Phosphoproteomics of CD2 signaling reveals AMPK-dependent regulation of lytic granule polarization in cytotoxic T cells.

Understanding the costimulatory signaling that enhances the activity of cytotoxic T cells (CTLs) could identify potential targets for immunotherapy. Here, we report that CD2 costimulation plays a critical role in target cell killing by freshly isolated human CD8+ T cells, which represent a challenging but valuable model to gain insight into CTL biology. We found that CD2 stimulation critically enhanced signaling by the T cell receptor in the formation of functional immune synapses by promoting the polarization of lytic granules toward the microtubule-organizing center (MTOC). To gain insight into the underlying mechanism, we explored the CD2 signaling network by phosphoproteomics, which revealed 616 CD2-regulated phosphorylation events in 373 proteins implicated in the regulation of vesicular trafficking, cytoskeletal organization, autophagy, and metabolism. Signaling by the master metabolic regulator AMP-activated protein kinase (AMPK) was a critical node in the CD2 network, which promoted granule polarization toward the MTOC in CD8+ T cells. Granule trafficking was driven by active AMPK enriched on adjacent lysosomes, revealing previously uncharacterized signaling cross-talk between vesicular compartments in CD8+ T cells. Our results thus establish CD2 signaling as key for mediating cytotoxic killing and granule polarization in freshly isolated CD8+ T cells and strengthen the rationale to choose CD2 and AMPK as therapeutic targets to enhance CTL activity.

Ectopic ILT3 controls BCR-dependent activation of Akt in B-cell chronic lymphocytic leukemia.

The high proportion of long-term nonprogressors among chronic lymphocytic leukemia (CLL) patients suggests the existence of a regulatory network that restrains the proliferation of tumor B cells. The identification of molecular determinants composing such network is hence fundamental for our understanding of CLL pathogenesis. Based on our previous finding establishing a deficiency in the signaling adaptor p66Shc in CLL cells, we undertook to identify unique phenotypic traits caused by this defect. Here we show that a lack of p66Shc shapes the transcriptional profile of CLL cells and leads to an upregulation of the surface receptor ILT3, the immunoglobulin-like transcript 3 that is normally found on myeloid cells. The ectopic expression of ILT3 in CLL was a distinctive feature of neoplastic B cells and hematopoietic stem cells, thus identifying ILT3 as a selective marker of malignancy in CLL and the first example of phenotypic continuity between mature CLL cells and their progenitors in the bone marrow. ILT3 expression in CLL was found to be driven by Deltex1, a suppressor of antigen receptor signaling in lymphocytes. Triggering of ILT3 inhibited the activation of Akt kinase upon B-cell receptor (BCR) stimulation. This effect was achieved through the dynamic coalescence of ILT3, BCRs, and phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase 1 into inhibitory clusters at the cell surface. Collectively, our findings identify ILT3 as a signature molecule of p66Shc deficiency in CLL and indicate that ILT3 may functionally contribute to a regulatory network controlling tumor progression by suppressing the Akt pathway.