eprintid: 33373
rev_number: 13
eprint_status: archive
userid: 7440
dir: disk0/00/03/33/73
datestamp: 2023-07-25 07:25:40
lastmod: 2024-06-17 11:52:48
status_changed: 2023-07-25 07:25:40
type: doctoralThesis
metadata_visibility: show
creators_name: Poelchen, Juliane
title: Generation of melanoma antigen-specific CD8+ T cells from human induced pluripotent stem cells for adoptive cell therapy
subjects: ddc-570
divisions: i-140001
adv_faculty: af-14
abstract: Adoptive cell therapy using tumor antigen-specific CD8+ T cells is a promising approach to treat
patients with advanced or metastasizing cancer including malignant melanoma. The general procedure
is based on the isolation, in vitro stimulation and expansion of tumor-infiltrating lymphocytes
(TILs). These activated and expanded TILs, which are specific for common tumor antigens are then
transferred back into the patients to attack and neutralize cancer cells. However, current studies report
disease relapse in treated patients because of T cell exhaustion after ongoing TCR stimulation
resulting in a decreased effector function and a loss of their replicative capacity.
The use of induced pluripotent stem cells (iPSCs) as a source for the generation of “off-the-shelf”
CD8+ T cells could enable T cell-based immunotherapy on a large scale. Similar to embryonic stem
cells, iPSCs have the potential to give rise to nearly unlimited amounts of any cell type, including
CD8+ T cells. The aim of this project was to develop strategies for the in vitro generation of
melanoma antigen-specific CD8+ T cells from human iPSCs.
First, iPSCs were transfected with S/MAR DNA vectors coding for a T cell receptor (TCR) against
MART-1 or a chimeric antigen receptor (CAR) directed against MCSP. S/MAR DNA vectors are
effective gene delivery systems since they contain a scaffold/matrix attachment region, which binds
to the nuclear matrix and ensures a persistent gene expression over hundreds of cell divisions. The
transfection of iPSCs with the S/MAR DNA vector did not alter the gene expression of the pluripotency
genes NANOG and OCT4 as well as other T cell-related differentiation genes.
Next, a 2D co-culture system with OP9 murine stromal cells was used to facilitate the differentiation
of CD34+ hematopoietic stem and progenitors cells (HSPCs) from iPSCs. Further differentiation
of CD34+ HSPCs into CD4+CD8+ double-positive (DP) T cells was achieved by long-term coculture
with bioengineered OP9 stromal cells expressing T cell-specific cytokines like FLT3-ligand,
CXCL12 and SCF as well as the Notch ligand DLL4 and supplementation of the growth medium
with recombinant IL7.
Since clinical applications require clean populations of effector T cells without contamination with
murine cells, the stromal cell based co-culture system was replaced with a commercially available
differentiation kit. Following the manufacturer’s instructions, I was able to generate a limited
amount of CD8+ single-positive (SP) T cells. Flow cytometric analysis of common T cell markers
revealed a low CD3 expression in generated T cells but a persistent MART-1 TCR or MCSP CAR
expression, respectively. In comparison to CD4+CD8+ DP T cells, CD8+ SP T cells demonstrated
the capability to produce cytokines, showed degranulation capacity after stimulation and exhibited
cytotoxic effects against melanoma cell lines.
Hence, these findings revealed the possibilities as well as the challenges connected with the use of
human iPSCs for the generation of CD8+ T cells. However, the use of S/MAR DNA vectors and
novel differentiation strategies could enable further clinical applications in cancer treatment and
help to improve common immunotherapies for melanoma patients.
date: 2024
id_scheme: DOI
id_number: 10.11588/heidok.00033373
ppn_swb: 1891371150
own_urn: urn:nbn:de:bsz:16-heidok-333731
date_accepted: 2023-06-05
advisor: HASH(0x559e37cfd938)
language: eng
bibsort: POELCHENJUGENERATION20240606
full_text_status: public
place_of_pub: Heidelberg
citation:   Poelchen, Juliane  (2024) Generation of melanoma antigen-specific CD8+ T cells from human induced pluripotent stem cells for adoptive cell therapy.  [Dissertation]     
document_url: https://archiv.ub.uni-heidelberg.de/volltextserver/33373/1/Thesis_Juliane%20Poelchen-1.pdf