High CD8+ and absence of Foxp3+ T lymphocytes infiltration in gallbladder tumors correlate with prolonged patients survival

Abstract

Background: Gallbladder cancer (GBC), although infrequent in industrialized countries, has high incidence rates in certain world regions, being a leading cause of death among elderly Chilean women. Surgery is the only effective treatment, and a five-year survival rate of advanced-stage patients is less than 10%. Hence, exploring immunotherapy is relevant, although GBC immunogenicity is poorly understood. This study examined the relationship between the host immune response and GBC patient survival based on the presence of tumor-infiltrating lymphocytes at different disease stages.

Methods: Tumor tissues from 80 GBC patients were analyzed by immunohistochemistry for the presence of CD3⁺, CD4⁺, CD8⁺, and Foxp3⁺ T cell populations, and the results were associated with clinical stage and patient survival.

Results: The majority of tumor samples showed CD3⁺ T cell infiltration, which correlated with better prognosis, particularly in advanced disease stages. CD8⁺, but not CD4⁺, T cell infiltration correlated with improved survival, particularly in advanced disease stages. Interestingly, a < 1 CD4⁺/CD8⁺ T cell ratio was related with increased survival. Additionally, the presence of Foxp3⁺ T cells correlated with decreased patient survival, whereas a ≤ 1 Foxp3⁺/CD8⁺ T cell ratio was associated with improved patient survival.

Conclusions: Depending on the disease stage, the presence of CD8⁺ and absence of Foxp3⁺ T cell populations in tumor tissues correlated with improved GBC patient survival, and thus represent potential markers for prognosis and management of advanced disease, and supports testing of immunotherapy.

Keywords: CD8+ T cells; Foxp3+ T cells; Gallbladder cancer; Patient survival; Tumor infiltrating lymphocytes.

Fig. 1

Immune cell infiltration in gallbladder cancer. a Representative photomicrographs of immunohistochemical staining T cells (CD3⁺, CD4⁺, CD8⁺, and Foxp3⁺) within GBC tissues (scale bar, 40 μm). b Quantified distribution of immune cell infiltration by photomicrograph field in GBC tissues. The frequency is displayed as the number of cases. The superimposed black lines show the fitted Gaussian distribution. Gaussian distribution was only found on CD4⁺ T cells (goodness of fit: R² = 0.796). c Quantified distribution of immune cell infiltration in GBC tissues according to GBC stage. Data represent the mean ± SD

Fig. 2

Increased CD3⁺ T cell infiltration in gallbladder cancer tissues correlates with improved late stage patient survival. Kaplan-Meier survival curves for patients with high and low CD3⁺ T cells infiltration in primary tumors based on Sturges’ Rule: (a) All stages; (b) Early stages (0 and I); and (c) Advanced stages (II, IIIA, IIIB, and IV). High CD3⁺ T cell infiltration was significantly associated with prolonged survival in advanced stage patients (log-rank p = 0.03). The survival proportions of patients after GBC diagnosis in a 5-year term are shown to the right of the curves

Fig. 3

Increased CD8⁺ T cell infiltration in gallbladder cancer tissues correlates with improved patient survival. Kaplan-Meier survival curves for patients with high and low CD8⁺ T cell infiltration in primary tumors based on Sturges’ Rule: (a) All stages; (b) Early stages (0 and I); and (c) Advanced stages (II, IIIA, IIIB, and IV). High CD8⁺ T cell infiltration was significantly associated with prolonged survival particularly in advanced stage patients (log-rank p = 0.0004). The survival proportions of patients after GBC diagnosis in a 5-year term are shown to the right of the curves

Fig. 4

Increased Foxp3⁺ T cell infiltration in gallbladder cancer tissues correlates with poor patient survival. Kaplan-Meier survival curves for patients with positive or negative Foxp3⁺ T cell infiltration in primary tumors for (a) All stages; (b) Early stages (0 and I); and (c) Advanced stages (II, IIIA, IIIB, and IV). Negative Foxp3⁺ T cell infiltration was significantly associated with prolonged survival in all stages and early stage patients (log-rank p = 0.03 and 0.04, respectively). The survival proportions of patients after GBC diagnosis in a 5-year term are shown to the right of the curves

Fig. 5

Lower CD4⁺/CD8⁺ and Foxp3⁺/CD8⁺ T cell ratios in gallbladder cancer tissues correlates with improved patient survival. Kaplan-Meier survival curves for patients by (a-c) CD4⁺/CD8⁺ T cell ratio or (d) Foxp3⁺/CD8⁺ T cell ratio in primary tumors for (a, d) All stages; (b) Early stages (0 and I); and (c) Advanced stages (II, IIIA, IIIB, and IV). A ≤ 1 CD4⁺/CD8⁺ T cell ratio was significantly associated with prolonged survival in advanced stage patients (log-rank p = 0.01). A ≤ 1 Foxp3⁺/CD8⁺ T cell ratio was significantly associated with prolonged survival in all stages patients (log-rank p = 0.002). The survival proportions of patients after GBC diagnosis in a 5-year term are shown to the right of the curves