Extramedullary hematopoiesis is increasingly recognized as an important mechanism by which peripheral tissues augment immune responses during inflammation and infection. However, the mechanisms governing recruitment, retention, and local differentiation of circulating hematopoietic stem and progenitor cells (HSPCs) within human tissues remain poorly understood due to the lack of physiologically relevant experimental models. Here, we developed a human skin-on-a-chip microphysiological platform to investigate the role of circulating hematopoietic stem and progenitor cells (HSPCs) in cutaneous immune responses and their potential contribution to extramedullary hematopoiesis. The device recapitulates key features of human skin, including a perfusable vascular endothelium, fibroblast-populated dermis, and keratinocyte epidermis. Upon stimulation with pro-inflammatory cytokines or a TLR2 agonist, endothelial activation significantly increased ICAM-1 and VCAM-1 expression and enhanced recruitment of both neutrophils (PMNs) and HSPCs. While PMNs readily underwent transendothelial migration into the dermal compartment, HSPCs remained localized to the vascular niche. Notably, HSPCs adhered robustly and persisted on inflamed endothelium independent of SDF-1/CXCR4 signaling. Under granulopoietic conditions, these retained HSPCs differentiated locally within the vascular compartment into PMN-like cells capable of phagocytosis and exhibiting pathogen-responsive gene expression profiles. These findings provide evidence in a human model that circulating HSPCs can contribute to host defense via localized granulopoiesis without tissue infiltration, and suggests that the vascular niche itself may serve as a site of immune cell production during infection. The platform further offers a foundation for developing HSPC-based therapeutic strategies to combat antibiotic-resistant skin infections.

Although the blood-brain barrier (BBB) restricts passage of most molecules, various naturally occurring and synthetic nanoparticles (NPs) are nonetheless found within the brain parenchyma. To study the mechanisms underlying this phenomenon, we developed a microfluidic BBB model (mBBB) using human cerebral microvascular endothelial cells (HCMECs) in direct contact with primary human astrocytes and pericytes within a physiologically relevant extracellular matrix. The horizontal architecture enables high-resolution imaging across the full barrier interface and allows direct assessment of nanoparticle transport and accumulation. This in vitro platform recapitulates key features of the BBB, including selective permeability, junctional protein expression, and receptor-mediated uptake pathways. Using this system, the trafficking and accumulation of structurally distinct nanoparticles, including liposomes, nanoplastics, and extracellular vesicles (EVs), were compared. Among these, heterologous EVs exhibit the highest transport efficiency. Analysis of nanoparticle properties suggest that ligand presentation and membrane composition, rather than size or stiffness, primarily govern BBB penetration. The mBBB platform provides a high-throughput, imaging-based framework to systematically interrogate nanoparticle trafficking across the BBB and offers a translational tool for both drug delivery and neurotoxicity screening.

Adoptive T-cell therapies rely on the identification and expansion of rare tumor-reactive T cells, yet current enrichment strategies are limited by the low abundance of these cells and complexity of their functional enrichment. Here, we present a microfluidic platform that exploits hydrodynamic shear as a controllable parameter for enriching antigen-specific T cells through peptide-major histocompatibility complex (pMHC)-mediated capture. An eight-channel microfluidic device was engineered to simultaneously interrogate a range of wall shear stresses while maintaining uniform cell delivery, enabling systematic identification of shear conditions that maximize antigen-specific enrichment. Using engineered MART-1-specific Jurkat cells, we demonstrate that T-cell capture is jointly regulated by wall shear stress and pMHC density, with intermediate shear preferentially enriching antigen-specific cells over nonspecific binders. Translation of the optimal operating condition to a high-throughput single-shear device enabled approximately 35-fold enrichment of antigen-specific T cells from peripheral blood mononuclear cells containing only 0.05% target cells. We further show that peptide-MHC complexes isolated directly from melanoma whole-cell lysates support shear-dependent enrichment comparable to recombinant pMHCs. Finally, primary MART-1-specific CD8⁺ T cells enriched using tumor-derived pMHCs retained the ability to recognize melanoma cells and upregulated the activation marker CD137 following antigen-specific stimulation. Together, these findings establish hydrodynamic shear as an orthogonal parameter for antigen-specific T-cell enrichment and provide a framework for integrating force-based selection with tumor-derived pMHCs to isolate functional antigen-specific T cells using tumor-derived pMHCs.

Chimeric antigen receptor (CAR)-T cell immunotherapy, effective in blood cancers, shows limited success in solid tumors, such as prostate, pancreatic, and brain cancers due, in part, to an immunosuppressive tumor microenvironment (TME). Immunosuppression affects various cell types, including tumor cells, macrophages, and endothelial cells. Conventional murine-based models offer limited concordance with human immunology and cancer biology. Therefore, we have developed a human "tumor-on-a-chip" (TOC) platform to model elements of immunosuppression at high spatiotemporal resolution. Our TOC features an endothelial cell-lined channel that mimics features of an in vivo capillary, such as cell attachment and extravasation across the endothelium and into the TME. Using 70 kDa dextran and fluorescence-recovery-after-photobleaching (FRAP), we confirmed physiologic interstitial flow velocities (0.1-1 μm s-1). Our device demonstrates that tumor-derived factors can diffuse in the opposite direction of interstitial flow to reach the endothelium up to 200 μm away, and at concentrations as high as 20% of those at the tumor margin. M2-like immunosuppressive macrophages and endothelial cells affect prostate tumor cell growth, clustering, and migration. M2-like macrophages also induce PD-L1 and inhibit ICAM-1 gene expression on the adjacent endothelium in a pattern that limits CAR-T cell extravasation and effector function. This observation is abrogated in the presence of the anti-PD-L1 drug atezolizumab. These results provide mechanistic insight for in vivo observations showing limited CAR-T cell extravasation and effector function in solid tumors. Furthermore, they point to a specific role of M2 macrophages in driving CAR-T cell migration into and within the TME and could prove useful in the development of novel therapies to improve solid tumor CAR-T cell therapies.

The immune protection of pancreatic β cells has three layers: anatomical, with their distribution in 1 million islets; central, with the thymic deletion of β cell-specific T cells; and peripheral, with inhibitory cellular networks. The failure of the latter leads to most spontaneous type 1 diabetes and all diabetes induced by checkpoint inhibitor therapy. Because CD4 T cells initiate disease, major histocompatibility complex (MHC) class II-expressing cells are central to the onset. In non-diabetic mouse and human islets, two such cells were detected outside of the islet boundaries near the efferent post-capillary venules: one related to the vasculature and a fibroblast referred to as a "vascular-associated fibroblast" (VAF). Functionally, primary VAFs spontaneously presented islet antigens to CD4 T cells and expressed high levels of inhibitory B7 receptors and no costimulatory receptors. VAFs induced anergy in primary pre-activated anti-islet CD4 T cells. VAFs are likely important to protect the endocrine pancreas from autoimmunity.

These are supplemental figures 1-5, which contain extra data supporting figures 1-7

Movie demonstrating DDR2-/- cells migration

Movie demonstrating K14-GFP migration

These are the movie legends and supplemental methods

A feature of severe COVID-19 is the onset of an acute and intense systemic inflammatory response referred to as the "cytokine storm". The cytokine storm is characterized by high serum levels of inflammatory cytokines and the subsequent transport of inflammatory cells to damaging levels in vital organs (e.g., myocarditis). Immune trafficking and its effect on underlying tissues (e.g., myocardium) are challenging to observe at a high spatial and temporal resolution in mouse models. In this study, we created a vascularized organ-on-a-chip system to mimic cytokine storm-like conditions and tested the effectiveness of a novel multivalent selectin-targeting carbohydrate conjugate (composed of DS - dermatan sulfate and IkL - a selectin-binding peptide, termed DS-IkL) in blocking infiltration of polymorphonuclear leukocytes (PMN). Our data shows that cytokine storm-like conditions induce endothelial cells to produce additional inflammatory cytokines and facilitate infiltration of PMNs into tissue. Treatment of tissues with DS-IkL (60 μM) reduced PMN accumulation in the tissue by >50%. We then created cytokine storm-like conditions in a vascularized cardiac tissue-chip and found that PMN infiltration increases the spontaneous beating rate of the cardiac tissue, and this effect is eliminated by treatment with DS-IkL (60 μM). In summary, we demonstrate the utility of an organ-on-a-chip platform to mimic COVID-19 related cytokine storm and that blocking leukocyte infiltration with DS-IkL could be a viable strategy to mitigate associated cardiac complications.

Extracellular vesicles (EVs) influence a host of normal and pathophysiological processes in vivo . Compared to soluble mediators, EVs are relatively large (~30-150 nm) and can traffic a wide range of proteins on their surface including extracellular matrix (ECM) binding proteins. We isolated EVs from the MCF10 series – a model human cell line of breast cancer progression – and demonstrated increasing presence of laminin-binding integrins α3β1 and α6β1 on the EVs as the malignant potential of the MCF10 cells increased. Transport of the EVs within a microfluidic device under controlled physiological interstitial flow (0.15-0.75 μm/s) demonstrated that convection was the dominant mechanism of transport. Binding of the EVs to the ECM enhanced the spatial concentration and gradient, which was partially mitigated by blocking integrins α3β1 and α6β1. Our studies demonstrate that convection and ECM binding are the dominant mechanisms controlling EV interstitial transport and should be leveraged in the design of nanotherapeutics.

Atrial fibrillation (AF) is the most common arrhythmia in the United States, affecting approximately 1 in 10 adults, and its prevalence is expected to rise as the population ages. Treatment options for AF are limited; moreover, the development of new treatments is hindered by limited (1) knowledge regarding human atrial electrophysiological endpoints (e.g., conduction velocity [CV]) and (2) accurate experimental models. Here, we measured the CV and refractory period, and subsequently calculated the conduction wavelength, in vivo (four subjects with AF and four controls), and ex vivo (atrial slices from human hearts). Then, we created an in vitro model of human atrial conduction using induced pluripotent stem (iPS) cells. This model consisted of iPS-derived human atrial cardiomyocytes plated onto a micropatterned linear 1D spiral design of Matrigel. The CV (34-41 cm/s) of the in vitro model was nearly five times faster than 2D controls (7-9 cm/s) and similar to in vivo (40-64 cm/s) and ex vivo (28-51 cm/s) measurements. Our iPS-derived in vitro model recapitulates key features of in vivo atrial conduction and may be a useful methodology to enhance our understanding of AF and model patient-specific disease.

The rational design of T Cell Receptors (TCRs) for immunotherapy has stagnated due to a limited understanding of the dynamic physiochemical features of the TCR that elicit an immunogenic response. The physiochemical features of the TCR-peptide major histocompatibility complex (pMHC) bond dictate bond lifetime which, in turn, correlates with immunogenicity. Here, we: i) characterize the force-dependent dissociation kinetics of the bond between a TCR and a set of pMHC ligands using Steered Molecular Dynamics (SMD); and ii) implement a machine learning algorithm to identify which physiochemical features of the TCR govern dissociation kinetics. Our results demonstrate that the total number of hydrogen bonds between the CDR2β-MHC⍺(β), CDR1α-Peptide, and CDR3β-Peptide are critical features that determine bond lifetime.

Although parallel plate flow chamber assays are widely performed, extraction of kinetic parameters is limited to specialized labs with mathematical expertise and customized video-microscopy tracking tools. The recent development of Trackmate has increased researcher accessibility to tracking particles in video-microscopy experiments; however, there is a lack of tools that analyze this tracking information. We report a software tool, compatible with Trackmate, that extracts Receptor Ligand Non-Equilibrium Kinetic (RLNEK) parameters from video-microscopy data. This software should be of particular interest to the community of researchers and scientists interrogating the target-specific binding and release of immune cells.

The development and physiologic role of small intestine (SI) vasculature is poorly studied. This is partly due to a lack of targetable, organ-specific markers for in vivo studies of two critical tissue components: endothelium and stroma. This challenge is exacerbated by limitations of traditional cell culture techniques, which fail to recapitulate mechanobiologic stimuli known to affect vessel development. Here, we construct and characterize a 3D in vitro microfluidic model that supports the growth of patient-derived intestinal subepithelial myofibroblasts (ISEMFs) and endothelial cells (ECs) into perfused capillary networks. We report how ISEMF and EC-derived vasculature responds to physiologic parameters such as oxygen tension, cell density, growth factors, and pharmacotherapy with an antineoplastic agent (Erlotinib). Finally, we demonstrate effects of ISEMF and EC co-culture on patient-derived human intestinal epithelial cells (HIECs), and incorporate perfused vasculature into a gut-on-a-chip (GOC) model that includes HIECs. Overall, we demonstrate that ISEMFs possess angiogenic properties as evidenced by their ability to reliably, reproducibly, and quantifiably facilitate development of perfused vasculature in a microfluidic system. We furthermore demonstrate the feasibility of including perfused vasculature, including ISEMFs, as critical components of a novel, patient-derived, GOC system with translational relevance as a platform for precision and personalized medicine research.

Abstract Collective cell migration is an adaptive, coordinated interactive process involving cell–cell and cell–extracellular matrix (ECM) microenvironmental interactions. A critical aspect of collective migration is the sensing and establishment of directional movement. It has been proposed that a subgroup of cells known as leader cells localize at the front edge of a collectively migrating cluster and are responsible for directing migration. However, it is unknown how and when leader cells arrive at the front edge and what environmental cues dictate leader cell development and behavior. Here, we addressed these questions by combining a microfluidic device design that mimics multiple tumor microenvironmental cues concurrently with biologically relevant primary, heterogeneous tumor cell organoids. Prior to migration, breast tumor leader cells (K14+) were present throughout a tumor organoid and migrated (polarized) to the leading edge in response to biochemical and biomechanical cues. Impairment of either CXCR4 (biochemical responsive) or the collagen receptor DDR2 (biomechanical responsive) abrogated polarization of leader cells and directed collective migration. This work demonstrates that K14+ leader cells utilize both chemical and mechanical cues from the microenvironment to polarize to the leading edge of collectively migrating tumors. Significance: These findings demonstrate that pre-existing, randomly distributed leader cells within primary tumor organoids use CXCR4 and DDR2 to polarize to the leading edge and direct migration.

Collective cell migration is an adaptive, coordinated interactive process involving cell–cell and cell–extracellular matrix (ECM) microenvironmental interactions. A critical aspect of collective migration is the sensing and establishment of directionalmovement. It has beenproposed that a subgroupof cells known as leader cells localize at the front edge of a collectively migrating cluster and are responsible for directing migration. However, it is unknownhowandwhen leader cells arrive at the front edge and what environmental cues dictate leader cell development and behavior. Here, we addressed these questions by combining a microfluidic device design that mimics multiple tumor microenvironmental cues concurrently with biologically relevant primary, heterogeneous tumor cell organoids. Prior to migration, breast tumor leader cells (K14þ) were present throughout a tumor organoid and migrated (polarized) to the leading edge in response to biochemical and biomechanical cues. Impairment of either CXCR4 (biochemical responsive) or the collagen receptor DDR2 (biomechanical responsive) abrogated polarization of leader cells and directed collective migration. This work demonstrates that K14þ leader cells utilize both chemical and mechanical cues from the microenvironment to polarize to the leading edge of collectively migrating tumors. Significance: These findings demonstrate that pre-existing, randomly distributed leader cells within primary tumor organoids use CXCR4 and DDR2 to polarize to the leading edge and direct migration.

SHIN, H.-W., C. M. ROSE-GOTTRON, D. M. COOPER, M. HILL, and S. C. GEORGE. Impact of High-Intensity Exercise on Nitric Oxide Exchange in Healthy Adults. Med. Sci. Sports Exerc. , Vol. 35, No. 6, pp. 995–1003, 2003. Purpose: After exercise, exhaled NO concentration has been reported to decrease, remain unchanged, or increase. A more mechanistic understanding of NO exchange dynamics after exercise is needed to understand the relationship between exercise and NO exchange. Methods: We measured several flow-independent NO exchange parameters characteristic of airway and alveolar regions using a single breath maneuver and a two-compartment model (maximum flux of NO from the airways, J' awNO , pL·s (cid:1) 1 ; diffusing capacity of NO in the airways, D awNO , pL·s (cid:1) 1 ·ppb (cid:1) 1 ; steady state alveolar concentration, C alv,ss , ppb; mean airway tissue NO concentration, C awNO , ppb), as well as serum IL-6 at baseline, 3, 30, and 120 min after a high-intensity exercise challenge in 10 healthy adults (21–37 yr old). Results: D awNO (mean (cid:2) SD) increased (37.1 (cid:2) 44.4%), whereas J' awNO and C awNO decreased ( (cid:1) 7.27 (cid:2) 11.1%, (cid:1) 26.1 (cid:2) 24.6%, respectively) 3 min postexercise. IL-6 increased steadily after exercise to 481% (cid:2) 562% above baseline 120 min postexercise. Conclusion: High-intensity exercise acutely enhances the ability of NO to diffuse between the airway tissue and the gas phase, and exhaled NO might be used to probe both the metabolic and physical properties of the airways. Words: