3. Role of PD-1-Expressing HCC-Specific CD8 T Cell Response in HCC Control

The tumor-associated antigen-specific CD8⁺ T cell response is essential for the control of solid tumors due to their ability to recognize tumor cells and to destroy them [40,41]. Exhausted CD8⁺ T cells are the main subset of TILs that perform anti-tumor effector functions [10,42]. Specifically, in HCC it is possible to find a high number of T cells infiltrating the tumor (Figure 2) and there is a correlation between the density of infiltrating lymphocytes and the prognosis of the disease [43]. Furthermore, after loco-regional treatment of HCC, a longer survival has been observed in those subjects in whom a specific CD8⁺ T response against HCC associated antigens is detected [44]. Therefore, large numbers of CD8⁺ TILs in HCC correlate with improved OS, longer relapse-free survival, and diminished disease progression [10,45,46]. HCC associated antigens comprise a heterogeneous set of autologous proteins that are rendered immunogenic in tumors by mutation [35] or aberrant expression [10,47] (Figure 2). The development of an effector cytotoxic T cell immune response against these antigens may provide a barrier to tumor progression. Nevertheless, most of the T cells targeting autologous aberrantly expressed epitopes are deleted or in a tolerogenic state [33,48], although some studies have shown a positive effect after PD-1 blockade in T cells targeting these kind of antigens [7]. Anyhow, the response to ICI could be more effective in T cells targeting HCC neo-antigens. A previous study has suggested that searching for host’s human leukocyte antigen (HLA) restricted tumoral neoantigens will permit to find T cells that after the appropriate treatment will exert tumor control [35]. HCC will commit mutations that will give rise to new epitopes that will be recognized as exogen antigens by T cells. Unfortunately, these cells will become exhausted during HCC progression, not being able to keep the tumor under control. These exhausted CD8⁺ T cells show a gene signature that is completely different from those of memory and effector T cells [49,50,51]. During the exhaustion process, T cell response progressively loses the effector functions and finally undergoes apoptosis [11,52]. The exhausted CD8 T cells are featured by the expression of different inhibitory IC, such as PD-1, cytotoxic T lymphocyte antigen-4 (CTLA-4), T cell immunoglobulin domain and mucin domain (Tim3), and lymphocyte-activation gene 3 (LAG3) that can be modulated to rescue T cell reactivity [21,53]. The ligands of these IC are expressed by resident liver cells. In fact, PD-L1 is expressed on hepatocytes [22], hepatic stellate cells [54], liver sinusoidal endothelial cell (LSEC) [55], and Kupffer cells [56,57], while PD-L2 expression is restricted to dendritic cells [58]. PD-1 and PD-L1 upregulation promotes CD8⁺ T-cell apoptosis and postoperative recurrence in hepatocellular carcinoma patients [59]. The in-vitro treatment of these T cells with antibodies against these myriad of inhibitory IC increases T cell proliferation and cytokine secretion.

Nevertheless, these exhausted CD8⁺ cells develop epigenetic imprints that steadily maintain the functional impairment, which is transmitted to the progeny, making temporary the effects of immunomodulatory treatments [49]. Even in the context of a PD-1 knockdown HCC-specific CD8 T cell model, although a tumor killing enhancement is initially observed, this effect is limited by compensatory engagement of alternative co-inhibitory and senescence programs upon repetitive antigen stimulation [60]. These epigenetic imprints induce the expression of specific transcription factors, such as the thymocyte selection-associated high mobility group box protein (TOX) that is up-regulated on CD8⁺ T cells in HCC and promotes their exhaustion by regulating endocytic recycling of PD-1 [61]. Additionally, the up-regulation of the ligands of these negative IC is induced in the HCC microenvironment. The regulation of PD-L1 expression could be related with the level of M2 macrophages that are recruited by the tumor cell-intrinsic osteopontin secretion. In HCC animal models, the level of M2 macrophages decreases after osteopintin blockade, which correlates with an increased CD8 effector response to PD-L1 blockade [62]. M2-like macrophages favor an immunosuppressive landscape by depleting CD8 T cells and inducing CD4⁺ T regulatory cells (Tregs) [63]. Additionally, M1 macrophages can induce PD-L1 expression on hepatocarcinoma cells by IL-1β effect [64]. These data suggest an important role of macrophages in modulating CD8 T cell exhaustion in HCC by promoting a immunotolerant environment [65] and by up-regulating themselves both PD-L1 and PD-L2 expression [66]. However, PD-L1 expression on macrophages could also have a positive input because it correlates with CD8 T cell infiltration and increased OS after treatment [67], probably in the case of M1-like macrophage infiltration [17]. Moreover, HCC-specific CD8 T cell itself induces PD-L1 up-regulation on hepatocytes, linked to a subsequent impairment of IFN-γ secretion by T cells [68]. There is a gradient of PD-1 expression on intra-tumoral CD8⁺ cells, which probably defines the progenitor (PD-1^dim) and effector (PD-1^high) pools [69]. The PD-1^high population can co-express positive co-stimulatory checkpoints, such as 4-1BB, that can be triggered to rescue these cells from exhaustion [70,71]. Nevertheless, to target the PD-1^dim subset could be more operative to get a more efficient response to ICI. The PD-1 level of the intrahepatic resident CD8⁺ cells inversely correlates with the expression of the transcription factor T-bet [72], which has been correlated with a late dysfunctional phenotype in the PD-1^high pool, since the effector late dysfunctional pool expresses the transcription factor Eomes and loses T-bet expression [73]. On the contrary, PD-1^dim expression is a marker of the progenitor pool, which is the subset that provides the proliferative burst after anti-PD-1 treatment [69]. Therefore, T cell restoration should focus on PD-1^dim CD8⁺ T cells. In order to improve both the precursor and the effector pools, searching for PD-1/PD-L1 based combinatory therapies, such as modulation of suppressive soluble mediators (interleukin (IL)-10, IL-17, TGF-β1), blocking suppressive cells (Tregs, myeloid derived suppressor cells (MDSC), M2 tumor associated macrophages (TAM)), triggering positive co-stimulation (tumor necrosis factor receptor superfamily member 9, 4-1BB), mitochondrial metabolic reprogramming, impairing neo-angiogenesis, inducing expression of HCC neo-antigens or epigenetic modulation by gamma-chain (γc) cytokines [11,12,14,74] could improve the response to PD-1/PD-L1 blocking monotherapy. Figure 3 summarizes the potential mechanisms involved in HCC-specific CD8 T cell impairment.

Figure 3. Scheme showing the potential mechanisms involved in HCC-specific CD8 T cell impairment. The graph also highlights the possible PD-1/PD-L1 blockade-based combination therapies to rescue effector and precursor HCC-specific cytotoxic T cell response. PD-1: programmed cell death protein 1, CTLA-4: cytotoxic T lymphocyte antigen-4, Tim-3: T cell immunoglobulin domain and mucin domain, LAG-3: lymphocyte-activation gene 3, T regs: CD4 T regulatory cells, Th: T helper, IL: interleukin, TFG: tumor growth factor, VEGF: vascular endothelial growth factor, 4-1BB: tumor necrosis factor receptor superfamily member 9, OX-40: Tumor necrosis factor receptor superfamily member 4, γc: gamma-chain.