Differential Regulation of Perforin Expression in CD4+ and CD8+ Cytotoxic T Lymphocytes.
Authors: Niiya Hironari , Lei Jin , Sakai Ikuya , Azuma Taichi , Uchida Naoyuki , Yakushijin Yoshihiro , Hato Takaaki , Fujita Shigeru , Yasukawa Masaki 
Published: October 2019

Background: Cytotoxic T lymphocytes (CTLs) play a crucial role in resistance against viral infections and cancer development. The conventional CTLs are MHC class I-restricted CD8+ T lymphocytes; however, MHC class II-restricted CD4+ CTLs directed against alloantigen and various kinds of virus and cancer are also present. We have reported recently that human CD4+ as well as CD8+ CTLs exert antigen-specific cytotoxicity through perforin-dependent cytolytic pathway (Blood95:2352–2355,2000; J. Immunol.170:2205–2213,2003). Although antigen-specific CD4+ CTLs can be generated generally in the in vitro culture systems, their significance in resistance against infections and malignancies in vivo is still obscure. Because perforin is the important cytolytic mediator of human CD4+ as well as CD8+ CTLs, understanding the regulatory mechanisms of perforin expression in CD4+ and CD8+ CTLs seems an important issue. Based on this background, we addressed the question of whether perforin expression is differentially regulated in CD4+ and CD8+ CTLs.

Methods: Herpes simplex virus (HSV)-specific and HLA class II-restriced CD4+ CTL clones and Epstein-Barr virus (EBV)-specific and HLA class I-restricted CD8+ CTL clones were established from healthy individuals. CTL clones were used for experiments after 3 to 5 days of viral antigen stimulation, when they were in activated phase and after 12 to 15 days, when they were in resting phase. The cytotoxic activity of activated and resting CTL clones was measured by standard 51Cr release assays. Perforin mRNA expression levels in CTLs were determined by quantitative RT-PCR. Expression of surface molecules on CTL clones was examined by flow cytometry. To compare the binding activity of STAT to the perforin promoter in CD4+ and CD8+ CTLs, electrophoretic mobility shift assay (EMSA) was performed using a STAT-binding probe.

Results: The degrees of antigen-specific cytotoxicity mediated by CD8+ CTLs were not significantly different in activated and resting phase; however, cytotoxicity of CD4+ CTLs in resting phase appeared to be significantly lower than that in activated phase. Similarly, perforin mRNA expression levels in activated and resting CD8+ CTLs were not significantly different, but activated CD4+ CTLs appeared to express abundant perforin compared to resting CD4+ CTLs. Expression levels of all surface functional molecules except CD25 are not significantly different between activated and resting CD4+ and CD8+ CTL clones. Interestingly, binding activity of STAT5 to promoter for perforin gene appeared to increase in activated CD4+ CTLs compared to that in resting CD4+ CTLs. In contrast, no significant difference of STAT5 binding activity to promoter for perforin gene was detected between activated and resting CD8+ CTLs.

Conclusion: The present study revealed the different control of perforin expression in CD4+ and CD8+ CTLs; that is, perfron is expressed constitutively in CD8+ CTLs but perforin expression in CD4+ CTLs is cell-activation-dependent.