Michelin Marcia A, Montes Leticia, Nomelini Rosekeila S, Abdalla Douglas R, Aleixo Andre A R, Murta Eddie F C
Oncology Research Institute (IPON), Federal University of Triângulo Mineiro, Uberaba, Minas Gerais 38025-440, Brazil ; Discipline of Immunology, Federal University of Triângulo Mineiro, Uberaba, Minas Gerais 38025-440, Brazil.
Oncology Research Institute (IPON), Federal University of Triângulo Mineiro, Uberaba, Minas Gerais 38025-440, Brazil.
Oncol Lett. 2015 Sep;10(3):1523-1526. doi: 10.3892/ol.2015.3470. Epub 2015 Jul 8.
The production of cytokines by helper T lymphocytes is a critical event in the immune response, as alterations in the regulation of this process may result in an appropriate immune response, persistent infection or the development of autoimmune disease. Previously, this group has used flow cytometry to demonstrate the expression of interleukin-12 (IL-12) in peripheral blood CD4+ T lymphocytes from patients and mice with advanced cancer. The aim of the present study was to investigate whether CD4+ T lymphocytes from the peripheral blood (PB) of patients with cancer produce IL-12, using molecular approaches, flow cytometry and cellular imaging techniques. CD3+ and CD4+ cells, and cells producing IL-12, were isolated from the PB obtained from patients with cancer, using a cell sorting flow cytometry technique. The positivity of cells for CD3, CD4 and IL-12, which were identified by cell sorting, was visualized using immunofluorescent cellular imaging. Total RNA was extracted from the CD3+CD4+IL-12+ cells, obtained by cell sorting, for confirmation of the presence of IL-12 mRNA, using reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR demonstrated the presence of IL-12 mRNA in all patients (n=14), in contrast to the control group, in whom IL-12 expression was not detected. Immunofluorescent analysis of CD4+ T lymphocytes showed positive intracytoplasmatic IL-12 staining. These results demonstrated that CD3+CD4+ T lymphocytes in the PB of patients with cancer have the capacity to synthesize and express IL-12.
辅助性T淋巴细胞产生细胞因子是免疫反应中的关键事件，因为这一过程调控的改变可能导致免疫反应不当、持续感染或自身免疫性疾病的发生。此前，该团队利用流式细胞术证明了晚期癌症患者和小鼠外周血CD4⁺ T淋巴细胞中白细胞介素12（IL - 12）的表达。本研究的目的是使用分子方法、流式细胞术和细胞成像技术，研究癌症患者外周血（PB）中的CD4⁺ T淋巴细胞是否产生IL - 12。使用细胞分选流式细胞术从癌症患者的PB中分离出CD3⁺和CD4⁺细胞以及产生IL - 12的细胞。通过细胞分选鉴定的CD3、CD4和IL - 12阳性细胞，使用免疫荧光细胞成像进行可视化。从通过细胞分选获得的CD3⁺CD4⁺IL - 12⁺细胞中提取总RNA，使用逆转录 - 聚合酶链反应（RT - PCR）来确认IL - 12 mRNA的存在。RT - PCR证明所有患者（n = 14）中均存在IL - 12 mRNA，而对照组未检测到IL - 12表达。对CD4⁺ T淋巴细胞的免疫荧光分析显示胞质内IL - 12染色呈阳性。这些结果表明，癌症患者PB中的CD3⁺CD4⁺ T淋巴细胞具有合成和表达IL - 12的能力。
