Reactivation of murine tumour-infiltrating lymphocytes with solid-phase anti-CD3 antibody: in vitro cytokine production is associated with in vivo efficacy.

Previously we described the use of solid-phase anti-CD3 monoclonal antibody (mAb) to stimulate murine tumour-infiltrating lymphocytes (TIL) and their subsequent expansion in recombinant interleukin 2 (rIL-2). In a pulmonary metastases model using the methylcholanthrene-induced sarcoma MCA-105 anti-CD3 activated TIL were capable of eradicating disease similar to TIL cultured in rIL-2 only. Here we extend these observations by characterizing the biological effects of sequential solid-phase anti-CD3 activation. TIL from MCA-105 tumour activated with solid-phase anti-CD3 on day 1 were reactivated on day 14, or day 26, or both and compared to TIL grown in rIL-2 only or TIL activated with anti-CD3 once on day 1. Reactivation enhanced in vitro proliferation 1.8- to 4-fold compared to TIL activated once with anti-CD3 (P < 0.05). In addition, the total lytic capacity of the cultures was enhanced after reactivation without changing the phenotype of the TIL cultures. Reactivation resulted in a greater in vivo efficacy when the TIL were administered within 72 h of reactivation. In contrast, TIL activated with anti-CD3 on day 1 and day 14 were least effective of all TIL cultures (P < 0.05). This correlated with in vitro cytokine production. The most effective TIL cultures in vivo produced 4- to 100-fold higher amounts of cytokines, especially interferon gamma (IFN gamma) and granulocyte macrophage colony stimulating factor (GM-CSF), than the other cultures. On the other hand, the least effective in vivo TIL culture, TIL activated with anti-CD3 on day 1 and 14, produced little or no cytokines. These data suggest that in vitro production of cytokines is indicative of in vivo efficacy of anti-CD3 activated TIL.