Biology subjects

Stepanek, O.

Publications and source records attributed to Stepanek, O..

17 recordsLinked to original sources

bioRxiv2026

Distinct activation programs in naive and memory CD8 T cells govern stemness and effector persistence of their progeny

Infection- and vaccination-induced memory CD8+ T cells provide protection upon subsequent exposure to cognate antigen through their increased abundance and robust per-cell responses. However, how prior antigen experience alters T-cell activation programs remains poorly understood. We longitudinally profiled gene expression in naive, central memory, and effector memory CD8+ T cells in response to cognate antigen across multiple models of acute infection. Naive T cells engaged TOX- and TCF7-centered programs and generated early stem-like central memory precursors. Their initially slow proliferation was followed by rapid expansion and the production of large numbers of short-lived effector cells. Central memory T cells rapidly triggered effector and proliferation programs while maintaining a smaller self-renewing population. Effector memory T cells expanded poorly and generated almost exclusively effector progeny. Effector progeny derived from both memory subsets survived contraction more efficiently than naive T-cell-derived progeny and established persistent effector memory populations. These data show that prior antigen experience does not simply accelerate CD8+ T-cell activation but redirects cell-intrinsic programs that shape progeny fate and persistence. These distinct programs may reflect adaptation to primary versus repeated antigen exposure and suggest that infection and vaccination history can shape the balance between stem-like memory and persistent effector populations.

immunology↗

bioRxiv2026

Adeno-Associated Virus Co-Precipitation with Extracellular Vesicles for Genome Editing in Rodent Embryo

Adeno-associated virus purification by density-gradient ultracentrifugation is labor-intensive and often results in substantial titer loss due to particle aggregation. Here, we present a scalable co-isolation strategy in which AAV is precipitated together with extracellular vesicles secreted by the producer cell line, completely bypassing density-gradient separation. The resulting AAV-EV preparations comprise free AAV, free EVs, and EV-associated AAV. Functionally, AAV-EV vectors (AAV2/1 serotype) support efficient ex vivo genome editing across multiple independent loci in mouse and rat zygotes, achieving a mean targeting efficiency of approximately 26%. Compared with gradient-purified AAV administered at matched doses, AAV-EV formulations yielded 2.34-fold higher embryo viability while maintaining equivalent transgene copy numbers. By leveraging EVs as a biological matrix, this approach enables ultracentrifugation-free AAV isolation without compromising vector functionality. Overall, AAV-EV represents an accessible and embryo-tolerant platform for rodent genome engineering that aligns with the principles of Replacement, Reduction, and Refinement (3R) principles.

bioengineering↗

bioRxiv2026

T cells restrain inflammation driven by TNF-induced cell death

TNF is a potent proinflammatory cytokine that can induce cell death by activating the kinase RIPK1. The adaptor proteins TANK and AZI2 protect against cell death by recruiting TBK1 to the TNF receptor signaling complex, thereby inhibiting RIPK1. While deficiency of either adaptor alone is well tolerated, combined loss of TANK and AZI2 results in partial embryonic lethality and severe TNF- and RIPK1-driven autoinflammation. Here, we show that TANK/AZI2-deficient mice exhibit a striking expansion of regulatory T cells (Tregs), most of which display an effector phenotype with high expression of immunosuppressive genes. Although thymic Treg generation is modestly increased, Tregs arise predominantly in the periphery through a largely cell-intrinsic mechanism. The marked accumulation of effector Tregs suggested that the T-cell compartment may limit TNF-driven pathology in this model. Supporting this, T cell ablation in TANK/AZI2-deficient mice markedly exacerbates disease progression and enhances TNF-driven, RIPK1-mediated inflammation. Similarly, T cells protect against acute TNF-induced systemic inflammatory response syndrome by limiting RIPK1-mediated cell death. Together, our findings identify TANK and AZI2 as negative regulators of Treg formation, demonstrate that T cells restrain TNF-driven inflammation by limiting RIPK1-dependent cell death, and suggest that this protective effect is mediated primarily by Tregs.

immunology↗

bioRxiv2025

LCK deficiency in CD8 T cells leads to reduced proliferation and increased effector T-cell formation in mice

LCK is an SRC-family kinase that mediates the initial steps in T-cell antigen receptor signaling and governs positive and negative selection during thymocyte development. While its developmental role is well established, its functions in peripheral T-cell responses remain poorly defined. Here, we investigated the responses of wild-type and LCK-deficient TCR-transgenic OT-I T cells across two infection models and an autoimmune diabetes model. LCK-deficient T cells exhibited reduced antigen-induced proliferation but, paradoxically, displayed enhanced effector differentiation in vivo. This phenotype likely reflects dysregulation of specific TCR signaling pathways, as LCK was more critical for ERK and NFAT activation than for NF{kappa}B, AP-1, or AKT/mTOR signaling. T cells deficient in a related kinase FYN also showed a slight increase in effector cell formation, suggesting that effector differentiation is regulated by their combined activity rather than distinct non-redundant roles. Our results reveal that LCK has two intrinsic roles in T-cell responses - promoting proliferation while restraining effector differentiation. These findings provide new insight into the molecular mechanisms of T-cell activation in vivo with implications for understanding the pathophysiology of LCK deficiency in humans and optimizing adoptive T-cell therapies.

immunology↗

bioRxiv2025

IFNG-producing self-reactive CD4+ T cells drive autoimmune adrenalitis in a mouse model of Addison's disease

Autoimmune Addisons disease (AD) is a rare but life-threatening disorder caused by immune-mediated destruction of the adrenal cortex, and progress in therapy has been limited by insufficient mechanistic insight. Here, we establish a model of Experimental Autoimmune Adrenalitis (EAA) that recapitulates key features of AD and reveals sex-dependent differences in disease manifestation within the model. Immunization with peptides derived from the adrenal self-antigen CYP11A1 induces corticosterone insufficiency. We show that autoimmune adrenalitis is driven by IFNG produced by self-reactive CD4 T cells, promoting granulomatous inflammation in the adrenal cortex. Together, these findings identify IFNG as a central effector of autoimmune adrenalitis and suggest that targeting the IFNG pathway may represent a potential therapeutic strategy for AD. SignificanceAddisons disease (AD) is a rare autoimmune disorder that destroys the adrenal cortex, yet its underlying mechanisms remain unknown. We developed a mouse model of Experimental Autoimmune Adrenalitis (EAA) that mirrors the hormonal and immunological features of AD. Our study reveals that IFNG-producing CD4 T cells drive adrenal inflammation and dysfunction, identifying IFNG as a key pathogenic factor and a potential therapeutic target.

immunology↗

bioRxiv2025

CMTM6 suppresses cell-surface expression of death receptor FAS in mice but not in humans

The transmembrane protein CMTM6 was shown to promote plasma membrane expression of immune checkpoint PD-L1, an important suppressor of anti-tumor immunity. Targeting of CMTM6 was proposed as a strategy to decrease surface PD-L1 and trigger cytotoxicity against tumors. In accord, ablation of CMTM6 in mouse cancer models was shown to efficiently suppress tumor growth in a manner partially independent of PD-L1, which suggested that CMTM6 might regulate other proteins involved in anti-tumor immunity. Using mass spectrometry, we discovered that mouse CMTM6 strongly associates with the cell death receptor FAS and negatively regulates its expression in mice. Deletion of CMTM6 promotes FAS membrane localization and renders murine cells sensitive to FASL-mediated cytotoxicity. However, the interaction between CMTM6 and FAS is lost in human cells due to the difference in three amino acids at the boundary of the FAS extracellular and transmembrane domains. Altogether, our data urge caution when transferring promising data regarding the targeting of CMTM6 from mouse cancer models to potential human therapies.

immunology↗

bioRxiv2025

Claudin 1-mediated positioning of DC1 to mTECs is essential for antigen transfer-coupled DC1 maturation and maintenance of central tolerance

The mechanisms of central tolerance, which rely on the presentation of self-antigens by medullary thymic epithelial cells (mTECs) and DCs, prevent autoimmunity by eliminating self-reactive T-cells. While mTECs produce self-antigens in an autonomous manner, DCs acquire them from mTECs via cooperative antigen transfer (CAT). Our recent data showed that preferential pairing occurs between distinct subsets of mTECs and DCs in CAT, providing a rationale for the existence of molecular determinants which control such pairing and the outcome of central tolerance. Here, we compared the transcriptomes of CAT-experienced and -inexperienced DCs and identified Claudin 1 as a molecule involved in CAT-coupled type 1 DC (DC1) maturation. By mapping thymic DC1 heterogeneity, we identified their early and late maturation states. DC1-specific ablation of Claudin 1 led to a reduction in CAT-experienced late mature DC1s and hampered DC1 maturation. These phenotypes correlated with the displacement of DC1s from the vicinity of mTECs. This translated into impaired Treg selection and clonal deletion of TRA-specific T-cells manifested via a break in tolerance and symptoms of multi-organ autoimmunity. Collectively, our results identify thymic DC1-derived Claudin 1 as a regulator of immune tolerance. One Sentence SummaryThe expression of Claudin 1 on type 1 dendritic cells regulates their proximity to mTECs, which is required for effective antigen transfer coupled with DC1 maturation and establishment of T-cell tolerance.

immunology↗

bioRxiv2024

An imbalance of naive and effector T-cell phenotypes in early type 1 diabetes across conventional and regulatory subsets

Type 1 diabetes (T1D) is an autoimmune disease caused by the loss of self-tolerance toward insulin-producing pancreatic {beta}-cells. Although the etiology of T1D is not fully understood, it is linked to dysregulation of the T-cell compartment. To identify T-cell signatures associated with T1D, we performed single-cell transcriptomic analysis of peripheral blood T-cells from newly diagnosed children, the same children after one year, and healthy donors. We observed reduced expression of genes related to effector and cytotoxic T-cell functions across conventional, unconventional, and regulatory T-cell subsets in diabetic children, particularly at the time of diagnosis. These findings were supported by flow cytometry analysis of the same cohort and by reanalysis of publicly available data. Overall, our results suggest that T1D is associated with impaired T-cell effector differentiation, which may contribute to immune dysregulation and loss of self-tolerance.

immunology↗

bioRxiv2024

Enhanced RNAi does not provide efficient innate antiviral immunity in mice in vivo.

In RNA interference (RNAi), long double-stranded RNA (dsRNA) is cleaved by Dicer endonuclease into small RNA interfering RNAs (siRNAs), which guide degradation of complementary RNAs. While RNAi mediates antiviral innate immunity in plants and many invertebrates, vertebrates adopted sequence-independent response and their Dicer produces siRNAs inefficiently because it is adapted to process small hairpin microRNA precursors in the gene-regulating microRNA pathway. Mammalian RNAi is thus a rudimentary pathway of unclear significance. To investigate its antiviral potential, we modified mouse Dicer locus to express a truncated variant (Dicer{Delta}HEL1) known to stimulate RNAi. Next, we analyzed how Dicer{Delta}HEL1/wt mice respond to four RNA viruses: Coxsackievirus B3 (CVB3) and encephalomyocarditis virus (ECMV) from Picornaviridae; tick-borne encephalitis virus (TBEV) from Flaviviridae; and lymphocytic choriomeningitis virus (LCMV) from Arenaviridae. Increased Dicer activity in Dicer{Delta}HEL1/wt mice did not elicit any antiviral effect. supporting insignificant antiviral function of endogenous mammalian RNAi in vivo. However, we also report that sufficiently high expression of Dicer{Delta}HEL1 suppressed LCMV in embryonic stem cells and in a transgenic mouse model. Altogether, mice with increased Dicer activity offer a new benchmark for identifying and studying viruses susceptible to mammalian RNAi in vivo.

molecular biology↗

bioRxiv2023

ABIN1 is a negative regulator of effector functions in cytotoxic T cells

T cells are pivotal in the adaptive immune defense, necessitating a delicate balance between robust response against infections and self-tolerance. Their activation involves intricate cross-talk among signaling pathways triggered by the T-cell antigen receptors (TCR) and co-stimulatory or inhibitory receptors. The molecular regulation of these complex signaling networks is still incompletely understood. We identified an adaptor protein, ABIN1 as a component of the signaling complexes of GITR and OX40 co-stimulation receptors. T cells lacking ABIN1 are hyper-responsive ex vivo, and exhibit enhanced responses to cognate infections, and superior ability to induce experimental autoimmune diabetes in mice. We observed that ABIN1 negatively regulates NF-{kappa}B and p38 pathways. The latter was at least partially responsible for the upregulation of key effector proteins, IFNG and GZMB in ABIN1-deficient T cells after TCR stimulation. Our findings reveal the intricate role of ABIN1 in T-cell regulation and its potential as a target for therapeutic fine-tuning of T-cell responses.

immunology↗

bioRxiv2023

BBSome-deficient cells activate intraciliary CDC42 to trigger actin-dependent ciliary ectocytosis

Bardet-Biedl syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of the BBSome, a cargo adaptor essential for export of transmembrane receptors from cilia. Although actin-dependent ectocytosis has been proposed as compensatory cellular process for defective cargo retrieval, the underlying molecular mechanism is poorly understood, particularly in the context of BBS pathology. In this study, we investigated how actin polymerization and ectocytosis are regulated within the cilium. Our findings reveal that ciliary CDC42, a RHO-family GTPase triggers in situ actin polymerization, ciliary ectocytosis, and cilia shortening in BBSome-deficient cells. Furthermore, activation of the Sonic Hedgehog pathway further enhances CDC42 activity in BBSome-deficient cilia, but not in healthy cells. Inhibition of CDC42 decreased the frequency and duration of ciliary actin polymerization events and lead to the accumulation of the GPR161 receptor in bulges along the axoneme in BBSome-deficient cells during Sonic Hedgehog signaling. Overall, our study identifies CDC42 as a key trigger of ciliary ectocytosis. Moreover, the hyperactivated ciliary CDC42-actin-ectocytosis axis in BBSome-deficient cells results in cilia shortening and loss of ciliary material, potentially impacting disease severity.

cell biology↗

bioRxiv2023

Matters Arising: FBXO38 does not control PD-1 stability

SKP1-CUL1-F-box protein (SCF) ubiquitin ligases are versatile protein complexes that mediate the ubiquitination of substrates, which are recognized by their F-box-domain- containing subunits1. One of these substrate receptors is FBXO38. Its gene has been found to be mutated in several families with early-onset distal hereditary motor neuronopathy2. SCFFBXO38 ubiquitin ligase controls the stability of ZXDB, a nuclear factor associated with the centromeric chromatin protein CENP-B3. Moreover, the loss of FBXO38 results in growth retardation and defect in spermatogenesis characterized by deregulation of the Sertoli cell transcription program and centromere integrity4. A report by Meng et al. proposed that SCFFBXO38 regulates the protein levels of the PD-1 inhibitory receptor (also known as CD279, PDCD1) in T cells5. Here, we have re-addressed the conclusions by Meng et al. using Fbxo38KO/KO mice and cell systems. We have found no evidence indicating that FBXO38 controls the abundance and stability of PD-1.

cell biology↗

bioRxiv2021

Regulatory T cells suppress the formation of super-effector CD8 T cells by limiting IL-2

Regulatory T cells (Tregs) are indispensable for maintaining self-tolerance by suppressing conventional T cells. On the other hand, Tregs promote tumor growth by inhibiting anti-cancer immunity. In this study, we identified that Tregs increase the quorum of self-reactive CD8+ T cells required for the induction of experimental autoimmune diabetes. Their major suppression mechanism is limiting available IL-2, an essential T-cell cytokine. Specifically, Tregs inhibit the formation of a previously uncharacterized subset of antigen-stimulated KLRK1+ IL7R+ (KILR) CD8+ effector T cells, which are distinct from conventional effector CD8+ T cells. KILR CD8+ T cells show a superior cell killing abilities in vivo. The administration of agonistic IL-2 immunocomplexes phenocopies the absence of Tregs, i.e., it induces KILR CD8+ T cells, promotes autoimmunity, and enhances anti-tumor responses. Counterparts of KILR CD8+ T cells were found in the human blood, revealing them as a potential target for immunotherapy.

immunology↗

bioRxiv2020

Phenotypic and clonal stability of antigen-inexperienced memory-like T cells across the genetic background, hygienic status, and aging

Antigen-inexperienced memory-like T (AIMT) cells are functionally unique T cells representing one of the two largest subsets of murine CD8+ T cells. However, differences between laboratory inbred strains, insufficient data from germ-free mice, a complete lack of data from feral mice, and unclear relationship between AIMT cells formation during aging represent major barriers for better understanding of their biology. We performed a thorough characterization of AIMT cells from mice of different genetic background, age, and hygienic status by flow cytometry and multi-omics approaches including analyses of gene expression, TCR repertoire, and microbial colonization. Our data showed that AIMT cells are steadily present in mice independently of their genetic background and hygienic status. Despite differences in their gene expression profiles, young and aged AIMT cells originate from identical clones. We identified that CD122 discriminates two major subsets of AIMT cells in a strain-independent manner. Whereas thymic CD122LOW AIMT cells (innate memory) prevail only in young animals with high thymic IL-4 production, peripheral CD122HIGH AIMT cells (virtual memory) dominate in aged mice. Co-housing with feral mice changed the bacterial colonization of laboratory strains, but had only minimal effects on the CD8+ T-cell compartment including AIMT cells.

immunology↗

bioRxiv2020

The BBSome assembly is spatially controlled by BBS1 and BBS4 in human cells

Bardet-Biedl Syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of primary cilia. Most BBS patients carry mutations in one of eight genes encoding for subunits of a protein complex, BBSome, which mediates the trafficking of ciliary cargoes. Although, the structure of the BBSome has been resolved recently, the mechanism of assembly of this complicated complex in living cells is poorly understood. We generated a large library of human retinal epithelial cell lines deficient in particular BBSome subunit and expressing another subunit tagged with a fluorescent protein. We performed a comprehensive analysis of these cell lines using biochemical and microscopy approaches. Our data revealed that the BBSome formation is a sequential process including a step of the pre-BBSome assembly at pericentriolar satellites nucleated by BBS4, followed by the translocation of the BBSome into the ciliary base mediated by BBS1.

cell biology↗

bioRxiv2020

Altered hematopoietic system and self-tolerance in Bardet-Biedl Syndrome

Bardet-Biedl Syndrome (BBS) is a pleiotropic genetic disease caused by dysfunction of primary cilia. The immune system of patients with BBS or another ciliopathy has not been investigated, most likely because hematopoietic cells do not form cilia. However, there are multiple indications that the impairment of the processes typically associated with cilia might influence the hematopoietic compartment and immunity. In this study, we analyzed clinical data of BBS patients as well as a corresponding mouse model of BBS4 deficiency. We uncovered that BBS patients have higher incidence of certain autoimmune diseases. BBS patients and animal models have elevated white blood cell levels and altered red blood cell and platelet compartments. Moreover, we observed that BBS4 deficiency alters the development and homeostasis of B cells in mice. Some of the hematopoietic system alterations were caused by the BBS-induced obesity. Overall, our study reveals a connection between a ciliopathy and the alterations of the immune system and the hematopoietic compartment.

genetics↗

bioRxiv2017

Strong homeostatic TCR signals induce formation of self-tolerant virtual memory CD8 T cells

Virtual memory T cells are foreign antigen-inexperienced T cells that have acquired memory-like phenotype and constitute for 10-20% of all peripheral CD8+ T cells in mice. Their origin, biological roles, and relationship to naive and foreign antigen-experienced memory T cells are incompletely understood. By analyzing TCR repertoires and using retrogenic monoclonal T-cell populations, we show that virtual memory T cells originate exclusively from strongly self-reactive T cells. Moreover, we show that the stoichiometry of the CD8 interaction with Lck regulates the size of the virtual memory T-cell compartment via modulating the self-reactivity of individual T-cell clones. We propose a so far unappreciated peripheral T-cell fate decision checkpoint that eventually leads to the differentiation of highly self-reactive T cells into virtual memory T cells. This underlines the importance of the variable level of self-reactivity in polyclonal T cells for the generation of functional T-cell diversity. Although virtual memory T cells descend from the highly self-reactive clones and acquire a partial memory program, they do not show higher capacity to induce autoimmune diabetes than naive T cells. Thus, virtual memory T cells are not generally more responsive than naive T cells, because their activity highly depends on the immunological context.\n\nSummaryWe conclude that virtual memory T cells are formed from self-reactive CD8+ T cells in a process regulated by CD8-Lck stoichiometry. Despite their self-reactivity and partial memory differentiation program, virtual memory T cells did not show a strong autoimmune potential.

immunology↗