Immunotherapy of bovine ocular squamous cell carcinoma: Isolation, culture and characterization of lymphocytes present in the tumor

Bovine ocular squamous cell carcinoma (BOSCC) is sensitive to intralesional immunotherapy with BCG or recombinant human IL-2 (rhIL-2). The mechanism of tumor regression is as yet unclear. Alterations in the concentration of IL-2 (and possibly other factors) in the tumor, due to regional injection or induction by BCG, may induce killer cell activity and thus tumor regression. To investigate this, lymphocytes were isolated by mechanical fractionation of biopsies of BOSCC. Growth, phenotypical, and functional characteristics were studied. TIL could be isolated and grown from all biopsies of BOSCC. An estimated increase in cell number of 50-150 fold was observed during 5-7 weeks of culture. FACS analysis of a limited number of the TIL cultures showed a characteristic shift in phenotypes until day 28 of culture. CD2⁺ cells (50-70%), and as a consequence of this CD2^- cells, remained stable in number. The number of CD8⁺ cells increased. CD4⁺ cells were detected in low numbers by day 28. Prolonged culture resulted in an increase of CD2^- ≤δ⁺ cells, CD2⁺4^-8^- cells, and occasionally of both CD8⁺ and CD2⁺ cells. In ⁵¹Cr release assays TIL showed cytotoxicity for BOSCC-derived tumor cell lines in general, which increased transiently by cocultivation with tumor cells. Killing of YAC-1, and P815 was far less efficient. Preferential killing of autologus cell lines was not seen. In conclusion, TIL from bovine ocular squamous cell carcinomas can be cultured in the presence of rhIL-2, which induces cytotoxic activity for BOSCC-derived tumor cells. Cells responsible for killing in vitro and potentially for regression of the tumor after immunotherapy with BCG or rhIL2 cannot yet be identified. Depletion and blocking experiments are being conducted in order to identify the cells (CD2⁺8⁺, CD2^-γδ⁺ or other CD2(+/-)) responsible for killing.