Biology subjects

Karhan, E.

Publications and source records attributed to Karhan, E..

5 recordsLinked to original sources

bioRxiv2024

Engineering programmable CAR-T cells with tunable controls as fail-safe mechanisms during cancer immunotherapy

Chimeric antigen receptor (CAR) T-cell therapies have demonstrated remarkable efficacy in treating various cancers, but significant risks of severe adverse effects remain. In this study, we present a synthetic biology toolbox for engineering CAR-T cells with tunable and controllable features to mitigate these risks. Using this toolbox, we programmed cytotoxic CAR-T cells to be turned on and off using synthetic genes with tetracycline response elements, referred to as iOnCAR and iOffCAR. We achieved temporal regulation of CAR expression using the synthetic Notch receptor system and "AND-gate" programs, which allow for additional control using doxycycline. We also engineered cooperation among T cells through CD4 help to CD8 T cells in targeting cancer cells via synthetic Notch receptors controlled by doxycycline treatment. Additionally, we developed a T cell fratricide system by triggering a genetic "Battle Royale" mechanism that enables CAR-T cells to eliminate each other. In this system, surviving T cells become activated, proliferate, and display high cytotoxicity to target cells. In conclusion, our synthetic biology toolbox provides targeted fail-safe solutions for improving the safety and potency of CAR-T cell therapies by integrating different genetic circuits with a commonly used antibiotic.

immunology↗

bioRxiv2022

Engineering human Mucosal Associated Invariant T (MAIT) cells with chimeric antigen receptors for cancer immunotherapy

Engineering immune cells with chimeric antigen receptors (CAR) is a promising technology in cancer immunotherapy. Besides classical cytotoxic CD8+ T cells, innate cell types such as NK cells have also been used to generate CAR-T or CAR-NK cells. Here we devised an approach to program a non-classical cytotoxic T cell subset called Mucosal Associated Invariant T (MAIT) cells into effective CAR-T cells against B cell lymphoma and breast cancer cells. Accordingly, we expressed anti-CD19 and anti-Her2 CARs in activated primary human MAIT cells and CD8+ T cells, expanded them in vitro and compared their cytotoxicity against tumor cell targets. We show upon activation through CARs, CAR-MAIT cells exhibit high levels of cytotoxicity towards target cells, comparable to CD8+ CAR-T cells, but interestingly expressed lower levels of IFN-{gamma} than conventional CAR CD8+ T cells. Additionally, in the presence of vitamin B2 metabolite 5-ARU, which is a conserved compound that activates MAIT cells through MHC-I related (MR1) protein, MAIT cells killed MR1-expressing target breast cancer and B cell lymphoma cell lines in a dose dependent manner. Thus, MAIT cells can be genetically edited as CAR-T cells or mobilized and expanded by MR1 ligands as an off-the-shelf novel approach to cell-based cancer immunotherapy strategies while being comparable to conventional methods in effectivity. Key PointsO_LIMAIT cells expressing CARs effectively kill CD19 and Her2 expressing targets C_LIO_LICAR-MAITs display strong cytotoxicity with lower TNF-{gamma} compared to CD8+ CAR-T cells C_LI

immunology↗

bioRxiv2022

Modulation of glucose metabolism by 2-Deoxy-D-Glucose (2DG) promotes IL-17 producing human T cell subsets

Activation and differentiation of T cells are closely linked to their cellular metabolic programs. Glycolysis and mitochondrial metabolism are thought to be critical in modulating T cell function. Here we asked to what extent inhibition of glycolysis, using 2-Deoxy-D-Glucose (2DG), regulate activation, effector function, or differentiation of human T cell subsets. We found that glycolysis is required for T cell receptor (TCR) -mediated activation and proliferation of human naive CD4+ T cells but had less of an impact on memory subsets. CD4+ T cells cultured in the presence of 2DG displayed higher level of IL-17-secreting cells (Th17) from memory or in vitro differentiated naive regulatory T cell (Tregs) subsets. Moreover, the mucosal associated invariant T (MAIT) cell subset survived or expanded better and secreted higher IL-17 in the presence of 2DG. Remarkably, we found that the 2DG effect was reversed by mannose but not by glucose. Collectively, these findings suggest that 2DG could enrich IL-17 secreting human effector T cell subsets and their cellular functions. Our finding provides a framework to manipulate glycolytic pathways in human T cells in infectious diseases such as COVID19 and in enhancing cancer immunotherapy.

immunology↗

bioRxiv2022

Development of 3D breast cancer models with human T cells expressing engineered MAIT cell receptors

Immunotherapy has revolutionized cancer treatment with the advent of advanced cell engineering techniques aimed at targeted therapy with reduced systemic toxicity. However, understanding the underlying immune-cancer interactions require development of advanced three-dimensional (3D) models of human tissues. In this study, we developed proof-of-concept 3D tumor models with increasing complexity to study the cytotoxic responses of CD8+ T cells, genetically engineered to express mucosal-associated invariant T (MAIT) cell receptors, towards MDA-MB-231 breast cancer cells. Homotypic MDA-MB-231 and heterotypic MDA-MB-231/human dermal fibroblast (HDF) tumor spheroids were primed with precursor MAIT cell ligand 5-amino-6-D-ribitylaminouracil (5-ARU). Engineered T cells effectively eliminated tumors after a 3-day culture period, demonstrating that the engineered TCR recognized MR1 expressing tumor cells in the presence of 5-ARU. Tumor cell killing efficiency of engineered T cells were also assessed by encapsulating these cells in fibrin, mimicking a tumor extracellular matrix microenvironment. Expression of proinflammatory cytokines such as IFN{gamma}, IL-13, CCL-3 indicated immune cell activation in all tumor models, post immunotherapy. Further, in corroborating the cytotoxic activity, we found that granzymes A and B were also upregulated, in homotypic as well as heterotypic tumors. Finally, a 3D bioprinted tumor model was employed to study the effect of localization of T cells with respect to tumors. T cells bioprinted proximal to the tumor had reduced invasion index and increased cytokine secretion, which indicated a paracrine mode of immune-cancer interaction. Development of 3D tumor-T cell platforms may enable studying the complex immune-cancer interactions and engineering MAIT cells for cell-based cancer immunotherapies.

immunology↗

bioRxiv2019

Perturbation of effector and regulatory T cell subsets in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS)

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating disorder of unknown etiology, and diagnosis of the disease is largely based on clinical symptoms. We hypothesized that immunological disruption is the major driver of this disease and analyzed a large cohort of ME/CFS patient or control blood samples for differences in T cell subset frequencies and functions. We found that the ratio of CD4+ to CD8+ T cells and the proportion of CD8+ effector memory T cells were increased, whereas NK cells were reduced in ME/CFS patients younger than 50 years old compared to a healthy control group. Remarkably, major differences were observed in Th1, Th2, Th17 and mucosal-associated invariant T (MAIT) T cell subset functions across all ages of patients compared to healthy subjects. While CCR6+ Th17 cells in ME/CFS secreted less IL-17 compared to controls, their overall frequency was higher. Similarly, MAIT cells from patients secreted lower IFN{gamma}, GranzymeA and IL-17 upon activation. Together, these findings suggest chronic stimulation of these T cell populations in ME/CFS patients. In contrast, the frequency of regulatory T cells (Tregs), which control excessive immune activation, was higher in ME/CFS patients. Finally, using a machine learning algorithm called random forest, we determined that the set of T cell parameters analyzed could identify more than 90% of the subjects in the ME/CFS cohort as patients (93% true positive rate or sensitivity). In conclusion, these multiple and major perturbations or dysfunctions in T cell subsets in ME/CFS patients suggest potential chronic infections or microbiome dysbiosis. These findings also have implications for development of ME/CFS specific immune biomarkers and reveal potential targets for novel therapeutic interventions.

immunology↗