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Volume 2 Supplement 2

Abstracts from the 1st Immunotherapy of Cancer Conference (ITOC1)

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S77. Proffered paper: In vitro induced response patterns of antileukemic T-cells – characterization by combination of functional assays, spectratyping and next generation sequencing


T-cell receptor (TCR) diversity is characterised by somatic alterations in the complementary determining region 3 (CDR3) of the human TCR β-chain. Complemented with the TCR alpha-chain, TCR diversity can hypothetically result in up to 1018 different TCR molecules.

Myeloid leukaemic cells can be induced to differentiate into leukaemia-derived dendritic cells (DCleu) regaining the stimulatory capacity of professional DCs while presenting the whole leukaemic antigen repertoire.

Our aim was to identify TCR Vβ-chain-rearrangements in T-cells stimulated with leukaemic blasts and DCleu in 3 patients with AML and furthermore to detect, amplify or monitor T-cell clones with defined Vβ-profiles in correlation with antileukaemic function, in vitro and in vivo.

Material and methods

HLA matched or HLA haplo-identical (allogeneic) donor- or autologous T-cells were repeatedly stimulated, either with leukaemic blasts or the corresponding DCleu from 3 different AML-patients. Cytotoxicity assay was carried out for measuring the lytic activity of effector T-cells, spectratyping was performed to identify the restriction of the TCR Vβ-repertoire in unstimulated and stimulated T-cells and Sanger sequencing to analyse the β-chain sequence information including the CDR3 regions. Additionally next generation sequencing (NGS) was established to analyse the accurate TCR sequence information of thousands of TCR β-chains with high coverage.


No significant differences in T-cell proliferation were observed. The T-cell mediated cytolytic response patterns showed blast lysis (n=1) and blast proliferation (n=2).

Spectratyping revealed a remarkable TCR Vβ-restriction of the CD4+- or CD8+-TCR repertoire of blast- or DC/DCleu-stimulated T-cells, independently of blast or DC/DCleu used as stimulators. Although in absolute terms, DC/DCleu stimulation induced the highest grade of restriction in the CD8+ T-cell subset, the CD4+ T-cells seemed to be relatively more affected.

In vitro stimulation with DC/DCleu resulted in an identical TCR (β-chain restriction pattern) as identified in vivo in a patient sample 3 months after allogeneic stem cell transplantation (SCT).


A combined strategy using spectratyping and NGS with functional tests may provide useful information about the specificity and efficacy of the intra-individual variable induced T-cell response.

Spectratyping allows the identification of a restricted Vβ-repertoire by Gaussian-like distribution, NGS allows sequencing of TCR repertoires with high coverage, novel software allows the analysis of the exact length and sequence composition (the combination of the Vβ- and Jβ-genes) of the β-chains, especially of the CDR3, and the exclusion of non-functional transcripts.

The identification of defined Vβ-T-cell clones may lead to selection procedures generating Graft-versus-Leukaemia reaction- but not Graft-versus-Host disease- mediating T-cells for adoptive immunotherapy after SCT.

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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

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Reuther, S., Krell, P., Schuster, F. et al. S77. Proffered paper: In vitro induced response patterns of antileukemic T-cells – characterization by combination of functional assays, spectratyping and next generation sequencing. j. immunotherapy cancer 2 (Suppl 2), I15 (2014).

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