Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro
Summary:
The authors of the paper tried to direct differentiation of embryonic stem cells into mature and functional T cells by co-culturing them with stromal cells which express Delta-Like 1 (DL1) ligand, initiating the Notch receptor pathways in the cell. In the experiment, embryonic stem cells were grown on two stromal cell lines (OP9-control and OP9-DL1, these cell lines were created by injecting either empty or DL1/GFP containing viral vectors into OP9 cells). Flow cytometry performed on the cells at days 8, 14, and 20 reveal the development and differentiation stages of the embryonic stem cells into lymphocytes. In the presence of DL1 ligand expression, ESCs differentiates into T cells. PCR analysis of these ESC-derived T cells shows that they rearranged during development and have a diverse repertoire of rearranged TCRs (T cell receptors). Upon further investigation, the authors, however, discovered that the majority of these ESC-derived T cells express CD8 only (CD8 identifies T cells as Cytotoxic T Lymphocytes or CTLs, which kill infected cells upon stimulation) and the other major group of T cells (CD4 T cells, which help activate B cells and CTLs), is absent. The experimenters then seeded T cell progenitors into irradiated Fetal Thymic Organ Cultures (FTOCs) and the progenitors were able to reconstitute the FTOC to produce both CD8 and CD4 T cells. These T cells were also shown to mount an antigen-specific response to the LCMV virus.
I am interested in this paper because it shows hope for patients with immunodeficiency, such as HIV, SCID, etc. One reason for HIV’s lethality is the virus’ potent attack against CD4 T cells or T helper cells. CD4 T cells serve one of the most important roles in our immune system including recruiting and stimulate proliferation of B cells and T killer cells. This paper shows that we can use ESCs and direct them to differentiate into functional and effective T cells (including CD4 if transplanted into a FTOC). Once the method is improved in in vitro reconstitution, ESC-derived T cells can be generated in vivo in implanted fetal thymic lobes. By allowing early T cell progenitor to grow in the body, these ESC-derived cells can undergo positive and negative selection. As a result, these cells become self-tolerant and can no longer raise inappropriate immune rejection.
6 comments:
It is interesting that the method they used to induce T cell differentiation was by co-culturing with another cell line expressing DL1. To reduce the number of confounding variables, why didnt they add the DL1 ligand directly to the ESC's? Perhaps the OP9 cell line itself had unseen effects on T cell differentiation.
As a hypothetical extension, perhaps transducing the ESC's directly with the DL1 construct would be a more effective/practical step towards an effective therapeutic for immunodeficiency syndromes. This would follow a classical gene therapy approach and I personally see this as being more effective than differentiating ESC's into T cells in vitro and injecting them into the patient for who knows how long?
The OP9 control cells serve as the feeder cells and have important roles in inducing differentiation. I personally doubt that transduction of ESCs with DL1 will allow the cells to differentiate into T cells. It does not seem that simple. Also, one of the reasons for in vitro induction and stimulation is that ESCs-derived precursors cannot grow in host thymus. For some reason, they just don't attach. That is why the authors had to seed them in a FTOC and then into the host thymus.
It is amazing to know that T-cells can be made to develop in vitro. However, I found it confusing when you stated: "In the presence of DL1 ligand expression, ESCs differentiates into T cells... Upon further investigation, the authors, however, discovered that the majority of these ESC-derived T cells express CD8 only." I wonder what is the mechanism that takes place so the final T cells solely express CD8 but not CD4 because I thought during T cell development, T cell develops from double negative to double positive(expressing both CD4 and CD8). Therefore, is DL1 the factor that drives most T cells into killer T cells (CD8) instead of into CD4 T cells?
Also, you stated later that "The experimenters then seeded T cell progenitors into FTOCs and were able to produce both CD8 and CD4 T cells." So I wonder if being exposed to FTOC, CD4 would be expressed? In other words, if we culture the T cells either with DL1 ligand expression or with FTOC, can we manipulate the final T cell to be expressing either CD8 or CD4?
Thanks.
This is similar to my paper about dendritic cells, that when pulsed with tumor RNA, can cause T cells to become active against that type of tumor cell. I wonder how scientists figured out which antigens are transferred to the T cells via this method, and what exactly causes the T cell to all of the sudden become cytotoxic. Also, how long does the process take?
To answer yisu's questions, OP9-control cells expressing DL1 present mostly MHC I molecules, which the author suspected to be the cause of CD8+ T cell production only.
FTOC should contain both MHC class I and class II molecules. Depending on which molecule T precursor interacts with, it will mature into either CD4+ or CD8+ T cells. For most immunodeficiency, both CD4 and CD8 T cells are deprived or destroyed. And a healthy immune system would need both mechanisms. Maybe there are diseases that require either one of these T cell types.
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