J ALLERGY CLIN IMMUNOL VOLUME 134, NUMBER 5

for classic allele imputation. Consequently, we analyzed only 56% of common classic alleles in this population (see Online Repository text). It is possible that we missed other interesting associations, as suggested by the fact that a weighted score including the 2 SNPs associated with asthma explained slightly higher phenotypic variance than did a model including the classic alleles (Nagelkerke’s R2 5 0.14 vs 0.10, respectively). In summary, a deeper examination of HLA genes has revealed a classic allele that shows pleiotropic effects for asthma and other immune-related diseases and likely constitutes a putative causal variant. Analysis of SNPs also revealed shared genetic risk factors between asthma and lipid levels. Finally, we provided evidence supporting the role of HLA polymorphisms in specific allergic sensitization. We thank Alexander Dilthey, Gonc¸alo Abecasis, and Christian Fuchsberger for their help with software tools, Servicio de Apoyo Inform
atico a la Investigaci
on (ULL) for the HPC support, and Katherine K. Nishimura for proofreading the manuscript. Mar
ıa Pino-Yanes, PhDa,b,c Almudena Corrales, CLT a,b Marialbert Acosta-Herrera, MSca,b,d Eva P
erez-Rodr
ıguez, MDe Jos
e Cumplido, MD f Paloma Campo, MD, PhDg Amalia Barreto-Luis, BSa,b Florentino S
anchez-Garc
ıa, MD, PhDh Tob
ıas Felipe, PhDi Inmaculada S
anchez-Mach
ın, MD j In
es Quintela, MSck Jos
e Carlos Garc
ıa-Robaina, MDe Jes
us Villar, MD, PhDa,d Miguel Blanca, MD, PhDg Angel Carracedo, MD, PhDl,m Teresa Carrillo, MD f Carlos Flores, PhDa,b,n From aCIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain; bResearch Unit, Hospital Universitario N.S. de Candelaria, Tenerife, Spain; c the Department of Medicine, University of California, San Francisco, Calif; dthe Research Unit, Multidisciplinary Organ Dysfunction Evaluation Research Network (MODERN), Hospital Universitario Dr Negrin, Gran Canaria, Spain; ethe Allergy Unit, Hospital Universitario N.S. de Candelaria, Tenerife, Spain; fAllergy Unit, Hospital Universitario Dr Negr
ın, Gran Canaria, Spain; gAllergy Service, Carlos Haya Hospital, Malaga, Spain; hthe Immunology Unit, Hospital de Gran Canaria Dr Negr
ın, Gran Canaria, Spain; iNorthWest Research Associates, Boulder, Colo; j the Allergy Unit, Hospital del T
orax, Complejo Hospitalario Universitario NS Candelaria, Tenerife, Spain; kGrupo de Medicina Xen
omica, CEGEN-ISCIIIUniversidade de Santiago de Compostela, Santiago de Compostela, Spain; lGrupo de Medicina Xen
omica, Fundaci
on Galega de Medicina Xen
omica (SERGAS)CIBERER-Universidade de Santiago de Compostela, Santiago de Compostela, Spain; m Center of Excellence in Genomic Medicine Research, King Abdulaziz University, Jeddah, Saudi Arabia; and nApplied Genomics Group (G2A), Genetics Laboratory, Instituto Universitario de Enfermedades Tropicales y Salud P
ublica de Canarias, Universidad de La Laguna, Tenerife, Spain. E-mail: [email protected]. This work was supported by grants from the Health Institute ‘‘Carlos III’’ (grant no. FIS PI08/1383 and grant no. FIS PI11/00623) and cofinanced by the European Regional Development Funds, ‘‘A way of making Europe’’ from the European Union, and by a specific agreement between Instituto de Salud Carlos III and Gobierno de Canarias (grant no. EMER07/001). M. Pino-Yanes was supported by a postdoctoral fellowship from Fundaci
on Ram
on Areces. M. Acosta-Herrera and A. Barreto-Luis were supported by fellowships from the Instituto de Salud Carlos III (grant no. FI11/00074 and grant no. FI12/00493, respectively). Disclosure of potential conflict of interest: M. Pino-Yanes has received funding from the Fundaci
on Ram
on Areces. A. Carracedo is employed at the University of Santiago, which has received funding from the Ministry of Justice, the Ministry of Health, and the European Union. The rest of the authors declare that they have no relevant conflicts of interest.

LETTERS TO THE EDITOR 1203

REFERENCES 1. Gregersen PK, Behrens TW. Genetics of autoimmune diseases–disorders of immune homeostasis. Nat Rev Genet 2006;7:917-28. 2. Ober C, Hoffjan S. Asthma genetics 2006: the long and winding road to gene discovery. Genes Immun 2006;7:95-100. 3. Hirota T, Takahashi A, Kubo M, Tsunoda T, Tomita K, Doi S, et al. Genome-wide association study identifies three new susceptibility loci for adult asthma in the Japanese population. Nat Genet 2011;43:893-6. 4. Li X, Howard TD, Zheng SL, Haselkorn T, Peters SP, Meyers DA, et al. Genome-wide association study of asthma identifies RAD50-IL13 and HLA-DR/ DQ regions. J Allergy Clin Immunol 2010;125:328-35.e11. 5. Moffatt MF, Gut IG, Demenais F, Strachan DP, Bouzigon E, Heath S, et al. A large-scale, consortium-based genomewide association study of asthma. N Engl J Med 2010;363:1211-21. 6. Dilthey A, Leslie S, Moutsianas L, Shen J, Cox C, Nelson MR, et al. Multi-population classical HLA type imputation. PLoS Comput Biol 2013;9: e1002877. 7. Pino-Yanes M, Sanchez-Machin I, Cumplido J, Figueroa J, Torres-Galvan MJ, Gonzalez R, et al. IL-1 receptor-associated kinase 3 gene (IRAK3) variants associate with asthma in a replication study in the Spanish population. J Allergy Clin Immunol 2012;129:573-5.e10. 8. Leslie S, Donnelly P, McVean G. A statistical method for predicting classical HLA alleles from SNP data. Am J Hum Genet 2008;82:48-56. 9. Li X, Ampleford EJ, Howard TD, Moore WC, Torgerson DG, Li H, et al. Genome-wide association studies of asthma indicate opposite immunopathogenesis direction from autoimmune diseases. J Allergy Clin Immunol 2012;130: 861-8.e7. 10. Noguchi E, Sakamoto H, Hirota T, Ochiai K, Imoto Y, Sakashita M, et al. Genome-wide association study identifies HLA-DP as a susceptibility gene for pediatric asthma in Asian populations. PLoS Genet 2011;7:e1002170. 11. Hinds DA, McMahon G, Kiefer AK, Do CB, Eriksson N, Evans DM, et al. A genome-wide association meta-analysis of self-reported allergy identifies shared and allergy-specific susceptibility loci. Nat Genet 2013;45:907-11. Available online July 14, 2014. http://dx.doi.org/10.1016/j.jaci.2014.05.031

Circulating endothelial cells as markers of endothelial dysfunction during hematopoietic stem cell transplantation for pediatric primary immunodeficiency To the Editor: Endothelial damage is the pathological hallmark of numerous acute complications occurring during hematopoietic stem cell transplantation (HSCT): sinusoidal obstruction syndrome (SOS), transplantation-associated thrombotic microangiopathy (TATMA), capillary leak syndrome (CLS), and pulmonary arterial hypertension (PAH).1 These disorders remain an important cause of morbidity and mortality, despite improvement in their prevention and their therapy. Many factors are thought to induce endothelial dysfunction: the underlying disease, the conditioning regimen, the immunosuppressive therapy, the intercurrent infections, and graft-versus-host disease (GVHD).1-3 Circulating endothelial cells (CECs) are specific and sensitive markers of endothelial dysfunction. They have been used in a variety of pathological conditions to monitor the occurrence of vascular damage.4 Because vascular complications impact the outcome of HSCT for several types of primary immunodeficiency (PID), we decided to monitor endothelial dysfunction during HSCT for PID through the measurement of CECs. Thirty-four pediatric patients treated in the department of Pediatric Immuno-Hematology at the Necker-Enfants Malades Hospital who received allogeneic HSCT after full-intensity conditioning regimen for PID were enrolled between April 2011 and December 2013. Informed consent for participation in the study was obtained from the children’s parents in accordance

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TABLE I. Hematopoietic transplantation characteristics

Sex ratio \/_ Age: median (range) Diagnosis SCID CID HLH Neutrophil disorders Donor Matched sibling donor MUD MMUD Haploidentical donor Defibrotide conditioning regimen Bu/Flu/ATG Bu/Flu/ThioTEPA/ATG Mel/Flu/Alemtuzumab Graft characteristics In vitro T-depletion No in vitro T-depletion CD34 cell dose (106/kg) Engraftment day Rejection GVHD Grade 1-2 Grade 3-4 Time onset (days) CMV systemic replication Severe sepsis Duration of hospitalization post-HSCT (days) Death

No vascular event (n 5 23)

Vascular event (n 5 11)

Total patients (n 5 34)

9/14 3 y (0.5-14 y)

3/8 0.9 y (0.25-9 y)

12/22 3 and 5 y (0.25-14 y)

6 8 5 3

2 5 3 1

8 13 8 5

7 7 4 5 8 16 6 1

0 5 2 4 9 6 5 0

7 12 6 9 17 22 11 1

5 18 8.2 (1.4-24.2) 19 (9-49) 2 8 6 2 16.5 (2-60) 4 2 78 (38-212) 3

6 5 13 (5-24.2) 18.5 (13-31) 1 3 1 2 27 (10-47) 3 1 92 (56-155) 5

11 23 10.5 (1.4-24.2) 19 (9-49) 3 11 7 4 17 (2-60) 7 3 83 (38-212) 8

For CD34 number, engraftment, time of onset of GVHD, and duration of post-HSCT hospitalization, median time is indicated with range in brackets. ATG, Antithymocyte globulin; Bu, busulfan; CID, combined immunodeficiency; CMV, cytomegalovirus; Flu, fludarabine; HLH, hemophagocytic lymphohistiocytosis; Mel, Melphalan; MMUD, mismatched unrelated donor; MUD, matched unrelated donor; SCID, severe combined immunodeficiency.

with French regulatory requirements and the Declaration of Helsinski. CECs were monitored on fresh total blood samples within 6 hours after the samples were taken. The quantification of CECs was performed according to the consensus isolation and enumeration protocol.5 The monitoring was performed at the time of transplant (day 0), day 15, day 30, and eventually day 60 posttransplant if the patient was still hospitalized. Blood samples were not collected within 5 days of a severe sepsis to avoid any confounding factor.6 Conditioning regimens were performed according to the guidelines of the European Group for Blood and Marrow Transplantation for primary immunodeficiency and metabolic disease. GVHD prophylaxis consisted of in vitro T-cell depletion in case of haploidentical or mismatched unrelated donor, or a combination of cyclosporine with mycophenolate mofetil in case of matched sibling or matched unrelated donor. All patients received ursodeoxycholic acid as SOS prophylaxis from the start of the conditioning regimen until day 60. Young patients (aged 2/hpf, negative Coombs test, and elevated blood pressure. CLS was defined as a clinico-biological state of hypotension, hypovolemia, generalized edema, and hypoalbuminemia. Statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, Inc, La Jolla, Calif) with Mann-Whitney U test and Fisher exact test. The median age at transplant was 3.5 years (the range was 3 months to 14 years), with a large male predominance. Indication for HSCT included severe, combined immunodeficiencies (8 patients), combined immunodeficiencies (13 patients), hemophagocytic lymphohistiocytosis (8 patients), and neutrophil disorders (5 patients). The patients’ characteristics are summarized in Table I. There was no significant difference between the patients with or without vascular events (VE) on univariate analysis, except for the younger age of the population presenting vascular complication (P 5 .04). Vascular complications occurred in 11 patients (32%) and are detailed in Fig 1, C. Of note, all patients who developed SOS were under defibrotide prophylaxis. Two patients developed CLS in association with grade IV GVHD. TA-TMA occurred in 2 patients and resolved in both after cyclosporine withdrawal. Patients with VE had significantly higher CEC counts (median 5 33/mL; range: 5.5-305) as compared to patients without VE (median 5 2/mL;

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LETTERS TO THE EDITOR 1205

FIG 1. Comparison of CEC counts (A) between patients presenting with or without VE at day 0, 15, and 30 post-transplant. CEC counts increase (B) significantly during the first month (DD0-D30) post-transplant in patients with VE as compared with patients without VE. Statistical analyses were performed with MannWhitney U test. VE characteristics (C). CEC count kinetics in patients with CLS (D). Vertical arrows symbolize onset of clinical symptoms. **P 5 .001-.01.

range: 0-34) at day 15 (P 5.005). The CEC count was persistently higher at day 30 in patients with VE (median 5 98/mL; range: 1-548) as compared to patients without VE (median 5 4/mL; range: 2-52; P 5 .002) (Fig 1, A). Moreover, the increase of CEC counts was significantly higher in patients with VE during the first month post-transplant (median DD0-D30 5 92; range: 16.5-547; P 5 .007) as compared to patients without (Fig 1, B). Among the patients without VE, all but 2 patients had a CEC count lower than 15/mL at day 15 (sensitivity: 55%, specificity: 91%). None of the patients without VE had a CEC level above 60/mL during the entire follow-up, while all patients who developed endothelial complication had at least 1 blood sample with CEC count above this threshold (sensitivity and specificity: 100%). Only 2 patients with VE had low CEC counts at day 30; they developed PAH at day 73 and 102 and had abnormal CEC counts measured at day 60 (77/mL and 125/mL). In our study, CEC counts failed to efficiently predict the onset of SOS. However, high CEC counts were detected before the onset of other vascular complications in 7 patients out of 8 (Fig 1, C). Of note, no correlations on univariate analysis were found between CEC counts and the following variables: underlying disease, intensity of conditioning regimen,7 occurrence of cytomegalovirus reactivation, defibrotide prophylaxis, GVHD, and type of complication or death.

In summary, we show that high CEC counts are associated with the occurrence of endothelial complications in children undergoing HSCT for PID. These encouraging preliminary results warrant the initiation of larger studies with tighter monitoring schedules to validate the extensive use of CEC enumeration as a predictive tool of vascular complication in high-risk patients. Fabien Touzot, MD, PhDa,b Despina Moshous, MD, PhDb,c Guilhem Cros, MDb,c Pierre Frange, MD, PhDd,e Maryline Chomton, MDb,c Marie-Louise Fr
emond, MDb,c B
en
edicte Neven, MD, PhDb,c Marina Cavazzana, MD, PhDa,b Alain Fischer, MD, PhDb,c,f St
ephane Blanche, MDb,c Dominique Helley, MD, PhDg,h From the aD
epartement de Bioth
erapie, Centre d’Investigation Clinique Int
egr
e en Bioth
erapies, H^opital Necker—Enfants Malades, Assistance Publique-H^opitaux de Paris, Paris, France; bUniversit
e Paris Descartes—Sorbonne Paris Cit
e, Institut Imagine, Paris, France; cUnit
e d’Immunologie-H
ematologie et Rhumatologie P
ediatrique, H^opital Necker-Enfants Malades, Assistance Publique-H^opitaux de Paris, Paris, France; dLaboratoire de Microbiologie, H^opital Necker—Enfants Malades, Assistance Publique-H^opitaux de Paris, Paris, France; eEA 3620, Universit
e Paris Descartes—Sorbonne Paris Cit
e, PRES Sorbonne Paris, Paris, France; fCollege de

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France, gService d’H
ematologie Biologique, H^opital George Pompidou, Assistance Publique-H^ opitaux de Paris, Paris, France; hUniversit
e Paris Descartes—Sorbonne Paris Cit
e, Paris, France. E-mail: [email protected]. This work was supported by the Assistance Publique-H^opitaux de Paris. Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest. REFERENCES 1. Carreras E, Diaz-Ricart M. The role of the endothelium in the short-term complications of hematopoietic SCT. Bone Marrow Transplant 2011;46:1495-502. 2. Palomo M, Diaz-Ricart M, Carbo C, Rovira M, Fernandez-Aviles F, Martine C, et al. Endothelial dysfunction after hematopoietic stem cell transplantation: role of the conditioning regimen and the type of transplantation. Biol Blood Marrow Transplant 2010;16:985-93. 3. Biedermann BC, Sahner S, Gregor M, Tsakiris DA, Jeanneret C, Pober JS, et al. Endothelial injury mediated by cytotoxic T lymphocytes and loss of microvessels in chronic graft versus host disease. Lancet 2002;359:2078-83. 4. Erdbruegger U, Dhaygude A, Haubitz M, Woywodt A. Circulating endothelial cells: markers and mediators of vascular damage. Curr Stem Cell Res Ther 2010;5:294-302. 5. Woywodt A, Blann AD, Kirsch T, Erdbruegger U, Banzet N, Haubitz M, et al. Isolation and enumeration of circulating endothelial cells by immunomagnetic isolation: proposal of a definition and a consensus protocol. J Thromb Haemost 2006;4:671-7. 6. Schlichting DE, Waxman AB, O’Brien LA, Wang T, Naum CC, Rubeiz GJ, et al. Circulating endothelial and endothelial progenitor cells in patients with severe sepsis. Microvasc Res 2011;81:216-21. 7. Woywodt A, Scheer J, Hambach L, Buchholz S, Ganser A, Haller H, et al. Circulating endothelial cells as a marker of endothelial damage in allogeneic hematopoietic stem cell transplantation. Blood 2004;103:3603-5. Available online July 16, 2014. http://dx.doi.org/10.1016/j.jaci.2014.05.039

Enhanced CD46-induced regulatory T cells suppress allergic inflammation after Dermatophagoides pteronyssinus–specific immunotherapy To the Editor: Induction of CD41 naturally occurring regulatory T (Treg) cells was associated with successful allergen-specific immunotherapy (SIT).1 Adaptive regulatory T (TR1) cells, which secrete immunosuppressive cytokines, might play an important role in the pathogenesis of asthma to maintain immune tolerance.1,2 Several reports have shown that the complement system is activated locally and systemically in patients with allergic asthma.3 The complement regulatory protein CD46 has been reported to be upregulated in certain activated leukocyte subsets to protect cells from autologous complement-mediated lysis at inflammatory sites.4 Cross-linking of CD46 during T-cell stimulation leads to strong CD41 T-cell proliferation and the synthesis of large amounts of IL-10, IFN-g, and granzyme B, similar to what is seen in inducible TR1 cells. Defects in CD46-costimulated TR1-like cells in regulating immune responses have been shown to be associated with autoimmune diseases, such as multiple sclerosis.5 It has also been demonstrated that Der p 2–activated human bronchial epithelial cells can recruit CD3/CD46-activated TR1-like cells to the airways to suppress airway inflammation.6 CD3/CD46-activated TR1-like cells differentiate toward a TH1 response, and competition for IL-2 as a growth factor might play a potential role in immune tolerance against allergic inflammation.4 Xu et al7 first demonstrated that the number of CD46-costimulated T cells resembling a TR1 phenotype was decreased in a small group of asthmatic patients. We further demonstrated diminished IL-10/granzyme B expression in CD3/CD46-activated T cells in asthmatic patients and that

TABLE I. Patients’ characteristics and clinical response with symptoms scores, FEV1, FENO, and D pteronyssinus–specific IgE changes after SIT

No. of patients Mean age (y) Sex (male/female)

SIT group

Control group

30 12.89 6 2.67 21:9

20 12.48 6 2.99 13:7

Before SIT

D pteronyssinus–specific IgE (kU/L) Asthma Control Test score Mean FEV1 (%) FENO (ppb)

73.62 24.4 75.49 27.9

6 6 6 6

14.38 2.3 13.13 19.7

After SIT

42.78 26.5 95.57 18.6

6 6 6 6

12.35* 1.4* 15.09* 10.9*

FENO, Fraction of exhaled nitric oxide. *P < .05 compared with baseline.

CD3/CD46-activated TR1-like cells could suppress miteinduced inflammation in bronchial epithelial cells.6 This study aimed to perform a quantitative and functional analysis of CD3/CD46-activated TR1-like cells in asthmatic patients during Dermatophagoides pteronyssinus SIT. The materials and methods used in this study are shown in this article’s Online Repository at www.jacionline.org. All of the asthmatic patients who received D pteronyssinus SIT had improved asthma scores and increased pulmonary function (FEV1) and fraction of exhaled nitric oxide levels after 1 year of SIT (P < .05, Table I). The number of CD41CD251 forkhead box protein 3 (Foxp3)–positive cells increased (4.09% 6 1.69% vs 7.91% 6 3.02%, P < .05) and the number of CD41IL-41 cells (15.04% 6 5.51% vs 8.15% 6 4.88%, P < .05) decreased after 1 year of SIT. Experiments were next performed to examine IL-10 and IFN-g expression in CD3/CD46-activated TR1-like cells. Increases in IL-10 (12.3% 6 8.9% vs 22.1% 6 10.1%, P < .05; Fig 1, A) and IFN-g (9.3% 6 3.3% vs 22.8% 6 7.3%, P < .05; Fig 1, B) expression in CD41 T cells after CD46 costimulation were noted after 1 year of SIT in the asthmatic patients. When cytokine secretion was quantified by using ELISA, a striking defect with CD46-mediated IL-10 and IFN-g production was observed in asthmatic patients when compared with that seen in control subjects (P < .05). We found increased IL-10 and IFN-g, GM-CSF, and soluble CD40 ligand levels from CD3/CD46activated TR1-like cells after 1 year of SIT (P < .05, Table II). To further investigate the mechanism for the loss of IL-10 secretion by CD3/CD46-activated CD41 T cells in asthmatic patients, we examined expression of the 2 cytoplasmic isoforms of CD46, Cyt1 and Cyt2, and its association with IL-10 production. Cyt1 inhibits inflammatory reactions, whereas Cyt2 augments inflammation, correlating with decreased IL-10 production, as has been shown.5 Quantitative RT-PCR analysis of Cyt1 and Cyt2 mRNA in the CD3/CD46-activated CD41 T cells revealed a reciprocal change in the increase in CD46-induced Cyt1 mRNA expression (Fig 1, C) associated with decreased CD46-induced Cyt2 mRNA expression (Fig 1, D). Purified CD41 T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with anti-CD3/CD46 or anti-CD3/CD28 in the presence of IL-2 to study CD3/CD46 cross-linking on human CD41 T-lymphocyte proliferation. The proliferation of CD3/CD46-activated T cells was not significantly different between the healthy control subjects and asthmatic