Development and evaluation of a limiting dilution analysis technique that can discriminate in vivo alloactivated cytotoxic T lymphocytes from their naive CTL precursors.

We describe a permutation of the conventional limiting dilution analysis (LDA) technique that allows, for the 1st time, the differential enumeration of alloantigen-specific CTL that have been activated by alloantigens in vivo. This technique does not detect nonactivated CTL precursors, even those with similar alloantigen specificity. Data are presented to validate this limiting dilution analysis technique. Using this LDA technique, we demonstrate that large numbers of the donor-reactive CTL arrive in sponge matrix allografts (f = 1/3,599 cells), most or all of which are in an activated state (f = 1/4,385 cells). In contrast, alloactivated CTL constitute only a small fraction (f = 1/57,208 cells) of the donor-reactive CTL in the regional lymph node (f = 1/1,873 cells) of the same sponge allograft recipients. As expected, regional lymph nodes from sponge isograft recipients contain DBA/2-reactive CTL precursors (f = 1/1,873 cells), but no activated DBA/2-reactive CTL (f less than 1/385,529 cells). This LDA technique should be useful in studies regarding activation and redistribution of alloreactive CTL caused by allograft implantation.