Specific Studies on In Vitro Generated Cytotoxic T Lymphocytes Directed Against Human Target Cells Grown in Monolayer Culture

Specific cytotoxic T lymphocytes (CTL) against HLA antigens of skin fibroblasts were generated with mixed leukocyte culture (MLC).

Nonspecific cytotoxicity (CTX) in 3H-proline microcytotoxicity test was detected as early as the 4th day by primary in vitro sensitization (IVS), and the activity continued until around the 8th day. The specific CTX by primary IVS was detectable from the 7th day to 12th day, and by secondary IVS, from around the 2nd to 4th day. However, obtainment of specific CTX only was rarely the case, and the probability of obtaining them did not increase until the 9th to 10th day by primary IVS and until the 3rd to 4th day by by secondary IVS. This is related to reduction of blastogenic response in MLC and suggests that inactivation of blast cells generated by MLC is of importance in obtaining specific CTX.

A CTX assay was conducted using cryopreserved effector cells, which revealed that in most cases nonspecific CTX either decreases or disappears altogether. On the other hand, specific CTX activity (more than 30 % reduction of only test target cells) remained and the specific CTX pattern was good and evident in many cases.

Specific CTL was generated against fibroblast antigens by adding allogenic lymphocytes as third party stimulators. Cryopreservation technique was effective in increasing the specificity of CTX.