Evasion of immune destruction is a hallmark of cancer and results in immune tolerance (1). Immune tolerance can be mediated through multiple pathways, including the immune checkpoint receptors cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD1) (2). A costimulatory signal exerted by CD28:B7 binding is necessary for T cell maturation; CTLA-4 belongs to the CD28 family of immunoglobulins and competitively binds to B7 to produce inhibitory signals that counteract stimulatory signals from CD28:B7 and TCR: MHC binding (3). CTLA-4: B7-1/2 (CD80/B7-1 and CD86/B7-2) binding suppresses several signaling cascades in T cells, including differentiation, proliferation, and survival through inhibits IL-2 accumulation and cell cycle progression etc. (4, 5). CTLA-4 maintains peripheral tolerance by enhancing regulatory T cell (Treg) functions; thus, undirected control of the effector T cells (6) and overexpression of CTLA-4 in tumor samples implicates poor prognosis in patients with melanoma (7). Because CTLA-4 inhibition results in increased activation of the immune system, Ipilimumab, an inhibitor of CTLA-4, was approved for the treatment of advanced or unresectable melanoma (8).

PD1 regulates the activation of T cells by binding to programmed death-ligand 1/2 (PD-L1/2). Activation of the PD1/PD-L1/L2 pathway inhibits T cell proliferation and secretion of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and IL-2, sustaining the immune inhibitory state of the tumor microenvironment (9). Clinical evidence supports aberrant PD-L1 expression in tumor cells, which aids in their escape from T cell immune attack in non-small-cell lung cancer (NSCLC), renal cell carcinoma, Hodgkin’s lymphoma, hepatocellular carcinoma (HCC), and intrahepatic cholangiocarcinoma (ICC) (10). Based on these findings, immune checkpoint inhibitors (ICIs) targeting PD1/PD-L1 are approved or being evaluated as a malignant tumor treatment in various tumors (11).

Although CTLA-4 and PD1/PD-L1 exert similar negative effects on T cell activity, their timing and mechanisms differ. CTLA-4 acts in the initial stage of the immune response, typically in lymph nodes, and the PD1/PD-L1 pathway regulates previously activated T cells at later stages primarily in peripheral tissues (12). Recent evidence reveals interactions between PD1/PD-L1 and CTLA-4 signals. For example, the tumor cell glycolytic rate is depressed by anti-PD-L1 therapy, and patients with low glucometabolic levels in tumors may benefit from CTLA-4 blockade (13, 14). ^A combination of anti-CTLA-4 and anti-PD1/PD-L1 therapies may have an additive or synergistic effect in the treatment of advanced malignancies. Preliminary results of a clinical study report that the combination of the anti-CTLA-4 antibody ipilimumab and anti-PD1 antibody nivolumab elevated the objective response rate (ORR) and the progression-free survival of patients with BRAF^WT metastatic or unresectable melanoma (15).

ICC accounts for 10%–15% of primary liver cancer cases, but its incidence has rapidly increased worldwide, and major ICC risk factors are hepatitis virus B (HBV) and C (HCV) infection along with hepatolithiasis (16, 17). ICC has a poor prognosis owing to local invasion and distal metastasis at first diagnosis (18). First-line therapy for ICC is gemcitabine-based chemotherapy as it is for other advanced biliary tract tumors, but even with treatment, the median overall survival (OS) is 11.7 months (19). Our previous study revealed elevated PD1/PD-L1 signals in tumor samples and distinct profiles of PD-1/PD-L1 in ICC patients with different risk factors (20), and the PD1 inhibitor Toripalimab, in combination with GEMOX (oxaliplatin and gemcitabine) chemotherapy and Lenvatinib, showed an ORR of 80% (24/30) and a 93.3% (28/30) disease control rate in treating advanced ICC (21). On the other hand, CTLA-4 expression and its relationship with tumor-infiltrating Tregs has not been characterized in ICC, and little is known about CTLA-4 and PD1/PD-L1 expression and interaction in ICC. This information could guide both diagnosis and treatment. To address this knowledge gap, we investigated the expression and interaction of CTLA-4 and PD1/PD-L1 in ICC and assessed their value as prognostic indicators in ICC.

We determined a profile of CTLA-4 expression with prognostic implication in a large cohort of patients with ICC. CTLA-4 was hyperactivated in tumor samples from patients with ICC, and the high density of CTLA-4⁺ TILs (TILs^{CTLA-4 High}) was significantly correlated with malignant characteristics. Clinically, the density of CTLA-4⁺ TILs was an independent risk factor for OS in patients with ICC, and we found that patients with TILs^{CTLA-4 High} showed an unfavorable prognosis. These data indicate that CTLA-4 expression in TILs is an important factor for sustaining the inhibitory immune microenvironment in the clinical setting of ICCs. Thus, this provides a rationale for anti-CTLA-4 therapy in ICCs, at least in a subset of patients. Treg cells are considered the strongest inhibitor of antitumor activity, and CTLA-4 expression is essential for the activation of FOXP3⁺ T cells. Our data also show a positive relationship between the density of CTLA-4⁺ TILs and FOXP3⁺ TILs, which provides indirect evidence to support a role for CTLA-4 in the inhibitory immune microenvironment of ICCs. Furthermore, plenty of studies suggest that CTLA-4 on both activated conventional T cells and FoxP3+ Tregs is important for immunology suppression (33, 34), and it is demonstrated that Treg CTLA-4 blockade alone could not induce antitumor immunity, but it could augment the antitumor responses induced by CTLA-4 blockade of conventional T cells by using selective blockade of CTLA-4 on Treg or conventional T cell (35). Here, we show that both hyperactivated CTLA-4 and FOXP3 are related to an unfavorable prognosis, and the amount of CTLA-4+ TILs is higher than FOXP3+ Tregs, which indicates that besides FOXP3+ Tregs, other CTLA-4+ TILs may be involved in antitumor immune disorders. Thus, the overexpression of CTLA-4 reflects a more global immunomodulatory effect, not just Treg infiltration.

CTLA-4 maintains immune homeostasis through complex mechanisms; the cell-intrinsic model of CTLA-4 function describes that the cytoplasmic tail of CTLA-4 affects intracellular posttranslational modifications and regulates cellular localization of CTLA-4, and the cell-extrinsic model describes CTLA-4 acting through Tregs to exert its function (5). CD28 can costimulate T cell functions by affecting cytokine production, reducing the TCR signaling threshold for T cell activation and enhancing T cell proliferation and survival (36). CTLA-4 may act as an antagonist of CD28–ligand interaction by competing for ligand binding. Recent studies show that CTLA-4 expression was overactivated in several malignant tumors, such as melanoma and spinal chordoma (7, 37). Here, we also demonstrate that the CTLA-4 signal was activated in tumor tissues of ICCs. Patients with early recurrence of ICC had a higher density of CTLA-4 expression than patients without early recurrence. Moreover, the density of CTLA-4⁺ TILs is related to the density of FOXP3⁺ TILs. Therefore, CTLA-4 acts as a central element of immunologic tolerance to lessen the immune response in the tumor microenvironment (38). Further, the density of CTLA-4⁺ TILs in ICC tissues is related to aggressive clinicopathologic features, such as preoperative serum CA19-9, larger tumor size, lymph node metastasis, and high TNM stage. The augmentation of CTLA-4 expression in T cells could reduce the secretion of IFN-γ (39) and then facilitate malignant phenotypes, such as tumorigenesis and metastasis (40). Hence, CTLA-4⁺ TILs may be involved in the invasive behavior of ICC cells. Our data indicate that overexpression of CTLA-4 in TILs promotes the invasion and metastasis of ICC and may be a prognostic indicator in patients with ICC.

However, CTLA-4 expressed in tumor cells was not related to the prognosis of ICC. The role of CTLA-4 in tumor cells is controversial, and a previous study suggests that elevated CTLA-4 expression in tumor cells of NSCLC is predictive of a good outcome (41). CTLA-4 is constitutively expressed in a variety of tumor cell lines, such as breast, colon, kidney, lung, ovarian, and uterine cancers and in melanoma cell lines, and elevated CTLA-4 expression is associated with the induction of apoptosis through sequential activation of caspase-8 and caspase-3 (42), but the exact role and mechanism of CTLA-4 in ICC remain to be fully elucidated.

Furthermore, we determined a distinct expression profile of CTLA-4 and PD1/PD-L1 in ICC. PD-L1 was overexpressed in tumor cells, and CTLA-4 was activated in TILs but not tumor cells. CTLA-4 acts as an antagonist of CD28–ligand interactions by competing for ligand binding with CD80. Meanwhile, a large amount of CD80 expressed in antigen-presenting cells (APCs) directly competes with PD1 on the overlapping interface on PD-L1 to disrupt the combination of PD-L1/PD-1 and its inhibitory function in T cell activation. Further, PD-L1 inhibition reduces the expression of CD80 on APCs, and the effect could be offset by the blockage of CTLA-4. This molecular basis has implications for the combination of anti-PD-L1 and anti-CTLA-4 in treating ICC (43, 44). In the present study, patients with coactivation of PD1/PD-L1 and CTLA-4 signals presented the worst prognosis among patients with ICC. Moreover, the density of CTLA-4⁺ TILs was determined as an independent predictor of OS, and PD-L1 expression in tumor cells was an independent predictor of cumulative recurrence. Interestingly, combined CTLA-4⁺ TILs and PD-L1⁺ in tumor cells showed better sensitivity for predicting prognosis of ICCs in terms of OS and cumulative recurrence than that of overexpression of either CTLA-4 or PD-L1 alone. These data indicate that CTLA-4 is a good assistant of PD1/PD-L1 in the inhibitory TEM of ICC.

Moreover, we found that Tumor^{PD-L1 High} patients have more Tregs infiltrating into the ICC tumor than Tumor^{PD-L1 Low} patients. A previous study revealed that PD-L1 could promote Treg development and enhance Treg function (45), which provides implications in the synergistic use of anti-PD-L1 and anti-CTLA-4 therapies.

Additionally, distinct expression of CTLA-4 and PD1/PD-L1 was observed among different risk factors in ICC. Our data suggest that CTLA-4 overactivation in hepatolithiasis-related ICC is likely the predominant factor involved in sustaining the inhibitory immune environment, providing a promising therapeutic target for such patients.

In conclusion, our findings reveal elevated CTLA-4 and FOXP3 in ICC; the combined overexpression of CTLA-4 and PD-L1 is a good marker for predicting poor prognosis in ICCs and presents a potential target for ICI treatment strategies. These findings will be further evaluated in our clinical trial (NCT04634058) about the combination of anti-PD-L1 and anti-CTLA-4 in treating ICC patients, which is already in progress.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by the Institutional Review Board of Zhongshan Hospital. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

Concept and design: X-YH, G-MS. Data collection: L-XY, YC, X-JG, J-CL, H-YZ, Q-MS, G-HY, A-WK, Y-HShi, JZ, and JF. Experiments: X-JG, J-CL, H-YZ, Y-ZP, X-LM, P-FZ, and P-XH. Data analysis and visualization: X-JG, G-MS, X-YH, J-CL, Q-MS, G-HY, Y-HShi, J-BC, and RZ. Writing article: X-JG, X-YH, and G-MS. All authors contributed to the article and approved the submitted version.

This study was supported by the National Key Research and Development Program of China (2019YFC1316000), the National Natural Science Foundation of China (81502028, 81972232, and 82072575), the Shanghai Municipal Natural Science Foundation (18410720700, 20JC1419103, and 21ZR1412200), the Clinical Research Plan of SHDC (SHDC2020CR1003A), and Sanming Project of Medicine in Shenzhen (No. SZSM202003009).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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