Increase in activated CD8+ T lymphocytes expressing perforin and granzyme B correlates with disease activity in patients with systemic lupus erythematosus.код для вставкиСкачать
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: email@example.com. 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. REFERENCES 1. 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