Differential Effects of Inteleukin-2 and CD3 Triggering on Cytokine Gene Transcription and Secretion in Cultured Tumor Infiltrating Lymphocytes

Abstract

The therapeutic potential of tumor-infiltrating lymphocytes (TIL) is currently under investigation. TIL have been reported to display characteristic functional defects, including impairments of activation mechanisms. In animal models, therapeutic efficacy of adoptive TIL transfer has been correlated with their capacity to produce cytokines rather than with their in vitro cytotoxic potential. In this work we assayed cytokine gene transcription and protein production in eight cultured TIL populations stimulated with recombinant human IL-2 (rhIL-2), solid-phase-bound anti-CD3 monoclonal antibodies (mAb), or a combination of the two. By using a sensitive reverse polymerase chain reaction technique, we observed that transcription of IL-5, GM-CSF, and TNF-α could be detected in unstimulated, IL-2-starved TIL from all cultures, while IL-4 and IFN-γ genes were found to be transcribed in two cultures out of eight. A 50 U/ml dose of rhIL-2 was sufficient to induce IL-4 and IFN-γ gene transcription in the remaining six cultures and in four more TIL populations, respectively. In contrast, rhIL-2 could not induce IL-2 gene transcription in five of eight TIL cultures. Furthermore, it could not induce IL- 10 gene transcription in any TIL population. Anti-CD3 mAb triggering, however, induced transcription of all these cytokine genes. On the other hand, IL-6, IL-7, or TGF-β 2 gene transcription could not be induced by any of the stimuli used, including the combination of anti-CD3 and rhIL-2. Despite detection of their gene transcripts, GM-CSF, IFN-γ, or TNF-α was never detectable, at the protein level, in unstimulated TIL. Stimulation by rhIL-2 induced GM-CSF secretion, albeit to different extents, in all TIL populations. In contrast, rhIL-2 induced IFN-γ and TNF-α production in three and one TIL population, respectively. CD3 triggering however, induced cytokine production in all TIL populations. Addition of rhIL-2 significantly increased production of GM-CSF, but not of IFN-γ and TNF-α. These data underline that stimulation with a moderate dose of rhIL-2 reveals considerable heterogeneity of TIL populations regarding cytokine gene transcription and secretion. On the other hand, triggering of the CD3-T-cell receptor complex induces transcription of an extended panel of cytokine genes in all TIL populations and secretion of GM-CSF, IFN-γ and TNF-α in virtually all cultures. Thus, if stimulated by properly presented antigenic peptides, TIL are capable of mounting an efficient response in terms of cytokine gene expression and protein production.