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23. Purtill D, Smith KA, Tonon J, et al. Analysis of 402 cord blood units to assess factors influencing infused viable CD34þ cell dose: the critical determinant of engraftment. Blood. 2013;122:296. 24. Brunstein CG, Gutman JA, Weisdorf DJ, et al. Allogeneic hematopoietic cell transplantation for hematological malignancy: relative risks and benefits of double umbilical cord blood. Blood. 2010;116: 4693-4699. 25. Scaradavou A, Smith KM, Hawke R, et al. Cord blood units with low CD34þ cell viability have a low probability of engraftment after double unit transplantation. Biol Blood Marrow Transplant. 2010;16: 500-508. 26. Georges GE, Lesnikov V, Baran SW, et al. A pre-clinical model of double versus single unit unrelated cord blood transplantation. Biol Blood Marrow Transplant. 2010;16:1090-1098.

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27. Gutman JA, Turtle CJ, Manley TJ, et al. Single-unit dominance after double-unit umbilical cord blood transplantation coincides with a specific CD8þ T-cell response against the nonengrafted unit. Blood. 2010;115:757-765. 28. Eldjerou LK, Chaudhury S, Baisre-de Leon A, et al. An in vivo model of double unit cord blood transplantation that correlates with clinical engraftment. Blood. 2010;116:3999-4006. 29. Barker JN, Weisdorf DJ, DeFor TE, et al. Rapid and complete donor chimerism in adult recipients of unrelated donor umbilical cord blood transplantation after reduced-intensity conditioning. Blood. 2003;102: 1915-1919. 30. Eapen M, Klein JP, Ruggeri A, et al. Impact of allele-level HLA matching on outcomes after myeloablative single unit umbilical cord blood transplantation for hematologic malignancy. Blood. 2014;123:133-140.

Tc1 Clonal T Cell Expansion during Chronic Graft-versus-Host DiseaseeAssociated Hypereosinophilia Emmanuel Clave 1, 2, Aliénor Xhaard 3, Corrine Douay 1, 2, Lionel Adès 4, Jean Michel Cayuela 5, Régis Peffault de Latour 3, Marie Robin 3, Antoine Toubert 1, 2, 6, Gérard Socié 1, 2, 3, * 1

Departement d’Immunologie, INSERM UMRS-940, Paris, France Université Paris Diderot, Sorbonne Paris Cité, Paris, France Service d’Hématologie-Greffe, AP-HP Hôpital Saint Louis, Paris, France 4 Service d’Hématologie, AP-HP Hôpital Avicenne, Paris, France 5 Service d’Hématologie Biologique, AP-HP Hôpital Saint Louis, Paris, France 6 Service d’Immunologie, AP-HP Hôpital Saint Louis, Paris, France 2 3

Article history: Received 11 February 2013 Accepted 21 January 2014 Key Words: Graft-versus-host disease Eosinophilia

a b s t r a c t Although hypereosinophilia (HE) associated with chronic graft-versus-host disease (GVHD) has long been recognized, biological data on this phenomenon are scarce. Here we compare patients with chronic GVHD with HE together with a clonal T cell expansion and control patients with acute or chronic GVHD but without HE. These clonal expansions share a CD8þ TC1 phenotype rather than a CD4þ Th2 profile. In contrast to the drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome, these allogeneic CD8þ clones do not recognize the epitopes of herpesviruses. Furthermore, these TC1 clones do not produce IL-17 as described in the DRESS syndrome. Ó 2014 American Society for Blood and Marrow Transplantation.

INTRODUCTION Chronic graft-versus-host disease (GVHD), the leading cause of nonrelapse mortality after allogeneic stem cell transplantation, is frequently associated with hypereosinophilia (HE). HE may precede or be present at the diagnosis of GVHD, or may reappear with flare-ups [1]. HE was specifically mentioned in the first published description of chronic GVHD [2] and has been added to the nondiagnostic criteria by the National Institutes of Health Consensus Conference on Chronic GVHD [3]. Despite this, however, the biology of HE associated with chronic GVHD has been poorly described to date [4,5]. In this report, we describe HE occurring together with a clonal T cell expansion of the TC1 phenotype.

Financial disclosure: See Acknowledgments on page 742. * Correspondence and reprint requests: Prof. Gérard Socié, Service d’Hématologie Greffe, Hôpital Saint-Louis, 1 Ave Claude Vellefaux, 75010 Paris, France. E-mail address: [email protected] (G. Socié) 1083-8791/$ e see front matter Ó 2014 American Society for Blood and Marrow Transplantation. http://dx.doi.org/10.1016/j.bbmt.2014.01.023

PATIENTS AND METHODS Patients We studied 6 adult patients (5 males and 1 female) with extensive chronic GVHD-associated HE and 4 adult controls with acute or chronic GVHD but without HE. Five patients with HE and 2 controls had undergone transplantation with peripheral blood stem cells. The donor was an HLAidentical sibling for 4 of the 5 patients and for both controls. All but 1 patient with HE underwent transplantation for a hematologic malignancy after a reduced-intensity conditioning regimen. At the onset of HE, all patients were in hematologic remission, with 100% donor chimerism based on quantitative PCR (qPCR) of variable number tandem repeat sequences. GVHD characteristics are summarized in Table 1. This study was approved by the Ethics Committee of Saint-Louis Hospital.

Methods T cell repertoire Peripheral blood mononuclear cells were separated using a lymphocyte separation medium (Eurobio, Les Ulis, France), and 5-10  106 cells were stored after lysis in TriReagent solution (Molecular Research Center, Cincinnati, OH) at 80 C. RNA was subsequently extracted following the manufacturer’s instructions. Then 2-5 mg of total RNA was reverse-transcribed into cDNA using the Superscript III first-strand synthesis system for RT-PCR (Invitrogen, Cergy-Pontoise, France). T cell receptor beta V qPCR amplification, beta chain variable and constant runoff using an internal beta chain constant fluorescent primer, gel running, and immunoscope analysis were performed as described previously [6].

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Table 1 GVHD Characteristics Patient

GVHD

Time from Transplantation to GVHD, d

Time from GVHD to Sampling, d

GVHD at Sampling

Treatment at Sampling

Ctrl1 Ctrl2 Ctrl3 Ctrl4 HE1 HE2 HE3 HE4 HE5 HE6

Chronic Acute Acute Chronic Chronic Chronic Chronic Chronic Chronic Chronic

257 134 43 174 359 423 140 195 570 118

84 52 160 189 571 1705 1407 126 1068 0

PR CR PR CR CR PR CR Flare Flare Flare

CSA Steroids (0.15 mg/kg) Steroids (0.15 mg/kg) Steroids Steroids (0.2 mg/kg) þ CSA þ MMF CSA þ local Local CSA Steroids (0.25 mg/kg) þ MMF CSA

Ctrl indicates control; CSA, cyclosporin A; MMF, mycophenolate mofetil; CR, complete response; PR, partial response.

Intracellular cytokine staining and flow Cytometry analysis Cells were thawed and left to rest overnight at 37 C in RPMI 10% medium with 10% human AB serum. They were then stimulated in 96-well plates using 50 ng/mL PMA (Phorbol 12-Myristate 13-Acetate), 1 mM ionomycin, and 1 mg/ mL brefeldin A (all from Sigma-Aldrich, Lyon, France). After 4 hours, cells were harvested, permeabilized using the Human FoxP3 Buffer Set (BD Pharmingen, Le Pont de Claix, France), and stained with the following antibodies: CD3 Amcyan, CD8 PerCP, PD1 (CD279) PE, CTLA4 (CD152) APC, IL5 PE, TNFa PE-Cy7, perforin FITC (all from BD Pharmingen), TCRVb3 FITC (Beckman-Coulter, Villepinte, France), and IL17 PE (eBioscience, Paris, France). Appropriate isotype-matched controls (BD Biosciences) were included. At least 10,000 lymphocytes were acquired with a FACSCanto II cytometer (BD Biosciences). Mass sequencing In 4 of the patients with HE (including donor, M4, M5, M6, and M24 for patient HE6) and all control patients, Vb3 and Vb6 were amplified from qPCR product with a primer containing the sequencing primer TitA, a 2-nucleotide patient-specific tag, the constant region primer HTCB3 (TitA_tag_HTCB3) (CCATCTCATCCCTGCGTGTCTCCGACTCAG-NN-CCTTTT GGGTGTGGGAGATCTC), and a Vb3- or Vb6-specific primer also containing the TitB sequencing primer (GCGCTCCTTCTTCTCTCTAGAGAC-CTGAGACT GCCAAGGCACACAGGGGATAGG, or GAAATTGGATCACACCTGAG-CTGAGA CTGCCAAGGCACACAGGGGATAGG, respectively). All PCR products were pooled and sequenced together in a single run on a GS FLXþ System with a Titanium XLþ Sequencing Kit (Roche, BoulogneBillancourt, France) by GATC Biotech, Mulhouse, France. After screening on the expected size and sorting according to patient tag, sequences were analyzed and aligned using Galaxy [7]. Analysis of the junctions and translation of CDR3 were done using IMGT V-Quest [8].

RESULTS AND DISCUSSION This study was initiated after we encountered a patient with peripheral T cell lymphoma who developed chronic GVHD and simultaneous HE. Because HE was present at the time of diagnosis of lymphoma, a relapse of the lymphoma was suspected, but this was ruled out by a different TCR rearrangement on Southern blot analysis. Five additional patients with HE were then analyzed for TCR d locus recombination by Southern blot analysis, which revealed clonal T cell expansion (data not shown). We studied these T cell expansions through quantitative determination of Vb usage using real time PCR and CDR3 size polymorphism (spectratyping or immunoscope analysis). Ten patients were studied, 6 of whom exhibited a clonal expansion profile. Comparisons of these 6 patients with HE and 4 controls showed massive expansions in the HE group (with >40% of families in 2 patients), especially within the Vb3 and Vb6 families (Figure 1A and Supplemental Figure S1). Moreover, for patients HE6 and HE4, we were able to test cells before and after onset of HE. Expansion was present (66% of all Vb at 6 months post-transplantation) and persisted long after resolution of HE (44% at 1 year posttransplantation). Follow-up of patient HE6 is shown in Figure 1B and C.

We then confirmed T cell clonality using mass sequencing, with >90% of the sequences corresponding to 1 clonotype. The same clonotype was found at 4, 5, 6, and 24 months posttransplantation, but was not detected in either the donor (85 independent sequences) or the 3 other patients with HE and the 4 controls (Supplemental Figure S1). A search for sequence homology with the other published CDR3 sequences was negative (Figure 1D). In 2 patients, we then characterized the clonal expansions using a Vb3-specific antibody, given that Vb6 antibody is not commercially available (Figure 2). Vb3 staining confirmed our qPCR data, and, unexpectedly, the expansion was restricted to the CD8þ T cells. On unspecific stimulation (with PMA-ionomycin), these Vb3 CD8þ T cells did not express IL-5 or IL-17A, but did express TC1 cytokines, IFN-g, and TNF-a. Furthermore, all CD8þ T cells were positive for perforin but negative for PD1 and surface CTLA4 (data not shown). Activated CD8þ T lymphocytes have recently been implicated in mediating the drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome [9]. In DRESS, these CD8þ T cells are largely directed against herpesviruses, particularly Epstein-Barr virus (EBV). Thus, we evaluated the link between cytomegalovirus (CMV) or EBV and chronic GVHDeassociated HE within the aforementioned Vb3 clone. In patient HE6, we detected a low EBV and high CMV CD8þ response (especially against the CMV antigen IE1), which was nevertheless independent of the Vb3 clone (Supplemental Figure S2). To summarize, we detected clonal T cell expansion in some patients with chronic GVHDeassociated HE. Although we would have expected to find an IL-5edriven Th2 phenomenon [10], we did find that the T cell expansions were CD8þ with a TC1 (IFN-g and TNF-a) phenotype, occurring mainly within the Vb3 and Vb6 families. As noted previously, CD8þ T cells with a TC1 phenotype have been described in DRESS syndrome as well [9]; however, unlike in DRESS syndrome, we found no evidence for a phenomena driven by herpesviruses, and a mechanical gap clearly persists between these clonal T cell expansions and the occurrence of HE (as is the case for DRESS). Although we cannot rule out the possibility that these clonal T cell expansions simply reflect allogeneic recognition [11] with no direct link to HE, we must note that alloreactivity was present in the patients with chronic GVHD without HE (Figure 1A). Another possibility is that HE in patients with chronic GVHD is driven by cytokines other than IL-5; however, the cytokines implicated in HE (i.e. IL-3, GM-CSF, and IL-33) are not T celle derived [10].

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Figure 1. Massive Vb expansion occurs in patients with chronic GVHD with HE. (A) Vb family usage measured by qRT-PCR in 10 patients with chronic GVHD, 6 with HE (in blue) and 4 without (in green). (B) Vb family usage for patient HE6 at months 4, 5 (as in A), 6, and 24 post-transplantation and for the corresponding donor (D). (C) Characterization of Vb3 expansion in patient HE6. Percentage of use was obtained by qPCR, and CDR3 size polymorphism (immunoscope analysis or spectratyping) and Vb3 sequences were obtained by mass sequencing. The last value indicates the percentage of independent sequences corresponding to the main clonotype (see Methods). (D) CDR3 nucleotide sequence and translation of the main Vb3 clonotype for patient HE6.

Figure 2. Phenotypic characterization and cytokine production of Vb3 T cell expansion in patient HE6. (A) Surface Vb3 staining of CD3þ cells at 4, 6, and 24 mo posttransplantation. Shown are percentages of cells as a proportion of total CD3þ cells (as gated in the left panel). (B) Intracellular cytokine staining of the CD3þCD8þ cells of patient HE6 at 24 mo post-transplantation. Cells were stimulated for 4 h with PMA-ionomycin before staining. Percentages in the top right of each plot refer to cytokine-positive cells as a proportion of total CD8þ Vb3þ cells.

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ACKNOWLEDGMENTS G.S. thanks Alain Ficher, MD and Bruce R. Blazar for helpful discussions. Conflict of interest statement: There are no conflicts of interest to report. Authorship statement: E.C. designed and performed the biological studies and wrote the manuscript. A.X. provided patient samples and wrote the manuscript. C.D. and J.M.C. performed experiments. L.A. initiated the study. R.P.L. and M.R. provided patient samples. A.T. contributed to the study design. G.S. designed and supervised the research and wrote the manuscript. All authors actively participated in manuscript preparation. Financial disclosure: The authors have nothing to disclose. SUPPLEMENTARY DATA Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.bbmt.2014.01.023. REFERENCES 1. Ahmad I, Labbe AC, Chagnon M, et al. Incidence and prognostic value of eosinophilia in chronic graft-versus-host disease after nonmyeloablative hematopoietic cell transplantation. Biol Blood Marrow Transplant. 2011; 17:1673-1678.

2. Shulman HM, Sullivan KM, Weiden PL, et al. Chronic graft-versus-host syndrome in man: a long-term clinicopathologic study of 20 Seattle patients. Am J Med. 1980;69:204-217. 3. Filipovich AH, Weisdorf D, Pavletic S, et al. National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease, I: Diagnosis and Staging Working Group report. Biol Blood Marrow Transplant. 2005;11:945-956. 4. McNeel D, Rubio MT, Damaj G, et al. Hypereosinophilia as a presenting sign of acute graft-versus-host disease after allogeneic bone marrow transplantation. Transplantation. 2002;74:1797-1800. 5. Daneshpouy M, Socie G, Lemann M, et al. Activated eosinophils in upper gastrointestinal tract of patients with graft-versus-host disease. Blood. 2002;99:3033-3040. 6. Clave E, Busson M, Douay C, et al. Acute graft-versus-host disease transiently impairs thymic output in young patients after allogeneic hematopoietic stem cell transplantation. Blood. 2009;113:6477-6484. 7. Blankenberg D, Von Kuster G, Coraor N, et al. Galaxy: a web-based genome analysis tool for experimentalists. Curr Protoc Mol Biol. 2010; 89:19.10.1-19.10.21. 8. Brochet X, Lefranc MP, Giudicelli V. IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis. Nucleic Acids Res. 2008;36(Web Server issue): W503-W508. 9. Picard D, Janela B, Descamps V, et al. Drug reaction with eosinophilia and systemic symptoms (DRESS): a multiorgan antiviral T cell response. Sci Transl Med. 2010;2:46ra62. 10. Akuthota P, Weller PF. Eosinophils and disease pathogenesis. Semin Hematol. 2012;49:113-119. 11. Michalek J, Collins RH, Hill BJ, et al. Identification and monitoring of graft-versus-host specific T cell clone in stem cell transplantation. Lancet. 2003;361:1183-1185.

Human Leukocyte AntigeneDO Regulates Surface Presentation of Human Leukocyte Antigen Class IIeRestricted Antigens on B Cell Malignancies Anita N. Kremer 1, 2, Edith D. van der Meijden 1, M. Willy Honders 1, Margot J. Pont 1, Jelle J. Goeman 3, J.H. Frederik Falkenburg 1, Marieke Griffioen 1, * 1

Department of Hematology, Leiden University Medical Center, RC Leiden, The Netherlands Department of Internal Medicine 5, Hematology and Oncology, University Hospital Erlangen, Erlangen, Germany 3 Department of Medical Statistics, Leiden University Medical Center, RC Leiden, The Netherlands 2

Article history: Received 10 July 2013 Accepted 6 February 2014 Key Words: T lymphocytes CD4þ T lymphocytes Allogeneic stem cell transplantation Leukemia Graft-versus-leukemia Human leukocyte antigen class II

a b s t r a c t Hematological malignancies often express surface HLA class II, making them attractive targets for CD4þ T cell therapy. We previously demonstrated that HLA class II ligands can be divided into DM-resistant and DMsensitive antigens. In contrast to presentation of DM-resistant antigens, presentation of DM-sensitive antigens is suppressed by HLA-DM but can be rescued by HLA-DO. We also showed that HLA-DO expression remains low in nonhematopoietic cells under inflammatory conditions, suggesting that DM-sensitive antigens may be ideal T cell targets with a low risk for graft-versus-host disease. Here, we demonstrated that B cell malignancies often express HLA-DO and that levels are in particular high in chronic lymphocytic leukemia. Moreover, we showed that surface presentation of DM-sensitive antigens is regulated by HLA-DO, and that DM-sensitive antigens are relevant T cell targets for B cell malignancies and, especially, chronic lymphocytic leukemia. These data open the perspective to target HLA class II ligands with specific processing and presentation behavior for CD4þ T cell therapy of hematological malignancies. Ó 2014 American Society for Blood and Marrow Transplantation.

Financial disclosure: See Acknowledgments on page 747. * Correspondence and reprint requests: Marieke Griffioen, PhD, Department of Hematology, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands. E-mail address: M.Griffi[email protected] (M. Griffioen) 1083-8791/$ e see front matter Ó 2014 American Society for Blood and Marrow Transplantation. http://dx.doi.org/10.1016/j.bbmt.2014.02.005

INTRODUCTION Allogeneic stem cell transplantation (aSCT) in the treatment of hematological malignancies is the most successful form of cellular immunotherapy [1]. The beneficial graft-versusleukemia (GVL) effect is mediated by donor-derived T cells recognizing residual leukemic cells of the patient [2]. These T cells, however, may also react against nonhematopoietic tissues of the patient, thereby inducing graft-versus-host

Tc1 clonal T cell expansion during chronic graft-versus-host disease-associated hypereosinophilia.

Although hypereosinophilia (HE) associated with chronic graft-versus-host disease (GVHD) has long been recognized, biological data on this phenomenon ...
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