Biology subjects

Marchuk, K.

Publications and source records attributed to Marchuk, K..

6 recordsLinked to original sources

bioRxiv2025

Submicron-Sampling of Living Cells by Macrophages

SUMMARY PARAGRAPHAn effective immune system must sample and appreciate healthy-self identity to prevent autoimmunity and to contrast to pathogenic insults1-3. Self-proteins are presented to T cells in the thymus during immune cell development2,3, and must be presented throughout the body to both maintain regulatory T cell populations4-6 and provide a tonic signal to maintain conventional T cells over time7-9. The ready observations of continuous apoptosis in some organs together with the ingestion of that material by myeloid populations has led to a conventional understanding of ongoing cell-death as a major source of self-antigens10, complemented in some situations by uptake of free-floating cell-derived vesicles. Here, we used a series of companion imaging and vesicular labeling technologies to reveal an alternate process undertaken by macrophages that results in non-destructive and direct sampling of living cells. The process requires cell-cell contact, does not require caspase activation, and takes place via a trogocytosis-like stretching of the target cell into the macrophage, leading to the generation of submicron-sized vesicles containing cytoplasm. Using a high-dimensional flow-based method for labeling vesicles ingested under this versus other conditions, we find that live-sampled material is distinctly processed, is poorly subject to fusion with lysosomes, and produces ensuing differential effects on the presentation of those to CD4 versus CD8 T cells. Disrupting this trafficking by redirecting antigen to the lysosome significantly reduced the associated macrophage-mediated priming of CD8 T cells. This demonstrates an important and substantial sampling of living cells by the immune system, with clear consequences for maintaining the border of immunity.

immunology↗

bioRxiv2022

T cells use distinct topological and membrane receptor scanning strategies that individually coalesce during receptor recognition

During immune surveillance, CD8 T cells scan the surface of antigen presenting cells using dynamic microvillar palpation and movements as well as by having their receptors pre-concentrated into patches. Here, we use real-time lattice light sheet microscopy to demonstrate the independence of microvillar and membrane receptor patch scanning. While T cell receptor (TCR) patches can distribute to microvilli, they do so stochastically and not preferentially as for other receptors such as CD62L. The distinctness of TCR patch movement from microvillar movement extends to many other receptors that form patches that also scan independently of the TCR. An exception to this is the CD8 co-receptor which largely co-migrates in patches that overlap with or are closely adjacent to those containing TCRs. Microvilli that assemble into a synapse contain various arrays of the engaged patches, notably of TCRs and the inhibitory receptor PD-1, creating a pastiche of occupancies that vary from microvillar contact to contact. In summary, this work demonstrates that localization of receptor patches within the membrane and on microvillar projections is stochastic prior to antigen detection and that such stochastic variation may play into the generation of many individually-composed receptor patch compositions at a single synapse. Significance statementMotile T cell microvilli palpate surfaces to facilitate surface scanning in a pattern that is independent of the movement of pre-formed patches of transmembrane antigen-receptors across those microvilli; once T cell receptors engage, the microvilli act to scaffold multiple receptors within a microvillar close-contact.

immunology↗

bioRxiv2021

Spatiotemporal co-dependency between macrophages and exhausted CD8+ T cells in cancer

T cell exhaustion is a major impediment to anti-tumor immunity. However, it remains elusive how other immune cells in the tumor microenvironment (TME) contribute to this dysfunctional state. Here we show that the biology of tumor-associated macrophages (TAM) and exhausted T cells (Tex) in the TME is extensively linked. We demonstrate that in vivo depletion of TAM reduces exhaustion programs in tumor-infiltrating CD8+ T cells and reinvigorates their effector potential. Reciprocally, transcriptional and epigenetic profiling reveals that Tex express factors that actively recruit monocytes to the TME and shape their differentiation. Using lattice light sheet microscopy, we show that TAM and CD8+ T cells engage in unique long-lasting antigen-specific synaptic interactions that fail to activate T cells, but prime them for exhaustion, which is then accelerated in hypoxic conditions. Spatially resolved sequencing supports a spatiotemporal self-enforcing positive feedback circuit that is aligned to protect rather than destroy a tumor. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/461866v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@1fb1c4aorg.highwire.dtl.DTLVardef@fe4571org.highwire.dtl.DTLVardef@1812f76org.highwire.dtl.DTLVardef@5317d5_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2021

Imaging-guided fine tuning of CAR synapse dynamics and T cell triggering

T cells typically recognize their ligands using a defined cell biology - the scanning of their membrane microvilli to palpate their environment - while that same membrane scaffolds T cell receptors (TCRs) that can signal upon ligand binding. Chimeric antigen receptors (CARs) present both a therapeutic promise as well as a tractable means to study the interplay between receptor affinity, microvillar dynamics and T cell function. CARs are often built using single-chain variable fragments (scFvs) with far greater affinity than that of natural TCRs. We used high resolution lattice lightsheet (LLS) and total internal reflection fluorescence (TIRF) imaging to visualize microvillar scanning in the context of variations in CAR design. This demonstrated that conventional CARs hyper-stabilized microvillar contacts relative to TCRs. Reducing the affinity and/or avidity of binding brought synapse microvillar dynamics into natural ranges, normalized synapse resolution and improved downstream effector function. This work highlights the importance of understanding the underlying cell biology when designing receptors for optimal antigen engagement.

immunology↗

bioRxiv2019

WAVE complex self-organization templates lamellipodial formation

How local interactions of actin regulators yield large-scale organization of cell shape and movement is not well understood. For example, why does the WAVE complex build lamellipodia, the broad sheet-like protrusions that power cell migration, whereas the homologous actin regulator N-WASP forms spiky finger-like actin networks? N-WASP is known to oligomerize into focal condensates that generate an actin finger. In contrast, the WAVE complex exhibits the linear distribution needed to generate an actin sheet. This linear organization of the WAVE complex could either arise from interactions with the actin cytoskeleton or could represent an ability of the complex to self-organize into a linear template. Using super-resolution microscopy, we find that the WAVE complex forms higher-order linear oligomers that curve into 270 nanometer-wide ring structures in the absence of actin polymer. These rings localize to the necks of membrane invaginations, which display saddle point geometries with positive curvature in one axis and negative curvature in the orthogonal axis. To investigate the molecular mechanism of saddle curvature enrichment, we show that the WAVE complex and IRSp53, a membrane curvature-sensitive protein, collaborate to recognize saddle curvature that IRSp53 cannot sense alone. This saddle preference for the WAVE complex could explain emergent cell behaviors, such as expanding and self-straightening lamellipodia as well as the ability of endothelial cells to recognize and seal transcellular holes. Our work highlights how partnering protein interactions enable complex shape sensing and how feedback between cell shape and actin regulators yields self-organized cell morphogenesis.

cell biology↗

bioRxiv2019

Tumors Exploit Dedicated Intracellular Vesicles to Program T cell Responses

In order to drive productive tumor-infiltrating lymphocyte (TIL) function, myeloid populations must direct antigens to the lymph node, including to resident antigen-presenting cells (APCs) that have never touched the tumor. It has long been supposed that APCs trade antigens with one another, but the dominant cell biology underlying that remains unknown. We used in vitro and in vivo assays together with lattice light sheet and multiphoton imaging to show that myeloid cells carry tumor antigen-laden vesicles that they trade with one another as they reach distant sites. This accounts for the majority of antigen displayed to T cells and provides tumors with a mechanism to access APCs that differentially direct T cell activation away from memory phenotypes. This work defines efficient cell biology that drives the first steps of TIL generation and represents a new frontier for engineering tumoral immunity.

immunology↗