Background: The cytokine network and the apoptotic pathway exhibit multiple regulatory interactions. In HIV-infected persons, both death receptor induced apoptosis and deprivation apoptosis are increased and have been proposed to contribute to T-cell deletion associated to this disease. Deprivation apoptosis is related to the defect of signaling via the IL-2-Rg chain by cytokines such as IL-2, IL-4 or IL-15 and alterations in the Th1/Th2 balance (in particuler increase in IL-4 expression) has been associated with disease progression. In this study, we report the identification of a new non secreted variant of IL-4 which expression is detected in T-cells from HIV-infected persons.

Methods: Deprivation apoptosis was induced in PBMC and Th clones by 24 h or 5 day incubation in culture medium respectively. AICD was induced by 16h polyclonal stimulation with PMA and ionomycin. Apoptosis and cytokine synthesis were simultaneously detected at the single cell level by flow cytometry. IL-4 was detected by ELISA, FACS, confocal microscopy or RT-PCR. Analysis of the run-off products was performed with the Immunosope software. Anti-IL4 mAbs included MP4-25D2 and 8D4-8 (Pharmingen, La Jolla).

Results: Following polyclonal stimulation of Th1 and Th2 clones, surprising intracellular staining was observed with anti-IL4 8D4 mAb since it stained both Th1 and Th2 clones, while anti-IL4 MP4 only stained the Th2 clone. While studying the relationship between intracellular cytokine expression and deprivation apoptosis in the Th clones, we found that MP4-reactive cells were mostly living whereas 8D4^-reactive cells were apoptotic (7-AAD⁺). The association between 8D4 reactivity and deprivation apoptosis was found in multiple cell types, including blood CD3 T-cells from HIV⁺ pts. We found that 8D4 mAb detects a new non-secreted variant of IL-4 (IL-4d₁₃), expressed in Th2 and Th1 clones, and in primary T-cells from HIV⁺ pts. This variant may result from an alternative IL-4 mRNA splicing and its expression is highly increased after induction of deprivation apoptosis. In blood samples from some pts, this variant was the only molecular form of IL-4 mRNA detected.

Conclusions: We report the identification of a new non-secreted variant of IL-4, which expression is detected in vivo in T-cells from HIV-infected pts and is tightly associated to deprivation apoptosis. These data may explain some discordances in studies analysing the Th1/Th2 balance in HIV-infection.