Volume: 56 Issue: 4
Year: 2025, Page: 650-658, Doi: https://doi.org/10.51966/jvas.2025.56.4.650-658
Received: Sept. 11, 2025 Accepted: Oct. 23, 2025 Published: Dec. 31, 2025
Tumour-infiltrating lymphocytes (TILs) are critical components of the tumour microenvironment (TME), comprising T cells, B cells and natural killer cells that influence tumour progression and clinical outcome. Among these, CD4+ and CD8+ T cells play pivotal roles, with CD4+ cells regulating immune responses and CD8+ cells mediating cytotoxic effects. Their distribution between stromal and intratumoural regions holds prognostic significance. The objective of the present study was to investigate the localisation of TILs in relation to histological grade, as well as to evaluate the prognostic relevance of the CD4+/CD8+ ratio. Immunohistochemistry was performed on 25 tumour samples, which revealed abundant CD4+ and CD8+ TILs in grade I tumours, which declined as grade advanced, whereas CD4+ T cells showed a slight predominance in high-grade tumours. Lymphocytes were primarily confined to stromal regions, followed by peritumoural areas, while intratumoural localisation was consistently sparse. Importantly, the CD4+/CD8+ ratio, increased with tumour grade, reflecting a relative predominance of CD4+ T cells in high-grade tumours and suggesting progression towards an immunosuppressive microenvironment. Kaplan–Meier survival analysis revealed that dogs with a low CD4+/CD8+ratio exhibited more favourable overall survival compared to those with a higher ratio (p< 0.05). The ratio between helper and cytotoxic T lymphocytes within the TME serves as an important determinant of disease outcome.
Keywords: Canine mammary tumours, tumour infiltrating lymphocytes, immunohistochemistry, CD4+/CD8+ ratio
Astuti, A., Miskad, U.A., Arsyadi, G., Zainuddin, A.A., Wahid, S. and Nelwan, B.J. 2022. CD8⁺ TILs expression in invasive breast carcinoma (no special type). Asian Pac. J. Cancer Prev. 23: 1813.
Bhimani, H., Parikh, P.V., Barot, H.M., Mahla, J.K. and Sodagar, B.P. 2024. Epidemiology of canine mammary gland tumours. Int. J. Vet. Sci. Anim. Husb. 9: 954-956.
Burrai, G.P., Baldassarre, V., Brunetti, B., Iussich, S., Maniscalco, L., Mariotti, F., Sfacteria, A., Cocumelli, C., Grieco, V., Millanta, F. and Paciello, O. 2022. Canine and feline in situ mammary carcinoma: A comparative review. Vet. Pathol. 59: 894-902.
Carvalho, M.I., Pires, I., Prada, J. and Queiroga, F.L. 2014. A role for T lymphocytes in human breast cancer and in canine mammary tumours. Biomed. Res. Int. 2014: 130894.
Chu, P.Y., Yeh, C.M., Yeh, K.T. and Liu, C.H. 2012. Tubulopapillary carcinoma: A frequent over-diagnosed entity in canine mammary gland tumours. Taiwan Vet. J. 38: 67-74
Chung, S.W., Xie, Y. and Suk, J.S. 2021. Overcoming physical stromal barriers to cancer immunotherapy. Drug Deliv. Transl. Res. 11: 2430-2447.
Clemente, M., Perez-Alenza, M.D., Illera, J.C. and Peña, L. 2010. Histological, immunohistological and ultrastructural description of vasculogenic mimicry in canine mammary cancer. Vet. Pathol. 47: 265-274.
Dhein, E.S., Heikkilä, U., Oevermann, A., Blatter, S., Meier, D., Hartnack, S. and Guscetti, F. 2024. Incidence rates of the most common canine tumours based on data from the Swiss Canine Cancer Registry (2008–2020). PLoS One. 19: e0302231.
Dolka, I., Czopowicz, M., Gruk-Jurka, A., Wojtkowska, A., Sapierzyński, R. and Jurka, P. 2018. Diagnostic efficacy of smear cytology and Robinson’s cytological grading of canine mammary tumours with respect to histopathology, cytomorphometry, metastases and overall survival. PLoS One. 13: e0191595.
Dias-Pereira, P. 2022. Morbidity and mortality in elderly dogs: A model for human ageing. BMC Vet. Res. 18: 457.
Dolina, J.S., Van Braeckel-Budimir, N., Thomas, G.D. and Salek-Ardakani, S. 2021. CD8⁺ T cell exhaustion in cancer. Front. Immunol. 12: 715234.
Estrela-Lima, A., Araújo, M.S., Costa-Neto, J.M., Teixeira-Carvalho, A., Barrouin-Melo, S.M., Cardoso, S.V. and Cassali, G.D. 2010. Immunophenotypic features of tumour infiltrating lymphocytes from mammary carcinomas in female dogs associated with prognostic factors and survival rates. BMC Cancer. 10: 256.
Flecher, M.C., Reys, M.P., Balabram, D., Nakagaki, K.Y. and Cassali, G.D. 2025. Tumour-infiltrating lymphocytes and neutrophils and their location in canine mammary neoplasms with a solid arrangement: A prognostic factor? Animals. 15: 287.
Fridman, W.H., Pagès, F., Sautès-Fridman, C. and Galon, J. 2012. The immune contexture in human tumours: Impact on clinical outcome. Nat. Rev. Cancer. 12: 298-306.
Goda, N., Sasada, S., Shigematsu, H., Masumoto, N., Arihiro, K., Nishikawa, H., Sakaguchi, S., Okada, M. and Kadoya, T. 2022. The ratio of CD8⁺ lymphocytes to tumour-infiltrating suppressive FOXP3⁺ regulatory T cells is associated with treatment response in invasive breast cancer. Discov. Oncol. 13: 82-95.
Goldschmidt, M., Peña, L., Rasotto, R. and Zappulli, V. 2011. Classification and grading of canine mammary tumours. Vet. Pathol. 48: 117-131.
Huang, Y., Ma, C., Zhang, Q., Ye, J., Wang, F., Zhang, Y. and Peng, G. 2015. CD4⁺ and CD8⁺ T cells have opposing roles in breast cancer progression and outcome. Oncotarget. 6: 17462.
Kim, J.H., Chon, S.K., Im, K.S., Kim, N.H. and Sur, J.H. 2013. Correlation of tumour-infiltrating lymphocytes to histopathological features and molecular phenotypes in canine mammary carcinoma: A morphologic and immunohistochemical morphometric study. Can. J. Vet. Res. 77: 142-149.
Koletsa, T., Kotoula, V., Koliou, G.A., Manousou, K., Chrisafi, S., Zagouri, F., Sotiropoulou, M., Pentheroudakis, G., Papoudou-Bai, A., Christodoulou, C., Xepapadakis, G., Zografos, G.C., Petraki, K., Pazarli, E., Koutras, A., Kourea, H.P., Bafaloukos, D., Chatzopoulos, K., Iliadis, A. and Fountzilas, G. 2020. Prognostic impact of stromal and intratumoural CD3, CD8 and FOXP3 in adjuvantly treated breast cancer: Do they add information over stromal tumour-infiltrating lymphocyte density? Cancer Immunol. Immunother. 69: 1549-1564.
Melssen, M.M., Sheybani, N.D., Leick, K.M. and Slingluff, C.L. 2023. Barriers to immune cell infiltration in tumours. J. Immunother. Cancer. 11: e006401.
Nair, S.S. 2021. Occurrence of canine mammary and skin/subcutaneous neoplasms in and around Thrissur district of Kerala during 2017–2020: A review of 265 cases. J. Vet. Anim. Sci. 52: 350–356.
Rech, A.J., Mick, R., Kaplan, D.E., Chang, K.M., Domchek, S.M. and Vonderheide, R.H. 2010. Homeostasis of peripheral FoxP3⁺ CD4⁺ regulatory T cells in patients with early and late stage breast cancer. Cancer Immunol. Immunother. 59: 599–607.
Rezaie, J., Chodari, L., Mohammadpour-Asl, S., Jafari, A. and Niknam, Z. 2024. Cell-mediated barriers in cancer immunosurveillance. Life Sci. 342: 122528.
Salgado, R., Denkert, C., Demaria, S., Sirtaine, N., Klauschen, F., Pruneri, G. and Loi, S. 2015. The evaluation of tumour-infiltrating lymphocytes (TILs) in breast cancer: Recommendations by an international TILs working group 2014. Ann. Oncol. 26: 259-271.
Śmiech, A., Kowal, K., Różańska, D. and Ślaska, B. 2023. Histopathological evaluation of canine mammary gland tumours: A study of 92 cases. Med. Weter. 79: 6771-2023.
Soudja, S.M., Wehbe, M., Mas, A., Chasson, L., Tenbossche, C.P., Huijbers, I. and Schmitt-Verhulst, A.M. 2010. Tumour-initiated inflammation overrides protective adaptive immunity in an induced melanoma model in mice. Cancer Res. 70: 3515-3525.
Suvarna, K.S., Layton, C. and Bancroft, J.D. 2019. Bancroft’s Theory and Practice of Histological Techniques. (8th Ed.). Amsterdam: Elsevier Health Sciences, Netherlands, 672p.
Vakkala, M., Pääkkö, P. and Soini, Y.J. 1999. Expression of caspases 3, 6 and 8 is increased in parallel with apoptosis and histological aggressiveness of the breast lesion. Br. J. Cancer. 81: 592–599.
Vihervuori, H., Autere, T.A., Repo, H., Kurki, S., Kallio, L., Lintunen, M.M. and Kronqvist, P. 2019. Tumour-infiltrating lymphocytes and CD8⁺ T cells predict survival of triple-negative breast cancer. J. Cancer Res. Clin. Oncol. 145: 3105-3114.
Whiteside, T.L. 2022. Tumour-infiltrating lymphocytes and their role in solid tumour progression. In: Klink, M. and Szulc-Kiełbik, I. (eds.), Interaction of Immune and Cancer Cells. Experientia Supplementum, Vol. 113. Springer, Cham, pp. 89–106.
Yang, X., Ren, H., Sun, Y., Shao, Y., Zhang, L., Li, H. and Fu, J. 2017. Prognostic significance of CD4/CD8 ratio in patients with breast cancer. Int. J. Clin. Exp. Pathol. 10: 4787–4793.
© 2025 Mohan et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Mohan, S.K., Krithiga., K., Sajitha., I.S., Prasanna., K.S., Divya., C., Anoop., S. and Joy, A. 2025. Immunohistochemical evaluation of CD4+ and CD8+ tumour-infiltrating lymphocytes and their prognostic significance in canine mammary tumours. J. Vet. Anim. Sci. 56 (4):650-658