**2.1 Immune cells**

The liver is a key immunological organ that receives antigen-rich blood from the gut via the portal vein. Cancer immunosurveillance and control rely heavily on the innate and adaptive immune systems. The conflicting actions of antitumor effectors and their suppressors in the TME determine immune activation or evasion, as shown in (**Figure 1**). Through highly conserved cytokine and chemokine-activated inflammatory reactions, adaptive and innate immune cells patrol the liver sinusoids to eliminate invading pathogens and endotoxins. The uninflamed liver generates a

**Figure 1.** *Homeostasis and immune cell composition of the liver.*

#### *Emerging Immunotherapy: Liver Cancer Microenvironment for Treatment DOI: http://dx.doi.org/10.5772/intechopen.106021*

tolerogenic milieu that suppresses both innate and adaptive immunity in order to maintain homeostasis and prevent chronic inflammation and tissue damage. The immune cell composition of the TME has a huge impact on HCC that can affect tumor initiation, progression, and therapeutic response. During hepatocarcinogenesis, several molecular processes cause distinct immune cell subsets to respond in ways that either cause inflammation or limit antitumor immunity. Herein, the immune cell landscape of HCC is discussed, with an emphasis on the role of innate immune cells and innate-like T cells in HCC, which may lead to the development of candidate immunotherapies that target the underlined cells.

#### **2.2 Cytokines**

HCC's genesis and progression have been linked to inflammation. TNF-α, a key inflammatory mediator, had previously been identified as a possible therapeutic target in a variety of malignancies. Furthermore, TNF-α, interleukin (IL)-1, and IL-6 levels in HCC patients' serum were found to be considerably greater than in healthy controls. CXCL5 and CCL15, which are produced by tumor cells, attract immunosuppressive neutrophils and monocytes, respectively, while CXCL13, which is produced by hepatic stellate cells, attracts B cells, which differentiate into protumor IgAproducing plasma cells in the context of NASH-associated HCC. IL-2, IFNγ, CXCL10, and CXCL9, on the other hand, recruit lymphocytes to mount an antitumor immune reaction. As a result, the immunological composition and response are determined by the balance of these stimuli. T helper 17 (Th17) cells generate the cytokine IL-17, which has been linked to the development and progression of inflammatory disorders. These findings have ramifications for patients who are undergoing immunotherapy. Activation of the IFN-γ signaling system predicts a favorable response to ICIs, according to the findings of two trials in patients with HCC. In HCC, the tumor microenvironment (TME) has a significant impact on cytokine production.

### **2.3 Signaling cascades lead to immune evasion**

HCC's immune microenvironment can be influenced by tumor intrinsic signaling pathways. Wnt ligands produced by HCC cells induce M2 polarization of TAMs, which results in tumor development, metastasis, and immunosuppression in HCC. β-Catenin signaling also inhibits MHC-independent immune responses facilitated by NK cells through decreased expression of NKG2D ligands on HCC cells. By reversing NK cell depletion or TGF- β1 reversing NK cell depletion, blocking the CD96 association restores NK cell immunity to tumors, implying that CD96 may considerably contribute to HCC. MYC may suppress PD-L1 expression in HCC, which suggested that treating HCC with a combination therapy targeting MYC and the PD-L1/PD-1 cascade could be beneficial. HCC is linked to TP53 gene mutations and immune cell heterogeneity, with TP53 mutations reported in roughly 40% of all HCCs. The TP53 mutation-associated immunotype is critical for better clinical outcomes and may have important implications for postoperative tailored follow-up and therapeutic decisionmaking. Furthermore, ARID1A mutations, which are a common driving factor in HCC, have a considerable contribution to antitumor immunity: they stimulate a positive response by suppressing mismatch repair, leading to an elevated TMB, and also reduce IFNγ signaling by lowering chromatin availability. In co-cultured CD4+ T cells, MDSC suppresses the immune system by generating CD4+ CD25+ Foxp3+ regulatory T cells. The IL6-STAT3-PDL1 signaling cascade is used by HCC-CAFs to

control neutrophil survival, stimulation, and function in HCC. Finally, T cell fatigue is caused by the upregulation of immunological checkpoint molecules such as PD-1, PDL1, CTLA4, LAG3, and TIM3 in HCC cells. PDL1 overexpression in Kupffer cells and leukocytes is also caused by prolonged HBV infection, which can boost the protein's level and enhance immunosuppression in HCCs of this etiology.

#### **2.4 Virus associated with HCC**

HCC is the main cause of death in cirrhotic patients, with HCV or HBV infection accounting for the majority of cases, especially in developing countries. HBV is a hepatotropic virus that causes liver inflammation. While another established risk factor for severe liver disease is HCV, a Flaviviridae virus. Long-term infection with HBV or HCV causes an inflammatory reaction in the liver that can develop into cirrhosis and, ultimately, HCC.

HBV and HCV-induced immune responses might be either procarcinogenic or anticarcinogenic. Platelets generate components that induce a necroinflammatory infiltration, *i.e.,* virus-specific CD8 + T cells, which drive HCC development and can be slowed down with antiplatelet medications. An effective HBV-specific T cell response, on the other hand, can aid in the control of HCC cells displaying HBV epitopes, as clinically demonstrated by tumor regressions accomplished with adoptive T cells genetically modified to produce TCRs targeting such epitopes. Despite this, persistent HBV infection causes multiple changes in the hepatic immune infiltrate, which promote a tolerogenic milieu, limiting effective antitumor immunity. B reg cells are a primary source of IL-10, an immunosuppressive cytokine that is increased during HBV flares. Furthermore, HBV-specific T cells are susceptible to BIM-induced apoptotic process and TRAIL + NKG2D + NK cell deletion. Chronic HBV infection also increases the production of inhibitory immunological checkpoint proteins on virus-specific T cells, mainly in the liver, restricting any T cells capable of attacking HCC cells. In patients with HBV-associated HCC, the presence of elevated suppressive PD-1 T reg cells is linked with worse survival outcomes, although CD8+ tissueresident memory T cells are correlated with a better outcome.

HCV has a single polyprotein genome, which is translated into structural as well as nonstructural proteins. These HCV proteins are targets for the host's innate and adaptive immune systems. The principal pattern recognition receptors that identify HCV PAMPs are RIG-I-like receptors and Toll-like receptors. The correlation stimulates a cascade of antiviral cytokines, including interferons. Perforin, as well as granzyme B, is secreted by CD8 + T cells and NK cells while interferon-gamma (IFN-γ) secreted by CD8 + T cells and NK cells causes noncytolytic HCV clearance. Moreover, the host-HCV interactions could make developing an HCV vaccine challenging. It is hard to neutralize a virus that typically has so many mutations in its E1 and E2 proteins, and attempts to do so clear the most abundant variants and leave replicative space for the expansion and continued replication of other quasispecies.

Understanding how virus-related HCCs regulate their metabolism could open up new avenues for immunotherapy. By decreasing arginine, granulocytic MDSCs accumulate in HBV-infected livers and can suppress HBV-specific T cells. High expression of the esterification enzyme sterol Oacyltransferase 1 (SOAT1) disrupts lipid homeostasis, promoting proliferative and migratory potential of the tumor cell in a subset of HBV-associated HCCs while reducing the activity of HBV/HCC-specific tumor-infiltrating lymphocytes.

#### **2.5 Nonviral HCC**

Hepatic steatosis and chronic necroinflammation in the liver can result from persistent alcohol exposure or high-calorie diets combined with a sedentary lifestyle, leading to HCC, which could be fatal. Moreover, NASH-induced inflammation is more frequently linked with diffuse inflammatory infiltrates.

New developments regarding the cellular and molecular cascades that drive NASH and the NASH-HCC transition have emerged in recent years. It has been revealed that platelets drive NASH and the NASH-HCC transition by fostering the first inflammatory reactions in the context of steatosis. Platelets interact with Kupffer cells and inflammatory monocytes through the platelet-specific glycoprotein Ibα (GPIbα). In rodents, and possibly also in humans, preventive and therapeutic antiplatelet treatment lowers the development of NASH and NASH-associated HCC. The study revealed that the usage of low-dose aspirin is linked with a considerably decreased risk of HCC and liver-associated death. NASH-related liver cancer is caused by a variety of immunological mechanisms. NKT cells mediate lipid uptake via LTβR activation on hepatocytes, and metabolic stimulation of intrahepatic CD8 + T cells and NKT cells, for example, has been demonstrated to induce NASH and HCC via hepatocyte cross talk. NKT cells primarily cause steatosis via secreted LIGHT, while CD8+ and NKT cells cooperatively induce liver damage. The metabolic machinery in hepatocytes is downregulated as a result of this cross talk, which involves direct interaction between immune cells and hepatocytes via Fas and release of porforins and granzymes as well as indirect communication via secreted substances (e.g., cytokines, chemokines), resulting in increased metabolic, endoplasmic reticulum, and mitochondrial stress. Surprisingly, human NASH has been found to have a considerable elevation in the number of intrahepatic CD8 + PD1 + T cells. As previously stated, metabolic imbalance triggers autoaggression in these CD8 + PD1 + T cells, culminating in MHC I independent cytotoxicity against hepatocytes and necroinflammation. This autoaggressive behavior of CD8 + PD1 + T cells has implications for patients receiving ICIs for NASH-associated HCC: nonviral HCCs, particularly NASHassociated HCCs, are less susceptible to these drugs than viral HCCs. This discrepancy has been linked to the autoaggressive intratumoral CD8 + PD1 + T cells losing their tumor surveillance function, resulting in a protumorigenic milieu.

Alcohol consumption is responsible for up to 30% of all HCC cases worldwide. The mucosal damage caused by alcohol might result in an impaired intestinal barrier function, enabling toxins of gut-inhabiting bacteria such as endotoxins to enter the systemic circulation and to contribute to liver injury after alcohol consumption. Alcohol increases gut permeability, allowing immunomodulatory microbiota-derived PAMPs such as LPS to enter the liver and decrease hepatic immune reactions, presumably through effects on resident macrophages. NASH is linked to an increase in protumorigenic, immunosuppressive granulocytic MDSCs in the liver, as well as a decrease in T cell migration to the liver. Furthermore, the neutrophils in the liver parenchyma are a hallmark of alcoholic hepatitis, which is thought to influence the hepatic immune landscape.

#### **2.6 The modulatory of the microbiota**

Dysbiosis has been seen in various phases of chronic liver injury, including HCC, according to several investigations. The gut microbiota increases HCC

development in the setting of chronic liver injury, presumably via microbial metabolites or PAMPs, according to in vivo studies using a mouse model. Through the primary to the secondary conversion of luminal bile acids, the microbiota of the stomach can reduce immunosurveillance and accelerate the progression of HCC. Furthermore, metabolized bile acids (deoxycholic acid, a secondary bile acid) have been demonstrated to cause senescence in hepatic stellate cells, leading to the production of numerous cytokines such as transforming growth factor-β1, angiotensin II, leptin) that enhance the progression of HCC. The relevance of the gut microbiota in influencing systemic immunity, including immunotherapeutic reactions and chemotherapy-induced immunological effects, is now widely recognized. The antigenic epitope tail length tape measure protein 1 (TMP1) in the genome of bacteriophage Enterococcus hirae had high similarity with the proteasome subunit beta type-4 (PSMB4) tumor antigen. They activated CD8+ T cells simultaneously and improved the efficacy of PD-1 blockade therapy. It has been demonstrated that the antigen epitope SVYRYYGL (SVY) expressed in the commensal bacterium Bifidobacterium breve was similar to the tumor-expressed antigen epitope SIYRYYGL (SIY), resulting in SVY-specific T cells recognizing SIY and inhibiting tumor growth. However, further research on the impacts on hepatic immunity is still needed.

#### **2.7 HCC immune classification**

Few studies have attempted to classify HCC according to its immunological status. The "Inflammatory" and "Lymphocyte Depleted" clusters were found prominent in HCCs, according to a pancancer analysis based on clustering of immune-associated gene expression profiles. Using a transcriptome deconvolution technique, the first thorough immune categorization of HCC was published in 2017, which found an "Immune" class (which accounts for 25% of HCCs). Immune tumors have an elevated level of immune infiltration, enhanced PD-1/PDL1 signaling, and signature enrichment that mimics the response to ICIs in other solid tumor types.

More recently, a modification of this classification established an "Inflamed" class of HCC, which accounts for about 30–35% of tumors, expanding the previously documented immune class with an additional subset of tumors labeled as an "Immune-like" subclass. This novel subclass is distinguished by the presence of CTNNB1 mutations and significant activation of interferon signaling and immunological activation. T-cell-inflamed tumors are characterized by type I interferon (IFN) activation, immune potentiating chemokines, antigen presentation, cytotoxic effector molecules, and activated CD8+ T cells. The inflamed tumor microenvironment is additionally characterized by IFN-induced inhibitory pathways such as programmed death-ligand 1 (PD-L1) and indoleamine-2, 3 dioxygenase and higher proportions of FOXP3+ regulatory T cells. Patients with HCC have a higher proportion of inflamed tumors, and those patients who responded to anti-PD-1/PDL1 antibodies were shown to be enriched in the inflamed class. In the "Noninflamed" class of HCCs, two subclasses have been evaluated based on the mechanisms of immune escape: (1) an "Intermediate" class with TP53 mutations, elevated levels of chromosomal instability, and frequent deletions in subcytobands harboring genes linked with interferon signaling or antigen presentation; and (2) an "Excluded" class with CTNNB1 mutations and immune desertification features.
