anaplastic lymphoma kinase

Clinical and Experimental Immunology

OR I GI NA L ARTI CLE

doi:10.1111/cei.12842

Analysis of nucleophosmin–anaplastic lymphoma kinase (NPM-ALK)reactive CD81 T cell responses in children with NPM-ALK1 anaplastic large cell lymphoma

V. K. Singh,*1 S. Werner,*1 H. Hackstein,† V. Lennerz,‡ A. Reiter,* T. W€ olfel,‡ C. Damm-Welk*2 and W. Woessmann*2 *Department of Pediatric Hematology and Oncology, †Institute of Clinical Immunology and Transfusion Medicine, Justus-LiebigUniversity, Giessen, Germany, and ‡Department of Internal Medicine III, University Medical Center, Johannes Gutenberg University, Mainz, Germany

Accepted for publication 12 July 2016 Correspondence: W. Woessmann, Department of Pediatric Hematology and Oncology, Justus-Liebig-University, Feulgenstraße 12, 35385 Giessen, Germany. E-mail: wilhelm.woessmann@paediat.med.unigiessen.de 1

These authors contributed equally to this study.

2

Co-senior authors.

Summary Cellular immune responses against the oncoantigen anaplastic lymphoma kinase (ALK) in patients with ALK-positive anaplastic large cell lymphoma (ALCL) have been detected using peptide-based approaches in individuals preselected for human leucocyte antigen (HLA)-A*02:01. In this study, we aimed to evaluate nucleophosmin (NPM)-ALK-specific CD81 T cell responses in ALCL patients ensuring endogenous peptide processing of ALK antigens and avoiding HLA preselection. We also examined the HLA class I restriction of ALK-specific CD81 T cells. Autologous dendritic cells (DCs) transfected with in-vitro-transcribed RNA (IVT-RNA) encoding NPM–ALK were used as antigen-presenting cells for T cell stimulation. Responder T lymphocytes were tested in interferon-gamma enzyme-linked immunospot (ELISPOT) assays with NPM–ALK-transfected autologous DCs as well as CV-1 in Origin with SV40 genes (COS-7) cells co-transfected with genes encoding the patients’ HLA class I alleles and with NPM–ALK encoding cDNA to verify responses and define the HLA restrictions of specific T cell responses. NPM–ALK-specific CD81 T cell responses were detected in three of five ALK-positive ALCL patients tested between 1 and 13 years after diagnosis. The three patients had also maintained anti-ALK antibody responses. No reactivity was detected in samples from five healthy donors. The NPM–ALK-specific CD81 T cell responses were restricted by HLA-Calleles (C*06:02 and C*12:02) in all three cases. This approach allowed for the detection of NPM–ALK-reactive T cells, irrespective of the individual HLA status, up to 9 years after ALCL diagnosis. Keywords: NPM–ALK, ALCL, CD81 T cell, IFN-g ELISPOT, immune

response

Introduction Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) is the third most common non-Hodgkin lymphoma in children and adolescents [1]. In paediatric patients, 90% of these ALK-positive ALCLs express the oncogenic nucleophosmin–anaplastic lymphoma kinase (NPM–ALK) fusion protein [2,3]. Early clinical and pathological observations indicated that ALK-positive ALCL is an attractive target for immune therapy [4,5]. The limited expression of ALK in immuneprivileged sites and the dependence of tumour cell survival on activated ALK signalling suggested that ALK fusion proteins would provide an almost ideal oncoantigen [6]. Studies on the immunogenicity of ALK in humans have shown 96

that patients with NPM–ALK-positive ALCL mount a humoral immune response against ALK [7–9]. The strength of the antibody titre correlates inversely with the risk of relapse, thus implicating the immune response against ALK in the final control of ALK-positive ALCL [8,9]. CD41 T cells reactive against an ALK-derived peptide selected by computer-based algorithm were detected in human leucocyte antigen (HLA)-DRB1-positive ALCL patients [10]. Proof-of-principle for a CD81 T cell response against ALK in humans was obtained in HLA-A*02:01-positive individuals using a peptide-based approach. ALK-derived HLA-A*02:01-binding peptides were selected using computer-based algorithms. The CD81 T cell repertoire in three HLA-A*02:011 healthy individuals contained T cells

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NPM-ALK-reactive CD81 T cells in ALCL

with specificity for ALK-derived peptides [11]. CD81 T cells reactive against these peptides were detected in 12 HLA-A*02:01-positive ALCL patients in two studies [12,13]. While the ALK peptide-reactive CD81 T cells in healthy donors exhibited a predominantly naive phenotype, effector and memory CD81 T cells were detected in ALK1 ALCL patients [12]. The possible clinical relevance of the ALK-specific CD81 T cell response was demonstrated in vaccination studies in mice, reported by Chiarle et al., who showed that the protection against ALCL growth conferred by vaccination with truncated ALK cDNA was mediated mainly by CD81 T cells [14]. As a prerequisite for pursuing immunotherapeutic approaches, we aimed to examine the CD81 T cell responses against NPM–ALK in ALCL patients across all individual HLA alleles. In addition, we wanted to provide an a priori guarantee for endogenous NPM–ALK peptide processing in presenting cells. To reach these goals, we chose autologous dendritic cells (DCs) as antigenpresenting cells (APCs) and in-vitro-transcribed RNA (IVT-RNA) coding for NPM–ALK was applied as antigen format [15,16]. To detect the predominant individual presenting HLA allele, in a second step responder CD81 T cells were tested for recognition of CV-1 in Origin with SV40 genes (COS-7) cells co-transfected with a patient’s individual HLA class I allele- and NPM–ALK-encoding cDNA [17]. Using this approach, we analysed the ALKspecific CD81 T cell responses of five patients with ALCL in remission for different lengths of time, including four patients with a high initial anti-ALK-antibody titre.

Materials and methods Patients and healthy controls The five NPM–ALK1 ALCL patients analysed herein had been included in the Non-Hodgkin Lymphoma Berlin– Frankfurt–M€ unster 95 (NHL-BFM 95) and ALCL 99 studies. They were treated with comparable BFM-type front-line therapy and were in clinical remission without relapse for 1–13 years at the time of T cell response analysis (Supporting information, Table S1). The selection criteria were: current patient age > 14 years, no infection or immunosuppressive therapy, no medical condition prohibiting blood drawing, a high pretherapeutic anti-ALK-antibody titre of 1 : 60 750 (plus one patient with low titre) and different lengths of time in clinical remission. The study was approved by the Ethics Committee of the medical faculty of the Justus-Liebig-University, Giessen, Germany (number: 193/11). Written informed consent for the study was obtained from all patients – and in those aged < 18 years, also from their legal guardians – after the patients (and their guardians) had been informed about the study orally and in writing by a study physician. At least 1 week was allowed for decision-making. Specimens from

cytomegalovirus (CMV)-seropositive healthy individuals were either collected from young adult volunteers or provided by the transfusion service of the University Hospital, Giessen, Germany, after written informed consent was obtained from the donors. One CMV-seronegative healthy donor was also included as the experimental control.

HLA class I genotyping, cloning of HLA I alleles and NPM–ALK-cDNA into an expression vector The HLA class I alleles of patients and healthy donors identified by high-resolution (four-digit) HLA class I genotyping are shown in Supporting information, Tables S1 and S2. Four ALCL patients as well as four healthy subjects were HLA-A*02:01-positive. HLA class I alleles not yet available in the laboratory (most HLA class I alleles had been cloned previously in other projects) were amplified from the cDNA of patients or healthy individuals via polymerase chain reaction (PCR) using the forward and reverse primers described by Ennis et al. [18] and cloned into the pcDNA3.1/V5-His TOPO TA expression vector system (Life Technologies, Darmstadt, Germany). Plasmid pcDNA3-NPM–ALK, encoding the NPM–ALK full-length fusion protein, was obtained from S.W. Morris [19]. The construction of plasmid pcDNA3.1.pp65 TOPO was described previously [16].

In-vitro transcription of antigen-encoding RNA pcDNA3-NPM–ALK was linearized with the restriction enzyme Xho I, and pcDNA3.1.pp65 with the restriction enzyme Apa I (both from New England Biolabs, Frankfurt, Germany). In-vitro transcription using the MESSAGE mMACHINE T7 Ultra Kit (Life Technologies, Darmstadt, Germany) and the polyadenylation of the resulting IVTRNA were performed according to the manufacturer’s guidelines.

T cell isolation and generation of APCs Acid citrate dextrose (ACD) anti-coagulated blood from patients and healthy individual leucocyte fractions were processed on the day of sample collection. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll/ Hypaque 1077 g/ml (Axis-Shield PoC AS, Oslo, Norway) density gradient centrifugation. CD81 T cells were purified from PBMCs using CD8 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The T cell fractions were frozen in Cryo-safe I (C.C.Pro GmbH, Oberdorla, Germany) medium for later stimulation and testing. Dendritic cells (FastDCs) were generated from monocytes as described previously [20]. The maturation status of the FastDCs [20] was determined by flow cytometry using cellsurface markers for human anti-CD83-allophycocyanin, -CD86-phycoerythrin (PE) and -CD209-peridinin chlorophyll (PerCP), anti-HLA-DR-PerCP (BD Biosciences, Heidelberg, Germany) and anti-CD14-PE, -CD80-fluorescein


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isothiocyanate (FITC), -CD40-APC and -CCR7-FITC (BD Pharmingen, Heidelberg, Germany). Mature FastDCs (Supporting information, Fig. S1) were irradiated with 10,000 rad and transfected with antigen-coding IVT-RNA using the nucleofection system (Lonza GmBH, Cologne, Germany). After 24 h, the transfected FastDCs were stained with human anti-NPM–ALK/ALK-PE antibody (BD Pharmingen) and nucleofection efficiency was measured by flow cytometry (Supporting information, Fig. S2). These RNA-transfected FastDCs were used as APCs in the subsequent in-vitro stimulation of T cells. In addition, portions of the transfected FastDCs were frozen in Cryo-safe I medium for later restimulation and testing.

In-vitro stimulation of CD81 T cells with FastDCs transfected with NPM–ALK-RNA Blood-derived CD81 T cells were plated at 1 3 105 per well in a 96-well U-bottomed plate in AIM-Vstim culture medium, consisting of AIM-V (Life Technologies, Darmstadt, Germany) supplemented with 5% human serum (Biochrom, Berlin, Germany), 20 U/ml interleukin (IL)-2 (Novartis Pharma, N€ urnberg, Germany) and 5 ng/ml IL-7 (Miltenyi Biotec). CD81 T cells were stimulated with autologous FastDCs transfected with NPM–ALK IVT-RNA, pp65 IVT-RNA or a mock control. From each patient donation six to eight NPM–ALK-stimulation microcultures, and from each healthy control donation eight to 16 NPM– ALK-stimulation microcultures, were initiated. All responder T cells were restimulated on days 7 and 14 under the same culture conditions using the FastDCs transfected with antigen-encoding IVT-RNA. Responder T cells were tested on day 19 in an interferon (IFN)-g enzyme-linked immunospot (ELISPOT) assay.

IFN-g ELISPOT assay Two separate IFN-g ELISPOT (20-h) assays were performed, as described previously [16]. Recognition of DCs expressing NPM–ALK fusion protein. To test the autologous anti-NPM–ALK responses, FastDCs (30–100 3 103 cells/well) transfected with IVT-RNAencoding NPM–ALK or control antigens were used as target cells. The target cells were resuspended in AIM-V/5% human serum and plated in ELISPOT plates as indicated. Recognition of COS-7/NPM–ALK transfectants. To assess the HLA restriction element responsible for NPM–ALK or control antigen presentation, COS-7 cells (obtained from the German Collection of Microorganisms and Cell Cultures, DSMZ) co-transfected with plasmids encoding the respective patient’s/donor’s HLA-I alleles and NPM–ALK or pp65 were used as APCs at a density of 20 3 104 cells/ well. COS-7 cells were transfected using LipofectaminTM 2000 (Invitrogen, Karlsruhe, Germany), according to the manufacturer’s guideline. Responder T cells (15–60 3 98

104/well) were added 4 h after FastDC transfection or 24 h after COS-7 transfection. After 20 h of co-culture, the cells were discarded and the ELISPOT plates were developed and prepared for IFN-g spot reading on an automated ELISPOT reader ImmunoSpot Series 5 Versa Analyzer (C.T.L. Europe, Bonn, Germany). Responses in a given microculture were defined as ‘positive’ when the mean number of IFN-g spot-forming T cells against the test antigen was at least two-fold higher than the background reactivity (baseline response against either antigen-negative target cells only or targets expressing an irrelevant antigen).

Ex-vivo analysis of NPM–ALK-reactive CD81 T cells in ALCL patients To detect the spontaneous T cell response prior to antigen stimulation in samples from ALCL patients, thawed CD81 T cells (10–20 3 105/well) were used directly in a 20-h IFN-g ELISPOT assay and tested for the recognition of autologous FastDCs (10–20 3 104/well) transfected with NPM–ALK IVT-RNA. To assess the restricting HLA I allele for NPM–ALK reactivity, unstimulated CD81 T cells were tested for the recognition of COS-7/NPM–ALK transfectants (20 3 104/well) co-transfected with the respective patients’ HLA I alleles.

ALK-antibody titre Anti-ALK-antibody titres were measured as reported previously [7].

Results Ex-vivo CD81 T cell response against the NPM–ALK fusion protein Blood samples from five NPM–ALK-positive paediatric ALCL patients in clinical remission for 1–13 years and from five healthy donors were analysed for their anti-NPM–ALK CD81 T cell responses. To detect T cell responses directed against the NPM–ALK fusion protein ex-vivo, blood-derived non-stimulated CD81 T cells were used directly in a 20-h IFN-g ELISPOT assay in which they were tested against autologous FastDCs expressing the NPM–ALK fusion protein. One ALCL patient (NHL-4JS2, Fig. 1) exhibited an ex-vivo CD81 T cell response against NPM–ALK. The ex-vivodetectable, NPM–ALK-responsive T cells comprised a maximum of 028% of the total CD81 T lymphocytes. None of the samples from healthy donors demonstrated ex-vivo NPM–ALK-specific CD81 T cell responses.

Detection of NPM–ALK-reactive CD81 T cells after short-term in-vitro stimulation NPM–ALK IVT-RNA-based in-vitro T cell stimulation was used to enrich antigen-specific CD81 T cells. Responder T cells were tested on day 19 after three stimulations for


NPM-ALK-reactive CD81 T cells in ALCL (a)

(b)

Fig. 1. Ex-vivo CD81 T cell response to autologous nucleophosmin–anaplastic lymphoma kinase (NPM–ALK)-transfected dendritic cells (FastDCs) and human leucocyte antigen (HLA) restriction. Unstimulated CD81 T cells of patient NHL-4JS2 were tested directly in an interferon (IFN)-g enzyme-linked immunospot (ELISPOT) assay. (a) Recognition of autologous FastDCs (10 3104/well) transfected with NPM–ALK invitro-transcribed RNA. Shown are ELISPOT filter scans and spot numbers determined via semi-automated computer-assisted video image analysis in a bar diagram. (b) Recognition of CV-1 in Origin with SV40 genes (COS-7) cells (20 3 104/well) co-transfected with the patient’s HLA class I alleles (except B*52:01) and NPM–ALK or control antigens (pp65) cDNA. The bars represent means of duplicates 6 standard deviation.

recognition of FastDCs transfected with IVT-RNA encoding NPM–ALK in an IFN-g ELISPOT assay. As a control for the chosen in-vitro short-term stimulation conditions and for the antigen format of IVT-RNA, anti-CMV-pp65 reactivity was confirmed in all four CMV-seropositive ALCL patients and healthy donors tested, but not in the patient and healthy donor, who were CMV-seronegative (Fig. 2). In two of the four ALCL patients (NHL-4JS2 and NHL7AKR), microculture responder T cells were able to recognize autologous FastDCs transfected with NPM–ALK IVT-RNA (Fig. 3). In a fifth patient, NHL-P1SU, the transfection efficiency of the target cells was below 15%, compromising the performance of the autologous NPM–ALK recognition assay. IFN-g spot numbers in positive microcultures ranged from three- to 100-fold above the background reactivity. Microcultures with the strongest reactivity against NPM–ALK were those from patient NHL-4JS2 with ex-vivo-detectable NPM–ALK-reactive CD81 T cells. NPM–ALK reactivity was not detected in the microcultures from the five healthy individuals. These results demonstrated that, using this short-term in-vitro

stimulation approach, NPM–ALK-reactive T cells in blood samples collected from NPM–ALK -positive ALCL patients in clinical remission could be enriched, and subsequently detected, after stimulation with NPM–ALK RNAtransfected FastDCs.

Identification of the restricting HLA class I allele for the anti-NPM–ALK T cell responses In parallel to the autologous NPM–ALK recognition test, the in-vitro-stimulated T cell microcultures of the five ALCL patients and healthy controls were also tested for their HLA restriction using COS-7 cells co-transfected with NPM–ALK and the patients’ individual HLA class I alleles as targets. HLA class I alleles restricting the NPM–ALK T cell responses were identified in samples from three of the five ALCL patients (NHL-4JS2, NHL-P1SU and NHL7AKR) (Fig. 4). In patients NHL-P1SU and NHL-7AKR, anti-NPM–ALK-specific T cell responses were restricted by the HLA-C*06:02 allele, whereas in patient 4JS2 the T cells were restricted by HLA-C*12:02 (Fig. 4). To obtain hints about possible potential NPM–ALK epitopes binding to


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(b)

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NPM-ALK-reactive CD81 T cells in ALCL Fig. 2. Anti-cytomegalovirus (CMV)/pp65 T cell responses after short-term pp65-RNA stimulation in blood samples from anaplastic large cell lymphoma (ALCL) patients and healthy donors. CD81 T cells (10 3 105) were stimulated with irradiated autologous dendritic cells (FastDCs) transfected with pp65 in-vitro-transcribed RNA. Microcultures were restimulated weekly for 2 weeks with the pp65-transfected FastDCs. On day 19, pp65 responder T cells were tested in a 20-h interferon (IFN)-g enzyme-linked immunospot (ELISPOT) assay for the recognition of autologous FastDCs expressing pp65 (3–10 3 103/well). Shown are representative microcultures against autologous pp65-transfected FastDCs from (a) four ALCL patients (NHL-P1SU could not be tested; only patient NHL-FVUD was CMV-seronegative) and (b) healthy donors (only NHL-96YC was CMV-seronegative). Bars represent the means of duplicates 6 standard deviation.

3 these HLA alleles, prediction algorithm-based Immune Epitope Database and Analysis Resource (IEDB) [21–23], NetMHC 4.0 [22,24] and NetMHCpan 3.0 [25,26] tools were used to predict NPM–ALK-derived peptides that bind to HLA-C*06:02 and C*12:02, respectively. For the HLA-C*06:02 allele, five of seven NetMHC 4.0 predicted peptides were also predicted by IEDB analysis within > 1000 nM threshold binding affinity (Supporting information, Table S3) and for the HLA-C*12:02 allele, three of five NetMHCpan 3.0 predicted peptides were also predicted by IEDB tool within > 500 nM threshold binding affinity (Supporting information, Table S4). After the autologous T cell response against pp65 was tested, the number of remaining CD81 T cells was high enough to determine the anti-pp65-restricting HLA I molecules of anti-pp65-reactive T cells in five of the eight CMV-seropositive individuals. In patients NHL-P1SU and NHL-4JS2, anti-pp65 T cell responses were restricted by

the alleles HLA-C*06:02 and -B*35:01, respectively. In healthy donors, anti-pp65 T cell responses were restricted by HLA-A*02:01 (NHL-K79N) and -B*35:01 (NHL-O6E5), as well as -A*02:01 and -B*07:02 (NHL-O5QZ).

Correlation of ALK-reactive T cell responses with ALK-antibody titres All three patients with measurable anti-NPM–ALK CD81 T cell responses, as determined by our approach, had high anti-ALK-antibody titres prior to therapy and a persistent titre between 1 : 750 and 1 : 2250 at the time of T cell response analysis (Supporting information, Table S1). Of the two patients in whom comparable responses could not be detected, one had a low antibody titre at the time of testing 13 years after diagnosis. The other had an initially low antibody titre and no detectable response when the blood sample was taken for T cell response determination.

Fig. 3. Anti-nucleophosmin–anaplastic lymphoma kinase (NPM–ALK) T cell responses after short-term NPM–ALK in-vitro-transcribed RNA stimulation in anaplastic large cell lymphoma (ALCL) patients. CD81 T cells were stimulated with irradiated autologous dendritic cells (FastDCs) transfected with NPM–ALK IVT-RNA, comparable to the pp65 stimulation. After two weekly restimulations, day 19 responder T cells were tested in a 20-h interferon (IFN)-g enzyme-linked immunospot (ELISPOT) assay for the recognition of FastDCs expressing NPM–ALK fusion protein. Representative microcultures against autologous NPM–ALK RNA transfected FastDCs from four ALCL patients are shown (patient NHL-P1SU could not be tested). Bars represent the means of duplicates 6 standard deviation.


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Fig. 4. Identification of human leucocyte antigen (HLA) class I-restriction alleles of CD81 T cells reactive against nucleophosmin–anaplastic lymphoma kinase (NPM–ALK) in NPM–ALK1 anaplastic large cell lymphoma (ALCL) patients. To assess the restricting HLA class I allele, day 19 responder T cells were tested in a parallel IFN-g enzyme-linked immunospot (ELISPOT) assay against CV-1 in origin with SV40 genes (COS7) cells (20 3 104/well) co-transfected with plasmids encoding the patients’ HLA class I alleles and NPM–ALK or cytomegalovirus (CMV)/pp65 antigen. Shown are representative microcultures reactive against COS-7 cells co-transfected with NPM–ALK and the individual HLA class I alleles from patients NHL-P1SU, NHL-4JS2 and NHL-7AKR, after short-term stimulation with autologous NPM–ALK transfected dendritic cells (FastDCs). Bars represent the means of duplicates 6 standard deviation.

Discussion An NPM–ALK-specific T cell response was described previously in HLA-A*02:01-preselected NPM–ALK-positive ALCL patients using ALK-derived peptides predicted to be presented by HLA-A*02:01. Our test system, using autologous DCs as presenting cells and IVT-RNA as the antigen format, allowed for the detection of NPM–ALK-reactive T cells across all individual HLA class I alleles in three of five patients. In the three reactive samples, the NPM–ALK specific CD81 T cell responses were restricted by HLA-C alleles (C*06:02 and C*12:02). 102

The initial antibody titres against NPM–ALK serve as a potential surrogate for the strength of a pre-existing immune response against ALK [8,9]. We therefore selected ALK-positive ALCL patients in first complete remission who had initially high ALK-antibody titres as well as one patient with a low titre in order to establish whether the DC- and RNA-based autologous stimulation procedure allowed for the detection of ALK-specific CD81 T cells and the definition of their HLA-restriction. In addition, patients with different remission durations were chosen so that the persistence of a T cell response could possibly be



judged. Similar to a previously described approach using ALK-derived HLA-A*02:01-restricted peptides in seven HLA-A*02:01 patients [13], our system detected persisting ALK-specific T cell and humoral responses in lymphoma patients up to 9 years after the diagnosis of an ALK-positive ALCL. Even if the patient number is too low to allow a statistical analysis, it is noteworthy that the three patients with detectable ALK-specific T cells had initially high ALK-antibody titres and a persisting titre at the time of T cell analysis, whereas the remaining two patients, without measurable T cell responses, had an initial titre of only 1 : 750 or had almost lost the antibody response at the time of T cell analysis, 13 years after ALCL diagnosis. DCs loaded with total tumour IVT-RNA or selected tumour antigen-encoding IVT-RNA are efficient stimulators of antigen-specific T cell responses and antitumour immunity in humans [27–32]. For immune monitoring, the main advantage of using autologous DCs transfected with full-length NPM–ALK IVT-RNA is the incorporation of all potential epitopes in the context of the complete individual HLA class I repertoire. The use of IVTRNA as an antigen format a priori ensures endogenous processing of all possible immunogenic peptides and circumvents the necessity to predict and synthesize peptides during the identification phase. As an alternative, overlapping peptides have been applied as an antigen format. Overlapping peptides were reported to allow for a more potent T cell stimulation [33–35] and to circumvent poor processing of some epitopes by DCs after IVT-RNA transfection [36,37]. In addition, the low transfection efficacy may hinder strong antigen presentation or prohibit analysis, as was the case in in one patient. HLA-C-restricted, but not HLA-A*02:01-restricted T cell responses were detected in all three NPM–ALK-reactive ALCL patients, even though they were HLA-A*02:01-positive. Compared to the direct stimulation with high concentrations of ALK peptide in the earlier studies [12,13], the RNA-based system stimulates and recognizes high-avidity antigen-specific T cells preferentially [27,38]. This difference could have resulted in the detection of lower-avidity T cells in the ALK peptide-based approaches [39,40]. Although cytotoxic T cells (CTLs) generated from ALKpositive patients by the HLA-A*02:01-binding peptides were able to lyse allogeneic HLA-A*02:01-positive ALKpositive ALCL cell lines, one limitation of the studies is the lack of a formal proof of endogenous processing of the ALK-p280-89 peptides [12,13]. Another possible explanation for the lack of a HLA-A*02:01-restricted T cell response may be that the HLA-A*02:01 binding epitopes are poorly or not processed from IVT-RNA-translated protein by the predominant immunoproteasomes expressed in mature DCs in contrast to the standard proteasome in other cells, including tumour cells [36,37].

In contrast to a study in which ALK-peptide (p280-89)reactive naive T cells were demonstrated in healthy individuals using a HLA-A*02:01/p280-89 tetramer [12], ALKspecific T cells were not detected in our study in healthy donors with the use of autologous IVT-RNA-transfected FastDCs for stimulation and recognition in an IFN-g ELISPOT assay. Our finding is in concordance with the lack of detection of ALK-specific T cells in another study, in which ALK-p280-89 was applied to an IFN-g ELISPOT-assay in six healthy volunteers [13]. The differences in the test system, i.e. ELISPOT-assay versus tetramer-assay, the lower number of analysed CD81 cells in a given healthy donor (08–16 3 106), and probably the use of IVT-RNA as the antigen format in our study, might have prohibited the detection of naive ALKspecific T cells. The estimated precursor frequency of naive ALK-specific CD81 T cells in the aforementioned study (02%) was higher than reported, even for strong viral or shared tumour epitopes [41–43]. In case the real frequency might be somewhat lower, the sensitivity threshold of our ELISPOT assay would preclude detection. As an additional potential explanation for the discrepancy, the short ALK peptide (p280-289) might not be processed from the whole NPM–ALK protein by the immunoproteasome active in DCs [36,37]. In summary, using IVT-RNA as antigen format and autologous DCs for antigen presentation, NPM–ALKreactive CD81 T cells could be identified in the blood samples of three of five NPM–ALK-positive ALCL patients in remission for up to 9 years after initial diagnosis. Recognition of NPM–ALK by CD81 T cells was restricted by HLAC alleles in the three reactive patients. As these patients were selected based on the strength of their initial humoral immune responses, further analysis of a larger unselected cohort of patients is planned to define whether the strength of the T cell response correlates with the ALK-antibody titre and clinical characteristics.

Acknowledgements This work was supported by a grant from the Von-BehringR€ ontgen-Stiftung (Project number 60-0011) to V. K. S., S. W., H. H., T. W., C. D. W. and W. W., V. K. S., S. W., C. D. W. and W. W. were supported additionally by the Forschungshilfe Peiper. The authors thank the patients and donors who participated in the study. We also thank Simone Schwalm, Jutta Schieferstein, and Katja M€ uller for expert technical assistance, and the physician at the NHL-BFM study centre, Stephanie Ruf, for visiting patients and collecting blood samples.

Disclosures The authors have no disclosures to declare.


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Author contributions V. K. S., S. W., C. D. W., A. R., T. W. and W. W. contributed to the design of the project. V. K. S. and S. W. performed the experiments. V. K. S., S. W., C. D. W. and W. W. analysed the data and wrote the manuscript. S. W., A. R. and W. W. collected patient blood samples and provided the clinical data. V. L. and T. W. contributed to the initial experimental setup and ELISPOT analysis. H. H. performed HLA-typing. W. W., C. D. W. and A. R. coordinated the study. All authors approved the final version of the manuscript.

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Supporting information Additional Supporting information may be found in the online version of this article at the publisher’s web-site: Fig. S1. Phenotypical analysis of dendritic cells (FastDCs). Mature FastDCs generated from CD141 cells were stained with anti-CD14-phycoerythrin (PE), -CD80-fluorescein isothiocyanate (FITC), -CD83-allophycocyanin (APC), CD86-PE, -CD40-APC, -CD209-peridinin chlorophyll

(PerCP), -CCR7-FITC and -human leucocyte antigen (HLA)-DR-PerCP antibodies and analysed by flow cytometry. Isotype controls for each antibody were used as a gating tool, as shown in grey. The relative fluorescence of labelled cells to background fluorescence was determined using FlowJo version 10.0 software. Values represent the percentage of FastDCs expressing the corresponding cellsurface markers. Representative FastDC maturation in an anaplastic large cell lymphoma (ALCL) patient and a healthy individual is shown. Fig. S2. Nucleophosmin–anaplastic lymphoma kinase (NPM–ALK) expression in mature dendritic cells (FastDCs) 24-h after transfection of in-vitro-transcribed RNA coding for NPM–ALK. 24 h after dendritic cell (FastDC)-nucleofection with NPM–ALK in-vitro-transcribed RNA, FastDCs (2.0 3 105) were stained with the commercially available human anti-NPM–ALK/ALK-phycoerythrin (PE) antibody and the nucleofection efficiency was measured by flow cytometry. FastDCs expressing NPM–ALK fusion protein are shown as the open histogram. An isotype control (mouse IgG3) is shown as the grey histogram. Values represent the percentage of FastDCs expressing the NPM–ALK fusion protein. Representative transfection efficiencies in one anaplastic large cell lymphoma (ALCL) patient and one healthy donor is shown. Table S1. Antigen-specific CD81 T cells and anaplastic lymphoma kinase (ALK)-antibody response in nucleophosmin (NPM)–ALK1 anaplastic large cell lymphoma (ALCL) patients in remission. Table S2. Antigen-specific CD81 T cells in healthy controls. Table S3. Prediction of computer algorithms-based human leucocyte antigen (HLA)-C*06:02 binding nucleophosmin–anaplastic lymphoma kinase (NPM–ALK)derived potential peptides IEDB analysis resource (http:// tools.iedb.org/mhci/); NetMHC 4.0 server (http://www. cbs.dtu.dk/services/NetMHC/). Table S4. Prediction of computer algorithms-based human leucocyte antigen (HLA)-C*12:02 binding nucleophosmin–anaplastic lymphoma kinase (NPM–ALK)derived potential peptides IEDB analysis resource (http:// tools.iedb.org/mhci/); NetMHCpan 3.0 server (http:// www.cbs.dtu.dk/services/NetMHCpan/).


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