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Antilymphocyte autoantibodies generate T cell–C4d signatures in systemic lupus erythematosus
Antilymphocyte autoantibodies generate T cell–C4d signatures in systemic lupus erythematosus CHAU-CHING LIU, SUSAN MANZI, and JOSEPH M. AHEARN PITTSBURGH, PENN
T cells bearing C4d, a complement activation product (CAP), have been shown to be highly sensitive and specific as diagnostic biomarkers for systemic lupus erythematosus (SLE). T cells bearing C4d are also functionally abnormal, suggesting a role for cell-bound CAPs in lupus pathogenesis. However, the mechanism responsible for generation of T-C4d has not been determined. The purpose of this cross-sectional and prospective study was to investigate the potential role of anti–T-cell autoantibodies in the generation of the T cell-bound C4d (T-C4d) signatures in SLE. Briefly, T cells from patients with SLE (n 5 326), patients with other inflammatory diseases (n 5 185), and healthy controls (n 5 48) were characterized for surface deposition of either or both of C4d and immunoglobulin (Ig) by flow cytometry. In vitro phenotype transfer experiments were performed to characterize Ig from patients with SLE for the capacity to generate T-C4d signatures in vitro. The results demonstrate that individual patients with SLE harbor specific signatures reflecting the presence of either or both of C4d and Ig on their T cells and T-cell subsets. In addition, SLE patientspecific signatures can be transferred in vitro to normal T cells by exposure to Ig purified from the signature donor. Complement activation does not proceed through the generation of C5b-9 (membrane attack complex) or cellular lysis, and T-C4d does not correlate with lymphopenia. In conclusion, these results suggest that patient-specific T-C4d signatures are generated by anti–T-cell autoantibodies that trigger sublytic complement activation, a previously unrecognized pathway in lupus pathogenesis. (Translational Research 2014;164:496–507) Abbreviations: ADCC ¼ antibody-dependent cellular cytotoxicity; ALA ¼ antilymphocyte autoantibodies; CB-CAPs ¼ cell-bound complement activation products; C4d ¼ complement C4 activation product C4d; Ig ¼ immunoglobulin; T-C4d ¼ T cell–bound C4d; SLE ¼ systemic lupus erythematosus
INTRODUCTION
ystemic lupus erythematosus (SLE), the prototypic systemic autoimmune disease, is characterized by myriad immune abnormalities including excessive autoantibody production, activation of the complement system, lymphocyte dysfunction, and lymphopenia.1-4 On activation of the complement system, proteolytic cleavage of C3 and C4 results ultimately
in the generation of C3d and C4d fragments that contain the highly reactive thioester moiety and can bind covalently to the surfaces of pathogens, cells, or immune complexes.5,6 We have recently reported that significant levels of C4d are present specifically on the surfaces of erythrocytes,7 reticulocytes,8 platelets,9 and lymphocytes10 of patients with SLE. A recent multicenter study validated cell-bound complement activation products (CB-CAPs) as diagnostic biomarkers for
From the Lupus Center of Excellence, Autoimmunity Institute, Allegheny-Singer Research Institute, Allegheny Health Network, Pittsburgh, PA; Temple University School of Medicine, Pittsburgh, PA.
Reprint requests: Chau-Ching Liu, Lupus Center of Excellence, Allegheny Health Network, 320 E North Avenue, Pittsburgh, PA 15212; e-mail: [email protected].
Submitted for publication May 1, 2014; revision submitted July 23, 2014; accepted for publication July 25, 2014.
1931-5244/$ - see front matter
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Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2014.07.007
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AT A GLANCE COMMENTARY Liu C-C, et al. Background
Anti-T cell autoantibodies were first reported in the 1970’s, however their role in the pathogenesis of SLE has not been determined. T cells bearing complement fragment C4d (T-C4d) have been shown to be highly sensitive and specific for a diagnosis of lupus, suggesting a link between anti-T cell autoantibodies and T-C4d. Translational Significance
This study identifies a previously unrecognized molecular and cellular pathway by which anti-T cell autoantibodies trigger sublytic complement activation on T cells in patients with SLE. Deposition of T-C4d through this pathway may render the target cells dysfunctional yet viable and circulating, suggesting a fertile source to mine for lupus biomarkers and targets for therapeutic intervention.
lupus,11 and additional reports have demonstrated their significant potential as biomarkers of lupus disease activity and stratification.12,13 In addition to their roles as lupus biomarkers, CBCAPs have been shown to confer functional abnormalities to circulating cells such as erythrocytes and T lymphocytes, suggesting a role in lupus pathogenesis.14-16 Elucidation of the cellular and molecular events whereby CB-CAPs are generated may lead to the identification of potential therapeutic targets by preventing, disrupting, or neutralizing the downstream effects of CB-CAP generation. One of the most intriguing potential links between CB-CAPs and lupus pathogenesis is the longstanding yet poorly understood observation that patients with SLE harbor circulating antilymphocyte autoantibodies (ALAs). ALAs in patients with SLE, particularly those specific for T cells, were discovered in the 1970s.17-22 Since then, numerous efforts have been made to characterize these ALAs. However, their role in disease pathogenesis has remained uncertain.23 Two primary types of anti–T-cell autoantibodies in SLE have been described previously. First, cold-reactive immunoglobulin M (IgM) antibodies, which bind optimally to T cells at 4 C, have been reported as common in patients with SLE.24,25 However, the in vivo significance of these antibodies is unclear because of the thermal difference between in vitro assays and in vivo pathogenic molecular and
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cellular mechanisms. Second, warm-reactive immunoglobulin G (IgG) anti–T-cell autoantibodies in lupus have been reported.25,26 These antibodies have been shown to have heterogeneous specificities against a variety of T-cell surface molecules including CD3, CD4, CD45, and interleukin (IL)-2R.24,27-30 Two distinct roles for these IgM vs IgG anti–T-cell autoantibodies in lupus pathogenesis have been suggested, both of which involve destruction of the cellular targets. IgM is 500-fold more effective in activation of the classical complement pathway, suggesting possible lytic attack of T cells.31,32 However, if binding of these coldreactive IgM molecules only occurs at low temperatures, this would eliminate this possibility in vivo. In addition, the presence of IgM anti–T-cell autoantibodies has not been shown to correlate with lymphopenia in SLE.33 IgG are less potent complement activators; however, their warm reactivity makes such an in vivo mechanism at least feasible.34,35 Other potential roles for anti–T-cell IgG have been suggested including antibody-dependent cellular cytotoxicity (ADCC)36,37 and modulation of T cell signaling and gene expression.29 Some reports have suggested that T lymphocyte dysfunction in lupus might be triggered by circulating IgM and IgG anti–T-cell autoantibodies rather than because of intrinsic defects, although the 2 possibilities are not mutually exclusive.23 Collectively, these prior reports have suggested that anti–T-cell autoantibodies are present in some patients with SLE and elucidation of their potential role(s) in disease pathogenesis should consider isotype, thermal amplitude of binding and cytotoxicity, and antigenic specificity. We hypothesized that anti–T-cell autoantibodies in SLE may be responsible for generating T cell–bound C4d (T-C4d) patient-specific signatures through sublytic complement activation, and this may be an important but previously unrecognized link in the complex interplay between autoantibodies and complement activation in lupus pathogenesis. METHODS Study participants. All study participants were aged 18 years or older and provided written informed consent. No one was excluded based on gender or ethnicity. Ethnicity was self-reported by study participants. The Institutional Review Boards of the University of Pittsburgh and the West Penn Allegheny Health System approved this study. Patients with SLE. Three hundred twenty-six patients who met the American College of Rheumatology classification criteria for definite SLE38 were recruited and followed during routine visits to the University of Pittsburgh Lupus Patient Care and Translational
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Research Center (January 2006-December 2009) or the West Penn Allegheny Health System Lupus Center of Excellence outpatient clinic (April 2010-December 2012). Patients with other diseases. A total of 185 patients with various non-SLE autoimmune diseases such as rheumatoid arthritis, Sj€ ogren’s syndrome, scleroderma, idiopathic inflammatory myopathy, primary antiphospholipid syndrome, and undifferentiated connective tissue disease were recruited during the same time period as were the SLE patient cohort. Healthy controls. A total of 48 healthy individuals were recruited through local advertisements. To confirm their healthy status, participants completed a brief questionnaire regarding existing medical conditions. Plasma and serum preparation, Ig isolation, and Ig depletion. At the time of each participant’s visit, a
4.5-mL sample of blood was collected into a Vacutainer tube containing EDTA as an anticoagulant (Becton Dickinson, Franklin Lakes, NJ). EDTA-anticoagulated blood samples were processed within 24 hours after the blood was drawn. Plasma was fractionated by centrifugation at 800g for 10 minutes and stored at 4 C (for immediate, short-term use) or 280 C (for future, longterm use). Normal human serum was prepared from the blood of a healthy control collected into a serum separation tube without anticoagulant, aliquoted, and stored at 280 C until use. IgG present in the plasma was isolated using Pierce ImmunoPure IgG purification kit (Thermo Scientific, Rockford, IL) following the manufacturer’s instruction. After collection of IgG, the flow-through from the protein A and G affinity column was further fractionated to enrich for IgM using the Pierce Nab Protein L Spin purification kit (Thermo Scientific) following the manufacturer’s instruction. The IgG and IgM fractions eluted from the respective affinity column were desalted, buffer changed into phosphate-buffered saline (PBS), and concentrated by centrifugation using Microcon centrifugal filters (molecular weight cutoff 30 kDa; EMD Millipore, Billerica, MA). To deplete Ig, the plasma sample was passed sequentially through the protein A and G column and protein L column twice. The final flow-through was collected, dialyzed against PBS, and concentrated back to the original volume using the Microcon device. To deplete lymphocyte-reactive Ig, the plasma sample (50 mL) was incubated with peripheral blood lymphocytes (107 cells; isolated from individual healthy controls) at 4 C for 30 minutes and then recovered by removal of cells by centrifugation. Isolation of peripheral blood mononuclear cells.
Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA-anticoagulated blood samples within 24 hours after the blood was drawn by gradient
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centrifugation. Briefly, blood samples were centrifuged to separate the plasma and cells. The cell fraction was diluted with 3 volumes of PBS, layered on top of Ficoll-Paque Plus solution (GE Healthcare BioSciences, Piscataway, NJ), and centrifuged at 400g at room temperature for 20 minutes. Mononuclear cells located at the interface between the plasma and Ficoll solution were carefully transferred into a fresh tube, washed extensively with PBS to remove contaminating platelets, and resuspended in PBS. Flow cytometric assays for T-C4d and T cell–bound Ig measurement. PBMCs prepared from EDTA-anticoagu-
lated blood were divided into equal-volume aliquots for the detection of CB-CAPs. Levels of C4d and Ig bound to T cells were measured using a multicolor flow cytometric assay recently developed,10 with some modifications. Lymphocytes, monocytes, and residual granulocytes were distinguished based on the expression of characteristic surface molecules and their unique features of forward (size) scattering and side (granularity) scattering. Phycoerythrin (PE)-, PE-Cy5-, or allophycocyaninconjugated mouse monoclonal antibodies (mAbs) reactive with lineage-specific cell surface markers (CD3, CD4, and CD8 for T cells; BD Biosciences, San Diego, CA) were used in conjunction with mouse monoclonal anti-human C4d mAb (mouse IgG1; reactive with C4d-containing fragments of C4; Quidel, San Diego, CA), anti-human IgG (mouse IgG1; BD Biosciences), or anti-human IgM (mouse IgG1; BD Biosciences) that had been labeled with Alexa Fluor dyes using the Zenon antibody labeling kit (Life Technologies, Carlsbad, CA). Alternatively, PE-conjugated anti-human Ig kappa chain or PE-conjugated antihuman Ig lambda chain (BD Biosciences) was used in combination with anti-C4d and anti-cell surface marker antibodies to identify the light chain subtypes of T cell–bound immunoglobulin (T-Ig). After staining, cells were analyzed using a FACSCalibur flow cytometer and CELLQuest software (Becton Dickinson Immunocytometry Systems). To ensure the specificity of the antibody staining detected, leukocyte aliquots from each patient stained with mouse IgG of appropriate isotypes were routinely included in all experiments. To ensure the day-to-day reliability of CB-CAP measurements, the FACSCalibur flow cytometer was calibrated daily using CaliBrite 3 beads and FACSComp software (Becton Dickinson Immunocytometry Systems). Levels of cell-bound C4d and Ig were expressed as specific median fluorescence intensity, which was calculated as the C4d (or Ig)-specific median fluorescence intensity minus the isotype control median fluorescence intensity. In vitro phenotype transfer experiments. PBMCs (106 cells in 50 mL of PBS), isolated from individual
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Fig 1. C4d can bind differentially to CD4 vs CD8 T cells in patients with SLE. Levels of C4d bound to T cells bearing CD3, CD4, or CD8 were measured simultaneously using a single blood sample obtained on the day of the study visit. Flow cytometric histograms from 6 representative patients with SLE are shown to illustrate the presence of similar (panels A and B) or distinct (panels C–F) levels of C4d on CD4 T and CD8 T cells in the same patient at a given time. Solid histograms represent Ig isotype control staining; open histograms represent C4d-specific staining. Ig, immunoglobulin; SLE, systemic lupus erythematosus; T-C4d, T cell–bound C4d.
healthy controls or patients with SLE with low T-C4d levels, were incubated with 50 mL of the plasma prepared from individual patients with SLE who had been identified as having the high T-C4d and T-Ig phenotype based on the flow cytometric analysis described previously. In some experiments, the SLE plasma was replaced with different amounts of purified IgG or IgM (100–1000 mg), Ig-depleted plasma, or lymphocytepreabsorbed plasma. After incubation at 4 C for 30 minutes, the PBMC suspension was washed twice with PBS, resuspended in 50 mL of gelatin veronal buffer (1% gelatin, 5 mM of Na veronal, 142 mM of NaCl, pH 7.3), and incubated with 50 mL of normal human serum (as a source of complement). In some cases, heat-inactivated normal human serum or C1q-depleted human serum (Quidel) was used as a negative control serum. After incubation at 37 C for 40 minutes, the cell suspension was washed twice with PBS and
subjected to multicolor flow cytometric assay described previously. RESULTS TCD3-C4d, TCD4-C4d, and TCD8-C4d patient-specific signatures are generated in SLE. We first conducted a
cross-sectional study to identify patients in whom levels of C4d on T cells were either similar or distinct between CD4 and CD8 subsets. In some patients, the C4d-staining histogram of CD31 T (TCD3) cells reflected a single population of cells and the C4d-staining histograms of CD41 T (TCD4) cells and CD81 T (TCD8) cells were indistinguishable, reflecting equal levels of C4d binding to each of the 2 cell subsets (Fig 1, panels A and B). However, biphasic TCD3-C4d histograms were observed in many of the patients with SLE (Fig 1, panels C–F). TCD4-C4d and TCD8-C4d assays demonstrated that these biphasic histograms
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Table I. Differential binding of C4d to T-cell subsets in patients with SLE over time Patient ID
Study date
CD3 T-C4d (SMFI)
CD4 T-C4d (SMFI)
CD8 T-C4d (SMFI)
SLE103343 SLE103343 SLE103343 SLE107395 SLE107395 SLE107395 SLE8383 SLE8383 SLE8383 SLE101601 SLE101601 SLE101601 SLE56525 SLE56525 SLE56525* SLE102763 SLE102763 SLE102763 SLE102683 SLE102683 SLE102683 SLE102357 SLE102357 SLE102357* SLE102357
03/05/09 12/01/09 11/04/11 05/04/12 05/26/12 02/01/13 05/18/05 01/04/06 09/25/07 09/01/09 03/06/12 12/06/12 04/03/08 10/10/11 01/24/12 01/17/08 07/01/08 10/16/08 12/14/10 06/16/11 12/06/11 04/14/11 12/01/11 04/19/12 01/29/13
309.44 417.70 1052.43 404.24 627.99 228.43 7.55 13.91 10.85 57.58 184.38 126.39 35.63 177.10 322.26 67.04 182.99 220.42 137.34 58.20 129.57 82.37 170.33 80.57 51.56
324.16 407.33 911.22 376.10 693.17 302.68 3.92 8.17 6.29 38.76 99.19 74.33 24.54 133.54 259.39 155.94 213.69 241.32 192.43 130.87 234.16 133.38 191.28 68.99 42.10
320.31 422.02 1086.94 411.57 602.55 236.17 231.71 258.09 152.80 108.67 250.36 250.74 95.08 451.16 169.35 30.95 125.35 129.61 66.04 31.99 34.54 39.60 44.54 147.62 94.00
Abbreviations: SLE, systemic lupus erythematosus; SMFI, specific median fluorescence intensity; T-C4d, T cell–bound C4d. Bold typeface indicates T-C4d levels preferentially elevated on CD4 T cels or CD8 T cells. *Indicate changes when the preferentially elevated T-C4d levels ‘‘converted’’ from one T cell subset to the other (see text).
were because of differences in the levels of C4d on these 2 cell subsets within individual patients. In some patients, considerably elevated levels of C4d were detected on TCD4 cells, compared with TCD8 cells (Fig 1, panels C and D). Conversely, significantly higher levels of C4d could be present on TCD8 cells compared with TCD4 cells in other patients (Fig 1, panels E and F). In general, the TCD4-C4d-specific or TCD8-C4d–specific preferential binding profile, or signature, of a given patient appeared to remain stable over time, although the actual levels of C4d present on the cells might fluctuate over time (Table I). There were exceptions to this pattern. A ‘‘conversion’’ of the binding pattern did occur in some patients during their disease course (eg, patients SLE56525 and SLE102357, Table I). As previously reported, complement C5b-9 (membrane attack complex) was not detected on the T cells.10 This observation that T-cell subsets could be preferentially targeted by C4d deposition and patient-specific T-C4d signatures could be identified suggested that specific mechanisms might
be responsible for generating these phenotypes. T-C4d signatures were not associated with lymphopenia (not shown). Ig and C4d are simultaneously present on T cells in SLE. Any mechanism responsible for T-C4d signatures
must account for differential deposition of C4d on TCD4 vs TCD8 cells in some patients and the most likely explanation would be via autoantibodies specific for TCD4 vs TCD8 cells. We investigated this possibility by flow cytometric analysis to examine and correlate the presence of C4d (T-C4d) and Ig (T-Ig) on the surface of T cells in patients with SLE. The results demonstrated that T cells from a significant fraction (30%) of our SLE patient cohort were indeed decorated simultaneously with both C4d and Ig at the study visit (Fig 2, A). The remainder of patients with SLE were either single positive for either C4d or Ig, or they were double negatives. In comparison, T cells of patients with other diseases and healthy controls were negative for both surface-bound Ig and C4d (Fig 2, B; data shown only for T-Ig). Of the 185 patients with other autoimmune diseases, only 2 patients, both with Sj€ogren’s syndrome, had significantly elevated levels of Ig present on the surface of their T cells. One of those 2 patients also had significantly elevated T-C4d levels. At any given time, T cells bearing both C4d and Ig (Fig 2, C, panel a), C4d alone (Fig 2, C, panel c), or Ig alone (Fig 2, C, panel e) could be detected in different patients with SLE. In some cases, distinct subpopulations of T cells bearing different levels of Ig and C4d could be detected simultaneously within the same patient (Fig 2, C, panels b and d). This latter finding suggested that these subpopulations may represent cells in transition from one stage to the next in a dynamic Ig/C4d binding process. Considering this possibility, we followed a selected group of patients with SLE and examined the presence of C4d and Ig on their T cells periodically over time ranging from 9 to 33 months. As shown in Fig 3, A, among individual patients, both the levels of T-C4d and T-Ig, as well as the frequencies and ratios of T cells bearing C4d and/or Ig varied considerably over time. In some patients, T-C4d and T-Ig levels were strongly correlated throughout all study visits (eg, patient #SLE102763, R2 5 0.7063; patient #SLE101592, R2 5 0.9551). However, in some patients, a general correlation between T-C4d and T-Ig levels was limited to a subset of the study visits (eg, visits 1, 3, and 5 of patient #SEL102326). These results further suggest a dynamic Ig/C4d binding process that may vary among individual patients. Furthermore, it was noted that the presence of surface-bound Ig on a specific subset of T cells correlated with increased levels of C4d on that particular T-cell subpopulation. For example, selectively higher
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Fig 2. C4d and Ig are simultaneously present on the surface of T cells in patients with SLE, but not in patients with other diseases or healthy controls. (A) Peripheral blood CD3 T cells prepared from 326 patients with SLE were characterized by flow cytometry for surface-bound Ig (T-Ig) and C4d (T-C4d). Note the presence of 4 distinct groups of patients with SLE: C4d2/Ig2 (blue diamonds), C4d1/Ig1 (red squares), C4d1/Ig2 (green triangles), and C4d2/Ig1 (aqua circles). (B) T-Ig levels on CD3 T cells from patients with SLE (n 5 326), patients with other diseases (n 5 185), and healthy controls (n 5 48) were measured by flow cytometry. The red horizontal line indicates the empirically determined cutoff for T-Ig positivity. (C) Levels of C4d and Ig bound on T cells were measured by flow cytometry using a single blood sample obtained on the day of the study visit. The representative results of 10 patients with SLE are shown. Note the concomitant or individual presence of C4d and Ig on T cells in different patients and on T-cell subsets in the same patients. Ig, immunoglobulin; SLE, systemic lupus erythematosus; T-C4d, T cell–bound C4d; T-Ig, T cell–bound immunoglobulin.
T-C4d levels on CD41 T cells corresponded with increased T-Ig levels selectively on CD41 T cells in a given patient (Fig 3, B, panels a and b), and vice versa (Fig 3, B, panels c and d). These observations suggested a causal link between the binding of Ig and the deposition of C4d on T cells in patients with SLE, most likely through activation of the classical complement pathway.
The T-C4d phenotype can be transferred by plasma from patients with SLE. Because Ig and C4d were detected
simultaneously on the surface of circulating T cells of some patients with SLE, we hypothesized a model in which binding of specific anti–T-cell autoantibodies may activate the complement system and generate C4d that deposits onto the surface of these cells. Therefore, we investigated whether the plasma of patients
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Fig 3. T-C4d and T-Ig levels fluctuate over time, but generally correlate on the surface of specific T-cell subsets in patients with SLE. (A) Levels of C4d (blue columns) and Ig (purple columns) bound on T cells were measured by flow cytometry using a single blood sample obtained on different study visits. Note the considerable changes in T-C4d/T-Ig phenotype and levels within each individual patient. A good correlation between T-C4d and T-Ig levels is noted in some, but not all patients. The inset within each histogram shows the corresponding correlation analysis. Blue columns, T-C4d; purple columns, T-Ig. (B) C4d and Ig present on the surface of CD4 (blue columns) and CD8 (purple columns) T cells of patients with SLE were measured using multicolor flow cytometric analysis. Note that when higher levels of Ig were detected on CD4 T cells than CD8 T cells in some patients (panel a), higher levels of C4d were also detected on CD4 T cells than on CD8 T cells (panel b). Similar correlations between increased levels of C4d (panel c) and Ig levels (panel d) were observed for CD8 T cells. Ig, immunoglobulin; SLE, systemic lupus erythematosus; T-C4d, T cell–bound C4d; T-Ig, T cell–bound immunoglobulin.
with SLE can bind and ‘‘transfer’’ the T-C4d phenotype to normal T cells in vitro. As shown representatively in Fig 4, normal T cells that were T-C4d negative (panel A)
acquired significant levels of C4d on their surface after being treated with the plasma prepared from donor patients with SLE who were known to have elevated
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T-C4d and T-Ig levels (panels B1 and C1). The patterns of T-C4d generated on the healthy recipient cells were remarkably similar to those present on the lupus donor cells in each experiment. In comparison, the plasma of a healthy control was not capable of generating C4d deposition on normal T cells (panel D1). To date, different plasma samples derived from our study cohort of patients with SLE, patients with other diseases, and healthy controls have been tested against T cells isolated from either healthy individuals or patients with SLE who had negligible levels of T-C4d. The results have consistently shown that only the plasma of SLE patient donors who exhibited the elevated T-Ig/T-C4d phenotype, but not the plasma of patients with SLE who exhibited the low T-Ig/T-C4d phenotype or the plasma of healthy controls, can transfer the T-C4d phenotype to normal T cells. Transfer of the T-C4d phenotype with plasma from lupus patients does not lead to the generation of C5b-9 or to a significant decrease in cell numbers (data not shown). The T-C4d phenotype can be transferred by Ig of patients with SLE. We next performed the phenotype
transfer experiment using Ig purified from the plasma of patients with SLE. The results demonstrated that Ig purified from the plasma of patients with SLE with high T-C4d levels was capable of transferring the T-C4d phenotype to normal T cells in vitro (Fig 4, panels B2 and C2; cf. Fig 4, panel A). Ig purified from either healthy controls (Fig 4, panel D2) or from patients with SLE with negligible T-C4d levels (not shown) could not transfer the T-C4d signature. Equal concentrations of IgM, compared with IgG, were more effective in generating the T-C4d signature, supporting a mechanistic role of classical complement pathway activation (Fig 5, A). Depletion of Ig from donor SLE plasma completely abolished its capacity to induce complement activation and C4d binding to T cells in vitro (Fig 5, B). Moreover, this capacity of the SLE plasma was significantly decreased if the potential lymphocyte-reactive humoral factors were removed by preincubation of the plasma with lymphocytes (Fig 5, B). Absorption with human platelets or fibroblasts had no effect (not shown). These results support the hypothesis that T-C4d signatures in patients with SLE are generated by anti–T-cell and anti–T-cell subset autoantibodies. DISCUSSION
We have previously demonstrated that CAPs, particularly C4d, bind at high levels specifically to circulating cells in patients with SLE,7-10 thereby generating CBCAP signatures that are highly sensitive and specific for a lupus diagnosis.13 Most patients with lupus harbor erythrocytes, lymphocytes, platelets, and other cir-
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culating cells bearing C4d or C4d and C3d in cellspecific patterns that are unique to individual patients. The mechanism or mechanisms responsible for generating these CB-CAP signatures have not been identified. We hypothesized in this study that anti–cell-specific autoantibodies may be responsible for CB-CAP deposition, and we focused specifically on the potential role of anti–T-cell autoantibodies. Here, we provide evidence to support the role of anti–T-cell autoantibodies in the generation of TCD3-C4d, TCD4-C4d, and TCD8-C4d signatures through sublytic complement activation and suggest that this is a previously unrecognized pathogenic mechanism in lupus. The results of this investigation may provide answers to several outstanding questions regarding lupus pathogenesis and may guide future efforts in lupus biomarker discovery. First, it has been demonstrated previously that more than 80% of patients with lupus bear CB-CAP signatures, which are highly patient-specific. The mechanism or mechanisms responsible for these signatures have not been previously determined. Autoantibodies would be natural suspects for generation of specific patterns of C4d deposition on cell surfaces; however, antibody-mediated activation of complement on cell surfaces is generally thought to result in destruction of the cell as observed in hemolytic anemia and in mAb-mediated B-cell depletion therapies with agents such as rituximab. We have demonstrated here that anti–T-cell autoantibodies are present on circulating T cells in lupus patients, and T-Ig/T-C4d patterns are consistent among individual patients over time. Furthermore, IgM and IgG purified from lupus patients bearing Ig and C4d on their T cells can transfer the T-C4d signature to healthy T cells. Collectively, these observations support that activation of the classical complement pathway on surfaces of T cells commonly occurs in patients with SLE. The lack of correlation between T-C4d signatures and lymphopenia and the lack of membrane attack complex on these T cells demonstrate that activation of the classical pathway does not proceed through significant generation of C5 convertases. Second, anti–T-cell autoantibodies in patients with SLE were first discovered more than 40 years ago yet no clear role in disease pathogenesis has been defined.39-44 One challenge has been to reconcile how cold-reactive IgM might be pathogenic at in vivo thermal conditions. We suggest from the studies in this report that low concentrations of IgM autoantibodies are capable of binding to T cells in vivo leading to the deposition of C4d without causing cell lysis. Third, the results of this study may explain a curious observation described in all reports of CBCAPs to date. Specific cell types bearing abnormal
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Fig 4. Plasma and Igs transfer the TC4d signature of the donor to normal T cells. PBMCs prepared from a healthy control were untreated (panel A; baseline phenotype), or incubated with the plasma (panels B1 and C1) or purified Ig (panels B2 and C2) of 2 T-C4d 1 SLE patients. As a negative control, the cells were incubated with the plasma (panel D1) or purified Ig (panel D2) of a healthy control. T cells within the treated cell populations were analyzed by flow cytometry. Note the high levels of C4d deposited on cell surfaces after treatment with T-C4d 1 SLE plasma and Ig, but not the healthy control plasma and Ig. Ig, immunoglobulin; PBMC, peripheral blood mononuclear cells; SLE, systemic lupus erythematosus; T-C4d, T cell–bound C4d.
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Fig 5. Anti–T-cell autoantibodies from patients with SLE can generate TC4d signatures of the donors on normal T cells. (A) IgG and IgM were purified from the plasma of patients with SLE (SLE102086, SLE101606, and SLE102763, with a high T-C4d levels), a patient with SLE (SLE102771, with a low T-C4d level), and healthy controls (HC2009 and HC2034). Purified Ig was used in the in vitro transfer experiment described in the text. (B) The plasma of patients SLE102086, SLE101919, and SLE128305 was depleted of IgG and IgM, or preabsorbed using a large number of leukocytes prepared from a healthy control to deplete lymphocyte-reactive Ig. Ig-depleted or leukocyte-preabsorbed plasma were then used in the in vitro phenotype transfer experiment. Ig, immunoglobulin; SLE, systemic lupus erythematosus; T-C4d, T cell–bound C4d.
levels of C4d are almost always uniformly positive in a given patient, as reflected by the flow cytometric histograms. For example, if the CB-CAP signature of a patient includes TCD3-C4d, all CD3 cells in that patient will be C4d-positive and the levels of C4d on each cell will be similar. This is in contrast to patterns generated by circulating immune complex deposition on cells, which might bind to only a subset of cells bearing receptors for the crystallizable fragment of Ig or CAPs. We propose from the results of the present investigation that circulating anti–T-cell autoantibodies in patients with lupus have the capacity to recognize all circulating T lymphocytes and thereby generate C4d deposition on each cell. This model explains the homogeneous CB-CAP patterns observed on T cells and perhaps other cells among CB-CAPs. Fourth, the observations in the present studies further support the hypothesis that CB-CAP signatures are not generated by systemic complement activation and deposition on innocent bystander cells but rather through a cell-specific targeting mechanism. The observations reported here also raise additional questions that require further investigation. For example, why were Ig and C4d simultaneously detected on T cells of only 30% of patients, with most others being single positive for either Ig or C4d? The T-Ig singlepositive patients might be explained by Ig binding to target T cells in a reversible manner during disease flares, catalyzing deposition of C4d on the T cells. C4d, once generated and bound covalently to T-cell surfaces, may remain stable even when the initiating au-
toantibodies have detached from T cells. Anti–T-cell autoantibodies of certain IgG subclasses (eg, IgG2 and IgG4) may be inefficient in triggering activation of the complement system, thereby creating a phenotype of binding of Ig without C4d. At present, we cannot exclude the possible presence of additional ALAindependent mechanisms whereby CAPs are generated and deposited on T cells. However, in view of the characteristic autoantibody overproduction in patients with SLE, we reason that the ALA-mediated mechanism is likely to play a dominant role in cultivating the celland patient-specific T-C4d phenotypes. CONCLUSIONS
We have shown that the deposition of C4d on T cells and T-cell subsets is generated by sublytic complement activation triggered by anti–T-cell autoantibodies in patients with SLE. This previously unrecognized pathway might be a vicious pathogenic circle whereby autoantibodies activate the classical complement pathway in a sublytic capacity. Deposition of C4d contributes to the dysfunction of these targeted cells, which remain in the circulation and drive further immune dysregulation, autoantibody production, complement activation, and tissue damage. This pathway should be further mined for lupus biomarkers and novel therapeutic targets. ACKNOWLEDGMENTS
Conflicts of Interest: All authors have read the journal’s policy on disclosure of potential conflicts of interest and have none to declare.
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This work was supported by grants from the National Institutes of Health (RO1 AI077591), the Department of Defense (Peer Reviewed Medical Research Grant W81XWH-06-2-0038), and the Lupus Research Institute. All authors have a financial interest as the inventors of the CB-CAPs’ lupus biomarker technology, which is owned by the University of Pittsburgh. S.M. and J.M.A. are consultants to Exagen Diagnostics, Inc, which has an exclusive license to the CB-CAPs’ technology. The authors would like to acknowledge our colleagues in Lupus Center of Excellence, Allegheny Health Network for providing patient blood samples for this study. We also thank Ms Nicole Wilson and Ms Margie Ruffling for coordinating the study.
REFERENCES
1. Schur PH. Complement and lupus erythematosus. Arthritis Rheum 1982;25:793–8. 2. Manderson AP, Botto M, Walport MJ. The role of complement in the development of systemic lupus erythematosus. Annu Rev Immunol 2004;22:431–56. 3. Cook HT, Botto M. Mechanisms of disease: the complement system and the pathogenesis of systemic lupus erythematosus. Nat Clin Pract Rheumatol 2006;2:330–7. 4. Tsokos GC. Systemic lupus erythematosus. N Engl J Med 2011; 365:2110–21. 5. Law SK. The covalent binding reaction of C3 and C4. Ann N Y Acad Sci 1983;421:246–58. 6. Law SK, Levine RP. Interaction between the third complement protein and cell surface macromolecules. Proc Natl Acad Sci U S A 1977;74:2701–5. 7. Manzi S, Navratil JS, Ruffing MJ, et al. Measurement of erythrocyte C4d and complement receptor 1 in systemic lupus erythematosus. Arthritis Rheum 2004;50:3596–604. 8. Liu CC, Manzi S, Kao AH, Navratil JS, Ruffing MJ, Ahearn JM. Reticulocytes bearing C4d as biomarkers of disease activity for systemic lupus erythematosus. Arthritis Rheum 2005;52: 3087–99. 9. Navratil JS, Manzi S, Kao AH, et al. Platelet C4d is highly specific for systemic lupus erythematosus. Arthritis Rheum 2006;54: 670–4. 10. Liu CC, Kao AH, Hawkins DM, et al. Lymphocyte-bound complement activation products as biomarkers for diagnosis of systemic lupus erythematosus. Clin Transl Sci 2009;2:300–8. 11. Kalunian KC, Chatham WW, Massarotti EM, et al. Measurement of cell-bound complement activation products enhances diagnostic performance in systemic lupus erythematosus. Arthritis Rheum 2012;64:4040–7. 12. Kao AH, Navratil JS, Ruffing MJ, et al. Erythrocyte C3d and C4d for monitoring disease activity in systemic lupus erythematosus. Arthritis Rheum 2010;62:837–44. 13. Liu CC, Manzi S, Kao AH, Navratil JS, Ahearn JM. Cell-bound complement biomarkers for systemic lupus erythematosus: from benchtop to bedside. Rheum Dis Clin North Am 2010;36:161–72. 14. Liu CC, Manzi S, Ahearn JM. Complement C4d deposition on T lymphocytes: mechanisms and significance in systemic lupus erythematosus (SLE) (Abstract). Mol Immunol 2007;44:206.
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15. Ghiran IC, Zeidel ML, Shevkoplyas SS, Burns JM, Tsokos GC, Kyttaris VC. Systemic lupus erythematosus serum deposits C4d on red blood cells, decreases red blood cell membrane deformability, and promotes nitric oxide production. Arthritis Rheum 2011;63:503–12. 16. Borschukova O, Paz Z, Ghiran IC, et al. Complement fragment C3d is colocalized within the lipid rafts of T cells and promotes cytokine production. Lupus 2012;21:1294–304. 17. Mittal KK, Rossen RD, Sharp JT, Lidsky MD, Butler WT. Lymphocyte cytotoxic antibodies in systemic lupus erythematosus. Nature 1970;225:1255–6. 18. Stastny P, Ziff M. Antibodies against cell membrane constituents in systemic lupus erythematosus and related diseases. I. Cytotoxic effect of serum from patients with systemic lupus erythematosus (SLE) for allogeneic and for autologous lymphocytes. Clin Exp Immunol 1971;8:543–50. 19. Wernet P, Kunkel HG. Antibodies to a specific surface antigen of T cells in human sera inhibiting mixed leukocyte culture reactions. J Exp Med 1973;138:1021–6. 20. Winfield JB, Winchester RJ, Wernet P, Fu SM, Kunkel HG. Nature of cold-reactive antibodies to lymphocyte surface determinants in systemic lupus erythematosus. Arthritis Rheum 1975;18: 1–8. 21. Okudaira K, Nakai H, Hayakawa T, et al. Detection of antilymphocyte antibody with two-color method in systemic lupus erythematosus and its heterogeneous specificities against human T-cell subsets. J Clin Invest 1979;64:1213–20. 22. Sakane T, Steinberg AD, Reeves JP, Green I. Studies of immune functions of patients with systemic lupus erythematosus. T-cell subsets and antibodies to T-cell subsets. J Clin Invest 1979;64: 1260–9. 23. Winfield JB. Antilymphocyte autoantibodies. In: Wallace DJ, Hahn BH, eds. Dubois’ lupus erythematosus. 5th ed. Baltimore: Williams and Wilkins, 1997:505–15. 24. Yamada A, Shaw M, Winfield JB. Surface antigen specificity of cold-reactive IgM antilymphocyte antibodies in systemic lupus erythematosus. Arthritis Rheum 1985;28:44–51. 25. Yamada A, Winfield JB. Inhibition of soluble antigen-induced T cell proliferation by warm-reactive antibodies to activated T cells in systemic lupus erythematosus. J Clin Invest 1984;74:1948–60. 26. Litvin DA, Cohen PL, Winfield JB. Characterization of warmreactive IgG anti-lymphocyte antibodies in systemic lupus erythematosus. Relative specificity for mitogen-activated T cells and their soluble products. J Immunol 1983;130:181–6. 27. Sano H, Kumagai S, Namiuchi S, et al. Systemic lupus erythematosus sera antilymphocyte reactivity: detection of antibodies to Tacantigen positive T cell lines. Clin Exp Immunol 1986;63:8–16. 28. Mimura T, Fernsten P, Jarjour W, Winfield JB. Autoantibodies specific for different isoforms of CD45 in systemic lupus erythematosus. J Exp Med 1990;172:653–6. 29. Juang YT, Wang Y, Solomou EE, et al. Systemic lupus erythematosus serum IgG increases CREM binding to the IL-2 promoter and suppresses IL-2 production through CaMKIV. J Clin Invest 2005;115:996–1005. 30. Lenert P, Lenert G, Senecal JL. CD4-reactive antibodies in systemic lupus erythematosus. Hum Immunol 1996;49:38–48. 31. Terasaki PI, Mottironi VD, Barnett EV. Cytotoxins in disease. Autocytotoxins in lupus. N Engl J Med 1970;283:724–8. 32. Winfield JB, Winchester RJ, Kunkel HG. Association of coldreactive antilymphocyte antibodies with lymphopenia in systemic lupus erythematosus. Arthritis Rheum 1975;18:587–94. 33. Bresnihan B, Grigor RR, Hughes GR. Lymphocytotoxic antibodies in systemic lupus erythematosus: their clinical significance. J Clin Pathol Suppl (R Coll Pathol) 1979;13:112–5.
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34. Okudaira K, Searles RP, Tanimoto K, Horiuchi Y, Williams RC Jr. T lymphocyte interaction with immunoglobulin G antibody in systemic lupus erythematosus. J Clin Invest 1982;69:1026–38. 35. Noguchi M, Iwamori M, Hirano T, et al. Autoantibodies to T and B cell lines detected in serum samples from patients with systemic lupus erythematosus with lymphopenia and hypocomplementaemia. Ann Rheum Dis 1992;51:713–6. 36. Kumagai S, Steinberg AD, Green I. Antibodies to T cells in patients with systemic lupus erythematosus can induce antibodydependent cell-mediated cytotoxicity against human T cells. J Clin Invest 1981;67:605–14. 37. Mahowald ML, Dalmasso AP. Lymphocyte destruction by antibody-dependent cellular cytotoxicity mediated in vitro by antibodies in serum from patients with systemic lupus erythematosus. Ann Rheum Dis 1982;41:593–8. 38. Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1997;40:1725. 39. Morimoto C, Abe T, Toguchi T, Kiyotaki M, Homma M. Studies of anti-lymphocyte antibody of patients with active SLE. I. Cause
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of loss of suppressor T-lymphocyte function. Scand J Immunol 1979;10:213–21. Morimoto C, Schlossman SF. Antilymphocyte antibodies and systemic lupus erythematosus. Arthritis Rheum 1987;30:225–8. Steinberg AD, Klassen LW, Budman DR, Williams GW. Immunofluorescence studies of anti-T cell antibodies and T cells in systemic lupus erythematosus. Selective loss of brightly staining T cells in active disease. Arthritis Rheum 1979;22:114–22. Morimoto C, Reinherz EL, Schlossman SF. Alterations in immunoregulatory T cell subsets in active systemic lupus erythematosus. J Clin Invest 1980;66:1171–4. Morimoto C, Reinherz EL, Distaso JA, Steinberg AD, Achlossman SF. Relationship between systemic lupus erythematosus T cell subsets, anti-T cell antibodies, and T cell functions. J Clin Invest 1984;73:689–700. Winchester RJ, Winfield JB, Siegal F, Wernet P, Bentwich Z, Kunkel HG. Analyses of lymphocytes from patients with rheumatoid arthritis and systemic lupus erythematosus: occurrence of cold-reactive antilymphocyte antibodies. J Clin Invest 1974;54: 1082–92.
T cells bearing C4d, a complement activation product (CAP), have been shown to be highly sensitive and specific as diagnostic biomarkers for systemic lupus erythematosus (SLE). T cells bearing C4d are also functionally abnormal, suggesting a role for cell-bound CAPs in lupus pathogenesis. However, the mechanism responsible for generation of T-C4d has not been determined. The purpose of this cross-sectional and prospective study was to investigate the potential role of anti–T-cell autoantibodies in the generation of the T cell-bound C4d (T-C4d) signatures in SLE. Briefly, T cells from patients with SLE (n 5 326), patients with other inflammatory diseases (n 5 185), and healthy controls (n 5 48) were characterized for surface deposition of either or both of C4d and immunoglobulin (Ig) by flow cytometry. In vitro phenotype transfer experiments were performed to characterize Ig from patients with SLE for the capacity to generate T-C4d signatures in vitro. The results demonstrate that individual patients with SLE harbor specific signatures reflecting the presence of either or both of C4d and Ig on their T cells and T-cell subsets. In addition, SLE patientspecific signatures can be transferred in vitro to normal T cells by exposure to Ig purified from the signature donor. Complement activation does not proceed through the generation of C5b-9 (membrane attack complex) or cellular lysis, and T-C4d does not correlate with lymphopenia. In conclusion, these results suggest that patient-specific T-C4d signatures are generated by anti–T-cell autoantibodies that trigger sublytic complement activation, a previously unrecognized pathway in lupus pathogenesis. (Translational Research 2014;164:496–507) Abbreviations: ADCC ¼ antibody-dependent cellular cytotoxicity; ALA ¼ antilymphocyte autoantibodies; CB-CAPs ¼ cell-bound complement activation products; C4d ¼ complement C4 activation product C4d; Ig ¼ immunoglobulin; T-C4d ¼ T cell–bound C4d; SLE ¼ systemic lupus erythematosus
INTRODUCTION
ystemic lupus erythematosus (SLE), the prototypic systemic autoimmune disease, is characterized by myriad immune abnormalities including excessive autoantibody production, activation of the complement system, lymphocyte dysfunction, and lymphopenia.1-4 On activation of the complement system, proteolytic cleavage of C3 and C4 results ultimately
in the generation of C3d and C4d fragments that contain the highly reactive thioester moiety and can bind covalently to the surfaces of pathogens, cells, or immune complexes.5,6 We have recently reported that significant levels of C4d are present specifically on the surfaces of erythrocytes,7 reticulocytes,8 platelets,9 and lymphocytes10 of patients with SLE. A recent multicenter study validated cell-bound complement activation products (CB-CAPs) as diagnostic biomarkers for
From the Lupus Center of Excellence, Autoimmunity Institute, Allegheny-Singer Research Institute, Allegheny Health Network, Pittsburgh, PA; Temple University School of Medicine, Pittsburgh, PA.
Reprint requests: Chau-Ching Liu, Lupus Center of Excellence, Allegheny Health Network, 320 E North Avenue, Pittsburgh, PA 15212; e-mail: [email protected].
Submitted for publication May 1, 2014; revision submitted July 23, 2014; accepted for publication July 25, 2014.
1931-5244/$ - see front matter
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Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2014.07.007
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AT A GLANCE COMMENTARY Liu C-C, et al. Background
Anti-T cell autoantibodies were first reported in the 1970’s, however their role in the pathogenesis of SLE has not been determined. T cells bearing complement fragment C4d (T-C4d) have been shown to be highly sensitive and specific for a diagnosis of lupus, suggesting a link between anti-T cell autoantibodies and T-C4d. Translational Significance
This study identifies a previously unrecognized molecular and cellular pathway by which anti-T cell autoantibodies trigger sublytic complement activation on T cells in patients with SLE. Deposition of T-C4d through this pathway may render the target cells dysfunctional yet viable and circulating, suggesting a fertile source to mine for lupus biomarkers and targets for therapeutic intervention.
lupus,11 and additional reports have demonstrated their significant potential as biomarkers of lupus disease activity and stratification.12,13 In addition to their roles as lupus biomarkers, CBCAPs have been shown to confer functional abnormalities to circulating cells such as erythrocytes and T lymphocytes, suggesting a role in lupus pathogenesis.14-16 Elucidation of the cellular and molecular events whereby CB-CAPs are generated may lead to the identification of potential therapeutic targets by preventing, disrupting, or neutralizing the downstream effects of CB-CAP generation. One of the most intriguing potential links between CB-CAPs and lupus pathogenesis is the longstanding yet poorly understood observation that patients with SLE harbor circulating antilymphocyte autoantibodies (ALAs). ALAs in patients with SLE, particularly those specific for T cells, were discovered in the 1970s.17-22 Since then, numerous efforts have been made to characterize these ALAs. However, their role in disease pathogenesis has remained uncertain.23 Two primary types of anti–T-cell autoantibodies in SLE have been described previously. First, cold-reactive immunoglobulin M (IgM) antibodies, which bind optimally to T cells at 4 C, have been reported as common in patients with SLE.24,25 However, the in vivo significance of these antibodies is unclear because of the thermal difference between in vitro assays and in vivo pathogenic molecular and
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cellular mechanisms. Second, warm-reactive immunoglobulin G (IgG) anti–T-cell autoantibodies in lupus have been reported.25,26 These antibodies have been shown to have heterogeneous specificities against a variety of T-cell surface molecules including CD3, CD4, CD45, and interleukin (IL)-2R.24,27-30 Two distinct roles for these IgM vs IgG anti–T-cell autoantibodies in lupus pathogenesis have been suggested, both of which involve destruction of the cellular targets. IgM is 500-fold more effective in activation of the classical complement pathway, suggesting possible lytic attack of T cells.31,32 However, if binding of these coldreactive IgM molecules only occurs at low temperatures, this would eliminate this possibility in vivo. In addition, the presence of IgM anti–T-cell autoantibodies has not been shown to correlate with lymphopenia in SLE.33 IgG are less potent complement activators; however, their warm reactivity makes such an in vivo mechanism at least feasible.34,35 Other potential roles for anti–T-cell IgG have been suggested including antibody-dependent cellular cytotoxicity (ADCC)36,37 and modulation of T cell signaling and gene expression.29 Some reports have suggested that T lymphocyte dysfunction in lupus might be triggered by circulating IgM and IgG anti–T-cell autoantibodies rather than because of intrinsic defects, although the 2 possibilities are not mutually exclusive.23 Collectively, these prior reports have suggested that anti–T-cell autoantibodies are present in some patients with SLE and elucidation of their potential role(s) in disease pathogenesis should consider isotype, thermal amplitude of binding and cytotoxicity, and antigenic specificity. We hypothesized that anti–T-cell autoantibodies in SLE may be responsible for generating T cell–bound C4d (T-C4d) patient-specific signatures through sublytic complement activation, and this may be an important but previously unrecognized link in the complex interplay between autoantibodies and complement activation in lupus pathogenesis. METHODS Study participants. All study participants were aged 18 years or older and provided written informed consent. No one was excluded based on gender or ethnicity. Ethnicity was self-reported by study participants. The Institutional Review Boards of the University of Pittsburgh and the West Penn Allegheny Health System approved this study. Patients with SLE. Three hundred twenty-six patients who met the American College of Rheumatology classification criteria for definite SLE38 were recruited and followed during routine visits to the University of Pittsburgh Lupus Patient Care and Translational
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Research Center (January 2006-December 2009) or the West Penn Allegheny Health System Lupus Center of Excellence outpatient clinic (April 2010-December 2012). Patients with other diseases. A total of 185 patients with various non-SLE autoimmune diseases such as rheumatoid arthritis, Sj€ ogren’s syndrome, scleroderma, idiopathic inflammatory myopathy, primary antiphospholipid syndrome, and undifferentiated connective tissue disease were recruited during the same time period as were the SLE patient cohort. Healthy controls. A total of 48 healthy individuals were recruited through local advertisements. To confirm their healthy status, participants completed a brief questionnaire regarding existing medical conditions. Plasma and serum preparation, Ig isolation, and Ig depletion. At the time of each participant’s visit, a
4.5-mL sample of blood was collected into a Vacutainer tube containing EDTA as an anticoagulant (Becton Dickinson, Franklin Lakes, NJ). EDTA-anticoagulated blood samples were processed within 24 hours after the blood was drawn. Plasma was fractionated by centrifugation at 800g for 10 minutes and stored at 4 C (for immediate, short-term use) or 280 C (for future, longterm use). Normal human serum was prepared from the blood of a healthy control collected into a serum separation tube without anticoagulant, aliquoted, and stored at 280 C until use. IgG present in the plasma was isolated using Pierce ImmunoPure IgG purification kit (Thermo Scientific, Rockford, IL) following the manufacturer’s instruction. After collection of IgG, the flow-through from the protein A and G affinity column was further fractionated to enrich for IgM using the Pierce Nab Protein L Spin purification kit (Thermo Scientific) following the manufacturer’s instruction. The IgG and IgM fractions eluted from the respective affinity column were desalted, buffer changed into phosphate-buffered saline (PBS), and concentrated by centrifugation using Microcon centrifugal filters (molecular weight cutoff 30 kDa; EMD Millipore, Billerica, MA). To deplete Ig, the plasma sample was passed sequentially through the protein A and G column and protein L column twice. The final flow-through was collected, dialyzed against PBS, and concentrated back to the original volume using the Microcon device. To deplete lymphocyte-reactive Ig, the plasma sample (50 mL) was incubated with peripheral blood lymphocytes (107 cells; isolated from individual healthy controls) at 4 C for 30 minutes and then recovered by removal of cells by centrifugation. Isolation of peripheral blood mononuclear cells.
Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA-anticoagulated blood samples within 24 hours after the blood was drawn by gradient
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centrifugation. Briefly, blood samples were centrifuged to separate the plasma and cells. The cell fraction was diluted with 3 volumes of PBS, layered on top of Ficoll-Paque Plus solution (GE Healthcare BioSciences, Piscataway, NJ), and centrifuged at 400g at room temperature for 20 minutes. Mononuclear cells located at the interface between the plasma and Ficoll solution were carefully transferred into a fresh tube, washed extensively with PBS to remove contaminating platelets, and resuspended in PBS. Flow cytometric assays for T-C4d and T cell–bound Ig measurement. PBMCs prepared from EDTA-anticoagu-
lated blood were divided into equal-volume aliquots for the detection of CB-CAPs. Levels of C4d and Ig bound to T cells were measured using a multicolor flow cytometric assay recently developed,10 with some modifications. Lymphocytes, monocytes, and residual granulocytes were distinguished based on the expression of characteristic surface molecules and their unique features of forward (size) scattering and side (granularity) scattering. Phycoerythrin (PE)-, PE-Cy5-, or allophycocyaninconjugated mouse monoclonal antibodies (mAbs) reactive with lineage-specific cell surface markers (CD3, CD4, and CD8 for T cells; BD Biosciences, San Diego, CA) were used in conjunction with mouse monoclonal anti-human C4d mAb (mouse IgG1; reactive with C4d-containing fragments of C4; Quidel, San Diego, CA), anti-human IgG (mouse IgG1; BD Biosciences), or anti-human IgM (mouse IgG1; BD Biosciences) that had been labeled with Alexa Fluor dyes using the Zenon antibody labeling kit (Life Technologies, Carlsbad, CA). Alternatively, PE-conjugated anti-human Ig kappa chain or PE-conjugated antihuman Ig lambda chain (BD Biosciences) was used in combination with anti-C4d and anti-cell surface marker antibodies to identify the light chain subtypes of T cell–bound immunoglobulin (T-Ig). After staining, cells were analyzed using a FACSCalibur flow cytometer and CELLQuest software (Becton Dickinson Immunocytometry Systems). To ensure the specificity of the antibody staining detected, leukocyte aliquots from each patient stained with mouse IgG of appropriate isotypes were routinely included in all experiments. To ensure the day-to-day reliability of CB-CAP measurements, the FACSCalibur flow cytometer was calibrated daily using CaliBrite 3 beads and FACSComp software (Becton Dickinson Immunocytometry Systems). Levels of cell-bound C4d and Ig were expressed as specific median fluorescence intensity, which was calculated as the C4d (or Ig)-specific median fluorescence intensity minus the isotype control median fluorescence intensity. In vitro phenotype transfer experiments. PBMCs (106 cells in 50 mL of PBS), isolated from individual
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Fig 1. C4d can bind differentially to CD4 vs CD8 T cells in patients with SLE. Levels of C4d bound to T cells bearing CD3, CD4, or CD8 were measured simultaneously using a single blood sample obtained on the day of the study visit. Flow cytometric histograms from 6 representative patients with SLE are shown to illustrate the presence of similar (panels A and B) or distinct (panels C–F) levels of C4d on CD4 T and CD8 T cells in the same patient at a given time. Solid histograms represent Ig isotype control staining; open histograms represent C4d-specific staining. Ig, immunoglobulin; SLE, systemic lupus erythematosus; T-C4d, T cell–bound C4d.
healthy controls or patients with SLE with low T-C4d levels, were incubated with 50 mL of the plasma prepared from individual patients with SLE who had been identified as having the high T-C4d and T-Ig phenotype based on the flow cytometric analysis described previously. In some experiments, the SLE plasma was replaced with different amounts of purified IgG or IgM (100–1000 mg), Ig-depleted plasma, or lymphocytepreabsorbed plasma. After incubation at 4 C for 30 minutes, the PBMC suspension was washed twice with PBS, resuspended in 50 mL of gelatin veronal buffer (1% gelatin, 5 mM of Na veronal, 142 mM of NaCl, pH 7.3), and incubated with 50 mL of normal human serum (as a source of complement). In some cases, heat-inactivated normal human serum or C1q-depleted human serum (Quidel) was used as a negative control serum. After incubation at 37 C for 40 minutes, the cell suspension was washed twice with PBS and
subjected to multicolor flow cytometric assay described previously. RESULTS TCD3-C4d, TCD4-C4d, and TCD8-C4d patient-specific signatures are generated in SLE. We first conducted a
cross-sectional study to identify patients in whom levels of C4d on T cells were either similar or distinct between CD4 and CD8 subsets. In some patients, the C4d-staining histogram of CD31 T (TCD3) cells reflected a single population of cells and the C4d-staining histograms of CD41 T (TCD4) cells and CD81 T (TCD8) cells were indistinguishable, reflecting equal levels of C4d binding to each of the 2 cell subsets (Fig 1, panels A and B). However, biphasic TCD3-C4d histograms were observed in many of the patients with SLE (Fig 1, panels C–F). TCD4-C4d and TCD8-C4d assays demonstrated that these biphasic histograms
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Table I. Differential binding of C4d to T-cell subsets in patients with SLE over time Patient ID
Study date
CD3 T-C4d (SMFI)
CD4 T-C4d (SMFI)
CD8 T-C4d (SMFI)
SLE103343 SLE103343 SLE103343 SLE107395 SLE107395 SLE107395 SLE8383 SLE8383 SLE8383 SLE101601 SLE101601 SLE101601 SLE56525 SLE56525 SLE56525* SLE102763 SLE102763 SLE102763 SLE102683 SLE102683 SLE102683 SLE102357 SLE102357 SLE102357* SLE102357
03/05/09 12/01/09 11/04/11 05/04/12 05/26/12 02/01/13 05/18/05 01/04/06 09/25/07 09/01/09 03/06/12 12/06/12 04/03/08 10/10/11 01/24/12 01/17/08 07/01/08 10/16/08 12/14/10 06/16/11 12/06/11 04/14/11 12/01/11 04/19/12 01/29/13
309.44 417.70 1052.43 404.24 627.99 228.43 7.55 13.91 10.85 57.58 184.38 126.39 35.63 177.10 322.26 67.04 182.99 220.42 137.34 58.20 129.57 82.37 170.33 80.57 51.56
324.16 407.33 911.22 376.10 693.17 302.68 3.92 8.17 6.29 38.76 99.19 74.33 24.54 133.54 259.39 155.94 213.69 241.32 192.43 130.87 234.16 133.38 191.28 68.99 42.10
320.31 422.02 1086.94 411.57 602.55 236.17 231.71 258.09 152.80 108.67 250.36 250.74 95.08 451.16 169.35 30.95 125.35 129.61 66.04 31.99 34.54 39.60 44.54 147.62 94.00
Abbreviations: SLE, systemic lupus erythematosus; SMFI, specific median fluorescence intensity; T-C4d, T cell–bound C4d. Bold typeface indicates T-C4d levels preferentially elevated on CD4 T cels or CD8 T cells. *Indicate changes when the preferentially elevated T-C4d levels ‘‘converted’’ from one T cell subset to the other (see text).
were because of differences in the levels of C4d on these 2 cell subsets within individual patients. In some patients, considerably elevated levels of C4d were detected on TCD4 cells, compared with TCD8 cells (Fig 1, panels C and D). Conversely, significantly higher levels of C4d could be present on TCD8 cells compared with TCD4 cells in other patients (Fig 1, panels E and F). In general, the TCD4-C4d-specific or TCD8-C4d–specific preferential binding profile, or signature, of a given patient appeared to remain stable over time, although the actual levels of C4d present on the cells might fluctuate over time (Table I). There were exceptions to this pattern. A ‘‘conversion’’ of the binding pattern did occur in some patients during their disease course (eg, patients SLE56525 and SLE102357, Table I). As previously reported, complement C5b-9 (membrane attack complex) was not detected on the T cells.10 This observation that T-cell subsets could be preferentially targeted by C4d deposition and patient-specific T-C4d signatures could be identified suggested that specific mechanisms might
be responsible for generating these phenotypes. T-C4d signatures were not associated with lymphopenia (not shown). Ig and C4d are simultaneously present on T cells in SLE. Any mechanism responsible for T-C4d signatures
must account for differential deposition of C4d on TCD4 vs TCD8 cells in some patients and the most likely explanation would be via autoantibodies specific for TCD4 vs TCD8 cells. We investigated this possibility by flow cytometric analysis to examine and correlate the presence of C4d (T-C4d) and Ig (T-Ig) on the surface of T cells in patients with SLE. The results demonstrated that T cells from a significant fraction (30%) of our SLE patient cohort were indeed decorated simultaneously with both C4d and Ig at the study visit (Fig 2, A). The remainder of patients with SLE were either single positive for either C4d or Ig, or they were double negatives. In comparison, T cells of patients with other diseases and healthy controls were negative for both surface-bound Ig and C4d (Fig 2, B; data shown only for T-Ig). Of the 185 patients with other autoimmune diseases, only 2 patients, both with Sj€ogren’s syndrome, had significantly elevated levels of Ig present on the surface of their T cells. One of those 2 patients also had significantly elevated T-C4d levels. At any given time, T cells bearing both C4d and Ig (Fig 2, C, panel a), C4d alone (Fig 2, C, panel c), or Ig alone (Fig 2, C, panel e) could be detected in different patients with SLE. In some cases, distinct subpopulations of T cells bearing different levels of Ig and C4d could be detected simultaneously within the same patient (Fig 2, C, panels b and d). This latter finding suggested that these subpopulations may represent cells in transition from one stage to the next in a dynamic Ig/C4d binding process. Considering this possibility, we followed a selected group of patients with SLE and examined the presence of C4d and Ig on their T cells periodically over time ranging from 9 to 33 months. As shown in Fig 3, A, among individual patients, both the levels of T-C4d and T-Ig, as well as the frequencies and ratios of T cells bearing C4d and/or Ig varied considerably over time. In some patients, T-C4d and T-Ig levels were strongly correlated throughout all study visits (eg, patient #SLE102763, R2 5 0.7063; patient #SLE101592, R2 5 0.9551). However, in some patients, a general correlation between T-C4d and T-Ig levels was limited to a subset of the study visits (eg, visits 1, 3, and 5 of patient #SEL102326). These results further suggest a dynamic Ig/C4d binding process that may vary among individual patients. Furthermore, it was noted that the presence of surface-bound Ig on a specific subset of T cells correlated with increased levels of C4d on that particular T-cell subpopulation. For example, selectively higher
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Fig 2. C4d and Ig are simultaneously present on the surface of T cells in patients with SLE, but not in patients with other diseases or healthy controls. (A) Peripheral blood CD3 T cells prepared from 326 patients with SLE were characterized by flow cytometry for surface-bound Ig (T-Ig) and C4d (T-C4d). Note the presence of 4 distinct groups of patients with SLE: C4d2/Ig2 (blue diamonds), C4d1/Ig1 (red squares), C4d1/Ig2 (green triangles), and C4d2/Ig1 (aqua circles). (B) T-Ig levels on CD3 T cells from patients with SLE (n 5 326), patients with other diseases (n 5 185), and healthy controls (n 5 48) were measured by flow cytometry. The red horizontal line indicates the empirically determined cutoff for T-Ig positivity. (C) Levels of C4d and Ig bound on T cells were measured by flow cytometry using a single blood sample obtained on the day of the study visit. The representative results of 10 patients with SLE are shown. Note the concomitant or individual presence of C4d and Ig on T cells in different patients and on T-cell subsets in the same patients. Ig, immunoglobulin; SLE, systemic lupus erythematosus; T-C4d, T cell–bound C4d; T-Ig, T cell–bound immunoglobulin.
T-C4d levels on CD41 T cells corresponded with increased T-Ig levels selectively on CD41 T cells in a given patient (Fig 3, B, panels a and b), and vice versa (Fig 3, B, panels c and d). These observations suggested a causal link between the binding of Ig and the deposition of C4d on T cells in patients with SLE, most likely through activation of the classical complement pathway.
The T-C4d phenotype can be transferred by plasma from patients with SLE. Because Ig and C4d were detected
simultaneously on the surface of circulating T cells of some patients with SLE, we hypothesized a model in which binding of specific anti–T-cell autoantibodies may activate the complement system and generate C4d that deposits onto the surface of these cells. Therefore, we investigated whether the plasma of patients
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Fig 3. T-C4d and T-Ig levels fluctuate over time, but generally correlate on the surface of specific T-cell subsets in patients with SLE. (A) Levels of C4d (blue columns) and Ig (purple columns) bound on T cells were measured by flow cytometry using a single blood sample obtained on different study visits. Note the considerable changes in T-C4d/T-Ig phenotype and levels within each individual patient. A good correlation between T-C4d and T-Ig levels is noted in some, but not all patients. The inset within each histogram shows the corresponding correlation analysis. Blue columns, T-C4d; purple columns, T-Ig. (B) C4d and Ig present on the surface of CD4 (blue columns) and CD8 (purple columns) T cells of patients with SLE were measured using multicolor flow cytometric analysis. Note that when higher levels of Ig were detected on CD4 T cells than CD8 T cells in some patients (panel a), higher levels of C4d were also detected on CD4 T cells than on CD8 T cells (panel b). Similar correlations between increased levels of C4d (panel c) and Ig levels (panel d) were observed for CD8 T cells. Ig, immunoglobulin; SLE, systemic lupus erythematosus; T-C4d, T cell–bound C4d; T-Ig, T cell–bound immunoglobulin.
with SLE can bind and ‘‘transfer’’ the T-C4d phenotype to normal T cells in vitro. As shown representatively in Fig 4, normal T cells that were T-C4d negative (panel A)
acquired significant levels of C4d on their surface after being treated with the plasma prepared from donor patients with SLE who were known to have elevated
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T-C4d and T-Ig levels (panels B1 and C1). The patterns of T-C4d generated on the healthy recipient cells were remarkably similar to those present on the lupus donor cells in each experiment. In comparison, the plasma of a healthy control was not capable of generating C4d deposition on normal T cells (panel D1). To date, different plasma samples derived from our study cohort of patients with SLE, patients with other diseases, and healthy controls have been tested against T cells isolated from either healthy individuals or patients with SLE who had negligible levels of T-C4d. The results have consistently shown that only the plasma of SLE patient donors who exhibited the elevated T-Ig/T-C4d phenotype, but not the plasma of patients with SLE who exhibited the low T-Ig/T-C4d phenotype or the plasma of healthy controls, can transfer the T-C4d phenotype to normal T cells. Transfer of the T-C4d phenotype with plasma from lupus patients does not lead to the generation of C5b-9 or to a significant decrease in cell numbers (data not shown). The T-C4d phenotype can be transferred by Ig of patients with SLE. We next performed the phenotype
transfer experiment using Ig purified from the plasma of patients with SLE. The results demonstrated that Ig purified from the plasma of patients with SLE with high T-C4d levels was capable of transferring the T-C4d phenotype to normal T cells in vitro (Fig 4, panels B2 and C2; cf. Fig 4, panel A). Ig purified from either healthy controls (Fig 4, panel D2) or from patients with SLE with negligible T-C4d levels (not shown) could not transfer the T-C4d signature. Equal concentrations of IgM, compared with IgG, were more effective in generating the T-C4d signature, supporting a mechanistic role of classical complement pathway activation (Fig 5, A). Depletion of Ig from donor SLE plasma completely abolished its capacity to induce complement activation and C4d binding to T cells in vitro (Fig 5, B). Moreover, this capacity of the SLE plasma was significantly decreased if the potential lymphocyte-reactive humoral factors were removed by preincubation of the plasma with lymphocytes (Fig 5, B). Absorption with human platelets or fibroblasts had no effect (not shown). These results support the hypothesis that T-C4d signatures in patients with SLE are generated by anti–T-cell and anti–T-cell subset autoantibodies. DISCUSSION
We have previously demonstrated that CAPs, particularly C4d, bind at high levels specifically to circulating cells in patients with SLE,7-10 thereby generating CBCAP signatures that are highly sensitive and specific for a lupus diagnosis.13 Most patients with lupus harbor erythrocytes, lymphocytes, platelets, and other cir-
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culating cells bearing C4d or C4d and C3d in cellspecific patterns that are unique to individual patients. The mechanism or mechanisms responsible for generating these CB-CAP signatures have not been identified. We hypothesized in this study that anti–cell-specific autoantibodies may be responsible for CB-CAP deposition, and we focused specifically on the potential role of anti–T-cell autoantibodies. Here, we provide evidence to support the role of anti–T-cell autoantibodies in the generation of TCD3-C4d, TCD4-C4d, and TCD8-C4d signatures through sublytic complement activation and suggest that this is a previously unrecognized pathogenic mechanism in lupus. The results of this investigation may provide answers to several outstanding questions regarding lupus pathogenesis and may guide future efforts in lupus biomarker discovery. First, it has been demonstrated previously that more than 80% of patients with lupus bear CB-CAP signatures, which are highly patient-specific. The mechanism or mechanisms responsible for these signatures have not been previously determined. Autoantibodies would be natural suspects for generation of specific patterns of C4d deposition on cell surfaces; however, antibody-mediated activation of complement on cell surfaces is generally thought to result in destruction of the cell as observed in hemolytic anemia and in mAb-mediated B-cell depletion therapies with agents such as rituximab. We have demonstrated here that anti–T-cell autoantibodies are present on circulating T cells in lupus patients, and T-Ig/T-C4d patterns are consistent among individual patients over time. Furthermore, IgM and IgG purified from lupus patients bearing Ig and C4d on their T cells can transfer the T-C4d signature to healthy T cells. Collectively, these observations support that activation of the classical complement pathway on surfaces of T cells commonly occurs in patients with SLE. The lack of correlation between T-C4d signatures and lymphopenia and the lack of membrane attack complex on these T cells demonstrate that activation of the classical pathway does not proceed through significant generation of C5 convertases. Second, anti–T-cell autoantibodies in patients with SLE were first discovered more than 40 years ago yet no clear role in disease pathogenesis has been defined.39-44 One challenge has been to reconcile how cold-reactive IgM might be pathogenic at in vivo thermal conditions. We suggest from the studies in this report that low concentrations of IgM autoantibodies are capable of binding to T cells in vivo leading to the deposition of C4d without causing cell lysis. Third, the results of this study may explain a curious observation described in all reports of CBCAPs to date. Specific cell types bearing abnormal
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Fig 4. Plasma and Igs transfer the TC4d signature of the donor to normal T cells. PBMCs prepared from a healthy control were untreated (panel A; baseline phenotype), or incubated with the plasma (panels B1 and C1) or purified Ig (panels B2 and C2) of 2 T-C4d 1 SLE patients. As a negative control, the cells were incubated with the plasma (panel D1) or purified Ig (panel D2) of a healthy control. T cells within the treated cell populations were analyzed by flow cytometry. Note the high levels of C4d deposited on cell surfaces after treatment with T-C4d 1 SLE plasma and Ig, but not the healthy control plasma and Ig. Ig, immunoglobulin; PBMC, peripheral blood mononuclear cells; SLE, systemic lupus erythematosus; T-C4d, T cell–bound C4d.
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Fig 5. Anti–T-cell autoantibodies from patients with SLE can generate TC4d signatures of the donors on normal T cells. (A) IgG and IgM were purified from the plasma of patients with SLE (SLE102086, SLE101606, and SLE102763, with a high T-C4d levels), a patient with SLE (SLE102771, with a low T-C4d level), and healthy controls (HC2009 and HC2034). Purified Ig was used in the in vitro transfer experiment described in the text. (B) The plasma of patients SLE102086, SLE101919, and SLE128305 was depleted of IgG and IgM, or preabsorbed using a large number of leukocytes prepared from a healthy control to deplete lymphocyte-reactive Ig. Ig-depleted or leukocyte-preabsorbed plasma were then used in the in vitro phenotype transfer experiment. Ig, immunoglobulin; SLE, systemic lupus erythematosus; T-C4d, T cell–bound C4d.
levels of C4d are almost always uniformly positive in a given patient, as reflected by the flow cytometric histograms. For example, if the CB-CAP signature of a patient includes TCD3-C4d, all CD3 cells in that patient will be C4d-positive and the levels of C4d on each cell will be similar. This is in contrast to patterns generated by circulating immune complex deposition on cells, which might bind to only a subset of cells bearing receptors for the crystallizable fragment of Ig or CAPs. We propose from the results of the present investigation that circulating anti–T-cell autoantibodies in patients with lupus have the capacity to recognize all circulating T lymphocytes and thereby generate C4d deposition on each cell. This model explains the homogeneous CB-CAP patterns observed on T cells and perhaps other cells among CB-CAPs. Fourth, the observations in the present studies further support the hypothesis that CB-CAP signatures are not generated by systemic complement activation and deposition on innocent bystander cells but rather through a cell-specific targeting mechanism. The observations reported here also raise additional questions that require further investigation. For example, why were Ig and C4d simultaneously detected on T cells of only 30% of patients, with most others being single positive for either Ig or C4d? The T-Ig singlepositive patients might be explained by Ig binding to target T cells in a reversible manner during disease flares, catalyzing deposition of C4d on the T cells. C4d, once generated and bound covalently to T-cell surfaces, may remain stable even when the initiating au-
toantibodies have detached from T cells. Anti–T-cell autoantibodies of certain IgG subclasses (eg, IgG2 and IgG4) may be inefficient in triggering activation of the complement system, thereby creating a phenotype of binding of Ig without C4d. At present, we cannot exclude the possible presence of additional ALAindependent mechanisms whereby CAPs are generated and deposited on T cells. However, in view of the characteristic autoantibody overproduction in patients with SLE, we reason that the ALA-mediated mechanism is likely to play a dominant role in cultivating the celland patient-specific T-C4d phenotypes. CONCLUSIONS
We have shown that the deposition of C4d on T cells and T-cell subsets is generated by sublytic complement activation triggered by anti–T-cell autoantibodies in patients with SLE. This previously unrecognized pathway might be a vicious pathogenic circle whereby autoantibodies activate the classical complement pathway in a sublytic capacity. Deposition of C4d contributes to the dysfunction of these targeted cells, which remain in the circulation and drive further immune dysregulation, autoantibody production, complement activation, and tissue damage. This pathway should be further mined for lupus biomarkers and novel therapeutic targets. ACKNOWLEDGMENTS
Conflicts of Interest: All authors have read the journal’s policy on disclosure of potential conflicts of interest and have none to declare.
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This work was supported by grants from the National Institutes of Health (RO1 AI077591), the Department of Defense (Peer Reviewed Medical Research Grant W81XWH-06-2-0038), and the Lupus Research Institute. All authors have a financial interest as the inventors of the CB-CAPs’ lupus biomarker technology, which is owned by the University of Pittsburgh. S.M. and J.M.A. are consultants to Exagen Diagnostics, Inc, which has an exclusive license to the CB-CAPs’ technology. The authors would like to acknowledge our colleagues in Lupus Center of Excellence, Allegheny Health Network for providing patient blood samples for this study. We also thank Ms Nicole Wilson and Ms Margie Ruffling for coordinating the study.
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