טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentLucy Leshansky
SubjectImmunomodulatory properties of Human Embryonic Stem Cells:
Mechanisms of Action and use in Transplantation
DepartmentDepartment of Medicine
Supervisors Professor Emeritus Itskovitz Joseph
Dr. Berrih Sonia
Full Thesis textFull thesis text - English Version


Abstract

Human ES cells are believed to be powerful tools for cell-transplantation therapies. However, their potential ability to provoke immune response in immunocompetent patients could limit their use.  hESCs express minimal levels of MHC-I, MHC-II, T cell costimulatory molecules B7.1 and B7.2, suggesting their inability to induce T-cell immune response via these pathways. However, little has been done to characterize human lymphocyte response to hESCs.

In the present study we investigate the cross-talk between established hESCs lines and normal human immune cells. We demonstrate for the first time that hESCs are able to inhibit the expression of Glytamine-Cysteine Ligase (GCL) in monocytes and CD4 T-cells leading to insufficient synthesis of Glutathione (GSH) in both cell types, altered differentiation of monocytes and as a result inhibition of CD4 T-cells activation, as demonstrated by three activation criteria: 1) lymphocyte size; 2) Fas expression and 3) the ratio of Naïve/Memory CD4 T-cells cells. Similar inhibitory effects are observed when PBMCs are cultured with hESCs’ conditioned medium or co-cultured with hESCs in Transwells, demonstrating that the effects were at least partially dependent upon a soluble factor. Furthermore, the activation of PBMCs is inhibited and down-regulated by switching to the hESCs’ conditioned medium, while PBMCs cultured in the presence of hESCs’ conditioned medium is activated when re-exposed to the control conditions. These findings indicate that both the activation and its inhibition are reversible, and most likely inhibition does not reflect anergy state of T-cells. We have also found that the ratio of Naïve/Memory CD4 T-cells decreases in control conditions while in the presence of hESCs it remains stable indicating that hESCs favor the Naïve phenotype. Moreover, hESCs’ conditioned medium when applied on HepG2 hepatocarcinoma cells, causes sharp decrease in proliferation despite the presence of L-cysteine (NAC). Normal proliferation is restored only by enhanced transcription of GCL catalytic subunit (GCLC) confirming specific effect of hESCs on GCL activity regardless the cell type used.

Our results demonstrate that hESCs are able to down-regulate the human immune system response by interfering with GSH synthesis pathway, specifically by inhibiting GCL activity, altering the GSH balance in the intra-cellular environment leading to the lack of CD4 T-cells activation.

Resolving the nature of soluble factor(s) and mapping the up-stream pathway responsible for down-regulation of GCLC expression by hESCs seem to be one of the most important challenges for further investigation.