- Invited speaker presentation
- Open Access
S77. Proffered paper: In vitro induced response patterns of antileukemic T-cells – characterization by combination of functional assays, spectratyping and next generation sequencing
© Reuther et al; licensee BioMed Central Ltd. 2014
Published: 12 March 2014
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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