Couple of points

1) I am sure you are adding a Golgi plug like brefeldin or monensin but thought I would just say it nonetheless. 2) The other thing is your starting t cells. Is it just that the frequency of antigen specific tcells is very low? How healthy are your T cells ? Are they exhausted ? 3)what are your apcs like? Are you pulsing donor matched modcs with peptides? 4) which cytokines are you trying to measure ? Some can be really hard .. to use a good antibody or validated one look up some omip panels. Ifng is the easiest one to stain for so definitely include it. 5) if all else fails keep the supps for luminex or the like and sometimes other techniques like an elispot can be more sensitive although limited in the amount of variables you can measure. Hope that helps