| Kate Wofford |
University of Pennsylvania |
Peripheral immune cell dysregulation following diffuse traumatic brain injury in pigs |
Traumatic brain injury (TBI) is a global health problem affecting millions of individuals annually, potentially resulting in persistent neuropathology, chronic neurological deficits, and death. However, TBI not only affects neural tissue, but also affects the peripheral immune systemÕs homeostasis and physiology. TBI disrupts the balanced signaling between the brain and the peripheral organs, resulting in immunodysregulation and increasing infection susceptibility. Indeed, secondary infections following TBI worsen neurological outcomes and are a major source of mortality and morbidity. Despite the compelling link between the damaged brain and peripheral immune functionality, little is known about how injury severity affects the peripheral immune system in closed-head diffuse TBI, the most common clinical presentation including all concussions. Therefore, we characterized peripheral blood mononuclear cells (PBMCs) and plasma changes over time and across injury severity using an established large-animal TBI model of closed-head, non-impact diffuse rotational acceleration in pigs. Although we could detect modest changes to plasma cytokine concentrations when values were normalized to pre-injury levels, changes to the PBMCs were much more robust and did not require normalization for detection. We observed the concentration and physiology of circulating PBMCs changed in an injury severity-dependent manner, with most cellular changes occurring 10 days following a high rotational velocity injury. Here, we report changes in the concentrations of myeloid and T cells, changes in PBMC composition, and changes in phagocytic clearance over time. Together, these data suggest that following a diffuse brain injury in a clinically relevant large-animal TBI model, the immune system exhibits perturbations that are detectable into the subacute timeframe. These findings invite future investigations into therapeutic interventions targeting peripheral immunity and the potential for peripheral blood cellular characterization as a diagnostic tool. |
| Tomas Gonzalez-Fernandez |
Lehigh University |
Enhancing Mesenchymal Stem Cell Immunomodulatory Function through CRISPR-Activated Cytokine Expression |
Mesenchymal stromal cells (MSCs) are adult multipotent cells with demonstrated immunomodulatory capacity in preclinical models for a wide range of therapeutic applications including graft-versus-host disease, autoimmune disorders, and musculoskeletal tissue repair under inflammatory conditions. Despite promising preclinical results, the clinical application of MSCs for immunomodulation remains elusive. This challenge primarily arises from significant variability among MSCs due to differences in tissue origin, isolation and culture protocols, and donor characteristics such as age and pre-existing health conditions. Our main objective was to enhance the MSC's ability to modulate the phenotype of innate immune cells by enriching their anti-inflammatory secretome through CRISPR-mediated activation of gene expression. Using a liposome-based CRISPR delivery strategy, we targeted the activation of anti-inflammatory cytokines interleukin-4 (IL-4) and interleukin-10 (IL-10), along with prostaglandin E2 (PGE2) in MSCs. This approach resulted in a 1.5- to 2-fold increase in PGE2 expression, a 1000-fold increase for IL-4, and a 2000-fold increase for IL-10. Despite the robust activation, when the conditioned media from CRISPR-modified cells was used to polarize primary macrophages, there was a significant increase of inflammation-related markers, including inducible nitric oxide synthase (iNOS) and CD86. Additionally, IL-10 activation accelerated MSC death compared to the IL-4 and PGE2 groups. Further analysis identified the delivery of guide RNA (gRNA) as the primary cause of the inflammatory secretome in edited MSCs. In summary, this study reveals that CRISPR activation design parameters can have unforeseen effects on MSCs and macrophages, highlighting the need for thorough investigation and optimization before clinical application. |
| Tina Tylek |
Drexel University, School of Biomedical Engineering, Science and Health Systems |
Biomaterial-enabled Macrophage Cell Therapy via Dexamethasone-loaded Microparticles for Pulmonary Fibrosis |
Idiopathic pulmonary fibrosis is a fatal disease for which currently no cure exists. The available treatments only slow the fibrosis progression but are associated with severe side effects. Thus, there is a major need for a therapy that can reverse established fibrosis to improve lung function. Macrophages are one of the only cell types capable of remodeling fibrotic tissue into functional tissue. Therefore, macrophage cell therapy is a promising approach to become a functional treatment for reversing established pulmonary fibrosis. However, macrophages are highly plastic cells and thus, a strategy is needed to control their phenotype in situ following administration. In previous work, we advanced the strategy of biomaterial-enabled intracellular control of macrophages using microparticles (MPs) encapsulated with the anti-fibrotic, anti-inflammatory drug dexamethasone (Dex) and showed that Dex-MP- macrophages withstand additional environmental stimuli and increase phagocytosis and collagen degradation in vitro. Therefore, in this study, we tested the treatment efficacy in a progressive, non-reversible pulmonary fibrosis model.
Host macrophages in the pulmonary fibrosis model increased the expression of CD163, CD206, CD301, MERTK and CD86, markers previously identified as drivers of fibrosis. Dex-MP-loaded macrophages decreased the expression of these markers in vivo compared to unloaded and blank MP-loaded macrophages. Furthermore, treatment of Dex-MP-loaded macrophages significantly reduced the expression of these markers in host macrophages compared to control animals up to 14 days, while unloaded macrophages and free Dex MPs did not affect host macrophage phenotype. Moreover, treatment with Dex-MP-macrophages significantly reduced total collagen content compared to untreated animals which could additionally be confirmed by microCT imaging. Furthermore, in vitro and in vivo studies showed that microparticles were transferred to bystander and host macrophages indicating a possible exploitable mechanism of how MP-loaded macrophages are able to modulate the host environment.
In conclusion, these results highlight that biomaterial-enabled intracellular control of macrophages for cell therapy is a promising strategy as it shows potential for phenotype maintenance and host cell control as well as therapeutic efficacy in a model of progressive pulmonary fibrosis. Furthermore, this approach allows the induction of a complex phenotype that can be adjusted for several applications by changing and modifying drug or biomaterial components. |
| Candice Cheung |
University of South Carolina |
Particle-based retinoic acid drug delivery accentuates myotube formation when cultured with macrophages and accelerates muscle recovery in mice with muscle atrophy. |
Muscle atrophy from disuse is caused by loss of contractile protein, resulting in smaller, weaker fibers. The recovery process is largely modulated by macrophages, an immune cell population that creates a local environment conducive for recovery. All-trans retinoic acid (ATRA) is a metabolite of vitamin A and has been researched as a regenerative agent for its muscle-supportive effects through macrophage modulation and direct impacts to muscle cells. We have encapsulated ATRA in poly(lactide-co-glycolide) microparticles (ATRA-PLG, 9-100µg ATRA per mg of particle, 2-3µm diameter) using single oil-in-water emulsion/solvent evaporation to improve bioavailability and stability of ATRA for extended release. ATRA-PLG incubated with RAW264.7 macrophages induced insulin-like growth factor-1 secretion into the cell media, a potent muscle-regenerative factor. Conditioned media from ATRA-PLG treated RAW264.7 increased myotube formation in C2C12 muscle cells compared to ATRA or RAW264.7 media alone. In a cast-immobilization mouse model of muscle atrophy, ATRA-PLG was administered to the triceps surae upon cast removal then allowed to reambulate for 3-7 days. Confocal microscopy of the solei indicated that inflammatory CD68+CD163-CD206- macrophages decreased with ATRA-PLG treatment at the 3-day timepoint compared to saline, whereas anti-inflammatory CD206+ and CD163+ macrophages were increased, indicating that inflammation was being attenuated earlier. Additionally, muscle fiber cross sectional area (CSA) of ATRA-PLG treated solei was almost doubled in size compared to saline at this timepoint, a measurement that is highly correlated to functional strength. In a focused, 14-day reambulatory study after casting atrophy, ATRA-PLG animals not only had larger CSA, but also had fewer centralized nuclei, an indication of accelerated recovery. These data indicate that the time frame for recovery after atrophy was accelerated with ATRA-PLG treatment. |
| Bryant Colin |
University of Chicago |
High-Throughput qPCR for T Cell Phenotyping: A Scalable and Efficient Alternative to Flow Cytometry |
With the increasing complexity of immune responses, scalable methods for T cell phenotyping are essential. Traditional flow cytometry, while effective, presents challenges in throughput and sample handling. To address these limitations, we developed a high-throughput qPCR platform for profiling CD4+ T cell subsetsÑincluding Th1, Th2, Th17, Treg, and T follicular helper (Tfh) cellsÑby measuring key transcription factors such as T-bet, GATA3, FOXP3, ROR_T, and Bcl6.
The qPCR platform offers multiplexed transcriptional profiling across hundreds of samples with enhanced scalability and precision. Using cytokine cocktails to induce specific T cell phenotypes, we validated transcriptional signatures through parallel cytokine analysis. This method provides a reliable and efficient alternative to flow cytometry for large-scale immune profiling.
Future work will explore the method's potential for identifying complex and hybrid phenotypes, such as Th1/Th17 cells, and benchmarking the platform directly against flow cytometry. This qPCR-based approach lays the foundation for comparative immune studies, including vaccine research and immunomodulation. With its scalable design, the platform holds promise for advancing therapeutic discovery and expanding the understanding of T cell responses across various conditions. |
| Juan Francisco Cortes |
Drexel University |
Immune-related Genes Linked with Wound Microbiota as Potential Biomarkers for Diabetic Foot Ulcer Healing |
Diabetes foot ulcers (DFU) are one of the major chronic complications of diabetes and are challenging to treat because the mechanisms behind impaired DFU healing are complex and unknown, and heterogeneity in patient responsiveness to treatment is poorly understood. These differences in patterns of treatment effectiveness make it particularly important to develop accurate and intuitive biomarkers for predicting wound healing that can be used to guide the most appropriate treatment decisions for optimal patient outcomes. Chronic inflammation and dysfunctional macrophage behavior impact healing in DFUs. Additionally, wound microbiota is a potential factor that tunes immune cell phenotypes in chronic wounds and is a strong factor that impairs wound healing. Hence, this study aimed to compare changes in inflammation- and macrophage phenotype-related genes over time in human healing and non-healing DFUs, to investigate the microbiome's influence as a potential mediator, and to uncover possible biomarkers of healing outcomes. Using a targeted panel of 227 human genes and 16S ribosomal RNA amplicon sequence, paired samples in two-time points from 27 subjects were analyzed to understand the changes over time and their correlation with wound microbial species. Significant differences in inflammation-related gene expression and correlations with the microbiome were observed between human healing and non-healing DFUs. While no individual genes predicted healing outcome, the ratio of C3AR1/CCL22 was identified as a unique biomarker that can accurately predict healing outcomes, which was validated in an independent cohort of 40 subjects. These results provide insight into the heterogeneity of human DFU healing and suggest that unique biomarkers can be useful for guiding treatment decisions. |
| Ajay Thatte |
University of Pennsylvania |
Exploring transient inhibition of Notch signaling via mRNA lipid nanoparticles as a new strategy to prevent graft-versus-host disease |
Graft-versus-host disease (GVHD) is a major complication that limits the efficacy and therapeutic benefits of allogeneic hematopoietic cell transplantation (allo-HCT). Within days after allo-HCT, donor T cells from the graft experience pathogenic Notch signals that promote T cell homing to the gut, production of inflammatory cytokines and target organ damage. Transient blockade of Notch signaling has emerged as a promising strategy to prevent GVHD in mouse and non-human primate models of allo-HCT. However, systemic Notch inhibition is associated with potential side effects even when targeted to individual Notch receptors and ligands. To bypass these limitations, we are exploring the use of mRNA lipid nanoparticles (LNPs) to prevent GVHD via transient Notch inhibition that is restricted to donor T cells. Demonstrated by the success of the Sars-CoV-2 vaccines, LNPs are safe, highly tunable non-viral vehicles that effectively encapsulate nucleic acid cargoes and promote their intracellular delivery. Here, we investigated targeted LNP formulations that facilitate effective ex vivo delivery of mRNA encoding a pan-Notch inhibitor to CD4+ T cells to prevent GVHD. So far, we have identified a lead LNP formulation engineered with anti-CD4 antibody fragments to facilitate mRNA delivery specifically to CD4+ T cells and have confirmed expression of the mRNA encoding the pan-Notch inhibitor. We have also performed allo-HCT experiments in mice in vivo where we have demonstrated that LNP-engineered Notch-deprived T cells are capable of protection against GVHD in the recipient. Specifically, transplanted T cells that express the pan-Notch inhibitor do not acquire an inflammatory phenotype Ð showcased by the downregulation of Notch-sensitive cytokines (e.g., IFN-_, TNF-_) and surface markers (e.g., CD43 1B11) Ð and do not cause GVHD. Ultimately, our work establishes a novel, targeted mRNA LNP platform for specifically engineering T cells in allo-HCT grafts to prevent the onset and progression of GVHD. |
| Kenneth Kim |
Drexel University College of Medicine |
Injectable Hydrogels for Localized Induction of Regulatory T cells |
Autoimmune diseases are associated with a deficiency in regulatory T cell (Treg) populations and increased autoreactive or pro-inflammatory T cells. Inflammatory environments further exacerbate the problem by impairing Treg function, hindering their ability to expand and adapt to an effector phenotype. Treg therapies have focused on adoptive cell transfer and in vivo induction using soluble factors (e.g., IL-2, TGF-_, and rapamycin). However, these approaches are limited by challenges in controlling the timing and rate of factor delivery and issues with Treg phenotype instability and loss of function. To overcome these challenges, we have developed an injectable granular hydrogel designed for the local presentation of an immune checkpoint, B7x, that is associated with increased Treg populations. The hydrogel is composed of hyaluronic acid modified with methacrylates (for photopolymerization) and DBCO (to anchor modified B7x via copper-free click chemistry). The hydrogel is then processed into granules (155±53µm) using extrusion fragmentation through 18-22G needles, resulting in an injectable and self-healing material optimized for local Treg induction. In vitro studies showed that modified B7x increased Treg populations dose-dependently. Subcutaneous injection of the B7x-conjugated granular hydrogel in C57BL/6J mice led to a two-fold increase in local Treg populations compared to hydrogels alone, as measured after 7 days from both the spleen and the injected hydrogels. We also observed a significant re-orientation of the T cell compartment in distal tissues (e.g., spleen) compared to treatment with soluble TGF-_, demonstrating the potential of localized checkpoint presentation for systemic Treg induction. Future studies will explore this systemÕs ability to regulate the inflammatory response (e.g., imiquimod-induced psoriasis) as well as in the context of promoting antigen-specific tolerance (e.g., experimental autoimmune encephalomyelitis). |
| Manthan Patel |
University of Pennsylvania |
Enabling non-viral DNA delivery using lipid nanoparticles co-loaded with endogenous anti-inflammatory lipids |
Lipid nanoparticles (LNPs) have transformed genetic medicine, recently shown by their use in COVID-19 mRNA vaccines. While loading LNPs with mRNA has many uses, loading DNA would provide additional advantages such as long-term transgene expression and availability of promoter sequences for cell-type specificity.
However, here, we find that plasmid DNA, unlike mRNA, delivered via LNPs induces acute inflammation in vivo, measured by a massive increase in various pro-inflammatory plasma cytokines such as IFN-_ and IL-6 and even mortality at therapeutically relevant doses. After screening a few innate DNA-sensors, we find that this acute response is primarily driven by the cGAS-STING pathway. Interestingly, many DNA viruses inhibit the cGAS-STING pathway for their successful replication. Inspired by this tactic, we co-loaded endogenous lipids that inhibit STING into pDNA-LNPs. Specifically, loading nitro-oleic acid (NOA) into pDNA-LNPs (NOA-pDNA-LNPs) ameliorates serious inflammatory responses in vivo which enables safe transgene expression for >1 month. Additionally, we demonstrate the ability to iteratively optimize NOA-pDNA-LNPsÕ expression by performing a small LNP formulation screen, driving up expression 50-fold. Thus, NOA-pDNA-LNPs, and pDNA-LNPs co-loaded with other bioactive molecules, will provide a major new tool in the genetic medicine toolbox, leveraging the power of DNAÕs long-term and promoter-controlled expression. |
| Tiffany Tran and Elyana A. Zewdie |
University of Virginia |
Perfusion Chip for Optimized Flow and Oxygenation to Improve Viability and Function of Ex Vivo Lymph Node Slice Cultures |
Lymph nodes (LN) are dynamic organs that constantly adapt to environmental changes, often initiating immunological responses. Ex vivo LN slices offer a valuable platform for studying these processes in short-term cultures; however, their functionality has shown to decline significantly after 48 hours, as characterized by reduced metabolic activity, decreased cytokine secretion, increased cell egress, decline in stromal health, and development of hypoxia. This limitation restricts their use in long-term applications, emphasizing the need for improved culturing methods. We hypothesized that controlled fluid flow, by mimicking biological shear stress and nutrient distribution of the lymphatic environment, will help maintain LN slice functionality in long-term culture.
To address this challenge, we developed a 3D-printed perfusion system to sustain LN slice functionality beyond 48 hours by simulating in vivo conditions. The system provides interstitial flow velocities of ~0.1Ð10 µm/s through the tissue by circulating fresh media and applying controlled shear stress. Preliminary results showed that LN slices remained undamaged following overnight perfusion with media supplemented with dextran to mimic physiological lymphatic viscosity (~1.7 mPaás) at the rate of 11 um/s. Currently, we are evaluating cytokine secretion, specifically CCL21, CXCL13, and IL-7, using ELISA and immunofluorescence imaging to study how mechanical forces influence immune signaling under different flow conditions. We are also exploring enhanced oxygenation through the chip to prevent hypoxia and assessing metabolic activity by measuring oxygen and glucose consumption.
Looking ahead, we also plan to test the impacts of targeted supplements to more effectively replicate in vivo conditions, providing essential signals and structural cues for maintaining cell function. Achieving long-term LN slice cultures would allow researchers to spatially map and monitor immune responses in LNs over time, significantly enhancing the study of complex immunological processes. This approach would also improve understanding of disease progression, immune aging, and therapeutic interventions. |
| Manav Jain |
Johns Hopkins University |
Evaluating chemical, physical, and immunological determinants of polymeric mRNA nanoparticles for simultaneous T cell activation and transfection |
While mRNA nanoparticles (NPs) are well suited for intracellular delivery due to their small size (~200nm in diameter) and ability to facilitate uptake/endosomal escape, these biomaterials are not optimized for T cell activation. Conversely, optimal T cell activating platforms are generally much larger (~5000nm) and present molecules like anti-CD3 (aCD3) and anti-CD28 (aCD28). Here, we leverage the modular nature of poly(beta-amino ester)s (PBAEs), a class of cationic polymers that can self-assemble with mRNA, to investigate the role of chemical, physical, and immunological nanoparticle properties on driving both T cell activation and transfection ex vivo.
We evaluated the role of polymer chemistry by synthesizing a library of 8 cationic poly(beta-amino ester)s with varying hydrocarbon sidechains and amine endcaps. After PBAE synthesis, T cell activating NPs were formed by mixing the polymers with mRNA, functional lipid, and aCD3/aCD28 antibodies. Nanoparticles were cultured with primary T cells, and transfection (%GFP+) and activation (proliferation/CD25 expression) was evaluated on D3. One formulation, F5, was selected for further testing due to its ability to achieve the highest T cell activation and robust transfection.
For physical testing, particle size was varied by modulating NP surface charge in a controlled manner, resulting in NPs that were either 200nm or 1000nm. These NPs were then conjugated to aCD3 and aCD28 antibodies in different conformations. Primary T cells were plated with these NPs and activation/transfection was evaluated on D3. Interestingly, both the 200nm and 1000nm NPs presenting aCD3/aCD28 stimulated T cells, but the 200nm formulation demonstrated higher transfection. Additionally, the 1000nm NPs with aCD3 demonstrated robust stimulation but low transfection, while the 200nm aCD3 only NP demonstrated poor stimulation and robust transfection. Through systematic evaluation, we identified optimal nanoparticle properties for simultaneous T cell activation and transfection that will next be evaluated in ex vivo CAR T cell engineering applications. |
| Sahana Sankaran |
University of Virginia |
DEVELOPMENT OF A CELL-LINKED SANDWICH IMMUNOASSAY TO VISUALIZE THE SPATIAL DISTRIBUTION OF CYTOKINES IN EX VIVO LYMPH NODE SLICES USING A COMMERICAL DUAL-AFFINITY REAGENT |
The lymph node (LN) is a spatially organized organ in which immune cells communicate by secreting small proteins called cytokines in response to external signals. Since most cytokines are believed to act locally, within a few hundred microns of their source cell, detection of the location of cytokine secretion is needed to understand cell-to-cell communication in this complex tissue. However, there are no current technologies to detect the spatial distribution of cytokines in live tissues except for genetically modified mice, which are expensive and not readily available for many cytokines. Current methods such as enzyme-linked immunosorbent assay (ELISA) and immunostaining of fixed tissue are limited by a loss of dynamic or organizational information.
Therefore, here we developed a cell-surface based sandwich immunoassay using a commercial antibody-based reagent to detect IFN-_, an important inflammatory cytokine, at its site of secretion within 300 µm thick murine LN tissue slices using confocal microscopy. The bispecific reagent binds IFN-_ on one side and a cell surface marker on the other. We determined that after a four-hour overlay, the reagent penetrated 48 ± 12 µm into a slice. We optimized the timing of anti-CD3_ stimulation and determined a suitable detection antibody clone for this sandwich immunoassay. Finally, we successfully visualized secreted IFN-_ within stimulated LN slices. This method showed that after stimulation with anti-CD3_, IFN-_ was located primarily in follicular regions within the slice.
Future work will focus on validating the assay against other methods used for cytokine detection. Genetically modified cytokine reporter mice will be used to validate spatial localization of the cytokine signal, while ELISA will be used to validate quantification. We envision that this assay will enable researchers to study the complex cytokine signaling pathways within both healthy and diseased tissue, allowing us to detect changes in cell communication. |
| Yufei Wang |
University of Pennsylvania |
Lipid Nanoparticle-Associated Inflammation is Mediated by Endosomal Damage Detectors |
Lipid nanoparticles (LNPs) have succeeded in nucleic acid delivery such as COVID-19 vaccines. However, LNPs have not advanced as therapeutics due to LNP-associated inflammation. we for the first time found that LNPsÕ endosomal escape not only releases mRNA payloads for expression, but causes endosomal membrane damage which eventually trigger rapid inflammation. Mice and pig lung models exhibited the increased BAL protein, cytokine release and leukocyte levels with LNPsÕ injections. In vitro screening of various LNP formulations in Raw264.7 macrophages showed that LNP-associated inflammation is highly dependent on ionizable lipids, and the severity of inflammation displays a strong correlation of mRNA expression: ionizable lipids driving higher mRNA expression (e.g., C12-200, 98N12-5 and cKK-E12) produce stronger cytokine release. These high-expressing ionizable lipids induce large endosomal holes that are detected by galectin proteins, which then activate inflammation. Notably, an ionizable lipid (4A3-SC8) attracting ESCRT machinery recruitment to repair endosomal membrane holes, exhibits high mRNA expression with minimal inflammation. Conclusively, we showed for the first time that galectins and ESCRT, the two cytosolic detectors recruiting to LNP-induced endosomal holes, are responsible for LNP-associated inflammation. |
| Jason Wickman |
Drexel University College of Medicine |
Immune Modulation of T Cell Dysfunction in Tibia Fracture Model of Complex Regional Pain Syndrome |
Background: Complex regional pain syndrome (CRPS) is a debilitating chronic pain disorder with no effective treatments. Increasingly, aberrant immune regulation has been recognized in the induction and maintenance of CRPS pathology. Less is known about the role of T cells in CRPS. More recently, resident memory T cells (Trm), which help define a local tissue environments response, have been implicated in autoimmune diseases. We hypothesize that systemic T cell dysfunction in CRPS contributes to pathological Trm development leading to disease development and persistence. Targeting T cell dysfunction through repurposing FDA approved immune modulating therapies may provide a novel treatment strategy for CRPS.
Methods: Mouse tibia fracture model (TFM) of CRPS was generated. Pain hypersensitivity was measured by von Frey and Hargreaves. Hind limb skin, lymph node, bone marrow, and PBMCs were collected at different time points. T cell populations were analyzed by flow cytometry. Drugs targeting T cell dysfunction were evaluated in fracture mice at 5 weeks post-fracture. Behavior studies utilizing T cell KO mice, T cell activation GFP reporter mice, and cytokine production assays were performed to assess drug efficacy and functional T cell phenotyping.
Results: TFM mice demonstrate increased presence of phenotypic Trm populations in the skin that from control mice. Trm demonstrate increased CD103+ populations. Signaling markers associated with Trm development were upregulated in pathological Trm, which were autoreactive measured by activation in GFP reporter mice. Systemic T cell dysfunction across other tissues was observed in TFM mice. Experiments evaluating the effect of pharmacological blockade of Trm development signaling and depletion on TFM development, as well as Trm effector function are ongoing.
Conclusions: Increases in autoreactive pathological skin Trm populations provide a novel avenue for therapeutic intervention, where blockade of signaling for Trm development or systemic T cell dysfunction could prove beneficial for attenuating pain in CRPS. |
| Michael J. Buckenmeyer |
National Cancer Institute |
Immune-competent 3D MatriSpheres: A Novel In Vitro Platform for High-Fidelity Immunotherapy Screening |
Immunotherapy has seen tremendous growth in the past decade due to its life saving potential for cancer patients. However, only 15-20% of these patients show a lasting response to treatment. One reason for immunotherapy resistance is the complex and heterogeneous tumor microenvironment (TME). Additionally, most preclinical in vitro tumor models lack immune-stromal interactions. To address this limitation, we developed immune-competent 3D ÒMatriSpheresÓ-extracellular matrix (ECM)-enhanced spheroids- to: (1) determine how the TME influences macrophage phenotype, and (2) evaluate the effects of the ECM in T cell therapy. Immune-competent colorectal cancer (CRC) MatriSpheres were prepared by mixing three mouse cell types: (1) MC38 CRC cells, (2) bone marrow-derived macrophages, and (3) fibroblasts with solubilized decellularized small intestine submucosa (SIS) ECM. Cells with/without SIS ECM were seeded onto ultra-low attachment 96-well plates and allowed to self-assemble for 7 days. SIS ECM was essential for MatriSphere formation, whereas cells alone failed to incorporate macrophages within a single cohesive structure. Picro-sirius red (PSR) staining confirmed that ECM assembly and collagen distribution within tumor MatriSpheres mimicked colorectal tumor morphology. Flow cytometry showed that MatriSphere-polarized macrophages increased PD-L1 (3.4-fold) and CD86 (4.3-fold) compared to unstimulated 2D macrophages. MatriSphere cytokine secretion at 7 days showed a two-fold reduction in CCL5 compared to macrophage-free spheroids. However, this phenotype was reversed in the presence of immune adjuvant, CDA (c-di-AMP), which resulted in a 3.5-fold increase in CCL5 and corroborated with in vivo cytokine response to CDA. To evaluate adoptive cell therapy, MC38-OVA-mKate2 MatriSpheres were established with/without SIS ECM to monitor OT-1 T-cell-specific killing. Using a 1:1 effector:target ratio, ECM-free spheroids (55.6%) showed a lower viability compared with MatriSpheres containing SIS ECM (66.9%), suggesting that ECM in solid tumors may inhibit T-cell killing. In summary, immune-competent MatriSpheres may provide a tunable, high-fidelity tool to screen immunotherapies for improved clinical efficacy. |
| Brooks A. Lane |
Drexel University |
Controlling Aortic Aneurysm Progression Through Macrophage Polarization |
Abdominal Aortic Aneurysms (AAA) are identified when a section of the aorta has an inner diameter exceeding 1.5-times that of a healthy vessel. AAAs are concomitant with chronic inflammation, indiscriminate extracellular matrix (ECM) degradation, and cellular dysfunction. Loss of structural integrity can lead to rupture, an almost invariably fatal event. Unfortunately, surgical intervention poses considerable risks while many AAAs stabilize independently. No effective pharmacological treatments are currently available for AAAs. Inflammation is implicated in AAA initiation but its mechanistic role in rupture or stabilization trajectories remains unclear. As part of the immune response, macrophage infiltrates present with both pro-inflammatory (M1-like) or pro-remodeling (M2-like) phenotypes in a context and time-dependent manner. As master regulators of the broader immune response we hypothesize that M1-like macrophage stimulation leads to protease-dependent AAA deterioration (i.e., expansion and rupture) and M2-like macrophage stimulation leads to fibrous reinforcement and stabilization. To examine this, we delivered either R848 (resiquimod, a TLR7/8 agonist) or celastrol (an anti-inflammatory quinone) to promote M1-like and M2-like macrophage phenotypes, respectively, using a prolonged-release drug-delivery system in a mouse model of AAA. Aortopathy was induced via periadventitial elastase exposure and administered blanks (no drug), R848, or celastrol from injectable granular hydrogels. Compared to non-aneurysmal shams, flow cytometry on postoperative Day-1 showed increased macrophages (F4/80+) in both blanks and R848-treated AAAs, with modest increases in celastrol-treated AAAs. R848-treated AAAs experienced a 3-fold increase in Nos2+ (pro-inflammatory) macrophages while celastrol-treated AAAs had a 1.22-fold increase in CD206+ (pro-remodeling) macrophages compared to blanks. At postoperative Day-14, AAAs treated with R848 were significantly dilated (1.27±0.18mm) compared to blanks (1.09±0.11mm; p=0.04) and celastrol (0.98±0.15mm; p<0.01) groups. Ongoing immunostaining supports elevated F4/80 and Nos2 using R848. Our data suggest macrophage-associated inflammation may deterministically influence AAA outcomes, making it a logical therapeutic target for future studies. |
| Paul Sagoe |
Syracuse University |
Nanoparticles in Microparticles (NiM) as Macrophage Targeting Immunomodulatory Drug Delivery System for Osteoarthritis Treatment |
Osteoarthritis (OA) is a painful degenerative joint disease with no known cure that affects over 600 million people worldwide. Mounting evidence indicates that joint inflammation, a major hallmark of OA pain and progression, is predominantly regulated by a specific macrophage subtype, notable for secreting proinflammatory and metabolic mediators upon activation. Thus, selectively targeting these pro-inflammatory macrophages holds promise as a potential disease-modifying strategy for OA treatment. Zoledronate (Zol), a potent bisphosphonate known for its anti-inflammatory potential and macrophage affinity, remains a promising candidate for reprogramming macrophages toward a reparative phenotype. However, Zol's clinical use is hindered by poor bioavailability, rapid synovial-clearance, non-specific targeting, and elevated cytotoxicity at uncontrolled dosage. To overcome this, we complexed Zol to calcium to form nanoparticles (CaZol-NP, ~80nm), which were subsequently loaded into PEG-PLGA microparticles via phase-separation technique to develop a novel macrophage-targeted drug delivery system, called nanoparticles in microparticles (NiM, ~7µm).
Incorporating folic acid (FA) to our system (NiM-FA) significantly enhanced uptake by RAW 264.7 cells, while blocking folate-receptor with free-FA reduced this uptake. We show that our NiM-system not only facilitates sustained release of Zol but also leverages pH-responsiveness, enabling lysosomal escape after uptake. By maintaining minimal cytotoxicity, NiM-FA allowed us to modulate inflammation, reprogram macrophage phenotypes, and scavenge reactive oxygen species (ROS). Particularly, in RAW 264.7 cells activated by lipopolysaccharides (LPS), NiM-FA treatment inhibited nuclear factor-_B (NF-_B), reduced ROS levels, and decreased the expression of the pro-inflammatory marker CD86, while increasing the expression of CD206, a pro-reparative marker, as shown by flow cytometry.
Building on our in vivo findings, which revealed the presence of folate receptor-expressing macrophages in the joint of a non-surgical post-traumatic OA mouse model, future studies will investigate the therapeutic efficacy of intra-articular delivery of NiM-FA in slowing disease progression and modulating inflammation in this clinically relevant PTOA mouse model. |
| Adam Glass |
Drexel University College of Medicine Department of Microbiology and Immunology |
Toll-Like Receptor Adaptors TRIF and MyD88: Coordinators of Macrophage Responses to Pathogenic Betacoronavirus |
Disease severity increases during highly pathogenic betacoronavirus (_-CoV) clearance, suggesting an immune response that is protective and pathogenic. CoV infections in human and mice are associated with M1-like macrophage infiltration to infected tissues, including lungs (SARS-CoV-2 and murine CoVs, MHVs) and the brain (MHV). However, the role of innate immune receptors, specifically toll-like receptors (TLRs), and their adaptors in macrophage sensing of _-CoVs remains unclear. MHVs are well-established prototypes for modeling _-CoV pathogenesis in the lungs and CNS. Using two MHV strains, the neurovirulent MHV-JHM and the neuroattenuated MHV-A59, we investigated the role of TLR adaptors TIR-domain-containing adapter-inducing interferon-_ (TRIF) and myeloid differentiation factor 88 (MyD88). Wild-type or adaptor-deficient cells were infected with either MHV strain alone or before TLR ligand stimulation. Plaque assays, immunoblotting, and enzyme-linked immunosorbent assays (ELISAs) were used to evaluate virus production, intracellular protein expression, and cytokine secretion, respectively. Replication and protein co-localization was visualized via immunofluorescence. Unexpectedly, CoV replication was suppressed in TRIF-deficient cells while expression of the RNA sensor melanoma differentiation-associated protein 5 (MDA5) was increased in TRIF- or MyD88-deficient cells. The ubiquitin-like interferon-stimulated gene 15 (ISG15) was induced by MHV and co-localized with MHV nucleocapsid. In adaptor-deficient cells, ISG15 was upregulated compared to wild-type cells, suggesting a regulatory role of TLR adaptors in ISG15 expression. Our findings demonstrate that macrophage inflammatory responses are MyD88-dependent, while TRIF negatively regulates CoV-induced macrophage antiviral responses. Defining the signaling pathways that govern macrophage responses to _-CoVs will be key for developing novel therapeutic strategies to counteract macrophage-driven hyperinflammation. |
| Emily Konopka |
Drexel College of Medicine |
Adenosine deaminase-1 improves immune responses to Clostridioides difficile DNA vaccination in an aged murine model |
Clostridioides difficile is a spore-forming gastrointestinal pathogen responsible for nearly half a million infections and up to 30,000 deaths in the U.S. annually. Aged individuals (>65) are among those with the highest susceptibility to severe infection, recurrence, and death. Current treatments for primary C. difficile infection (CDI) predominantly involve antibiotic use, but these may increase risk for recurrent infection. To avoid this, efforts to develop an effective vaccine for those at greater risk of infection, particularly the elderly, are necessary. Previously, our lab has developed a DNA vaccine construct targeting receptor binding domains of the C. difficile virulence factors toxins A and B. When immunized twice over 30 days with RBDA/RBDB-expressing plasmids (5ug/plasmid/mouse) followed by orogastric challenge, aged mice exhibit a 40% increase in survival rate compared to 0% without vaccination. To improve responses to the vaccine in aged mice, a plasmid encoding adenosine deaminase-1 (ADA-1) was added to the vaccine (10ug/mouse) to act as an immune modulator and subsequent adaptive immune responses were evaluated. Examination of antigen-specific CD4 T cell responses using intracellular cytokine staining revealed improved IFNy, TNFa, and IL-2 production in young and aged CD4 T effector memory cells following peptide stimulation. Flow cytometry analysis using a toxin B-specific tetramer also showed that ADA-1 improved quantity of antigen-specific CD4 T cells in aged mice. IFNy ELISpot analysis indicated an increase in IFNy production following peptide stimulation in aged mice vaccinated with ADA-1. Together, this data indicates that in an aged murine model, ADA-1 can improve both quantity and quality of antigen-specific CD4 T cell responses following two doses. Future studies will aim to investigate the impact of ADA-1 on antibody neutralization and affinity, Tfh/B cell responses in the lymph node, and susceptibility to challenge. |
| Arielle M. D'Elia |
Drexel University |
Development of Avidity-Controlled Cytokine Delivery Systems for the Treatment of Acute Kidney Injury |
Biotherapeutics exhibit excellent target specificity and are used to treat a range of diseases from cancer to autoimmune dysfunction. Their use benefits from sustained local delivery to concentrate drugs at the site of action and prolong therapeutic effects. Here, we develop locally-injectable hydrogels composed of a supramolecular host, _-cyclodextrin, that undergoes guest-host complexation with guest-modified proteins to enable their tunable release. Dextran and _-cyclodextrin were methacrylated by reaction with glycidyl methacrylate and co-polymerized (5mM LAP, 10mW/cm2) to yield hydrogels (G'~15kPa) with a high host concentration. Hydrogels were processed into microgels (34.0±16.7µm) by extrusion fragmentation and centrifuged to yield an injectable granular hydrogel. Guests (adamantane, Ad) were conjugated to bovine serum albumin (BSA, a model biomolecule) and cytokines via EDC chemistry in near-quantitative yield (1-10 Ad:BSA), with maintenance of guest-host affinity (12.0±1.81µM, isothermal calorimetry); avidity was assessed by surface plasmon resonance and release monitored in vitro. The avidity of Ad-BSA conjugates increased with the extent of guest-modification (kd=1.58±0.36_10-4 to 3.95±0.94_10-5s-1). Guest-modification attenuated burst release (>5-fold) and sustained BSA release for over one month with release rates inversely related to avidity. Guest-modification of cytokines (IL-10, IL-4, IFNy) did not alter their bioactivity (qPCR, bone marrow-derived macrophages) and enabled their controlled release (>2-fold less cumulative release, relative to unmodified controls). Cytokine-loaded hydrogels remained bioactive at the endpoint. Biocompatibility of the granular hydrogel was examined in healthy BalbC mice (15µL kidney subcapsular injection) relative to saline injection controls; kidney function was assessed (transdermal glomerular filtration rate, blood urea nitrogen, serum creatinine, and fibrosis) over 28 days. Hydrogel injection did not negatively impact kidney function. In sum, the bioconjugation proceeds under mild conditions, maintains therapeutic bioactivity, and is therefore applicable to a range of biotherapeutics (e.g., chemokines, cytokines, and antibodies) that may benefit from sustained local delivery, including for tissue repair following acute kidney injury. |
| Biplab Sarkar |
Drexel University |
Myeloid-targeted TLR agonists inhibit tumor growth and complement CTLA-4 blockade |
Tumor-associated macrophages (TAMs) not only support tumor growth and immune evasion, but also reduce the efficacy of immunotherapy. Here, we aimed to induce a robust anti-tumor phenotype in TAMs using agonists of their toll-like receptors (TLRs). First, we used host-functionalized poly-glucose nanoparticles (macrins) with high macrophage affinity to sequester and specifically deliver immunostimulatory drugs (such as the TLR7/8 agonist resiquimod) in a site (tumor)-specific, cell (TAM)-specific, and organelle (endosome)-specific manner. Robust pro-inflammatory reprogramming (elevated Nos2 and Il12; suppressed Arg1 and Mrc1 levels) resulted from resiquimod treatment. Intravital microscopy in IL-12-eYFP reporter mice demonstrated higher IL-12 expression in drug-targeted TAMs. In a MC38 tumor model, drug-loaded nanoparticles reduced tumor growth but did not eliminate the tumors, motivating us to next target intratumoral T cells. Apart from TAMs, regulatory T cells (Tregs) also contribute to the immunosuppressive tumor microenvironment. We hypothesized that TAMs can also be re-programmed to deplete Tregs. We scrutinized TLR expression in TAMs (within MC38 tumors) using single-cell RNA sequencing, identifying high expression levels of TLR2 (>10-fold higher than other TLRs). A TLR2 agonist (CU-T12-9) promoted inflammatory TAM re-polarization (increased Il12 and Nos2; decreased Mrc1) but was ineffective against MC38 tumors as a monotherapy. In contrast, anti-CTLA4 antibody plus CU-T12-9 markedly improved therapeutic response in mice, associated with a 2-fold higher intratumoral abundance of CD8+ T cells relative to Tregs. Our data support a model that the adjuvant capability of the TLR2 agonist resulted not only from inflammatory reprogramming of TAMs, but also from (a) the resulting increase in Fc_RIV expression in TAMs and (b) depletion of anti-CTLA-4-bound Tregs by Fc_RIV+ TAMs. Furthermore, tumor development was attenuated in a model of melanoma (B16.F10) metastasis to the lung. In sum, therapeutic TAM re-polarization is a powerful tool for improving the efficacy of cancer immunotherapy. |
| Akash Gupta |
Massachusetts Institute of Technology |
Engineering Delivery Vehicles and RNA-design to develop potent vaccines and immunotherapies |
RNA-based vaccines and immunotherapies offer a powerful tool for immunomodulation, showing immense potential in developing effective vaccines and cancer immunotherapies. RNA-based therapies typically comprise of two major components - (i) mRNA sequence for immunostimulation and antigen production, and (ii) lipid nanoparticles for RNA delivery. While mRNA transcripts can be engineered to stimulate novel immune pathways and generate novel antigen constructs. LNPs can be engineered to overcome delivery challenges and generate desired adaptive immune response. Here, I have engineered both the delivery vehicle and the RNA component to develop potent vaccines and immunotherapies. I utilized combinatorial chemistry to synthesize a library of 100 ionizable lipids, followed by high-throughput screening approaches to evaluate mRNA delivery. The molecular structure of the leading ionizable lipid was further optimized using medicinal chemistry and correlated with structure-immunogenicity studies to identify the most potent vaccine LNP formulations (AMG lipids). These AMG LNP formulations elicited more robust humoral and cellular responses than the current FDA-approved mRNA vaccines (SM-102). This enhanced efficacy was attributed to increased mRNA transfection and improved lymph node drainage. Next, we engineered mRNA transcripts to stimulate innate immune cells through unique pathways (ADJ-mRNA). These ADJ-mRNAs acted as versatile vectors for immunotherapy, targeting key immune signaling nodes rather than directly expressing cytokines or co-stimulatory receptors/ligands. ADJ-mRNA demonstrated high anti-tumor efficacy in several tumor models, generating long-term memory response. Additionally, when combined with OVA mRNA, ADJ-mRNAs significantly enhanced antigen-specific CD8 T-cell responses, sustaining a lasting memory response for up to 3 months, preventing the growth of B16-OVA tumors in vaccinated mice. ADJ-mRNAs markedly boosted adaptive immune responses when used with influenza and SARS-CoV-2 antigens, highlighting their broad applicability in cancer and infectious disease therapeutics. Overall, our study offers novel insights into engineering of delivery vehicles and immunostimulatory pathways to generate broad and durable adaptive immunity. |
| Benjamin Nachod |
The University of Pennsylvania |
Corticosteroid-Incorporated Lipid Nanoparticles for Anti Inflammatory Delivery of mRNA |
Lipid nanoparticles (LNPs) have emerged as a leading platform for delivering therapeutic RNAs, demonstrated by the FDA approval of the siRNA drug Onpattro and the success of ModernaÕs COVID-19 mRNA vaccines. mRNA delivery holds potential for various therapies, such as immunotherapies and protein replacement. LNPs are especially effective for intracellular delivery of mRNA, promoting endosomal escape and mRNA translation. However, LNPs can induce inflammation, which limits their therapeutic effectiveness in inflammation related diseases. Managing this response is critical to expanding LNP applications in inflammation-sensitive diseases.
One promising strategy is incorporating corticosteroids into LNPs to reduce inflammation. Studies have shown that co-delivering anti-inflammatory steroids with mRNA can suppress inflammation by inhibiting proinflammatory genes. Further research has explored LNPs encapsulating steroids to reduce inflammation, but only limited formulations were tested. In this study, we investigate incorporation of additional steroids into LNPs to mitigate inflammation and enhance mRNA delivery.
Steroid-incorporated LNPs with varying Dexamethasone substitution for cholesterol (0%, 10%, 50%, 100%) were tested against free steroid delivery. LNPs, containing ionizable lipid C12-200, cholesterol, DSPC, DMG-PEG, and steroids, were mixed with Firefly luciferase mRNA. The LNPs were evaluated for toxicity and anti-inflammatory activity using a SEAP reporter assay in RAW BLUE cells. The 50% substitution ratio was identified as optimal and further screened with six steroids. All LNPs were characterized for size, polydispersity, zeta potential, encapsulation efficiency, and pKa.
The DEX-incorporated LNPs effectively reduced inflammation, particularly at the 50% substitution ratio, which balanced mRNA delivery and anti-inflammatory effects. Further screening with different steroids showed that most formulations reduced SEAP expression, indicating decreased inflammation. Notably, the TRI-LNP had the highest luminescence and greatest anti-inflammatory effects. Future work will involve testing in various cell types, optimizing formulations, and assessing in vivo effects. These findings suggest that steroid-incorporated LNPs can mitigate LNP-induced inflammation while efficiently delivering mRNA. |
| Yash Agarwal |
Drexel University College of Medicine |
Assessing HIV infection of iPSC-derived human immune-competent , vascularized cortical organoids and midbrain organoids |
Despite antiretroviral therapy, numerous neurologic symptoms persist in nearly 50% of persons with HIV (PWH) and these are collectively known as neuroHIV. Low levels of inflammation and altered innate immune response in the central nervous system (CNS) can increase HIV neuropathogenesis in dopamine-rich brain regions including the striatum and cortex. This could be amplified in conjunction with substance misuse, a common comorbidity in PWH, as use of all addictive substances increases CNS dopamine and our prior data show that dopamine can increase inflammation and HIV kinetics of iPSC-derived microglia in vitro. Substance misuse is associated with worsened neurological outcomes, increased viremia, and innate immune dysregulation, suggesting that the dopamine-associated effects seen in vitro could, in part, be influencing HIV neuropathogenesis. To better assess the dopaminergic effects on HIV neuropathogenesis, we developed syngeneic forebrain cortical and midbrain organoids. The cortical organoids encompass endothelial cells, astrocytes, microglia, and excitatory cortical neurons by two months of differentiation and we show that they support infection with R5-tropic HIV. The midbrain organoids showed development of dopaminergic neurons (immuostaining, qPCR), and demonstrated dopamine release as assessed by GRAB-DA cells, a type of dopamine ÒsnifferÓ cell using D2 receptor driven GFP expression. These midbrains release dopamine in response to KCl, glutamate and glycine co-treatment, and methamphetamine, suggesting functional neurotransmitter release. Together, our data suggest that HIV infection can be studied in vitro in regionally patterned brain assembloids to assess the neuroinflammatory consequences of a hyperdopaminergic environment as seen in PWH using stimulants. Ongoing research will explore more responses to HIV, such as astrogliosis and synaptic changes in cortical neurons, and examine co-culture midbrain and forebrain assembloids to assess the impacts of stimulant-mediated dopamine release on neuropathology of HIV infected forebrains. |
| David Joyner |
Drexel University College of Medicine |
Adenosine Deaminase enhances HIV specific immune responses in pre-clinical nucleic acid vaccine model |
Despite decades of work, the development of a prophylactic HIV vaccine remains an elusive goal. While attempts have been made employing numerous strategies, no vaccine candidate brought to trials have produced lasting HIV immunity. Development of SOSIP native like trimers that contain stabilizing mutations yet still closely resemble the wild-type trimeric protein increase the likelihood of developing a neutralizing immune response after vaccination. These trimers can be delivered as a nucleic acid immunogen by using the triple tandem trimer (SOSIP-TTT). The immune responses can further be improved by employing the immunomodulatory effects of Adenosine Deaminase-1 (ADA) to enhance the Tfh Âpopulation. This cell population mediates key immune responses important to maintain control of HIV including an increased magnitude and durability of humoral and cellular immune responses. Tfh cell populations are also associated with the formation and maintenance of larger germinal centers leading to increased rate of somatic hypermutation and B cell proliferation that improve functional characteristics of resulting antibodies. We hypothesize that immunization with a BG505 SOSIP-TTT derivative, germline trimer 1.1 (pGT1.1-TTT), adjuvanted with ADA (pADA) will enhance humoral and cellular HIV specific immune responses in a murine vaccination model. To evaluate the extent ADA alters these immune responses our ongoing work uses the GT1.1 trimer, modified from BG505 to improve development of CD4 binding site antibodies. Our studies show how co-immunization with GT1.1 and pADA elicits anti-HIV-specific IgG following two immunizations. We found that ADA and a membrane bound GT1.1 antigen induces a robust HIV-specific T cell response as evaluated by activation induced marker and intracellular cytokine staining assays. This data supports the use of pADA co-immunization to enhance HIV-specific T cell immunity in a preclinical model which is important for the formation of durable vaccine immunity. |
| Mara Lanis |
Johns Hopkins University |
Investigation of CD4+ T Cells Using Artificial Antigen Presenting Cells for Cancer Immunotherapy |
Decades of encouraging yet incomplete responses from cytotoxic (CD8) T-cell-based cancer immunotherapies have invigorated recent efforts to harness helper (CD4) T-cells for cancer immunity. CD4 T-cells not only provide essential support to CD8 T-cells by enhancing their cytotoxic function and memory formation, but they also produce cytokines and recruit other immune cells, making them a critical component of an effective antitumor response. With this in mind, we have modified our labÕs CD8 T-cell activating nanoparticle platform, referred to as an artificial antigen presenting cell (aAPC), for use with CD4 T-cells. These aAPCs use conjugated signal 1 (peptide-MHC complex), along with soluble signal 2 (anti-CD28) and signal 3 (cytokines). The goal for this project was to use these aAPCs to study how different environmental factors affect CD4 phenotype and differentiation, ultimately aiming to optimize conditions that drive the development of T-cell subsets capable of enhancing antitumor immunity.
First, T-cells were activated in the presence of known CD4 cytokine skewing milieus and flow cytometry was used to confirm subset skewing. From here, we further probed the TH1 subset to see the how different dosages of IL-2 specifically affected activation, memory, and effector functions. Interestingly, higher amounts of IL-2 not only increased the percentage of effector cells, but also coincided with increases in granzyme showing cytotoxic function that is separate from canonical CD4 helper functions. Additional tests confirmed the presence of proteins required for cytotoxic function in CD4s. Finally, we began to examine potential mechanisms of cytotoxic induction starting with the role of mechanical nuclear depression by measuring the correlation between cellular aAPC internalization and granzyme B. Future work will continue to investigate the relevant signaling pathways that are engaged by our aAPCs to induce cytotoxic function in CD4 T-cells that can be harnessed to improve the efficacy of cancer immunotherapies. |
| Matthias Recktenwald |
Rowan University |
Peptide Responsive Transmembrane Receptor for Reversible Mammalian Cell-Biomaterial Guided Interactions |
In native biology there is a well-regulated interplay between cells and ligands within their extracellular environment. Synthetic transmembrane receptor systems which can reversibly respond to non-native signals within a synthetic biomaterial can be used to produce living materials with synthetic homeostatic feedback and therapeutic cytokine production. The transmembrane receptor platform developed throughout this work, termed Extracellular Peptide-ligand Dimerization Actuator (EPDA), consists of stimulatory or inhibitory receptor pairs that come together upon extracellular peptide dimer binding with corresponding monobody receptors. Intracellularly, Stimulatory EPDAs phosphorylate a substrate that merges two protein halves, whereas Inhibitory EPDAs revert split proteins back to their unmerged, inactive state via substrate dephosphorylation. To identify ligand-receptor pairs, over 2,000 candidate monobodies were built in silico using PETEI, a novel computational algorithm we developed. The top 30 monobodies based on predicted peptide binding affinity were tested experimentally, and monobodies that induced the highest change in protein merging (green fluorescent protein, GFP) were incorporated in the final EPDA receptor design. In soluble form, stimulatory peptides induce intracellular GFP merging in a time and concentration-dependent manner, and varying levels of green fluorescence were observed based on stimulatory and inhibitory peptide-ligand dosing. Materials formed with thiol-norbornene chemistry are amenable to thiol-peptide patterning. We show EPDA-programmed cells encapsulated in thiol-norbornene hydrogels patterned with stimulatory and inhibitory domains exhibited 3D activation or deactivation based on their location within peptide-patterned hydrogels. EPDA receptors can recognize a myriad of peptide-ligands bound to 3D materials, can reversibly induce cellular responses beyond fluorescence, and are widely applicable in biological research and immunoengineering. Future research is focused on developing this platform for transcriptional activation upon ligand exposure and expanding the cell types to those relevant for autoimmune regulation such as macrophages and regulatory T cells. |
| Ryan Tannir |
University of Pennsylvania |
Engineering Strategies for Enhanced Lentiviral Gene Therapy |
Lentiviral vectors are a widely used tool that facilitate gene delivery to desired cell types and are commonly used in routine laboratory research and therapeutic cell engineering. However, the lack of proper entry receptors on many cell types including hematopoietic stem cells, na•ve human T cells, B cells, and murine T cells, often results in poor gene delivery. Here, we present a simple paired virus-cell engineering approach drastically promoting lentiviral gene delivery into mammalian cells. We dual-pseudotyped lentiviruses (LVs) with vesicular stomatitis virus glycoprotein (VSV-G) and a chimeric envelope single chain variable fragment (scFv) specifically recognizing a small molecule Fluorescein (FITC) to target cells transiently labeled with FITC. Chemically attaching FITC onto the cell surface created surrogate receptors for lentivirus attachment and entry into a broad range of cell types. The synthetic interaction between FITC-labeled cells and FITC-binding LVs enables efficient LV docking, viral entry, and stable transgene expression in a range of mammalian cell lines and primary human T cells. We showed that our paired virus-cell approach can successfully deliver a human CD19-specific chimeric antigen receptor (CAR) into na•ve human T cells that are naturally refractory to LVs and produce fully functional CAR T cells that can eradicate CD19+ leukemic cells. This paired virus-cell engineering approach may serve as a universal method for engineering synthetic virus-cell interactions to improve lentivirus-based gene delivery to mammalian cells. |
| Christine Hamadani |
The University of Mississippi |
Elucidating the Physicochemical Interactions of Ionic Liquid-Coated Polymeric Nanoparticles with Red Blood Cells |
Bioinspired ionic liquids (ILs) are liquid salts under 100 _ composed of bulky and asymmetric bio-derived cations and anions. IL choline trans-2-hexenoate (CA2HA 1:2) can coat polymeric NPs (IL-NPs) and redirect affinity to red blood cell (RBC) membranes in situ, extending circulation half-life and directing biodistribution post-intravenous (IV) injection across in vivo models. Because the anion in the outermost coating layer drives RBC hitchhiking, we engineered anionic identity around 2-hexenoic acid within choline carboxylate ILs to reveal two new candidates (trans-2-butenoate (CA2BE 1:1) and heptanoate (CAHPA 1:1)) to study the anionÕs structure role in the underlying chemical origin of in situ RBC attachment. Anion structure-to-function relationship of all 3 IL-NPs (loaded with far-red DiD dye) was examined using fluorescent plate reader and fluorescence activated cell sorting (FACS) in human and murine whole blood systems via: 1) temperature kinetics (37¡C vs 4¡C over 2hrs) and NP-dose dependent (1:20- 1:2 v/v) RBC binding, 2) RBC membrane anion transport inhibition assays (saline, 0.06% DMSO, 100 uM CHC-_ to inhibit monocarboxylate transporter 1 (MCT1), 0.4 uM Cytochalasin B to inhibit glucose transporter (GLUT1-4), and 0.5uM DIDS to inhibit anion exchange transporter 1 (AE1)), and 3) low (12 RPM) and high-shear (0.1-6.0 Pa) rheology at 37¡C to evaluate NP-RBC binding strength at 50% hematocrit (1 mm gap, 40 mm cone-plate geometry).
While PLGA NPs weakly mechanically bound RBCs and were affected by GLUT inhibition, IL-NPs chemically bound RBCs: 2BE primarily docked through AE1, 2HA bound via MCT1, and HPA interacted with both MCT1 and AE1. IL-NPs redirected to different blood components in whole blood (2HA: lymphocytes vs. 2BE: monocytes vs. HPA: platelets) only when the anionÕs preferred RBC docking point (MCT1 or AE1) was inhibited. Chemical RBC hitchhiking was supported by a binding plateau despite increasing IL-NP dosage in whole blood from 1:5 - 1:2 v/v at 37¡C. As membrane anion transporter dynamics are differentially affected by temperature, transitioning to 4¡C most diminished MCT1-dependent 2HAÕs binding, while AE1-dependent 2BE continued slowly binding. RBC rheology under low shear in serum revealed strongest membrane binding via HPAÕs longest flexible alkyl tail, while 2HAÕs highly rigid and shorter alkyl tail bound more weakly and showed proportional IL-NP binding loss with increasing shear strength (half-dose loss at 0.4 Pa). |
| Jennifer A. Simonovich |
University of Florida |
Localized versus Systemically Delivered Immunomodulation for Treatment of Psoriasis |
Indoleamine 2,3-dioxygenase (IDO) is an enzyme that catalyzes tryptophan into kynurenine, and plays a critical role in the promotion of immune tolerance and restoration of homeostasis in many inflammatory and autoimmune diseases. Our group has developed two different therapeutic delivery methods; (1) a localized delivery of fusion protein IDO-Galectin3 (Gal3), and (2) systemic delivery of IDO conjugated with poly(ethylene glycol) (PEG). We have compared the therapeutic efficacy of localized versus systemic delivery in a mouse model of psoriasis.
PEG-IDO circulation half-life is dependent on the length of the conjugated polymer chain. 10kDa PEG-IDO had a circulation half-life of 21 hours and 30kDa PEG-IDO persisted past 7 days, while WT IDO was barely detectable 2 hours post injection. IMQ-induced psoriasis mice treated with IDO-Gal3 showed significantly lower cumulative clinical scores from day 6 onwards. Mice treated with 10kDa PEG-IDO required two doses, one at day 3 and a follow up at day 8, to maintain decreased clinical scores. 30kDa PEG-IDO, which persisted longer in circulation, only required one treatment on day 3 to reduce clinical scores for the entirety of the experiment, suggesting that circulation time directly impacts the length of therapeutic efficacy of PEG-IDO. Clinical scores were comparable after any IDO treatment, demonstrating that systemic delivery of IDO has comparable efficacy as localized. For IDO-Gal3 treated mice, there was a reduction in neutrophils in the epidermis at day 7, as well as fewer activated TCR__+ cells of the dermis in the IDO-Gal3 treated mice. Neutrophils and TCR__+ T cells are both pathogenic in psoriasis, and the observed reduction in cell population may be responsible for the decrease in clinical scores after IDO-Gal3 treatment. This also suggests that IDOÕs immunomodulation affects both myeloid and lymphoid cells. Future work will be done to determine the immune cell population of PEG-IDO treated mice. |
| Jia Nong |
University of Pennsylvania |
Core-Then-Shell Lipid Nanoparticles for Next Generation Nucleic Acid Delivery: Improving Gene Delivery by Enhanced DNA Encapsulation |
Lipid nanoparticles (LNPs) have successfully delivered RNA, but RNA therapeutics face challenges like transient protein expression and lack of cell-type specificity. DNA delivery via LNPs could solve these issues, offering sustained expression and high specificity through promoter design. However, DNA-LNPs cause inflammatory side effects and present unique encapsulation and delivery problems. Our group has improved DNA-LNP safety by co-loading with nitro-oleic acid, a STING inhibitor. Yet, standard LNP synthesis methods fail to load nucleic acid in 40%-80% of LNPs, and empty LNPs damage endosomes, causing more inflammatory injury. Due to limited dosing, protein expression levels induced by DNA-LNPs remain low.
I introduce a new synthesis method: multi-stage mixing (MSM) to form a core-then-shell (CTS) lipid nanoparticle. This approach creates a cationic lipid/helper lipid/DNA lipoplex core, which forms inverse hexagonal phase complexes with DNA to enhance endosome escape, and then rapidly wrap with a lipid shell containing ionizable lipid, helper lipid, cholesterol, and PEG-lipid to stabilize the unstable lipoplex core. The resulting CTS LNPs have a ~150nm diameter, polydispersity index <0.1, neutral surface charge, and >2 weeks stability at 4¡C. Cryo-EM confirms the hypothesized core-then-shell structure. CTS LNPs achieve more homogenous nucleotide loading, with 95% containing payload versus <10% for standard LNPs. Luciferase expression induced by pDNA in CTS LNPs is 2 orders of magnitude higher than standard LNPs, comparable to lipofectamine transfection in Hela cells. CTS LNP-induced expression is also significantly higher in vivo after intravenous injection. This MSM system develops next-generation LNPs with enhanced DNA encapsulation (safe) and gene expression (effective) profiles, potentially revolutionizing cell and gene therapy. |
| Beatriz Hernaez-Estrada |
Drexel University |
The polarization state of human macrophages affects their crosstalk with tissue engineered blood vessels |
Introduction: A potential solution to the integration of engineered tissues is the implantation of pre-vascularized biomaterials. Macrophages are known to be key regulators for the vascularization and integration of implanted biomaterials. Even though macrophages can exist as different phenotypes, they are able to switch phenotypes very easily. Upon implantation, in the first phase, pro-inflammatory macrophages are predominant. However, in the second phase, the regenerative phenotype predominates. It has been previously described that pro-inflammatory macrophages are able to repolarize into regenerative phenotype (pro-inflammatory ˆ regenerative) that differ from the mentioned regenerative phenotype (undifferentiated phenotype ˆ regenerative). The diversity of these populations when they crosstalk with blood vessels are unknown. Thus, the purpose of this study is to assess the phenotypic heterogeneity of different human primary macrophage phenotypes, when cultured with tissue engineered blood vessels.
Method: Engineered blood vessels were formed by the co-culture of endothelial cells and mesenchymal stromal cells in a 3D collagen scaffold. Undifferentiated macrophages (M[MSCSF]), proinflammatory macrophages [M(LPS,IFN)], pro-regenerative macrophages [M(IL4,IL13)] and pro-inflammatory macrophages repolarized to regenerative macrophages [M(LPSIFN ˆ IL4IL13)] were seeded alone, or in coculture with endothelial cells, mesenchymal stromal cells or engineered blood vessels in the 3D scaffold [3]. Subsequently, after digestion of the scaffold, the single cell suspension was stained with the macrophage general (CD45), pro-inflammatory (CD80, CD38, PDL1, CCR7, HLA-DR), and regenerative (CD206, CD209, CD163, CXCR4) markers. Dimensionality reduction, clustering and pseudotime analysis was conducted.
Results: Polarized macrophages were homogenous in 2D culture, however, just after culturing alone in 3D system, the presence of different clusters expressing different intensity levels for each marker was observed. On the other hand, culturing macrophages with the blood vessels or mesenchymal stromal cells caused macrophages to upregulate CD163 and CD209 regenerative markers. When macrophages were seeded alone in the 3D scaffold or cocultured with endothelial cells, they upregulated CD209, a different regenerative marker. These effects were different depending on their prior polarization state.
Conclusions: These findings help to understand the role of biomaterials and other cell types on macrophage phenotype, which is important for the design of strategies to modulate this crosstalk. |
| Gabrielle Lohrenz |
Drexel University |
Sex-Dependent Immuno-Mechanical Properties of Central Arteries from a Mouse Model of Early Life Stress |
The brain and the cardiovascular system work in syncytium through a complex network of nerves, hormones, and feedback mechanisms. Stimulation of the sympathetic nervous system, for example, during the fight-or-flight response, alters blood pressure, volume, heart rate, contractility, vasomotor tone, and blood distribution to different organs. Interestingly, early life stress (ELS), such as childhood trauma, neglect, abuse, family instability, loss of a caregiver, or exposure to violence, chronically increases the risk of cardiovascular diseases later in life. The reasons for this are multifactorial but we hypothesize that ELS itself may lead to endothelial dysfunction, inflammation, and arterial stiffening, which are key clinical and biological markers of vascular aging. As a surrogate for the human condition, we used a limited bedding and nesting (LBN) mouse model of ELS. LBN represents a form of neglect characterized by fragmented and unpredictable maternal care. Briefly, 4-day-old pups in the ELS group were placed in a cage with limited bedding materials for 7 days and then returned to standard housing while control animals (CTRL) were left undisturbed in their cage with the dam. At 13-16 weeks of age mice were euthanized and the descending thoracic (DTA) and infrarenal abdominal (IAA) aortas were excised for biaxial inflation-extension testing, histology, and immune cell characterization. Analysis revealed a pathological ELS phenotype that was both sex and location-dependent. That is, the DTAs of ELS mice were dilated, and the IAAs stiffer in male, but not female mice compared to CTRLs. Immunostaining of general (F4/80+) and pro-inflammatory macrophages (iNOS/NOS2+) revealed higher iNOS+ cells in the ELS group than in CTRLs. Furthermore, aortas taken from ELS mice did not demonstrate fibrosis or altered smooth muscle content. Collectively, these findings suggest hypertrophic outward remodeling and premature vascular aging of central arteries from ELS mice, which may carry significant clinical implications. |
| Samuel Sung |
Drexel University |
Designing phenotype-modulating biomaterials for macrophage-based cell therapy in volumetric muscle loss |
Macrophage cell therapy is an emerging field that uses living macrophages to repair or replace damaged tissues. Macrophages are excellent cell therapy candidates because they are central to supporting several other cell types throughout the resolution of inflammation and healing. However, current macrophage cell therapy treatments face two challenges: retention at the site of injury and sustained maintenance of the desired phenotype once transplanted.
To address these challenges, we conducted a parallel in vitro/in vivo study to understand how transplanted macrophages change phenotype and how biomaterial carriers influence these changes in vitro and in vivo in a murine volumetric muscle loss model of impaired tissue regeneration. We embedded macrophages that were pre-polarized to a reparative phenotype using interleukin-4 within microporous-gelatin Surgifoam scaffolds or nanoporous-gelatin methacryloyl (GelMA) hydrogels. We compared macrophage viability and phenotype in vitro and in vivo over 7 days. Additionally, we assessed the response of host immune cells to the macrophage carriers in vivo. We hypothesized that the biomaterial carrier would influence the changing phenotype of macrophages in vitro and in vivo, and furthermore that microporous scaffolds would facilitate more interactions with host cells compared to the nanoporous hydrogels.
In vitro, nanoporous hydrogels consistently promoted a pro-inflammatory phenotype by day 1 with evidence of a phenotype switch towards a reparative phenotype by day 7, which mimics the natural phenotypic switch observed in healthy tissue regeneration. However, by day 7 cell viability in nanoporous hydrogels was significantly decreased to 56.3%±10.6%, whereas microporous scaffolds maintained macrophage viability at 94.5% ±1.7%. In vivo, we improved macrophage retention significantly with microporous macrophage retention being 7 times higher than nanoporous hydrogels ; however, we still recovered less than 10% of transplanted macrophages after 24 hrs in both vehicles. Notably, vehicles had considerable phenotype differences between the vitro/vivo environment and host cell interactions were largely driven by the biomaterial. Microporous scaffolds supported B and dendritic cell interactions, whereas nanoporous hydrogels significantly increased the recruitment of monocytes. |
| Salwa Ahmed |
Drexel University |
Single-cell sequencing reveals differential responses by monocytes from HAMP/TSP patients versus asymptomatic carriers of human T-lymphotropic virus type 1 |
Infection with human T-cell lymphotropic virus type 1 causes HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a neuroinflammatory demyelinating condition of the spinal cord that is influenced by peripheral inflammation. Identifying elements that contribute to HAM/TSP development can offer useful insights to develop better therapeutics to treat this disorder. Hence, we performed Honeycomb single cell sequencing of total PBMCs from the asymptomatic carrier (AC) and the HAM/TSP patient by utilizing innovative HoneyComb technology to determine differential gene expression patterns. Upon extensive cellular and gene analysis, we observed significant gene level differences between AC and HAM/TSP patient, albeit, only in monocytes, which have been the least explored cell type in HTLV-1 pathogenesis thus far. A total of 101 genes were identified to be differentially expressed by HAM/TSP monocytes of which 38 genes were upregulated and 63 genes were downregulated. The Ingenuity pathway analysis (IPA) revealed that these genes belonged to various pathways, some of which are highly relevant to HAM/TSP pathogenesis (i.e. Macrophage Classical Activation Signaling, cytokine storm signaling pathway, etc.) while others have never been studied in this context (i.e. Granulocyte Adhesion and Diapedesis Signaling pathway, etc.), which makes the result of this analysis significant and innovative. Ongoing studies are focused on validating top 10 up- and down-regulated genes in the monocytes of many carriers and patients to confirm the role of newly identified genes/pathways in the development of HAM/TSP as well as other neuro-inflammatory diseases potentially opening doors for future therapies. |
| Winni Gao |
Drexel University College of Medicine |
Investigating the Role of Celastrol as an Immunomodulatory Agent in Monocyte-mediated Inflammation |
Monocytes (Mo) and macrophages (MF) play crucial roles in tissue injury (e.g., myocardial infarction). Upon injury, Mo residing in the bone marrow are recruited to the injury site by inflammatory chemokines, where they can differentiate into MF to exhibit either pro-inflammatory (M1-like) or reparatory (M2-like) phenotypes. Classical Mo (Ly6Chi CCR2+ CX3CR1-) primarily accumulate in the ischemic heart, while non-classical Mo (Ly6Clow CCR2- CX3CR1+) may better mediate anti-inflammatory responses post-injury. The lab has previously identified celastrol as a potent immunomodulatory compound that inhibits M1-like MF activation and promotes M2-like polarization. Our objective is to investigate the effects of celastrol on Mo phenotypes. To achieve this, bone marrow was isolated from C57BL/6 mice, Mo isolated by negative selection, and cells treated with celastrol to quantify Mo population subsets and other myeloid markers (e.g., F4/80 and Ly6G expression) using flow cytometry. Mo were treated at varying drug concentrations (0.1-10µM) to assess cell viability and phenotype. Lowest drug concentrations were non-cytotoxic. Notably, celastrol treatment significantly reduced classical Mo populations and co-treatment with LPS significantly increased non-classical Mo compared to treatment with celastrol or LPS alone, indicative of the drugÕs immunomodulatory effects in the setting of inflammation. Additionally, celastrol treatment significantly increased Ly6G and decreased F4/80 expression, potentially indicating induction of recently-reported Ly6G+ reparatory myeloid cells. Overall, celastrol exhibited diverse immunomodulatory effects on Mo populations, and these findings suggest that targeting Mo could be a viable therapeutic strategy to shift damaging inflammatory responses toward a reparatory orientation. |
| Morgan-Elizabeth R. McKnight |
University of Virginia |
Reduced Tumor Cell Invasion into Remodeled Tumor Draining Lymph Nodes in Murine Breast Cancer Model |
In breast cancer, lymph node (LN) metastasis is indicative of poor prognosis; however, the mechanisms behind tumor invasion into the Tumor Draining Lymph Node (TDLN) remain poorly understood. This project aimed to establish the characteristics of early (pre-metastatic) remodeling of TDLNs and quantify the impact of early remodeling on tumor cell invasion into TDLNs. To establish hallmarks of early remodeling in TDLNs, we utilized a murine breast cancer model (BRPKp110 expressing GFP) known to metastasize to TDLNs. We predicted that TDLNs would show stromal network expansion, increased secretion of chemokines, and increases in the number and area of blood vessels prior to the onset of metastasis. At 5 days post- tumor cell inoculation, we collected TDLNs and imaged with anti-GFP to confirm the absence of metastasis. At this time point, the stromal network, which is critical for immune cell trafficking, showed a significant increase in pore size. The secretion of the chemokines CCL19 and CCL21, signaling molecules which draw leukocytes to the LN, were increased in TDLN versus naive LNs. Additionally, TDLNs had a 3-fold increase in the number and a 5-fold increase in the area of blood vessels. To assess the impact of TDLN remodeling on tumor cell invasiveness, we utilized an ex vivo model (outside the body) of tumor cell invasion into cultured slices of murine LN. Unexpectedly, we found that tumor cells exhibited 1.3-fold reduced invasion into pre-metastatic TDLN. In addition, TDLNs had an average cell infiltration depth of 30+/- 14 µm compared with 65+/- 24 µm in na•ve LNs. In conclusion, our results suggest that pre-metastatic remodeling of TDLNs limits tumor cell invasion. We hypothesize that reduced tumor invasion may be due to immune activation in TDLNs; future research will test this hypothesis. |
| Ted Keunsil Kang |
Drexel University Chemical and Biological Engineering |
HIV-targeted?Nanotrap Therapeutic Vaccine - Modified Liposome with HIV Capturing Molecule |
HIV is a global health problem due to its high mutation rate, making antigen variability a major challenge for HIV vaccine design. Therefore, we are introducing our novel HIV therapeutic vaccine, nanotrap therapeutic vaccine (NTV) to provide a personalized vaccine. NTV consists of 3 components: a bulk lipid to generate liposomes, immune stimulating adjuvants loaded into the core, and HIV capturing gp120 bininding lipids. Unlike to other HIV therapeutic vaccines that guess at prospective HIV antigens, NTV is a liposomal platform which will not only inactivate HIV by binding to the major HIV antigen, gp120, by multivalently presenting already developed fusion inhibitor molecules, but also will facilitate HIV-NTV complex uptake by innate immune cells to eventually induce adaptive immunity against HIV.
As NTV is a gp120 bindable liposome, our first objective was to design different NTV-lipid that consists of CD4-mimetic compound (CJF-III-288), combination of amino acids/PEG spacers and lipid tails. CJF is an indoline scaffold HIV inactivator which has high potency to broad range of HIV-1 strains. Utilizing solid phase peptide synthesis, three different gp120 binding lipid linkers were synthesized, purified via HPLC, and validated via MALDI, which contains lysine, arginine, or PEG spacers. Using SPR, we monitored that gp120 binding affinity of HIV capturing molecule was not lost during chemical conjugation in NTV-lipid, and attaching multiple number of lysine on NTV-lipid showed the highest Kd value. NTV liposomes displaying 1% NTV lipid demonstrated in vitro infection inhibition like free CJF, indicating that binding was not affected by presentation on liposomal surface.
In the future, we will also load various Th1 skewing adjuvants into NTV core to induce specific adaptive immunity against HIV with the goal of using NTVs as a therapeutic vaccine. |
| Valerie Lallo |
Villanova University |
Tracking Endosomal Escape of Polymer Nanoparticles for Knockdown of Inflammatory Genes |
Endothelial cells are promising targets for immunomodulation, as they are key regulators in the inflammatory response in the tissue. These cells line all blood vessels and thus are in contact with blood, where they can recruit other immune cells as well as produce pro-inflammatory cytokines. We propose that non-viral drug delivery systems, such as cationic polymer nanoparticles (NPs), can transport therapeutics into these cells with precision and efficacy, controlling the inflammatory activity. Physical and chemical properties of NPs can control the release of therapeutics; but it is difficult to directly track the delivery of the cargo once it enters the cell, specifically its endosomal escape. Endosomal escape of the therapeutic can determine the success of a treatment, and therefore, a method is needed to follow drug release from the endosome accurately and directly. To do this, primary human umbilical vein endothelial cells (HUVECs) were transfected to create GFP-Rab-5 and RFP-LAMP1 on endosomes and lysosomes, respectively. Different variations of poly(amine-co-ester) (PACE) NPs containing fluorescent nucleic acids were added to these cells and live-imaged using fluorescence microscopy over the course of 48 hours. These images were analyzed to quantify the colocalization of the NPs with vesicles overtime, thus tracking endosomal escape of the nucleic acids. Using this knowledge of NP location over time, various PACE NPs containing siRNA for TNFRSF1A were added to cells and gene knockdown was correlated at corresponding time points. To further link NP location with therapeutic activity, the reduction of TNF_ induced E-selectin expression was quantified using flow cytometry. These data suggest that this fluorescent imaging method can accurately measure the colocalization of endocytic vesicles with the PACE NPs over time and be used to determine the ideal polymer composition for maximum reduction in inflammatory activity. |
| Binh Ha |
Drexel Universit College of Medicine |
Adjuvant CCL5/CCR5 axis inhibition for the treatment of BRAF-mutant melanoma |
Melanoma is the model pathology for targeted therapies due to remarkable responses seen particularly in patients carrying the BRAFV600E mutation after combination BRAFi+MEKi treatment. However, most patients eventually develop resistance to treatment stemming from minimal residual disease (MRD). Our data indicates that treatment with BRAFi+MEKi recruits myeloid-derived suppressive cells (MDSCs) to tumor sites favoring the establishment of MRD. There are two types of described MDSCs, granulocytic/polymorphonuclear MDSCs (PWN-MDSC) and monocytic MDSCs (M-MDSC) both have distinct inhibitory capabilities. MDSCs have potent immunosuppressive functions that inhibit immune effector responses by secreting suppressive molecules such as ROS, PGEs, NO, and cytokines such as TGF§, and IL-10, among others. In drug-resistant pre-clinical models of melanoma and in patient samples, MDSC recruitment is mediated by the upregulation of the chemokine CCL5, which recruits CCR5+ MDSCs. In Yumm 1.7 mouse BRAFV600E mutant melanoma, treatment with BRAFi+MEKi demonstrates an accumulation of CCR5+ cells and CCL5+ cells over time. In vitro, we observe that CCL5 is not significantly produced in tumor cells across 5 different human BRAFV600E mutant melanoma cell lines. However, results from a large in vivo flow cytometry screen indicate CCL5 is mainly produced by tumor cells. Identifying the source of CCL5 in BRAFi+MEKi resistant melanoma and disrupting the CCL5/CCR5 axis uncovers a valuable therapeutic target for boosting antitumor response by disrupting the recruitment of immunosuppressive MDSCs. |
| Asheley Chapman |
Scripps Research, MIT |
Targeted deletion of clonal germinal center B cells using antigen-drug conjugates |
The benchmark of an as-yet successful HIV vaccine is its ability to produce broadly neutralizing antibodies (bnAbs) capable of neutralizing the many strains of the highly mutable virus. VRC01 is a bnAb that targets the CD4 binding site (CD4bs) of the gp120 HIV envelope protein and arises from rare germline VRC01-class B cells. However, VRC01-class B cells are outcompeted in germinal centers (GC) by more abundant immunodominant B cells that recognize non-neutralizing epitopes. We hypothesize that elimination of competitor GC B cells using a novel antigen-drug conjugate approach will allow rare VRC01-class B cells to expand and undergo somatic hypermutation, resulting in a broadly neutralizing response. Our approach is to prime with an Òon-targetÓ antigen (germline targeting trimer MD39 VGT3.1 or eOD-60mer) bearing the neutralizing epitope of interest followed by targeted depletion of Òoff-targetÓ competitor B cells using an antigen lacking said epitope carrying a chemotherapeutic drug.
VRC01-class antigens eOD-60mer or MD39-VGT3.1 were modified using bioconjugation chemistries to bear cytotoxic small molecule payloads forming antigen-drug conjugates (AgDCs) characterized by immunoassays and spectroscopy. Antigen drug conjugates bearing four different FDA-approved chemotherapeutic payloads were toxic against antigen-specific VRC01+ Ramos B cells in vitro, however primary VRC01+ splenocytes were found to be resistant to all payloads except one: PBD dimers. In vivo, antigen drug conjugates bearing PBD dimers were observed to be highly potent in deleting antigen-specific B cells in both VRC01+ adoptive transfer systems and in VH1-2 humanized mouse models where off-target, non-neutralizing germinal center B cells were targeted using epitope knockout AgDCs.
Selective elimination of distracting GC B cells has the potential to remodel the output of vaccine induced immunity in cases like HIV where immunodominance thwarts protective responses. |
| Arden Edgerton |
Drexel University College of Medicine, Microbiology and Immunology Program |
Targeting the Adenosine Pathway: ADA-1 as a Novel Strategy to Reinvigorate Exhausted CD8+ T Cells in Chronic Infection and Cancer |
CD8+T cells play a crucial role in the immune response against viral infections and cancer. However, persistent antigen exposure in chronic infection and cancer leads to T cell exhaustion. Consequently, restoring T cell function has become a primary goal in developing new immunotherapies. Despite early success with strategies aimed at rejuvenating CD8+T cells, their efficacy remains limited in cases such as HIV and solid tumors, highlighting the need for alternative targets and approaches. The adenosine pathway plays a key role in controlling inflammation and preventing tissue damage during inflammatory conditions. However, this pathway can become dysregulated in chronic infection and the tumor microenvironment, leading to excessive extracellular adenosine (eADO) accumulation. eADO binding to A2aR (the predominant, eADO receptor on T cells) leads to inhibition of TCR activation, effector function and cytokine production. Adenosine deaminase-1 (ADA-1) regulates levels of eADO, catalyzing the irreversible deamination of adenosine to inosine. ADA-1 also acts as a membrane bound ectoenzyme facilitating immune synapse formation, increasing T cell stimulation and proliferation. We hypothesize that ADA-1 will restore the function of exhausted CD8+T cells ex-vivo and in vitro by decreasing concentrations of adenosine, increasing concentrations of inosine as an alternative fuel source/driver of stemlike phenotype, and enhancing CD8+T cell function. Ex-vivo we will utilize primary human samples from a cohort of HIV+ patients to investigate if ADA-1 treatment can rescue exhausted CD8+T cell function in the context of HIV. In vitro, we will use an established T cell exhaustion model with chronic stimulation of CAR-T cells to assess if ADA-1 treatment can restore the function exhausted CAR-TÕs. Reversed exhaustion in both models will be measured by increased (1) stemlike phenotype, (2) cytokine secretion, (3) antigen specific proliferation and (4) killing of target cells. |
| Hannah Yamagata |
University of Pennsylvania |
mRNA lipid nanoparticle platform with aromatic ionizable lipids confers vaccine protection with reduced off-target liver delivery |
While mRNA lipid nanoparticles (LNPs) have received clinical approval as a vaccine platform for COVID-19, their tendency to accumulate in the liver represents a significant challenge for vaccine applications due to the potential for hepatic toxicity. As it has been well established that the ionizable lipid component in the LNP formulation plays a tremendous role in efficacy and tropism, we hypothesized that altering lipid tail length and regiochemistry will affect vaccine performance. Here, a library of novel ionizable lipids (ILs) that are highly modular in their tail lengths and regiochemistries will be presented. The ILs in this structurally diverse library also have bioreducible disulfide bonds, which may aid in decreasing LNP toxicity. We found that LNPs in this platform demonstrate comparable vaccine performance to SM-102, ModernaÕs COVID-19 vaccine lipid, yet have decreased hepatic delivery. |
| Liuqian Wang |
University of Pennsylvania, Department of Bioengineering |
Amelioration of LNP-Associated Inflammation through Targeted Inhibition of Signaling Pathways |
Lipid nanoparticles (LNPs) have emerged as a key technology for delivering nucleic acids, notably in the COVID-19 vaccines. However, LNP-associated inflammation (LAI) limits their broader therapeutic applications. Even empty LNPs can trigger an innate immune response, leading to the secretion of pro-inflammatory cytokines such as IL-6 and TNF-_. This immune activation is largely caused by endosomal membrane damage, which happens during the endosomal escape of LNPs. The resulting damage provokes severe inflammation and exacerbates existing inflammatory conditions. There is a need for strategies to mitigate this inflammation to enhance the safety and efficacy of LNP-based therapeutics. We hypothesized that LAI is triggered by endosomal membrane damage, and subsequently activates multiple inflammatory pathways, including galectin-mediated recognition, NF-_B activation, etc.
Our study investigates various peptide and small molecule inhibitors targeting these key pathways, such as Olitigaltin (galectin inhibitor), NLRP3/AIM2-IN-3 (inflammasome inhibitor), E-64 (Cathepsins inhibitor), U0126 and Trametinib (MEK pathway inhibitors). We assess the ability to reduce LAI using an in vitro RAW 264.7 macrophage model by measuring the concentrations of pro-inflammatory cytokines such as IL-6 and TNF-_ through ELISAs and bead-based immunoassays and quantifying NF-_B activity via a reporter cell line. Inhibitors were tested both in free form and when co-loaded into LNPs to evaluate their impact on mRNA delivery efficiency and inflammatory responses.
Preliminary results indicate that inhibitors U0126, Trametinib, and Olitigaltin significantly reduce inflammation while maintaining effective mRNA expression and low cytotoxicity. Additional promising candidates will be tested in vitro, and the most effective candidates will be evaluated in vivo using a mouse model to assess their efficacy in mitigating LAI across different administration routes.
This research identifies potential inhibitors that can reduce LAI and their mechanisms and could lead to safer, more effective LNP-based therapies by mitigating LAI, particularly in patients with pre-existing inflammatory conditions. |
| Jarelis Cabrera |
Drexel University |
Developing Functionalized Liposomes for Depletion of Microglia Following Traumatic Brain Injury |
Traumatic brain injury (TBI) occurs when external forces impact the brain. TBIs are a consequence of motor vehicle accidents, injuries sustained in contact sports, or experiences encountered by combat veterans. Current medical treatment of TBI primarily focuses on alleviating symptoms rather than addressing the root cause of the injury and long-term effects. One emerging hypothesis is that aspects of long-term TBI symptoms may trace back to neuro-immune response following injury. The goal of this project is to develop functionalized nanoliposomes loaded with a membrane-impermeant toxin, chlodronate, such that they can be used to deplete the activated microglial we hypothesize are at the root of some long-term TBI symptoms. Here, we develop chlodronate loaded liposomes with surface-exposed, carboxylic acid-terminated, PEG-2000 spacers. These allow coupling of proteins, such as antibodies targeting specific receptors on our target microglial population, using EDC/NHS chemistry. Once the liposome preparation protocol is optimized, I will use these liposomes to treat mouse models of TBI and assess changes in brain structure and function using IHC and behavioral measurements. |
| Seung-Hyun B. Ko |
University of Pennsylvania |
Exosome-driven PD-1 localization as a mechanism for suppressing cytotoxic T-cells |
Solid tumors establish and develop within microenvironments capable of potently suppressing host immune responses against the tumor. Recent studies demonstrate that cancerous cell-secreted exosomes contribute to tumor immunosuppression by carrying upregulated levels of the immune checkpoint molecule PD-L1. However, given the limited capacity of exosomes to carry such cargo, it is unclear what contributes to their potent immunosuppression observed experimentally. Our collaborators demonstrated that the adhesion molecule ICAM-1 is necessary for exosomes to successfully bind and suppress cytotoxic T-cells. Furthermore, our lab previously developed a biophysical Monte Carlo model and demonstrated that the potency of nanoparticle/cell interactions is dependent on multivalent binding and clustering of ICAM-1. Here, we use this model to simulate the exosome/T-cell interface in conjunction with STORM superresolution microscopy and find that exosomes alter the localization of PD-1 on the T-cell membrane. Specifically, we find that bound PD-1 localizes in tighter clusters than T-cell LFA-1 molecules bound by exosomal ICAM-1. Furthermore, we combine these simulations with a stochastic model of T-cell signaling and find that such clustering patterns lead to significantly lower levels of phosphorylated AKT, a critical kinase for cytotoxic T-cell activation and development. These results provide novel insight into how PD-1 clustering may be a mechanism by which cancerous exosomes potently suppress recipient T-cells. |
| Margaret Billingsley |
MIT |
Layer-by-Layer Nanoparticles for Cytokine Therapy in Ovarian Cancer Minimal Residual Disease |
After receiving initial therapeutic interventions including aggressive chemotherapies and debulking surgeries, over 85% of high grade serous ovarian cancer patients will relapse with incurable disease. These relapses are attributed to minimal residual disease (MRD)Ñthe persistence of small populations of resistant cancer cells that remain after frontline treatments. In an effort to eliminate the remaining ovarian cancer MRD and prevent recurrence in these cancer patients, recent work has begun exploring the use of cytokines and chemokines to recruit immune cells and target them toward these rare cancer cell populations. Though these interventions hold promise, the translation of cytokine-based therapies is limited by their high toxicity and rapid clearance. Thus, in this work, layer-by-layer nanoparticles (LbL-NPs) are explored as a delivery platform that allows for targeted delivery with improved pharmacokinetics. Specifically, the therapeutic cytokine or chemokine is conjugated to the surface of a liposomal core that is then modified with polymeric layers to allow for specific anchoring on the surface of ovarian cancer cells. Here, we explore the use of varied conjugation strategies with a range of stabilities to fine-tune the in vivo release kinetics of therapeutic cytokinesÑsuch as IL-12, IFN-alpha, and XCL1--from the surface of these LbL-NPs following IP administration. Ultimately, by optimizing the loading strategy of each cytokine to best suit its optimal release kinetics, this work aims to maximize therapeutic efficacy while minimizing off target toxicities in the MRD disease state. |
| Sarah O'Neill |
University of Pennsylvania |
Novel Decoy Oligodeoxynucleotide-LNP System for Inhibition of NF-kB Activity In Vitro |
With the limitations of small molecule inhibitors (limited specificity, require an active site, difficult to load into nanocarriers), the nanomedicine community has looked to nucleic acid-based cargo, such as siRNA, to limit the gene expression of multiple disease targets. Double-stranded decoy oligonucleotides (ODNs) are a promising option that bind to the consensus sequence of a specific transcription factor, immediately preventing it from binding to and activating transcription of multiple genes that drive a disease pathology. The nuclear factor kappa B (NF-kB) transcription factor family, a key regulator of innate and adaptive immune functions, has been a popular target for decoy ODNs with the goal of treating various inflammatory diseases since the 1990s. However, their efficacy was severely limited by ineffective delivery vehicles. Several decoy ODN-based applications have since attempted in vivo delivery using liposomes. As the next generation of nanocarrier platforms, LNPs have more complex internal architecture, better stability, and can be targeted to specific locations in the body. Here, we have added these decoy ODNs into our LNPs to reduce NF-kB activity in vitro as a preliminary proof of concept for a novel nanomedicine-based platform, which can be easily tailored to any transcription factor by altering the ODNÕs short sequence. |
| Lindsay Barger |
Drexel College of Medicine |
MDSC-mediated immune suppression as a driver of HIV and Melanoma co-morbidity |
The lifelong use of antiretroviral therapies (ART) can manage HIV infections to significantly extend the lifespan of PLWH. As PLWH are aging, there is an increase in age-related non-AIDS defining cancers. Compared to uninfected age-matched individuals, there is a four-times greater mortality rate to cutaneous melanoma in PLWH. PLWH are challenged with chronic inflammation that may impair immune responses to cancer. Resulting from chronic inflammation, PLWH have an increase of a highly heterogenous and immunosuppressive population of cells, called myeloid-derived suppressor cells (MDSCs) that are not attenuated with ART. Elevated MDSCs positively correlate with tumor burden and poor therapeutic responses in metastatic disease of individuals without HIV. However, the contribution of high levels of MDSCs in PLWH at baseline to melanoma outcomes has not yet been investigated.
We have completed the first-ever immune-focused spatial transcriptomics analysis of untreated melanoma tumors and tumor microenvironments from PLWH. This investigation revealed a significant differential expression of genes associated with MDSC recruitment, T-cell exhaustion, and Tregs compared to healthy controls with melanoma. To explore these aspects further, we have established the first mouse model of HIV and melanoma co-morbidity using the chimeric EcoHIV virus. Immunofluorescent analysis of EcoHIV-infected lymphoid tissues revealed an increase of MDSCs compared to controls, consistent with human pathology. In addition, EcoHIV-infected mice developed and succumbed to syngeneic melanoma tumor injection more quickly than uninfected mice and showed altered immune infiltration to tumors. Overall, our data indicate that PLWH with melanoma present with an immune suppressive/exhaustive tumor microenvironment which is mirrored in our immune-competent model of HIV and melanoma co-morbidity and is potentially druggable with MDSC-directed strategies in combination with standard-of-care immunotherapies. |