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The large granular lymphocyte syndrome with rheumatoid arthritis. immunogenetic evidence for a broader definition of felty's syndrome

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Number 9, September 1994, pp 1326-1330
0 1994, American College of Rheumatology
Immunogenetic Evidence for a Broader Definition of Felty’s Syndrome
Objective. To assess whether the HLA-DR4 association found in rheumatoid arthritis (RA) is also seen in
the large granular lymphocyte (LGL) syndrome.
Methods. HLA-DR genotyping was performed
using restriction fragment length polymorphism and
polymerase chain reaction analysis.
Results. LGL syndrome patients with RA showed
the same HLA-DR4 association seen in RA/Felty’s
syndrome (FS), while LGL syndrome patients without
arthritis did not.
Conclusion. It is proposed that FS and the LGL
syndrome represent different variants of a broader
syndrome comprising RA, neutropenia, LGL expansions, HLA-DR4 positivity, and splenomegaly.
The association between rheumatoid arthritis
(RA) and the HLA antigens DR4 and/or DR1 is now
well described (1). One rare variant of RA, Felty’s
syndrome (FS), originally characterized by the triad of
RA, neutropenia, and splenomegaly , exhibits a particularly strong genetic association: -90% of patients are
HLA-DR4 positive (2). Based on studies using molecular techniques, it has been proposed that this association with RA is derived from a relatively conserved
amino acid sequence of the polymorphic third hyperSupported in part by the Arthritis and Rheumatism Council,
UK. Dr. Bowman’s work was supported by an MRC Training
S . J. Bowman, MRCP: UMDS, London, UK; M. Sivakumaran, MRCP: Leicester Royal Infirmary; N. Snowden, MRCP:
University of Manchester, Manchester, UK; M. Bhavnani, MRCPath: Royal Albert Edward Infirmary, Wigan, UK; M. A. Hall,
PhD: UMDS; G. S. Panayi, FRCP: UMDS; J. S. Lanchbury, PhD:
Address reprint requests to J. S. Lanchbury, PhD, Division
of Medicine, UMDS, Guy’s Hospital, London SEI 9RT, UK.
Submitted for publication November 1, 1993; accepted in
revised form March 3 1, 1994.
variable region of the HLA-DRP1 chain that forms
part of the antigen binding site (3,4).
Particular subtypes of HLA-DR4 have been
grouped according to those that do (Dw4 [DRB1*04011,
Dw14 [DRB1*0404/8], Dw15 [DRB1*0405]) and those
that do not (DwlO [DRB1*0402], Dw13 [DRB1*0403/7])
show a positive association with RA (3). The other,
rare, subtypes of HLA-DR4 have not been formally
ascribed in this way. HLA-DR1 (DRB1*0101/2, but
not the rarer DRB1*0103), HLA-DR10 (DRB1*1001),
and one rare variant of HLA-DR6 (DRB1*1402) are
also similar in the third hypervariable region, and are
positively associated with RA in some populations (3).
However, the observation that among patients with FS,
Dw4,Dw14 (DRB1*0401,DRB1*0404/8) compound
heterozygosity is particularly frequent, and the lack of
an independent role for DR1 (2), suggest that these
alleles are not functionally equivalent. Since antigen is
presented to T cells in association with HLA molecules, and T cells appear to be important in the
pathogenesis of RA, it has been proposed that HLADR4 is particularly predisposed to present a “rheumatoid antigen” to T cells at some stage in the pathogenesis of the disease (5).
Recently it has become clear that the degree of
splenomegaly in FS is extremely variable and that
patients with RA and neutropenia without splenomegaly share the same clinical and immunogenetic features as patients with classic FS (6). Thus, the key
definition of FS has subtly changed to RA with neutropenia, with splenomegaly no longer an obligatory
feature and with the HLA-DR4 association a consistent feature of potentially etiologic relevance.
Over the last 15 years, one other new feature of
this syndrome has became apparent, due to the identification of a form of chronic lymphoproliferative
disease of large granular lymphocytes (LGLs) as a
distinct clinical entity (7). LGLs comprise 5-15% of
peripheral blood mononuclear cells. They are a heterogeneous population, but the 2 main groupings are
natural killer cells expressing CD16 and CD56 cell
surface markers, and cytotoxic T cells expressing
CD3, CD8, and CD57. The T cell LGL syndrome is by
far the most common form and is often clonal in nature
(7). Up to one-third of patients with LGL syndrome
have RA (7), and a significant proportion have neutropenia and splenomegaly, thus resembling patients with
classic FS. Furthermore, 19-35% of patients with FS
have clonal expansions of LGLs (ref. 8 and Bowman
SJ et al: unpublished observations). This close relationship between FS and the LGL syndrome is suggestive of a common etiologic process. To investigate
this further, we sought to establish whether the LGL
syndrome shares the same HLA-DR4 association as is
found in W S .
Two LGL patient groups and 3 control groups were
studied. All patients were of English Caucasoid origin,
representing a relatively homogeneous ethnic group. All of
the LGL syndrome patients studied (n = 38) had an absolute
peripheral blood LGL count of >1.0 x 109/liter (range
1.8-13.8) for at least 6 months. This was defined morphologically (7) in all except 1 patient, and confirmed by immunophenotyping in all except 4 patients. Twenty-eight of the 38
had an absolute lymphocyte count of >4 x 10’iliter. The
majority of the patients had presented to hematologists in
northern England; additional patients were recruited via the
British Society for Haematology newsletter.
Group 1 consisted of 19 patients with LGL syndrome
and an associated arthropathy; 17 had RA and 2 had a
symmetric polyarthropathy that failed to meet the American
College of Rheumatology (formerly, the American Rheumatism Association) 1987 criteria for RA (9). Of this group, 8
were male and 1 1 were female, with a mean age of 68 years
at the time of the study (range 40-90). Fourteen of the 19
were rheumatoid factor positive.
Group 2 comprised 19 LGL syndrome patients who
had no arthropathy (although 3 had a history of giant cell
arteritis [GCA]). Of this group, 10 were male and 9 female,
with a mean age of 65 years (range 40-87).
Group 3 consisted of 23 FS patients, mainly from
the southeast of England, who had not previously been
HLA-DR genotyped. Patients with an abnormal peripheral
blood CD4:CD8 ratio, an increased CD16, CD56, or CD57
count, or clonal lymphocyte expansions were excluded from
this group. Group 3 included 10 male and 13 female patients,
with a mean age of 59 years (range 22-78 years).
The healthy control groups consisted of a panel of 64
unrelated normal subjects (group 4a) who had previously
been HLA-DR genotyped at UMDS, and 32 HLA-DR4
positive normal controls (group 4b) whose subtyping has
been described elsewhere (2). No significant difference in the
frequency of HLA-DR4 has been reported among normal
populations across the catchment area of this study.
HLA-DR genotyping from DNA samples was performed in most cases by analysis of restriction fragment
length polymorphisms (RFLP) using Tuq I enzyme, and
HLA-DR and HLA-DQa complementary DNA (cDNA)
probes as previously described (2). This was complemented
by HLA class I1 oligotyping for HLA-DR3, DR9, and DRlO
using a panel of standard probes (lo), with additional oligotyping where necessary. The latter panel was also used for
HLA-DR genotyping of samples for which only a small
amount of DNA was available. DR4 subtyping for
DRB 1*0401-O412 was carried out according to a previously
published method (2,ll). The presence of clonality was
assessed using standard RFLP techniques. This involved
digestion of DNA by Hind 111 and either Bum HI or Eco RI
enzymes, Southern blotting, and analysis using a radiolabeled cDNA for T cell receptor C p , and in some cases, Jy
and JS, probes (12). The significance of differences between
groups was analyzed by chi-square test with Yates’ correction, and further correction of P values for differences in
HLA-DR distribution by a factor of 10, reflecting the number of HLA-DR types analyzed.
Neutropenia was present in 12 of the 19 LGL
syndrome patients with arthropathy (group 1) (63%)
and 10 of the 19 LGL syndrome patients without
arthropathy (group 2) (53%). Clonal expansions were
found in 8 of 19 (42%) of those with arthropathy and 11
of 18 (61%) of those without (not tested in 1 patient).
(A negative test result, however, does not always
exclude the presence of a clonal population 1111.)
Neutropenia was significantly associated with clonal
disease, since 16 of the 19 patients with clonal disease
(84%) were neutropenic, compared with 5 of the 18
(28%) without (P = 0.002).
Immunophenotyping data were available on 34
of the 38 LGL syndrome patients. Groups 1 and 2
demonstrated similar phenotypes. In group 1, 17 of 18
patients (94%) were CD3+,CD8+,CD57+, and 11 of
18 (61%) were CD16+ (1 patient was CD3-). In group
2, 14 of 16 patients (88%) were CD3+, 15 of 16 (94%)
were CD8+, 12 of 13 (92%) were CD57+, and 8 of 11
(73%) were CD16+. Expression of CD56 was more
frequent in group 2 (5 of 9; 56%) than in group 1 (3 of
14; 21%), although no tests of significance could be
applied. These results are consistent with previous
data (7).
The HLA-DR status of the groups is shown in
Table 1. As seen, the LGL syndrome patients with
arthropathy had a statistically significant increase in
Table 1. HLA-DR genotyping results in the groups studied*
Group I
(LGL with
(n = 19)
Group 2
(LGL without
(n = 19)
Group 3
(Felty 's
(n = 23)
DR 1
Group 4a
(n = 64)
14 (22)
24 (38)
14 (22)
18 (28)
9 (14)
21 (33)
17 (27)
6 (9)
0 (0)
0 (0)
* Values are the number (%). LGL = large granular lymphocyte syndrome.
t P = 0.0002, P,,, = 0.002, versus group 4a.
t P = 0.005, P,,,, < 0.05, versus group 4a.
= 0.05,
P,,,, not significant, versus group 4a.
HLA-DR4 frequency (15 of 19; 79%) compared with
normal controls (18 of 64; 28%) (P = 0.0002, P,,, =
0.002). This frequency was similar to that in the FS
patients (20 of 23; 87%). In contrast, LGL patients
without arthropathy had a similar HLA-DR4 genotype
frequency (6 of 19; 32%) to that seen in the control
In group 1, 9 of 11 patients with neutropenia
(82%) and 6 of 8 without neutropenia (75%) were
HLA-DR4+, while in group 2, these respective figures
were 3 of 7 (43%) and 3 of 12 (25%). LGL patients with
arthropathy and FS patients showed a reduction in the
frequency of HLA-DR2 compared with normal controls as previously described in FS/RA (13), although
only in the former group did this remain significant
after correcting for the number of HLA alleles exam-
ined. HLA-DR6 was also reduced in frequency among
FS patients compared with normal controls, although
this was not seen in the LGLlarthropathy patients and
the finding in FS patients was not significant after
correction. HLA-DR genotype frequencies among
LGL patients without arthropathy were similar to
those in normal controls.
Although there was a tendency toward an increase in HLA-DRUlO in the LGL patients, this did
not reach statistical significance. Only 1 of the 3
patients with GCA was HLA-DR4 positive.
There was an increase in the number of HLADR4 homozygotes among the FS patients (60%) compared with the HLA-DR4 positive controls (16%) (P=
0.003, P,,,, = 0.03) (Table 2). This was not seen in the
LGL syndrome patients with arthropathy (20%), al-
Table 2. HLA-DR4 subtyping results in the groups studied*
(DRB1*0401-041 2)
HLA-DR4 homozygotes
Dw4,4 (*0401,0401)
Dw4,14 (*0401,0404/8)
Dw4,IO (*0401,0402)
Dw4,13.2 (*0401,0407)
HLA-DR4 heterozygotes
Dw4 (*0401)
Dw14 (*0404/8)
Dw13.1 (*0403)
Dw15 (*0405)
Group 1
(LGL with
(n = 15)
Group 2
(LGL without
(n = 6)
Group 3
(n = 20)
Group 4b
(HLA-DR4 +
(n = 32)
3 (20)
12 (80)
3 (50)
3 (50)
12 (60)t
8 (40)t
5 (16)
27 (84)
* Values are the number or the number (%). LGL = large granular lymphocyte syndrome. No patients
with DRB1*0406 or *0409-0412 were found in this study.
t P = 0.003, PcOrr
= 0.03, versus group 4b.
though the numbers were small. However, it is important to note that, as previously described (2), patients
with FS who were HLA-DR4 heterozygotes largely
had the Dw4 (DRB1*0401) subtype (and occasionally
the other susceptible subtypes Dw14 (DRB 1*0404/8)
or Dw 15 (DRB 1*0405), while HLA-DR4 homozygotes
showed an unexpected bias toward Dw4,Dw14
(DRB1*0401,DRB 1*0404/8) compound heterozygosity. The same feature was seen in the LGL patients,
both in group 1 and group 2.
The increased prevalence of RA among patients
with the LGL syndrome has been described in a
number of reports (7). Initially, some authors attempted to distinguish RA with neutropenia and the
LGL syndrome from FS, proposing for example that
LGL patients have a milder arthritis of shorter duration. In practice, there is considerable overlap, with
many LGL patients having longstanding severe destructive RA (14) while many patients with classic FS
show occult clonal expansions of LGLs (ref. 8 and
Bowman SJ et al: unpublished observations). The
presence of clonality was found in this study to be
closely associated with neutropenia, although not all
LGL patients with neutropenia had clonal disease.
One hypothesis is that clonal disease is present in all
patients, but in some cases is below the level of
detection with the technique used. Alternatively, a
similar underlying process may be present even in the
absence of clonality. The same concept may be applied to the majority of FS patients without overt
LGL expansions. The results of this study of the
immunogenetics of the LGL syndrome in a relatively
large series of patients with this rare condition provide
further evidence of a close linkage between FS and the
LGL syndrome in patients with RA. The LGL syndrome per se, in the absence of RA, was not associated with HLA-DR4 in this study and could be due to
quite separate factors in these patients.
LGLs are known to be increased in certain viral
infections, particularly cytomegalovirus. Their presence in RA could represent the consequence of
chronic viral infection, either as a causative agent of
RA itself, or as a consequence of abnormal immune
function or drug therapy in RA. This process could
then predispose RA patients to clonal transformation
of these cells. Alternative theories focus on the B
cell hyperactivity seen in both RA and the LGL syndrome (14): there is evidence that these cells have a
regulatory/suppressor role that in some systems is
HLA-DR restricted (15), and they can suppress B cell
proliferation in vitro. CD3+ LGLs can produce interferon-y(16), and thus may function to shift the balance
of immune activity toward cell-mediated immunity and
dampen the influence of the hyperactive humoral
immune system.
In view of the close overlap between FS and the
LGL syndrome, in terms of both clinical features and
the immunogenetic data reported here, we propose
that they represent variations of a single syndrome,
although not every feature is present in any individual
patient. The original definition of FS no longer fully
reflects these features and represents just one variant
of this broader syndrome comprising RA, LGL expansions, neutropenia, HLA-DR4 positivity, and splenomegaly. From a clinical perspective it is becoming
clear that, provided the bone marrow is active, treatment of both the arthritis and the neutropenia should
not differ substantively between the FS and the LGL
variants of this syndrome (7).
The authors thank all the clinicians and patients who
participated in this study.
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immunogenetics, large, felt, syndrome, evidence, broader, arthritis, definitive, granular, lymphocytes, rheumatoid
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