Hi Dimitry. Did you look at pre-TCR? Even when you "knockout" Va genes to insert your Va of interest, the pre-alpha can remain. You can actually see "T cells" in those mice. The T cells arent functional but will be detected by flow.

Several questions (things to check) and potential answers come to mind. What kind of TCRa gene did you use? Can it pair with beta? and is there TCRbeta rearrangement and expression? Perhaps the premature rearranged TCRalpha expression blocks the beta rearrangement and you get no proper full(mature) TCR expression? Does the pTA cassette allow for expression in all T cell developmental stages, or is it restricted to DN3 and a few further stages? Then it is unlikely you get expression inn mature T cells.

David, thank you very much! I believed, that preTCR did not appear on the cell surface. It is associated with ER. I can not understand, how CD3 could appear on T cell surface without correctly paired and folded mature TCR.

How many founders that you have, have this phenomenon?

Dear Frank, thank you very much! Our TCR alpha transgen was amplified from genomic DNA of T cell hybridoma and verified by transfection into 4G4 together with beta chain of original TCR and coreceptors. TCR together with CD3 appeared on the cell surfaceIt and worked perfect. In mice: Full sequencing of transgene has shown that it was "in frame". TCRs formed by endogenous alpha and beta chains develop normally, for exception some lymphopenia in thymus. pTA allows expression even in early stages. I think, this may be major problem, because there are some reports, showing development of gamma/delta like TCR alpha/beta T cells if the expression of TCRalpha chain begins too early. I am surprized, because in parallel, we made TCR alpha trangenic mice by the same way from another TCR, but we did not see these problems with expression of alpha chains and 20-30% of peripheral T cells successfully expressed alpha-transgen.

Dear Lei Huang, at present, only one. Initially, primary trangenics were obtained on complex genetical background (progenies of F1(CBA x C57BL/6) mice). In first generations we detected expression of transgene on peripheral T cells. After transfer onto B10.D2(R101) background (on which initial TCR was selected) expression gradually disappeared and we had great doubts to perform back-crossing of mice further.

Hi Dmitry, It could be that this particular TCRa is so efficient that it binds to the very first rearranged beta chains, prevents further TCRB rearrangements and indeed has properties of gd T cells. Do you see abnormalities with the DN compartment? For instance CD3+ cells in the DN2 or early DN3 thymocytes?

It is a typical problem in TCR trangenisis, but usually other way around. Because TCR are typically coned from B6 or B10 or Balb, it may not be selected well in B6/CBA F1. I actually had this problem before. Once crossed to B6 or B10 more, they will getting better. Your problem is somehow different. If you got good TCR in your original founder, it means no problem in your TCR expressing, so it is possible that it is a thymic selection problem, but I don't think so because your Tg is only for TCR alpha chain (if I understood correctly). So I don't really know why. This may be something very interesting in fundamental T cell biology.

Dear Frank, we did not analyse DN T cells in details before. Thank you very much! May be another idea to be productive? This single alpha chain successfully competed with preTCRa, but ineffective during alpha chain selection? Thank you!!

I don't know if this helps... I read that in mice during T cell development there could be expression of CD3dimers in the abscence of any TCR chains

http://www.jimmunol.org/content/162/5/2589.long

Dear Beatriz, thank you! Cited article suggests low levels of CD3 in RAG-2 deficient mice. These levels were demonstrated by immunoprecipitation of iodinated surface proteins. In this article, I could not see undoubted evidence for real location of these proteins on the surface of cells. The method of isolation represented obtaining the total membrane fraction after labeling with 125I by the lactoperoxidase method and subsequent extraction in 1% digitonin or 0.5% Triton X-100. There are no FACS data or microscopy. I think, too low levels of CD3 could not be detected by FACS. We see at some extent different picture based on FACS and functional assays. CD3 does not appear on the surface of 4G4 cells, and they stay unresponsive to plate bound anti-CD3, until full mature TCR alpha/beta will be expressed on the cell surface. In normal mice, expression of CD3 on thymocytes is strongly correlated with DP and subsequent developmental stages. So, we were surprised to see bright CD3 DN non gamma/delta T cells in transgenic mice. It may be, my position is wrong. I would greatly appreciate alternative substantiated points of view. Thank you!

Dear Frank! In transgenic mice we really see 3x increase in CD3+ DN2 and 8x increase in CD3+DN3 thymocytes as compared with wild type animals. We see also slight increase in percent of DN1 cells and decrease in DN2 and DN3 thymocytes. You are right! Thank you!!! You can see dataset here https://www.researchgate.net/publication/244993510_CD3_on_DN_thymocytes_in_TCR_alpha_transgenic_mice?ev=prf_pub

Data CD3 on DN thymocytes in TCR alpha transgenic mice

Thank you very much to all! Up to now I have not explanation for the absence of TCRalpa on CD3+ DN T cells. It seems we will need to study IEL compartment on periphery.

CD3 can be present on the surface on DN cells in the absence of any TCR even in Rag2-/- mice. The CD3 can then be crosslinked with antibodies which induces development that includes the upregulation of CD4 and CD8

That is an excellent point Luise!

Thank you very much, Luise! I should agree with you, David and Beatriz that some quantity of CD3 (not detectable by FACS in normal mice up to DN3 stage) can exist on the surface of DN cells. It seems, this can mediate biological effects of anti-CD3 mAbs on developing thymocytes. Would you be so kind, have you explanation for the shift of CD3 expression to DN2 stage in TCR alpha transgenics (you can see this in presented dataset)?

I have three possible explanations for you that are not mutually exclusive: 1. the classical CD44 vs. CD25 subdivision of DN1-4 is oftentimes sufficient for preliminary T-cell developmental analysis. However the DN2 subcompartment is ill defined by those two markers. I recommend additional staining with c-kit and CD24.

2. The introduction of transgenic TCRs or their subcomponents can screw up DN development. HY transgenic mice for example will have no DN2 or DN3 cells because the transgenic TCR is expressed in the DN1 compartment and classical beta-selection is no longer necessary.

3. As one of your colleagues already suggested, the signaling induced by transgenic TCRa expression is "too strong", therefore the cells develop into gamma-delta like T -cell starting at the DN2. Since the signal strength and not the identity of the TCR expressed at the DN stage is believed to direct development, DN precursor that receive strong signals will develop along the gamma delta lineage even if the signals was induced by an alpha (preTCRalpha) beta TCR. To further investigate this phenomenon, I recommend GL3 (TCRyd antibody) staining, You can also stain for CD5 which is normally used as a readout for TCR signal strength but also assesses the signaling strength of the pre-TCR.

Please indicate what lineage cocktail you used in your posted analysis, because other non-Tcell thymic cells types may express small amounts of CD3 as well.

Sincerely, Luise

Dear Luise, thank you very much for excellent answer!!! 1) Thank you for advice to use c-kit and CD24! 2) I think, our transgenics need beta-selection step, because have transgenic alpha chain only. It seems, DN stages are nor screwed significantly. Development of T cells with endogenous alpha/beta TCRs also seems normal. 3) We used GL3 antibodies and we have seen only small increase in DN gamma/delta+ in TCR alpha transgenics. Vast majority of DN CD3 cells do not express gamma/delta TCR. Thank you for advice to use CD5! 4) Analysis was performed after simple exclusion of CD4 and CD8 expressing cells. No additional staining (for exception of indicated on pictures) was performed. So, I can not exclude possible involvement of non T cells, and, especially, peripheral T cells. Thank you!

Use Thy1 to gate for thymocytes (true T cells), in the DNs if you want to exclude alternative lineages, than you have a positively identifying markers (otherwise everything is negative, CD4, CD8, lin) If you want to explore the DN subsets further you can use Ton Rolink's (Basel) markers, including CD24, c-kit and others. I would check rearrangements of TCRB as a of what is very insightful measurement of what is going on in your mice.

Thank you very much, Frank! This is very clear and useful programme! Cordially!

Most beta -selection and T-cell development papers use CD11c, CD11b, B220, Ter119, Gr1, and a NKcells marker in their Lin stain, Thy1 as Frank Staal suggested is also a good idea.

Thank you, Luise! Your answers are excellent and very helpful. I think, it is good time to start discussion about T cell markers on myeloid cells (or myeloid markers on T cells). Are they functional or represent side effect of T cell differentiation, when the T cell "decide" to change lineage? I frequently see TCR on neutrophils, CD4 on dendritic (CD11c+) cells. I was greatly intrigued (almost shocked) by recent works published by Wolfgang E. Kaminski and co-authors. I would greatly appreciate feedback on this issue from community.

Good news! The expression of transgenic alpha chain is restored on the surface of CD8 T lymphocytes after injection of tumor cells expressing Kb (the ligand to which initial TCR 1D1 was specific).

Nice, I was wondering if there was a small population of TCRa+ cells "hidden" in the thymus between many cells hat were negative for the Tg?

Hi, Frank! Happy to hear from you! I meant peripheral (intraperitoneal) CD8 T cells arising in response to EL4 injection. Evidently, we are on the start of big and very interesting work. Certainly, now I should be especially careful in doing any claims.