Biology subjects

Yuzefpolskiy, Y.

Publications and source records attributed to Yuzefpolskiy, Y..

3 recordsLinked to original sources

bioRxiv2026

Querying functional drivers in primary murine naive and memory T cells using RNP-mediated CRISPR-Cas9 Gene Deletion

Cytotoxic CD8 T lymphocytes (CTL) are critical for the clearance of pathogenic cells via antigen-dependent cell lysis, thus providing protection against infectious diseases and cancers. CTLs represent one of the key targets of immunotherapies and vaccine design. While microarray and single-cell RNA sequencing of effector and memory CD8 T cells have identified several promising gene targets that may modulate CD8 T cell responses, their development is slowed down by the financial and time constraints related to the generation of germline knockout mice for further validation studies. Here we present a protocol for conducting efficient deletion of genes from activated effector, as well as resting naive and memory primary murine CD8 T cells using RNP-based Crispr/Cas9 technology. This CRISPR modification of CD8 T cells was then adapted to study the effects of gene deletion in the context of acute memory differentiation as well as chronic exhaustion of CD8 T cells. To this end, we have titrated the necessary dose of antigen-specific CD8 T cells to study their differentiation in acute and chronic infections and confirmed the model by demonstrating rapid expansion of CRISPR mediated PD-1 ablated CD8 T cells in a chronic viral infection. Finally, by combining this methodology with a murine model of subcutaneous tumor challenge, this study provides a unique screening system for genes critical for mediating clearance of malignant tumor cells by CTLs. This study expands current technical capabilities for rapid evaluation of functional role of any candidate gene in CTL responses to infections and cancer through targeted gene deletion at any stage of CD8 T cell differentiation without the need for germline gene deletion mouse models.

immunology↗

bioRxiv2026

MicroRNA-17~92 drives metabolic programming of virus-specific effector CD4 and CD8 T cell responses

The miR-17[~]92 microRNA cluster drives oncogenesis by inducing proliferation and survival of cancer cells. A similar pro-proliferative role of miR-17[~]92 has also been identified in pathogen- and tumor-reactive T cells. However, the metabolic underpinnings of miR-17[~]92-drivenT cell expansion and effector differentiation remain undefined. Using a murine model of conditional miR-17[~]92 deletion or constitutive overexpression in viral antigen-specific CD4 or CD8 T cells, here we show that miR-17[~]92 drives terminal differentiation of CD4 and CD8 T cells through sustained activation of mTOR and glycolytic bioenergetics pathways. Constitutively increased expression of miR-17[~]92 led to a universal increase in the numbers of antigen-specific T follicular helper (TFH), T helper 1 (TH1) and cytotoxic T lymphocyte (CTL) effector subsets. During early stages of T cell activation, miR-17[~]92 overexpression was associated with increased glucose uptake and heightened glycolytic and oxidative metabolism to sustain increased proliferation in both CD4 and CD8 T cells. However, prolonged overexpression of miR-17[~]92 led to a loss of polyfunctionality and compromised metabolic fitness (glycolysis and mitochondrial respiration) by the peak of the effector responses. These studies establish miR-17[~]92 as a critical driver of T cell proliferation and effector differentiation through metabolic regulation of both effector CTL and CD4 T cell subsets (TFH and TH1) that are critical for combating viral infections. These studies form the basis for future manipulation of miR-17[~]92 gene/metabolic regulatory network to commandeer T cell immunity during infection, vaccination or cancer immunotherapy.

immunology↗

bioRxiv2017

Development of a novel Francisella tularensis Live Vaccine Strain expressing ovalbumin provides insight into Francisella tularensis-specific CD8+ T cell responses.

Progress towards a safe and effective vaccine for the prevention of tularemia has been hindered by a lack of knowledge regarding the correlates of protective adaptive immunity and a lack of tools to generate this knowledge. CD8+ T cells are essential for protective immunity against virulent strains of Francisella tularensis, but to-date, it has not been possible to study these cells in a pathogen-specific manner. Here, we report the development of a tool for expression of the model antigen ovalbumin (OVA) in F. tularensis, which allows for the study of CD8+ T cell responses to the bacterium. We demonstrate that in response to intranasal infection with the F. tularensis Live Vaccine Strain, pathogen-specific CD8+ T cells expand after the first week and produce IFN-{gamma} but not IL-17. Effector and central memory subsets develop with disparate kinetics in the lungs, draining lymph node and spleen. Notably, F. tularensis-specific cells are poorly retained in the lungs after clearance of infection. We also show that intranasal vaccination leads to more pathogen-specific CD8+ T cells in the lung-draining lymph node compared to scarification vaccination, but that an intranasal booster overcomes this difference. Together, our data show that this novel tool can be used to study multiple aspects of the CD8+ T cell response to F. tularensis. Use of this tool will enhance our understanding of immunity to this deadly pathogen.

immunology↗