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

Abstract

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.

CD2 Immunobiology

“…Preliminary evidence analyzing the kinome of CD2-mediated signaling in CTLs confirmed these findings and elucidated a broad network of CD2-induced signaling which influences IS assembly, cell polarization, T cell activation and metabolic regulation which overlapped only partially with known TCR/CD3-or CD28-mediated signaling pathways (62). Generally, CD2 costimulation has been shown to act additively with TCR/CD3-and CD28-mediated signaling and to induce strong mTOR signaling but weak NF-κB activity when compared to CD28 costimulation (62,69,71,72). While the majority of the literature investigating CD2 signaling has been conducted in T cells, there are indications that CD2-mediated signaling in NK cells utilizes the same pathways as T cells (73)(74)(75).…”

“…Additionally, CD2 can induce activation signaling via the PLCγ1/calmodulin/calcineurin/NFAT and PLCγ1/Vav1/PKCθ/Dok/FAK/Pyk2/JNK axes (57,68,71). Preliminary evidence analyzing the kinome of CD2-mediated signaling in CTLs confirmed these findings and elucidated a broad network of CD2-induced signaling which influences IS assembly, cell polarization, T cell activation and metabolic regulation which overlapped only partially with known TCR/CD3-or CD28-mediated signaling pathways (62). Generally, CD2 costimulation has been shown to act additively with TCR/CD3-and CD28-mediated signaling and to induce strong mTOR signaling but weak NF-κB activity when compared to CD28 costimulation (62,69,71,72).…”

“…Consequently, reorganization of the actin cytoskeleton and polarization of the microtubule organizing center (MTOC) toward the IS can occur. Indeed, CD2 and CMS facilitate polarization of the actin cytoskeleton and T cells expressing truncated CD2 mutants lacking the C-terminal 20 amino acids of the cytoplasmic tail do not display polarization toward the IS (42,62). Furthermore, CD2BP1 also binds SH3binding domain 4 and locates to the IS upon cell activation where it links CD2 or CMS and Wiskott-Aldrich syndrome protein (WASp) to facilitate actin cytoskeleton rearrangement (54).…”

“…Considering the evidence discussed above, co-localization of CD2 and TCR/CD3 may, at least in part, be mediated through adaptor proteins and the actin cytoskeleton. Future research should investigate the interaction of CD2 and the actin cytoskeleton through adaptor proteins more closely, as it seems to play an important role in IS formation (28) and transport of lytic granules [CTLs; (62)]. Another unanswered question is whether CD2 similarly influences transport of secretory cytokines.…”

Coordinating Cytoskeleton and Molecular Traffic in T Cell Migration, Activation, and Effector Functions

“…Lytic granule translocation to the cytotoxic synapse may occur in the absence of centrosome polarization, and CTLs may simultaneously kill several target cells (Wiedemann et al, 2006;Bertrand et al, 2013). Conversely, human B cells, by inducing weak CD2 signaling, may trigger non-polarized granule exocytosis by the CTLs, although the centrosome is at the synapse (Kabanova et al, 2016;Zurli et al, 2020). Finally, centriole deletion has no effect on lytic granule polarized secretion, but reduces killing efficiency by impairing lytic granule biogenesis and actin-induced forces at the synapse (Tamzalit et al, 2020).…”