Biology subjects

Stepanov, A. V.

Publications and source records attributed to Stepanov, A. V..

5 recordsLinked to original sources

bioRxiv2025

The earliest changes in the translatome upon human T cell activation

Stimulation of resting T cells triggers a rapid transition into an activated state, characterized by significant changes in phenotype, metabolism and secretory function. To explore the earliest responses, we examined transcriptional and translational changes in isolated human T cells within the first four hours post-stimulation. Polyclonal stimulation initially upregulates cytokine genes while downregulating certain transcription factors and regulators. Subsequently, selective mRNA translation occurs on 5-terminal oligopyrimidine (TOP) motif-containing mRNAs and ATF4, alongside global transcriptional reprogramming involving alternative transcription and splicing. Notably, both stimulated and unstimulated T cells undergo dynamic gene expression changes over time, reflecting adaptation to in vitro conditions and the loss of in vivo homeostatic signals. This includes heightened translation initiation stringency, particularly in non-stimulated cells. Thus, stress responses induced by standard T cell isolation protocols must be considered when investigating T cell signaling and activation.

immunology↗

bioRxiv2023

Targeting CD45 by gene-edited CAR-T cells for leukemia eradication and hematopoietic stem cell transplantation preconditioning

Hematopoietic stem cell transplantation (HSCT) is widely used to treat patients with life-threatening hematologic and immune system disorders. The currently used nontargeted chemo-/radiotherapy conditioning regimens cause tissue injury and induce an array of immediate and delayed adverse effects, which limits the use of this potentially curative treatment. The growing demand to replace canonical conditioning regimens has led to the development of alternative approaches based on antibody-drug conjugates, naked antibodies and CAR T cells. Here, we propose a preconditioning strategy based on targeting CD45 on hematopoietic cells with CAR45 T cells. To avoid fratricide of CD45 CAR T cells, targeted genomic disruption of the CD45 gene was performed in human CD45 CAR T cells in combination with dasatinib treatment. CD45{Delta}CAR45 T cells showed impressive activity in terms of target cell elimination in vitro and depletion of tumor cells in vivo or human hematopoietic cells in humanized immunodeficient mice engrafted with human blood-derived HSCs. CD45{Delta}CAR45 NK cells also exhibited potent killing activity against tumor cell lines and human hematopoietic cells. Therefore, fratricide-resistant CAR45 T and NK cells have the potential to provide the benefits of full myeloablative conditioning and therapy for hematologic malignancies. Thus, we provide the proof of concept for the generation and preclinical efficacy of CAR T cells directed against CD45-expressing cells.

molecular biology↗

bioRxiv2021

Antigen-specific stimulation and expansion of CAR-T cells using membrane vesicles as target cell surrogates

Development of CAR-T therapy led to immediate success in the treatment of B cell leukemia and lymphoma. It also raised an opportunity to design new protocols to target solid tumors. Manufacturing of therapy-competent functional CAR-T cells needs robust protocols for ex vivo/in vitro expansion of modified T-cells. This step is challenging, especially if non-viral low efficiency delivery protocols are used to generate CAR-T cells. Modern protocols for CAR-T cell expansion are based on incubation with high doses of recombinant cytokines to support proliferation, non-specific stimulation with surface-bound antibodies to induce TCR cross-linking, or co-cultivation with antigen-expressing feeder cell lines. These approaches are imperfect since non-specific stimulation results in rapid outgrowth of CAR-negative T cells, and removal of feeder cells from mixed cultures necessitates additional purification steps. In an effort to develop a specific and improved protocol for CAR-T cell expansion, we took advantage of cell-derived membrane vesicles, and the simple structural demands of the CAR-antigen interaction. Our approach was to make antigenic microcytospheres from common cell lines stably expressing surface-bound CAR antigens (antigenic vesicles, AVs), and then use them for stimulation and expansion of CAR-T cells. We developed a rapid, simple, efficient, and inexpensive protocol to generate, stabilize and purify AVs. As proof-of-concept we tested the efficacy of our AV constructs on several CAR-antigen pairs. The data presented in this article clearly demonstrate that our protocol produced AVs with the capacity to induce stronger stimulation, proliferation and functional activity of CAR-T cells than is possible with existing protocols. We predict that this new methodology will significantly improve the ability to obtain improved populations of functional CAR-T cells for therapy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/435976v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@abbe23org.highwire.dtl.DTLVardef@15007dforg.highwire.dtl.DTLVardef@134e506org.highwire.dtl.DTLVardef@1a7f1a2_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2019

Soluble cyanobacterial carotenoprotein as a robust antioxidant nanocarrier and delivery module

To counteract oxidative stress, antioxidants including carotenoids are highly promising, yet their exploitation is drastically limited by the poor bioavailability and fast photodestruction, whereas current delivery systems are far from being efficient. Here we demonstrate that the recently discovered nanometer-sized water-soluble carotenoprotein from Anabaena (termed CTDH) transiently interacts with liposomes to efficiently extract carotenoids via carotenoid-mediated homodimerization, yielding violet-purple protein samples amenable to lyophilization and long-term storage. We characterize spectroscopic properties of the pigment-protein complexes and thermodynamics of liposome-protein carotenoid transfer and demonstrate the highly efficient delivery of echinenone form CTDH into liposomes. Most importantly, we show carotenoid delivery to membranes of mammalian cells, which provides protection from reactive oxygen species. The described carotenoprotein may be considered as part of modular systems for the targeted antioxidant delivery.\n\nSignificance statementCarotenoids are excellent natural antioxidants but their delivery to vulnerable cells is challenging due to their hydrophobic nature and susceptibility to degradation. Thus, systems securing antioxidant stability and facilitating targeted delivery are of great interest for the design of medical agents. In this work, we have demonstrated that soluble cyanobacterial carotenoprotein can deliver echinenone into membranes of liposomes and mammalian cells with almost 70 % efficiency, which alleviates the induced oxidative stress. Our findings warrant the robustness of the protein-based carotenoid delivery for studies of carotenoid activities and effects on cell models.

biophysics↗

bioRxiv2017

The photocycle of orange carotenoid protein conceals distinct intermediates and asynchronous changes in the carotenoid and protein components

The 35 kDa water-soluble Orange Carotenoid Protein (OCP) is responsible for photoprotection in cyanobacteria. It acts as a light intensity sensor that simultaneously serves as efficient quencher of phycobilisome excitation energy as well as of reactive oxygen species. Photoactivation triggers large-scale conformational rearrangements to convert OCP from the orange OCPO state to the red active signaling state OCPR, as demonstrated by various structural methods. Eventually, such rearrangements imply complete yet reversible separation of structural domains (C- and N-terminal domain) and significant translocation of the carotenoid cofactor. Very recently, dynamic crystallography of OCPO crystals suggested the existence of photocycle intermediates with small-scale rearrangements that may trigger further transitions in the protein. However, the currently existing gap between the ultra-fast picosecond and 100 millisecond time scale of spectroscopic and structural data precludes knowledge about distinct intermediate states. In this study, we took advantage of single 7 ns laser pulses to study carotenoid absorption transients in OCP on the time-scale from 100 ns to 10 s, which allowed us to detect a red intermediate state preceding the red signaling state OCPR. In addition, time-resolved fluorescence spectroscopy and following assignment of carotenoid-induced quenching of different tryptophan residues revealed a novel orange intermediate state, which appears during back-relaxation of photoactivated OCPR to OCPO. Our results show asynchronous changes in the carotenoid and protein components and provide refined mechanistic information about the OCP photocycle as well as introduce new kinetic signatures for future studies of OCP photoactivity and photoprotection.\n\nSignificance statementCyanobacteria utilize the Orange Carotenoid Protein (OCP) to protect their photosynthetic apparatus from the harmful effects of intense sunlight. OCP is a blue light-triggered photoswitch, which undergoes photoconversion from its dark adapted orange to the active red state, the latter being able to interact with the phycobilisome antennae and quench their fluorescence, thus avoiding excessive energy flow to the photosystems. With the help of the fluorescence recovery protein (FRP), OCP detaches from phycobilisomes and can return faster into the orange state. Until now, only the thermodynamically stable orange state and the metastable red state are established in a primitive photocycle. In this work, we apply transient absorption and fluorescence spectroscopy and identify two novel photocycle intermediates of physiological relevance.

biophysics↗