Introduction: Ex vivo expansion of CD3-activated T cells is a promising approach to correct the impaired immunity, caused by growing tumors and progressive dysregulation of CD4+ T helper (Th) cells. Although CD4+ cells possess limited direct cytotoxicity against bystander tumor cells, they play a central role in stimulation of CD8+ cytotoxic T cells. In this study, we examined whether activated naïve and expanded CD4+ cells may radiosensitize bystander tumor cell through the release of IFN-gamma, a major mediator of Th1 immunity.

Methods: Spleen cells from Balb/c mice were primed with bound anti-CD3 plus soluble anti-CD28 antibodies and expanded in the presence of IL-2 for 7-10 days. CD4+ cells were selected from naïve or expanded splenocytes by immunomagnetic MACS beads and analyzed for intracellular IFN-γ by FACS. Activation of naïve and expanded CD4+ cells was performed by IL12/IL18 and anti-CD3/CD28 respectively. The expression of the IFN-γ gene and the secreted protein were estimated by RT-PCR and ELISA respectively. To access radiosensitivity with regard to the hypoxic tumor micoenvironment, CD4+ cells were incubated together with syngeneic EMT-6 tumor cells for 16 h in 1% oxygen, and afterwards mixed cultures were irradiated at 6 to 18 Gy, and examined for tumor cell survival by colony formation assay. Alternatively, conditioned medium from activated CD4+ cells was used in radiosensitizing experiments. The expression of inducible nitric oxide synthase (iNOS), a downstream target of IFN-γ in tumor cells, was analyzed by RT-PCR and Western blotting.

Results: Non-stimulated CD4+ cells contained 0.2-0.5% IFN-positive cells, which secreted low levels of IFN-γ (<1 ng/ml) and did not affect the hypoxic radioresponse of EMT-6 tumor cells. Upon stimulation of naïve CD4+ cells, the level of IFN-γ secretion in hypoxia and normoxia increased up to 60 and 130 ng/ml respectively, with a frequency of IFN-γ positive cells of 3-4%, resulting in a moderate tumor cell radiosensitization by 1.2-1.4-times. Expanded CD4+ cells contained more than 50% of IFN-γ positive cells, and secreted up to 5000 ng/ml of IFN-γ, which drastically increased tumor cell radiosensitivity by 1.4-1.8 times, and was associated with a 40-times activation of the iNOS gene. Radiosensitization was abrogated by neutralizing anti-IFN-γ antibodies and by the metabolic iNOS inhibitor aminoguanidine respectively, therefore identifying IFN-γ as a radiosensitizing effector of activated CD4+ cells towards tumor cells.

Conclusions: This study demonstrates that expanded CD4+ cells possess radiosensitizing properties, thus indicating a rationale for combining immunostimulatory and radiosensitizing strategies.

This research was funded by grants G.0075.05 and G.0386.07 from the “Fonds voor Wetenschappelijk Onderzoek - Vlaanderen”.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA