TAM-ing T cells in the tumor microenvironment: implications for TAM receptor targeting

TAM receptors are reported to be overexpressed in both solid and hematological malignancies, and high expression of the TAM receptors has been associated with poor patient survival in a variety of cancers [7, 8]. Oncogenic TAM receptor signaling results in increased proliferation, cell survival and metastasis (reviewed in [7]). Many tumors express not only the receptors, but also the ligands PROS1 and GAS6 [9, 10]. In combination with the abundant presence of apoptotic PtdSer [11], cancer cells are capable of TAM receptor auto-signaling. This subsequently results in the activation of various downstream pathways, including ERK, MAPK, AKT and others [12, 13]. Interestingly, TAM receptors have been implicated in therapy resistance to MAPK, PI3 K and EGFR inhibitors [13]. Mechanistically, a study from Kotenko et al. showed that TAM receptor expression leads to upregulation of PD-L1, possibly contributing to therapy resistance [14].

As a result, TAM receptors seem an attractive target for cancer therapy, not in the least to overcome therapy resistance. Many small molecule inhibitors have been developed, which are usually reactive to all three TAM receptors [15]. Currently, AXL inhibitors are in clinical testing for NSCLC and pan-TAM inhibitors are in clinical testing for the treatment of metastasized solid tumors [8, 16]. In addition, many inhibitors are currently in the preclinical phase where they show anti-tumor activity (reviewed in [8]). Finally, MERTK is frequently overexpressed in leukemias, including T-ALL [15, 17]. Mouse studies show promising results where MERTK inhibition favors tumor regression [18]. In conclusion, TAM receptor targeting is a promising strategy for cancer therapy. However, since many other cell types express TAM receptors, inhibition of this integral receptor should be considered in the wider context of tumor-associated immune responses. It will be interesting to see how TAM receptor inhibition for cancer treatment develops, and caution might be warranted due to adverse immunological side effects.

Efferocytosis is the process of apoptotic cell clearance by antigen-presenting cells (APCs), including macrophages and dendritic cells (DCs). This is an essential process for tissue homeostasis. Due to regular cellular turnover, billions of apoptotic cells are removed by efferocytosis every day in the human body [19]. All three TAM receptors have essential functions in efferocytosis in APCs, which has been reviewed in detail by Rothlin and colleagues [20]. In short, TAM receptors in APCs are essential for efferocytosis and subsequent immune-suppressive signals. TAM receptor knockout mice have significant defects in the efferocytosis of apoptotic cells by APCs [21]. As a result, these mice show hyperactivation of APCs and subsequent autoimmunity.

TYRO3, AXL, and MERTK have distinct roles on APCs. Seitz et al. reported MERTK to be the main receptor for efferocytosis on macrophages, whereas AXL and TYRO3 were most essential for DCs [22]. In addition, Zagorska et al. showed MERTK to be a tolerogenic receptor in macrophages and during immunosuppression [23]. Correspondingly, they reported AXL to be an inflammatory response receptor on DCs, induced by proinflammatory stimuli. The molecular basis of TAM receptor signalling in APCs has been elucidated and functions mainly via downstream SOCS ubiquitin ligases and inhibition of Toll-like receptor signalling (reviewed in detail by Rothlin and colleagues [20]).

NK cells are important components of the innate immune system, as they provide rapid responses to both pathogens and tumors. TAM receptors play an important role in the differentiation of these cells. Caraux et al. showed that all three TAM receptors were expressed by maturing NK cells in mice [24]. This TAM receptor expression by NK cells was deemed essential for mouse NK cell maturation and subsequent expression of both inhibitory and activating receptors. It was also reported that AXL was essential for human NK cell development from hematopoietic progenitor cells [25]. The TAM receptors reportedly regulate NK cell development via IL-15, c-kit and T-BET [24, 25]. Furthermore, Paolino et al. reported that ubiquitination of the TAM receptors on mouse NK cells inhibited their proliferation and cytotoxicity in a tumor metastasis model [26].

Finally, MERTK has been reported to be functionally expressed by mouse NKT cells [27]. Genetic deletion of MERTK resulted in impaired cytokine secretion after antigen-induced activation. This was due to an intrinsic NKT cell defect, as the source of antigen-presenting cells (wild-type or MERTK-deficient) did not alter the impaired MERTK-deficient NKT cell response [27]. Taken together, all studies point toward an essential role for the TAM receptors in the functional maturation and cytotoxicity of NK(T) cells.

In 2008, Shao et al. reported MERTK to be functionally expressed by wildtype mouse B cells. In their study, B cells upregulated MERTK upon chronic GvHD, while MERTK-deficient mice were protected against GvHD [28]. A few years later, the same group showed that upon MERTK depletion in mouse B cells, B cells decreased their antibody production and subsequent T cell activation [29]. Consistent with a stimulatory role for TAM receptors in B cells, both AXL and TYRO3 are reported to be overexpressed in B cell chronic lymphocytic leukemia patient samples [30, 31]. Correspondingly, AXL inhibition decreased B leukemia cell survival in vitro [30]. In conclusion, only a handful of researchers have studied the function of TAM receptors on B cells. The overall hypothesis links TAM receptor signaling to healthy B cell responses. Hence, dysregulation of TAM receptor signaling could lead to aberrant B cell proliferation and leukemia, although more research is needed.

TAM receptors on platelets have been well studied, and all three TAM receptors are expressed by both mouse and human platelets [2, 32, 33]. Various combinations of single, double, and triple TAM receptor knockouts have shown that all three TAM receptors are individually essential for normal platelet function [33]. Deletion of one of the TAM receptors resulted in mild dysfunctional platelets. Deletion of any two of the three TAM receptors did not affect platelet counts, but impaired hemostasis and platelet function. Lastly, absence of all three TAM receptors resulted in thrombocytopenia, severely impaired hemostasis and platelet function, and defective megakaryocytopoiesis [33]. In this regard, small-molecule MERTK-inhibition decreased mouse platelet activation and subsequent thrombosis [34]. Taken together, TAM receptor expression by platelets is essential for regular platelet function in both human and mouse.

T cells were long believed to be negative for TAM receptors. For the ligands, however, results have consistently showed expression by activated T cells. In 1997, it was shown for the first time that PROS1 expression was induced in mouse lymphocytes (both CD4⁺ and CD8⁺) upon TCR activation [35]. IL-4 addition further upregulated PROS1 expression and subsequent secretion. Almost two decades later, PROS1 expression in TCR-activated CD4⁺ T cells was confirmed by Carrera Silva et al. in both mouse and human [36]. Subsequently, Chan et al. showed that PROS1 expression in mouse CD4⁺ T cells was limited to the TH2 subset, and that PROS1 expression was dependent on IL-4 [37]. Regarding CD8⁺ T cells, our group recently verified PROS1 expression by human CD8⁺ T cells, as they expressed PROS1 upon TCR stimulation from day 2 onwards [38]. A more detailed overview of PROS1 expression in lymphocytes is found in Table 1. In all studies, resting T cells remained negative. Taken together, these studies prove that the TAM receptor ligand PROS1 is expressed and secreted by TCR-activated T cells. Interestingly, PROS1 was surface-bound in both ours and Carrera Silva’s study. This indicates that PROS1 is bound to either PtdSer or the TAM receptors, which could both be expressed by T cells. As for the other TAM receptor ligand GAS6, no expression by T cells has been reported.

In the early 2000s, two studies reported that T cells did not express the TAM receptors. Both studies reported no MERTK expression after two-day activation of mouse splenocytes with αCD3, or two-day activation of human T cells with PHA/PMA [17, 27]. In 2014, a study which reported increased MERTK and TYRO3 expression on CD4⁺ T cells from SLE patients went rather unnoticed [39]. The following year, Cabezon et al. convincingly showed that TCR-activated human CD4⁺ T cells expressed MERTK from day 3 onwards [40]. In addition, it was reported that murine CD4⁺ regulatory T cells expressed both AXL and MERTK, without in vitro or in vivo stimulation [41]. Regarding CD3⁺ T cells, Yokoyama et al. suggested that (mouse) CD45⁺ TILs could be responsible for increased MERTK levels in the tumor-microenvironment [42]. Finally, our group recently verified TAM receptor expression on human CD3⁺ and CD8⁺ T cells. We demonstrated on three different levels (RNA, protein, surface expression) that MERTK was expressed on TCR-activated human CD8⁺ T cells and CD3⁺ T cells [38]. In addition, we did not detect AXL and only a low amount of TYRO3.

The discrepancy of all later reports with the two earliest studies could be explained by the chosen species, timepoint, or stimulation method (a definitive overview is found in Table 1). Based on these studies, whether mouse T cells do or do not express any TAM receptor is until now not definitively proven. In humans, TAM receptor expression is better studied, especially regarding MERTK. Both Cabezon and our study showed that MERTK expression is only induced by TCR-mediated (e.g. via CD3 or peptide) activation and only detectable after 2 or 3 days [38, 40]. This could explain why Graham et al. found human T cells negative, as these were stimulated with non-TCR-specific PHA/PMA and the experiment did not go beyond 48 h [17].

According to our knowledge, only four studies have been published on MERTK expression on human T cells in the past 25 years (Table 1). The three most recent studies consistently found a varying amount and subset of T cells MERTK-positive. Combined with the independent and varying investigation methods used, these are compelling arguments for MERTK expression on primary T cells. Taken together, we conclude that TCR-activation leads to MERTK expression on both CD4⁺ and CD8⁺ human T cells. Combined with the T cell’s expression of PROS1, it becomes necessary to elucidate in what functional capacity the TAM receptors and ligands are expressed by T cells.

Shortly after PROS1 was described to be expressed by mouse T cells, PROS1’s function on T cells was studied by the same group. Their study initially suggested that receptors for PROS1 transduced proliferative signals [43]. As the function and expression pattern of the TAM receptors was at that moment unknown, they attributed any positive or negative role to the anti-coagulant functions of PROS1 [43]. Their initial suggestion, however, that an Fc-TAM receptor competed with T cells for the ligand PROS1, proved to be correct two decades later. In this later study, Cabezon et al. added Fc-MERTK to CD4⁺ T cells. Subsequent PROS1 ligand depletion resulted in inhibition of T cell proliferation and activation [40]. Accordingly, adding exogenous PROS1 increased cytokine secretion and proliferation. This corresponds with our data on CD8⁺ T cells, where PROS1 positively regulated proliferation and cytokine secretion. We validated PROS1 signal transduction through MERTK using MERTK-inhibitors and knockdown of MERTK on CD8⁺ T cells [38]. As for GAS6, it has been reported that exogenous GAS6 could increase the suppressive properties of mouse CD4⁺ regulatory T cells via T cell-expressed AXL [41]. Furthermore, Keating et al. overexpressed MERTK in mouse T lymphocytes [44]. Their results showed that dysregulation of MERTK on T cells caused T cell leukemia due to uncontrolled division and proliferation. This highlights MERTK as a stimulatory T-cell molecule which, when dysregulated, results in disproportionate stimulatory and proliferative signals.

Since T cells have been believed not to express MERTK, previous results might need to be re-interpreted. To this end, it was previously shown that treatment of wildtype immunocompetent mice with MERTK-inhibitors decreased PD-1 expression on T cells [45]. PD-1 is solely expressed by previously activated T cells, is dependent on TCR signal strength and avidity, and is an identifying marker for tumor-reactive T cells [46]. T cells lacking PD-1 upon MERTK-inhibition are therefore likely to be less activated, non-tumor-reactive and correspondingly, less likely to respond to PD-1 immunotherapy. In agreement, we have shown with a MERTK-specific inhibitor that MERTK inhibition decreased T cell proliferation and cytokine secretion [38].

Finally, both Cabezon et al.’s and our study have indications that MERTK could regulate the T cell memory response. As MERTK seems to be a ‘late’ costimulatory molecule on T cells (only detectable from day 2–3 up to day 7—and possibly longer—after TCR activation), this is a tempting explanation. It will be exciting to see what future research uncovers. All in all, it becomes increasingly clear that the PROS1-MERTK axis has a functional impact on (human) T cells. Contrary to previous dogma, T cells do express TAM receptors and most importantly, MERTK seems a strong costimulatory receptor upon T cell activation (Fig. 1). This opens up exciting new areas in research for the TAM receptor field, as T cells are now added to the long list of TAM receptor-expressing cells. As availability of the ligands in vivo is not limitless, it will be important to pinpoint the net effect of TAM receptor signaling on the various immune subsets and cancer cells.