The molecular and functional characterization of clonally expanded CD8 + TCR BV T cells in eosinophilic granulomatosis with polyangiitis (EGPA)

YCLIM-07272; No. of pages: 12; 4C: Clinical Immunology (2014) xx, xxx–xxx

available at www.sciencedirect.com

Clinical Immunology www.elsevier.com/locate/yclim

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Monica Boita a,g , Giuseppe Guida b,⁎, Paola Circosta c,d , Angela Rita Elia c,d , Stefania Stella e , Enrico Heffler a,g , Iuliana Badiu a , Davide Martorana f , Sara Mariani g , Giovanni Rolla a,g,1 , Alessandro Cignetti c,d,1

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Allergology and Clinical Immunology, University of Torino, Italy Internal Medicine II - Birago di Vische Hospital - ASL TO2, Torino, Italy c University Division of Hematology and Cell Therapy, AO Mauriziano, Torino, University of Torino, Italy d Molecular Biotechnology Center (MBC), University of Torino, Italy e Immunohematology and Transfusional Medicine - Giovanni Bosco Hospital - ASL TO2-Torino, Italy f Molecular Genetics Unit, University Hospital of Parma, Italy g Medical Science Department, University of Torino, Italy

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The molecular and functional characterization of clonally expanded CD8 + TCR BV T cells in eosinophilic granulomatosis with polyangiitis (EGPA)

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Received 28 December 2013; accepted with revision 3 March 2014

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Abstract In eosinophilic granulomatosis with polyangiitis (EGPA) clonally expanded T cells might concur in granuloma formation and vascular injury. The TCR β-variable (BV) chain repertoire and third complementarity determining region (CDR3) of peripheral CD4 + and CD8 + cells in EGPA patients and age-matched controls and the expression of cytokines and chemokine receptors were investigated. The CD8 + lymphocytes of EGPA patients showed an increased frequency of BV expansion with a skewed profile of BV CDR3 lengths, increased CCR5 and CXCR3 expression and increased INFγ and TNFα production. In two patients, the TCR CDR3 cDNA sequences of the expanded BV family were identified. The CD4 + lymphocytes of EGPA patients revealed a higher expression of CRTH2 and increased production of IL-5.

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Vasculitis; EGPA; TCR BV; CD8 + lymphocytes; Proinflammatory cytokines

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KEYWORDS

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Abbreviations: ANCA, anti-neutrophil cytoplasmic autoantibody; CRTH2, chemoattractant receptor-homologous molecule expressed on Th2 cells; TEM, effector memory T cell; EGPA, eosinophilic granulomatosis with polyangiitis; GV, gamma variable; GPA, granulomatosis with polyangitis; HC, healthy controls; IC, intracytoplasmatic; MoAb, monoclonal antibodies; CDR3, third complementarity determining region; BV, Beta chain Variable gene. ⁎ Corresponding author at: Medicina Interna II - Ospedale Birago di Vische - ASL TO2, Corso Svizzera 164, 10149 Torino, Italy. Fax: +39 0114393706. E-mail addresses: [email protected] (M. Boita), [email protected] (G. Guida), [email protected] (P. Circosta), [email protected] (A.R. Elia), [email protected] (S. Stella), [email protected] (E. Heffler), [email protected] (I. Badiu), [email protected] (D. Martorana), [email protected] (S. Mariani), [email protected] (G. Rolla), [email protected] (A. Cignetti). 1 These authors equally contributed to the study.

http://dx.doi.org/10.1016/j.clim.2014.03.001 1521-6616/© 2014 Published by Elsevier Inc. Please cite this article as: M. Boita, et al., The molecular and functional characterization of clonally expanded CD8+ TCR BV T cells in eosinophilic granulom..., Clin. Immunol. (2014), http://dx.doi.org/10.1016/j.clim.2014.03.001

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M. Boita et al.

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In conclusion, CD4 + T cells display a Th2 profile and CD8 + T cells are clonally expanded in EGPA and have a proinflammatory phenotype, suggesting their pathogenic role in vasculitic damage. © 2014 Published by Elsevier Inc.

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2.2. TCR BV flow cytometry

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2. Materials and methods

Peripheral blood mononuclear cells (PBMCs) were separated from whole blood by density gradient centrifugation (Lymphoprep, Nycomed, Roskilde, Denmark) and analyzed by 4-color flow cytometry, using a panel of monoclonal antibodies (MoAb) to 24 different TCR BV families (IOTest Beta Mark TCR-Vβ- Repertoire Kit, Beckman Coulter, Miami, FL). The percentage of positive cells for each BV family was determined by gating on CD8+ and CD4+ cells. A BV family was considered expanded when the percentage of BV positive cells was 3 standard deviations (SDs) higher than reference values given by Beckman Coulter as well as 3 SDs higher than the values obtained in a cohort of 42 age-matched healthy blood donors (mean age 63, range 45–82) [4,6].

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2.1. Patients

2.3. TCR GV rearrangement

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Eight patients with confirmed Churg–Strauss syndrome, diagnosed according to the American College of Rheumatology

An analysis of TCR gamma variable (GV) [14] rearrangement by heteroduplex PCR was performed on PBMCs. DNA was

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Eosinophilic granulomatosis with polyangiitis (EGPA), previously defined as Churg–Strauss syndrome, is a multisystemic disorder observed in patients with allergic rhinitis, asthma, and hypereosinophilia. EGPA is classified as an anti-neutrophil cytoplasmic autoantibody (ANCA)-associated small vessel vasculitis (AAV) [1] and is characterized by eosinophil-rich inflammation, granulomas and vasculitis, commonly involving the lung, skin and peripheral nerves [2]. The pathogenic role of T cells in EGPA has been suggested by the discovery of T cells in biopsies of active vasculitic lesions and by the demonstration of upregulated Th2/Th17 markers in peripheral T cells [3]. The expanded beta chain variable gene (BV) families of CD8 + lymphocytes, are characterized by an effector memory phenotype and have previously been reported by our group [4]. They have been described among the CD8 + and/or CD4 + cells found in vasculitides [5,6]. The immune phenotype of peripheral T cells from patients with EGPA was previously examined by Kiene et al., who noted the increased expression of both Th1 and Th2 markers, with a predominant type 2 cytokine production pattern [7]. More recently, Jakiela et al. observed Th2/Th17 markers, with an increased Th17/Treg ratio [3]. These observations indicate that the Th2-polarized immune response coexists with a Th17 immune response, with different contributions of individual T-helper cell lineages to the pathogenesis of EGPA, with the role of the CD8 + lineage less well characterized. One approach to studying the TCR repertoire within expanded BV families is to utilize spectratyping analysis [8], which determines the length distribution profile of the third complementarity determining region (CDR3) of each BV family TCR. By using this technique, clonally expanded CD8+ and CD4+ T cells have been reported in Kawasaki disease [9] and granulomatosis with polyangiitis (GPA, formerly Wegener granulomatosis), respectively [10]. Clonally expanded T cells, which were not phenotyped, were also found in the tissue and peripheral blood of EGPA patients by Muschen et al. [11]. In the present study, we investigated in detail the TCR repertoire of CD4+ and CD8+ T cells from EGPA patients by three methods: i) flow cytometry to detect TCR BV expanded families; ii) spectratyping of all BV families to estimate the degree of clonality; iii) cDNA sequencing of TCR-alpha and TCR-beta chains to confirm that clonal expansion was occurring at the molecular level. Chemokine receptor expression and cytokine production by CD4+ and CD8+ cells of the same patients were also investigated.

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1. Introduction

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classification (ACR) [12], were included in the study. All patients could be classified as having eosinophilic granulomatosis with polyangiitis (EGPA) according to the 2012 revised nomenclature for vasculitides [1]. All patients were recruited from the Allergy and Clinical Immunology Division, University of Torino. All patients had asthma, hypereosinophilia, rhinosinusitis and clinical manifestations consistent with systemic vasculitis, confirmed by tissue biopsy in four patients. Venous blood samples from each patient were collected at two different time points throughout 3–32 months of follow up, with the exception of patients #1 and #7, in whom all the analyses were performed simultaneously. At the first time point, only 1/8 patients exhibited active vasculitis and was receiving high dose oral prednisone, whereas the remaining seven patients, five of whom were receiving low dose prednisone and two of whom were receiving methotrexate, had persistent disease according to the Birmingham Vasculitis Activity Score (BVAS3) item list (Table 1) [13]. Most patients had received more intensive immunosuppressive therapy before the first time point, as reported in Table 1. At the second time point, two patients showed active disease, four showed persistent disease, and two were considered to be in remission, apart from persistent asthma and/or neurologic sequelae. Perinuclear anti-neutrophil cytoplasmic autoantibodies (P-ANCA) were present in 5/8 patients. No patient had laboratory evidence of CMV and EBV reactivation. Patient clinical data, including HLA haplotype, are summarized in Table 1. Seven out of the eight patients had also been recruited for a previous study [4]. All the subjects gave their informed consent to participate to the study, which was approved by the local ethics committee. All experiments were carried out in accordance with the Declaration of Helsinki.

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Please cite this article as: M. Boita, et al., The molecular and functional characterization of clonally expanded CD8+ TCR BV T cells in eosinophilic granulom..., Clin. Immunol. (2014), http://dx.doi.org/10.1016/j.clim.2014.03.001

92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122

125 126 127 128 129 130 131 132 133 134 135 136

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Table 1

Clinical and laboratory data of EGPA patients.

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p-ANCA Eosinophils Involved organs (anti-MPO cell/mm3 (%WBC) a UI/ml).

Patient Sex Age HLA

EGPA-1 M

59

t1:6

EGPA-2 F

59

t1:7

EGPA-3 M

57

t1:8

EGPA-4 M

67

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HLA-A: *02-*02 HLA-B: *18-*18 HLA-DRB1: *01-*11 DRB3* HLA-A: *01-*29 HLA-B: *08-*41 HLA-DRB1: *03-*11 DRB3* HLA-A: *02-*26 HLA-B: *27-*38 HLA-DRB1: *01-*07 DRB4* HLA-A: *02-*33 HLA-B: *14-*50 HLA-DRB1: *04-*07 DRB4* HLA-A: *02-*26 HLA-B: *18-*40 HLA-DRB1: *04-*08 DRB4* HLA-A: *03-*26 HLA-B: *07-*35 HLA-DRB1: *01-*08 HLA-A: *32-*69 HLA-B: *18-*55 HLA-DRB1: *03-*11 DRB3* HLA-A: *24-*24 HLA-B: *44-*51 HLA-DRB1: *01-*16 DRB5*

–

14060 (37)

Previous Treatment

BVAS-3 Eosinophils Ongoing therapy

BVAS-3 Eosinophils Ongoing therapy

1st time point (TCRVbeta and spectratyping analysis)

2nd time point (functional analysis)

PNS, CCS, CYC, AZA 5 (P) gallbladder, ileum

570

MP 12 mg alternate

PNS

CCS, AZA

2 (P)

690

PDN 5 mg

0 (R)

720

PDN 5/0 mg

R

PNS, lung (DAH), kidney

CCS, CYC, MTX 1 (P)

190

MTX 7.5 mg/week

0 (R)

250

none

PNS, lung (DAH), kidney

CCS, CYC

590

PDN 10/0 mg

10 (A)

1180

PDN 12.5/0 mg

PNS, skin, kidney, eye

CCS, CYC

840

PDN 15/0 mg

6 (A)

1690

MP 16 mg

5500 (26)

PNS, skin, joints

CCS, AZA, MTX 5 (P)

420

650

MTX 12.5 mg/week

–

17500 (50)

PNS, lung, heart, skin

CCS, MTX

7 (P)

110

+(63)

25000 (40)

PNS, skin, joints

CCS, CYC

10 (A)

200

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–

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22230 (59)

Same time

R

+(46)

6920 (30)

+(32)

9990 (37)

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4 (P)

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t1:9

EGPA-5 M

t1:10

EGPA-6 M

60

t1:11

EGPA-7 F

85

t1:12

EGPA-8 M

38

t1:13 t1:14 t1:15 t1:16 t1:17 t1:18

Venous blood samples of each patient were collected at 1st time point for flow cytometric analysis and spectratyping; at 2nd time point for functional analysis (cytokine production and chemokine receptor analysis). p-ANCA: perinuclear anti neutrophil cytoplasmic antibodies; MPO: myeloperoxidase; BVAS: Birmingham Vasculitis Activity Score: (P) = persistent; (A) = active; (R) = remission; PNS: peripheral neuron system; CNS: central neuron system; DAH: diffuse alveolar hemorrhage; AZA: azathioprine; CCS: corticosteroids; MTX: methotrexate; CYC: cyclophosphamide; PDN: prednisone; MP: methylprednisolone. a At diagnosis.

55

+(28)

+(35)

15630 (50)

4 (P)

Characterization of clonally expanded CD8+ TCR BV T cells in EGPA

Please cite this article as: M. Boita, et al., The molecular and functional characterization of clonally expanded CD8+ TCR BV T cells in eosinophilic granulom..., Clin. Immunol. (2014), http://dx.doi.org/10.1016/j.clim.2014.03.001

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MTX 12.5 mg/week 1 (P)

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PDN 62.5 mg

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PDN 10 mg

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CD4+, CD8+ and CD8+/BV+ lymphocyte subpopulations were isolated by using immunomagnetic methods (MACS Cell Separation Microbeads, Miltenyi). PBMCs from healthy subjects and EGPA patients were resuspended in PBS with 0.5% BSA, 2 mM EDTA and incubated with microbeads for CD4+ or CD8+ populations. To isolate CD8+/BV+ expanded families from EGPA patients, the CD8+ population was isolated from PBMC by negative selection, and the CD8+ fraction was then incubated with anti-fluorescein isothiocyanate (FITC) conjugated monoclonal antibody (MoAb) specific for the expanded BV family of each patient and then with anti-FITC magnetic microbeads in order to obtain the CD8+BV+ fraction.

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2.5. Spectratyping

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Total RNA was extracted from both unfractionated PBMC and from purified CD8+ and CD4+ subpopulations after lysis in Trizol Reagent (Invitrogen Life Technologies). Five EGPA patients and 5 age-matched healthy controls were analyzed. RNA samples were reverse transcribed at 42 °C with the Reverse Transcription System (Promega, Madison, WI, USA), according to the manufacturer's instructions. Complementary DNA (cDNA) quality controls were carried out by PCR analysis of beta-2-microglobulin transcripts. Spectratyping with a twostep PCR reaction, capillary electrophoresis and software analyses were performed following the methods described by Mariani et al. [8]. From four to seven BV-specific primers and one consensus BC primer were multiplexed in five PCR reactions. The PCR mixture contained 1× Buffer (Promega), 4 mM MgCl2 (Promega), 200 mM deoxyribonucleotide triphosphates (Pharmacia, Uppsala, Sweden), 0.5 mM reverse consensus BC1 primer (MWG-Biotech AG, High Point, NC, USA), and 2 IU Taq DNA polymerase (Promega). DNAse- and RNAsefree distilled water, BV primers (MWG-Biotech AG) and the appropriate amount of T-cell derived cDNA were added to a final volume of 50 μl. The grouping and concentration of BV primers used in the multiplex polymerase chain reaction analysis have been previously described in detail [8]. Samples were amplified in a PTC-100 Thermal Cycler (MJ Research, Watertown, MA, USA) with the following PCR conditions: 10 minute denaturing time at 95 °C, 40 amplification cycles each consisting of 30 s at 94 °C, 90 s at 50 °C, and 90 s at 68 °C, followed by a final extension time of 10 min at 72 °C and cooling at 4 °C. PCR products were then stored at −20 °C. Two microliters of multiplex PCR product from each set was added to 10 μl fresh PCR reagent mix. BV-BC run-off reactions were performed using 1.3 mM BC2 primer. This was labeled with Ned, 6-Fam (Fam), or Hex and used as follows: Ned-BC2 primer for sets 1 and 3, Fam-BC2 primer for sets 2 and 4, and Hex-BC2 primer for set 5. BV-BJ run-off reactions were performed using 0.2 mM 13 separate Fam-BJ primers. Run-off reactions were run for 15 cycles under the same conditions described previously.

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For capillary electrophoresis, equal volumes of fluorescent BV-BC run-off products were pooled from sets 1 and 2, from sets 3 and 4, and from set 5. Four microliters of these pooled products or 4 μl of monoplex BV-BJ run-off products were diluted in 10.5 μl deionized formamide and 0.5 μl GeneScan500 ROX size marker (both from Applied Biosystems, Applera Italia, Monza, Italy), heated at 95 °C for 10 min for cDNA denaturation and immediately cooled on ice. Electrophoresis was run for 24 min at 60 °C for each tube through a POP-4 polymer on an ABI Prism 310 capillary sequencer (Applied Biosystems). Data were acquired on GeneScan platform and compensated for with a standard D-Set matrix (Applied Biosystems) that specifically distinguishes Ned, Fam, Hex, and Rox colors. Finally, CDR3 length profiles were determined by GeneScan or Genotyper software analyses (Applied Biosystems) through appropriate fluorophore channels.

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2.6. Spectratyping complexity scoring

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The data are reported as complexity score, which was determined by counting the number of discrete peaks per BV subfamily, and graded between 0 and 8 [15]. The overall complexity within a BV subfamily was determined by counting the number of discrete peaks per subfamily. Subfamilies were graded on a score of 0 to 8 based on the degree of complexity. Normal complexity is characterized by a Gaussian distribution of transcript sizes, which reflects the presence of polyclonal cDNA species and contains 8 to 10 peaks for each BV subfamily. A score of 0 was assigned if a subfamily was absent. A score of 1 was given if a subfamily demonstrated only a single monoclonal peak. A score of 2 was given for a biclonal profile. A subfamily was designated with a score of 3 if 3 peaks were present, etc. Finally, a score of 8 denoted a normal-appearing spectratype of 8 to 10 peaks with a complex, diverse, and polyclonal appearance. The overall spectratype complexity score per sample was calculated as the summation of the number of subfamilies per score category, with a maximum possible score of 176 (22 BV × 8). Scoring was calculated blindly without any prior knowledge regarding which group the patient belongs to. The inter-observer variability in spectratyping complexity scoring was less than 5%, as determined by a blinded review of three individual cases by two different investigators. An example of CDR3 region peaks distribution in BV families, as they appear in PBMCs and in purified CD8+ and CD4+ cells, in one patient with EGPA and in a healthy control are reported in Fig. 1.

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2.7. Molecular analysis of BV families

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Aliquots of cDNA from purified CD8 + T cells were amplified in a 20 μl reaction using primer pairs specific for BV families. Ten microliters of each PCR product was run on a non-denaturating polyacrylamide gel, stained with ethidium bromide and visualized under ultraviolet light. The migration pattern appeared as a sharp band when a dominant clone was present in the BV family; in contrast, the migration pattern appeared as a homogeneous smear when the BV population was polyclonal. To determine the BV sequence, cDNA from CD8 + T cells was re-amplified by PCR with specific BV primer pairs. The PCR product was purified and sequenced as

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extracted using the QIAamp DNA Blood Mini Kit. PCR was used to amplify the fragment of the rearranged TCR GV chain. Primer sequences, PCR cycles and the revealing method are extensively explained elsewhere [4].

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Please cite this article as: M. Boita, et al., The molecular and functional characterization of clonally expanded CD8+ TCR BV T cells in eosinophilic granulom..., Clin. Immunol. (2014), http://dx.doi.org/10.1016/j.clim.2014.03.001

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Characterization of clonally expanded CD8+ TCR BV T cells in EGPA

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described above. The length of CDR3 was determined according to Moss et al. [16].

2.8. Cloning

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The expanded families of patients EGPA-5 and EGPA-8 were cloned by limiting dilution after purification of the relevant family with magnetic microbeads. CD8 + BV + cells were plated in a 96-well plate with RPMI with 10% FBS at a concentration of 1 cell/well with an allogeneic feeder made of irradiated PBMC (105) and an irradiated lymphoblastoid cell line (2 × 104). Then, to every well, 2% PHA (GIBCO, Invitrogen), 100 U/ml IL2 (Peprotech, Inc.) and 10 ng/ml IL7 (Peprotech, Inc.) was added. The proliferating clones were subsequently expanded in RPMI medium with 10% FBS, 100 U/ml IL-2 and 10 ng/ml IL7 and restimulated every four weeks with the same protocol. After expansion, twenty CD8 + clones were selected and stained with a MoAb directed against the appropriate BV family, i.e., BV1 for patient EGPA-5 and BV8 for patient EGPA-8. Nine clones from each patient were then randomly selected for molecular analysis, and the CDR3 region of their TCR BV chain was sequenced and analyzed.

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2.9. Cytokine production

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Cytokine production was determined both by intracytoplasmatic (IC) staining and by analysis of culture supernatant. Samples were obtained from 6 EGPA patients and 6–8 age-matched healthy donors, as indicated. Peripheral blood mononuclear cells (PBMCs) were separated from whole blood by density gradient centrifugation (Lymphoprep, Nycomed, Roskilde, Denmark). For IC staining, PBMC were plated in AIM-V medium

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at 2 × 106 cells/ml and stimulated at 37 °C with phorbol 12-myristate 13-acetate (PMA) at 25 ng/ml plus ionomycin (IONO) at 500 ng/ml. Brefeldin A at 10 μg/ml was added after 5 h, and samples were incubated at 37 °C for 24 h. After incubation, cells were stained with surface markers (as indicated) and then fixed with PBS containing 2% formaldehyde. After fixation, cells were permeabilized with PBS containing 5% saponin and stained with MoAbs specific for INFγ, TNFα, TGFβ, IL10, IL4 and IL13. Samples were acquired by flow cytometry (Facs Calibur (BD Biosciences, San Josè, CA) and analyzed on the software CellQuest (BD Biosciences). For analysis of cytokine production in culture supernatants, cells were purified into CD4+ and CD8+ positive fractions, plated in AIM-V medium at 2 × 106 cells/ml and stimulated with PMA and ionomycin at 25 ng/ml and 500 ng/ml, respectively. After 24 h incubation, culture supernatants were collected and stored at −20 °C. Then, supernatant were analyzed for release of IL1b, IL2, IL5, IL-6, IL17, GM-CSF by a Multiplex Bio-Plex Technology by Bioclarma (Turin, Italy) using a Bio-Rad Multiplex Bio-Plex kit (Bio-Plex Cytokine Assay). The assay was performed according to the manufacturer's instructions.

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2.10. Chemokine receptors

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Staining for chemokine receptors was performed directly on whole blood of 6/8 patients using a panel of monoclonal antibodies (MoAbs) followed by four-color flow cytometry (Facs Calibur (BD Biosciences, San Josè, CA) and analysis using CellQuest software (BD Biosciences). The MoAbs used were CXCR3-PE (BD Pharmingen) and CCR5-PE (BD Pharmingen) for Th1 polarization and CCR4-PE (BD Pharmingen) and CRTH2 (CD294 biotin, Miltenyi, and PE-labeled streptavidin, Southern

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Figure 1 Three different BV families, as they appear by spectratyping in PBMCs and in purified CD8 + and CD4 + cells of one EGPA patient (EGPA-8) and one healthy control are reported. Number of peaks is reported in brackets. A low complexity score is observed in CD8 + cells of the EGPA patient indicating a skewed profile.

Please cite this article as: M. Boita, et al., The molecular and functional characterization of clonally expanded CD8+ TCR BV T cells in eosinophilic granulom..., Clin. Immunol. (2014), http://dx.doi.org/10.1016/j.clim.2014.03.001

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305 306 307 308 309 310 311

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2.12. Statistical analysis

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All statistical analyses were performed using GraphPad InStat software (GraphPad Software, San Diego, CA). The normal distribution of variables was assessed according to KolmogorovSmirnov's test of normality. In the case of non-normal distribution, the variables were computed as logarithmic. Comparisons between continuous variables were estimated by

326 327 328 329 330 331

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t2:1 t2:2 t2:3

Table 2

t2:4

Patient #

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3.1. TCR BV and GV repertoire

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PBMC from 8 EGPA patients were collected at two different time points. Flow cytometric analysis of TCR BV families was performed on peripheral CD4 + and CD8 + T cells at the first time point, as indicated in Table 1. The expansion of one or more BV families, as well as clonal TCR GV rearrangements, were observed in all patients (Table 2), suggesting that several BV families are monoclonal or oligoclonal. In all patients, the stability of TCR BV expansions over time has been observed with a mean follow-up of 26 months.

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3.2. Skewed TCR BV repertoire

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The degree of clonality of the whole T cell repertoire was investigated in 5 EGPA patients and in 5 age-matched healthy controls by spectratyping. The data are reported as a complexity score, which was determined by counting the number of distinct peaks and by summing all 22 individual TCR BV family-specific single scores. The complexity score was significantly lower (p = 0.016) in the purified CD8+ subpopulation of EGPA patients compared to healthy controls, whereas the difference in complexity score of whole PBMC and purified CD4+ cells was not significant (Fig. 2). The low complexity score of CD8+ BV families in EGPA patients was due to the high

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TCR BV expansions and the related molecular analysis of TCR GV rearrangement from PBMCs of EGPA patients. CD8 +

R

320

Expanded

R

319

343

F

333

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3. Results

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Genomic DNA was extracted from EDTA (5 ml)–treated peripheral blood samples using the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA) and stored at −20 °C until further use. Low-resolution genotyping for HLA-A, HLA-B, and HLA-DR loci was performed by polymerase chain reaction (PCR) using sequence-specific primers (One Lambda, Canoga Park, CA); 21 alleles in HLA-A genes, 35 in HLA-B genes, and 13 in HLA-DR genes were investigated. The laboratory in which the HLA genotyping was performed is certified by the European Foundation for Immunogenetics.

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paired and unpaired Student's t-tests or the Mann–Whitney test, depending on the distribution of variables. Categorical variables were compared with Fisher's exact test.

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Biotechnology Associates Inc.) for Th2 polarization, combined with a FITC-labeled specific antibody of the different numerically expanded Vβ family of each patient (Immunotech), with a CD4-PE-Cy7 (PE-Cyanin 7, Beckman Coulter) and a CD8-APC (Miltenyi). All the samples were analyzed by flow cytometry by gating on light scatter parameter in Region 1 (R1) to discriminate lymphocytes within PBMCs. The R1 gate was combined with a gate on CD8 +/BV+ cells (R2) to better analyze every single BV family. Six normal age-matched controls were similarly analyzed and compared to patients.

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t2:5

CD4 +

TCR GV-J clonality

Expanded

Rearrangement

BV family

BV family

BV4 BV20 BV5.3 BV3 BV18

–

Vγ9 Jγ1/2

– –

BV13.1 BV18

BV4

BV1 BV16

BV18 BV22 BV12 BV14 BV23 –

Vγ10–11 Jγ1/2 Vγ1–8 Jγ1/2 Vγ1–8 JP1/P2 Vγ9 Jγ1/2 Vγ1–8 Jγ1/2 Vγ1–8 JP1/P2 Vγ10–11 Jγ1/2 Vγ10–11 JP1/P2 Vγ1–8 Jγ1/2 Vγ1–8 JP1/P2

1

t2:7 t2:8

2 3

t2:9

4

t2:10

5

t2:11

6

BV23

t2:12

7

t2:13

8

BV1 BV13.6 BV8

t2:14

A BV family was considered expanded when the percentage of BV positive cells was three standard deviations higher than the reference values4.

Q3 t2:15

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N C O

t2:6

–

Vγ1–8 JP1/P2 Vγ1–8 Jγ1/2 Vγ1–8 JP1/P2 Vγ1–8 JP1/P2 Vγ10–11 Jγ1/2

Please cite this article as: M. Boita, et al., The molecular and functional characterization of clonally expanded CD8+ TCR BV T cells in eosinophilic granulom..., Clin. Immunol. (2014), http://dx.doi.org/10.1016/j.clim.2014.03.001

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Characterization of clonally expanded CD8+ TCR BV T cells in EGPA

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To confirm the clonality of expanded TCR BV families, the purified BV+/CD8+ T cells of two patients (BV1 for EGPA-5 and BV8 for EGPA-8) were cloned by limiting dilution (Table 2). Twenty CD8 + clones were selected and expanded after checking the correct expression of the BV family by staining with a specific MoAb. Nine clones from each patient were then randomly selected, and the CDR3 region of TCR BV chain was sequenced and analyzed [17–20]. Seven out of nine clones of patient EGPA-5 showed the same VDJ rearrangement of BV chain, and 4/5 clones had the same VJ rearrangement of AV chain, i.e., they had an identical sequence of their CDR3 region (Table 3). Additionally, in patient EGPA-8, 8/9 clones had identical CDR3 sequence of TCR BV chain, and 3/3 selected clones with identical BV had also identical VA chain (data not shown). Similar to individual T cell clones, the cDNA obtained from whole PBMCs of patient EGPA-5 (Table 3) and EGPA-8 (not shown) was also amplified with primers specific for the relevant BV family and then sequenced. The sequence obtained was identical to that observed in each T-cell clone. These data indicate the presence of a dominant clonotype, molecularly detectable, within the expanded BV families of EGPA patients.

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To determine whether BV families with a skewed repertoire were the result of a clonal expansion, 11 BV families of patient EGPA-5, which had a complexity score b 5 by spectratyping, were selected. cDNA from CD8 + purified cells was amplified with specific primer pairs and run on a polyacrylamide gel. Gel analysis revealed the presence of five discrete bands for the following BV families: BV1 (the BV family expanded at flow

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Cytokine production and chemokine receptor expression were explored on peripheral CD4 + and CD8 + T cells at the 2nd time point, as indicated in Table 1. The intracellular production of cytokines, which is reported as the percentage of CD4 + and CD8+ cells expressing each cytokine, is displayed in Figs. 4, panels A, B and C. Compared to healthy controls, a significantly higher percentage of both CD8+ and CD4 + cells producing INFγ and TNFα (Fig. 4, panel A) and a significantly higher percentage of CD4 + cells producing IL10 (Fig. 4, panel C) was observed in EGPA patients. Cytokine measurements in the culture supernatant of purified CD4 + and CD8 + cells showed a significant increase of IL5 in CD4 + cells obtained from EGPA patients compared to healthy controls (see Fig. 4, panel D). All other cytokines analyzed did not reveal any significant differences compared to healthy controls. Interleukin 17 was markedly increased in two patients, but there was a great variability in IL17 production even among healthy controls, which hampered a meaningful statistical analysis (Fig. 4, panel D).

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The expression of CCR5, CXCR3, CRTH2 and CCR4 observed in CD8 +/BV + expanded cells and in total CD8 + and CD4 + cells are reported in Fig. 5. The mean expression of CCR5 was significantly higher in CD8 + cells as well as in the expanded CD8 +/BV + fraction of EGPA patients, compared to agematched healthy controls (p = 0,020 and p = 0.006, respectively). No difference between chemokine receptors expression of expanded CD8 + cells compared to total CD8 + cells of EGPA patients had been found. Mean expression of CRTH2 was significantly higher in CD4 + cells of EGPA patients than in controls (p = 0.034).

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cytometry), BV3, BV16, BV18 and BV22. Gel purification and sequencing allowed us to determine that BV1 and BV22 had a clonal VDJ rearrangement of the CDR3 region. As expected, the sequence obtained from amplified BV1 of CD8+ cells was the same as that identified by sequencing CD8+/BV1+ T-cell clones (see Table 3).

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number of BV families with a skewed profile. A representative spectratyping analysis of the TCR repertoire in CD8+ cells of one EGPA patient (EGPA-7) compared to one age-matched healthy subject is reported in Fig. 3. BV6, 13, 18 and 22 were the most commonly observed skewed families (data not shown). All the CD8 + BV families that were expanded at flow cytometry (see Table 2) were also found to be skewed by spectratyping.

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Figure 2 Means and single values of the complexity score of spectratyping of BV families. Complexity score is reported for PBMCs, CD8 + and CD4 + purified subpopulation of EGPA patients and healthy controls (CTRL). Please cite this article as: M. Boita, et al., The molecular and functional characterization of clonally expanded CD8+ TCR BV T cells in eosinophilic granulom..., Clin. Immunol. (2014), http://dx.doi.org/10.1016/j.clim.2014.03.001

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Figure 3 Representative spectratyping analysis of TCR repertoire in CD8 + and CD4 + populations from one EGPA patient (EGPA-7) compared to one age matched healthy subject. A high number of BV families show a skewed profile in the CD8 + but not in the CD4 + cells from the EGPA patient. Please cite this article as: M. Boita, et al., The molecular and functional characterization of clonally expanded CD8+ TCR BV T cells in eosinophilic granulom..., Clin. Immunol. (2014), http://dx.doi.org/10.1016/j.clim.2014.03.001

Characterization of clonally expanded CD8+ TCR BV T cells in EGPA

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t3:1 t3:2 t3:3

Table 3

t3:4

Clone

V-gene

D-gene

J-gene

CDR3 aa sequence

t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 t3:13 t3:14 t3:15

3C5 4B9 4D11 4E6 5B8 9F6 10F9 10G9 11D9 PBMC

TRBV9*01 TRBV9*01 TRBV9*01 TRBV9*01 TRBV9*01 TRBV9*01 TRBV9*01 TRBV9*01 TRBV9*01 TRBV9*01

TRBD1*01 TRBD1*01 TRBD1*01 TRBD1*01 TRBD1*01 TRBD2*01 / TRBD1*01 TRBD1*01 TRBD1*01

TRBJ2-2*01 TRBJ2-2*01 TRBJ2-2*01 TRBJ2-2*01 TRBJ2-2*01 TRBJ2-1*01 TRBJ1-1*01 TRBJ2-2*01 TRBJ2-2*01 TRBJ2-2*01

CASSPFRDRGTGELFF CASSPFRDRGTGELFF CASSPFRDRGTGELFF CASSPFRDRGTGELFF CASSPFRDRGTGELFF LCQQQLPMSSFF CASSPPRSTEAFF CASSPFRDRGTGELFF CASSPFRDRGTGELFF CASSPFRDRGTGELFF

BV and AV CDR3 region sequence of clones from purified BV1 cells in patient #5 (EGPA-5).

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chainsequence sequence AVAVchain

t3:17

Clone Clone

V-geneV-gene

J-gene

J-gene

t3:18 t3:19 t3:20 t3:21 t3:22

3C5 3C5 4E6 4E6 11D9 11D9 4D11 4D11 4B9 4B9

TRAV35*02 TRAV35*02 TRAV35*02 TRAV35*02 TRAV35*02 TRAV35*02 TRAV35*02 TRAV35*02 TRAV35*02 TRAV35*02

TRAJ58*01 TRAJ15*01 TRAJ58*01 TRAJ58*01 TRAJ58*01

TRAJ58*01 TRAJ15*01 TRAJ58*01 TRAJ58*01 TRAJ58*01

t3:23 t3:24 t3:25 t3:26 t3:27

7/9clones cloneshad hadthe thesame sameVDJ VDJrearrangement rearrangementofoftheir theirBV BVchain; chain; 4/5 4/5 clones clones (with (with the same VB sequence) had 7/9 had the the same sameVJ VJrearrangement rearrangementof of theirAVAVchain. chain.This Thismeans meansthat thatthe the4 4clones cloneshad hadananidentical identicalsequence sequenceofoftheir theirCDR3 CDR3region. region. their PCR productsobtained obtainedfrom fromwhole wholePBMC PBMCamplified amplifiedwith withBV1 BV1were weresequenced. sequenced.We Wefound foundthat thatthe thesequence sequenceobtained obtainedfrom fromthe theamplification amplificationof PCR products of BV1 the BV1 family identical to that obtained T-cell clone. the family was was identical to that obtained fromfrom eacheach T-cell clone. IMGTnomenclature: nomenclature:reference referenceno. no.16–19. 16–19. IMGT

The principal original result of our study is that CD8+ T cells from EPGA patients are clonally expanded and have a proinflammatory profile, according to chemokine receptor expression and cytokine production. Alternately, CD4+ T cells are less clearly polarized, but a significant fraction of CD4 T cells produce IL5 and express CRTH2, a marker of Th2 differentiation, in agreement with previous reports [3]. These results may implicate a pathogenic role of CD8 + T cells in vasculitic damage and a role of CD4+ T cells in driving eosinophilia and eosinophilic tissue infiltration. Recently, the increased production of IgG4 in EGPA as a consequence of the Th2 response has been demonstrated [21]. In the present study, we extend our previous observation on expanded BV families of CD8+ cells from EPGA patients, which were found to have TCR GV rearrangements suggestive of clonality [4]. In all patients stability of TCR BV expansions over time have been observed with a mean follow-up of 26 months, despite immunosuppressive therapy. The degree and extent of clonality have now been analyzed at a deeper level. First, by spectratyping, we evaluated the CDR3 length distribution in each BV family [8]. In EGPA patients, the BV complexity score of CD8+ purified cells, but not of CD4+ cells, was significantly lower than in healthy donors, confirming a clonal drift of the CD8 + population. In fact, several CD8+/BV+ families showed one or more dominant peaks at spectratyping, suggesting a broader disruption of the T cell repertoire and a higher degree of clonality than initially expected. The data obtained by spectratyping, though, are insufficient to prove

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CAGRETSGSRLTF CAGRETSGSRLTF CAGRETQVASRLTFCAGRETQVASRLTF CAGRETSGSRLTF CAGRETSGSRLTF CAGRETSGSRLTF CAGRETSGSRLTF CAGRETSGSRLTF CAGRETSGSRLTF

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CDR3 aa sequenceCDR3 aa sequence

true clonality, because a dominant peak may originate either from the expansion of a single clone expressing a specific TCR or from a polyclonal population of cells with different TCRs of the same CDR3 length. The sequencing of multiple TCR-BV derived from individual T cell clones was therefore performed to establish the presence of a unique CDR3 nucleotide rearrangement within a BV skewed family and an identical CDR3 sequence was found in the majority of the randomly derived clones. Moreover, the identical CDR3 sequence was also found in the whole PBMC population. These data indicate the presence of a dominant clonotype within expanded BV families (BV1 and BV8 for patient EGPA-5 and EGPA-8, respectively). Interestingly, we also identified a dominant clonotype in a skewed but non-expanded BV family (BV22 of patient EGPA-5), suggesting the presence of other unsuspected clonal expansions. Whether the loss of heterogeneity in the CD8 + T cell repertoire may be related to previous immunosuppressive therapies cannot be excluded, as we have no data on treatment-naïve patients. Indeed, T cell clonal expansions should be diminished by immunosuppressive treatment, as reported in two cases by Muschen et al. [11]. The clonal TCR of expanded BV families suggests that an antigen-driven selection has occurred [22]. In five out of seven EGPA patients, Muschen et al. reported clonally expanded T cells, all using a BV-gene from the BV21 family with similar CDR3 motifs, suggesting the predominance of T cell clones of a similar specificity in the EGPA patients [11]. Nevertheless, the overall heterogeneity of expanded BV families observed in patients with different HLA genotypes

Please cite this article as: M. Boita, et al., The molecular and functional characterization of clonally expanded CD8+ TCR BV T cells in eosinophilic granulom..., Clin. Immunol. (2014), http://dx.doi.org/10.1016/j.clim.2014.03.001

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Figure 4 Means and single values of intracellular (panel A: INFγ and TNFα; panel B: IL13 and IL4; panel C: IL10 and TGFβ) and supernatant (panel D: IL5 and IL17) production of cytokines by total CD8 + and CD4 + cells in EGPA patients compared to healthy controls (CTRL). Please cite this article as: M. Boita, et al., The molecular and functional characterization of clonally expanded CD8+ TCR BV T cells in eosinophilic granulom..., Clin. Immunol. (2014), http://dx.doi.org/10.1016/j.clim.2014.03.001

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The authors declare that there are no conflicts of interest.

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reported higher expression of NKG2D receptor and CD107a on circulating CD8+ T cells and BV expanded CD8+ in EGPA suggesting a cytotoxic role of these cells in target tissues, through an NK cell-like mechanism [29]. Our results suggest that circulating CD8+/BV+ TEM cells of EGPA patients are antigen-driven cells, which may infiltrate target organs and participate in the inflammatory cascade of both vasculitic and granulomatous lesions. Alternatively, the CD4 + lymphocytes of our patients have been shown to be polarized toward a Th2 profile, as indicated by CRTH2 expression and by their increased production of IL5. These data confirm the role of CD4 + lymphocytes in promoting eosinophilia. The role of CD4 + cells in EGPA has been extensively analyzed by Jakiela et al. [3], who found not only increased levels of IL5 and CCR4 but also of IL17, suggesting Th17 cells could be responsible for maintaining inflammatory reaction in EGPA. We found increased levels of IL17 only in two patients (EGPA-4 and EGPA-8), both showing active or persistent disease, while low levels of IL-17 were observed in the patients (EGPA-2 and EGPA-3) who were in remission at the time of cytokine analysis. Therefore, our data also suggest that IL17 plays an important role during active disease. In conclusion, clonal restricted effector CD8 + lymphocytes with a proinflammatory profile have been described for the first time in patients with EGPA, where they may be involved in vasculitic and granulomatous lesions. In the same patients, as expected, CD4 + cells with a Th2 profile were observed, confirming their role in promoting eosinophilic inflammation and IgG subclass switching from IgG1 to IgG4, as recently reported [21].

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(see Table 1) suggests that different antigens have shaped the BV repertoire, promoting clonal expansion of different TCRs. Based on the recently proposed pathogenic mechanisms of AAV [23], a role of bacterial fragments, apoptotic bodies from inflammatory cells or endothelial cells can be hypothesized [24]. Further, in vitro studies on T cell clones and on the antigen-presenting cells of EGPA patients could support this hypothesis. Analysis of cytokine production and chemokine receptor expression revealed a proinflammatory functional profile of CD8 + T cells in our patients, which was not specific of the expanded CD8 + T cells, as it was observed also in the whole CD8 + T cells of our patients, suggesting the changes in CD8 + T cells are not limited to antigen-specific cells. Upregulated CCR5 was observed both in BV expanded and in total CD8 + cells, which were the source of INFγ and TNFα. This is the first observation of proinflammatory polarized CD8 + cells reported in EGPA. Our hypothesis is that mono/oligoclonal expanded populations of effector CD8 + T cells are involved in vasculitic and granulomatous damage observed in the disease. In granulomatosis with polyangiitis (GPA), T cells have been shown to be the major source of IFNγ and TNFα, crucial cytokines for granulomatous inflammatory reaction [25]. Most of these cells are memory T cells, express CCR5 and CCR3 [26], and migrate from the circulation to the target tissue, where they act as effector cells [27]. We also found that in EGPA, CD8 + cells producing IFNγ and TNFα have an effector memory phenotype (membrane expression of CXCR3 and CCR5). We have previously shown that in EGPA, the CD8 +/BV+ expanded populations had an effector memory phenotype (TEM), as demonstrated by CD45RA membrane expression, combined with CD62L, CCR7 or CD28 [4]. TEM cells are antigenexperienced T cells, characterized by the low expression of CD62L and CCR7, which are suitable for migration into peripheral and inflammatory sites [28]. We have recently

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Figure 5 Means and single values of the expression of CCR5, CXCR3, CRTH2 and CCR4 in CD8 +/BV + expanded cells and in total CD8 + and CD4 + cells of EGPA patients compared to healthy controls (CTRL).

Please cite this article as: M. Boita, et al., The molecular and functional characterization of clonally expanded CD8+ TCR BV T cells in eosinophilic granulom..., Clin. Immunol. (2014), http://dx.doi.org/10.1016/j.clim.2014.03.001

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