We try to study the immune cell training in our project. While the primary human immune cell has a limited life which is hard to be employed in some medical applications, thus we try to extend the lifespan of cells and then train the immune cells.

Cell immortalization can be achieved by activating human telomerase reverse transcriptase (hTERT) to maintain sufficient telomere lengths to avoid replicative senescence. The conventional methods such as overexpressing SV40T antigens or suppressing tumor suppressor genes, will randomly integrate the hTERT transgene into the genome via viral vectors and then lead to insertional mutagenesis and genome instability. To circumvent these issues, we utilize CRISPR/dCas9-based epigenetic modifiers (p300 histone acetyltransferase and TET1 DNA demethylase) and transcriptional activators (VPH and VPR) to activate the endogenous TERT gene in unstimulated T cells in the peripheral blood mononuclear cells (PBMCs) by rewiring the epigenetic marks of the TERT promoter. Importantly, we have successfully expanded resting T cells and delayed their cellular senescence for at least half a year through TERT reactivation, without affecting the function of T lymphocytes or inducing malignant transformation. We have also demonstrated the effectiveness of these CRISPR tools in HEK293FT and THP-1-derived macrophages. We compare the proliferation rate and cell cycle state with Jurkat cells (T lymphoblast isolated from an acute T-cell leukemia patient) to further prove that our immortalized cell lines have the same characterization as primary cells.

We know the Extended lifespan of immune cells may help maintain long-term immune memory. Long-lived immune cells can continue to survive and maintain the memory of previously exposed antigens, providing longer-lasting immune protection. In addition, the Extended lifespan of immune cells may lead to more durable immune responses. This may manifest as longer periods of cellular activation and secretion of immune factors, thereby enhancing the immune response to infection or disease.

After modifying the immune cells, we try to improve the ability of immune cells. We utilized the microfluidic chip to pair the immune cells with cancer cells. To be specific, we employed a microfluidic chip to force the membrane contact and detect cytokine secretion to further prove the immune response of immune cells.

The microfluidic chip can be utilized in the temporal and spatial control of cells, which enables to mimicking of the truth of cellular interaction and cellular microenvironment. In this project, we co-culture between immune cells (monocyte and monocyte-derived macrophage) and breast cancer cells (MDA-MB-231). We evaluated immune cell function by two applications: high-throughput longitudinal secretory profiling of single cell line, and integrated macrophage-tumor cell interactions. We established a microfluidic chip based on a trap array to exert precise control over the positioning of single cells for cell pairing and a microsieve array for dynamic profiling of cytokine secretion by capturing cytometric beads (CBA). We quantify the fluorescence signal from these microbeads to measure the cytokine concentration. Our results proved that co-culture is an effective way to promote the immune response and cytokine secretion. These measurements of secretion from single cells provided a new biological and technological insight into cancer immunotherapy.