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Quantitative analysis of lymphocyte exhaustion markers in patients with localized clear cell renal cell carcinoma

  • Authors:
    • Raquel González‑Garza
    • Adrián Gutiérrez‑González
    • Nallely López‑López
    • Raquel Garza‑Guajardo
    • Juan Pablo Flores‑Gutiérrez
    • Mario César Salinas‑Carmona
    • Víctor Manuel Oyervides‑Juárez
    • Marco Alberto Ocaña‑Munguía
    • José Héctor Elizondo‑Jasso
    • Manuel Mejía‑Torres
  • View Affiliations / Copyright

    Affiliations: Service of Immunology, University Hospital ‘José Eleuterio González’, Autonomous University of Nuevo León, Monterrey, Nuevo León 64460, Mexico, Service of Urology, University Hospital ‘José Eleuterio González,’ Autonomous University of Nuevo León, Monterrey, Nuevo León 64460, Mexico, Service of Pathology, University Hospital ‘José Eleuterio González’, Autonomous University of Nuevo León, Monterrey, Nuevo León 64460, Mexico, Service of Oncology, University Hospital ‘José Eleuterio González’, Autonomous University of Nuevo León, Monterrey, Nuevo León 64460, Mexico
    Copyright: © González‑Garza et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 499
    |
    Published online on: August 27, 2025
       https://doi.org/10.3892/ol.2025.15245
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Abstract

 Clear cell renal cell carcinoma (ccRCC) is a neoplastic disease associated with poor prognosis. Localized disease is successfully treated with nephrectomy; however, advanced disease often requires the combined use of immunotherapy and targeted therapy. To the best of our knowledge, there is no validated method to predict immunotherapy response and there is a lack of knowledge regarding the expression kinetics of exhaustion receptors in the early stages of ccRCC. In the present study, immunohistochemistry and flow cytometry were performed to evaluate the expression levels of five biomarkers associated with immune dysfunction [programmed cell death protein‑1 (PD‑1), lymphocyte activation gene 3 (LAG‑3), T‑cell immunoglobulin and mucin domain containing‑3 (TIM‑3), cytotoxic T lymphocyte‑­associated protein‑4 (CTLA‑4) and programmed death ligand‑1 (PD‑L1)] in 20 patients diagnosed with localized ccRCC. Immunohistochemistry demonstrated that ccRCC tissue was highly positive for CD3, with low expression of PD‑L1 and CTLA‑4. Based on flow cytometry, the infiltrating leukocytes were mostly CD8+ lymphocytes and monocytes. In tumors, decreased expression levels of LAG‑3, TIM‑3, CTLA‑4 and PD‑L1 were reported; however, expression levels of PD‑1 remained unchanged. Sample stratification according to the nuclear grade demonstrated no changes in the evaluated markers. Finally, the 12‑month follow‑up of blood biomarkers revealed no change in the receptors, except for CTLA‑4. The results of the present study suggested that immune exhaustion, defined as increased expression of exhaustion receptors in lymphocytes, was not present in tissues from patients with ccRCC with localized disease. 
View Figures

Figure 1

ccRCC tumors are highly positive for
CD3 but mostly negative for CTLA and PD-L1. The tumor tissue from
20 patients was analyzed for (A) CD3, (B) CTLA-4 and (C) PD-L1
expression using immunohistochemistry. The percentage of positive
tissue was quantified using two approaches: Clinical analysis by a
group of pathologists and digital analysis from scanned slides
using QuPath software. ccRCC, clear cell renal cell carcinoma;
PD-L1, programmed death-ligand 1; PD-1, programmed cell death
protein-1; LAG-3, lymphocyte activation gene-3; TIM-3, T-cell
immunoglobulin and mucin domain-containing protein-3; CTLA-4,
cytotoxic T lymphocyte-associated protein-4.

Figure 2

In tumor tissue, cellularity is
dominated by CD8+ lymphocytes and monocytes. The
percentage of leukocytes (A) and lymphocytes (B) were evaluated in
the blood and tumors using flow cytometry. For blood, there were no
differences between healthy controls (control) and patients
(blood). In tumors, there was an increased percentage of monocytes
and CD8+ lymphocytes, a decreased percentage of
granulocytes and almost no CD4+ lymphocytes, compared
with blood samples. Mean ± SD n=10 (control); n=14-21 (blood and
tumor). One-way ANOVA with Bonferroni's post hoc test. *P<0.05;
**P<0.01; ***P<0.001. ccRCC, clear cell renal cell carcinoma;
Gran, granulocytes; Mono, monocytes; Lymph, lymphocytes.

Figure 3

Compared with circulating leukocytes,
tumor-infiltrating leukocytes exhibit decreased expression of the
exhaustion receptors LAG-3, TIM-3, CTLA-4 and PD-L1 based on flow
cytometry. All receptors were evaluated in blood leukocytes from
healthy controls (control), blood leukocytes from patients (blood)
and tumor-infiltrating leukocytes (tumor). PD-1 was evaluated in
(A) CD3+, (B) CD4+ and (C) CD8+
lymphocytes. LAG-3 was evaluated in (D) CD3+, (E)
CD4+ and (F) CD8+ lymphocytes. TIM-3 was
evaluated in (G) CD3+, (H) CD4+ and (I)
CD8+ lymphocytes. CTLA-4 was evaluated in (J)
CD3+, (K) CD4+ and (L) CD8+
lymphocytes. PD-L1 was evaluated in (M) granulocytes and (N)
monocytes. The tumor-infiltrating leukocytes exhibited decreased
expression levels of LAG-3, TIM-3, CTLA-4 and PD-L1 compared with
blood leukocytes, but no changes were observed in PD-1 expression.
Data are expressed as MFI ± SD. Control group, n=10; blood group,
n=7-12; tumor group, n=18. Data were analyzed using one-way ANOVA
with Bonferroni's post hoc test (except panels B, E, H and K, which
were analyzed using an unpaired Student's t-test). *P<0.05;
**P<0.01; ***P<0.001. PD-L1, programmed death-ligand 1; PD-1,
programmed cell death protein-1; LAG-3, lymphocyte activation
gene-3; TIM-3, T-cell immunoglobulin and mucin domain-containing
protein-3; CTLA-4, cytotoxic T lymphocyte-associated protein-4;
MFI, mean fluorescence intensity.
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Copy and paste a formatted citation
Spandidos Publications style
González‑Garza R, Gutiérrez‑González A, López‑López N, Garza‑Guajardo R, Flores‑Gutiérrez JP, Salinas‑Carmona MC, Oyervides‑Juárez VM, Ocaña‑Munguía MA, Elizondo‑Jasso JH, Mejía‑Torres M, Mejía‑Torres M, et al: Quantitative analysis of lymphocyte exhaustion markers in patients with localized clear cell renal cell carcinoma. Oncol Lett 30: 499, 2025.
APA
González‑Garza, R., Gutiérrez‑González, A., López‑López, N., Garza‑Guajardo, R., Flores‑Gutiérrez, J.P., Salinas‑Carmona, M.C. ... Mejía‑Torres, M. (2025). Quantitative analysis of lymphocyte exhaustion markers in patients with localized clear cell renal cell carcinoma. Oncology Letters, 30, 499. https://doi.org/10.3892/ol.2025.15245
MLA
González‑Garza, R., Gutiérrez‑González, A., López‑López, N., Garza‑Guajardo, R., Flores‑Gutiérrez, J. P., Salinas‑Carmona, M. C., Oyervides‑Juárez, V. M., Ocaña‑Munguía, M. A., Elizondo‑Jasso, J. H., Mejía‑Torres, M."Quantitative analysis of lymphocyte exhaustion markers in patients with localized clear cell renal cell carcinoma". Oncology Letters 30.5 (2025): 499.
Chicago
González‑Garza, R., Gutiérrez‑González, A., López‑López, N., Garza‑Guajardo, R., Flores‑Gutiérrez, J. P., Salinas‑Carmona, M. C., Oyervides‑Juárez, V. M., Ocaña‑Munguía, M. A., Elizondo‑Jasso, J. H., Mejía‑Torres, M."Quantitative analysis of lymphocyte exhaustion markers in patients with localized clear cell renal cell carcinoma". Oncology Letters 30, no. 5 (2025): 499. https://doi.org/10.3892/ol.2025.15245
Copy and paste a formatted citation
x
Spandidos Publications style
González‑Garza R, Gutiérrez‑González A, López‑López N, Garza‑Guajardo R, Flores‑Gutiérrez JP, Salinas‑Carmona MC, Oyervides‑Juárez VM, Ocaña‑Munguía MA, Elizondo‑Jasso JH, Mejía‑Torres M, Mejía‑Torres M, et al: Quantitative analysis of lymphocyte exhaustion markers in patients with localized clear cell renal cell carcinoma. Oncol Lett 30: 499, 2025.
APA
González‑Garza, R., Gutiérrez‑González, A., López‑López, N., Garza‑Guajardo, R., Flores‑Gutiérrez, J.P., Salinas‑Carmona, M.C. ... Mejía‑Torres, M. (2025). Quantitative analysis of lymphocyte exhaustion markers in patients with localized clear cell renal cell carcinoma. Oncology Letters, 30, 499. https://doi.org/10.3892/ol.2025.15245
MLA
González‑Garza, R., Gutiérrez‑González, A., López‑López, N., Garza‑Guajardo, R., Flores‑Gutiérrez, J. P., Salinas‑Carmona, M. C., Oyervides‑Juárez, V. M., Ocaña‑Munguía, M. A., Elizondo‑Jasso, J. H., Mejía‑Torres, M."Quantitative analysis of lymphocyte exhaustion markers in patients with localized clear cell renal cell carcinoma". Oncology Letters 30.5 (2025): 499.
Chicago
González‑Garza, R., Gutiérrez‑González, A., López‑López, N., Garza‑Guajardo, R., Flores‑Gutiérrez, J. P., Salinas‑Carmona, M. C., Oyervides‑Juárez, V. M., Ocaña‑Munguía, M. A., Elizondo‑Jasso, J. H., Mejía‑Torres, M."Quantitative analysis of lymphocyte exhaustion markers in patients with localized clear cell renal cell carcinoma". Oncology Letters 30, no. 5 (2025): 499. https://doi.org/10.3892/ol.2025.15245
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