- TYRO3, AXL and MER (TAM) are co-expressed in macrophages, dendritic cells (DCs) and natural killer (NK) cells.
- TAM kinases are functionally redundant in all cell lineages where there is endogenous expression of more than one TAM family member, including macrophages, DCs, NK cells, megakaryocytes and Sertoli cells.
- Due to the functional redundancy, inhibition of all three family members has been required to see significant modulation of TAM-dependent immune system regulation.
TAM Family of Receptor Tyrosine Kinases
The TAM family of receptor tyrosine kinases includes TYRO3, AXL and MER and represents a unique set of anti-tumor targets. In addition to their role as direct tumor drivers1, TAM receptors participate in key immune system checkpoints, acting as inhibitory receptors in the tumor microenvironment to suppress innate immune responses. TAM RTKs have a broad expression pattern with highest expression reported in the nervous, reproductive, vascular and immune systems2. Co-expression of two or three TAM family members in a given cell type is common, and many TAM-positive cells also express one or both TAM ligands, GAS6 and Protein S2. Here, we will focus on the role of TAM receptors within the immune system.
TYRO3, AXL and MER are co-expressed in a variety of immune cell lineages, including macrophages, DCs and NK cells2. Characterization of mice lacking expression of all three TAM receptor family members highlights the important role of TAM receptors in regulating the innate immune response by DCs and macrophages and in regulating NK cell cytotoxicity3. In addition, TAM receptors play a role in modulating the cytokine milieu of the tumor to limit effective CD8+ T cell recruitment and to polarize macrophages toward an M2 anti-inflammatory state4.
Role of TAM in Regulating Macrophages
Phagocytosis of apoptotic cells, termed efferocytosis, is essential during development and for maintaining normal tissue homeostasis5. Macrophage ingestion of apoptotic cells triggers an anti-inflammatory M2 macrophage polarization6–8 and results in decreased production of pro-inflammatory cytokines and upregulation of immunosuppressive factors4. TAM receptors play a critical role in regulating efferocytosis9,10 with germline loss of TYRO3, AXL and MER resulting in progressive chronic inflammation and autoimmunity due to aberrant lymphocyte activation and defects in efferocytosis9. TYRO3, AXL and MER co-expression is seen in both monocytes and differentiated macrophages responsible for efferocytosis11. TAM receptors appears to be compensatory such that animals lacking one or two family members have no or modest impairment in homeostatic regulation while animals with germline deletion of all three family members develop a severe lymphoproliferative disorder9. MER has been implicated as the primary TAM receptor involved in efferocytosis; however, both AXL and TYRO3 are required for apoptotic cell ingestion by macrophages and DCs and appear to be important for phosphorylation of MER and subsequent downstream signaling12.
Role of TAM in Regulating Dendritic Cells
TAM receptors play an important role in negative regulation of DCs. DCs serve a critical role in the innate immune system by acting as messengers between the innate and the adaptive immune systems, as well as by fostering immune tolerance13. In addition to their function in the innate immune response, DCs act as potent stimulators of adaptive immunity through their role as antigen presenting cells. In response to activation through Toll-like receptors (TLR), DCs upregulate MHC class II and co-stimulatory molecules at the cell surface and present processed antigens to T cells to activate the adaptive immune response13. TLR engagement also stimulates DC production of type I interferons (IFN) and other pro-inflammatory cytokines. This powerful pro-inflammatory response is under strict negative regulation via the TAM receptors, and TLR engagement results in rapid upregulation of AXL expression on DCs14. TAM signaling negatively regulates both TLR activation and inflammatory cytokine production through the type 1 interferon receptor (IFNAR)-STAT1 pathway14. The rapid negative regulation of the type 1 IFN response is critical to avoiding the consequences of uncontrolled inflammation; however, such an immune suppression can blunt anti-tumor immune responses. As in the case of macrophages, expression of all three TAM family members is seen in DCs and each may play compensatory roles in the regulatory control of DCs2,9,12.
Role of TAM in Regulating Natural Killer Cells
TAM receptors also appear to play a regulatory role in NK cells. NK cell target recognition and killing activity depends on the expression of both inhibitory and activating receptors. In addition, NK cells release pro-inflammatory cytokines1. TAM receptors have been shown to be essential for the functional maturation of NK cells during development and for the normal expression of inhibitory and activating NK cell receptors15. In mature NK cells, TAMs have a negative regulatory function such that stimulation of TAM receptors with GAS6 decreases NK cell proliferation and IFNg secretion and reduces NK cell cytotoxicity16. All three TAM family members are expressed on NK cells2 and likely exert redundant functions as they do in NK cell development15, DC activation and macrophage-dependent efferocytosis9,10.
TAM Receptors in Oncogenesis
In the context of oncogenesis, the negative immunomodulatory roles of the TAM receptors serve to blunt innate and adaptive immune responses to the tumor, thus promoting tumor growth and metastases8,16–18. TAM receptor-dependent efferocytosis of apoptotic tumor cells triggers production of immunosuppressive cytokines favoring tumor-promoting polarization of macrophages6–8. TAM receptor signaling downstream of TLR-induced production of type I IFNs inhibits the activation of intratumoral DCs and ultimately impairs the priming and maintenance of anti-tumor cytotoxic T cells14. Finally, TAM receptor signaling decreases NK cell proliferation and IFNg secretion and reduces NK cell cytotoxicity16.
Blockade of TAM receptor signaling with a pan-TAM inhibitor is predicted to increase pro-inflammatory cytokine expression, suppress tolerogenic cytokine production and promote anti-tumor M1 macrophage polarization in the tumor microenvironment. Additionally, TAM receptor inhibition is expected to reduce tumor resident Treg cells, enhance DC-dependent antigen presentation and subsequent engagement of the adaptive immune response, and enhance NK cell cytotoxicity. Overall, this would result in maintenance of a robust anti-tumor innate immune response combined with an enhanced adaptive immune response and may allow for effective immune attack of tumors. Given the functional redundancy within the TAM family, a pan-TAM inhibitor capable to concurrently targeting TYRO3, AXL and MER is likely to be required to block TAM receptor signaling and obtain optimal stimulation of anti-tumor immune responses.
Redundancy of TAM Receptor Function
As discussed above, homeostatic regulation of the immune system requires TAM expression. Animals that are singly or doubly mutant in TYRO3, AXL or MER develop normally with no or modest impairments in immune responses. In contrast, loss of all three TAM receptors results in aberrant and progressive growth of peripheral lymphoid organs due to hyperproliferation and constitutive activation of B and T cells induced by constitutively activated macrophages and DCs9. Similar redundancy of TAM kinase function is seen in megakaryocytopoiesis19 and spermatogenesis20. The mechanism of this redundancy is unclear but it has been noted that disruption of signaling through one TAM receptor by knockdown or knockout can affect the expression and therefore the signaling of another TAM receptor21. Additionally, TAM receptors and/ or TAM ligands can form heterodimers2,22.
Rationale for Pan-TAM Inhibitor
Overall, the published literature supports the need to concomitantly block all three TAM receptors to effectively modulate their immunosuppressive functions, mobilize innate immunity, and promote engagement of adaptive immunity in the tissue microenvironment. In addition to addressing TAM receptor redundancy within the immune system, a pan-TAM inhibitor may allow one to target multiple anti-tumor pathways with a single molecular entity. For example, targeting the oncogenic driver AXL on tumor cells23, MER on infiltrating M2 macrophages to promote M1 polarization6–8 and TYRO3 on maturing DCs to maximize antigen presentation and engagement of the adaptive immune response12 may maximize the anti-tumor efficacy of a pan-TAM inhibitor24.