A quite interesting question popped up to me while doing CD4+ T cell proliferation assay. I am a "newbie" in such experiment so I would appreciate your help :)

I harvested lymph nodes from C57BL/6 mice and isolated CD4+ T cells through MACS. After labeling those cells with CFSE, I seeded them into anti-CD3 coated cell culture plate and analyzed them with FACS on Day 2 and 3.

When I gated CD4/CD8, I could clearly see two distinct CD4+ populations: the first being the majority with quite high CD4 expression and the second being the minority with relatively low CD4 expression. I further analyzed the two populations with CFSE histogram and the minority population seemed to proliferate more (this could be inferred by their low CFSE values, some of them being almost 0). In addition, percentage of IFN-r producing proliferated cells were almost 10 fold larger in the minority group (even if I consider the small absolute cell number of this group, it is quite significant).

My supervisor has told me that this population is not significant in the first place and it could be just due to some mistakes during antibody labeling or any step during the experiment. But I think this specific population which proliferate to a greater extent and express less CD4 did not appear just because of some error. What I have proposed is as CD4+ T cells proliferate, one of their surface protein, CD4, would decrease in number and the number would recover as they mature. Thus, I thought the cells that proliferated at last would have less surface CD4 and would be distinguished from the others who have normal level of surface CD4.

This could be a silly question but I hope I get ideas and answers from you. Thank you!