Immune-mediated Liver Injury caused by Checkpoint Inhibitors (ILICI)

Posted on: May 5, 2022

-Contributed by Abi Kasberg, PhD

The immune checkpoint is a vital regulatory component that prevents the immune system from targeting healthy cells or developing autoimmunity. It does so via immunoreceptors that suppress T cell activity in mechanisms that protect normal, healthy cells from destruction. However, prolonged expression of immune checkpoint proteins can lead to T cell exhaustion which is commonly seen in cancer and persistent infections (De Martin et al. 2020). Tumor cells can leverage immune checkpoints to elude immune surveillance and inactivate T cell activity, thus preventing the immune system from detecting and killing antigen-specific cancer cells (Shojaie et al. 2021). The main regulators of the immune checkpoint system are programmed cell death (PD-1) and its ligand PD-L1, along with cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and its ligands B7-1 (CD80) and B7-2 (CD86) (Shojaie et al. 2021). These molecules are located on the cell surface making them accessible targets for cancer immunotherapies.

Immune checkpoint inhibitors (ICIs) are monoclonal antibodies that bind and block immune checkpoints to restore T cell activity and antitumor responses (De Martin et al. 2020, Shojaie et al. 2021). ICIs approved for clinical use include the CLTA-4 inhibitor ipilimumab, PD-1-inhibitors nivolumab, pembrolizumab, and cemiplimab, and PD-L1-inhibitors atezolizumab, avelumab, and durvalumab (Regev et al. 2020). ICI therapies have significantly improved survival rates and long-term clinical outcomes for several types of cancer including metastatic melanoma, lung cancers, Hodgkin’s lymphoma, head and neck cancers, and renal cell carcinoma (Simsek et al. 2019). Despite these benefits, the favorable outcomes of ICI anti-tumor therapy do not come risk-free. ICIs are associated with a loss of self-tolerance that can lead to serious immune-related adverse events (irAEs). irAEs that are observed with ICI therapies include severe organ toxicities and an increased frequency of immune-related inflammation and autoimmune events (De Martin et al. 2020). Liver injury, pericarditis, myocarditis, colitis, and skin rashes can accompany ICI immunotherapy (Regev et al. 2020).

Due to the incredible success of immunotherapies at treating tumors, there is a dramatic increase in the number of ICI immunotherapy cases, which is also driving an increase in the frequency of irAEs. Risks of ICI-induced toxicity include direct cytotoxicity to healthy tissues, depletion of regulatory T cells, and cross-reactivity between tumor cell antigens and normal cells resulting in paraneoplastic syndromes (De Martin et al. 2020). Organ toxicities and irAEs can become severe to the point that ICI therapy must be paused or terminated. Combination therapies that utilize both anti-PD-1 and anti-CTLA-4 antibodies to target complicated tumors have a higher incidence of toxicity (De Martin et al. 2020). The remainder of this article will focus on the symptoms, diagnosis, and mechanisms of ICI-associated hepatotoxicity in the liver, a disorder named immune-mediated liver injury caused by checkpoint inhibitors (ILICI).

ILICI

Occurrence

ILICI is more common in individuals treated with combination therapy of anti-PD-1 and anti-CTLA-4 (9.2%) compared to monotherapy of either anti-CTLA-4 (1.7%) or anti-PD-1 (1.1%) (Clinton et al. 2021). The onset of liver toxicity is variable during ICI therapy and may even appear following the end of ICI therapy (De Martin et al. 2020). ILICI may occur during ICI treatment of liver cancers and non-hepatic cancers. ICI therapy increases the risk of graft rejection following liver transplant, suggesting that ICIs should not be administered to treat HCC or other tumors following liver transplant (Clinton et al. 2021).

Diagnosis

Patients with liver toxicity caused by ICIs, termed ILICI, can present with a fever, skin rash, jaundice, or may be asymptomatic. ILICI can symptomatically associate with co-toxicity of other organs including colitis and hypophysitis of the pituitary gland (De Martin et al. 2020). ILICI is diagnosed following the exclusion of other causes of liver hepatitis and can be characterized by elevated blood levels of AST, ALT, and total bilirubin (De Martin et al. 2020). Histological analysis of liver biopsies is the recommended method for diagnosing hepatitis, but there are currently no ICI-specific biomarkers available to distinguish ILICI from other causes of hepatitis (De Martin et al. 2020).

ILICI is mediated by ICI therapies and develops as an indirect result of changes to immune-mediated activity within the liver. This differs from autoimmune hepatitis (AIH), idiosyncratic drug-induced liver injury (DILI), and direct hepatotoxicity (Regev et al. 2020). Idiopathic AIH is associated with autoimmune antibodies, which is not commonly observed with ILICI (Clinton et al. 2021). Idiosyncratic DILI encompasses unique adverse liver reactions in susceptible individuals following drug treatment (Teschke and Uetrecht 2021). ILICI can be differentiated from DILI and AIH by distinct histological characteristics, such as an absence of plasmacytes and confluent necrosis that is rarely observed (De Martin et al. 2020, Zen and Yeh 2018).

Treatment

Hepatotoxicity in the liver stemming from ICIs is typically graded using the Common Terminology Criteria for Adverse Events (CTCAE), which is a standard classification scale to grade adverse events to cancer therapy. Hepatotoxicity can range from grade 1 being minimal elevations of liver enzymes to grade 4 which reflects severe liver enzyme elevations and treatment with steroids. Grade 5 corresponds to death.

Recommended treatment can vary, but in general, ILICI graded toxicity of 1, 2, and 3 requires a pause in ICI therapy (DeMartin et al. 2020). Hepatotoxicity grade 3 and above requires the permanent discontinuation of ICIs accompanied by administration of corticosteroids (Shojaie et al. 2021). Additional treatment using immunosuppressives such as mycophenolate mofetil (MMF), azathioprine, or calcineurin inhibitors may be administered if liver enzyme levels do not drop following the discontinuation of ICIs (De Martin et al. 2020).

ILICI Terminology

Published literature is filled with inconsistent terminologies used to describe the adverse hepatic events that occur related to ICI immunotherapy. To address this, the IQ DILI Immunotherapy Working Group recommends that the preferred nomenclature used moving forward be “immune-mediated liver injury caused by checkpoint inhibitors”, shortened to ILICI (Regev et al. 2020).

Other terms that have been published in literature include, “immune-related liver injury”, “immune-related hepatotoxicity”, “ICI-associated immune-mediated hepatitis”, “ICI-induced liver injury”, “checkpoint-induced liver injury”, “autoimmune-like hepatitis”, “immune-induced hepatitis”, along with many other names. (Regev et al. 2020). The interchangeability and range of use can be problematic and challenging to follow (Regev et al. 2020). Some of these names are associated with implied mechanisms or histological conclusions, such as hepatitis, that may not be accurate (Regev et al. 2020). Adhering to the selected ILICI nomenclature will mitigate confusion surrounding ICI-induced adverse events in the liver.

Mechanisms of ILICI

As ICI immunotherapy use increases and the number of patients experiencing irAEs and ILICI grows, it is crucial to better understand the mechanisms driving immune toxicities in the liver so that improved treatment options can be established. The liver has innate mechanisms of immunotolerance that enable the liver to process the abundant and diverse array of antigens that pass through the liver daily without becoming a pro-inflammatory environment (Tiegs and Lohse 2010). Liver immunotolerance is mediated by a variety of immune cells, signaling pathways, liver cells, and immune checkpoint regulators (Shojaie et al. 2021). Here, the function of immune checkpoint regulators during liver immunotolerance is discussed along with what is known about the mechanisms driving ILICI.

Immune Checkpoint Function and Expression

PD-1 and CTLA-4 both negatively regulate T cell activities but are distinct from each other in that they function through distinct signaling pathways and have unique patterns of expression. PD-1 is expressed in peripheral tissues on activated T cells, B cells, and myeloid cells. The PD-1 ligands are widely distributed on leukocytes and non-hematopoietic cells (Buchbinder and Desai 2016). Interestingly, inflammatory cytokines and tumorigenic signaling pathways can induce PD-1 ligand expression on tumor cells, tumor-infiltrating lymphocytes, additional immune cells, and even nonimmune cells (Buchbinder and Desai 2016).

In contrast, CTLA-4 expression is typically limited to the T cells of lymph nodes and CTLA-4 functions to regulate T cell proliferation. The ligands for CTLA-4 are restricted to antigen presenting cells residing in lymph nodes and the spleen (Buchbinder and Desai 2016). However, in tumor microenvironments, CTLA-4 can also be expressed on infiltrating regulatory T cell (Tregs) populations and on tumor cells (Shojaie et al. 2021).

Within the liver, immune checkpoint proteins provide critical protection from autoimmune responses triggered by the high quantity of antigens present in the liver. PD-L1 is normally expressed on non-parenchymal liver hepatic stellate cells and Kupffer cells (Shojaie et al. 2021). During states of chronic liver inflammation, such as autoimmune and viral hepatitis, immune checkpoint markers are upregulated in hepatocytes, Kupffer cells, and intrahepatic lymphocytes (Shojaie et al. 2021). The increased presence of immune checkpoint markers is a compensatory effort to promote immune tolerance in an environment that is riddled with potentially damaging autoimmune and immune activity.

Due to the differences in PD-1 and CTLA-4 expression and mechanisms of activity, ICI combination therapy utilizing both anti-CTLA-4 and anti-PD-1 antibodies provide a synergistic and longer-lasting antitumor response (Buchbinder and Desai 2016). The differences between immune checkpoint protein expression and pathways may contribute to the mechanistic variability observed in ILICI.

Fig 1: The pathogenesis of Immune-mediated liver injury caused by checkpoint inhibitors (ILICI) can be characterized with the following three attributes: 1- Administration of immune checkpoint inhibitors (ICIs), 2- T-cell mediated activation of the immune system, and 3- injury to the liver resulting in hepatocyte death.

T cell-mediated immune activity

ILICI can be described as having the following three characteristics: immune checkpoint inhibitor therapy, T cell-mediated immune activity, and hepatocyte cell death (Fig 1). ICI-induced blockage of PD-L1, PD-1, or CTLA-4 triggers the activity of CD8+ cytotoxic T lymphocytes (CTLs) (Quezada and Peggs 2013). CD8+ CTLs target a broad spectrum of self-antigens which in turn activates a complex array of autoimmune events (Shojaie et al. 2021). It has been suggested that the activation of CTLs contributes to irAEs from ICIs, but the signaling pathways that cause liver-specific injury and ILICI is not well understood (Shojaie et al. 2021). T helper cell populations are also affected by ICIs. ICIs alter the recruitment and expansion of T helper cell populations, which results in the increased production of proinflammatory cytokines.

One circulating cytokine in particular, tumor necrosis factor (TNF), has been suggested to be important for mediating ILICI. After ICI therapy, elevated TNF levels can activate CTLs and innate immune cells (Shojaie et al. 2021). TNF can also bind the death receptor TNFR1 that activates cytotoxic signaling pathways that trigger cell death (Wajant and Siegmund 2019). This presents the possibility that TNF could be driving irAEs through activation of innate immunity or through the direct activation of death receptor pathway signaling resulting in liver cell death (Shojaie et al. 2021).

Tregs function to suppress the immune system to prevent autoimmune disease. Tregs do so through the secretion of anti-inflammatory cytokines and by regulating the immune activity of dendritic cells, macrophages, and neutrophils (Okeke and Uzonna 2019, Shojaie et al. 2021). Treg depletion occurs when anti-CTLA-4 antibodies bind CTLA-4 receptors on Tregs resulting in cell-mediated cytotoxicity (Shojaie et al. 2021). Following anti-CTLA-4 therapy, the number of Tregs is diminished in tumor cells in a negative correlation with irAEs (Shojaie et al. 2021).

Alterations to T cell populations and activities cause disruptions to the production and secretion of proinflammatory and anti-inflammatory cytokines. Following ICI administration, T cell-mediated immune activity is altered, driving an increase in pro-inflammatory cytokines that can mediate cell death and injury in the liver. It is unclear whether the injury to the liver is a result of direct and/or indirect T cell activity.

Cell Death

Liver cell death is a key feature of irAEs and ILICI, however the mechanisms that drive cell death in the liver has not yet been explored (Shojaie et al. 2021). The main indicators of ILICI are elevated liver enzymes (ALT, AST, and bilirurubin), suggesting that hepatic cell death may be a key contributor to the irAEs associated with ILICI. It has been observed in ILICI patients that CD163+/CCR2+ macrophages and CD8+ T cells are enriched and co-localize to sites of liver inflammation (Gudd et al. 2021). This suggests that during ILICI, T cells infiltrate the liver and accumulate at sites of liver injury and cell death.

It has been speculated that the innate and adaptive immune systems may be co-contributing to ICI-derived hepatotoxicity (Shojaie et al. 2021). A possible explanation could be that cytotoxic T cells are directly mediating apoptosis (Affolter et al. 2019, Shojaie et al. 2021). It is unknown whether hepatotoxicity downstream of immune activation is directly targeted to hepatocytes or if non-parenchymal cells that express PD-L1 are targeted as well (Shojaie et al. 2021). Cell death could also be an indirect result of altered Tregs populations or the increased expression of inflammatory cytokines (Shojaie et al. 2021). The signaling pathways and cytokines that mediate the hepatoxicity downstream of cytotoxic T cell and macrophage activation have not been fully identified or understood.

Animal Models of ILICI

Mouse models of ILICI have been developed that block or knockout immune checkpoints PD-1 and CTLA-4 (Affolter et al. 2019). These models demonstrate liver injury and the infiltration of immune cells (Affolter et al. 2019). The immune cells that infiltrate the liver are primarily CD8+ T cells that associate with sites of hepatocyte necrosis (Affolter et al. 2019). Gene expression analyses suggest that both apoptotic and necrotic pathways are activated in ILICI animal models (Affolter et al. 2019).

Future ILICI research directions

Due to the significance of immune checkpoints in supporting immunotolerance in the liver, it is not surprising that ICI therapies disrupt the delicate immune homeostasis of the liver (Shojaie et al. 2021). This highlights the importance of monitoring for hepatotoxicity and the onset of ILICI concurrent to ICI administration. Individuals with pre-existing chronic liver diseases such as viral hepatitis, alcoholic liver disease (ALD), and non-alcoholic steatohepatitis (NASH) have stressed liver environments and may be more susceptible to adverse liver reactions by ICIs. Additional studies examining the effects of ICI therapies on pre-existing chronic liver disease and inflammation needs further examination.

As can be said of many cancer treatments, ICI immunotherapies exist in a complex clinical space. The life-saving potential of ICI cancer treatments must be considered and balanced against the risk of developing organ toxicities and other adverse immune events. The increasing and widespread use of ICI immunotherapies to treat challenging cancers has created a new field of liver injury that urgently needs additional research and clinical investigation. There is an immediate need to better understand the causes of ILICI following ICI therapies, particularly during chronic liver diseases such as HCC.

Current areas of ILICI research and ICI drug development that directly need consideration and further investigation include:

Why do ICIs induce ILICI in some patients, but not all?
Is there an increased risk for patients with underlying liver diseases to develop ILICI following ICI treatment?
ILICI can develop with non-specific or an absence of clinical symptoms. Predictive liver toxicity biomarkers are needed to identify individuals at-risk for developing ILICI.
What are the specific mechanisms that drive hepatic injury following ICI therapies?
The pathways leading to hepatocyte cell death during ILICI need to be defined. Biomarkers of cell death, such as CK18, could be used to characterize the mechanisms that lead to ILICI.
ICI-specific biomarkers and diagnostic tools are needed to distinguish liver toxicity induced by ICIs from other causes of hepatitis.
Pre-clinical investigations assessing ICI-induced liver toxicity during early stages of ICI drug development should be considered.
Clinical trial monitoring of early stages of ILICI development following treatment with mono or combination ICI drugs needs to be prioritized and documented.

Further research in these areas will answer the unknowns pertaining to the causes of ILICI, the mechanisms of ILICI, and how best to prevent and treat ILICI. The forecasted increase of ICIs is hastening the need for more research in this new and growing liver field.