Phytohemagglutinin-Induced NF-κB/MAPK Activation Stimulates a Broad Immune Checkpoint-Positive T Cell Model

Jurkat cells serve as alternative cellular models to costly transgenic mice for immune checkpoint antagonist toxicology studies but require activation to express immune checkpoints. Although phytohemagglutinin (PHA) activates Jurkat cells and upregulates several checkpoints, its effects on broad-spectrum checkpoint expression and underlying mechanisms remain unclear. This study aimed to investigate PHA-induced broad-spectrum immune checkpoint expression in Jurkat cells and the underlying signaling mechanisms to establish an optimized <i>in vitro</i> model for toxicological evaluations of immune checkpoint modulators. The cytotoxic effects of PHA on Jurkat cells were evaluated by Cell Counting Kit-8 (CCK-8) assay, which yielded IC50 values of 40.41, 32.04, and 21.06 μg/mL at 24, 48, and 72 h, respectively. Transcriptional changes in immune checkpoint genes were assessed via RT-qPCR, revealing that PHA stimulation upregulated mRNA levels of multiple co-inhibitory and co-stimulatory immune checkpoints by 1.3 to 10 fold. Concurrently, flow cytometry analysis demonstrated significantly increased secretion of IL-2, IL-8, IL-10, and TNF-α. Western blot analysis showed that PHA treatment enhanced the phosphorylation of IKKα/β, IκBα, p65, JNK, ERK, and p38 by 1.2 to 3.5 fold, indicating activation of both NF-κB and MAPK signaling pathways. This was accompanied by the elevated expression of immune checkpoint proteins, which persisted even at 72 h post-PHA removal. Pharmacological inhibition of these pathways attenuated PHA-induced immune checkpoint upregulation, with immunofluorescence results corroborating these findings. We demonstrate PHA dose-dependently upregulates broad-spectrum immune checkpoints in Jurkat cells via NF-κB/MAPK activation, establishing an optimized model for <i>in vitro</i> toxicological and mechanistic studies of immune checkpoint-targeting therapeutics.