Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2025 Jun 6:16:1611365.
doi: 10.3389/fimmu.2025.1611365. eCollection 2025.

Lymphocyte exhaustion in hepatocellular carcinoma: a dynamic evolution across disease stages

Carla Fuster-Anglada  1   2   3 , Josep Corominas  2   3 , Aida Marsal  2   3 , Neus Llarch  2   3   4   5 , Gemma Iserte  2   3   4 , Marco Sanduzzi-Zamparelli  2   3   4 , Alejandro Forner  2   3   4   5 , Joana Ferrer-Fábrega  2   3   5   6 , Victor Holguin  2   6 , Albert Morales  2   3   7 , Carolina Saavedra  8 , Maria Reig  2   3   4 , Loreto Boix  2   3 , Montserrat Marí  2   3   7 , Alba Díaz  1   2   3   5
Affiliations

Lymphocyte exhaustion in hepatocellular carcinoma: a dynamic evolution across disease stages

Carla Fuster-Anglada et al. Front Immunol. .

Abstract

Background: Immune checkpoint inhibitors (ICIs) have transformed cancer therapy. However, their efficacy in hepatocellular carcinoma (HCC) is limited, highlighting the need to further explore immune microenvironments and novel biomarkers. This study examined lymphocyte populations and immune checkpoint dynamics in early, advanced, and post-progression HCC to better understand immune dynamics in HCC and to help identify predictive biomarkers and immune modulation strategies.

Methods: Tumoral and non-tumoral liver tissues were analyzed from HCC patients across early (n=25), advanced (n=22), and advanced-beyond-progression (n=15) stages. Lymphocyte profiling was performed using immunohistochemistry and flow cytometry, focusing on NK cells, T cells, and immune exhaustion markers. An exploratory analysis of this profile and its association with disease progression and recurrence was conducted.

Results: Early HCC exhibited higher liver-resident NK (lrNK) cell densities in non-tumor regions, which diminished with advanced stages. Increased CD56+ cell infiltration in the tumor core was associated with recurrence. Tumor region showed elevated PD-1, NKG2A, and CD39 expression in CD4+ and CD8+ T cells, indicating progressive immune exhaustion. Advanced HCC stages demonstrated altered NK cell phenotypes, with reduced cytotoxic activation (CD16) and increased residency markers (CXCR6/CD69) in tumor-isolated lymphocytes.

Conclusions: Progressive immune exhaustion and dysregulation of lrNK and T cells in HCC reflect the evolution of the immune microenvironment originating in the tumor and leaking into the non-tumoral liver, progressively diminishing the cytotoxic capacity of NK and T cells. CD56+ cell density and immune checkpoint profiles are potential biomarkers for therapeutic response and disease monitoring, underscoring the need for personalized immunotherapy strategies.

Keywords: NK cells; hepatocellular carcinoma; immune exhaustion; lymphocytes; sorafenib.

PubMed Disclaimer

Conflict of interest statement

MR: Employment: Head of Liver Oncology Unit. Hospital Clinic Barcelona. Director of BCLC group at IDIBAPS/CIBEREHD. Consultant or Advisory Role: AstraZeneca, Bayer, BMS, Eli Lilly, Geneos, Ipsen, Merck, Roche, Universal DX, Boston Scientific, Engitix Therapeutics, Parabilis Medicines Inc. Research Funding: Yes ISCIII, CIBER. Speaking: AstraZeneca, Bayer, BMS, Eli Lilly, Gilead, Roche, Biotoscana Farma. Travel support: Astrazeneca, Roche, Bayer, BMS, Lilly, Ipsen. Principal or sub-Investigator of drug under development: Abbvie, BMS, Adaptimmune, Nerviano, Medivir, Bayer, Ipsen, Astrazeneca, Terumo, Incyte, Roche, Boston Scientific, Medivir. Grant Research Support to the institution: Bayer, Ipsen. Educational Support to the institution: Bayer, Astrazeneca, Eisai- Merck MSD, Roche, Ipsen, Lilly, Terumo, BMS, Next, Boston Scientific, Ciscar Medical, Eventy C3 LLC Egypt. MS-Z: Employment: Hospital Clinic Barcelona. Research Funding: Yes ISCIIII; CIBER. Speaking: Bayer, Roche and AstraZeneca. Travel support: Bayer, BTG, MSD-Eisai and Roche. AF: Employment: Hospital Clinic Barcelona. Consultant or Advisory Role: AstraZeneca, Roche, Boston Scientific. Research Funding: Yes ISCIIII. Speaking: Boston Scientific, Terumo, Roche and AstraZeneca. Travel support: Boston Scientific, AstraZeneca and Roche. NL: Consultant or Advisory Role: Bayer, AstraZeneca, Universal DX. Speaking: Roche, AstraZeneca. Educational Support: Congress fees: Eisai, Travel fees: Bayer. The remaining 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. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1
Representative IHC from eHCC (A) whole tissue slide displaying CD68 positive staining in tumor, peritumor and non-tumor eHCC; (B) inset amplification of CD163 staining in tumor, peritumor and non-tumor eHCC; and (C) PD-L1 positive staining in tumoral and peritumoral area.
Figure 2
Figure 2
Expression of DNAM-1, CD96 and TIGIT in eHCC patient’s liver-isolated lymphocytes. (A) DNAM-1, (B) CD96 and (C) TIGIT expression was assessed on T and NK TILs and snTILs of eHCC patients (n=14). Lymph, Lymphocytes; eHCC, early HCC patients; snTILs, surrounding non-tumor isolated lymphocytes; TILs, tumor isolated lymphocytes.
Figure 3
Figure 3
Differences between TILs and snTILs of all groups of patients. TILs from all groups of patients had increased amounts of (A) T CD4+ cells and (B) regulatory T cells (Treg). PD-1 expression is increased in tumor (C) T CD4+ and (D) CD8+ cells, compared to non-tumor, in eHCC patients, but not in advanced patients (n = 25). Lymph, Lymphocytes; eHCC, early HCC patients; aHCC, advanced HCC patients; aHCCbp, advanced HCC patients beyond progression; snTILs, surrounding non-tumor isolated lymphocytes; TILs, tumor isolated lymphocytes.
Figure 4
Figure 4
Differences in immune checkpoints expression between TILs and snTILs of eHCC patients. NKdim and NKdim-like CD3+ cells isolated from tumor samples of eHCC patients had higher expression of (A) NKG2A (n=13) and (B) CD96 (n=11) compared to their non-tumor counterparts. Both T CD4+ and CD8+ cells isolated from tumor contained higher amounts of (C) CD39+ cells (n=9). Lymph, Lymphocytes; eHCC, early HCC patients; MFI, Mean Fluorescence Intesity; snTILs, surrounding non-tumor isolated lymphocytes; TILs, tumor isolated lymphocytes.
Figure 5
Figure 5
Liver resident NK cells are more abundant in non-tumor tissues and decrease with progression. The presence of lrNK cells was assessed using CXCR6/CD69. (A) eHCC (n=11) snTILs exhibit higher numbers of NKdim and NKbr liver-resident cells. (B) In aHCC patients (n=9), this difference is observed specifically in NKdim cells. However, (C) in aHCCbp patients (n=15), no significant differences in lrNK cells are found between snTILs and TILs. Lymph, Lymphocytes; eHCC, early HCC patients; aHCC, advanced HCC patients; aHCCbp, advanced beyond progression HCC patients; snTILs, surrounding non-tumor isolated lymphocytes; TILs, tumor isolated lymphocytes.
Figure 6
Figure 6
Decrease of cytotoxic lymphocytes and increase of immune exhaustion in snTILs of aHCC patients. (A) Percentage of CD8+ and NKdim cells isolated from non-tumor tissues of eHCC (n=25), aHCC (n=22) and aHCCbp (n=15) patients. Percentage of (B) T CD4+ cells and NK-likedim CD3+ PD-1+ cells, (C) CD16+ cells and (D) NKbr CD69+ cells. eHCC, early HCC patients; aHCC, advanced HCC patients; aHCCbp, advanced HCC patients beyond progression; snTILs, surrounding non-tumor isolated lymphocytes; TILs, tumor isolated lymphocytes; NKbr, NKbright.
Figure 7
Figure 7
Changes in tumor immune cells of aHCC patients with progression. Tumor NK cells further change their (A) CD3+/CD3- balance (aHCC/aHCCbp, n=22/15), (B) NKG2D expression (n=3/6), (C) CD69/CXCR6 expression (n=20/9) and (D) CD16 expression (n=21/12) with progression. aHCC, advanced HCC patients; aHCCbp, advanced HCC patients beyond progression; TILs, tumor isolated lymphocytes; MFI, Mean Fluorescence Intensity.
Figure 8
Figure 8
Liver-resident T CXCR6+CD69+ cells have a differential immune checkpoint expression pattern. Liver-resident T CXCR6+CD69+ cells from eHCC patients have lower (A) DNAM-1 expression in the tumor area (n=9). In the surrounding non-tumor area, (B) PD-1, (C) TIM-3, and (D) LAG-3 expression is higher in liver resident T cells across all HCC groups (eHCC/aHHC-nt/aHCCbp, n=10/22/9), as compared to non-resident liver T cells, with higher presence of immune exhaustion markers displayed in lymphocytes from advanced patients. Lymph, Lymphocytes; eHCC, early HCC patients; aHCC, advanced HCC patients; aHCCbp, advanced HCC patients beyond progression; snTILs, surrounding non-tumor isolated lymphocytes; TILs, tumor isolated lymphocytes; MFI, Mean Fluorescence Intensity.
Figure 9
Figure 9
Immune evolution and clinical implications across hepatocellular carcinoma (HCC) progression. Early HCC: Characterized by preserved liver-resident NK (IrNK) cells (CXCR6-CD69+) and low expression of exhaustion markers (PD-1, NKG2A, CD39) in both NK and T cells. This immunologically active profile aligns with curative-intent treatments such as surgical resection or local ablation. If immunotherapy is considered, anti-PD-1 monotherapy may be appropriate at this stage. Advanced HCC (aHCC): Marked by a decline in lrNK cells and increased expression of checkpoint receptors on both NK and T cells, indicating immune dysfunction. Patients typically receive systemic therapies such as tyrosine kinase inhibitors (TKIs) or immune checkpoint inhibitors (ICIs). Combination immunotherapy (e.g., anti-PD-1 plus another ICI, such as anti- NKG2A) may be more effective in addressing emerging exhaustion phenotypes. Advanced HCC beyond progression (aHCCbp): Immune exhaustion is pronounced, with severe reduction in cytotoxic markers and expansion of dysfunctional NK-like cells. These patients are often treatment-refractory and may benefit from clinical trials exploring novel immunotherapeutic strategies and/or strategies to restore NK/T cell function.
See this image and copyright information in PMC

References

    1. Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer. (2012) 12:252. doi: 10.1038/nrc3239 - DOI - PMC - PubMed
    1. Ventola CL. Cancer immunotherapy, part 1: current strategies and agents. P T Peer-Rev J Formul Manage. (2017) 42:375–83. - PMC - PubMed
    1. Arafat Hossain M. A comprehensive review of immune checkpoint inhibitors for cancer treatment. Int Immunopharmacol. (2024) 143:113365. doi: 10.1016/j.intimp.2024.113365 - DOI - PubMed
    1. European Association for the Study of the Liver . Electronic address: easloffice@easloffice.eu; European Association for the Study of the Liver EASL Clinical Practice Guidelines on the Management of Hepatocellular Carcinoma. J Hepatol. (2025) 82:315–74. doi: 10.1016/j.jhep.2024.08.028 - DOI - PubMed
    1. Fortuny M, García-Calonge M, Arrabal O, Sanduzzi-Zamparelli M, Castano-Garcia A, Gemma Iserte G, et al. Profile of immune-related adverse events and treatment outcomes in first-line immunotherapy for hepatocellular carcinoma. European Association for the Study of the Liver: Liver Cancer Summit; (2024).