Divergent fates of activated CD4+ T cells can be tracked with gradients of cellular size following activation.

<p><b>a-b)</b> Splenocytes from 5c.c7 Rag^–/—mice were stimulated with 0.5ug/ml PCC peptide for 24hrs and then sorted into CD4+CD69+ populations with FSC/SSC “big” mTOR^hi or FSC/SSC “small” mTOR^lo profiles. Cells were cultured in media supplemented with IL-2 for 3 days. <b>a)</b> 3 days after sort, mTOR^lo cells express higher mRNA transcript, and <b>b)</b> surface protein expression of CD25 than sorted mTOR^hi cells. The bar graph to the right depicts the fold increase in CD25 MFI expression in mTOR^lo cells compared to mTOR^hi cells from 6 replicate experiments, with expression of mTOR^hi cells set to a value of 1. Error bars represent ± S.D, n = 3 (a) or n = 6 (b). <b>c-d)</b> 5c.c7 Rag^–/—splenocytes were stimulated with PCC for 24hrs and activated (CD69+) CD4+ cells were sorted based on 4 size profiles. Cells were sorted into 10% smallest (Q1) and largest (Q4) populations in addition to the next 25% smallest (Q2) and largest (Q3) populations. <b>c)</b> Representation of sorting scheme utilized. <b>d)</b> After the sort, cells were cultured separately in IL-2 supplemented media for 3 days. On day 3, expression of CD25 and CD62L was assessed by flow cytometry. An illustration of cell size after sort is depicted above flow plots for clarification of sorted quartile populations. <b>e)</b> Purified CD4+ T cells from 5c.c7 Rag^–/—mice were stimulated by plate bound anti-CD3 and anti-CD28 for 24hrs. Cells were sorted as indicated in <b>c,</b> and mRNA transcript expression of CD25 and Bcl-2 was determined from sorted populations after a 3 day culture in IL-2 supplemented media. Bar graphs show relative expression ± S.D. from 3 independent experiments. The data are representative of 3 independent experiments or show cumulative results.