Immune checkpoint receptors: homeostatic regulators of immunity

The PD-1 pathway belongs to the CD28/B7 family of T-cell co-receptors. PD-1/CD279 is probably the most studied checkpoint receptor in the field of T-cell exhaustion. This receptor was first identified in apoptotic T-cell lines (hence the name “programmed death 1”) but was soon characterised as a negative immunoregulator [36‐39]. PD-1 has two known ligands, namely PD-L1/B7-H1/CD274 and PD-L2/B7-DC/CD273 [40‐43]. PD-L1 is ubiquitously expressed at low levels and is strongly induced by proinflammatory signals [44, 45], while PD-L2 displays a more restricted expression profile [43, 46, 47]. Upon engagement, PD-1 sequesters intracellular factors involved in the TCR signalling, stopping T-cell activation [40‐43]. PD-1/PD-L1 signalling appears to be bidirectional: PD-L1-expressing cancer cells possibly receive anti-apoptotic signals upon interaction with PD-1-expressing T cells [48, 49], but it is not known if this happens also in the context of T-cell interactions with antigen-presenting cells (APCs). PD-L1 also binds CD80, triggering inhibitory signals within PD-L1-expressing cells [50‐53]. Amongst T cells, PD-1 is mostly expressed on primed T cells and is strongly upregulated upon TCR-mediated antigen-specific T-cell activation in peripheral tissues. Therefore, the PD-1 pathway is believed to play a role in the establishment and maintenance of peripheral tolerance [54]. It has been demonstrated that the PD-1 pathway can modulate immune cells other than T cells. The effect of PD-1 engagement on causing B cell exhaustion, for instance, is well-described [28, 29] and PD-1 expression on NK cells has also been linked to NK-cell functional suppression [31, 32]. In a study investigating immune exhaustion in HIV patients, contact with bacterial products induced monocyte expression of PD-1 and these monocytes secreted suppressive IL-10 upon PD-1 engagement. Furthermore, T-cell exhaustion in these patients could be reversed by blocking either PD-1 or IL-10 receptor [55]. Monocyte activation by bacterial endotoxin was also shown to physiologically cause increased secretion of suppressive IL-10 and upregulation of PD-1 and TIM-3 on T cells, and simultaneous blockade of TLR4 and CD14 abolished IL-10 secretion and inhibited T-cell checkpoint upregulation [21]. These findings link the PD-1 pathway to the regulation of antibacterial immunity, which is key in ALD patients.

TIM-3/CD366 was first described as a marker of activated IFNγ-producing T cells [56]. TIM-3 binds to Galectin-9 causing suppression of cytokine production, cell cycle arrest and even cell death [57, 58]. TIM-3 is widely expressed on several tissues and is promptly upregulated on T cells in response to both TCR-dependent and TCR-independent stimulation by common-gamma-chain cytokines (IL-2/7/15/21) [58‐60]. Galectin-9 is widely expressed and is further induced by proinflammatory cytokines [61]. TIM-3 is important for T-cell exhaustion both in chronic viral infections [62‐64] and in cancer [65‐67]. TIM-3 is often co-expressed with PD-1 on severely exhausted T cells [25], where both receptors act synergistically to suppress immune functions [68, 69] and it has been demonstrated that blockade of TIM-3 partially restores these impaired T-cell functions [58, 66, 70]. However, there are still unanswered questions regarding paradoxical effects of the TIM-3 pathway observed in bacterial infection, for instance in tuberculosis where activation of this pathway seems to favour immune activation and disease control [71‐73] but also suppression of T-cell functions and disease persistence [74]. Patients with SAH compared to healthy individuals display increased plasma Galectin-9 and higher TIM-3 expression on several immune subsets, together with production of suppressive IL-10 and reduced antibacterial functions, suggesting activation of the TIM-3 pathway in these patients [21].

CTLA-4/CD152 is another member of the CD28/B7 family of T-cell co-receptors and is the first negative immune checkpoint studied in depth. CTLA-4 binds to CD80 and CD86 with approximately 20 times greater affinity than CD28 [40, 75], competing for CD80/CD86 binding and lowering the probability of T-cell costimulation by preventing activating interactions with APCs. Second, upon engagement the cytoplasmic tail of CTLA-4 sequesters factors involved in the TCR signalling, shutting down TCR-mediated T-cell activation [54, 76, 77]. Furthermore, CTLA-4 binding with CD80/CD86 can induce transendocytosis, effectively removing B7 molecules from the surface of APCs [78]. CTLA-4 can act bidirectionally, inducing the production of immunosuppressive IDO by APCs, which metabolically inhibits bystander T-cell functions [51]. CTLA-4 can also bind to another B7 family member called B7-H2, which is the only known ligand for the activatory receptor ICOS, possibly preventing ICOS-mediated T-cell costimulation [79]. Amongst T cells, CTLA-4 expression is stronger in naïve T cells and Tregs, and therefore it is believed that CTLA-4 in comparison to PD-1 may be more relevant in the initial phases of immune activation, preventing immune priming and the establishment/maintenance of central tolerance [54].

LAG-3/CD223 is a molecular homolog of CD4 [80], first described as a regulator of Treg activity [81]. LAG-3 binds uniquely to MHC-II molecules, which are upregulated during inflammation. The exact mechanisms of action of LAG-3 are still unclear. LAG-3 is strongly expressed on anergic and on exhausted T cells, often in strong association with PD-1 [82, 83]. Neutralizing antibodies against LAG-3 can only partially reverse anergy and rescue immune dysfunction [54, 84], but combined anti-LAG-3/PD-1 approaches have demonstrated stronger immune restoration [85], suggesting that LAG-3 inhibitory activity alone may be gentler than other inhibitory checkpoints.

Many pathogens have developed strategies to exploit immune checkpoint regulation as a way to facilitate immune escape/masking. For instance, viruses such as HIV, HCV and HBV, which establish chronic infections in humans, have evolved the ability to manipulate the PD-1 pathway to favour viral persistence [86‐92]. In patients with chronic HBeAg + HBV infection, for example, we previously found dramatic T-cell dysfunctions associated with upregulation of PD-1 on virus-specific T cells [93]. PD-1 expression correlated directly with viremia and decreased progressively during antiviral treatment. In these patients, T cells displayed a skewed cytokine production with lack of antiviral IFNγ and predominant suppressive IL-10. During antiviral treatment, HBeAg + patients who achieved HBeAg seroconversion, which requires the presence of immunologically active T cells, appeared to be those with a more prominent loss of T-cell PD-1, suggesting immune reactivation, and the decreased PD-1 expression was accompanied by normalisation of cytokine production and IFNγ/IL-10 ratio. Higher expression of PD-1 on HBV-specific T cells has also been linked to failure to spontaneously eradicate the virus during acute infections, determining an immune milieu favourable to viral persistence [90, 94], while increased expression of PD-1 on B cells has been linked to B cell functional suppression in HIV infection [28, 29]. Blockade of the PD-1 pathway has been suggested as a possible host-targeted strategy to reactivate antiviral immunity and immune-mediated viral control in HBV, HIV or HCV infections (Figs. 1b, 2c) [86‐92].

Blockade of negative checkpoints first received FDA approval in the context of anticancer treatments [54]. The tumor microenvironment expresses high levels of negative checkpoints and their ligands, which translates into a strong local suppression of anticancer responses. For instance, cancer cells of different origins express high levels of PD-L1 and IDO [95‐100], either as a direct effect of cancer-related intracellular pathways [101, 102] or as a result of IFNγ stimulation by infiltrating immune cells [103‐107]. Therefore, they can suppress tumor-infiltrating cancer-specific T cells by PD-1 engagement and by depleting the local milieu of essential tryptophan metabolites. Additionally, tumor-infiltrating lymphocytes have high expression of multiple checkpoint markers, with increasing numbers of receptors correlated to the severity of immune impairment [26]. Lastly, PD-1 is not only expressed on tumor-infiltrating T cells, but also on NK and B cells [54], suggesting a farther-reaching effect for a broader immune modulation.

In vitro checkpoint receptor blockade has demonstrated efficacy at rescuing exhausted tumor-specific T cells, favouring increased breadth and magnitude of effector functions and T-cell survival (Figs. 1b, 2c) [108] and an increasing number of clinical trials for new anticancer treatments based on immune checkpoint blockade are showing objective therapeutic responses in several patients, limiting disease progression and in some cases arresting or even reverting tumor growth. The most studied pathway in this context has been the B7 pathway [54, 109‐112].

In pre-clinical studies, anti-CTLA-4 treatment showed successful reactivation of pre-existent anti-tumor immunity, alone or in combination with other immunomodulatory agents (such as GM-CSF) [54]. In clinical trials, treatment of melanoma with the FDA-approved anti-CTLA-4 antibody ipilimumab improved clinical outcome and survival both short term and long term, with relatively contained immune-related adverse events [113‐116].

In a number of pre-clinical studies, PD-1/PD-L1 expression in the tumor microenvironment has been linked to immune dysfunction and PD-1/PD-L1 blockade has achieved immune rescue [54]. Clinically, anti-PD-1 and anti-PD-L1 have been tested in several cancers displaying good outcomes and a relatively good safety profile [111, 112]. The anti-PD-1 antibodies nivolumab and pembrolizumab have demonstrated better safety profiles and far greater success rates in comparison to current standard treatments (such as sorafenib to treat advanced hepatocellular carcinoma, HCC) [110] and also in comparison to anti-CTLA-4 strategies [54] in several clinical trials for different cancers [111, 117, 118]. In the ‘CheckMate 040’ trial for the use of nivolumab (humanised anti-PD-1 antibody) in advanced HCC 20% of patients had an objective response (OR), and up to 64% of them achieved disease control (DC), compared to < 1%OR and 43%DC, respectively, with sorafenib as standard treatment [110, 119]. PBMC and plasma/serum samples from the ‘CheckMate 040’ patients are being collected and stored in our laboratories and currently used to investigate a robust biomarker of treatment response in this cohort.

Inhibitory checkpoint antibodies are currently being tested alone or in combination with other neutralizing antibodies in cancer to achieve a stronger immune reconstitution by blocking several checkpoint receptors at once [54]. Inhibitory checkpoint antibodies currently in clinical development or tested in clinical trials as new anticancer agents are illustrated in Fig. 3 [120‐124].

According to the Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3), sepsis is a “life-threatening organ dysfunction caused by a dysregulated host response to infection (…) that arises when the body’s response to an infection injures its own tissues and organs” and septic shock is a “subset of sepsis in which particularly profound circulatory, cellular, and metabolic abnormalities are associated with a greater risk of mortality than with sepsis alone” [35]. The original biphasic description of sepsis and septic shock included a first phase during which an overwhelming systemic inflammatory response would develop upon pathogen detection (systemic inflammatory response syndrome, SIRS), characterised by uncontrolled immune activation, cytokine storm and the occurrence of immune-mediated tissue and organ damage, with potentially lethal consequences. A second phase would then ensue during which the hyper-active SIRS response would subside and compensatory anti-inflammatory mechanisms would complete pathogen clearance and tissue repair (compensatory anti-inflammatory response syndrome, CARS) [125‐127], characterised by the induction of physiological mechanisms of immune shut-down and the activation of the healing response, with the acquisition of a strong endogenous immunosuppressed state. The current description of sepsis, however, has changed. It is now clear that SIRS and CARS are not two distinct sequential entities and the immune derangement that occurs during sepsis represents the contrasting combination of concurrent immune activation and immune exhaustion, where a state of persistent immunosuppression arises in parallel with the initial immune activation and drives the underlying immune dysfunction long term [128‐132]. Indeed, septic patients who survive the initial inflammatory phase of sepsis present with an increased susceptibility to secondary opportunistic infections long term, indicating that immunosuppression and immune impairments are maintained over time [133, 134]. Immunosuppression rather than hyper-immunity drives the response to sepsis, as also supported by the evidence that clinical trials focussed on reducing hyper-immunity/SIRS have provided conflicting and disappointing results [135‐138].

Negative immune checkpoints (including PD-1, PD-L1, TIM-3, CTLA-4, LAG-3 and others) play a causal role in this persistent immunosuppression [139‐143]. Their expression on both innate and adaptive immune cells is greatly increased in septic patients, correlating with loss of immune functions (including innate antibacterial activities from monocytes, macrophages or neutrophils and T-cell production of cytokines and cytotoxic factors), immune cell apoptosis, reduced pathogen clearance and increased patient mortality [34, 141, 143‐146]. Most of these immune dysfunctions can be at least partially restored by blocking checkpoint pathways (Figs. 1b, 2c) [34]. This strategy is currently being investigated in several pre-clinical and clinical ex vivo studies with promising results in septic patients, suggesting that host-targeted immunotherapy may rescue suppressed antimicrobial immunity, reduce susceptibility to infection and improve patient survival [34]. The first clinical trial to determine the safety profile and efficacy of treatment with the anti-PD-1 antibody nivolumab in patients with severe sepsis or septic shock has been completed (NCT02960854), and results of this trial are eagerly awaited.