CD3/CD28 costimulation-induced NF-KB activation, is not mediated by protein metallothionein 2A and FAS associated death domain

Abstract

Objectives: The immune response depends on T cell activation, which is triggered by signals from receptors such as CD28 and TCR/CD3, resulting in T cell proliferation and programmed cell death (AICD). This control avoids disorders like immunity and cancer. These receptors are stimulated by Antigen-Presenting Cells (APCs), which set off a signaling cascade that activates the important transcription factor NF-κB. Degrading inhibitory proteins is necessary for NF-κB activation, which permits it to reach the nucleus and regulate gene expression. Methods: Semi-quantitative RT-PCR was used to assess the levels of metallothionein 2A mRNA in primary T cells to validate the results of the microarray analysis. Five- to six-week-old male C57BL/6 wild-type mice were used in the investigation. Mouse primary T cells from lymph nodes were suspended aseptically, and anti-CD3/CD28 was used to activate the cells. The cells were transfected with plasmid DNA using a Gen Pulser, and the T cells were separated by magnetic cell sorting. Following the synthesis of cDNA from total RNA, microarray analysis was used to assess variations in gene expression. The microarray results were validated by RT-PCR. Western blotting was used to confirm protein expression, and flow cytometry with CD69 was used to measure cell death. Immunoreactive bands were visible in co-immunoprecipitation assays using monoclonal anti-MT2A and FADD antibodies. Results: This work focused on FAS-associated Death Domain (FADD), MT2A, and NF-κB gene expression profiles in mouse primary T cells before and after anti-CD3/CD28 stimulation, utilizing microarray analysis. The results shed light on these genes' functions in AICD and T cell activation. The cascade triggers the activation of transcription factors necessary for T cell proliferation and cytokine production, such as NF-κB. Degradation of inhibitory IκB proteins is necessary for NF-κB activation, which permits NF-κB to reach the nucleus and control gene transcription. The involvement of PLC-γ1 in CD3/CD28-induced NF-κB activation has been highlighted by recent studies. Conclusion: The processes that determine whether T cells divide or undergo apoptosis are still unknown, despite advances. To compare the gene expression of FADD, MT2A, and NF-κB in mouse primary T cells before and after anti-CD3/ CD28 stimulation, this work used microarray analysis. The purpose of our research is to shed light on these genes' functions in T cell activation and activation-induced cell death (AICD).