There are different isoforms of CD45, CD45RA, CD45RB, CD45RC, CD45RAB, CD45RAC, CD45RBC, CD45RO, CD45R (ABC). CD45RA is located on naive T cells and CD45RO is located on memory T cells. CD45 is also highly glycosylated. CD45R is the longest protein and migrates at 200 kDa when isolated from T cells. B cells also express CD45R with heavier glycosylation, bringing the molecular weight to 220 kDa, hence the name B220; B cell isoform of 220 kDa. B220 expression is not restricted to B cells and can also be expressed on activated T cells, on a subset of dendritic cells and other antigen-presenting cells. So maybe you have an antibody that recognize all forms of B220. I do not know what those cells. But try to do Perforin staining and CD28, or anti apoptotic BCL2 to see the functional status and the grade of differentiation.

Good luck with your research,

rafa

The B220 is likely a marker of apoptotic/pre-apoptotic T cells (http://www.ncbi.nlm.nih.gov/pubmed/9521064). Not sure what to make of the NK1.1 stain. A small subset of NKT cells are CD8+, but dead/dying cells can also be quite "sticky", and bind antibodies non-specifically. It is also possible for cells to pick up membrane proteins from other cells. Although the conditions under which that sort of thing happens in vivo aren't very well defined, it's quite common in vitro, especially if you leave the cells overnight before staining or running flow. Were the cells run immediately post processing? Also, any chance these cells were the targets of NK cell-mediated lysis in vivo?

I could see NK1.1 expression by subsets CD8 T cells, this has been shown before in B6 mice.  I think the B220 expression is more surprising.  The apoptotic T cell blast could be a potential, but unlikely since those are largely DN cells in a unique setting.  

There are a few papers by Zuhair Ballas and Wendy Rasmussen on lymphokine-activated killer cells that may be of interest in relation to NK1.1 and B220 expression.

One confusing thing is that your figure with the gating strategy says that you gated on B220- cells after the live gate so you shouldn't have any B220+ cells in the end (this a typo?).  Also while I understand that there are multiple isoforms of CD45 (including the B220 variant), it's not the other way around.  So an antibody specific for B220 should not bind other CD45 isoforms (different antibody clones and such).  

Hi Cory,

I didn't explain my gating clearly. I excluded B220+CD3- cells (B cells essentially) but kept all the rest. So there are are some B220+CD3+ cells further down the line. From what I understand B200 is the largest splice variant of CD45 and this antibody binds specifically to it.

Ah makes more sense now and yes I agree on the B220 specificity.  As I mentioned before the only paper I know of with NK1.1 and B220 on CD8+ T cells (though we're talking ex vivo cells) is the 1993 J Immuno paper by Ballas and Rasmussen.  

It would be interesting to think that these cells are perhaps more functional and better killers?  But I guess they are clearly not enough to prevent tumor progression.

I'm very intrigued by this population because of its relation with tumour mass.

While overall CD8 infiltration has a negative correlation with tumour mass I saw strong positive correlation between mass and the frequency of CD8+B220+NK1.1+ suggesting they are either a product of exhausted cytotoxic effort or are somehow immunosuppressive.