Therapeutics, Laboratory of Sensory Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health (NIH),
Bethesda, MD; 2Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD; 3Leprosy Laboratory,
Oswaldo Cruz Institute, FIOCRUZ, Manguinhos, Rio de Janeiro, Brazil; 4Medical Oncology Branch, National Cancer Institute, NIH, Bethesda, MD; and
5Immunology Service, Department of Laboratory Medicine, NIH Clinical Center, Bethesda, MD
Patients with thymic malignancy have
high rates of autoimmunity leading to a
variety of autoimmune diseases, most
commonly myasthenia gravis caused by
anti-acetylcholine receptor autoantibodies. High rates of autoantibodies to cytokines have also been described, although
prevalence, spectrum, and functionality
of these anti-cytokine autoantibodies are
poorly defined. To better understand the
presence and function of anti-cytokine
autoantibodies, we created a luciferase
immunoprecipitation system panel to
search for autoantibodies against 39 different cytokines and examined plasma
from controls (n ⴝ 30) and patients with
thymic neoplasia (n ⴝ 17). In this screen,
our patients showed statistically elevated,
but highly heterogeneous immunoreactivity against 16 of the 39 cytokines. Some
patients showed autoantibodies to multiple cytokines. Functional testing proved
that autoantibodies directed against interferon-␣, interferon-␤, interleukin-1␣ (IL-
1␣), IL-12p35, IL-12p40, and IL-17A had
biologic blocking activity in vitro. All patients with opportunistic infection showed
multiple anti-cytokine autoantibodies
(range 3-11), suggesting that anti-cytokine
autoantibodies may be important in the
pathogenesis of opportunistic infections
in patients with thymic malignancy. This
study was registered at http://clinicaltrials.
gov as NCT00001355. (Blood. 2010;
116(23):4848-4858)
Introduction
Anti-cytokine autoantibodies cause several important and emerging diseases ranging from pulmonary alveolar proteinosis, caused
by anti–granulocyte-macrophage colony-stimulating factor (anti–
GM-CSF) autoantibodies,1,2 to pure red cell aplasia, caused by
anti-erythropoietin autoantibodies,3,4 to opportunistic infections
caused by anti–interferon-␥ (anti–IFN-␥) autoantibodies.5-8 Anticytokine autoantibodies may also have benefits, such as dampening
inflammation through neutralizing anti–tumor necrosis factor-␣
(anti–TNF-␣) autoantibodies in rheumatoid arthritis.9 However,
there has been no comprehensive method to detect the prevalence
and functional significance of anti-cytokine autoantibodies.
Thymic malignancies are associated with a high frequency of
autoimmune phenomena, likely due to dysregulation of central
immune tolerance in the thymus. Approximately 10%-15% of
patients with myasthenia gravis, due to autoantibodies to the
acetylcholine receptor or other proteins present at the neuromuscular junction, have thymoma, and an additional 70% have thymic
hyperplasia. Conversely, 40% of thymoma patients will develop an
autoimmune condition, approximately half of which will be
myasthenia gravis.10,11 Many other autoimmune diseases have been
described in association with thymoma, ranging from pure red cell
aplasia to systemic lupus erythematosis.12,13 In patients with
thymoma, myasthenia gravis or both, autoantibodies to IFN-␣,
IFN-␭, IFN-␻, and interleukin-12 (IL-12) occur and may neutralize
cytokine signaling in vitro.14,15 However, the role of these anticytokine autoantibodies in disease pathogenesis is not established.
A method known as luciferase immunoprecipitation systems
(LIPS) quantitatively measures antibodies to a wide range of
infectious agents,16-19 as well as to a variety of human autoantigens.20-22 LIPS is a liquid phase immunoassay that uses antigens
directly tagged with Renilla luciferase, which can sensitively and
quantitatively detect antibodies. Using LIPS, highly informative
autoantibody profiles can be detected in cancer20 and autoimmune
conditions such as stiff-person syndrome21 and Sjögren syndrome.22 Therefore, we used this sensitive technique to explore
autoantibodies to cytokines in patients with thymic neoplasia
without and with opportunistic infections.
Submitted May 24, 2010; accepted August 9, 2010. Prepublished online as
Blood First Edition paper, August 17, 2010; DOI 10.1182/blood-2010-05-286161.
Presented in abstract form at the 1st International Conference on Thymic
Malignancy, Bethesda, MD, August 21, 2009.
*P.D.B. and S.K.B. contributed equally to this study.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
4848
Methods
Subjects
Patients were seen at the NIH for treatment of thymic malignancy (n ⫽ 16)
or for pulmonary Mycobacterium avium complex infection (n ⫽ 1). All
patients gave informed consent in accordance with the Declaration of
Helsinki under Internal Review Board–approved National Institute of
Allergy and Infectious Diseases protocol 93-I-0119. Patients had history
and physical data recorded on a standard form, including specific questions
BLOOD, 2 DECEMBER 2010 䡠 VOLUME 116, NUMBER 23
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BLOOD, 2 DECEMBER 2010 䡠 VOLUME 116, NUMBER 23
about infections, temporal relationship to immunosuppressive chemotherapy, treatment of associated autoimmune diseases, and the use of
corticosteroids for myasthenia gravis. Normal samples were obtained
though the NIH Blood Bank under appropriate protocols.
Antibodies
Blood was studied for immunoglobulin levels and lymphocyte markers
including total T cells (CD3; BD Pharmingen); total CD4 (Immunotech) or
CD8 (Immunotech); and total B cells (CD20; BD Pharmingen). Naive
T cells with CD4⫹ or CD8⫹ CD45RA (Immunotech), and memory subsets
measured by CD4⫹ or CD8⫹ CD45RO (Dako) as well as memory B cells
measured by CD20⫹CD27⫹ (BD Pharmingen) were determined. Natural
killer cells were defined as CD3⫺ and CD16⫹ or CD56⫹ (BD Pharmingen),
whereas natural killer T cells were defined as CD3⫹ and CD16⫹ or CD56⫹.
LIPS analysis for anti-cytokine autoantibodies
LIPS harnesses light-emitting Renilla luciferase (RUC) recombinant antigen fusion proteins to quantitatively measure patient antibody titers. We
generated 39 different C-terminal cytokine fusions using the pREN2
mammalian expression vector.20 Briefly, human cDNA clones (Open
Biosystems) of the following genes were amplified by polymerase chain
reaction (PCR) using gene-specific primers as described previously20:
IFN-␣1, IFN-␤1, IFN-␥, IFN-ε, IFN-␭1, IFN-␻, IL-1␣, IL-1␤, IL-1
receptor antagonist, IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12p35,
IL-12p40, IL-15, IL-17A, IL-18, IL-21, IL-22, IL-23p19, IL-27p28, IL-32,
EBI3/IL-27␤, GM-CSF, G-CSF, stem cell factor, TNF-␣, TNF-␤, BAFF,
APRIL, FasL, CD40 ligand, erythropoietin, EBI1/CCL19, and transforming growth factor-␤. For almost all these cytokines, the mature cytokine
coding sequences without the signal sequences were generated for fusions
at the C terminus of Ruc with a stop codon included at the end of the coding
sequence. DNA sequencing was used to confirm the integrity of all DNA
constructs. Plasmid DNA was prepared from the different pREN2 expression vectors (QIAGEN). After transfection into COS1 cells, crude protein
extracts were obtained as described.23
Plasma was obtained from heparinized venous whole blood by centrifugation and stored in aliquots at ⫺80°C. All samples were coded to prevent
observer bias before assay. For LIPS testing, a master plate was first
constructed by diluting patient sera 1:10 in assay buffer A (50mM Tris, pH
7.5, 100mM NaCl, 5mM MgCl2, 1% Triton X-100) in a 96-well polypropylene microtiter plate, which was then used for dispensing diluted plasma
samples for testing in a 96-well plate format as described.23 For evaluating
antibody titers by LIPS, 40 ␮L of buffer A, 10 ␮L of diluted human sera
(1 ␮L equivalent), and 1 ⫻ 107 light units (LUs) of Ruc-antigen Cos1 cell
extract, diluted in buffer A to a volume of 50 ␮L, were added to each well of
a polypropylene plate at room temperature for 1 hour. The mixture was then
transferred to 96-well filter plates containing protein A/G beads for 1 hour.
After washing, the LUs were measured in a Berthold LB 960 Centro
microplate luminometer (Berthold Technologies;) using coelenterazine
substrate mix (Promega). All LU data were obtained from the average of at
least 2 independent determinations. Antibody titer values less that 10 000 LUs
typically represent low-level signals and/or background binding.
Statistical analysis of LIPS antibody titers and heatmap
GraphPad Prism Version 5.0c was used for statistical analyses. Due to the
overdispersed nature of autoantibody titers, controls are reported as the
geometric mean titer (GMT) and 95% confidence interval (CI). The
nonparametric Mann-Whitney U test was used for comparison of antibody
titers between groups. For determining the cutoff limits for each of the LIPS
tests, the mean value of the 30 control samples plus 3 and 5 standard
deviations (SDs) was used. Data transformation and heatmap plots were
used to visualize the autoantibody profiles. Autoantibody titer values for
each antigen-antibody measurement greater than the control mean plus
3 SD were color-coded with different shades of green to red to signify the
relative number of SDs above these cutoff values (see Figure 3).
ANTI-CYTOKINE AUTOANTIBODIES AND THYMIC NEOPLASIA
4849
Cell culture and stimulation
Peripheral blood mononuclear cells (PBMCs) were obtained by density
gradient centrifugation as described.24 PBMCs were cultured at 106 cells/mL in
complete medium consisting of RPMI 1640, 2mM glutamine, 20mM HEPES
(N-2-hydroxyethylpiperazine-N⬘-2-ethanesulfonic acid), 100 U/mL penicillin,
100 ␮g/mL streptomycin with 10% patient or normal plasma. Cells were left
unstimulated or stimulated with phytohemagglutinin (PHA 1%; Invitrogen) plus
IL-12 (1 ng/mL; R&D Systems) or IFN-␥ (1000 U/mL; Intermune) plus
lipopolysaccharide (LPS, 200 ng/mL; Sigma-Aldrich) for 48 hours. Supernatants
were collected and stored at ⫺20°C. For evaluation of IL-1␣ signaling, PBMCs
were stimulated as above in the presence of commercial anti–IL-1␣ antibody
(2 mg/mL; R&D Systems). In addition, human foreskin fibroblast (HFF)–1 cells
were incubated in the presence of 10% plasma from healthy volunteers with or
without commercial IL-1␣ antibody or with 10% patient plasma and left
unstimulated or stimulated with IL-1␣ (10 ng/mL) for 4 hours with cell lysates
collected for isolation of RNA.
For stimulation of the IL-12 receptor, lymphoblasts were prepared as
described previously.25
For stimulation of the IL-17 receptor, HFF-1 cells (ATCC SCRC-1041)
were used as described previously,26 and cultured in complete medium
consisting of Dulbecco modified Eagle medium with 15% FCS, 2mM
glutamine, 20mM HEPES, and 0.01 mg/mL penicillin/streptomycin in 5%
CO2. Cells (5 ⫻ 105) were cultured for 48 hours, and media were removed
and replaced with complete Dulbecco modified Eagle medium containing
either 10% normal or patient plasma. HFF-1 cells were then left non
stimulated or stimulated overnight with IL-17 (100 ng/mL; R&D Systems)
when culture supernatants were harvested and stored at ⫺80°C until use.
Detection of phosphoSTAT-1 and STAT-4 by flow cytometry
For detection of phosphoSTAT-1, PBMCs (5 ⫻ 105 cells) were cultured in
complete RPMI media containing normal or patient plasma (10%) and left
unstimulated or stimulated with IFN-␥ (1000 U/mL) or IFN-␣ (1000 U/mL,
IFN-␣ 2b; Schering) or IFN-␤ (1000 U/mL; PBL Interferon Source) for
15 minutes at 37°C. Cells were fixed and permeabilized as previously
described8 and stained with phosphoSTAT-1 (Y701) antibody (BD Pharmingen). Data were collected using FACSCanto (BD Biosciences) and
analyzed using FlowJo Version 9.1 (TreeStar).
For detection of phosphoSTAT-4, prepared blasts were washed and
resuspended in complete RPMI with 10% normal or patient plasma and
either left unstimulated or stimulated with IL-12 (100 ng/mL) or IFN-␣
(1000 U/mL) for 15 minutes. Cells were fixed and permeabilized as above
and incubated with anti–phosphoSTAT-4 (Y693) antibody (BD Pharmingen) at 4°C for 30 minutes.
Immunoblotting
For immunoblot analysis, PBMCs (3 ⫻ 106 cells/well) cultured in RPMI medium in the presence of 10% normal or patient plasma, were stimulated or not
with IFNs (IFN-␣, IFN-␥, or IFN-␤; 1000 U/mL) for 30 minutes. Cell lysates
were obtained using whole cell lysis buffer (Cell Signaling) containing
protease and phosphatase inhibitors (Calbiochem). Whole PBMC lysates
were separated under reducing conditions on 10% sodium dodecyl sulfate
polyacrylamide gel electrophoresis gels (Invitrogen) at 200 volts for 60 minutes.
Proteins were transferred electrophoretically to a polyvinylidene fluoride
membrane (Invitrogen), the membranes were blocked for 1 hour in nonfat
dry milk solution in 0.1% phosphate-buffered saline Tween-20 and
incubated with 1:1000 dilution of primary rabbit anti-phosphoSTAT1
(Y701) antibody (Cell Signaling Technology). After additional washing, the
membranes were incubated with 1:10 000 secondary mouse anti–rabbit
antibody conjugated to horseradish peroxidase (Amersham) and developed
with enhanced chemiluminescence plus (Amersham). Blots were stripped
and reprobed with anti–total STAT1 (Cell Signaling) and anti–␤-actin
antibodies (Abcam) to ensure equivalent protein loading among samples.
Quantitative real-time PCR
For evaluation of gene expression, PBMCs isolated from healthy donors
were cultured in complete RPMI-1640 medium (Gibco BRL) containing
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4850
BLOOD, 2 DECEMBER 2010 䡠 VOLUME 116, NUMBER 23
BURBELO et al
Table 1. Demographics, histology, and autoantibody-associated phenomena for 17 patients with thymic neoplasm
Age at
diagnosis/sex
Age at
study
enrollment
1
34/M
40
B2
CMC, dVZV
2
58/F
61
B2
CMC, S apiospermum, and M avium
3
18/F
23
B2
Patient
WHO
classification
Opportunistic infections
Other
paraneoplastic conditions
No. of
anti-cytokine
autoantibodies
8
11
sinopulmonary disease
Myasthenia gravis, type 1 diabetes mellitus
2
with anti–GAD-65 autoantibodies
4
44/F
53
Thymoma*
5
45/M
48
B2/B3
6
51/M
57
B3
0
7
48/F
50
B1
6
8
67/F
71
C
9
36/M
45
B3
CMC
Myasthenia gravis; eosinophilic fasciitis
2
Myasthenia gravis
5
6
Disseminated cryptococcosis
(lung, blood, liver)
Coombs positive hemolytic anemia;
6
Sweet syndrome
10
34/F
35
B3
11
36/M
42
B2/B3
Myasthenia gravis
0
8
12
53/F
53
C
0
13
41/F
41
B2
14
27/M
31
C
15
39/M
39
C
16
55/F
57
B3
17
59/M
66
B3
6
Cushing syndrome
0
0
1
dVZV
3
CMC indicates chronic mucocutaneous candidiasis; and dVZV, disseminated varicella zoster virus
*World Health Organization (WHO) classification not available.
10% normal or patient plasma at 37°C and were unstimulated or treated
with IFN-␥ (400 or 1000 U/mL) or IFN-␣ (1000 U/mL) for 3 hours, at
which time cells were harvested for RNA isolation. Total RNA was
extracted using the RNeasy kit according to the manufacturer’s protocols
(QIAGEN). RNA (1 ␮g) was used for reverse transcription by oligo-dT
primer (Invitrogen) and the resulting cDNA amplified by PCR using the
ABI 7500 Sequence detector (Applied Biosystems). Amplification was
performed using TaqMan expression assays (Applied Biosystems). Glyceraldehyde 3-phosphate dehydrogenase was used as normalization control,
and results are expressed as fold induction.
Gene expression was also assayed in HFF-1 cells in response to IL-1␣
(2 ng/mL; R&D Systems) cultured for 4 hours in the presence of 10%
normal plasma, patient plasma or the commercial anti-IL-1␣ antibody.
RNA was harvested and processed as above.
Cytokine determination
Culture supernatants from stimulated and nonstimulated PBMCs and
HFF-1 cells obtained as described above were analyzed for cytokine levels
using a custom bead-based cytokine assay detecting IL-1␤, IL-6, IL-10,
IL-12p70, IFN-␥, and TNF-␣ (Bio-Plex assay; Bio-Rad Laboratories)
processed according to the manufacturer’s specifications.
Results
Subjects with thymic malignancy
We enrolled 17 patients with thymic malignancies, 8 males and
9 females ranging from 23 to 71 years (mean 48 years; Table 1).
Sixteen patients were identified through their referral for treatment
of thymic malignancy. Patient 2 was referred for treatment of
pulmonary M avium infection. Based on the World Health Organization histopathologic grading scale,27 our patients’ diagnoses
were: 1 type B1; 4 type B2; 2 type B2/B3; 5 type B3; 1 thymoma
without World Health Organization classification; and 4 type C or
thymic carcinoma (Table 1). Patients (5 of 17) had current or
previous opportunistic infections including: 1 chronic mucocutaneous candidiasis (CMC) alone; 1 disseminated varicella zoster virus
infection (dVZV); 1 CMC and dVZV; 1 disseminated cryptococco-
sis; and 1 with CMC with pulmonary M avium and pulmonary and
sinus Scedosporium apiospermum. No patients were on active
immunosuppression at time of onset of opportunistic infection.
Lymphocyte phenotyping was performed on all patients with the
following exceptions: memory markers were not collected on
patient 1, and phenotyping was not performed on patients 7, 9, and
12 (Figure 1). Interestingly, none of the patients with CD4 counts
below 200 (patients 4, 6, 10, 15, and 16) had opportunistic
infections. The most notable abnormality was that all 13 patients
tested had low absolute numbers of memory B cells (CD20⫹/
CD27⫹), ranging from 0-7/␮L (normal range, 16-118/␮L); 4 of the
patients fell within the low-normal range for percentages, with
patients ranging from 0%-1.2% of total lymphocytes (normal
range, 0.7%-6.3%). In addition, 11 of the 13 patients had low CD4⫹
T cells as well, but CD4⫹ lymphopenia did not correlate with
opportunistic infections. Thus, no abnormalities in lymphocyte
phenotyping were noted that were unique to patients with opportunistic infection. However, the abnormalities seen in the memory B
and CD4⫹ T-cell compartment may point to mechanisms underlying autoantibody production.
LIPS profiling anti-IFN autoantibodies in thymic malignancy
LIPS was used to search for autoantibodies against 39 different
cytokines. We first screened for autoantibodies to 6 different IFNs
including IFN-␣, IFN-␤, IFN-ε, IFN-␥, IFN-␭, and IFN-␻. We
used a cutoff based on the mean plus 3 SD of the 30 controls
(Figure 2A). Ten patients had anti-IFN-␣ autoantibody titers with
values of approximately 1.5 million LU, approximately 1000⫻
higher than the GMT of anti–IFN-␣ antibodies in the controls
(2347 LUs [95% CI; 2080-2649]). We did not find any difference in
anti-IFN-␣ antibody titers between patients with and without
infection (P ⬍ .08, Mann Whitney U test; Figure 2B). The same
10/17 patients also had high titer antibodies to IFN-␻ (Figure 2C).
Of the other type I IFNs tested, 5/17 patients were seropositive for
IFN-␭ (Figure 2D), 4/17 patients were seropositive for IFN-␤
(Figure 2E), and 2/17 patients were seropositive for IFN-ε (Figure
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BLOOD, 2 DECEMBER 2010 䡠 VOLUME 116, NUMBER 23
ANTI-CYTOKINE AUTOANTIBODIES AND THYMIC NEOPLASIA
4851
Figure 1. Quantitative immunoglobulins (Igs) and lymphocyte phenotyping for all patients enrolled on study. (A) IgG, IgA, and IgM levels. Lymphocyte subsets in
(B) absolute and (C) percentage of total lymphocyte count; memory lymphocyte subsets in (D) absolute and (E) percentage of total lymphocyte count based on complete blood
count with differential performed on the same day.
2F). None of the anti-IFN autoantibodies were statistically different
between patients with and without opportunistic infections. Therefore, consistent with previous reports, high-titer anti–type I IFN
autoantibodies are common in patients with thymic neoplasia,14,15
and though they may play a role in the development of opportunistic infection, they are not sufficient. None of the patients had
detectable autoantibodies against IFN-␥.
Other anti-cytokine autoantibodies in thymic malignancy
Anti–IL-12p40 autoantibodies were found in 9/17 patients, correlated with opportunistic infection (P ⬍ .03; Figure 2G). Similarly,
7/17 patients showed anti–IL-12p35 autoantibodies that were
generally 20-100⫻ higher than the GMT of anti–IL-12 autoantibodies in the controls (Figure 2H). Although anti–IL-12p35 autoantibodies were found in patients both with and without opportunistic
infections, they were significantly higher in those with opportunistic infections (P ⬍ .005; Figure 2H). Three patients showed high
titer anti–IL-17A autoantibodies (Figure 2I), and 2 had anti-BAFF
autoantibodies (Figure 2J). Although high titer autoantibodies to
IL-1␣ were detected in 2 patients, 1 healthy volunteer also showed
high titer autoantibodies to this cytokine (Figure 2K). In addition,
1 patient had anti-TNF-␣ autoantibodies (Figure 2L), and several
of the patients showed single positive anti-cytokine autoantibodies
to IL-6, IL-18, APRIL, and EBI1 (not shown). No significant
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BURBELO et al
BLOOD, 2 DECEMBER 2010 䡠 VOLUME 116, NUMBER 23
Figure 2. Anti-cytokine autoantibodies in patients with thymic neoplasia. Shown are results from 30 healthy volunteers (HVs), 12 patients with thymic neoplasia without
opportunistic infection (Thym/OI⫺), and 5 patients thymic neoplasia with opportunistic infection (Thym/OI⫹). Each symbol represents a sample from one patient. The antibody
titers in LU for (A) anti–IFN-␥, (B) anti–IFN-␣, (C) anti–IFN-␻, (D) anti–IFN-␭, (E) anti–IFN-␤, and (F) anti–IFN-␧, (G) anti–IL-12p40 (H) anti–IL-12p35, (I) anti–IL-17,
(J) anti-BAFF, (K) anti–IL-1␣, and (L) anti–TNF-␣ antibody titers are plotted on the y-axis using a log10 scale. The geometric mean antibody titer for each group is shown by the
short line. The dashed and solid lines represent the cutoff levels for determining seropositivity and are derived from the mean ⫹ 3 SDs and mean ⫹ 5 SDs of the antibody titer
of the 30 controls, respectively. P values for the different groups were calculated using the Mann-Whitney U test.
autoantibodies were detected against IL-1␤, IL-1 receptor antagonist, IL-2, IL-3, IL-4, IL-7, IL-8, IL-10, IL-15, IL-18, IL-21,
IL-23p19, IL-24, IL-27, IL-32, or EBI3 (not shown). In addition,
no significant autoantibodies were detected against erythropoietin,
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BLOOD, 2 DECEMBER 2010 䡠 VOLUME 116, NUMBER 23
ANTI-CYTOKINE AUTOANTIBODIES AND THYMIC NEOPLASIA
4853
Figure 3. Heatmap analysis of anti-cytokine autoantibody titers for plasma from 17 patients with thymic neoplasia and 30 healthy volunteers. Shown are the 16
cytokines that had any positive result of the 38 cytokines tested. Titer values greater than the mean plus 3 SDs of the 30 healthy volunteers were color-coded from green to red
to signify the relative number of SDs above these reference values. The patient codes colored blue indicate the thymoma patients with OI: patients 1 and 5 had CMC, patient 2
had CMC pulmonary M avium and sinopulmonary S apiospermum, patients 1 and 17 had dVZV, and patient 9 had disseminated cryptococcosis.
GM-CSF, CSF-1, stem cell factor, and transforming growth
factor-␤ (not shown).
Anti-cytokine autoantibody profiles in thymic malingancy and
clinical phenotypes
The heatmap shows that patients with thymic malignancy may
have a wide range of autoantibodies to different cytokines (Figure
3). Twelve of the 17 patients had at least one significant autoantibody. The most frequent autoantibodies were those against IFN-␣
and IFN-␻, which were both present in the same 10 patients.
Anti–IL-12p40 autoantibodies occurred in 9/17 patients, followed
by IL-12p35 autoantibodies in 7/17 patients. Of the 12 patients
with autoantibodies, only patient 16 had an autoantibody to a single
cytokine (IFN-␭). At the other end of the spectrum, patient 2 had
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BURBELO et al
autoantibodies to 11 different cytokines. As expected, only a few
single weak signals were detected in the healthy volunteer controls.
Anti–type I IFN autoantibodies are biologically active in vitro
The ability of patient plasma to affect IFN-␣ or IFN-␤ signaling
was evaluated by flow cytometry (supplemental Figures 1 and 2A,
available on the Blood Web site; see the Supplemental Materials
link at the top of the online article), immunoblot (supplemental
Figure 2B), and quantitative real-time PCR (supplemental Figure
2C). Normal PBMCs or lymphoblasts were incubated in 10%
normal or patient plasma unstimulated or stimulated with IFN-␣,
IFN-␤, or IFN-␥ for 15 minutes. Plasma from healthy volunteers
and all patients without anti–IFN-␣ or anti–IFN-␤ autoantibodies
demonstrated normal type I IFN signaling as measured by production of inducion of pSTAT-1 production and expression of type I
IFN-responsive genes. Conversely, all 10 patients with anti-IFN-␣
autoantibodies by LIPS (Figure 3) prevented IFN-␣–induced
pSTAT-1 and pSTAT-4 production (supplemental Figure 1) as well
as expression of IFN-␣–responsive genes (supplemental Figure
2C). Of the 4 patients with IFN-␤ autoantibodies (patients 2, 8, 9,
and 11), 3 patients prevented IFN-␤ signaling (patients 8, 9, and
11), while patient 2 did not (supplemental Figure 2C). All patients
with IFN-␤ autoantibodies had neutralizing IFN-␣ autoantibodies,
although patients with only IFN-␣ but no IFN-␤ autoantibodies
inhibited IFN-␣ signaling while preserving IFN-␤ signaling.
Further specificity of these signaling pathways was demonstrated
using commercial anti-IFN-␣ and anti-IFN-␤ antibodies in the
same set of flow, immunoblot, and quantitative real-time PCR
experiments (data not shown).
Anti–IL-12 autoantibodies are biologically active in vitro
We evaluated the ability of patient plasma to affect IL-12 signaling,
using flow cytometry PBMC–derived lymphoblasts and the cytokine inhibition on normal PBMCs incubated with patient or normal
plasma (supplemental Figure 3). Normal PBMC-derived lymphoblasts were incubated with 10% control or patient plasma left
unstimulated or stimulated with IFN-␣ or IL-12 and examined for
STAT-4 phosphorylation. Although 10 patients had anti–IL-12p35
and/or anti–IL-12p40 autoantibodies, only 4 patients (patients 1, 5,
7, and 11), all copositive for p35 and p40 autoantibodies (Figure 3),
inhibited IL-12–dependent STAT-4 phosphorylation (supplemental
Figure 3A). Interestingly, plasma from 2 other patients with both
anti–IL-12p35 and anti–IL-12p40 autoantibodies did not inhibit
IL-12–induced STAT-4 phosphorylation, demonstrating that differences in titer or epitope specificity affect the biologic activity of
autoantibodies, including those targeting a common antigen.
To further assess the biologic activity of anti-IL-12 autoantibodies, normal PBMCs were incubated with control or patient plasma
and stimulated or not with PHA plus IL-12 or IFN-␥ plus LPS
(supplemental Figure 3B-C). Despite differences in the ability of
these autoantibodies to prevent IL-12–induced phosphorylation of
STAT-4, all patients with autoantibodies to either IL-12p35 or
IL-12p40 subunits inhibited detection of IL-12 produced by the
cultured PBMC as well as IL-12 exogenously added for stimulation
(supplemental Figure 3B). In addition, all plasma from patients
with any IL-12 autoantibodies prevented IL-12–mediated induction of IFN-␥ (supplemental Figure 3C). Interestingly, plasma from
patient 3 inhibited detection of IL-12p70 despite lack of detectable
autoantibodies to either IL-12p35 or IL-12p40 by LIPS (supplemental Figure 3C). Additionally patients 3 and 16, both without
anti-IL-12 autoantibodies by LIPS, prevented IL-12–induced IFN-␥
BLOOD, 2 DECEMBER 2010 䡠 VOLUME 116, NUMBER 23
production. These patients may have an autoantibody specific to an
IL-12p70 epitope, or target the IL-12 receptor. It is also notable that
although STAT-4 is the critical downstream signal transduction
molecule for IL-12,28-30 plasma from some patients with anti-IL-12
autoantibodies allowed phosphorylation of STAT-4 while still
preventing IL-12–induced cytokine production.
Anti–IL-1␣ antibodies are biologically active in vitro
Inbibition of IL-1␣ signaling by plasma containing anti–IL-1␣
autoantibodies in patients 1 and 7 (LIPS; Figure 3) was evaluated
by bio-plex cytokine determination and measurement IL-1␣–
induced gene expression. Because IL-1␣ supports proliferation of
T cells,31 the ability of plasma containing IL-1␣ autoantibodies to
prevent PHA-induced IFN-␥ production was measured. PBMCs
were incubated in RPMI containing 10% normal plasma, normal
plasma with commercial IL-1␣ antibody, or plasma from patients 1
and 7. The commercial anti–IL-1␣ antibody, as well as plasma
from patient 1 prevented IL-1␣–dependent PHA-induced IFN-␥
production, while normal plasma and plasma from patient 7 had no
effect (supplemental Figure 4A). In addition, in HFF-1 cells
stimulated with IL-1␣, expression of IL-1␣–induced genes, CXCL11
and ICAM-1,32 were reduced by both plasma from patient 1 and
commercial anti–IL-1␣ antibody, while signaling remained intact
when cells were stimulated in the presence of plasma from healthy
volunteers and patient 7 (supplemental Figure 4B).
Anti–IL-17 autoantibodies are biologically active in vitro
To determine biologic activity of anti-IL-17 autoantibodies HFF-1
cells, which express the IL-17 receptor26 were used. Cells were
incubated with 10% control or patient plasma and left unstimulated
or stimulated overnight with IL-17. Supernatants were tested for
the production of IL-6 (supplemental Figure 4C). The 2 patients
with the highest titers of anti–IL-17 autoantibodies, patients 1 and
2, inhibited IL-17–induced IL-6, while patient 13 with the lowest
titers did not inhibit (LIPS; Figure 3). Interestingly, both patients
1 and 2 with neutralizing anti–IL-17 autoantibodies had CMC,
while patient 13 with nonneutralizing IL-17 autoantibodies did not
have opportunistic infections (Table 1).
Anti–TNF-␣ autoantibodies are not biologically active in vitro
Based on LIPS (Figure 3), patient 8 showed anti–TNF-␣ autoantibodies. To assess their biologic function, normal PBMCs were
incubated with control or patient plasma with or without PHA plus
IL-12 or LPS plus IFN-␥, and cytokine production was monitored
48 hours later. Neither the patient nor the control plasma blocked
detection of TNF-␣ (data not shown).
Association of autoantibodies with occurrence of opportunistic
infection
Five of our 17 patients (29.4%) had histories of opportunistic
infections, but none had laboratory evidence of Good syndrome,
the combination of opportunistic infection and hypogammaglobulinemia with variable lymphopenia (Figure 1).33 All patients with
opportunistic infections had multiple autoantibodies (range, 3-10;
Table 1 and Figure 3), with biologic activity of these autoantibodies
demonstrated using in vitro functional assays (Table 2). On the
other hand, patients 7, 8, 11, and 13 had biologically active
autoantibodies without opportunistic infection, suggesting that the
patients with opportunistic infection may have additional factors
contributing to their immune deficiency, including autoantibodies
not assessed in our panel.
All nonneutralizing
All nonneutralizing
IFN-␣
IFN-␤
IL-12p35 and
All nonneutralizing
All nonneutralizing
All nonneutralizing
All nonneutralizing
IL-17
TNF-␣
1
3
2
and p40 (6)
(supplemental Figure 3B-C)
All nonneutralizing
anti–IL-12p35
All nonneutralizing
IL-1␣
anti–IL-12p40 (3);
IL-12–induced IFN-␥ production
Anti–IL-12p35 (1);
4
10
and detection of IL-12
Plasma from 2 patients prevented
All nonneutralizing
All nonneutralizing
Number of
patients with
autoantibody
0
2 (patients 1 and 2)
1 (patient 1)
10
3 (patients 8, 9, and 11)
10
Patients with
neutralizing
autoantibody
1 (patient 8)
1 (patient 13)
1 (patient 7)
0
1 (patient 2)
0
Patients with
nonneutralizing
autoantibodies
Comments on neutralization assays and results
cytokine determination (data not shown)
detection of TNF-␣ produced by PBMCs by luminex-based
Anti-TNF-␣ autoantibodies in patient plasma did not prevent
(supplemental Figure 4C)
(supplemental Figure 4A-B)
(supplemental Figure 3)
IL-12–induced pSTAT-4 production by flow cytometry
antibodies against both IL-12p35 and p40 prevented
and detection of IL-12. Plasma from 4 of 6 patients with
All 10 plasmas prevented IL-12–induced IFN-␥ production
(supplemental Figure 2)
IFN-␤–inducible gene expression by qRT-PCR
flow cytometry and immunoblot and production of
Plasma prevented IFN-␤–induced pSTAT-1 production by
(supplemental Figures 1 and 2C)
of IFN-␣–inducible gene expression by qRT-PCR
production by flow cytometry and immunoblot, and production
Plasma prevented IFN-␣–induced pSTAT-1 and pSTAT-4
86% similarity between these IFN-␣ species
IFN-␣1 used for LIPS but IFN-␣2b used for functional assays;
BLOOD, 2 DECEMBER 2010 䡠 VOLUME 116, NUMBER 23
IL-12p40
Control plasma
All nonneutralizing
Autoantibody
Patients without
detectable autoantibodies
Table 2. Evaluation of ability of anti-cytokine autoantibodies to prevent their respective cytokine signaling in vitro
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ANTI-CYTOKINE AUTOANTIBODIES AND THYMIC NEOPLASIA
4855
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
4856
BURBELO et al
All 5 patients with opportunistic infection had thymoma grade
B2 or B3, while none of the patients with thymic carcinoma, a
clinically more aggressive tumor, had opportunistic infections. The
range of opportunistic infections included mucosal (CMC) and
invasive disease (disseminated varicella, M avium complex, Scedosporium, and cryptococcosis; Table 1).
At the time of submission, patients 1, 9, 14, and 15 have
expired, patients 1, 14, and 15 from progressive disease. Patient 9
had widely metastatic disease with severe paraneoplastic complications including severe Sweet syndrome, transfusion-dependent
hemolytic anemia, disseminated cryptococcosis (blood, central
nervous system, lung, and liver), liver failure and dialysisdependent kidney failure.
Discussion
We describe an extensive screen for autoantibodies against cytokines in patients with thymic malignancy, which detected 16 distinct
anti-cytokine autoantibodies in 17 patients. Consistent with previous reports, high antibody titers to IFN-␣, IFN-␻ and IL-12 were
common.14,15 We also identified autoantibodies to IFN-ε, IL-1␣,
IL-6, IL-18, TNF-␣, APRIL, BAFF, and EBI1, which have not been
previously described in thymoma or thymic carcinoma. Eleven of
12 patients with histologically proven thymoma had autoantibodies
up to 2000 SDs above the mean for healthy persons, while only 1 of
4 patients with thymic carcinoma had autoantibodies (Table 1).
Interestingly, although high-titer anti–GM-CSF, anti-erythropoietin,
and anti–IFN-␥ autoantibodies have been described and are known
to cause pulmonary alveolar proteinosis, pure red-cell aplasia, and
disseminated nontuberculous mycobacteria, respectively, none of
these autoantibodies were detected (Figure 3).
Autoantibodies to IFN-␣, IFN-␤, IL-12 components, IL-1␣, and
IL-17 have varying degrees of biologic activity in vitro (supplemental Figures 1-3). While anti–IFN-␣ autoantibodies consistently
inhibited IFN-␣–stimulated STAT-1 and STAT-4 phosphorylation
and IFN-␣–induced gene transcription, other autoantibodies were
more variable. Three plasmas with anti–IFN-␤ autoantibodies
(patients 8, 9, and 11) inhibited IFN-␤–stimulated STAT-1 phosphorylation and RNA expression, while patient 2 did not (supplemental
Figure 2). Likewise, only some patients with IL-12 autoantibodies
inhibited IL-12–induced STAT-4 phosphorlylation, while all of
them inhibited IL-12–induced cytokine elaboration (supplemental
Figure 3). Furthermore, 2 patients without measurable IL-12
autoantibodies inhibited IL-12 induction of cytokines, implying
other autoantibodies or functions not directly measured. Therefore,
identification of autoantibodies is insufficient; functionality should
be evaluated. Complicating matters further, the presence of different gene family members for certain cytokines may provide
redundancy in signaling pathways or complicate diagnosis. For
example, IFN-␣1 used in LIPS analysis and IFN-␣2b used for the
functional assays show 86% similarity, and we cannot rule out
subtle differences in immunoreactivity between these 2 forms.
Pulmonary alveolar proteinosis,1,2,34 pure red cell aplasia,3,4 and
mycobacterial disease from anti–IFN-␥ autoantibodies5-8 demonstrate clear relationships between autoantibodies and disease. The
spectrum of infections we identified in thymic neoplasm suggests
that combinations of different autoantibodies may contribute to
unique patterns of susceptibility. Because patients with opportunistic infections had higher numbers of autoantibodies, it is plausible
that combinations of autoantibodies may have additive or synergistic effects. For example, patient 2 had autoantibodies to 11 of the
BLOOD, 2 DECEMBER 2010 䡠 VOLUME 116, NUMBER 23
39 cytokines along with the highest number of opportunistic
infections in the cohort (CMC, pulmonary nontuberculous mycobacteria, and sinopulmonary S apiospermum). The presence of of
anti-cytokine autoantibodies against nearly 30% of the cytokines
tested, suggests that she may have other clinically relevant
anti-cytokine (or anti-cytokine receptor) autoantibodies, as well.
Puel et al35 and Kisand et al36 demonstrated strong associations
between anti-IL-17 and anti-IL-22 autoantibodies and mucocutaneous candidiasis in patients with autoimmune polyendocrinopathy,
candidiasis ecodermal dystrophy (APECED) syndrome. In addition, Kisand et al identified high-titer IL-22 autoantibodies in
2 patients with CMC and thymoma.36 Similarly, we found that 2 of
the 3 patients with CMC had autoantibodies to both IL-17 and
IL-22 (patients 1 and 2; Table 1 and Figure 3), and the IL-17
autoantibodies prevented IL-17–induced IL-6 elaboration in vitro
(supplemental Figure 4C). The third patient with CMC had
anti-IL22 autoantibodies only. Importantly, in the one patient with
anti-IL-17 autoantibodies without CMC (patient 13), the IL-17
autoantibodies were not functional in vitro (supplemental Figure
4C). IL-17 is important in protection against CMC as shown by
STAT3 deficiency (hyper IgE or Job syndrome), dectin-1 deficiency, CARD9 deficiency and, to a lesser extent, IL-12 receptor
␤1 deficiency,37-41 which have dysfunction of the IL-17 pathway.
APECED syndrome, caused by recessive mutations in the
autoimmune regulator (AIRE) gene, exhibits a classic triad of
hypoparathyroidism, adrenal failure, and mucocutaneous candidiasis along with other autoimmune phenomena.42 AIRE is important
for ectopic expression of peripheral self-antigen and as such is
critical for central thymic T-cell education and deletion of autoreactive clones.43 APECED patients share with thymoma patients high
titer anti–IFN-␣, anti–IL-17, and anti–IL-22 autoantibodies, along
with increased occurrence of CMC. The absence of AIRE mRNA
and expression in tissue biopsies of thymic neoplasm implies that
AIRE is an important factor in the autoimmunity and, in particular,
anti-cytokine autoantibodies seen in thymoma.44,45
B-cell dysregulation in our cohort was evidenced by B-cell
lymphopenia, anti-cytokine autoantibodies, and occurance of other
autoantibody-mediated processes including myasthenia gravis,
type 1 diabetes mellitus, and Coombs-positive hemolytic anemia
(Table 1). The critical role for the thymus in T-cell education
implies that B-cell dysfunction in thymic neoplasm results from
T-cell dysfunction. Why the spectrum of autoantibodies remains
relatively limited and disease-specific remains unclear. For example, no patients with thymic neoplasm had anti–IFN-␥ or
–GM-CSF autoantibodies; conversely, patients with disseminated
nontuberculous mycobacterial infection have high-titer IFN-␥
autoantibodies but not against IFN-␣, and patients with anti–GMCSF autoantibodies and pulmonary alveolar proteinosis have no
autoantibodies against IFN-␣ or IFN-␥ (data not shown). Perhaps
thymic neoplasm and associated dysfunction results in high
cytokine levels and autoimmunity similar to the prototypical
autoimmune condition, systemic lupus erythematosus.46,47 Production of anti-cytokine autoantibodies may be a counter regulatory
effect. Wildbaum et al demonstrated development of anti–TNF-␣
antibodies in mouse models and humans with rheumatoid arthritis.9
The high frequency of biologically active anti-cytokine autoantibodies in thymic neoplasm suggests that other, yet unidentified,
anti-cytokine or anti-cytokine receptor autoantibodies exist. Adult
onset immunodeficiency may be an important cohort in which to
look for these, particularly because their diagnosis opens novel
possibilities for treatment by targeting the underlying immune
dysregulation beyond the resulting secondary infection. Multiple
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BLOOD, 2 DECEMBER 2010 䡠 VOLUME 116, NUMBER 23
ANTI-CYTOKINE AUTOANTIBODIES AND THYMIC NEOPLASIA
inhibitors of cytokine signaling in diverse or overlapping cytokine
pathways may generate phenotypes of immunodeficiency that are
unique to individual patients. Ultimately, the important consequences
of autoimmunization in thymoma should shed light on the fundamental mechanisms underlying both autoimmunity and infection.
Acknowledgments
Financial support of this work was supported in part by the
Division of Intramural Research, the National Institute of Allergy
and Infectious Diseases, the National Cancer Institute, and the
National Institute of Dental and Craniofacial Research, all at the
National Institutes of Health.
4857
Authorship
Contribution: P.D.B., S.K.B., E.P.S., R.Z., E.K., L.D., K.H.C., and
C.M.K. performed experiments; P.D.B., S.K.B., E.P.S., J.F.B., and
A.P.H. analyzed results and made the figures; G.G., S.K.B., A.R.,
and A.B. identified patients and provided clinical information; and
P.D.B., S.K.B., M.J.I., and S.M.H. designed the research and wrote
the paper.
Conflict-of-interest disclosure: P.D.B. has a patent application
submitted using the LIPS technology. The remaining authors
declare no competing financial interests.
Correspondence: Sarah K. Browne, CRC B3-4141, MSC 1684,
Bethesda, MD 20 892-1684; e-mail: [email protected].
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2010 116: 4848-4858
doi:10.1182/blood-2010-05-286161 originally published
online August 17, 2010
Anti-cytokine autoantibodies are associated with opportunistic infection
in patients with thymic neoplasia
Peter D. Burbelo, Sarah K. Browne, Elizabeth P. Sampaio, Giuseppe Giaccone, Rifat Zaman, Ervand
Kristosturyan, Arun Rajan, Li Ding, Kathryn H. Ching, Arlene Berman, Joao B. Oliveira, Amy P. Hsu,
Caitlin M. Klimavicz, Michael J. Iadarola and Steven M. Holland
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