CD25 is the 55 kDa α chain of the IL-2 receptor that is expressed on activated B and T lymphocytes. Thus, CD25 positive CD4 negative could be CD8 positive T cells as well as B cells. Note, CD25 is also expressed in the early phase (CD4-CD8-) of thymic T cell development and at a lower level on activated monocytes.

I think they are thymocytes or undifferentiated T-Lymphocytes

CD25 is the 55 kDa α chain of the IL-2 receptor that is expressed on activated B and T lymphocytes. Thus, CD25 positive CD4 negative could be CD8 positive T cells as well as B cells. Note, CD25 is also expressed in the early phase (CD4-CD8-) of thymic T cell development and at a lower level on activated monocytes.

I would be inclined to think that the population you observe could be pre B cells or developing T cells, as Fuad and Bernhard says, because all of them express CD25 and they should be smaller in size than activated B cells or activated monocytes.

I believe some mast cell might give some reactivity, although I am not certain of the their surface expression level compared to T cells. It would be very informative if you would let us know what organs are you looking at and the level of expression compared to other populations.

It is certainly important to know what tissue are you analyzing. As others mentioned, a variety of cells express IL-2Ralpha, and that includes type 2 innate lymphoid cells which of course by far less frequent than cell types.

What about other lineage markers? Beyond CD4 and CD8 T cells, CD25 has been shown to be expressed also on some subpopulations of innate lymphoid cells (ILC) which can be either CD4+ or CD4- and in FSC, SSC they are very similar to lymphocytes. Obviously, it would be of interest to know where you are observing this population (e.g. in peripheral blood or tissues).

Definitely ILCs (innate lymphocytes) and/or eosinophils.

Thank you all for your logical explanation. Actually, those cells are from specific antigen immunized mouse spleen. Around 70 percent of total cd25 positve from live gated cells (macrophages and other bigger cells than lymphocytes excluded) are CD4 negative. Do you think such big population of CD25 cells are all ILC in present in spleen?

You should add an additional marker such as CD90 (Thy-1), which stains ILCs.

I think you might massively expand CD8 T cells then. I find very unlikely that those are ILC especially because ILC are very few in WT mice as compared with mice deficient in T and B cells. I would add a CD3 CD8 staining just to confirm it

I agree, you first need to exclude the obvious including CD8, NKs, some NKTs, gamma-delta,...before going after more rare type of cells.

My first port of call when questions like this come up is the immgen app. You can find out more about the protocols used to collect the data and download the app at the following website: http://www.immgen.org/ Whether you're in the pub or in lab meeting you'll find it useful.

Activated CD8+ T cells or NK cells would be my first bet. You need to include a few more markers.

Some B cell subsets in mice express CD25. http://www.ncbi.nlm.nih.gov/pubmed/17032240

CD8+ cells. not only in mice but also in men, exist CD8+CD25+FoxP3+ cells in significant amount.