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Study of human T cell differentiation using the OP9-DL1 co-culture system as a model

(2011)
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Abstract
While most hematopoietic lineages develop in the bone marrow, waves of hematopoietic precursor cells (HPC) need to travel from the bone marrow to the thymus, to allow T cell development. The unique thymic microenvironment provides all elements necessary for both the initiation of T cell differentiation and terminal differentiation. While several models have been developed to study human T cell differentiation in vitro, the only model that allows for the in vitro generation of mature T cells in the absence of a thymic microenvironment, is the OP9-Delta ligand (DL) 1 co-culture system. OP9-DL1 co-culture generated T cells could be of great therapeutic value for both the treatment of malignant disease in an immunotherapeutic setting and to restore T cell based immunity in immunodeficient patients. However, the clinical use of OP9-DL1 o-culture generated T cells has been impeded, partly because of the lack of proof that mature T cells thus generated are functional major histocompatibility complex (MHC) class I and class II restricted T cell receptor (TCR) alpha beta+ T cells or functional TCRgammadelta+ T cells. In this work we present evidence that both unconventional and conventional T cells are generated on OP9-DL1. A prominent unconventional CD8alpha alpha single positive (SP) TCRalphabeta+ population was observed that was interleukin (IL)-15 responsive and we believe this population is the in vitro equivalent of the gut intraepithelial lymphocyte (IEL) population. CD8alphabeta SP and CD4 SP TCRalphabeta+ cells did not only present a mature phenotype, but also displayed conventional CD8alphabeta SP and CD4 SP TCRalphabeta+ function. Although this suggests that generation of these cells is dependent on MHC class I and class II induced positive selection mechanisms, similar to those operative in the thymus, we show that functional maturation of these cells is independent of the presence of MHC class I and class II. MHC class I and class II independent selection may impede the use of in vitro generated T cells, because of the risk of the generation of auto-reactive T cells. Therefore, further investigation will be needed in order to better understand selection mechanisms in OP9-DL1 co-cultures. We also present a detailed precursor-progeny relationship between the different TCRgammadelta+ populations that are present in human thymus. We show that, in contrast to the murine situation, human mature TCRgammadelta+ cells are generated along 2 different pathways: a Notch independent double negative (DN) pathway generating functional mature DN and CDB alpha alpha SP TCRgammadelta+ cells and a Notch dependent double positive (DP) pathway generating functional mature CD8alphabetaSP TCRgammadelta+ cells. Maturation of TCRgammadelta6 expressing DN and DP T cell precursors is enhanced by TCR ligation. We furthermore show that DP TCRgammadelta6+ cells are not lineage committed, since they not only give rise to mature CD8alphabeta SP TCRgamma delta6+, but also to TCRalphabeta + cells. These data open new challenges in the search for functional differences between ontogenetically different TCRgammadelta+ populations. In conclusion, our data contribute to the understanding of both in vitro and in vivo T cell differentiation. However, prior to the use of in vitro generated T cells in a clinical setting, selection mechanisms in the absence of a thymic microenvironment will have to be further clarified in order to allow for the efficient production of a self-tolerant T cell repertoire.

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Citation

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Chicago
Van Coppernolle, Stefanie. 2011. “Study of Human T Cell Differentiation Using the OP9-DL1 Co-culture System as a Model”. Ghent, Belgium: Ghent University. Faculty of Medicine and Health Sciences.
APA
Van Coppernolle, S. (2011). Study of human T cell differentiation using the OP9-DL1 co-culture system as a model. Ghent University. Faculty of Medicine and Health Sciences, Ghent, Belgium.
Vancouver
1.
Van Coppernolle S. Study of human T cell differentiation using the OP9-DL1 co-culture system as a model. [Ghent, Belgium]: Ghent University. Faculty of Medicine and Health Sciences; 2011.
MLA
Van Coppernolle, Stefanie. “Study of Human T Cell Differentiation Using the OP9-DL1 Co-culture System as a Model.” 2011 : n. pag. Print.
@phdthesis{3135981,
  abstract     = {While most hematopoietic  lineages develop in the bone marrow, waves of hematopoietic precursor cells (HPC) need to travel from the bone marrow  to the thymus, to allow T cell development. The unique thymic microenvironment provides all elements necessary for both the initiation of T cell differentiation and terminal  differentiation. While several models have been developed to study human T cell differentiation in vitro, the only model that  allows for the in vitro generation of mature T cells in the  absence of a thymic microenvironment, is the  OP9-Delta ligand  (DL) 1 co-culture  system. OP9-DL1 co-culture generated T cells could be of great therapeutic  value for both  the treatment of malignant disease  in  an  immunotherapeutic  setting   and  to   restore  T  cell  based  immunity   in immunodeficient patients. However, the clinical use of OP9-DL1 o-culture generated T cells has been impeded, partly because of the lack of proof that mature T cells thus generated are functional  major  histocompatibility complex  (MHC) class I and  class II restricted    T cell receptor (TCR) alpha beta+ T cells or functional TCRgammadelta+ T cells. In this work we present evidence that both  unconventional  and conventional  T cells are  generated  on  OP9-DL1. A prominent unconventional  CD8alpha alpha  single  positive  (SP) TCRalphabeta+ population   was  observed  that  was interleukin  (IL)-15 responsive and we believe this population  is the in vitro equivalent of the gut intraepithelial lymphocyte  (IEL) population. CD8alphabeta SP and CD4 SP TCRalphabeta+ cells did not only present a mature  phenotype, but also displayed conventional CD8alphabeta  SP and CD4 SP TCRalphabeta+ function. Although this suggests that generation of these cells is dependent on MHC class I and class II induced positive selection mechanisms, similar to those operative in the thymus, we show that functional maturation of these cells is independent  of the presence of MHC class I and class II. MHC class I and class II independent  selection may impede the use of in vitro generated T cells, because of the risk of the generation of auto-reactive  T cells. Therefore, further  investigation  will  be needed  in  order  to  better  understand  selection mechanisms in OP9-DL1 co-cultures.
We also present a detailed  precursor-progeny  relationship  between  the  different TCRgammadelta+ populations  that  are present  in human thymus. We show that, in contrast  to the murine situation, human mature  TCRgammadelta+ cells are generated  along 2 different  pathways: a Notch independent  double negative (DN) pathway generating functional mature DN and CDB alpha alpha SP TCRgammadelta+ cells and a Notch  dependent  double  positive  (DP) pathway  generating  functional mature CD8alphabetaSP TCRgammadelta+ cells. Maturation of TCRgammadelta6 expressing DN and DP T cell precursors is  enhanced by TCR  ligation. We furthermore show that  DP TCRgammadelta6+ cells are not  lineage committed, since they not  only give rise to mature  CD8alphabeta SP TCRgamma delta6+, but  also to TCRalphabeta + cells. These data open new  challenges in the  search for  functional  differences  between ontogenetically different  TCRgammadelta+ populations.
In conclusion, our data contribute  to the understanding  of both  in vitro and in vivo T cell differentiation. However, prior  to the use of in vitro generated T cells in a clinical setting, selection mechanisms in the absence of a thymic microenvironment will have to be further clarified in order to allow for the efficient production of a self-tolerant T cell repertoire.},
  author       = {Van Coppernolle, Stefanie},
  language     = {eng},
  pages        = {144},
  publisher    = {Ghent University. Faculty of Medicine and Health Sciences},
  school       = {Ghent University},
  title        = {Study of human T cell differentiation using the OP9-DL1 co-culture system as a model},
  year         = {2011},
}