PI3Kδ is indispensable for CTL-mediated cytotoxicity
- Eva M Putz†2,
- Michaela Prchal†3,
- Olivia Simma1,
- Florian Forster4,
- Xaver Koenig1,
- Roland Piekorz5,
- Michael Freissmuth1,
- Veronika Sexl2 and
- Eva-Maria Zebedin1Email author
© Putz et al; licensee BioMed Central Ltd. 2011
Published: 5 September 2011
The expression of catalytic phosphoinositol-3-kinase isoform δ (PI3Kδ) is restricted to the haematopoetic compartment. Accordingly, PI3Kδ serves as a drug target to eliminate leukaemic cells. However, we previously showed that PI3Kδ is indispensable for the function of natural killer (NK)-cells . Thus, the therapeutic success of PI3Kδ inhibitors is likely to be compromised by unintended side effects on the immune system. Besides NK-cells, CD8+ cytotoxic T-cells (CTLs) are well-known key players in natural host response against developing tumours and viral infections. In this study, we examine the role of PI3Kδ for CTL function and CTL-mediated tumour surveillance.
PI3Kδ−/− animals have been described in . Flow cytometric lymphocyte characterization, the in vivo CTL-assay and MC-38 tumour model were done as outlined in . Membrane capacitance and degranulation were measured by patch-clamp recordings and flow cytometry, respectively ; the mixed lymphocyte reaction was monitored as in . In vitro, expanded CTLs were generated by stimulation with an anti-CD3ε antibody (0.5 µg/µL; BDPharmingen) and cultured for 3 days in T-cell medium containing 100 U/mL IL-2 prior to FACS analysis. For the in vitro cytotoxicity assay mice were immunized twice with the peptide SINFEKL. Peptide-reactive T-cells were generated by co-culturing splenocytes derived from immunized and control mice with SINFEKL-pulsed, irradiated splenocytes for 5 days. Peptide-reactive T-cells were co-cultured with CFSE-stained EL4 or EG7 cells in different effector:target-ratios. After 18 h peptide-specific killing was quantified via FACS.
Antigen-specific cytotoxicity of PI3Kδ−/− CTLs was significantly reduced in vivo and in vitro as compared to wild-type. This defect translated into severely impaired CTL-mediated MC-38 tumour surveillance: tumours derived from PI3Kδ−/− recipients were significantly bigger. PI3Kδ was required for full activation of CTLs and interfered with essential stages in the canonical killing pathway of CTL, e.g. with the endowment of their lytic machinery with key cytolytic molecules, with the production of interferon-γ and the fusion of lytic granules with the cellular membrane.
Our findings are of particular interest for the clinical development, because specific inhibitors of PI3Kδ are entering clinical trials. Our observation shows that PI3Kδ is indispensable for CTL effector functions. Accordingly, long-term drug safety monitoring ought to include adequate measures to identify side effects resulting from impaired surveillance of viral infections and tumour cells.
This work was supported by grants from SFB JAK-STAT and the Austrian Academy of Science (DOC-forte fellowship to EMP).
- Zebedin E, Simma O, Schuster C, Putz EM, Fajmann S, Warsch W, Eckelhart E, Stoiber D, Weisz E, Schmid JA, Pickl WF, Baumgartner C, Valent P, Piekorz RP, Freissmuth M, Sexl V: Leukemic challenge unmasks a requirement for PI3Kδ in NK cell-mediated tumour surveillance. Blood. 2008, 112: 4655-4664. 10.1182/blood-2008-02-139105.View ArticlePubMedGoogle Scholar
- Jou ST, Carpino N, Takahashi Y, Piekorz R, Chao JR, Carpino N, Wang D, Ihle JN: Essential, nonredundant role for the phosphoinositide 3-kinase p110δ in signaling by the B-cell receptor complex. Mol Cell Biol. 2002, 22: 8580-8591. 10.1128/MCB.22.24.8580-8591.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Simma O, Zebedin E, Neugebauer N, Schellack C, Pilz A, Chang-Rodriguez S, Lingnau K, Weisz E, Putz EM, Pickl WF, Felzmann T, Müller M, Decker T, Sexl V, Stoiber D: Identification of an indispensable role for tyrosine kinase 2 in CTL-mediated tumour surveillance. Cancer Res. 2009, 69: 203-211.View ArticlePubMedGoogle Scholar
- Chen JC, Chang ML, Muench MO: A kinetic study of the murine mixed lymphocyte reaction by 5,6-carboxyfluorescein diacetate succinimidyl ester labelling. J Immunol Meth. 2003, 279: 123-133. 10.1016/S0022-1759(03)00236-9.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.