CTLA-4, PD-1:PDL-1, TIGIT, VISTA, Tim-3 and other inhibitory receptors in cancer therapy:

Given that CTLA4 is an alternate ligand for CD80/86, and unlike the activating ligand CD28, induces regulation of T cell responses rather than T cell activation, it was no surprise that neutralization of CTLA4 showed a beneficial effect on anti-tumor immunity [15,16], with such neutralizing antibodies being the first approved inhibitors of checkpoint blockade to be used clinically in melanoma [17,18] and subsequently in solid tumors [19]. More recent studies have focused on the possible use of combinations of reagents targeting different checkpoints to improve tumor immunotherapy eg. CTLA4 and PD-1 (below) blockade [20], as well as the use of checkpoint blockade along with vaccination (see below). This latter strategy is well documented in studies showing that patients with advanced prostate cancer responding to treatment with anti-CTLA4 therapy developed enhanced responses to a number of other tumor-related antigens, both patient specific and disease specific, compared with non-responders [21].

As note above, a current evolving trend has been to use anti-CTLA4 therapy in association with other therapies, either immunotherapy (vaccination) or other checkpoint blockade reagents. Huang et al, in a murine model of ovarian cancer [22] found that multiple immune checkpoint molecules were expressed in tumor-associated or tumor-infiltrating cells in ovarian tumorbearing mice, but simple blockade of any one of PD-1, LAG-3, or CTLA-4 alone using blocking antibodies or knockout mice merely led to an up-regulation of the other checkpoint pathways with failure to attenuate tumor outgrowth. However, dual antibody blockade against PD-1/CTLA-4 or triple blockade against PD-1/LAG-3/CTLA-4 led to tumor-free survival in ~20% of treated mice [22]. Interestingly, dual blockade of LAG-3 and CTLA-4 pathways in PD-1 knockout mice resulted in tumor-free survival in ~40% of treated mice, implying the existence of a hierarchy in the importance of immune checkpoints in some tumors, though this remains to be explored in depth. Similar results with combination checkpoint inhibitor blockade has been reported by others [23,24].

Focus on the importance of the PD-1:PDL-1 dyad [25] in cancer growth was stimulated by evidence that up-regulation of PDL-1 was often seen on cancer cells [26] and the ligand PD-1 was in turn expressed on T cells [27]. At least one potential important effect of blockade of PDL-1 signaling may involve augmentation of DC maturation, proliferation, and IL-12 secretion, with resultant improved immunity induced by DCs [28]. Results from such model systems are borne out by positive clinical trial results with antibodies targeting the PD-1:PDL-1 pathway in metastatic melanoma, prostate cancer, non-small-cell lung cancer (NSCLC), renal cell carcinoma (RCC) and colorectal cancer(CRC) [29,30]. Such therapy is often used in combination with additional treatments, including CAR-T cell therapy [31], tumor vaccine administration [32], and manipulation of exogenous/endogenous cytokines [33,34]. It is interesting in this context too that there are reports that high levels of soluble PD-L1 are potentially a useful biomarker of poorer prognosis in renal cell carcinoma/multiple myeloma/melanoma patients [35], analogous to the observation we had reported earlier in regards to soluble serum CD200 [36,37].

The T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT), which binds two ligands, CD155 (PVR) and CD112 (PVRL2, nectin-2) expressed by tumor and other cells in the tumor microenvironment, has also been acknowledged as another potential target for cancer immunotherapy. Expression of TIGIT is increased on activated T cells, natural killer cells, and regulatory T cells, and there is evidence that combined PD-1/ TIGIT blockade increased tumor antigen-specific CD8⁺ T cell expansion/function and promoted tumor rejection in mouse models [38]. Similarly, targeting V-domain immunoglobulin suppressor of T-cell activation (VISTA), a molecule expressed on hematopoietic cells, and on myeloid cells infiltrating tumors [39] has been reported to prove effective in attenuating tumor growth in combination with PD-1 blockade [40], with preliminary data lending credence to a potential value in clinical therapy in gastric cancer patients [41]. More recently it has been suggested that targeting Tim-3, a molecule expressed on Foxp3⁺Tregs (along with other cells of the innate immune system), has a role in leading to augmentation of anti-tumor immunity [42].

Another interesting target molecule is CD47, a (relatively) ubiquitously expressed glycoprotein of the immunoglobulin superfamily, over-represented on many solid and hematologic cancer cells, with high expression correlated with poor clinical prognosis. CD47 is implicated in the regulation of self-recognition [43] through interaction with its receptor, SIRPα, expressed on myeloid cells. This interaction in turn activates cytoplasmic immunoreceptor tyrosine-based inhibition motifs, recruitment of Src homology 2 domain-containing tyrosine phosphatases, and delivery of the anti-phagocytic-”don’t eat me”-signal. Thus CD47SIRPα acts as a negative checkpoint bridging innate immunity and adaptive immunity, and CD47 blocking antibodies diminish tumor growth in several animal models [43]. While to date no clinical studies targeting this pathway are available, there are a number of animal model studies which suggest there may be clinical utility in targeting such a molecule [44,45]. As is discussed next, there is independent precedence for such an approach in a plethora of data investigating a role for targeting a similar molecular dyad, the CD200:CD200R axis, in cancer therapy.

The importance of CD200:CD200 interactions in controlling tumor growth

CD200 is a molecule known to be relatively ubiquitously expressed, while expression of the receptors (CD200R1-5 in mouse; CD200R1,R2 in man) is seen mainly on cells of the myeloid and macrophage lineage. There are a number of studies which suggest that blockade of CD200:CD200R interactions attenuates inflammatory (innate) immune reactions while augmenting development of acquired immunity [46], which in turn promoted investigations into the potential role of regulation of CD200:CD200R interactions in tumors [47].

Regulation of hematopoietic tumor growth by CD200:CD200R

Abundant data indicates expression of CD200 both by activated normal B cells, by tumor cells in patients with B lymphoid malignancies, and even by precursor stem cells for B cell malignancies. Expression of CD200 is generally a poor prognostic indicator in B cell malignancies [48], and therapies aimed at neutralizing CD200 expression favour modulation of tumor growth [49-52], including in both pediatric AML and ALL [53,54]. There is now too a growing body of evidence implicating a role for CD200 expression levels in regulation of growth of CLL [55-57]. Interestingly, often the effects seen in different patients, at least in CLL, were not reflective of relative expression levels of CD200 by tumor cells, suggesting that the mechanism of action was not simply one of neutralization of expressed CD200 [58].

Wong et al reported [36] that soluble CD200 found in the serum of CLL patients was essential for promotion of CLL growth in a NOD.SCID humanized mouse model. There are now similar reports of the prognostic significance of sCD200 expression in patients with CLL [59]. Newer evidence suggests that CD200 expression controls Treg expansion and disease progression in both acute myeloid leukemia and CLL [60]. In a study with 14 CLL patients, CD200 blockade using an anti-CD200 antibody we described earlier [36] ledto disruption of T cell suppression as measured in autologous MLCs using CD40 ligand (CD40L)stimulated CLL cells as antigen-presenting cells (APCs) [60], a mechanism inferred by Wong et al in studies in NOD.SCID mice with CLL cells [36]. A similar mechanism has also been suggested as contributing to the mechanism behind increases in Foxp3⁺T cell numbers in PTLD patients following overexpression of CD200

[61].

An alternate mechanism of action of CD200:CD200 blockade was suggested by Poh et al. [62], who reported that checkpoint blockade augmented cytotoxicity of cytokine-induced killer cells against human myeloid leukemia blasts. Interestingly, CD200 blockade has also been reported to improve regulation of AML growth by Tr1cells engineered to overexpress IL-10 [63]. This is in turn reminiscent of the report that Ibrutinib, an inhibitor of Bruton’s tyrosine kinase (BTK) and IL-2-inducible T cell kinase (ITK), which is used in treatment of CLL, may impact CLL induced immunosuppression by modulating CD200 expression [64]. Zhu et al (personal communication) more recently combined a vaccination approach to CLL (using PMA and ionomycin stimulated CLLs as vaccine) in concert with blockade using anti-human CD200 antibody, to attenuate both local disease and secondary organ spread of CLL cells in a NOD.SCID mouse model.

Before concluding this section on discussion of the importance of CD200:CD200R in control of myeloid tumors, it is worthy of note that even in studies where there was little direct evidence for a role of attenuating CD200 expression in tumor growth control, there has been reported value in monitoring expression in both the diagnosis of B cell malignancies [65,66], and in monitoring dynamic changes during the response to treatment [67]. This is the case also in high risk myelodysplastic patients [68]. A final comment, which will be elaborated on below in a discussion of the effect of CD200:CD200R interactions in solid tumors, concerns the potential importance of this dyad, beyond any direct effect on tumor cells themselves, in controlling tumor growth through regulation of the tumor microenvironment (TME) [69]. Tumor associated myeloid cells (TAMCs) are important in cancer immunotherapy, and CD200-CD200 receptor (CD200R) interactions are reported to be implicated in regulating the TME by affecting TAMCs [70]. Studies in an in vitro/in vivo model exploring control of growth of CLL, showed that bone marrow mesenchymal stem cells (BMMSCs) and the cytokines they produce, including IL-6, are important components of the TME in CLL, and that BMMSCs support the survival of CLL cells in vitro through an IL-6 dependent mechanism [70]. IL-17, which can induce IL-6 generation in many cells, increased production of IL-6 both in CLL cells and BMMSCs in vitro. It must be acknowledged that the effect of CD200-target therapy in this particular system was not investigated.

Regulation of solid tumor growth by CD200:CD200R

The role of CD200:CD200R as a crucial checkpoint for immunity in solid tumors was implied from an earlier study by Moreaux et al [71], who described overexpression of CD200 in renal carcinoma, head and neck carcinoma, testicular cancer, malignant mesothelioma, colon carcinoma, myeloma, and CLL, all compared to their normal cells or their tissue counterparts. These data also showed that CD200 expression was associated with tumor progression in many cancers. Similarly, Siva et al [72] reported expression of CD200 on cell lines derived from ovarian cancer, melanoma, neuroblastoma and renal carcinoma cell patients. Addition of CD200⁺ but not CD200^- tumor cells to MLCs led to a decline in Th1 cytokine production, and this effect was attenuated by anti-CD200 antibody [72]. Increased staining of CD200 and CD200R protein expression was also found on liver tissue specimens in subjects with hepatocellular carcinoma compared with healthy controls [73], which was correlated with tumor size, alpha-fetoprotein levels, and pathological tumor grade compared with healthy controls. Overall and recurrencefree survival rates were lower in patients with high CD200R [73] expression compared to those with low CD200R expression. These data are consistent with those from Matsuo et al, who investigated immune responsiveness in patients with colorectal liver metastasis [74]. CD4⁺, CD8⁺, and CD45RO⁺ tumor-infiltrating lymphocytes were significantly higher in the low-CD200 group than in the highCD200 group , and overall survival was significantly worse in patients in the high-CD200 group who had received preoperative chemotherapy. Similarly, in a rat model of hepatocellular carcinoma where treatment involved thermal ablation of tumor, Huang et al concluded that the CD200/CD200R pathway participated in tumor growth and the expression of CD4⁺T cell subsets in cancer tissue, and that thermal ablation treatment inhibited cancer recurrence through a mechanism potentially involving this same CD200:CD200R axis [75].

Multiple studies have investigated the importance of checkpoint inhibition in lung cancer besides the antagonism of the PD1:PDl-L and CTLA4 axes discussed above (see earlier). Vathiotis et al found that CD200:CD200R was frequently expressed in non small cell lung cancer patients (NSCLCs), and that expression was correlated with PD-L1 expression, with implications for targeting this pathway alone, or in combination with PD1:PDL1 in NSCLCs [76]. In an animal model system Wang et al [77] also concluded that the CD200:CD200R and PD-1:PDL-1 axes might interact favourably to regulate tumor growth, while Yoshimura et al [78] independently argued for the potential importance of assessing CD200:CD200R expression in NSCLCs. CD200 expression was reported by Zhang et al as one of six signature marker genes whose expression was a prognostic predictor of survival in NSCLCs [79]. The favourable interaction between CD200:CD200R and PD-1:PDL-1 in regulating tumor growth was also highlighted in studies if pancreatic adenocarcinomas by Choueiry et al [80], who concluded that one of the primary mechanisms of action involved modulating MDSCs, known to express CD200R, and possibly their expansion in response to local cytokines in the TME. The role of CD200 expression in modifying the local TME, in turn resulting in regulation of tumor growth, was also noted by Zhang et al in studies of cervical cancer [81], and by Feng et al in prostate cancer [82]. Interestingly, in a study of metastatic pancreatic cancer, liver metastases in both animal models and humans were reported to contain highly anergic T cells and MHCIIloIL10+ macrophages which could not present tumor-antigen, along with CD24+CD44CD40- B cells [83]. These B cells were apparently recruited to the metastatic milieu by Muc1hiIL18hi metastatic tumor cells, and were implicated in driving macrophage-mediated immunetolerance through CD200 and BTLA. These authors reported that depletion of these B cells and targeting CD200/BTLA led to enhanced macrophage and T-cell immunogenicity with subsequent effective immunotherapeutic of liver metastases [83]. A similar result showing therapeutic efficacy of CD200 blockade in reducing Merkel cell carcinoma tumor growth was seen in tumors which were infiltrated by suppressive CD200+ macrophages and Tregs [84]. Along similar lines, in animal models of lung cancer, within the TME there was preferential differentiation of DCs away from CD103+ DC1s, which have the ability to induce tumor immunity, towards CD103loCD11b+ DCs, expressing the DC2 markers IRF4 and Sirpα, along with high levels of T-cell inhibitory molecules PD-L1/2 and CD200 [85]. The effect of CD200 blockade on DC differentiation within the tumor was not investigated in these studies-see also [86]. A model which might help explain much of these data is suggested by recent studies of adrenocortical carcinoma patients [87], which were subdivided into low/high hypoxia risk scores (HRS) by gene expression data. The low hypoxia risk score group showed a “fit” to an inflammatory immunophenotype, while the high HRS group represented a noninflammatory immunophenotype. Importantly, high HRS correlated with expression of immune checkpoint molecules including PDL1, CD200, CTLA-4, and TIGIT, consistent with data that patients with low HRSs responded better to immunotherapy. 

The potential importance of the CD200:CD200R axis in regulation of tumor growth independent of tumor expression of CD200 (or CD200R) was also described by Rygiel et al in animal studies exploring regulation of skin malignancies induced in an animal model of chemical carcinogenesis [58] . CD200KO mice were resistant to tumor induction, an effect which was correlated with an alteration in the Th17:Treg ratio, and which occurred independent of any CD200 expression by tumor cells themselves. In stark contrast to these data are those of Talebain et al, investigating the effect of CD200 blockade in a CD200+ Yumm1.7 mouse melanoma model [88]. It is known that Yumm1.7 cells bear Braf/ Pten mutations resembling human melanoma, yet they observed that anti-CD200 therapy failed to show efficacy either alone or in combination with checkpoint inhibitors such as anti-PD-1 or anti-CTLA4 in inhibiting Yumm1.7 tumor growth, and that both CD200R-deficiency and anti-CD200 treatment resulted in reduced T cell responses in the TME-they highlighted these studies as indicating some caution in using blockade of CD200 as an immunotherapy for melanoma [88]. Somewhat analgous to studies above indicating the potential importance of CD200:CD200-R interactions in regulating the TME are reports by Belkin et al, who explored the ability of this same dyad to modulate squamous cell carcinoma tumor diapedesis at the endothelium of blood vessels [89]. CD200 expression on local blood vessels was thought to promote tumor progression by suppressing CD200R myeloid cells during diapedesis leading to immune evasion by SCC, and again suggesting a novel therapeutic target for control of metastasis of

SCC.

There has been intense interest in the past decade on the role of CD200 blockade in cancers of the brain, which had generally been poorly responsive to tumor-derived vaccines and chemotherapy, yet are known to express CD200 [90,91]. These poor results may reflect the effect of expression of immune-checkpoint ligands including PD-L1 and CD200 by glioblastoma (GBM) cells which interact with their respective receptors on tumor-infiltrating effector T cells/myeloid cells thus curtailing the activation of anti-GBM immunity. Studies of expression of genes involved in autophagy, apoptosis and necrosis in 518 GBM patients from The Cancer Genome Atlas (TCGA) database revealed a cohort of 16-genes grouped into a cell death index (CDI), with patients subsequently clustered into either a high risk or low risk group according to the CDI score, with overall survival subsequently correlated with higher infiltration of neutrophils, macrophages, Treg, T helper cells, and lower expression levels of immune checkpoint molecules PDCD1 and CD200 [92]. Subsequent studies by Olin’s group confirmed that overexpression of CD200 attenuated the efficacy of a tumor derived vaccine in glioblastoma (GBM) [93], which could in turn be blocked using a CD200 checkpoint inhibitor peptide [94].The mechanism of action of this peptide inhibitor, CD200AR-L was hypothesized to reflect the targeting of a CD200 activation receptors (CD200AR) to overcome tumor-induced immunosuppression [95]. The same group subsequently used a proteomics approach to produce a humanized CD200AR-L, which was shown to induce a unique chemokine response, stimulating immature dendritic cell differentiation and significantly enhancing an antigen-specific response, which also led to downregulation of the expression of CD200 inhibitory and PD-1 receptors [95]. More recent studies showed that CD200AR-L/CD200AR binding induced early activation of both the DAP10&12 pathways, followed by a decrease in activity within 30 min, followed by reactivation via a positive feedback loop [96]. In vivo studies with DAP10&12KO mice showed the importance of DAP10, not DAP12, for tumor control. This immunotherapy aimed at modulating immune checkpoints in GMB is currently being analyzed in a phase I adult trial [96]. An interesting variant of a similar approach was described by Kobayashi et al [97], who described expression of both full-length CD200 (CD200L) and a truncated form, CD200S, in human carcinoma tissues. In a murine transplant model, we previously reported that this truncated variant could itself antagonize immunosuppression induced by full-length CD200, likely through engaging alternate receptors and/or by acting as a competitive inhibitor to full-length CD200 at an inhibitor receptor [98]. Kobayashi used rat C6 glioma cell lines that expressed either CD200L or CD200S and showed that CD200S activated TAMs to become DC-like antigen presenting cells, resulting in activation of CD8(⁺) cytotoxic T lymphocytes and subsequent apoptotic elimination of tumor cells. They suggest this may in itself highlight a novel therapeutic modality for the treatment of carcinomas [97].

A focus of my own group, besides the CLL studies discussed earlier, has been to investigate the role of CD200:CD200R as an immune checkpoint in breast cancer. Two quite different BALB/c mouse models have been characterized. The one, 4T1 is a highly aggressive, metastasizing, poorly immunogenic tumor, while EMT6 in contrast is an example of a moderately immunogenic, poorly metastasizing, and slower growing tumor [99]. In the case of the former, an inflammatory cascade is growth promoting, while for EMT6 tumors, suppression of inflammation results in development of T cell immunity and delayed tumor growth-similar heterogeneous findings have been made with human breast cancer [100-104]. Consistent with this dichotomy, over-expression of CD200 led to improved control of growth of 4T1 tumors, while augmenting growth of EMT6 cells [99]. In contrast, in the absence of endogenous expression of CD200, or more particularly CD200R, tumor bearing mice showed markedly reduced local and metastatic EMT6 growth and could be immunized for sterile immunity with resistance to further tumor challenge, again with the opposite effects seen for 4T1 tumor [105]. It was clear that an active immune process developed in EMT6 tumor-injected mice in association with CD200 (or CD200R) blockade since we showed that conventional chemotherapy acted synergistically with CD200 blockade to cure wild-type mice and produce immune mice resistant to re-challenge and/or metastasis even at 1 year post treatment-such an effect was not seen with chemotherapy alone [106].

It has also become clearer over the past few years that checkpoint inhibitors modulate not simply the innate or adaptive immune responses to the tumor, but can, as already mentioned, have a equally important effect on the TME [69,107,108]. This dual effect likely helps explain some anomalies observed in the effect of either CD200 or CD200R blockade on tumor growth [109], where, for example, Pilch et al in a mouse melanoma observed that blockade of endogenous CD200 expression prevented the tumorigenic effect of CD200R-expressing myeloid cells in the tumor microenvironment, although agonistic anti-CD200R had no effect on tumor development. A further effect of manipulation of CD200:CD200R signaling to influence tumor growth indirectly through the TME was reported from studies of papilloma tumors in mice [110]. Toll-like receptor 7 (TLR7) stimulation is known to augment immunity through activation of myeloid cells, decreasing papilloma growth, an effect antagonized by CD200R stimulation which suppresses TLR7 signaling. However, the immediate antitumor effects of TLR7 signaling alone were independent of lymphocytes, with similar results seen in wildtype and Rag1^tm1Mom mice [110]. Moreover, TLR7 stimulation, particularly in combination with anti-CD200R, changed the phenotype of intratumoral myeloid cells, decreasing expression of the macrophage markers F4/80, CD206, CD86, CD115, and the ability to produce IL1β, suggesting a shift in the composition of intratumor myeloid cells. Indeed, CD11b+ myeloid cells, isolated from the tumors of mice receiving combined treatment inhibited tumor outgrowth on adoptive transfer suggesting that administration of agonistic anti-CD200R improved the antitumor effects of TLR7 signaling and altered the local TME to become one less supportive of tumor progression [110]. More recently an intriguing effect of a CD200 mimetic aptamer given in conjunction with pegylated doxorubicin was reported in regulation of growth of breast cancer cells in a mouse model [111]. PEG-M49 and PegDox co-treatment induced complete tumor regression and loss of macroscopic lung metastasis in four out of seven BALB/c mice [111]. Synergism was associated with Peg-M49-induced inhibition of Gr1+CD11b+ cells and Peg-Dox-induced increases in tumorinfiltrating CD8+ and CD8+CD4+ cells. Importantly, similar changes were observed in CD200R1-/- mice suggesting that the primary effects of Peg-M49 were mediated by non-inhibitory CD200R receptors. Moreover, tumor growth, metastasis, and tumor infiltrating GR1+ CD11b+ cells were markedly increased in CD200R1-/- mice, implying that there existed an anti-inflammatory and protective role for CD200. The authors concluded that in fact CD200 mimetics might be safe and effective therapeutics, in conjunction with classical chemotherapy, in treatment of aggressive metastatic breast carcinoma. In this regard it might be noted that a recent study of T cells engineered following singlecell mRNA sequencing (scRNA-seq) with T-cell receptors (TCRs) from naturally occurring tumor antigen-specific T cells (TAS cells) in different patients, presumably targeting personal TSAs in that individual’s tumor, were observed to express CXCL13 as a unique marker for both CD4+ and CD8+ TAS cells [112]. TCR-T cells expressing TCRs from TAS cells attenuated growth of autologous patient-derived xenograft tumors, with intratumoral TAS cell levels (CXCL13 expression) predicting the response to immune checkpoint blockade. Importantly, CD200 was a surface marker for CD4+which enabled the isolation of TAS cells from tumor by FACS sorting, implying a non-suppressive (overall) effect in this model.

Summary

There can be little doubt that immune checkpoint blockade has transformed cancer immunotherapy producing some quite remarkable and long-lasting effects, particularly in melanoma and some hematopoietic tumors and now expanding to multiple solid tumor studies. As has been highlighted, the use of genomic expression analysis has both guided characterization of the molecules involved, and helped gain insight into their mechanism(s) of action [65,68,73,74,79,87,92,101-103]. As but one further example, analysis of sorted CD4⁺CD26^- and CD4⁺CD26⁺ T cells from classical Hodgkins Lymphoma lymph node cell suspensions (HLN) by RNA sequencing and T cell receptor variable gene segment usage analysis showed that while CD4⁺CD26^- T cells were antigen experienced, they were not clonally expanded [113]. In part at least this is explained by the over-expression of exhaustion associated transcription factors TOX and TOX2, immune checkpoints PDCD1 and CD200, and the chemokine CXCL13, which were all significantly enriched genes compared with the CD4⁺CD26⁺ T cells. This overwhelming dominance of a CD4⁺CD26^- T cell population in HLNs which is antigen experienced, polyclonal, and exhausted likely is itself predictive of, and contributes to, the very high response rates to immune checkpoint inhibitors in such patients [113].

With experience in use of single agents for checkpoint blockade it is also becoming increasingly apparent that the overall response rate for the currently approved reagents used alone is limited. This has encouraged studies using multiple checkpoint blockade reagents in combination (see Figure 1 and Table 1 suggesting the potential for use of checkpoint blockade of T cell expressed and myeloid expressed markers), and/or with additional immunotherapy, as well as further biomarker analysis (gene expression data) which might predict both the success of these therapies; can monitor their efficacy [114]; and may even highlight new molecules for consideration [115]-see also studies on VISTA/ TIGIT/CD47 highlighted above, and more recent reports on use of combined checkpoint blockade for NK-mediated tumor killing [116]. In this regard, the very fact that some 40% of humans and Tasmanian devils (Sarcophilus harrisii) develop cancer in their lifetime, compared to less than 10% for most species, and that CD200:CD200R is one of the immune checkpoint interactions which has been conserved across 160,000,000 years of evolution, suggests that CD200, which is highly expressed on transmissible tumor cells, may be an important molecule to understand [117].

The final, though by no means the least important, observation to be made concerns the demographics of the at-risk population, and those reflected in clinical trials. Studies of cancer diagnosis and death by age groups shows that elderly patients (≥65 years) are over-represented with even further increases expected over the next decade, for example for non-small cell lung cancer [118]. Nevertheless, for a variety of reasons this population is quite under-represented in clinical trials, despite the fact that it has been acknowledged for many years that ageing per se is associated with both qualitative and quantitative changes in immunity [119,120]. It would not be surprising to find that many of the reagents used as checkpoint inhibitors in younger subjects might be more/less efficacious in elderly ones and may produce quite unexpected responses in the two different cohorts. Moreover, there may be novel molecules which take on important roles in immunoregulation, or TME control, in the elderly which have yet to be identified. It seems self-evident that that over the next decade, with more attention paid to this discrepancy, an improved understanding of checkpoint blockade in aging; more specific clinical trials for elderly cancer patients; and increased use of combination immunotherapies will likely further improve cancer survival across multiple disease types.

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