Objectives <p>T lymphocytes (T cells) are crucial important types of immune cells which are responsible for adaptive immune response during the progression of bacterial infectious diseases. Different subtypes of T cells can participate in regulating the progress of human periapical lesions. This study investigates the co-localization of different types of T cells by multiplex immunofluorescence staining (mIHC) from different types of human periapical lesions (PLs) which might elucidate the potential clinical relevance of T cell infiltration with PLs.</p> Materials and methods <p>20 PLs tissues and 6 healthy samples from healthy periodontal ligament tissues were collected for subsequent experiments. Histological examination and immunohistochemical analysis were performed using hematoxylin-eosin (H&amp;E) staining, immunohistochemical staining (IHC) and mIHC. Data analysis involved quantifying the number of positive cells of CD3, CD4, GATA3, Tbet and Foxp3 using QuPath software. In addition, the co-localization of CD4, GATA3, Tbet and Foxp3 was also assessed.</p> Results <p>Radicular cysts (RCs) (<i>n</i> = 10) and periapical granulomas (PGs) (<i>n</i> = 10) were identified by histological evaluation. The number of CD3⁺ cells in RCs was significantly higher compared to PGs and healthy controls. Additionally, the infiltration numbers of CD3, CD4, GATA3, T-bet and Foxp3 cells were greater in RCs than in PGs.</p> Conclusions <p>This study demonstrates the different expression levels of T cells in PLs and indicates the distinct inflammatory states between the two types of PLs.</p>