CTLA-4-(CD152)-induced signalling pathways as regulators of CD8+ T lymphocytes (SFB854 B14)

CD8⁺ T cells play a crucial role in immunity to viral infection and cancer. We had previously shown that CD8⁺ T cell differentiation is regulated by CTLA-4. In order to specify proximal signal transduction pathways, which are under the control of CTLA-4 such as IFN- production, a phosphoproteome analysis using iTRAQ mass spectrometry and PepChip was performed. These approaches revealed distinct changes in post-translational modifications, pointing out novel regulatory mechanisms. Specifically, in the 2^nd funding period we demonstrated that CTLA-4 activates FoxO1, which initiated the expression of the translational inhibitor PDCD4 and the transcription factor TCF-1, acting downstream of the canonical Wnt pathway that is involved in the formation of effector and memory CD8⁺ T cells. Indeed, the CTLA-4-mediated inhibition of IFN- production of CD8⁺ T cells was mediated by PDCD4. In addition, PDCD4 connects CTLA-4 with the restriction of the metabolic process of glutaminolysis, in particular by regulating the rate-limiting enzyme glutaminase. Furthermore, iTRAQ analysis led to the identification of a CTLA-4-dependent phosphorylation of the junctional adhesion molecule family protein JAM-L (AMICA1), a surface receptor localized at the immunological synapse (IS). In addition, identification of reduced pSTAT1 and enhanced pSTAT3 accumulation in CTLA-4-deficient CD8⁺ T cells demonstrated that CTLA-4 is indeed able to modulate the 3^rd signal of T cell stimulation, namely cytokine signaling.

In the 3^rd funding period, we will mainly focus on the role of the CTLA-4-FoxO1-PDCD4 axis in controlling the metabolic capacity of CD8⁺ T cells regarding the glutamine catabolism involved in regulation of T cell functions. Additionally, we will characterize the identified FoxO1-PDCD4-pathway as a central signaling hub of inhibitory surface receptors by monitoring temporal changes during protein synthesis in response to ligation of the inhibitory surface molecule PD-1 by using tagged amino acid incorporation (BONCAT). As a further aim, we will determine the role of CTLA-4-FoxO1-TCF-1 signaling in the effector and memory responses of CD8⁺ T cells using a Listeria infection model and aDEC-OVA-immunization with aCD40 treatment. Regarding the CTLA-4-regulated surface receptor JAM-L, we will analyze its function as a costimulatory molecule of CD8⁺ T cells by generating JAM-L-mutants and performing cytotoxic assays in vitro and in vivo. To confirm relevance of CTLA-4-regulation of STATs in vivo, we will follow Tc17 cells of OT-I-STAT1^-/- and STAT1^+/+ mice with/without CTLA-4 blockade using specific antibodies in the Listeria infection model, for fate tracking combined with ex vivo pSTAT3/5-analysis. Together, these experiments will give us a comprehensive picture of CTLA-4-modulated signaling pathways in CD8⁺ T cells.