I believe your reasoning is correct. Here a list of Jurkat derived cell lines deficient in some TCR subunits. I hope it helps

JGN / CD3γ / Geisler 1992

R3.20.11a / TCRα and TCRβ / Arnaud et al. 2001

JR3.11 / TCRα / Arnaud et al. 2001

JRT-T3.1 / TCRα / Weiss and Stobo 1984

18.B3 / TCRα / Saito et al. 1987

J76.2 / TCRβ / Arnaud et al. 2001

JRT3-T3.5 / TCRβ / Weiss and Stobo 1984

JBN / TCRβ / Saito et al. 1987 

As far as I understand how these things work, your reasoning is correct. I believe the Jurkat-76 cell line (derived from Jurkat) is deficient in both alpha and beta TCR subunits, so these cells might be better for your experiments.

I believe your reasoning is correct. Here a list of Jurkat derived cell lines deficient in some TCR subunits. I hope it helps

JGN / CD3γ / Geisler 1992

R3.20.11a / TCRα and TCRβ / Arnaud et al. 2001

JR3.11 / TCRα / Arnaud et al. 2001

JRT-T3.1 / TCRα / Weiss and Stobo 1984

18.B3 / TCRα / Saito et al. 1987

J76.2 / TCRβ / Arnaud et al. 2001

JRT3-T3.5 / TCRβ / Weiss and Stobo 1984

JBN / TCRβ / Saito et al. 1987 

I think your reasoning is correct.

An alternative solution to avoid undesired pairing of the introduced TCR chains with the endogenous ones is to mutate the constant part of the alpha and the beta chain. The details can be found in Voss et al, J Immunol 2008, 180: 391-401.

We are also working to express functional human TCRs in human T cell lines and are trying to adapt a method using the 58a-b- hybridoma T cell line (original citation: “Derivation of a T cell hybridoma variant deprived of functional T cell receptor α and B chain transcripts reveals a non-functional a-mRNA of BW5147 origin in Eur. J. Immunol., 1989. 19:2269-2274”)- we have found that some cell lines that should work for this type of experiment actually still do not signal or have a functional TCR response. You might see if this cell line would be helpful.

I agree that your reasoning is correct. To add to what has been said here, I would re-assess expression of both chains by flow cyometry to check their surface expression levels every time you are re-establishing a cell line in your lab,.And also, as a suggestion, do this every some weeks to check for consistency of levels of expression within different time points, experiments, etc, as this could very strongly impact your results if you are looking at TCR signaling events. be very tight with your growing medium composition and conditions, as T cells that are proliferating or under stress will vary their TCR expression levels quite a bit. Good luck!

Dear Colleagues,

Thank you ever so much for your helpful answers. They contain great stuff to look into, and also very helpful technical tips.

Best wishes to all,

Tomas

We have had no luck with JRT3-T3.5 that we used to introduce a chimeric human variable-Mouse constant domain hybrid construct of an Ag-specific TCR. The NFkB reporter works in a specific way when Ag is presented exogenously as a peptide on T2 cells. The transfectants also bind Ag presented as a HLA-A2 pentamer. However, these cells do not recognize HLA-A2+ Ag+ tumor cell lines or a similar cross-presented Ag by DCs. Introduction of CD8 in the JRT3s did not result in enhanced effector activity. The conclusion is that while these cells are highly transfectable but the TCRs are not functional in the physiological sense. Does anyone on this forum know whether JRT3s are missing downstream signaling molecules like CD3 zeta? BTW, our construct was designed to prevent endogenous mispairing of the TCR alpha chain.

Any line can be purchased from commercial source?