Biology subjects

Filipp, D.

Publications and source records attributed to Filipp, D..

7 recordsLinked to original sources

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↗

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↗

bioRxiv2025

HSCs and Tregs cooperate to preserve extramedullary hematopoiesis under chronic inflammation

Hematopoietic stem cells (HSCs) are localized within specialized niches of the bone marrow (BM). However, during hematological disorders or infections, the functionality of HSCs in the BM is compromised, leading to extramedullary hematopoiesis (EMH). Chronic inflammation drives EMH, yet its impact on HSCs outside the BM is poorly understood. Using a mouse model of chronic autoinflammatory disease, we demonstrated the presence of extramedullary HSCs in blood, spleen, and inflamed tails and paws. Single-cell transcriptomics revealed a unique expression profile in extramedullary HSCs, with significant upregulation of Cd53, MHCII-associated, and immunosuppressive genes. We further demonstrated that extramedullary CD53+ HSCs act as antigen-presenting cells, promoting the development of regulatory T cells (Tregs) to control chronic inflammation at extramedullary sites. Conversely, Tregs exert a protective role on extramedullary HSCs. Altogether, our findings revealed a mutually supportive relationship between a unique subset of HSCs and T cells in inflamed tissues during chronic inflammation.

immunology↗

bioRxiv2024

The fate of secretory cells during intestinal homeostasis, regeneration, and tumor formation is regulated by Tcf4

The single-layer epithelium of the gastrointestinal tract is a dynamically renewing tissue that ensures nutrient absorption, secretory and barrier functions and is involved in immune responses. The basis for this homeostatic renewal is the Wnt signaling pathway. Blocking this pathway can lead to epithelial damage, while its abnormal activation can result in the development of intestinal tumors. In this study, we investigated the dynamics of intestinal epithelial cells and tumorigenesis using a conditional mouse model. Using single-cell and bulk RNA sequencing and histological analysis, we elucidated the cellular responses following the loss of specific cell types. We focused on the fate of cells in the lower parts of the intestinal crypts and the development of colon adenomas. By partially inactivating the transcription factor Tcf4, a key effector of the Wnt signaling pathway, we analyzed the regeneration of isolated hyperproliferative foci (crypts). Our results suggest that the damaged epithelium is not restored by a specific regeneration program associated with oncofetal gene production, but rather by a standard homeostatic renewal pathway. Moreover, disruption of Tcf4 in secretory progenitors resulted in a significant shift in the cell lineage from Paneth cells to goblet cells, characterized by morphological changes and loss of Paneth cell-specific genes. We also found that hyperactivation of the Wnt signaling pathway in colonic adenomas correlated with the upregulation of genes typical of Paneth cells in the intestine, followed by the emergence of secretory tumor cells producing the Wnt3 ligand. The absence of Tcf4 led to a phenotypic shift of the tumor cells towards goblet cells. Our study presents a new model of epithelial regeneration based on the genetically driven partial elimination of intestinal crypts. We highlight the critical role of Tcf4 in the control of cell lineage decisions in the intestinal epithelium and colon tumors. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=160 HEIGHT=200 SRC="FIGDIR/small/603019v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1d8e5a0org.highwire.dtl.DTLVardef@cb85f7org.highwire.dtl.DTLVardef@1c84ed3org.highwire.dtl.DTLVardef@1af36f3_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

Epithelial antigen presentation controls commensal-specific intraepithelial T-cells in the gut

The expression of MHCII by intestinal epithelial cells (IEC) determines the severity of intestinal immunopathological reactions. However, the function of MHCII on IEC under homeostatic conditions remains elusive. Here we report that MHCII expression on IECs is a hallmark of an adaptive wave of homeostatic intestinal immune responses to commensal segmented filamentous bacteria (SFB). Focusing on SFB-driven responses, we describe the expression pattern of MHCII and the associated antigen processing machinery among IEC subpopulations along with the cellular network that regulates MHCII induction. Furthermore, we show that SFB induce the accumulation of SFB-specific intraepithelial lymphocytes (IELs) that originate from conventional CD4+ T-cells. Importantly, induced IELs are dependent on the epithelial MHCII. Finally, we demonstrate that both epithelial MHCII and the IEL functionality regulate the epithelial turnover. This study describes the organization of a commensal-targeted, IEL-driven immune response that is controlled by IEC antigen presentation and ultimately regulates IEC turnover.

immunology↗

bioRxiv2021

A model of preferential pairing between epithelial and dendritic cells in thymic antigen transfer

Medullary thymic epithelial cells (mTECs) which produce and present self-antigens are essential for the establishment of central tolerance. Since mTEC numbers are limited, their function is complemented by thymic dendritic cells (DCs), which transfer mTEC-produced self-antigens via cooperative antigen transfer (CAT). While CAT is required for effective T cell selection, many aspects remain enigmatic. Given the recently described heterogeneity of mTECs and DCs, it is unclear whether the antigen acquisition from a particular TEC subset is mediated by preferential pairing with specific subset of DCs. Using several relevant Cre-based mouse models controlling the expression of fluorescent proteins, we found that in regards to CAT, each subset of thymic DCs preferentially targets distinct mTEC subset(s) and importantly, XCR1+ activated DCs represented the most potent subset in CAT. Interestingly, one thymic DC can acquire antigen repetitively and of these, monocyte-derived DCs (moDC) were determined to be the most efficient in repetitive CAT. moDCs also represented the most potent DC subset in the acquisition of antigen from other DCs. These findings suggest a preferential pairing model for the distribution of mTEC-derived antigens among distinct populations of thymic DCs.

immunology↗

bioRxiv2020

Lyl-1 marks and regulates primitive macrophages and microglia development

During ontogeny, macrophages (M{Phi}) populations emerge in the Yolk Sac (YS) via two distinct progenitor waves, prior to hematopoietic stem cell development. M{Phi}-progenitors from the primitive/"early EMP" and transient-definitive/"late EMP" waves both contribute to various resident-M{Phi} populations in the developing embryonic organs. Identifying factors that modulates early stages of M{Phi}-progenitor development may lead to a better understanding of defective function of specific resident-M{Phi} subsets. Here we show that primitive macrophage (M{Phi}Prim) progenitors in the YS express Lyl-1, a bHLH transcription factor related to SCL/Tal-1. Transcriptomic analysis of YS M{Phi}-progenitors indicated that M{Phi}Prim progenitors present at embryonic day (E) 9 are clearly distinct from those present at later stages. Disruption of Lyl-1 basic helix-loop-helix domain led initially to an early increased emergence of M{Phi}Prim progenitors, and later to their defective differentiation. These defects were associated with a disrupted expression of gene sets related to embryonic patterning and neurodevelopment. Lyl-1-deficiency also induced a reduced production of mature M{Phi}/microglia in the early brain, as well as a transient reduction of the microglia pool at midgestation and in the newborn. We thus identify Lyl-1 as a critical regulator of M{Phi}Prim and microglia development, which disruption may impair resident-M{Phi} function during organogenesis. Key points1- Yolk sac primitive macrophage progenitors and microglia/Border Associated macrophages express Lyl-1. 2- Lyl-1-deficiency impairs primitive macrophage and microglia development and leads to the up-regulation of gene sets related to embryo patterning and neuro-development.

developmental biology↗