Research

JAMA Ophthalmology | Original Investigation

Association of T and B Cells Infiltrating Orbital Tissues

With Clinical Features of Graves Orbitopathy
Giovanna Rotondo Dottore, MD; Liborio Torregrossa, MD; Patrizio Caturegli, MD; Ilaria Ionni, MD;
Angela Sframeli, MD; Elena Sabini, MD; Francesca Menconi, MD; Paolo Piaggi, PhD, MD;
Stefano Sellari-Franceschini, MD; Marco Nardi, MD; Francesco Latrofa, MD; Paolo Vitti, MD;
Claudio Marcocci, MD; Fulvio Basolo, MD; Michele Marinò, MD

Invited Commentary
IMPORTANCE Graves orbitopathy (GO) responds to immunosuppressive treatments when
clinically active but poorly when inactive. In other autoimmune diseases, response has been
ascribed to a reduction in lymphocytes infiltrating the target organ. It is not known whether
active vs inactive GO differs in this regard, which would help in understanding the link
between GO immunologic features and clinical behavior.

OBJECTIVE To investigate the association between orbital lymphocytic infiltrate and GO

clinical features.

DESIGN, SETTING, AND PARTICIPANTS A cohort study aimed at assessing the extent and
immunohistochemical phenotype of orbital lymphocytes and associating it with the
ophthalmologic features of GO, especially its clinical activity score (CAS), was conducted at a
tertiary referral center. Twenty consecutive patients with GO who underwent orbital
decompression were included. The study was conducted from January 1 to May 31, 2017.

EXPOSURES Orbital tissue histology and immunohistochemistry testing as well as

ophthalmologic evaluation.

MAIN OUTCOMES AND MEASURES Association between CAS and orbital lymphocytes,
analyzed as total number of lymphocytes and main lymphoid subsets.

RESULTS The patient population included 8 men and 12 women, all of white race, with a mean
(SD) age of 46 (13) years. With an established cutoff value of 300 lymphoid cells per tissue
sample, lymphocytes above this value were found in orbital tissues of 9 of 20 patients (45%),
often organized into distinct foci. The lymphocytes comprised a mixture of T (CD3-positive)
and B (CD20-positive) cells, suggesting a mature, polyclonal autoimmune response. In a
simple linear regression model, the total number of lymphocytes, as well as the number of
CD3- and CD20-positive subsets, correlated with CAS (R = 0.63; 95% CI, 0.27-0.84; P = .003;
R = 0.59; 95% CI, 0.20-0.82; P = .006; and R = 0.65; 95% CI, 0.30-0.85; P = .002,
respectively). In a multiple linear regression model, lymphocytes maintained their effect on
CAS when adjusted for 2 additional variables that were correlated with CAS—smoking and GO
duration—highlighting even more the important role of orbital lymphocytes in affecting CAS
(total number: R = 0.58; 95% CI, 0.18-0.82; P = .01; CD3-positive: R = 0.58; 95% CI,
0.17-0.82; P = .01; and CD20-positive: R = 0.59; 95% CI, 0.19-0.83; P = .01).

CONCLUSIONS AND RELEVANCE This study shows a correlation between T and B lymphocytes
infiltrating orbital tissues and the activity of GO, possibly enhancing our understanding of the
association between GO immunologic features and clinical expression.
Author Affiliations: Author
affiliations are listed at the end of this
article.
Corresponding Author: Michele
Marinò, MD, Department of Clinical
and Experimental Medicine,
Endocrinology Unit I, University of
Pisa and University Hospital of Pisa,
JAMA Ophthalmol. doi:10.1001/jamaophthalmol.2018.0806 Via Paradisa 2, 56124, Pisa, Italy
Published online April 19, 2018. (michele.marino@med.unipi.it).

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 Research Original Investigation Association of T and B Cells Infiltrating Orbital Tissues With Clinical Features of Graves Orbitopathy
Introduction
Graves orbitopathy (GO) is a chronic inflammatory disease as-
sociated with Graves hyperthyroidism in 95% of patients, with
hypothyroid autoimmune thyroiditis in 3% to 4% and ob-
served cases in 1% to 2% of patients who have thyroid autoim-
munity but without overt thyroid dysfunction.1-3 Graves orbi-
topathy has an autoimmune pathogenesis, whereby the
thyrotropin (TSH) receptor is considered the major autoanti-
gen, thus establishing a direct link between the thyroid and
orbital tissues.4 Unlike Graves hyperthyroidism, which is a
classic example of humoral autoimmunity caused by TSH re-
ceptor–stimulating autoantibodies,5 GO is believed to reflect T-
cell–mediated autoimmunity, although B-cell– and antibody-
mediated autoimmunity, to some extent, may also participate
in the pathogenic process.4,6,7 The role of B lymphocytes in the
pathogenesis of GO is suggested by the finding that rituximab,
a monoclonal antibody that destroys B cells by binding to CD20
on their surface, may ameliorate GO activity.8 The mechanism
through which destruction of antibody-producing B cells ame-
liorates a T-cell–mediated disease is unknown but has been as-
cribed to the role that B cells have as professional antigen-
presenting cells.
Rituximab has been used in other autoimmune diseases,
such as type 1 diabetes, 9 in which patients are known to
be heterogeneous in terms of CD20-positive lymphocytes
infiltrating the pancreatic islets.10 We hypothesized that some-
thing similar occurs in the orbits of patients with GO, a hetero-
geneity that may shed light on the link between GO immuno-
logic features and clinical expression and possibly explain the
variable response to immunosuppressive drugs. We thus de-
signed this study to characterize in a relatively large cohort of
patients the presence and composition of lymphocytes infil-
trating orbital tissues and relate them to the clinical features
of GO.
Methods
Patient Characteristics and Study Design
We arbitrarily chose a sample size of 20 consecutive patients
with GO who underwent orbital decompressive surgery. In all
patients, we recorded smoking habits; analyzed thyroid func-
tion through measurement of free thyroxine, free triiodothy-
ronine (Vitros Immunodiagnostics), and TSH (Immulite 2000;
Siemens Healthcare); measured the presence of anti-TSH
receptor antibodies (TRAbs) (Brahms); and performed a de-
tailed ophthalmologic evaluation that included 6 factors: (1)
exophthalmometry, (2) clinical activity score (CAS) (range, 0-7;
GO active for CAS values ≥3),11 (3) diplopia according to the
Gorman score (from absent to constant),11 (4) corneal status,
(5) ocular fundus, and (6) visual acuity.
The study was conducted from January 1 to May 31, 2017.
Written and signed informed consent was obtained from all
patients. The study was approved by the local ethics commit-
tee (Comitato Etico Area Vasta Nord Ovest). The participants
did not receive financial compensation.
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Key Points
Question Does the phenotype of Graves orbitopathy, especially
its activity, reflect the extent and features of the lymphocytic
infiltrate of orbital tissues?
Findings In this cohort study conducted in 20 consecutive
patients with Graves orbitopathy, an association between the
clinical activity score and orbital lymphocytes was found, analyzed
as total number and main lymphoid subsets, both in a simple and
multiple regression model.
Meaning These findings suggest a correlation between T and B
lymphocytes infiltrating orbital tissue and Graves orbitopathy
activity, possibly enhancing understanding of the association
between Graves orbitopathy immunologic features and clinical
expression.
Tissue Assays
Orbital tissues collected at surgery were fixed in formalin and
embedded in paraffin for routine hematoxylin and eosin
histology and immunohistochemistry (Ventana Benchmark sys-
tem; Ventana Medical Systems) testing. Archival paraffin-
embedded thyroid tissue samples from patients with Graves
hyperthyroidism as well as archival orbital and abdominal fi-
broadipose tissue samples from patients without Graves hyper-
thyroidism and GO were used as controls. Sections 3- to 5-μm
thick were deparaffinized in xylene, dehydrated, and processed
using a diaminobenzidine detection system (Ventana), accord-
ing to the manufacturer’s instructions. CD3, a pan T-lymphocyte
marker, was detected using a rabbit monoclonal antibody (im-
munoglobulin [Ig]G) against the nonglycosylated ε chain of the
CD3 molecule expressed by human T lymphocytes (CONFIRM
anti-CD3, clone 2GV6; Ventana). CD20, a pan B-lymphocyte
marker, was detected using a mouse monoclonal antibody
(IgG2a κ) (CONFIRM anti-CD20, clone L26; Ventana). After
hematoxylin-eosin immunostaining, the entire orbital adipose
tissue was scanned at low power ( × 2.5 magnification) and then
analyzed at ×20 magnification to count the total number of in-
filtrating lymphocytes and T and B cells in 4 representative
fields. The final number of lymphoid cells per patient was ex-
pressed as the sum of the cells in all 4 fields. More than 300 lym-
phocytes per tissue sample were found in 9 of 20 patients (45%).
The same number of patients had more than 100 CD3-positive
cells, and 8 of 20 patients (40%) had more than 200 CD20-
positive cells. These cutoff values were obtained using the fi-
nite mixture models, assuming the sum of 2 gaussian curves as
a model. Thus, the finite mixture model is a convex combina-
tion of 2 or more probability density functions, and mixture
models are capable of approximating value distributions by
combining the properties of the individual probability density
functions.12 Once the parameters of the 2 curves (mean [SD])
were established, cutoff levels were calculated as the values on
which the probability of the 2 curves was equal. Thus, the re-
ported cutoff values were those that best discriminated the 2
normal distributions identified with the finite mixture model
analysis. The pathologists (L.T. and F.B.) who examined the tis-
sue sections and performed the cell counting were masked (ie,
they were not aware of the clinical features of the patients).
jamaophthalmology.com
 Association of T and B Cells Infiltrating Orbital Tissues With Clinical Features of Graves Orbitopathy Original Investigation Research

Statistical Analysis Table 1. Demographic and Clinical Features of the Patient Population
Data were summarized as mean (SD) or median and interquar-
tile range. Lymphocytes were expressed in a logarithmic scale Characteristic Value
because their distribution was not gaussian. Groups were com- Sex, No. (%)

pared by simple or multiple linear regression, χ2 test, t test, or Male 8 (40)

Mann-Whitney test, as appropriate. Level of significance was Female 12 (60)
set at P ≤ .05; testing was 2-tailed and unpaired. SPSS, ver- Age, mean (SD) [range], y 46 (13)
[16-67]
sion 25 (IBM Corp) was used for analysis.
Smoking habits, No. (%)
Smoker 5 (25)
Ex-smoker 2 (10)
Results Nonsmoker 13 (65)
Thyroid treatment, No. (%)
Clinical Features of Patients
Methimazole 3 (15)
The patient population included 8 men and 12 women, all of
white race, with a mean (SD) age of 46 (13) years. Most of the Radioiodine 10 (50)

20 patients were nonsmokers (Table 1). Graves orbitopathy was Thyroidectomy 6 (3)

associated with Graves hyperthyroidism in 19 patients and oc- None 1 (5)

curred alone (euthyroid GO) in the remaining patient. All of the FT3, mean (SD) [range], pg/dL 0.38 (0.04)
[0.32-0.47]
patients with Graves hyperthyroidism had received a form
TSH, median (IQR) [range], μU/mL 0.6 (0.1-1.9)
of treatment (radioiodine in 10, thyroidectomy in 6, and me- [0.003-4.2]
thimazole in 3) and were euthyroid at the time of orbital de- TRAbs, median (IQR) [range], U/L 3.9 (2.3-5.7)
[0.3-9.8]
compression. This surgery was performed for severe, disfig-
GO duration, median (IQR) [range], mo 34 (18-51)
uring proptosis in 17 patients or for optic neuropathy in 3 [2-85]
patients. Four patients with GO were untreated, whereas the Indication for OD, No. (%)
remaining patients had received glucocorticoids alone or with Optic neuropathy 3 (15)
orbital radiotherapy, the latter being performed in 5 patients. Severe proptosis 17 (85)
The median time elapsed since the last glucocorticoid admin- GO treatment, No. (%)
istration was 4 months. Three patients had been treated with
Intravenous GC and radiotherapy 4 (20)
glucocorticoids in the 3 months preceding orbital decompres-
Intravenous GC alone 11 (55)
sion. Most patients had moderately severe GO, and the eye dis-
Oral GC and radiotherapy 1 (5)
ease was variably active, as shown by CAS values reported in
None 4 (20)
Table 1.
Months since last GC administration, median (IQR) [range] 4 (3-18.5)
[0-36]
Infiltrating Lymphocytes and Their Correlation With CAS Exophthalmometry, mean (SD) [range], mm 24.6 (2.7)
Nine patients (45%) had a total number of orbital infiltrating [17-28]
lymphocytes above the 300 cutoff value. Two representative Clinical activity score, median (IQR) [range]a 4 (3-5)
[1-7]
cases, 1 with a scarce and 1 with a marked infiltrate, are shown Diplopia (Gorman score), No. (%) of patients
in Figure 1. Lymphocytes comprised a mixture of both CD3-
Absent 8 (40)
positive T cells and CD20-positive B cells, without an overall
Intermittent 1 (5)
predominance of 1 subset over another (Figure 1). Lympho-
Inconstant 5 (20)
cytes that stained positive for either CD3 or CD20 were also de-
Constant 6 (30)
tected in thyroid tissue samples from patients with Graves hy-
Visual acuity (Snellen), mean (SD) [range] 18.6/20
perthyroidism, used as positive controls (not shown). In (2.4/20)
contrast, no lymphocytes and, consequently, no CD3 and CD20 [12/20-20/20]
staining were observed in archival orbital and abdominal Abbreviations: FT3, free triiodothyronine; GC, glucocorticoid; GO, Graves
fibroadipose tissue samples from patients without Graves orbitopathy; IQR, interquartile range; TRAbs, anti-thyrotropin (TSH) receptor
antibodies.
hyperthyroidism or GO, used as negative controls.
SI conversion factor: To convert FT3 to picomoles per liter, multiply by
In a simple linear regression model that assessed the over-
0.0154.
all effect of orbital lymphocytes on CAS, lymphocytes posi-
a
Range, 0 to 7; GO active for clinical activity score values of 3 or higher.
tively correlated with CAS (Table 2 and Figure 2A). For every
10-fold increase in the total number of orbital lymphocytes,
CAS increased by 1.52 points (R = 0.63; 95% CI, 0.27-0.84;
P = .003). The correlation was similar when lymphocytes were 0.30-0.85; P = .002). Lymphocytes did not correlate with
analyzed separately as T cells (Table 2, Figure 2B) or B cells age, sex, smoking habits, GO duration, thyroid treatment, pre-
(Table 2, Figure 2C). For every 10-fold increase in CD3- vious GO treatments, thyroid function, levels of TRAbs, ex-
positive cells, CAS increased by 1.68 points (R = 0.59; 95% ophthalmometry, eyelid aperture, diplopia, and visual acu-
CI, 0.20-0.82; P = .006). For every 10-fold increase in CD20- ity, overall suggesting that their number is mainly an indicator
positive cells, CAS increased by 1.2 points (R = 0.65; 95% CI, of GO activity. Of the various patient features under examina-

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 Research Original Investigation Association of T and B Cells Infiltrating Orbital Tissues With Clinical Features of Graves Orbitopathy

Figure 1. Immunohistochemical Staining for CD3 and CD20 in Orbital Tissues

A Patient with scarce infiltrate B Patient with marked infiltrate

CD3

CD20

CD3 and CD20 staining in orbital

tissues of 2 representative patients
with Graves orbitopathy with scarce
(A) and marked (B) infiltrate
(hematoxylin-eosin, original
magnification ×112).

Table 2. Correlation Between Clinical Activity Score of Patients With GO and the Indicated Featuresa

Linear Regression Multiple Regressionb

Variable R (95% CI) P Value R (95% CI) P Value
Total No. of lymphocytesc 0.63 (0.27 to 0.84) .003 0.58 (0.18 to 0.82) .01
CD3-positive cells 0.59 (0.20 to 0.82) .006 0.58 (0.17 to 0.82) .01
CD20-positive cells 0.65 (0.30 to 0.85) .002 0.59 (0.19 to 0.83) .01 Abbreviations: GO, Graves
Smoking habitsd −0.63 (−0.84 to −0.25) .003 NA NA orbitopathy; NA, not applicable.
a
vs Total No./GO duration NA NA −0.43 (−0.73 to 0.01) .08 Population included 20 white
patients (8 men, 12 women; mean
vs CD3/GO duration NA NA −0.43 (−0.73 to 0.01) .09 [SD] age, 46 [13 years]).
vs CD20/GO duration NA NA −0.44 (−0.74 to 0.003) .08 b
Multiple regressions were
GO duration −0.70 (−0.88 to −0.36) <.001 NA NA performed individually.
c
vs Total No./smoking NA NA 0.68 (−0.86 to −0.34) .002 For lymphocytes, log10 values were
used.
vs CD3/smoking NA NA −0.69 (−0.87 to −0.36) <.001
d
Smoking habits were converted into
vs CD20/smoking NA NA −0.68 (−0.86 to 0.34) <.001
continuous values.

tion, apart from orbital lymphocytes, smoking and GO dura-
tion correlated with CAS (Table 2).
As reported in Table 2, in a multiple linear regression model
that analyzed the effect on CAS not only of the orbital lym-
phocytes but also of smoking and GO duration, orbital lym-
phocytes maintained their action on CAS when adjusted for
the other covariates, highlighting even more the important role
of orbital lymphocytes in affecting CAS.
Features of Patients According to the Orbital
Lymphocytic Infiltrate
In confirmation of the findings reported above, when pa-
tients were grouped based on the total number of orbital in-
filtrating lymphocytes, those with counts above the cutoff
value had a greater CAS than those below the cutoff value
(Figure 3D). In addition, patients with counts above the cut-
off value were more often smokers (4 smokers, 1 ex-smoker,
and 4 nonsmokers vs 1 smoker, 1 ex-smoker, and 9 nonsmok-
ers in patients with a number of lymphocytes below the
cutoff level; P = .007), which was in line with the correlation
between smoking and CAS. The remaining features (age, sex,
GO duration, thyroid treatment, previous GO treatments, thy-
roid function, levels of TRAbs, exophthalmometry, eyelid ap-
erture, diplopia, and visual acuity) did not differ significantly
between the 2 groups.
The absence of a correlation between the lymphocytic in-
filtrate in orbital tissues and TRAbs was surprising in view of
the knowledge that TRAbs are correlated with GO activity and
severity.13 Individual levels of TRAbs according to the pres-
ence of a relevant orbital lymphocytic infiltrate are shown in

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 Association of T and B Cells Infiltrating Orbital Tissues With Clinical Features of Graves Orbitopathy Original Investigation Research

Figure 2. Orbital Lymphocytes and Clinical Activity Score (CAS) in White Patients (8 Men, 12 Women; Mean [SD] Age, 46 [13] Years)
With Graves Orbitopathy

A Total lymphocytes B CD3-positive lymphocytes

8 8

6 6
CAS, Points

CAS, Points
4 4

2 2

0 0
0 1 2 3 4 0 1 2 3 4
No. of Lymphocytes, log10 No. of CD3-Positive Cells, log10

C CD20-positive lymphocytes D Lymphocytic infiltrate

8 8

6 6
CAS, Points

CAS, Points
4 4

2 2

0 0
0 1 2 3 4 ≤300 >300
No. of CD20-Positive Cells, log10 Orbital Lymphocytes, No. of Cells per Tissue Sample

A, Correlation between total lymphocytes and CAS (P = .003). B, Correlation interquartile range) according to the presence a relevant (>300 cells in 4 fields)
between CD3-positive lymphocytes and CAS (P = .006). C, Correlation lymphocytic infiltrate.
between CD20-positive lymphocytes and CAS (P = .002). D, CAS (median and

Figure 3. The findings probably reflect the relatively long GO
duration (median, 34 months) and especially the fact that most
patients had undergone an ablative thyroid treatment that, with
the exception of a known transient increase in TRAbs after
radioiodine,14 is generally followed in the long term by a re-
duction of these autoantibodies.15 This reduction may ex-
plain the relatively low levels and, consequently, the lack of
correlation with orbital lymphocytes. Overall, our findings do
not exclude with certainty a general correlation between or-
bital infiltrating lymphocytes and antibodies to the TSH re-
ceptor; some studies have shown that thyroid-stimulating
immunoglobulins may be more sensitive than TRAbs in cor-
relating with GO features.16
Discussion
In the present study, we show that the lymphocytic infiltrate
of orbital tissues in patients with GO correlates with GO activ-
ity, thereby shedding light on an association between GO
immunologic features and clinical expression and possibly
posing an immunopathologic basis for future studies aimed at
investigating the association between the orbital lympho-
cytic infiltrate and the response of GO to immunosuppres-
sive treatments.17,18 Results supporting our conclusions can be
summarized as follows.
We examined orbital tissue samples from 20 patients with
GO who underwent orbital decompression. At histology exami-
nation in 45% of the patients, several focal areas of distinct lym-
phocytic infiltration could be seen in orbital tissues. On immu-
nohistochemistry testing, lymphocytes stained positive for both
CD3 and CD20, indicating that both T and B cells were repre-
sented, with variable predominance. The total number of in-
filtrating lymphocytes as well as the number of CD3- and CD20-
positive cells correlated with CAS, which in turn correlated with
smoking and GO duration. Multivariate analyses confirmed the
correlation between CAS and the total number of infiltrating
lymphocytes as well as CD3-positive and CD20-positive infil-
trating cells, indicating that orbital infiltrating lymphocytes cor-
relate with GO activity independently. In confirmation of these
findings, CAS was greater in patients with a total number of or-
bital infiltrating lymphocytes above a cutoff level of 300 in 4

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 Research Original Investigation Association of T and B Cells Infiltrating Orbital Tissues With Clinical Features of Graves Orbitopathy

age and the lymphocytic infiltration in orbital tissues. On the

Figure 3. Individual Levels of Serum Anti–Thyrotropin Receptor
Antibodies (TRAbs) in White Patients (8 Men, 12 Women; Mean [SD]
same line, 5 patients had received orbital radiotherapy, but
Age, 46 [13] Years) again there was no correlation between orbital radiotherapy
and the findings reported above. Our results may suggest a pos-
10
sible role of infiltrating lymphocytes in the pathogenesis of
GO, although data are not sufficient to prove it, which is a limi-
8 tation of our study. In this regard, it should be taken into ac-
count that lymphocytes likely represent only 1 of the many
causative cell elements involved, in addition to orbital fibro-
TRAbs, U/L

6
blasts and fibrocytes.4 We observed a persistent lymphocytic
4 infiltration that correlates with CAS even in chronic, long-
standing cases of GO. The mechanism underlying this obser-
vation is unknown. It remains to be established whether lym-
2
phocytes are somehow “trapped” in orbital tissues or whether
they continue to marginate; further studies are needed. An-
0
≤300 >300 other limitation of our study is that CAS was not originally in-
Orbital Lymphocytes, No. of Cells per Tissue Sample tended as a long-term factor for GO evaluation (although it is
widely used in this manner), instead being intended as an in-
Levels of serum TRAbs according to presence of a relevant (>300 cells in 4
dicator for the early stages of the disease.11
fields) orbital lymphocytic infiltrate.
As mentioned above, our findings may pose the basis for
future studies aimed at investigating the association be-
microscopic fields, established using a finite mixture model,
tween response to immunosuppressive treatments, in par-
compared with patients who had levels of orbital infiltrating
ticular glucocorticoids, GO activity, and immunohistologic fea-
lymphocytes equal to or below the cutoff value. Patients with
tures of GO.8 In this regard, we considered the possibility that
a relevant orbital lymphocytic infiltrate were more often smok-
the variable presence of orbital infiltrating lymphocytes and,
ers, thereby indirectly confirming the correlation between smok-
in particular, CD20-positive cells explains the discrepant find-
ing and CAS observed here and in agreement with the notion
ings on the action of rituximab in patients with GO.8 Thus, ri-
that smoking is a GO risk factor.11
tuximab was shown to determine an improvement of GO in
Apart from studies in which the phenotype of orbital T cells
patients with an eye disease of recent onset but not in those
in culture was investigated,19,20 to our knowledge, 1 previous
with long-standing GO.8 However, we did not find a correla-
study on the same issue was conducted in a smaller number
tion between CD20-positive lymphocytes infiltrating orbital
of patients (n = 14).21 In that study, orbital T cells were de-
tissue and disease duration, suggesting that the variable re-
tected to a greater extent in patients with GO of recent onset
sponse to rituximab unlikely reflects a different expression of
compared with those with long-standing GO, which, in view
these cells in orbital tissues. However, Salvi et al23 showed that
of the knowledge that GO is more active in its early phases,22
CD20-positive lymphocytes infiltrating orbital tissues were de-
is indirectly in line with our findings. However, in the same
pleted after administration of rituximab in a small number (2)
study, the extent of B cell involvement was negligible, both in
of patients with active GO who responded to the treatment,
early and late GO.21 This apparent discrepancy with our find-
which suggests that an association between CD20-positive lym-
ings may reflect the fact that, in that previous investigation,
phocytes and response to rituximab may exist. In any case, our
patients were selected and not consecutive, the number (5) of
investigation may not be relevant to treatment with ritux-
subjects with early and presumably active GO was limited, and
imab considering that most of our patients had long-standing
2 of these 5 patients had undergone immunosuppressive treat-
GO, which may not be the proper population to be treated with
ment (glucocorticoids and, in 1 patient, also azathioprine) in
the anti-CD20 monoclonal antibody.
the month preceding tissue harvesting.21

Limitations
Most patients (16 [80%]) from whom tissues were taken had
been treated with glucocorticoids before orbital decompres-
sion, and it could be argued that this intervention may have
affected the results. However, the median time that elapsed
since the last glucocorticoid administration was longer than
3 months, suggesting that glucocorticoids unlikely affected the
lymphocytic infiltration of orbital tissues. There was no cor-
relation between previous glucocorticoid treatment or dos-
Conclusions
Our study provides evidence for a correlation between both T
and B cells in the activity of GO and establishes a basis for un-
derstanding the association between the immune system and
clinical features of GO, as well as for future studies aimed at
the comprehension of the response of GO to immunosuppres-
sive treatment based on its activity.

ARTICLE INFORMATION Published Online: April 19, 2018. Author Affiliations: Department of Clinical and
Accepted for Publication: February 15, 2018. doi:10.1001/jamaophthalmol.2018.0806 Experimental Medicine, Endocrinology Unit I,
University of Pisa and University Hospital of Pisa,

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 Association of T and B Cells Infiltrating Orbital Tissues With Clinical Features of Graves Orbitopathy
Pisa, Italy (Rotondo Dottore, Ionni, Sabini, Menconi,
Latrofa, Vitti, Marinò); Department of Surgical,
Medical and Molecular Pathology, Pathology Unit,
University of Pisa and University Hospital of Pisa,
Pisa, Italy (Torregrossa, Basolo); Department of
Pathology, Division of Immunology, Johns Hopkins
School of Medicine, Baltimore, Maryland
(Caturegli); Department of Surgical, Medical and
Molecular Pathology, Ophthalmopathy Unit I,
University of Pisa and University Hospital of Pisa,
Pisa, Italy (Sframeli, Nardi); Obesity and Diabetes
Clinical Research Section, Phoenix Epidemiology
and Clinical Research Branch, National Institute of
Diabetes and Digestive and Kidney Diseases,
Phoenix, Arizona (Piaggi); Department of Surgical,
Medical and Molecular Pathology, Endocrinology
Unit I, University of Pisa and University Hospital of
Pisa, Pisa, Italy (Sellari-Franceschini); Department
of Clinical and Experimental Medicine,
Endocrinology Unit II, University of Pisa and
University Hospital of Pisa, Pisa, Italy (Marcocci).
Author Contributions: Drs Rotondo Dottore and
Marinò had full access to all of the data in the study
and take responsibility for the integrity of the data
and the accuracy of the data analysis.
Study concept and design: Caturegli, Sframeli,
Sabini, Nardi, Latrofa, Vitti, Marcocci, Marinò.
Acquisition, analysis, or interpretation of data:
Rotondo Dottore, Torregrossa, Ionni, Sframeli,
Sabini, Menconi, Piaggi, Sellari-Franceschini,
Latrofa, Basolo, Marinò.
Drafting of the manuscript: Rotondo Dottore,
Torregrossa, Ionni, Sframeli, Sabini, Piaggi, Marinò.
Critical revision of the manuscript for important
intellectual content: Caturegli, Sabini, Menconi,
Sellari-Franceschini, Nardi, Latrofa, Vitti, Marcocci,
Basolo, Marinò.
Statistical analysis: Piaggi, Marinò.
Administrative, technical, or material support:
Torregrossa, Menconi, Sellari-Franceschini, Latrofa,
Basolo, Marinò.
Study supervision: Caturegli, Menconi, Nardi,
Latrofa, Vitti, Marcocci, Marinò.
Conflict of Interest Disclosures: All authors have
completed and submitted the ICMJE Form for
Disclosure of Potential Conflicts of Interest and
none were reported.
Funding/Support: The study was funded by the
University of Pisa Fondi di Ateneo (Dr Marinò).
Role of the Funder/Sponsor: The funding source
had no role in the design and conduct of the study;
collection, management, analysis, and
interpretation of the data; preparation, review, or
approval of the manuscript; and decision to submit
the manuscript for publication.
jamaophthalmology.com
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