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Lymphocytes in the skin of patients with progressive systemic sclerosis.

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Quantification, Subtyping, and Clinical Correlations
Mononuclear cells (MNC) present in the dermis
of the forearm and in the blood of patients with progressive systemic sclerosis (PSS) were quantified and analyzed for subsets using monoclonal antibodies. The
findings were correlated with cutaneous and systemic
features of the disease. Total T lymphocytes and their
subsets, B cells, and macrophages were enumerated in
the skin samples of 21 patients with PSS. The dermal
MNC infiltrates consisted mostly of activated T lymphocytes with a mean T helper/T suppressor (T4/T8)
ratio of 2.4 2 1.3 SD. Few B1-positive or TCpositive
cells (macrophages) were observed. There was no correlation between the skin or blood T4/T8 ratios and the
degree of skin thickening. On histologic examination, 58
of 115 (50%) untreated patients with PSS had prominent dermal MNC infiltration. Significant correlations
between the degree of MNC infiltration and both the
degree (P < 0.05) and progression (P < 0.05) of skin
thickening were observed. No correlations with other
systemic disease features of PSS were noted. These
From the Division of Rheumatology & Clinical Immunology, Department of Medicine and the Division of Clinical Immunopathology, Department of Pathology, University of Pittsburgh School
of Medicine, Pittsburgh, Pennsylvania.
Supported by grants from the US Public Health Service
(FR-00056), National Institutes of Health (AM24019 and AM21393).
the RGK Foundation, Austin, Texas, and the Arthritis Foundation,
Western Pennsylvania Chapter.
Alan D. Roumm, MD: Fellow in Rheumatology; Theresa L.
Whiteside, PhD: Associate Professor of Pathology; Thomas A.
Medsger, Jr., MD: Associate Professor of Medicine.
Dr. Rodnan is deceased.
Address reprint requests to T. L. Whiteside, Division of
Immunopathology, 404 Scaife Hall, University of Pittsburgh School
of Medicine, Pittsburgh, PA 15261.
Submitted for publication July 8, 1983; accepted in revised
form February 13, 1984.
Arthritis and Rheumatism, Vol. 27, No. 6 (June 1984)
results suggest that cutaneous T lymphocytes may play a
role in mediating dermal sclerosis in PSS.
Progressive systemic sclerosis (PSS) is a generalized connective tissue disorder of unknown etiology
with vascular abnormalities, cutaneous and visceral
fibrosis, and a variety of serum antinuclear antibodies.
Its most characteristic feature is cutaneous sclerosis
attributable to increased synthesis of collagen, glycosaminoglycans (GAG), and other connective tissue
substances by dermal fibroblasts (1). Cellular immune
mechanisms are believed to play a major role in the
pathogenesis of PSS (2). Mononuclear cell (MNC)
infiltrates have been identified in many biopsy samples
from sclerodermatous skin (3,4). Recent in vitro studies have shown that soluble products of MNCs (cytokines) can stimulate the production of collagen and
GAG by cultured fibroblasts derived from skin of
normal individuals (5,6). Thus, it appears that MNCs
accumulating in the skin of patients with PSS may play
a direct role in mediating dermal sclerosis. We were
therefore interested in characterizing cellular infiltrates in the skin of patients with PSS, using histopathology as well as immunohistologic techniques with
monoclonal antibodies. We enumerated and identified
subsets of MNCs in the skin and peripheral blood of
patients with PSS and correlated the results with
cutaneous, systemic, and serologic features of the
Patients and disease classification. For the quantification of skin MNC, only patients with PSS who were first
evaluated at the University of Pittsburgh from 1976-1980
and who had forearm skin punch biopsies were considered.
Patients were excluded if they were receiving corticosteroids, D-penicillamine, or cytotoxic drugs at the time of the
biopsy or if no followup evaluations were performed. The
final study group consisted of I15 patients.
For lymphocyte subtyping, 35 patients were selected
from among the PSS patients first evaluated between November 1981 and January 1983. Again, individuals receiving
corticosteroids, D-penicillamine. or other cytotoxic drugs
were excluded from participation in this study. Twenty-one
of these 35 patients had both peripheral blood lymphocyte
studies and sufficient lymphocytes in the dermal biopsy
( > S O ) to obtain subset counts. Most patients were evaluated
early in the course of their disease; 15 of 21 had disease
duration of <1.5 years from the onset of the first symptom
attributable to PSS.
Patients in both of the above groups were classified
according to whether they had PSS with diffuse scleroderma
or PSS with the CREST syndrome (calcinosis, Raynaud’s
phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) (7). PSS with diffuse scleroderma was defined as
widespread cutaneous thickening with rapidly advancing
visceral involvement, and PSS with the CREST syndrome
was distinguished by more restrictcd skin changes, often
confined to the fingers and face, and the passage of prolonged periods of time before the development of characteristic internal manifestations. All patients satisfied published
criteria for classification as definite systemic sclerosis (8).
Clinical evaluations. Each patient had a complete
history and physical examination. Skin evaluation included
the degree of cutaneous thickening, estimated by palpation,
in each of 26 anatomic areas. The sum of these involvements
(0 = no thickening; 4 = severe thickening) constituted the
total skin score (‘TSS) as previously described (9). TSS was
determined and recorded prior to, and independent of, the
skin biopsy results. We have found the TSS to be reproducible with good interobserver correlation (unpublished data).
Close correlation of forearm skin score (FSS) and corepunch skin biopsy weights has been documented previously
Chest radiographs, cineesophagrams with small bowel radiograph series, and pulmonary function tests were
routinely performed. Data from these evaluations were
entered and stored in the scleroderma databank of the
American Rheumatism Association Medical Information
Service facility for later retrieval and analysis. Assessment
of the progression of skin disease was made by calculating
the difference between the TSS at the time of the biopsy visit
and the average of the TSS values recorded during the
subsequent 6-18 months.
Biopsy and histologic examination. A 7-mm diameter
punch skin biopsy sample for weight and a 6-mm punch
biopsy sample for histologic and immunohistologic study
were obtained from the dorsal aspect of the mid-forearm of
each patient. In order to assess the consistency of the
findings, an additional biopsy specimen was obtained 6 cm
distal to the initial biopsy area in 7 patients who had
lymphocyte subtyping performed. The mid-forearm was
chosen as the biopsy site because it was most often the zone
of transition between clinically normal and abnormal skin.
Sections of skin stained with hematoxytin and eosin
were reviewed for all patients and graded for degree of MNC
infiltration without knowledge of the clinical findings. Figure
1 illustrates the grading system used: grade I = few scattered
MNC; grade I1 = maximum number of MNC per collection
<lo; grade 111 = maximum number of MNC per collection
10-50; and grade IV = >50 MNC per collection. The highest
grade in 20 biopsies of normal skin was II, thus PSS patient
specimens with MNC grades >I1 were considered abnormal.
Immunopathologic studies. Monoclonal antibodies
were purchased from Ortho Pharmaceutical Corp., Raritan,
NJ (OKT3, OKT4, OKT8, OKT6, OKT9, M1) and from
Coulter Electronics, Inc., Hialeah, FL (TI 1, T4, T8, BI, 12).
Biotinylated anti-mouse IgG and avidin-biotin horseradish
peroxidase complex were obtained from Vector Laboratories, Inc., Burlingame, CA. The chromogen 3-amino-9ethylcarbazole (AEC) and Mayer’s hematoxylin were purchased from Sigma, St. Louis, MO. Aqua-mount, used for
mounting the stained sections, was obtained from Lerner
Laboratories, Greenwich, CT.
The monoclonal antibodies to cell surface antigens
have been shown in previous studies to react with the
following subsets of mononuclear cells: OKT3 and TI 1 with
total T lymphocytes; OKT4 and T4 with T helperhnducer;
OKT8 and T8 with T supprcssorkytotoxic; OKT6 with
Langerhans’ epidermal macrophages; BI with B lymphocytes; MI with monocytes/macrophages; I2 with B cells,
macrophages. and activated T lymphocytes; OKT9 with the
transferrin receptor found on proliferating lymphocytes ( 1 117).
One-half of each 6-mm punch skin biopsy specimen
taken for immunohistologic examination was embedded immediately in Tissue Tek OCT medium and stored at -40°C.
Frozen serial 4-pm sections were cut in an Ames cryostat,
air dried for 12-24 hours at room temperature, and stored at
-20°C until staining was performed. Prior to staining, sections were fixed in acetone for 10 minutes, air dried, and
washed in phosphate buffered saline (PBS), pH 7.4.
The avidin-biotin-peroxidase complex (ABC) technique (IS), with minor modifications, was used for immunostaining. Working dilutions of the primary antibodies were
determined by titration using sections of human tonsils. The
tissues were overlaid with the appropriately diluted primary
antibody and incubated for 15 minutes in a humidified
chamber at room temperature. Sections were next washed in
3 changes of PBS and incubated with biotinylated antimouse IgG for 15 minutes. After another PBS wash, ABC
was added for 15 minutes. The sections were then incubated
in AEC with 0.01% hydrogen peroxide for 10 minutes to
develop the color, washed in PBS, counterstained with
Mayer’s hematoxylin for 10 minutes, and mounted in Aquamount for light microscopy.
For negative controls, tissues were processed as
described except that the primary antibody was omitted.
Normal tonsillar tissue served as a positive control. To
check for the presence of endogenous peroxidase activity,
tissues were incubated with the substrate alone.
Enumeration of cells in tissue sections. Mononuclear
cells were counted in the dermis in serially cut sections, each
stained with a different monoclonal antibody. Cells in 20
separate high-power fields (magnification x 250) of each
biopsy were enumerated. Two different observers performed
the counts on coded slides, and the results were averaged.
Figure 1. Hematoxylin and eosin staincd sections of forearm skin biopsy specimens of a patient with progressive systemic sclerosis
(magnification x 250). Grades were based on degree of lyrnphocytic infiltration per high-power field. A, Grade I: few scattered lymphocytes
(<5); B, Grade 11: collection of 5-9 lymphocytes; C. Grade 111: collection of 10-50 lymphocytes: D.Grade IV: collection of 5O+ lymphocytes.
Cytofluorographic analysis. T cells in the peripheral
blood were quantified by direct immunofluorescence using
standard procedures. Peripheral blood obtained at the time
of skin biopsy was incubated with iron filings for 45 minutes
to remove monocytes. Mononuclear cells were then isolated
by centrifugation on Ficoll-Hypaque gradients. The recovered cells were checked for viability using trypan blue
(>95%), diluted to an appropriate concentration with medi-
um, and stained with fluoresceinated monoclonal antibodies.
The analysis was performed using an FACS IV flow cytometer (Bccton-Dickinson, Sunnyvale, CA).
Statistics. Differences between proportions were examined using chi-square contingency table analysis, and
differences between mean values by Student’s t-test. Linear
regression analysis was used to derive correlation coefficients.
Table 1. Comparison of clinical features in 1 I5 patients with progressive systemic sclerosis
according to degree of skin mononuclear cell (MNC) infiltration
Grade Ill-IV MNC
infiltration (n = 58)
Clinical features*
D 8 u s e sclerodermdCREST
Disease duration (years)
Forearm skin score
Total skin score
Raynaud’s phenomenon
Esophageal hypomotility
Pulmonary fibrosis
Cardiac involvement
Renal involvement
DLco (% predicted)
WBC count ( x I03/mm3)
Lymphocyte count ( x I03/mm3)
ANA positive (any titer)
Grade 1-11 MNC
infiltration ( n = 57)
79. I
I .7
0. I
I .6
0. I
* CREST = calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia; DLco = carbon monoxide diffusion capacity; WBC = white blood cells; A N A = antinuclear
t P < 0.05.
t P < 0.01.
forearm and total skin thickening scores. There were
no significant differences for the other clinical or
laboratory variables noted.
Table 2 compares the forearm skin biopsy MNC
grade with several measures of dermal sclerosis using
linear regression analysis. There were significant positive correlations for all variables listed, with the
strongest association being between biopsy MNC
grade and biopsy weight ( r = 0.59; P < 0.01). Of note,
a higher MNC biopsy grade served to predict an
increase in total skin score over the subsequent 6-18
Subtyping of dermal mononuclear cells. Subtyping of the dermal MNC with monoclonal antibodies
to surface antigens indicated that almost all cells
counted were T lymphocytes (Table 3). B lymphocytes
were found only rarely in the biopsy specimens. Since
Grading of mononuclear cell infiltrates. Fiftyeight of 115 (50%) untreated patients with PSS had
abnormal (grade 111 or IV) MNC infiltration in the
dermis on examination of the forearm skin biopsy
sample. These infiltrates were usually found in a
perivascular location predominantly in the mid and
deeper portions of the dermis. Table 1 compares the
clinical features of patients who had grade 111-IV
infiltration with those who had grade 1-11 (normal)
MNC numbers. Patients with excessive cutaneous
MNC infiltration were significantly more often classified as having PSS with diffuse scleroderma than those
who had normal numbers of skin MNCs. Also, patients with grade 111-IV MNC infiltration more frequently had disease of short duration with greater
Table 2. Significant correlations between skin biopsy mononuclear cell grade (MG) and clinical
evidence of skin involvement by linear regression analysis (r = Pearson’s rho) of patients with
progressive systemic sclerosis (PSS)
MG versus skin biopsy weight
MG versus forearm skin score
at time of biopsy
MG versus total skin score at
time of biopsy
MG versus change in total skin
score in subsequent 6-18
r value
P < 0.01
P < 0.01
P < 0.05
P < 0.05
Table 3. Numbers of mononuclear cell populations stained with monoclonal antibodies in
consecutive sections of skin biopsies from patients with progressive systemic sclerosis. Data
presented as mean counts 2 SD
No. of cells
T6 +
162 5 93
+ T8+
174 -t 88
monoclonal antibody BI does not react with plasma
cells, we cannot exclude the presence of plasma cells
in the dermal infiltrates. Although Langerhans’ macrophages (OKT6-positive cells) were found in the epidermis of many biopsy samples of PSS patients, they
were rarely found in the dermis or among infiltrating
MNC. In fact, there was no relationship between the
OKT6-positive cells and the lymphocytic infiltrates in
the dermis. Further analysis of the biopsy specimens
from 3 patients showed that 86 -t 8% (mean SD) of
the T lymphocytes were la-positive, and 70
2 2 3
were OKTPpositive, suggesting that the majority of
the T cells in the dermis were activated.
The mean T4/T8 ratio in dermal infiltrates was
2.4 2 1.3. This figure was based on the average counts
of 2 observers. The correlation between counts obtained by the 2 observers was r = 0.86 (P < 0.01). An
example 3f one patient’s skin biopsy stained for T4and TS-positive cells by the ABC method is shown in
Figure 2.
The T4/T8 ratios in simultaneously obtained
peripheral blood and skin for the 21 PSS patients,
Figure 2. Sequentially studied biopsy sections of scleroderma skin stained by the avidin-biotin-peroxidase complex technique (magnification x
250). A, OKT4 infiltrations; 8 , OKT8 infiltrations. Inserts show the infiltrating cells at higher magnification ( ~ 5 0 0 ) .
the mean control value. This elevated ratio was due to
a decrease in the absolute number of T8-positive
(suppressor) lymphocytes, as shown in Table 4. There
was no increase in T8-positive cells in the skin of
patients who had depressed T8 counts in the peripheral blood.
Correlations between the T4R8 ratio in the skin
of the forearm and the degree of cutaneous sclerosis,
as measured by FSS and TSS, were both weak, as
shown in Table 5. In addition, there was poor correlation between the T4R8 ratio in the skin and peripheral
blood in individual patients (r = 0.24; P > 0.1).
Analysis of multiple clinical and laboratory variables
similar to those shown in Table 1 failed to, reveal
significant differences between groups of patients divided on the basis of either peripheral blood or skin
T4/T8 ratios, except for serum antinuclear antibodies.
All 7 patients with elevated peripheral blood T4R8
ratios had serum antinuclear antibodies on rat liver
substrate comDared with 6 of 14 Datients with normal
peripheral blood T4R8 ratios (P 2 0.05). Correlations
were made between 2 sections of the same biopsy
specimen and between 2 biopsy specimens on the
same forearm, 6 cm apart, for both lymphocyte grade
and T4R8 ratio (Table 6). These findings suggest that
the experimental results are reproducible but that the
histologic findings may change over a short distance
between biopsy sites.
Figure 3. T4/T8 ratios in the skin and peripheral blood of patients
with progressive systemic sclerosis (PSS), and the peripheral blood
of simultaneously tested age- and sex-matched controls.
along with the control values for the peripheral blood,
are shown in Figure 3. We were unable to calculate T4/
T8 ratios in normal skin biopsies due to a lack of
lymphocytic infiltrates in the dermis of normal controls. The mean T4/T8 ratio of 3.4 ? 1.7 in the
peripheral blood of PSS patients was higher than the
control value of 2.5 2 1.0, but the difference was not
statistically significant. Seven (one-third) PSS patients had a peripheral blood T4/T8 ratio >2 SD above
Cutaneous cellular infiltrates are a frequent
histopathologic finding in progressive systemic sclerosis (PSS). Fleischmajer and coworkers (4) reported
that 49% of PSS patients had either diffuse or perivascular cellular infiltrates in the dermis or subcutaneous
tissue. They found no association with disease dura-
Table 4. Comparison of T4- and T8-positivc cells in the skin and peripheral blood of patients with
prograssive systemic sclerosis (PSS)
Peripheral blood*
PSS: high T4R8 ratio in
blood (n = 7)
PSS: normal T4/T8 ratio
in blood (n = 14)
Normal controls (n = 21)
7.6 2 4.0
5.1 2 0.8
1.3 2 0.6
8.0 2 3.9
2.4 r 1.2
1.6 -t 0.9
0.8 f 0.4
8.8 c 3.8
3.9 f 1.8
2.9 2 1.6
* Mean number of stained cells/mm7 x lo2 ? SD.
t Mean number of stained cells/20 high-power fields x lo2 -C SD.
t The comparison of PSS patients who have high T4E8 ratios with PSS patients who have normal T4/
T8 ratios is significant, P < 0.02. The comparison of patients who have high T4E8 ratios with normal
controls is significant, P < 0.01.
65 1
PSS and then compared the MNC infiltration grade
with cutaneous, visceral, and serologic features of the
disease. As shown in Table 1, one-half of our 115
patients with PSS had prominent dermal MNC infiltration. In contrast, none of the 20 normal (control) skin
biopsy specimens contained impressive MNC accumulations. The degree of MNC infiltration correlated
significantly with both the seventy and progression of
cutaneous sclerosis, suggesting that MNC-fibroblast
interactions in the skin are important in PSS. Although
edema theoretically could contribute to skin thickness,
our previous studies showed that the water content of
sclerodermatous and normal skin was similar (10).
It may also be argued that such local accumulations of MNC represent a secondary response to tissue
damage. Since in our experience these infiltrates were
noted early in the course of disease and correlated
with progression of skin thickening, it is more likely
that the skin-localized MNCs play an integral role in
the pathogenesis of the dermal sclerosis. The lack of
adequate numbers of MNCs in the forearm biopsy
samples of 50% of our patients with PSS may, in part,
be explained by the inclusion of some patients with
either disease of long duration or without concurrent
forearm skin involvement at the time of the biopsy.
We found no correlation between the skin MNC
infiltration grade and other visceral or serologic features of PSS. This lack of association is not surprising
since products of MNCs usually exert their effects
locally. The correlation studies shown in Table 5
suggest that observations about skin MNC may apply
to a limited area of skin. In fact, there were variations
in both MNC grade and T4/T8 ratios only 6 cm away
from the primary biopsy site. Therefore, one would
not expect the number of skin MNCs in one location to
reflect pathologic processes in more distant tissues.
Table 5. Correlations between T4/T8 ratios in skin and other
findings by lincar regression analysis ( r = Pearson's rho), in patients
with progressive systemic sclerosis (PSS)
Skin T4R8 versus
Skin T4/1'8 versus
skin score
Skin T4/1'8 versus
ar cell grade
Skin T4/T8 versus
blood T4/T8
* NS
r value
total skin
not significant.
tion but made no attempt to correlate cellular infiltrates with disease severity or activity. In other studies, these infiltrates were found to be either
predominantly T lymphocytes (3), or composed of T
lymphocytes and monocytes/macrophages (19).
Several in vitro studies have shown that soluble
products of mononuclear cells (cytokines) can stimulate fibroblasts to synthesize increased amounts of
collagen and other connective tissue substances (5,6).
Furthermore, we have recently shown that normal
dermal fibroblasts outgrown from an explant in the
presence of cytokines obtained from in vitro activated
lymphocytes acquire scleroderma-like characteristics
which persist even after the cytokines are removed
(Worrall J. Whiteside T, Prince R, Buckingham R,
Rodnan GP: unpublished data). In view of this evidence, it appears that mononuclear cells accumulating
in the skin early in the disease may play a major role in
initiating and mediating the dermal sclerosis characteristic of PSS through their effects on dermal fibroblasts.
To obtain additional evidence for the validity of
this concept, we first quantified the MNC infiltrates in
the forearm skin biopsy specimens of patients with
Table 6. Comparison of results in 2 separate forearm biopsy sites in the same progressive systemic
sclerosis patient*
Mononuclear cell grade/
Same biopsy, sectiogls
100 pm apart
Two biopsies 6 crn apart
Randomly paired biopsies
T4R8 ratio
Same biopsy, sections
100 pm apart
Two biopsies 6 cm apart
Randomly paired biopsies
* The r value for randomly
No. of
vat ient s
P < 0.05
P < 0.05
+O. I4
+o. 12
paired biopsies is shown for comparison. NS
not significant
Several authors (20-23) have reported abnormalities of T lymphocyte subpopulations in the peripheral blood of patients with PSS. The most consistent
finding has been an elevated T helper/suppressor (T4/
T8) ratio due to a decrease in the number of suppressor
cells. Our results confirm these findings. Seven of 21
(33%) of our patients with PSS had an elevated T4/T8
ratio in the peripheral blood, due primarily to a decrease in T8-positive cells. As had been noted in
systemic lupus erythematosus (24), these patients
were significantly more active serologically, at least
with respect to the frequency of serum antinuclear
antibody, than the group of PSS patients with normal
T4/T8 ratios. We were unable to document a selective
accumulation of suppressor cells in the skin of PSS
patients, as might be expected had cutaneous compartmentalization been responsible for the T8-positive cell
deficiency in the peripheral blood.
Further immunohistologic analysis of the dermal MNCs showed them to be mostly T lymphocytes,
with a mean T4/T8 ratio of 2.4 ? 1.3 SD. Bl-positive
and OKT6-positive cells were rarely seen. Kurosaka
and Ziff (25) have reported variations in the T4K8 ratio
of lymphocytes in different areas of rheumatoid synovium, with T4-positive cells accumulating predominantly in lymphocyte-rich areas. We found no differences in the T4R8 ratios in the lymphocyte-rich or
lymphocyte-depleted areas of the skin biopsy specimens of PSS patients.
McCain and Banejee (26) have shown the
presence of increased numbers of activated (anti-DRpositive) T lymphocytes in rheumatoid synovial tissues. We also found that T lymphocytes accumulating
in the dermis of patients with PSS are activated. In
contrast to the results of Meijer and coworkers (27),
who studied the synovium from patients with rheumatoid arthritis, we found no association between PSS
dermal lymphocytes and Langerhans’ dermal macrophages (OKT6-positive cells). Thus, the role of cutaneous macrophages in the pathogenesis of PSS remains unknown. We found no correlation between T4/
T8 ratios in the skin or peripheral blood with cutaneous or other visceral features in patients with PSS.
Because all of the patients examined for T4/T8 ratios
were followed for only 18 months, information on how
T4/T8 ratios relate to the long-term course of PSS or
survival is not yet available.
Several authors have noted the similarity between chronic graft-versus-host disease and progressive systemic sclerosis (28,29). Lampert et a1 (30)
reported a predominance of suppressor (T8-positive) T
cells in the skin biopsy specimens of 4 patients who
had this complication of bone marrow transplantation.
Our studies showed a predominance of helper (T4)
lymphocytes in 20 of 21 patients with PSS. This finding
suggests that the cutaneous immunopathology in these
two disorders may be different.
We conclude that T lymphocyte infiltrates correlate with several characteristics of cutaneous involvement in patients with progressive systemic sclerosis. Our results support the concept that the dermal
lymphocyte plays an important role in the pathogenesis of the disease. Future therapeutic approaches
designed to decrease numbers of cutaneous lymphocytes and/or interfere with their functions might prove
useful in the management of cutaneous involvement in
progressive systemic sclerosis.
The authors wish to thank Dr. Edward Abell of the
Department of Dermatology for assistance in development
of the lymphocyte infiltrate grading scale, Robin A. Almendinger, MA for assistance in performing lymphocyte subset
staining, and Joan Neitznick for typing the manuscript.
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