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Increase in activated CD8+ T lymphocytes expressing perforin and granzyme B correlates with disease activity in patients with systemic lupus erythematosus.

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ARTHRITIS & RHEUMATISM
Vol. 52, No. 1, January 2005, pp 201–211
DOI 10.1002/art.20745
© 2005, American College of Rheumatology
Increase in Activated CD8⫹ T Lymphocytes Expressing
Perforin and Granzyme B Correlates With Disease Activity in
Patients With Systemic Lupus Erythematosus
Patrick Blanco,1 Vincent Pitard,1 Jean-François Viallard,2 Jean-Luc Taupin,1
Jean-Luc Pellegrin,2 and Jean-François Moreau1
Objective. Cytotoxic T lymphocyte–mediated killing using granzyme B has recently been proposed to be
a preferential and selective source of autoantigens in
systemic autoimmune diseases, including systemic lupus erythematosus (SLE), while other reports have
indicated that cytolytic activity in SLE patients was
decreased. The aim of this study was to examine the
phenotypic and functional status of the CD8ⴙ T cells in
SLE patients.
Methods. Phenotype analysis of CD8ⴙ T cells was
carried out using flow cytometry. The cytotoxic potential
of CD8ⴙ T cells and its consequences were examined in
redirected-killing experiments. SLE patients with quiescent disease (n ⴝ 41) were compared with SLE
patients with active disease (n ⴝ 20), normal individuals (n ⴝ 36), and control patients with vasculitis (n ⴝ
14). Cytotoxic CD8ⴙ T cell differentiation was examined by coculture with differentiated dendritic cells
(DCs) in the presence of SLE patient sera.
Results. Patients with disease flares were characterized by higher proportions of perforin- and/or granzyme B–positive lymphocytes with a differentiated effector phenotype (CCR7ⴚ and CD45RAⴙ). The frequency
of these cells in peripheral blood correlated with clinical
disease activity as assessed by the SLE Disease Activity
Index. These cells generated high amounts of soluble
nucleosomes as well as granzyme B–dependent unique
autoantigen fragments. Finally, the activation of DCs
with serum from a patient with active lupus induced
granzyme B expression in CD8ⴙ T lymphocytes.
Conclusion. DCs generated in the presence of
sera from SLE patients with active disease could promote the differentiation of CD8ⴙ effector T lymphocytes
that are fully functional and able to generate SLE
autoantigens. Our data disclose a new and pivotal role
of activated CD8ⴙ T lymphocytes in SLE pathogenesis.
Systemic lupus erythematosus (SLE) is a systemic
autoimmune disease with multiorgan involvement characterized by an immune response against nuclear components (1). SLE patients experience a waxing and
waning disease course and a wide array of clinical
manifestations reflecting the systemic nature of the
disease. The skin, kidneys, joints, and central nervous
system may become the target of SLE-induced inflammation at its onset or during the course of the disease.
Environmental triggers such as viruses (2) may act in the
context of susceptibility genes, including genes involved
in antigen/immune complex clearance, lymphoid signaling, and apoptosis among several others (3), explaining
why the pathogenesis of this disease remains largely
unknown.
The autoimmune response in SLE patients was
recently found to be driven by unabated activation of
myeloid dendritic cells (DCs) through interferon-␣
(IFN␣) produced by another subset of DCs (i.e., plasmacytoid DCs) (4). The professional antigen-presenting
cells capture, process, and present autoantigens to T
cells, thereby initiating the full autoimmune response. In
this respect, much of the attention is now focused on
1
Patrick Blanco, MD, Vincent Pitard, MSc, Jean-Luc Taupin,
PhD, Jean-François Moreau, MD, PhD: CNRS–UMR5164 and
IFR66, Université de Bordeaux 2, Bordeaux, France; 2Jean-François
Viallard, MD, PhD, Jean-Luc Pellegrin, MD, PhD: Hôpital du HautLévêque, CHU de Bordeaux, Bordeaux, France.
Address correspondence and reprint requests to Patrick
Blanco, MD, CHU de Bordeaux, Place Amélie Raba Léon, Bordeaux
33076, France. E-mail: patrick.blanco@u-bordeaux2.fr.
Submitted for publication June 15, 2004; accepted in revised
form September 21, 2004.
201
202
BLANCO ET AL
how these autoantigens that drive the autoimmune
response are produced and/or selected in order to
understand the pathophysiology of SLE (5–8).
Apoptotic and necrotic cells are strong candidates as a source of such autoantigens (9,10). An
overload on the capacity to clear dying cells may allow
for an unrestricted availability of autoantigens for DCs
(11). In normal mice, the intravenous administration of
high numbers of apoptotic thymocytes was shown to
determine, by itself, the generation of antinuclear and
antiphospholipid antibodies (12). Moreover, a recent
report from Casciola-Rosen et al proposed cytotoxic T
lymphocyte–mediated killing to be a preferential and
selective source of autoantigens (13). That group reported the finding from in vitro studies that an exclusive
property of autoantigens is their ability to be cleaved by
granzyme B, a serine protease released by cytotoxic T
lymphocytes (13). However, the question remains
whether cleavage by granzyme B could be operating in
vivo in SLE, since no studies showing this have yet been
reported. The results obtained in the present study
allowed us to better delineate a still-unknown role for
CD8⫹ cytotoxic T lymphocytes in SLE pathogenesis and
perpetuation.
PATIENTS AND METHODS
Patients and controls. Sixty-one consecutive SLE patients were included in the present study between September
2001 and June 2003. Patients met at least 4 of the American
College of Rheumatology 1982 revised criteria for SLE (14).
All clinically and biologically relevant information concerning
the patients is shown in Table 1.
Clinical disease activity was scored using the SLE
Disease Activity Index (SLEDAI) (15,16). Two groups of
patients were defined. The active-disease group included 20
patients with a flare of disease, defined either as a minimal
3-point increase in the SLEDAI score compared with the score
at the previous examination or as a score ⬎6 for patients at
diagnosis. The quiescent-disease group included 41 patients
with a SLEDAI score ⱕ6 and with no variations throughout
the entire followup period. A third group comprised 14 control
patients with vasculitis.
For patients who presented with a disease flare (n ⫽
20), the concomitant or the closest biologic variables measured
within 2 weeks of diagnosis were considered for statistical
analyses. For patients with quiescent disease throughout the
entire followup period (n ⫽ 41), the last biologic variables were
used for statistical analyses. Healthy individuals from our staff
(30 women and 6 men) were studied as a control group. All
blood samples were obtained after the patients and control
subjects had given their informed consent.
Flow cytometric analysis. In all cytometric analyses, a
total of at least 5,000 lymphocytes from SLE patients or control
subjects were analyzed using a 4-color flow cytometer
(FACSCalibur; Becton Dickinson, Mountain View, CA). Specific antibodies directed at surface markers included anti-CD3,
anti-CD8, anti-CD4, anti-CD45, anti-CD45RA, anti–HLA–
DR, and anti-CCR7 (all from Becton Dickinson). Antibodies
were incubated in whole blood before red blood cells were
lysed using fluorescence-activated cell sorting lysing solution
(Becton Dickinson). For intracellular staining, cells were first
labeled with anti-CD3, anti-CD8, and anti–HLA–DR for 30
minutes and were then resuspended in Permeafix (Becton
Dickinson) for 30 minutes at room temperature before labeling with anti–granzyme B (Tébu, Paris, France), antiperforin
(Becton Dickinson), or an isotype control antibody (Becton
Dickinson).
Purification of CD8ⴙ T lymphocytes. CD8⫹ T lymphocytes were purified from peripheral blood mononuclear
cells (PBMCs; obtained after Ficoll-Hypaque gradient centrifugation) by using a magnetic cell separation method (MACS;
Miltenyi Biotec, Bergisch Gladbach, Germany). In all experiments, purity was subsequently checked and always exceeded
97%.
Anti-CD3–redirected killing assay and nucleosome
quantification assay. The assay was carried out as previously
described (17). Briefly, Fc receptor–bearing K562 cells labeled
with 51Cr were seeded in the presence of either anti-CD3
(OrthoClone OKT3; Janssen-Cilag, Boulogne-Billancourt,
France) or an IgG2a isotype-matched control antibody (antiCD19; Becton Dickinson) at a final concentration of 10 ␮g/ml.
In a second step, purified CD8⫹ T lymphocytes from patients
with active or quiescent disease were added in each well at
various target:effector ratios. For nucleosome release, K562
cells were incubated with purified CD8⫹ T lymphocytes at a
target:effector ratio of 1:20, and supernatants were harvested
at 6, 12, and 24 hours. Soluble nucleosome quantification was
done using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Cell Death Detection ELISA
Plus; Roche Diagnostics, Mannheim, Germany).
Monocyte-derived DC generation and mixed lymphocyte reaction. Plastic-adherent monocytes were cultured in
6-well plates (106/well) for 4 days in RPMI 1640 supplemented
with 10% fetal calf serum and with 100 ng/ml of recombinant
human granulocyte–macrophage colony-stimulating factor
(GM-CSF) (Leucomax; Novartis Pharma, Rueil-Malmaison,
France) along with 20 ng/ml recombinant human interleukin-4
(IL-4) or supplemented with 25% fresh lupus patient sera. On
day 5, monocyte-derived DCs were cultured at 104/well with
105 allogeneic lymphocytes for 6 days. At specified time points,
proliferation was checked by standard 3H-thymidine incorporation, and T lymphocyte phenotypes were analyzed by flow
cytometry.
Cleavage of endogenous autoantigens after in vitro
incubation of Fas-negative K562 cells with purified CD8ⴙ T
lymphocytes. K562 cells were incubated for 4 hours
with purified CD8⫹ T lymphocytes as previously described
(13) at a target:effector cell ratio of 1:5 in the absence or
presence of a 50 ␮M final concentration of Z-IETDfluoromethylketone (Z-IETD-FMK; Calbiochem, Fontenay
Sous Bois, France). Cell extracts were separated by 10%
ACTIVATED CD8⫹ T LYMPHOCYTES IN SLE PATHOGENESIS
203
Table 1. Summary of the demographic, clinical, and biologic characteristics and treatments of SLE patients in the study*
Patient/age/sex
With quiescent disease
1/30/F
2/27/F
3/23/M
4/34/F
5/21/F
6/28/F
7/22/F
8/45/F
9/52/F
10/33/M
11/45/F
12/37/F
13/40/F
14/32/F
15/25/F
16/50/F
17/40/F
18/24/F
19/20/F
20/60/F
21/17/F
22/23/F
23/18/F
24/56/F
25/36/F
26/16/F
27/43/F
28/30/F
29/25/F
30/25/F
31/43/F
32/38/F
33/64/F
34/70/F
35/16/F
36/17/F
37/41/F
38/22/F
39/32/F
40/25/F
41/38/F
With active disease
101/28/F
102/28/F
103/32/F
104/66/F
105/40/F
106/33/F
107/70/M
108/26/F
109/47/F
110/67/F
111/28/F
112/36/F
113/46/F
114/37/F
115/38/F
116/70/F
117/13/F
118/28/F
119/16/F
120/36/F
Lymphocytes/ml
(% CD8⫹,HLA–DR⫹)
Disease flare
manifestations†
SLEDAI score‡
Treatment‡
1,154 (1.40)
3,411 (10.62)
2,004 (13.52)
1,154 (1.40)
2,423 (2.96)
1,396 (2.74)
1,759 (3.23)
1,950 (3.3)
2,045 (10.33)
1,231 (4.72)
1,344 (5.72)
1,528 (3.66)
1,682 (8.96)
1,161 (9.95)
3,530 (14.01)
1,640 (8.57)
1,027 (4.74)
3,784 (0.96)
1,428 (4.08)
1,196 (1.88)
2,194 (3.58)
2,538 (3.97)
2,006 (2.64)
2,063 (5.55)
1,383 (3.50)
886 (7.94)
434 (3.1)
988 (5.27)
1,700 (3.34)
1,976 (2.47)
4,226 (9.9)
314 (4.64)
1,332 (11.18)
2,823 (12.00)
1,577 (7.63)
1,031 (6.19)
1,072 (7.87)
1,722 (8.22)
2,124 (10.2)
1,590 (1.82)
1,833 (5.6)
A, Sk
Bl, A
Sk, Bl, P, A
A, Bl
A, Sk
Sk, A
P, A, Sk
P, A, Bl
A, Sk
A, NP
Bl, A
A, Sk
A, Sk, Bl
Sk, A
K, Sk, A
A, P
Bl, APS, Sk
A, P
K, A
K, A
A, Sk
A, K, P
A, Sk
P, A
Bl, A, NP
Sk, A, APS
A, Sk, Bl
P, Bl
APS, A, P
A, P, Bl
APS, NP
Sk, A, Bl
Sk, Bl, APS
Sk, A, Bl
SK, A, Bl
Sk, A
Bl, A, P
A, Sk
A, P, Bl, Sk
A, Sk
Bl, NP, A
1
2
4
4
3
3
0
2
2
2
2
3
2
5
0
4
3
4
0
2
2
5
2
2
5
2
6
2
3
2
5
2
5
3
4
3
5
3
5
2
5
Pred., HCQ
Pred.
Pred., AZA
None
Pred., HCQ
Pred.
None
Pred.
None
Pred.
Pred., HCQ
Pred., AZA
Pred., HCQ
Pred., HCQ
None
None
Pred., AZA
Pred., HCQ
Pred.
None
None
None
None
None
Pred.
Pred.
Pred., MMF
HCQ
Pred.
None
HCQ
Pred.
Pred.
Pred.
None
HCQ, Pred.
Pred.
None
HCQ, Pred.
None
Pred., HCQ
1,144 (34.76)
668 (26.34)
946 (21.53)
216 (19.27)
3,073 (29.23)
2,053 (28.96)
1,292 (26.33)
1,254 (28.29)
1,291 (16.70)
1,005 (39.45)
1,507 (22.06)
382 (26.09)
655 (26.84)
1,599 (18.31)
2,693 (12.81)
1,460 (22.30)
511 (25.72)
932 (22.4)
1,224 (25.2)
542 (19.00)
K, A, Sk
A, P, K
Sk, A, P
Sk, A, P, Bl
Sk, A, Bl, P
A, Sk
A, K, Sk
A, K
P, A
P, A, Sk
A, Sk, P, Bl
A, Sk
A, Sk, K
Bl, NP, P
P, A, K
A, Sk, NP
A, Sk, K
K, P, Bl, NP
Bl, A, NP
A, Sk
12
7
9
8
9
7
15
8
8
13
8
12
12
8
12
8
12
10
8
7
Pred.,
None
Pred.,
Pred.
None
Pred.,
None
Pred.,
Pred.
None
Pred.
Pred.,
None
Pred.
None
None
None
None
HCQ
None
AZA
HCQ
HCQ
MMF
AZA
* SLE ⫽ systemic lupus erythematosus; ACR ⫽ American College of Rheumatology; SLEDAI ⫽ SLE Disease Activity Index; A ⫽ musculoskeletal
system; Sk ⫽ mucocutaneous lesions; Pred. ⫽ prednisone; HCQ ⫽ hydroxychloroquine; Bl ⫽ hematologic abnormality; P ⫽ pericarditis; AZA ⫽
azathioprine; NP ⫽ neuropsychiatric disorders; K ⫽ renal disease; APS ⫽ antiphospholipid syndrome; MMF ⫽ mycophenolate mofetil.
† Organs or organ systems affected or syndromes occurring during a disease flare in patients who had previously been diagnosed as having SLE
according to the ACR criteria.
‡ At time of blood sampling.
204
BLANCO ET AL
sodium dodecyl sulfate–polyacrylamide gel electrophoresis
under reducing conditions and then electroblotted onto
nitrocellulose membranes. To ensure that equal amounts of
proteins were loaded per lane, protein concentrations for
each condition were determined using the bicinchoninic
acid/copper sulfate method following the instructions of the
manufacturer (Sigma, Pont-de-Claix, France). Incubation
with monospecific SLE patient anti–U1–70 kd serum at a
final dilution of 1:2,000 was used to identify autoantigen
fragments. We observed that incubation of either K562 cells
or CD8⫹ T lymphocytes alone did not affect the autoantigens analyzed (data not shown).
Statistical analysis. All T cell subpopulation percentages and absolute cell counts between groups were compared
using the nonparametric Mann-Whitney U test, with a level of
significance of P ⫽ 0.05. We used the Spearman test to
determine the correlation between percentages of CD8⫹
perforin- or granzyme B–positive T cells and the SLEDAI
score. The tests were carried out using Statistica statistical
software (StatSoft, Tucson, AZ).
RESULTS
Activation of a high proportion of CD8ⴙ T
lymphocytes in SLE patients. Following our preliminary
observations, we confirmed that activated T cells in SLE
patients are confined to the CD8⫹ compartment and
increase during disease flare (18). The phenotypes of T
lymphocytes in 61 consecutive patients followed up from
December 2000 to June 2003 were evaluated. As mentioned in Patients and Methods, we defined a group of
patients with active disease (n ⫽ 20) and a group with
quiescent disease (n ⫽ 41). In addition, those 2 groups
were compared with a group of age- and sex-matched
healthy control volunteers (n ⫽ 36) who were free of any
autoimmune disease and/or infection, as well as with a
group of control patients with vasculitis (n ⫽ 14).
As we reported recently (18), and as shown in
Figure 1A, patients with disease flares were characterized by a statistically significant increase in the percentage of CD8⫹ T lymphocytes expressing HLA–DR (P ⬍
10⫺6) compared with the other groups, whereas the
percentages of total CD3⫹, CD3⫹,CD4⫹, or
CD3⫹,CD8⫹ T lymphocytes were not found to be
statistically different among all 4 groups. This activated
phenotype was virtually restricted to the sole CD3⫹,CD8⫹
compartment and mildly affected CD3⫹,CD4⫹ T cells
(P ⫽ 0.01), which therefore barely accounted for the
increase in CD3⫹,HLA–DR⫹ cells seen in the course of
disease acceleration. It also suggested that CD8⫹ cells
bearing an activated T cell phenotype might have acquired a cytotoxic phenotype. We therefore examined
their intracellular expression of perforin and gran-
zyme B by flow cytometry (Figure 1B). We noted an
⬃3-fold increase in the median percentages of perforinor granzyme B–positive CD8⫹ T cells in the activedisease group compared with the quiescent-disease
group (P ⬍ 10⫺6 for both comparisons, by MannWhitney U test). The intracellular expression of perforin
and granzyme B in the quiescent-disease group was
similar to that in the healthy controls (P ⫽ 0.3 and P ⫽
0.06, respectively).
Figure 1C shows the T cell phenotype of 4
representative individuals from 3 of the 4 groups. Not all
HLA–DR⫹ T cells expressed also perforin or granzyme
B, and while a majority of cells displayed this phenotype,
some CD8⫹ cells were found to be positive for only
HLA–DR or perforin or granzyme B, reflecting the
differential kinetics of the expression of these markers at
the cell surface and/or of their recirculation capacity at
the periphery. However, among the 3 groups, the most
stringent difference by far concerned the percentage of
double-positive CD8⫹ T cells (HLA–DR and perforin
or granzyme B positive), suggesting a true increase in
recently cytotoxic and activated effector T cells only in
patients with disease flares. Because it has recently been
found that subpopulations of effector cells in the resting
memory T cell compartment can be distinguished based
on their expression of CCR7/CD45RA (19), we examined the expression of these 2 cell markers at the surface
of the blood CD8⫹ T cells (Figure 1D). Compared
with the normal control or quiescent-disease groups, a
marked
decrease
in
the
percentages
of
CCR7⫹,CD45RA⫹,CD8⫹ (naive) T cells, associated
with a reciprocal increase in CCR7⫺,CD45RA⫹,CD8⫹
and CCR7⫺,CD45RA⫺, CD8⫹ effector T cells, was
noted in the SLE patients with disease flares.
Taken together, these data—the percentages as
well as the absolute cell counts (data not shown)—
demonstrated that SLE patients with disease flares are
specifically characterized by an altered differentiation of
their CD8⫹ T cells toward a cytotoxic T lymphocyte
phenotype. These data disclose a hitherto ignored but
potentially important role for the cytotoxic CD8⫹ T
cells in the pathogenesis of SLE.
Correlation between SLE activity and an increased proportion of perforin- or granzyme B–expressing
CD8ⴙ T lymphocytes in the blood. In order to formally
demonstrate the relationship between the increased
proportion of cytotoxic CD8⫹ T cells and the fluctuations of clinical manifestations associated with SLE,
we plotted the percentages of peripheral blood
ACTIVATED CD8⫹ T LYMPHOCYTES IN SLE PATHOGENESIS
205
Figure 1. Skewing of CD8⫹ T cells toward a cytotoxic effector phenotype in systemic lupus erythematosus (SLE) patients with disease flares. A,
Comparisons of percentages of CD3⫹,HLA–DR⫹, CD3⫹,CD4⫹,HLA–DR⫹, and CD3⫹,CD8⫹,HLA–DR⫹ lymphocytes among total lymphocytes from healthy controls, SLE patients with active disease, SLE patients with quiescent disease, and control patients with vasculitis. Squares,
triangles, and diamonds inside boxes represent the median value for each group. Boxes include the 25th–75th percentiles; bars outside boxes
represent the 10th and 90th percentiles; open circles represent values beyond the 10th and 90th percentiles. P values were determined by
Mann-Whitney U test. B, Comparisons of intracellular expression of perforin and granzyme B in peripheral blood CD8⫹ T cells from healthy
controls, SLE patients with active disease, and SLE patients with quiescent disease. Squares and triangles inside boxes represent the median value
for each group. Percentiles and outlying values are as described in A. P values were determined by Mann-Whitney U test. C, Flow cytometric analysis
showing the T cell phenotype of 4 representative individuals from 3 of the 4 groups. Active 1 and active 2 represent 2 patients from the group with
active disease. Percentages on the y-axis of positive cells among total peripheral blood lymphocytes, as defined by size, cell content, and CD45
expression, are indicated in the double-positive dot-plot quadrant. D, Flow cytometric analysis of the expression of CCR7 and CD45RA on the
surfaces of peripheral blood CD8⫹ T cells from healthy controls, SLE patients with active disease, and SLE patients with quiescent disease.
Percentages on the y-axis are expressed among CD8⫹ T cells. Squares, triangles, diamonds, and circles inside boxes represent the median value for
each group. Percentiles and outlying values are as described in A. P values were determined by Mann-Whitney U test. ISO ⫽ isotype control
antibody.
perforin-positive (Figure 2A) or granzyme B–positive (Figure 2B) CD8⫹ T cells against the SLEDAI scores for each
SLE patient (n ⫽ 61) at the time of flow cytometric
evaluation of that patient’s cells. The percentages of
perforin- or granzyme B–positive CD8⫹ T cells correlated
strongly with the SLEDAI scores. The Spearman statistical
test yielded high values for the correlation coefficients,
similar in both instances (R ⫽ 0.731 and R ⫽ 0.733), with
a very high degree of significance (P ⬍ 10⫺6). This
demonstrated that the increase in circulating perforin- or
granzyme B–positive CD8⫹ T cells thoroughly reflected
the activity of the disease.
206
BLANCO ET AL
following positive anti-CD8–coated bead selection)
from the blood of patients with active disease (n ⫽ 3)
displayed 3-fold higher cytotoxic activity than the purified CD8⫹ T lymphocyte fraction from the blood of
patients with quiescent disease (n ⫽ 3). All 3 patients
with disease flares had percentages of CD3⫹,CD8⫹,
HLA–DR⫹,granzyme B–positive cells ranging from
Figure 2. Correlation between percentages of cytotoxic effector T
cells circulating in the blood and Systemic Lupus Erythematosus
Disease Activity Index (SLEDAI) scores in SLE patients. A, Percentages of CD8⫹,perforin-positive T lymphocytes among total
CD3⫹,CD8⫹ lymphocytes plotted against SLEDAI scores. B, Percentages of CD8⫹,granzyme B–positive T lymphocytes among total
CD3⫹,CD8⫹ lymphocytes plotted against SLEDAI scores. In both
instances, correlation coefficients and P values were obtained using the
Spearman test (see Patients and Methods).
Peripheral blood CD8ⴙ T cells from SLE patients acting as functional cytotoxic effectors in vitro
generating soluble nucleosomes. In the absence of nominal antigens, we carried out a nonspecific, anti-CD3–
dependent, redirected killing to test whether circulating
CD8⫹ T lymphocytes from SLE patients were functional cytotoxic effectors. This was accomplished by
using Fas-negative K562 erythroleukemic human cells,
which abundantly express Fc␥ receptors at their cell
surfaces, as target cells. In this 4-hour chromium release
assay, target cells bound anti-CD3 through its Fc domain, leaving the F(ab⬘)2 domain free to activate the
effector T cells nonspecifically.
As shown in Figure 3A, the fresh and enriched
(⬎97% pure) CD8⫹ T lymphocyte fraction (sorted
Figure 3. Cytotoxicity of, and induction of high levels of soluble
nucleosomes by, CD8⫹ T cells from systemic lupus erythematosus
(SLE) patients with disease flares. A, Redirected killing of Fasnegative K562 cells by freshly purified CD8⫹ T lymphocytes from
SLE patients with quiescent (triangles) or active (squares) disease in
the absence (open triangles or squares) or presence (solid triangles or
squares) of 10 ␮g/ml anti-CD3. Values are the mean and SD specific
51
Cr release (target:effector ratios of 1:1, 1:5, 1:10, and 1:20) obtained
from 3 separate 51Cr release assays performed on cells from 3 SLE
patients with quiescent disease and 3 SLE patients with active disease.
B, Soluble nucleosome generation following anti-CD3–redirected killing of K562 cells by CD8⫹ T lymphocytes (target:effector ratio 1:20)
from SLE patients with quiescent (squares) or active (diamonds)
disease in the absence (open squares or diamonds) or presence (solid
squares or diamonds) of 10 ␮g/ml anti-CD3. Values are the mean
optical densities (ODs) obtained at 6-hour, 12-hour, and 24-hour
coincubation times from 1 experiment representative of 3.
ACTIVATED CD8⫹ T LYMPHOCYTES IN SLE PATHOGENESIS
42% to 60% of total lymphocytes, whereas the corresponding percentages among the 3 patients with quiescent disease were ⬍15%. Because CD8⫹ sorted lymphocytes may contain natural killer (NK) cells, and
because K562 cells are exquisitely sensitive to killing by
NK cells, control experiments were carried out in the
absence of anti-CD3 antibody. Under these conditions,
the killing remained at the baseline level, indicating that
all the cytotoxic effects on K562 cells revealed in the
presence of anti-CD3 were attributable to CD3⫹,CD8⫹
T lymphocytes, the percentage of which increased dramatically in patients with disease flares.
Soluble nucleosomes are found in high amounts
in sera from SLE patients (20,21) and are thought to
play a key role in the autoimmune reaction against
nuclear components. We therefore asked whether
CD8⫹ T lymphocytes from patients had the capacity to
generate nucleosomes when mixed with target cells in
vitro. Again, freshly purified CD8⫹ cells from SLE
patients were added to K562 cells in the presence or
absence of anti-CD3, and a fraction of the cell supernatant was harvested at 6, 12, and 24 hours of coincubation
time. The concentrations of soluble nucleosomes
present in the supernatants were then determined using
a commercially available semiquantitative ELISA. Data
from a representative experiment are shown in Figure
3B and demonstrate that supernatants from cultures
containing CD8⫹ T lymphocytes from patients with
active disease generated soluble nucleosomes earlier
and in higher amounts than CD8⫹ T lymphocytes from
patients with quiescent disease. Together, these data
confirmed the presence of a greater number of functional cytotoxic effector cells among CD8⫹ T cells from
SLE patients with disease flares.
Cleavage by cytotoxic effector T cells of autoantigens recognized by autoantibodies found in SLE patients. Casciola-Rosen et al recently demonstrated that
granzyme B cleavage in target cells during cytotoxic
lymphocyte granule–induced cell death led to the production of unique peptide fragments exclusively from
autoantigens in patients with systemic autoimmune diseases including SLE. To ascertain whether similar autoantigen fragments could be preferentially generated by
CD8⫹ T lymphocytes from SLE patients, we cocultured
K562 cells with purified CD8⫹ T lymphocytes from SLE
patients with active or quiescent disease and analyzed
the cleavage of autoantigens through immunoblotting
using patient sera. In order to avoid confusing the
interpretation of the data, we used a monospecific SLE
207
Figure 4. Granzyme B–specific autoantigens generated by freshly
purified CD8⫹ T lymphocytes from systemic lupus erythematosus
(SLE) patients with active disease. K562 cells were coincubated (lanes
2–4) or not (lane 1) with freshly purified CD8⫹ T lymphocytes
(target:effector ratio 1:5) from SLE patients with quiescent disease
(lane 4) or from SLE patients with active disease in the presence
(lane 3) or absence (lane 2) of granzyme B inhibitor (Z-IETDfluoromethylketone). Effectors were also incubated in the absence of
K562 cells (lanes 5 and 6) as controls. After a 4-hour incubation, cells
were lysed in loading sodium dodecyl sulfate–polyacrylamide gel
electrophoresis buffer before being electrophoresed. U1–70-kd autoantigen was detected by Western blotting using a monospecific SLE
patient serum. Large solid arrow indicates intact antigen; small solid
and open arrows indicate granzyme B–specific and caspase-specific
autoantigen fragments, respectively.
patient serum directed against U1–70-kd autoantigen to
detect its fragments potentially produced by cytotoxic T
cells.
As shown in Figure 4, purified CD8⫹ T lymphocytes were able to spontaneously generate granzyme
B–specific fragments of U1–70-kd autoantigen (small
solid arrow). In contrast, the production of the unique
granzyme B fragment was abolished by the addition of
50 ␮M Z-IETD-FMK, a known inhibitor of granzyme B,
to the reaction wells (lane 3), and the granzyme B
fragment was undetectable using CD8⫹ T cells purified
from the blood of patients with quiescent disease (lane
4). K562 cells alone (lane 1) or purified CD8⫹ T cells
alone (lanes 5 and 6), both in the presence of anti-CD3,
did not lead to any granzyme B–specific U1–70-kd band.
Taken together, these data demonstrated that purified
CD8⫹ T lymphocytes from SLE patients with active
disease have the capacity to generate unique granzyme
B fragments that may subsequently be the target of the
autoimmune response.
208
Unique ability of DCs generated in the presence
of SLE serum to induce the expression of granzyme B in
CD8ⴙ T lymphocytes. In order to demonstrate which
factors could account for such an activation of the
CD8⫹ T cell compartment, we first incubated allogeneic
PBMCs (obtained by Ficoll-Hypaque gradient centrifugation) with SLE serum from patients with active and
quiescent disease. We did not observe any difference in
terms of intracellular perforin and granzyme B expression after 10 days of culture under these conditions, as
tested by flow cytometry (data not shown), suggesting
that soluble factors present in SLE patient sera (therapeutic agents, cytokines, etc.) could not be the direct
causative agent for the altered differentiation of CD8⫹
T cells.
Since investigators in our group and others have
recently demonstrated that unabated induction of DCs
by IFN␣ may drive the autoimmune response in SLE
(4,8,22), we addressed the question of whether DCs
generated in the presence of sera from SLE patients
with active or quiescent disease (hereafter referred to as
active SLE DCs or quiescent SLE DCs, respectively) had
the capacity to specifically induce the expression of
intracellular perforin and granzyme B in allogeneic
naive CD8⫹ T cells. To this end, we cultured normal
monocytes as described elsewhere (4) with SLE sera
from patients with active or quiescent disease for 4 days.
Alternatively, DCs differentiated in the presence of
GM-CSF and IL-4 were used as a control. In this first
step, we confirmed that monocytes cultured in the
presence of sera from SLE patients with active disease
differentiated into DCs, whereas sera from patients with
quiescent disease or from normal individuals were ineffective. In the second step, allogeneic peripheral blood
lymphocytes were added to the original washed cultures
and monitored for their proliferation and cell marker
expression over time. As expected, DCs differentiated in
the presence of GM-CSF and IL-4 or active SLE DCs
induced a robust proliferation of lymphocytes, whereas
quiescent SLE DCs were unable to do so (ref. 4 and data
not shown).
The intracellular staining for granzyme B of
CD8⫹ T cells from cocultures was analyzed on days 0, 3,
and 6 (Figure 5A). Clearly, active SLE DCs had the
unique ability to induce the intracellular expression of
granzyme B in CD8⫹ T cells. Indeed, after 6 days of
coculture with active SLE DCs, ⬎40% of allogeneic
CD8⫹ T lymphocytes expressed intracellular granzyme
B, as compared with 20% with quiescent GM⫹IL-4 DCs
(Figure 5A). Moreover, this effect could be inhibited
BLANCO ET AL
Figure 5. Unique ability of dendritic cells (DCs) generated in the
presence of serum from patients with systemic lupus erythematosus
(SLE) to drive the differentiation of allogeneic T lymphocytes toward
functional cytotoxic effector cells. A, Induction of intracellular granzyme B expression in CD8⫹ allogeneic T lymphocytes. Monocytes
were either cultured in the presence of recombinant human
granulocyte–macrophage colony-stimulating factor (GM-CSF) and
interleukin-4 (IL-4) or were cultured with sera from SLE patients with
active disease in the absence or presence of blocking anti–interferon-␣
(anti-IFN␣) monoclonal antibody. On day 4, differentiated DCs were
harvested and cocultured with allogeneic lymphocytes from normal
individuals. On days 0 (open bars), 2 (striped bars), and 6 (solid bars),
CD8⫹ T lymphocytes were labeled for intracellular expression of
granzyme B. Shown are mean percentages of granzyme B–positive
cells among CD8⫹ T lymphocytes from 1 experiment representative of
3. B, Freshly isolated CD8⫹ T lymphocytes from SLE patients with
active disease act in a manner similar to that of allogeneic CD8⫹ T
lymphocytes primed in vitro by SLE DCs in a cytotoxic functional
assay. Shown is redirected killing of Fas-negative K562 cells by freshly
purified CD8⫹ T lymphocytes from SLE patients with active disease
(squares) or by in vitro SLE DC–primed allogeneic CD8⫹ T lymphocytes (triangles) in the absence (open squares or triangles) or presence
(solid squares or triangles) of 10 ␮g/ml anti-CD3. Values are the mean
and SD specific 51Cr release (target:effector ratios of 1:1, 1:5, and 1:10)
obtained from triplicates from 1 representative experiment of 3.
by adding 10 ␮g/ml of blocking anti-IFN␣ monoclonal antibody during the first step of active SLE DC
ACTIVATED CD8⫹ T LYMPHOCYTES IN SLE PATHOGENESIS
generation. Intracellular perforin staining gave similar
results (data not shown). As a control, normal lymphocytes cultured with quiescent SLE DCs expressed neither intracellular perforin nor granzyme B (data not
shown).
In addition, we compared the cytotoxic potential
of these in vitro–primed CD8⫹ T lymphocytes with that
of their counterparts found in the blood of SLE patients
with active disease (Figure 5B). The CD3-dependent
redirected killing against K562 cells did not disclose any
difference between the 2 types of T lymphocytes, confirming that CD8⫹ T lymphocytes not only acquired
cytotoxic phenotypes when cultured in the presence of
SLE DCs, but also became fully functional and indistinguishable from freshly purified blood CD8⫹ T lymphocytes from SLE patients with active disease. These data
demonstrate that activation of DCs by IFN␣ in SLE
patients with active disease is sufficient to drive the
differentiation of CD8⫹ T cells toward fully active
cytotoxic effector T lymphocytes.
DISCUSSION
Recent studies have focused on the role of apoptosis and the antigen-presenting function of DCs in the
pathophysiology of human SLE. The role of cytotoxic
CD8⫹ T lymphocytes in the defense against viral agents
or organ-specific autoimmune diseases is well documented, but it is still unexplored in SLE. The present
study reveals a quantitative and functional increase in
CD8⫹ cytotoxic T lymphocytes that is highly correlated
with SLE disease activity and that may be responsible for
the increased production of autoantigens. In addition,
following stimulation with IFN␣ derived from sera from
SLE patients with active disease, monocyte-derived DCs
acquired the unique ability to induce the differentiation
of naive CD8⫹ T lymphocytes toward a functional
cytotoxic phenotype identical to that observed in vivo.
Thus, our data imply a previously ignored role of CD8⫹
T lymphocytes in the generation of high amounts of
nuclear autoantigens which, as a consequence, may
overwhelm the physiologic clearance pathway.
Owing to their central role in the humoral response against autoantigens, CD4⫹ cells are thought to
be the primary T lymphocyte subpopulation involved in
lupus autoimmune response (23). Although some reports of studies in humans suggested that cell cytotoxicity is impaired in SLE (24,25), studies in several rodent
models indicate that CD8⫹ T lymphocytes may also
209
contribute to this response, either directly, as a noxious
element of the cellular response, or indirectly, by providing supplies for overcoming mechanisms of tolerance
to autoantigens. It is significant that in a rat model of
autoimmune glomerulonephritis (i.e., Goodpasture’s
syndrome), in which anti–glomerular basement membrane antibodies are pathogenic, CD8⫹ cell depletion
can prevent or treat the renal disease without affecting
serum levels of autoantibodies (26). Mice deprived of
CD8⫹ T cells following deficiency in major histocompatibility complex class I antigen expression are resistant
to experimental SLE (27). NZB mice deficient in ␤2microglobulin had a lower incidence and a delayed onset
of antierythrocyte autoantibody production compared
with that seen in normal NZB mice (28). More recently,
NZB mice deficient in type I IFN receptor were shown
to have a significant decrease in splenic CD8⫹ cells and
a reduced lupus-like disease (29).
In vitro studies reported by Casciola-Rosen et al
indicate that apoptosis involving granzyme B may be
important for autoantigen generation (13), but no direct
links between cytotoxic T lymphocytes and autoantigen
generation have ever been demonstrated in SLE patients. In those studies, the effective generation of
granzyme B autoantigen fragments was dependent on
the relative exogenous inhibition of the caspase pathway.
That is not the case in our study, since CD8⫹ T
lymphocytes freshly isolated from the peripheral blood
of SLE patients with active disease had the intrinsic
capacity to generate nontolerized granzyme B autoantigen fragments without any inhibition of the caspase
pathway (U1–70 kd and topoisomerase I), suggesting a
peculiar status for these cells in patients with active SLE
(Figure 4 and data not shown). Among several possibilities, this discrepancy may rely on the fact that we are
dealing with in vivo–activated T lymphocytes, whereas
Andrade et al dealt with lymphokine-activated killer
cells cultured for 4 days (30).
In this view, the circumstances of T lymphocyte
activation by DCs in vivo may be of paramount importance but difficult to investigate in humans. However,
our in vitro experiments suggest that normal T lymphocytes are converted to an activated and functional phenotype only when cocultured with monocyte-derived DCs
in the presence of sera from patients with active SLE.
Inhibition of IFN␣ in SLE sera by blocking antibody led
to the abrogation of this process, a finding that emphasizes and allows us to better understand the involvement
of IFN␣ in the pathogenesis of SLE.
210
BLANCO ET AL
The mechanism(s) leading to the wide activation
of CD8⫹ T lymphocytes is still elusive, but we suggest
that it is a consequence of the DC system activation
found in SLE (4). In this regard, the increased level of
immune complexes leading preferentially to crosspresentation (31) and/or a direct presentation of viral
antigens (2) may considerably increase the proportion of
activated T cells.
In summary, the present data support the hypothesis of an existing vicious circle initiating and perpetuating SLE disease, in which IFN␣-activated DCs strongly
alter the differentiation of CD8⫹ T lymphocytes, generation of nontolerized autoantigens, and efficient “antigenic feeding” of DCs. IFN␣ is indispensable in this
process by acting on DCs, and it represents a key target
for SLE therapy.
ACKNOWLEDGMENTS
We are indebted to N. Berrié, J. C. Carron, M. Garcie,
and F. Saussais, and to all members of the Laboratory of
Clinical Immunology at the Centre Hospitalier Régional de
Bordeaux who skillfully contributed to this study. We thank
Drs. F. Halary and J. Dechanet-Merville for critically reading
the manuscript and for their helpful suggestions.
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