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  • 1
    In: Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 3364-3364
    Abstract: Background Immunotherapy for CLL with new antibodies or T-cells with modified TCR relies on attractive targets. ROR1 is such a promising target since it is highly overexpressed in CLL. Chimeric antigen receptor engineered T cells and antibodies directed against the extracellular part of ROR1 have already been developed and tested in vitro or in animal models, but still there is no MHC-class I presented peptide serving as target structure for CD8+ T cells (with or without a genetically modified T cell receptor) available. Aim The aim of this study was (1) to identify an immunogenic MHC-class I presented ROR1 peptide, (2) to generate respective ROR1 peptide specific CD8+ T cell clones, and (3) to analyze the nucleotide sequence of the CDR3 region of the expressed alpha and beta T cell receptor chain. Results In mass spectrometric-based analyses of the HLA-ligandome a HLA-B*07 presented ROR1 peptide was identified in primary CLL cells of two patients. Six T cell clones specific for this particular ROR1-peptide were generated from single CD8+ T cells from 2 healthy individuals with 3 T cell clones generated from each donor. Functionality and specificity of those T cell clones were tested in cytotoxicity assays. All 6 dextramer+ CD8+ T cell clones lysed peptide loaded and HLA-B*07+ transduced K562 cells (kindly provided by Lorenz Jahn, [Jahn et al., Blood, 2015 Feb 5;125(6):949-58]). Two selected clones (XD8 and XB6) were tested for their cytotoxic potential against 2 ROR1+ HLA-B*07+ tumor cell lines (with the ROR1 peptide identified by mass spectrometry for both of them) and against 2 primary CLL cell samples. Tested clones showed a significant lysis of the respective target cells. CDR3 regions of the alpha and beta T cell receptor chain were sequenced on a single cell level. The CDR3 alpha region from each of the 3 ROR1 specific T cell clones from donor A showed some similarities to T cell clones derived from donor B (Table 1). Conclusion For the first time a MHC-class I presented ROR1 peptide antigen is reported. ROR1 positive CLL cells can be targeted by specific HLA-B*07 restricted CTLs. Respective CD8+ T cell clones with anti-leukemic activity from 2 donors share some amino acid motifs of the CDR3 alpha and beta regions. In conclusion, this information provides the possibility of generating ROR1 specific CD8+ T cells with genetically modified T cell receptors for immunotherapy and for tracking those cells after administration with next generation sequencing in peripheral blood samples of patients. Furthermore, data suggest the existence of public TCR motifs in leukemia antigen specific CTLs, which needs to be proven in follow-up experiments with larger cohorts of donors and patients. Finally, the presented strategy to identify leukemia specific peptide antigens for CD8+ T cells might be an attractive method for similar projects. Table 1 Amino acid sequences of CDR3 alpha and beta regions of the TCR of ROR1 specific CD8+ T cell clones. When comparing two clones, matching amino acids are depicted in red. The aromatic amino acids phenylalanine (F) and tyrosine (Y) are shown in blue when situated at the same position. Gaps inserted during the sequence alignment process are indicated by a hyphen '-'. Table 1. Amino acid sequences of CDR3 alpha and beta regions of the TCR of ROR1 specific CD8+ T cell clones. When comparing two clones, matching amino acids are depicted in red. The aromatic amino acids phenylalanine (F) and tyrosine (Y) are shown in blue when situated at the same position. Gaps inserted during the sequence alignment process are indicated by a hyphen '-'. Disclosures Middeke: Sanofi: Honoraria. Schetelig:Sanofi: Honoraria.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2016
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 2
    In: Haematologica, Ferrata Storti Foundation (Haematologica), Vol. 102, No. 11 ( 2017-11), p. e460-e464
    Type of Medium: Online Resource
    ISSN: 0390-6078 , 1592-8721
    Language: English
    Publisher: Ferrata Storti Foundation (Haematologica)
    Publication Date: 2017
    detail.hit.zdb_id: 2186022-1
    detail.hit.zdb_id: 2030158-3
    detail.hit.zdb_id: 2805244-4
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  • 3
    Online Resource
    Online Resource
    Frontiers Media SA ; 2022
    In:  Frontiers in Immunology Vol. 13 ( 2022-2-2)
    In: Frontiers in Immunology, Frontiers Media SA, Vol. 13 ( 2022-2-2)
    Abstract: Graft-versus-host disease (GvHD) of the skin is a severe allo-immune reaction and complication following allogeneic stem cell transplantation. Over the past years, intensive pre-clinical research has led to an improved understanding of the pathophysiology of acute and to a lesser extend chronic GvHD. This has translated into the approval of several new agents for the treatment of both forms of GvHD. This review summarizes the most recent advances in underlying pathomechanisms, clinical trials and newly approved agents for GvHD, with a special focus on skin involvement.
    Type of Medium: Online Resource
    ISSN: 1664-3224
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2022
    detail.hit.zdb_id: 2606827-8
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  • 4
    In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 5488-5488
    Abstract: CMV-infection is a serious complication in patients after allogeneic stem cell transplantation (SCT) where immunosuppressive therapy and impaired T cell reconstitution result in a high risk for viral infections. Monitoring of CMV-virus load by PCR and preemptive therapy are important tools to prevent CMV disease. However, CMV specific cytotoxic T cells (CMV-CTLs) are needed to successfully control CMV-infections. CMV-specific multimers composed of the patients HLA Class I molecule bound to CMV pp65 epitopes give the possibility to monitor CMV-CTLs. Here, we present the case of CMV-reactivation following SCT for AML. Methods The percentage of CMV-specific CD8+ T cells was determined by flow cytometry and mapped to clinical and laboratory parameters of the patient. CD8+ T cells were detected using CD8-fluorescein isothiocyanate (FITC, Beckman Coulter) antibody and CD3 as a T-cell marker was labeled with CD3-allophycocyanin (APC, MACS Miltenyi Biotec) antibody. CMV-specific CD8+ T cells were detected using the CMV major histocompatibility complex (MHC) with Strep-Tactinphycoerythrin (PE) conjugate (Streptamers, IBA GmbH). Case A 60 years old male patient was diagnosed with acute myeloid leukemia (AML) with 95% myeloid blasts in the bone marrow and extramedullary AML manifestations at the time of diagnosis. Following induction therapy the patient was transplanted from a matched unrelated donor. The stem cell recipient as well as his donor had been tested sero-positive for CMV prior to SCT. Within the first month following transplantation, the patient developed an effective CMV specific immunity as seen by high levels of CMV-specific T cells (Figure 1). About three months following transplantation the patient was diagnosed with intestinal GVHD requiring high-dose glucocorticoid treatment. Following steroid exposure, levels of CMV-CTLs dropped and shortly thereafter rising CMV-copy numbers were observed which was accompanied by clinical signs of CMV enteritis. With the administration of antiviral treatment the CMV specific virus load decreased. However, levels of CMV-CTLs remained low, presumably as a result of ongoing steroid exposure. Discussion High levels of CMV-CTLs appeared to control CMV, as seen by a non-detectable virus load in standard PCR testing. The close correlation between the drop in CMV-CTL count and CMV activation highlights the potential of this method to monitor and understand immune responses to CMV following SCT. Of note, early presence of high frequencies of CMV-CTLs did not guarantee CMV-control under steroid exposure as seen in our case. Previous reports have suggested that high dose glucocorticoids may impact CMV-CTLs survival. This is supported by our case, where we see a rapid drop in CMV-CTLs following glucocorticoid exposure. However, the exact molecular mechanisms and more importantly, the predictive value of this finding remain elusive. Furthermore, these data suggest, that patients with ongoing high steroid exposure may not benefit from a transfer of CMV-specific T-cells to control CMV disease. Conclusion Further investigations to clarify the potential of CMV-CTL measurements and to understand the effect of steroid exposure at the functional level are warranted. Studies to correlate CMV-CTL counts with the level of immunosuppression and their influence on controlling CMV-disease will follow. In future, this tool could provide a chance to select patients at high risk of CMV reactivation who could profit from an individualized monitoring and early treatment. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 5
    In: Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 5718-5718
    Abstract: Mutated nucleophosmin (NPM1) was identified as a promising leukemia-specific antigen for cytotoxic T lymphocytes (CTL). NPM1 is a multifunctional nucleocytoplasmic shuttling phosphoprotein. In AML patients with normal cytogenetics NPM1 mutations are the most frequent molecular genetic abnormalities, accounting for up to 60% of the patients. The peptide (AIQDLCLAV) derived from the mutated NPM1 (NPM1mut) has been described to elicit a CTL response restricted to HLA-A*02:01. We observed that NPM1mut multimer+ T cells were very rare in peripheral blood. The limitation of the multimer technology is the absence of a positive control; nevertheless it is an attractive tool to generate antigen positive T cell clones. The goal was to compare strategies for the generation of NPM1mut multimer+ T cell clones systematically. For this purpose we analyzed blood samples from two patients with AML after transplantation and six different healthy donors. We explored different strategies to isolate HLA-A*02:01 restricted NPM1mut multimer+ T single cells. The first strategy was to isolate multimer+ T cells directly from the blood without any supplements by single cell sorting. The second strategy was to sort multimer+ T cells which were previously CD8+ enriched supplementing the media either with or without IL-21. Published by Yongqing et al.IL-21 enhances the generation of human antigen-specific CD8+ T cells. A further strategy was to previously enrich CD14+ cells for the generation of autologous monocyte-derived dendritic cells (MoDCs). The co-cultivation of MoDCs loaded with the NPM1mut peptide and CD8+ cells were performed either with or without IL-21, as well. We expanded the last strategy by a second round of NPM1mut-specific stimulation. So far it was not possible to generate NPM1mut-specific T cell clones based on the advanced strategies and consistently there is no data published on NPM1mut multimer+ T cell clones. This fact raises the question why NPM1mut specific clones display such low frequencies. We want to point out that although we varied the strategies and we used eight different donors the isolation of NPM1mut-specific T cells restricted to HLA-A*02:01 apparently is challenging. Greater efforts, e.g. a larger number of donors or the use of immunological checkpoint inhibitors during cell culture are needed. Disclosures Thiede: AgenDix: Employment, Other: Ownership. Schetelig:Sanofi: Honoraria.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
    RVK:
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2016
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 6
    In: Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 3495-3495
    Abstract: Purpose Counselling of patients with AML about allogeneic hematopoietic stem cell transplantation (alloHSCT) is still an ambitious task in the light of the potential curative perspective after alloHSCT, poor outcomes after non-transplant approaches but a high risk of transplant-associated complications and still a significant risk of relapse even after HSCT. Several scores have been developed to predict outcome after HSCT, such as the HCT-CI and the Pre-transplant Assessment of Mortality (PAM) score. The PAM score has been revised recently, thereby acknowledging the shift to more frequently used reduced intensity conditioning. This score utilizes information on pts.age, donor type, disease risk, theserostatus for the CMV of pts.and donor, and the forcedexspiratory volume in the 1 second (FEV1). The aim of this study was to analyze the predictive power of the PAM score in an independent, large cohort of AML pts.who receivedalloHSCT within the last 12 years. Patients and Methods We selected all adult AMLpts.whoreceived the firstalloHSCTat the University Hospital of Dresden, a tertiary care hospital with a large transplant program, from January, 1, 2003 to July, 1, 2015.Pts.withhaplo-identical donors or after cord-blood transplantation were excluded. All patients gave their informed consent on analysing data. The PAM score was calculated as published (Au et al., BBMT 2015) and stratified into 4 groups: scores 〈 17,scores17 to 〈 24, scores 24 through 30, and scores 〉 30. Overall survival (OS), event-free survival (EFS), cumulative incidence of relapse (CIR) and non-relapse-mortality (NRM) after alloHSCTwereanalyzed according to several factors known to impact outcome using the log-rank test for univariate comparison. Age, AML type (de novo vs. sAMLvs. t-MN), sex match (female donor/male recipient vs. all other), CMV match (negative/negative vs. all other), donor type (sibling vs. matched unrelated vs. mismatched unrelated), ELN risk classification, type of conditioning (RIC vs. MAC), disease stage (CR1 vs. primary induction failure vs. 〉 = first relapse) and the PAM score as a continuous variable were selected a priori for multivariate Cox regression analyses. Results Overall, 544 pts.metthe inclusion criteria and were analyzed,the median age was 57 years (range, 18 to 76). Two-hundred-three pts.(37%) were treated with standard myeloablative conditioning (MAC) regimens while the remaining pts. received reduced intensity conditioning (RIC). Donors were siblings in 120 (22%), matched unrelated in 295 (54%) and mismatched unrelated donors in 129 (24%) pts. With a median follow up of 47 months (range, 1 to 161), the estimated OS for the whole cohort at five years was 43%, with a CIR of 30% and a NRM of 31% up to that time-point. The probability for OS at five years for pts.in PAM score group 0, 1, 2, and 3 was 65%, 50%, 33%, 22%, respectively (log-rank test, p= 〈 .001). Both the CIR and NRM increased with increasing PAM scores (gray-tests, p= .005 and p= 〈 .001, respectively). Notably, the PAM score contributed significantly to the prediction of OS even when added to a multivariate regression model which contained the single components of the score. In the final multivariate model, age (HR 1.02 per year, p= .004), disease stage (primary induction failure versus CR1, HR 1.5, p= .03), and the PAM score (HR 1.04, p= .03) had a significant impact on OS. Conclusion We validated the revised PAM for the prediction of OS after HLA-compatiblealloHSCTin a large, well characterised cohort of AMLpts.treatedat a large German transplantcenter. To the best of our knowledge, this is the first external validation of the revised PAM score. OS prediction based on this tool will be useful for counselling of futurepts.withAML. Figure OS after HSCT according to the PAM score Figure. OS after HSCT according to the PAM score Disclosures Middeke: Sanofi: Honoraria. Thiede:AgenDix: Employment, Other: Ownership. Schetelig:Sanofi: Honoraria.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
    RVK:
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2016
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 7
    Online Resource
    Online Resource
    Elsevier BV ; 2023
    In:  The Lancet Diabetes & Endocrinology Vol. 11, No. 1 ( 2023-01), p. 2-3
    In: The Lancet Diabetes & Endocrinology, Elsevier BV, Vol. 11, No. 1 ( 2023-01), p. 2-3
    Type of Medium: Online Resource
    ISSN: 2213-8587
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2023
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  • 8
    Online Resource
    Online Resource
    Springer Science and Business Media LLC ; 2023
    In:  Bone Marrow Transplantation Vol. 58, No. 3 ( 2023-03), p. 239-246
    In: Bone Marrow Transplantation, Springer Science and Business Media LLC, Vol. 58, No. 3 ( 2023-03), p. 239-246
    Abstract: Graft-versus-host disease (GvHD) remains one of the major complications following allogeneic haematopoietic stem cell transplantation (allo-HSCT). GvHD can occur in almost every tissue, with the skin, liver, and intestines being the mainly affected organs. T cells are implicated in initiating GvHD. T cells identify a broad range of antigens and mediate the immune response through receptors on their surfaces (T cell receptors, TCRs). The composition of TCRs within a T cell population defines the TCR repertoire of an individual, and this repertoire represents exposure to self and non-self proteins. Monitoring the changes in the TCR repertoire using TCR sequencing can provide an indication of the dynamics of a T cell population. Monitoring the frequency and specificities of specific TCR clonotypes longitudinally in different conditions and specimens (peripheral blood, GvHD-affected tissue samples) can provide insights into factors modulating immune reactions following allogeneic transplantation and will help to understand the underlying mechanisms mediating GvHD. This review provides insights into current studies of the TCR repertoire in GvHD and potential future clinical implications of TCR sequencing.
    Type of Medium: Online Resource
    ISSN: 0268-3369 , 1476-5365
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    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2023
    detail.hit.zdb_id: 2004030-1
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  • 9
    In: Bone, Elsevier BV, Vol. 158 ( 2022-05), p. 115684-
    Type of Medium: Online Resource
    ISSN: 8756-3282
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2022
    detail.hit.zdb_id: 1496324-3
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  • 10
    In: Haematologica, Ferrata Storti Foundation (Haematologica), Vol. 104, No. 3 ( 2019-03), p. 622-631
    Type of Medium: Online Resource
    ISSN: 0390-6078 , 1592-8721
    Language: English
    Publisher: Ferrata Storti Foundation (Haematologica)
    Publication Date: 2019
    detail.hit.zdb_id: 2186022-1
    detail.hit.zdb_id: 2030158-3
    detail.hit.zdb_id: 2805244-4
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