| Lindsay Barger |
Drexel University College of Medicine |
Of the 38.4 million HIV infections, 75% of People Living with HIV (PLWH) have access to standardized antiretroviral therapy (ART). Although ART inhibits intracellular replication of HIV, preventing CD4+ T-cell death, the integration of HIV establishes a lifelong chronic infection. This infection induces persistent low-grade inflammation, unablated by ART. Consequently, PLWH have high numbers of exhausted T cells and immunoregulatory cells, particularly Myeloid Derived Suppressor Cells (MDSCs). MDSCs are potent immunosuppressive cells often implicated in “cold” tumor formation, i.e., an immune evasive/suppressive tumor microenvironment. Cold tumors inherently reduce therapeutic efficacy and immune responses, notably in melanoma patients. As PLWH who develop melanoma are four times more likely to succumb to cancer, there is a pressing need to develop treatments for the unique immunological dynamics of PLWH. This project characterizes MDSCs in PLWH as a driving factor to cold tumor formation and HIV-associated mortality. To understand the mechanisms of MDSC-mediated immune suppression, we established a mouse model of HIV and melanoma co-morbidity using the chimeric EcoHIV virus. Immunohistochemistry analysis of EcoHIV-infected lymphoid tissues revealed an increase in MDSCs compared to controls, consistent with human pathology. Additionally, EcoHIV-infected mice developed and succumbed to tumors more quickly than uninfected mice, suggesting dysregulated responses to cancer. To further characterize MDSCs in this model, we will detail immune infiltration in tumor-bearing EcoHIV mice. We will validate our findings through a co-culture model of patient derived MDSCs, CD8+ T cells and melanoma cancer cells to identify novel druggable targets of MDSCs in HIV and melanoma co-morbidity. |
| Ajay Thatte |
University of Pennsylvania |
Cell-based therapies for autoimmune diseases have gained significant traction, with several approaches centered around the regulatory T cell (Treg) – a well-known immunosuppressive cell characterized by its expression of the transcription factor Foxp3. Unfortunately, due to low numbers of Tregs available in circulation, harvesting and culturing Tregs remains a challenge. It has been reported that delivering Foxp3 to CD4+ T cells can result in a Treg-like phenotype; however, current delivery methods result in inefficient engineering of these cells. Here, we develop an ionizable lipid nanoparticle (LNP) platform to effectively deliver Foxp3 mRNA to CD4+ T cells. We successfully engineer CD4+ T cells into Foxp3-T (FP3T) cells that transiently exhibit an immunosuppressive phenotype and functionally suppress the proliferation of effector T cells. These results demonstrate the promise of an LNP platform for engineering immunosuppressive T cells with potential applications in autoimmunity therapies. |
| Ricardo Whitaker |
Drexel University |
"Volumetric Muscle Loss (VML) is a debilitating condition defined by the rapid loss of muscle mass, leading to permanent impairment. The current standard of care is highly inefficient and clinical trials using decellularized matrix to fill the defect showed only a 37% improvement in muscle force. Furthermore, the molecular/cellular processes governing VML repair are poorly understood, hindering treatment development. The goal of this study is to uncover the dysfunctional immune response following VML to instruct better treatments. We employed a murine model of VML using subcritical size injuries (Regenerative), and critical size injuries (Fibrotic). Fibrotic macrophages displayed a higher inflammatory and less reparative phenotype, observed via flow cytometry. Interestingly, splenic macrophages displayed a similar phenotype, suggesting systemic repercussions from this local injury. NanoString of FACS-sorted macrophages indicated a broad and indiscriminate gene downregulation of Fibrotic macrophage at Day 1after injury. This suggests that changes in macrophage phenotype occur soon after injury, and prior to onset of fibrosis. NanoString and Luminex analyses of whole-muscle and blood, respectively, displayed a higher expression of neutrophil chemokines and G-CSF after Fibrotic injuries. Muscle flow cytometry analysis confirmed an increase in neutrophils in Fibrotic injuries compared to Regenerative ones. These results point to neutrophils as possible macrophage modulators. Similar analyses on neutrophil demonstrated an increase in apoptosis in Fibrotic neutrophil, with little differences in gene expression compared to Regenerative neutrophils. Partial neutrophil depletion improved macrophage phenotype by decreasing inflammatory and increasing reparative markers expression, at the gene and protein level. However, no improvement in muscle structure was observed. These results demonstrate that although partial neutrophil depletion improves macrophage phenotype, that is not enough to leverage tissue healing. We have performed, to our knowledge, the most thorough macrophage characterization, locally and systemic, in VML. These novel results can instruct the development of efficient therapies for VML." |
| Victoria Nash |
Drexel University |
Immunomodulatory proteins are difficult to add to engineered tissue, however utilizing biotin-avidin interactions to conjugate these proteins to tissues could be the solution. Biotin and avidin are conjugated to proteins, biomaterials, and even cells, without effecting their intrinsic properties. However, its use as a drug delivery system has not been explored. Therefore, the goal of this study was to 1) define how avidin variants can influence macrophage phenotype 2) determine how bioconjugation parameters control biotin-avidin interactions to deliver an immunomodulatory cytokine from a model biomaterial and 3) characterize how presentation of that cytokine via biotin-avidin interactions effect macrophages’ response to the modified biomaterial. First, we characterized the effect avidin and its variants have on macrophages using RNASeq. Surprisingly, gene ontology analysis showed that avidin, which historically was used as a vaccine adjuvant, did not significantly affect any pathways. Then, we demonstrated control over bioconjugation parameters of biotin density and avidin association with a model biomaterial. We highlighted how a biotinylated immunomodulatory cytokine, interleukin 4 (IL4), was released from biotin-CaptAvidin modified scaffolds for up to ten days when free biotin was in the media. Finally, we conducted a pilot study to compare how biotin-avidin mediated presentation of biotinylated IL4 differs from other delivery methods, such as adsorption or directly conjugating IL4 to the biomaterial. Interestingly, biotin-CaptAvidin mediated presentation may promote a hybrid macrophage phenotype, while direct conjugation of IL4 may promote fibrosis, indicated by multiplex gene expression analysis. Overall, this work suggests that biotin-avidin affinity interactions can be leveraged as a platform technology to alter tissue engineered constructs by demonstrating control over bioconjugation properties for a model biomaterial. In its entirety, these results begin to describe a platform technology that can promote integration of engineered tissue into native tissue through the utilization of immunomodulatory cytokine delivery via biotin-avidin affinity interactions. |
| Gina Cusimano |
Drexel University College of Medicine |
"Human immunodeficiency virus (HIV) remains a prominent global health threat for which no prophylactic vaccine is available. HIV vaccine development has proven to be challenging due to the metastability and heavy glycosylation of the envelope protein which aids in HIV immune evasion. Recent HIV vaccine efforts have focused on utilizing trimeric immunogens that more closely mimic the native envelope trimer instead of monomeric envelope proteins. One such trimeric immunogen known as BG505-SOSIP has been engineered to increase the stability, exposure of neutralizing epitopes and subsequent immunogenicity of HIV envelope making it a promising HIV vaccine antigen. We have demonstrated that adenosine deaminase-1 (ADA-1) is critical to the T follicular helper (TFH) function and survival. In fact, ADA-1-induced TFH function that improved the magnitude and durability of both cellular and humoral vaccine immune responses in vivo. We therefore hypothesized that ADA-1 combined with the improved HIV envelope antigen BG505-SOSIP would result in enhanced qualitative and quantitative humoral and cellular HIV specific responses. Mice co-immunized with plasmid encoded ADA-1 (pADA) and BG505-SOSIP (pSOSIP) displayed increased SOSIP specific antibody titers with enhanced affinity as well as increased frequencies of SOSIP specific memory B cells when compared to non-adjuvanted pSOSIP. Additionally, pADA co-immunized mice exhibited enhanced antigen specific T cell activation, polyfunctionality, and degranulation following stimulation with BG505 peptide pools. pADA elicits a dose sparing effect as exhibited by enhanced immune responses with pSOSIP doses as low as 1ug. These data suggest that pADA enhances both cellular and humoral immunity to BG505-SOSIP making it a promising adjuvant for HIV targeting vaccines. |
| Cassandra Villicana |
Stanford University |
Introduction: While most biomaterials on tissue regeneration focus on stem cell differentiation, recent studies showed tuning biomaterials compositions can induce divergent immune responses to impact tissue regeneration. Tissue derived extracellular matrix (tdECM)-based hydrogels have shown promise in inducing regenerative immune response when injected in soft tissues. However, their application for bone regeneration remains underexplored. Furthermore, most tdECM hydrogels are nanoporous, yet macroporosity is desirable for bone regeneration in vivo. The goals of the present study are to develop macroporous microribbon scaffolds containing bone-derived ECM (bECM), and assess the effect of tuning bECM on macrophage polarization and MSC osteogenesis in vitro and in vivo. Methods: A wet-spinning process was used to make microribbons using a mixture of gelatin with varying ratio of bECM (0% to 50%). Scaffold was characterized using biochemical assays, mass spectrometry, fluorescence labeling and imaging, and mechanical testing. Effects of varying bECM on MSC osteogenesis and macrophage polarization was tested in vitro and in vivo using a critical-sized mouse cranial defect model. Results and Discussions: Fluorescence imaging confirmed incorporation of bECM into microribbons, which crosslinked into 3D macroporous scaffolds, and supported high cell viability. In vitro cell studies showed varying bECM ratio modulated MSC osteogenesis and macrophage polarization in a dose-dependent manner. When implanted in vivo, 15% bECM scaffold synergized with a low dosage of mineral particle coating to induce robust endogenous bone regeneration, with over 50% bone defect filled by week 2. Flow Cytometry revealed that increased bone bone formation is associated with decreased M1 macrophage recruitment but increased recruitment of endogenous stem cells. Histology showed 15% bECM led to most robust bone formation, accompanied by increased vascularization, as shown by CD31+ staining. In summary, we report a method to incorporate bECM into microribbon hydrogels, which provides a promising new macroporous scaffold for bone regeneration via immunomodulation. |
| Sophia Kioulaphides |
Georgia Institute of Technology |
Type 1 Diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin-producing β-cells in the pancreas. The most promising therapy thus far is the transplantation of cadaveric islets, but there are significant issues surrounding the lack of a cell source, poor engraftment, and significant immune responses to the graft. Our lab has previously developed a poly(ethylene glycol) (PEG)-based hydrogel tethered with vascular endothelial growth factor (VEGF) that leads to engraftment of islets in the gonadal fat pad (gFP) of diabetic mice. In recent efforts, stem cell-derived β (SC-β) cells have become a promising scalable source of insulin-producing cells. Preliminary results have shown success when SC-β cells are delivered to the gFP of male diabetic, immune deficient SCID-beige mice, which possess defective T, B, and natural killer (NK) cells, but still possess myeloid cells. We are using imaging mass cytometry (IMC) as a tool for spatial immune profiling of the gFP’s of diabetic SCID-Beige mice that were transplanted with SC-β cells with or without PEG-VEGF hydrogels. IMC allows the user to stain up to 40 markers, providing extremely valuable data on tissue structure and immune cell infiltration, and early results from our lab have validated IMC by imaging the infiltration of innate and adaptive immune cells to the pancreases of NOD mice. SCID-Beige gFP samples will be stained and analyzed according to IMC protocols with a customized antibody panel containing markers for SC-β cell maturation, innate immune cells, and tissue architecture. These images provide valuable data on the immune protection from the hydrogel carrier, an understanding of myeloid infiltration, maturation of SC-β cells, and vascularization at the transplant site. All these results establish a base for future studies using IMC to analyze transplants with immune modulated PEG-VEGF hydrogels in diabetic immune competent mice and larger animal models. |
| Shreya Soni |
Drexel University |
Macrophages exist across a spectrum of phenotypes broadly characterized as M1-like (inflammatory) and M2-like (reparatory), the former of which underlies chronic inflammation and failed tissue healing. The use of small molecule immunotherapeutics to modulate macrophage phenotype and promote tissue repair remains limited by poor solubility, rapid blood clearance, and an inability to target these cells. We have therefore developed a macrophage-targeted drug delivery vehicle (cyclodextrin nanoparticle, CDNP), composed of β-cyclodextrin (CD) to improve drug solubility and bioavailability necessary to achieve M1 to M2 re-education. CDNPs were prepared by crosslinking succinylated-CD with L-lysine; particle diameter (25-125nm) was controlled via the concentration of EDC/NHS catalyst. Increasing CDNP size improved uptake kinetics and the extent of CDNP accumulation at 24 hrs in RAW264.7 cells. The largest CDNP (123.3±4.3nm) was therefore selected for continued study. Antibody blockade of mannose and scavenger receptors reduced CDNP uptake, indicating their pivotal role in macrophage-targeted delivery. A dual-stage drug screen examining a range of immunomodulatory drugs revealed that celastrol robustly suppresses the damaging M1-like phenotype (IC50<100nM for NF-κB inhibition, RAW-BlueTM reporter assay) while concurrently upregulating M2-associated genes by 10-fold. CDNPs were loaded with celastrol through supramolecular guest-host interactions (Keq=0.474mM, SPR) by simple mixing in water. Celastrol loading did not inhibit CDNP uptake, and treatment of M1-polarized RAW-BlueTM cells exhibited dose-dependent knockdown of inflammatory signaling that was improved relative to soluble drug alone. Intravenous injection of CDNP in a murine model of myocardial ischemia-reperfusion injury resulted in a >2.5 times increase in myocardial uptake relative to sham controls due to macrophage accumulation, which persisted over 28 days. CDNPs are a promising macrophage-targeted drug delivery vehicle that can be used to modulate the post-injury immune environment, including for the treatment of myocardial infarction. |
| Tina Tylek |
Drexel University |
Macrophages are key players of the innate immune system and the primary initiators of inflammatory responses. They differentiate from monocytes infiltrating the wound site and are involved in different wound healing and tissue repair stages, making them an attractive choice for cell therapy applications. However, macrophage cell therapy has been limited by their tendency to change phenotype in response to local cues rapidly. Therefore, control over macrophage phenotype is critical for their use in cell therapy applications. Hence, in this study, we investigated the controllability of uptake and efficacy of PLGA microparticles (MP) encapsulating dexamethasone (Dex), an anti-inflammatory and antifibrotic drug, to control the macrophage phenotype intracellularly. Microparticle uptake by murine macrophages was increased with increasing dose and incubation time and was higher for blank MPs. Furthermore, increasing the dose of Dex MPs led to dose-dependent phenotype changes with increasing expression of CD206 (pro-reparative), CD9 (fibrotic), MHC2 (Antigen presentation) and decreasing expression of CD163, CD301b (pro-reparative), and CD86 (pro-inflammatory), demonstrating the importance of dose control. Overall, Dex-MP-loaded macrophages exhibited an anti-inflammatory, antifibrotic macrophage phenotype (downregulation of TNFa, IL1b, CD86; upregulation of CD163, CD206, MMP8), which could be maintained even when cultivated with various environmental stimuli (LPS/IFNg, IL4/IL13, IL10, IL17a) over seven days. Upon administration directly to inflamed or fibrotic lungs of mice, macrophages containing Dex-MPs exhibited a phenotypic profile that was distinct from in vitro cultivated Dex-MP macrophages but also host macrophages. Furthermore, compared to blank-loaded and control macrophages, upregulation of CD206 and CD301b and downregulation of MH2 and CD86 were maintained. These results show that the intracellular release of Dex did affect macrophage phenotype but was also affected by the microenvironment. In conclusion, intracellular control of macrophages for cell therapy with Dex is a promising strategy controllable by cell MP loading, leading to a phenotype that can be (partly) maintained in various environments in vitro and in vivo. |
| Marzieh Daniali |
Department of Pharmacology and Physiology, Drexel University |
Macrophages play a pivotal role in immune homeostasis and stimuli that alter macrophage functions can have a substantial impact on the innate immune response. Although primarily studied in the context of reward, motor control and learning, the neurotransmitter dopamine has been shown to have substantial immunoregulatory capacity in many types of immune cells, including macrophages. Our previous studies show that dopamine can drive the translocation of NF-B, priming the NLRP3 inflammasome in increasing the transcription and translation of NLRP3, PYCARD and pro-IL-1β. These effects result in a more robust release of IL-1 cytokines, such as IL-1β, in response to inflammasome activation. There are several different types of inflammasomes, which serve as critical sensors of different types of cellular stress, damage, or pathogen invasion. These multi-protein complexes consist of several proteins, including an effector protein such as NLRP3, NLRC4 or AIM2, procaspase-1 and an adaptor protein called PYCARD or ASC. Based on our prior studies, we hypothesize that activation of dopamine receptors on myeloid cells alters the formation and activation of inflammasomes in macrophages. To examine this, primary human monocyte-derived macrophages (hMDM) were derived from deidentified human blood, matured in vitro for six days, and treated with dopamine (10-6M). The hMDM were lysed at several different timepoints (3, 4.5, 6 and 7.5 hrs post treatment) then evaluated for changes in transcription and expression of inflammasome component, caspase-mediated signaling activation and changes in cellular morphology. Our results show distinctive inflammasome formation kinetic profiles relative to LPS (positive control), along with changes in both morphology and the emergence of 'speck' formations. These findings shed light on the interplay between dopamine signaling and inflammasome activation, underscoring their potential implications for immunomodulation therapeutic strategies. |
| Ni Su |
Stanford University |
Introduction. The aged population suffers from a higher incidence of bone injuries and delayed bone healing, yet most research on bone regeneration to date is limited to using cells from young donors and using young animal models. Previous patient data and animal studies suggest aging is associated with decreased stem cell differentiation and increased inflammation. However, how immune-stem cell crosstalk modulates bone healing during aging remains largely unknown. Given the short supply of aged animals, there remains a critical need for in vitro experimental models that can recapitulate key features of bone healing during aging. Our group has previously reported microribbon (µRB) hydrogels as 3D macroporous scaffolds that support robust bone regeneration in vitro and in vivo by promoting regenerative immune-stem cell crosstalk. Here, we seek to harness µRB hydrogels to develop 3D in vitro models that mimic key features of aging-associated delayed bone healing and immune cell-stem cell crosstalk. We hypothesize that culturing macrophage (Mφ) and mesenchymal stem cells (MSCs) from aged or young donors in µRB scaffolds will mimic aging-associated changes in inflammation and bone healing. Methods. Mφ and MSCs were isolated from young (5 weeks) and aged mice (56 weeks). Effects of aging on Mφ phenotype or MSC osteogenesis were evaluated first using a 3D monoculture model. To mimic the Mφ/MSC crosstalk, a conditioned medium (CM) treatment model was used. Serum from young or aged mice was also supplemented. Results and discussions. In 3D monoculture, MSCs from aged mice exhibited delayed proliferation and mineralization, and Mφ from aged mice showed increased inflammation compared to young cells. CM from aged MSC triggered more inflammatory cytokine release from Mφ, suggesting senescence-associated secretory phenotype (SASPs) from aged MSC may promote inflammation. Furthermore, serum from the aged mice significantly inhibited the mineralization of young MSCs, indicating aging-associated changes in serum composition also contribute to delayed bone healing during aging. Together, these results demonstrate our 3D models recapitulate key features of aging-associated delayed bone regeneration, offering a unique tool for mechanistic exploration and drug discovery by targeting immune cell-stem cell crosstalk. |
| Juan Cortes-Troncoso |
Drexel University, School of Biomedical Engineering, Science and Health Systems |
"Diabetes foot ulcers (DFU) are one of the major chronic complications of diabetes and the main cause of disability and death in the USA. The mechanisms behind impaired DFU healing are complex, but chronic inflammation is a major contributor. After recognizing that transition from the early inflammatory to the late resolution phase is required for successful healing, we developed a composite biomarker using the ratio of 4 early-stage pro-inflammatory gene markers to 3 late-stage inflammation-resolution biomarkers, referred to as the Inflammation Index (II). The II is an indirect measurement of the wound’s healing stage. Our previous studies measured the II via RT-qPCR using RNA extracted from debrided wound tissue. To evaluate the expression of biomarkers that comprise the II, quality RNA is essential. The chronic wound environment is particularly damaging for RNA because of its high levels of enzymes and cellular debris containing RNases. Therefore, our goal in this project was to optimize biomarker detection and determine the minimum sample quality and quantity in which the II can be reliably detected. Furthermore, we also optimized the RNA extraction procedures to maximize RNA quantity and quality yield. By using an experimental model of intact and partially degraded RNA from in vitro-cultivated macrophages derived from human primary monocytes, we prove how quality control (QC) metrics affect biomarker expression. We determine that degradation-influenced shifts of threshold values (Ct-values) can be compensated by calculating delta-Ct values between test genes and the mean values of several control genes. We demonstrate that the II can be measured on samples with even low-quality and quantity RNA. Additionally, we validate how sample storage/shipping conditions affect RNA QC metrics. Based on these results, we conclude that by using controllably degraded cell samples in vitro to model tissue wounds, the measurement of the II in DFU samples was appropriately optimized. |
| Sridatta V. Teerdhala |
University of Pennsylvania |
Chimeric antigen receptor T-cell (CAR-T) therapy has shown remarkable success in treating certain hematological malignancies. However, challenges such as cytokine release syndrome and antigen escape have prompted the need for alternative approaches. Natural killer (NK) cells possess the ability to recognize and eliminate cancerous cells without prior sensitization. As a result, CAR-NK cell therapies are being developed to harness NK cells’ innate cytotoxicity and reduced risk of graft-versus-host disease. Incorporating both CAR-T and CAR-NK therapies could offer a very promising synergistic approach that combines the strengths of adaptive and innate immune responses while countering their respective weaknesses; however, to achieve a combined therapy, effective CAR delivery is essential. Current methods use viral delivery vectors, which induce permanent expression, causing potential severe adverse effects. Lipid nanoparticles (LNPs) are effective delivery vectors and induce transient expression due to their proven success in delivering mRNA (e.g., COVID-19 vaccines). Thus, by encapsulating CAR mRNA constructs within LNPs, it may be possible to simultaneously modify CAR-T and CAR-NK cells and subsequently produce a combined therapy. Here, LNPs were designed for mRNA delivery to NK-92 and primary human T cells. A library of 24 ionizable lipids was synthesized, formulated into LNPs using two different excipient ratios, and screened for luciferase mRNA delivery, revealing a single LNP, C12i-200, that had enhanced mRNA delivery in both cell types. This LNP will be used for CD19 CAR mRNA delivery to primary human T cells and NK-92 cells, after which, the combined cellular therapy will be evaluated in a co-culture assay with Nalm-6 acute lymphoblastic leukemia cells, and finally CAR mRNA delivery in mice models to test for cancer-killing activity. Positive future results would open exciting possibilities for the development of a novel and highly effective combined CAR-T and CAR-NK cell therapy approach for the treatment of hematological malignancies. |
| Arielle M. D'Elia |
Drexel University |
Biotherapeutics exhibit excellent target specificity and are used to treat a range of diseases from cancer to autoimmune dysfunction. Their use can benefit from local delivery systems (to concentrate drugs at the site of action) and sustained release (to afford prolonged therapeutic presentation). Here, we develop locally-injectable granular hydrogels that undergo guest-host complexation with synthetically modified proteins. The interaction of the host-modified hydrogels with guest-modified proteins enables their sustained local delivery, with release rates tunable through hydrogel avidity. To form the hydrogels, dextran and β-cyclodextrin were methacrylated (DexMA, MeCD) via esterification with glycidyl methacrylate. Co-polymerization (5mM LAP, 10mW/cm2) of MeCD (10%w/v) with DexMA (5%w/v) yielded solid bulk hydrogels (G'~15kPa) with a high host concentration. Hydrogels were processed into microgels (34.0 +/-16.7µm) by sequential extrusion fragmentation (18-30G needles) and jammed via centrifugation to yield a shear-thinning injectable granular hydrogel. Bovine serum albumin (BSA, a model biomolecule) was conjugated to supramolecular guests (adamantane, Ad) via EDC in near quantitative yield (1-10 eq. Ad per BSA), with maintenance of guest–host affinity (12.0 +/- 1.81µM, by isothermal calorimetry), relative to soluble Ad. The avidity of Ad-BSA conjugates increased with the degree of modification, as assessed by surface plasmon resonance. Guest-modified BSA (0, 2.5, 5 eq. Ad per BSA) release was monitored by conjugation with orthogonal fluorescent labels; guest modification attenuated burst release and sustained BSA release for greater than one month, with release rate dependent upon the degree of guest modification. Furthermore, the modification of cytokines (IL-10, IL-4, IFNy) with Ad did not affect bioactivity (PCR, treated bone marrow-derived macrophages) and enabled controlled release for greater than two weeks. The bioconjugation proceeds under mild conditions, is non-selective, and is therefore applicable to the conjugation of a range of biotherapeutics (e.g., chemokines, cytokines, and antibodies) that may benefit from sustained local delivery. |
| David A. Christian |
University of Pennsylvania |
"In the peritoneal cavity, the omentum contains fat-associated lymphoid clusters (FALCs) whose role in response to infection is poorly understood. After intraperitoneal immunization with Toxoplasma gondii, conventional type 1 dendritic cells (cDC1s) were critical to induce innate sources of IFN-γ and cellular changes in the FALCs. Unexpectedly, infected peritoneal macrophages that migrated into the FALCs primed CD8+ T cells. Although T cell priming was cDC1 independent, these DCs were required for maximal CD8+ T cell expansion. An agent-based computational model and experimental data highlighted that cDC1s affected the magnitude of the proliferative burst and promoted CD8+ T cell expression of nutrient uptake receptors and cell survival. Thus, although FALCs lack the organization of secondary lymphoid organs, cDC1s resident in this tissue coordinate innate responses to microbial challenge and provide secondary signals required for T cell expansion and memory formation."
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| Christopher Pierce |
Oklahoma University |
Neurological disorders are the leading cause of disability and the second leading cause of death worldwide. Toward the goal of intracerebral delivery of disease-modifying therapeutics, we created an injectable brain-tissue inspired hydrogel depot named “Parenchygel”. As a key first step in development of this novel delivery device, the present study sought to characterize the systemic immune response, local brain tissue response, and neurological consequences of Parenchygel injection. We hypothesized that Parenchygel would not stimulate a systemic inflammatory immune response or cause neurological damage in a healthy rat model. Parenchygel was synthesized by cross linking an optimized blend of chemically modified hyaluronic acid, heparin, poly (ethylene glycol), and PLGA nanoparticles. The hydrogel immunogenicity was evaluated using healthy rats with a treatment group (Parenchygel, n = 5) and a control group (saline, n = 5). The rats were injected once with 20 µl of Parenchygel or saline, respectively, and monitored for 6 weeks. We evaluated systemic toxicity by analyzing concentrations of inflammatory and anti-inflammatory cytokines from blood plasma, isolated on days 7, 14, and 28. Cytokines were quantified using a Biorad® Th1 Th2 multiplex assay. We performed hematoxylin and eosin (H&E) staining to evaluate the local immune response around intracerebral injection site. 2-way ANOVA revealed no statistically significant differences in blood plasma cytokine composition or concentration between treatment and control groups at any time point, indicating that intracerebral Parenchygel injection does not induce a systemic immune response. Behavioral analysis did not indicate any motor, sensory, or reflex neurological consequences caused by the hydrogel. Taken together, these results indicate that a single intracerebral injection of Parenchygel is safe in healthy animals. |
| Sydney Yang |
University of Maryland |
LL37 is a cationic antimicrobial peptide (AMP) exhibiting broad-spectrum activity against bacterial pathogens making LL37 a promising therapeutic agent to overcome antibiotic resistance. However, the short half-life of LL37 limits treatment efficacy, minimizing therapeutic effect to combat infection. AMPs naturally produced in mucosal tissues are known to bind to mucus through electrostatic interactions with mucins suggested to protect AMPs from degradation. This provides motivation to develop synthetic mucus (SM) biomaterials as localized AMP delivery vehicles for prolonged release to enhance antibiotic resistant infection treatment. SM hydrogels were formulated using purified porcine gastric mucins and 4-arm polyethylene glycol thiol. LL37 was added to hydrogel solution prior to gelation for loading. Hydrogels were mechanically characterized via multiple particle tracking microrheology. Release profiles and release kinetics of LL37-SM hydrogels were assessed. The effect of LL37-SM treatment on Pseudomonas aeruginosa (PAO1) was evaluated via planktonic and biofilm growth and viability. When loaded with LL37, SM hydrogels form elastic dominant gels and, at loaded concentrations greater than 50 µg/mL LL37, degrade completely over 24 hours. LL37-SM hydrogels demonstrated diffusion dominant release of LL37 over 8 hours. As hypothesized, these results indicate LL37 is strongly associated and retained within SM biomaterials allowing for slow release over time. Compared to treatment with only LL37, SM and LL37-SM hydrogels inhibited 85% of PAO1 growth over 12 hours. LL37-SM hydrogel treatment continuously inhibited planktonic PAO1 viability over 6 hours resulting in 77% viability reduction. In addition, LL37-SM treatment inhibited biofilm growth and reduced viability over 24 hours. In combination, LL37-SM hydrogels demonstrated enhanced antimicrobial activity, suggesting LL37 activity is preserved, and bioavailability is prolonged. These findings motivate future studies to assess the effect of LL37-SM treatment on macrophage polarization and phagocytic activity in vitro and in combating bacterial infections at topical wound sites. |
| Leonor Teles |
University of Miami |
Autoimmune diseases (AD) like type 1 diabetes are caused by the disruption of T-cell tolerance to self-antigens, thereby unleashing autoreactive T-cells that attack self-tissues. AD affect 8% of the US population, and their incidence is increasing. Current clinical treatments are limited to systemic T-cell approaches with adverse side effects, so there is a need for localized, antigen-specific (Ag) therapies. A promising strategy may lie in immunomodulation mediated by Fibroblastic Reticular Cells (FRCs). FRCs are lymph node (LN) stromal cells that build 3D reticula and act as non-professional antigen-presenting cells with immunomodulatory effects. Upon inflammation, FRCs expand the LN to enable immune cell infiltration and upregulate Ag-presenting machinery (MHC class I and II) and immune checkpoint molecules (PD-L1) with limited (low CD80) co-stimulatory molecule expression. In artificial Ag-specific models, FRCs induced Ag-specific naïve T-cell deletion rather than activation, indicative of their potential for selective regulation of Ag-specific T-cells. However, the therapeutic role of FRCs in Ag-specific immunomodulation against AD has not been tested. This project focuses on designing a 3D biomaterial platform for localized, FRC-mediated immunomodulation using type 1 diabetes as an AD model. Our platform using macroporous gelatin scaffolds is biocompatible (trichrome staining, inflammatory panel ELISA, and immunohistochemistry), promotes host cell infiltration, and enables FRC viability, reticula formation, and retention in a murine subcutaneous transplant site for at least 21 days (IVIS imaging of luciferase-expressing FRC). We have also studied in vitro FRC and CD8+ T-cell interactions and confirmed Ag-specific engagement of mouse NY8.3 T-cells via proliferation (CellTrace dilution) and CD44 upregulation after culture with 3D FRCs and IGRP peptide antigen. We are currently assessing FRC interactions with mouse Ag-specific BDC2.5 CD4+ T-cells. Our platform provides a local, retrievable, Ag-specific immunomodulation to treat ADs like type 1 diabetes by selectively suppressing and/or regulating Ag-specific autoreactive T-cells. |
| Ankur Sikder |
Stony Brook University |
CAR-T therapy is a promising immunotherapy that reprograms a patient's T-cells to target cancer. For Cancers that CAR-T therapy is indicated for (DLBCL, follicular lymphoma, etc.) the average age is over 65 years old. This becomes an issue for CAR-T therapy as older patient cells are known to be less functional, as they become less sensitive to activation signals. Leading to less-than-ideal CAR-T outcomes. T-BET is an important gene that controls the expression of these inhibitory signals. Mechanical stimulation has been a major focus in our lab with mesenchymal stem cells (MSCs) being the main target, recently the lab has shown similar results in T-cells. Mechanical stimulation has been shown to have differences in effect based on age. This effect was examined in T-cells. Blood collected from patients aged 65 and 23 were isolated for T-cells. Experimental samples were treated with IL-2 and dyna beads (1:10) and exposed to 0.7g (g= 9.8 m/s^2) at 30Hz LIV stimulation. Experimental samples were stimulated for one hour, twice daily, separated by a two-hour rest period, for 5 days. Control samples were treated with the same IL-2 concentration, dyna bead ratio, but without LIV stimulation. All cell samples were counted during cell passaging. Real Time rtPCR of the T-BET gene was performed on day 5 with a GAPDH endogenous control. In the older patient sample, there was a 57% improvement in proliferation, in the younger patient sample there was an insignificant improvement in proliferation. Furthermore, T-BET expression analysis suggests that there is no significant change in the expression of T-BET between Control and LIV groups. This pilot study indicates that there may be a method to improve the proliferation of older patients (65+) T-Cells while retaining functionality with mechanical stimulation. |
| Madeline Dunsmore |
Rowan University |
Preeclampsia is a leading cause for maternal and neonatal mortality and morbidity worldwide, affecting approximately 4% of pregnancies in the US. Historically a poorly understood condition, the role of immune response disruption in the progression of preeclampsia is becoming increasingly prevalent. In addition, it is widely considered that uteroplacental hypoxia leads to these maternal manifestations of preeclampsia, with recent findings suggesting an overexpression of hypoxia-inducible factor 2 (HIF-2ɑ), but not HIF-1ɑ in placentas from people with preeclampsia. Understanding and harnessing the molecular mechanisms of HIF-1ɑ and HIF-2ɑ in immune cells could lead to potential treatment and prevention of preeclampsia. To test HIF-ɑ dynamics, we engineered a three-part, constitutively-active intracellular biosensor. The biosensor consists of: the active domain of a kinase linked to half a leucine zipper; the substrate of the kinase linked to both the cognate half of the zipper and split mNeonGreen; and the SH2 domain of the phosphorylated substrate linked to the cognate of mNeonGreen. The activation of the biosensor initiates an orthogonal phosphorylation cascade that produces a fluorescent protein. Truncated oxygen-degradation domains (ODDs) of HIF-1ɑ and HIF-2ɑ were cloned into the substrate portion of the biosensor. HEK293FT cells underwent chemical transient transfection and were subjected to hypoxic or normoxic conditions, 1% or 21% oxygen, respectively. Cells were harvested 24 hours post-transfection and analyzed using Flow Cytometry to measure mean fluorescent intensity (MFI). HIF-2ɑ performed better than expected with an approximately 40-fold increase in hypoxic conditions compared to without the ODD, unlike HIF-1ɑ. However, a 200 fold decrease in MFI using HIF-1ɑ ODD compared to no ODD in normoxic conditions was also observed. These results indicates the potential utilization of HIF-1ɑ and HIF-2ɑ to develop a novel biosensor for the treatment and prevention of preeclampsia. |
| Yash Agarwal |
Drexel University College of Medicine |
Macrophages and microglia are myeloid lineage immune cells that exert several innate homeostatic and pathophysiologic functions. One of the most important functions is phagocytosis, which helps to clear cellular debris, repel pathogens, and remodel synaptic connections in the central nervous system. Studies show that proteins regulating autophagy – a process well-known for recycling and degrading intracellular material –contribute to the phagocytosis in myeloid cells, and inhibition of autophagy can increase cytokine secretion and limit phagocytosis. Several studies found that HIV viral proteins such as Nef and Tat can negatively regulate autophagy in neurons, macrophages, and astrocytes, suggesting that HIV could dysregulate other autophagy-associated processes. Currently, the majority of individuals infected with HIV use antiretroviral drugs (ARV), but the effects of ARV on autophagy, downstream inflammatory signaling, and phagocytic function are not well established. Here we optimized an in vitro phagocytosis assay using high content imaging to examine the associations between autophagy inhibition and phagocytic capacity in human monocyte-derived macrophages (hMDMs). We then used this assay in induced pluripotent stem cell-derived microglia (iMg) infected with HIVADA and treated with ARV, examining changes in LC3B expression, a regulator of autophagolysosome formation, and phagocytosis. Results show that LC3B levels increase with phagocytosis in hMDMs, and that inhibition of autophagy by chloroquine pre-treatment does not significantly alter phagocytosis. HIV infection attenuated phagocytosis in iMg, but ART treatment did not restore full recover phagocytic activity. Future studies will explore the effects of ART in co-cultures of iMg with iPSC-derived neurons and astrocytes to define intercellular interactions that may regulate other autophagy-related functions such as inflammation and synapse remodeling. |
| Leah Davis |
Rowan University |
Chimeric antigen receptors (CARs) have shown remarkable efficacy in treating hematological malignancies. However, several challenges, including off-target toxicity, the potential for relapse, and the need for adaptation to treat solid cancers, persist. The optimization of CAR structure is pivotal for enhancing their performance, safety, and overall therapeutic efficacy. The clinical trial BrianChild-01 underscored this importance when it demonstrated that modifying the hinge length of their CAR could improve its targeting of the juxtamembrane antigen HER2. Computational methods, such as protein modeling and protein-protein docking, offer an alternative approach to fine-tuning CAR structures and predicting their efficacy before experimental testing. In this study, we employed a computational approach to model a CAR with three different hinge lengths and compared the docking scores of each CAR with the same antigen. To construct the CAR, derived from NCT03500991, we incorporated five domains: scFv (single-chain fragment variable), HI (hinge), TM (transmembrane), CS (costimulatory), and SI (stimulatory). While the crystal structure of the anti-HER2 scFv (PDB: 1N8Z) was available, other domains were built using iTASSER and AlphaFold. These included TM (CD28, Uniprot: P10747), SI (4-1BB, UniProt: Q07011), CS (CD3ζ, UniProt: P20963), and the full HER2 protein (UniProt: P04626). The HI varied among experiments, featuring short, medium, and long hinges derived from IgG4 (UniProt: P01861). The complete structures were inserted into a cell membrane to ensure steric hindrance and native conformation was addressed to the fullest extent possible. Protein-protein docking simulations of the anti-HER2 CAR, which varied in hinge length, and full HER2 revealed that a medium hinge length yielded the most optimal docking score for HER2 targeting, corroborating the findings of BrainChild-01. This study underscores the potential of computational oncology as a potent tool for enhancing existing adoptive cell therapies and expediting their development. |
| Beatriz Hernaez-Estrada |
Drexel University |
Introduction: The persistent inflammatory and stress responses generate in burn patients have a strong effect on the immune system. Macrophages that are activated in response to burns polarize towards an immunosuppressive phenotype commonly known as M2b. Furthermore, due to sepsis, the chronic exposure to the pro-inflammatory environment also leads to an exhausted macrophage phenotype. Together these effects compromise macrophage behavior that influences the healing outcome. In clinical trials, bioengineered allogeneic cellular constructs (BACCs) have shown potential in promoting healing in patients with deep partial thickness burns. However, the mechanism(s) behind these successful effects are not totally understood yet. For this reason, the aim of this study is to investigate how the crosstalk with the BACC influence different burn-related macrophage phenotype.Methods: Primary human macrophages (N=4 healthy donors) were co-cultured with the BACC or collagen controls. Macrophages were isolated over 6 days of co-culture and their RNA was extracted for gene expression analysis, and conditioned media was collected to analyze the secretion of proteins. To assess phenotype changes, we performed gene set analysis using single sample gene set enrichment. To investigate changes in the phenotype of macrophages relevant to burn wounds macrophages were polarized to the immune suppressive M2b phenotype or LPS-exhausted phenotypes before co-culturing them with the BACC. Undifferentiated macrophages (M0) were used as controls. Results: Gene expression analysis revealed a shift in undifferentiated macrophage phenotype towards an M2 phenotype by day 3 and 6 in co-culture as demonstrated by an increase in the expression of multiple M2 genes, including MRC1 and CCL18. Secretory analysis revealed a decrease in the secretory of inflammatory proteins, like interleukin 6, and increased secretion of a growth factor and remodeling enzyme. Conclusions: Collectively these results demonstrate that BACCS promote the transition of macrophage phenotype towards a pro-reparative M2 phenotype which may contribute to the construct’s pro-healing capabilities. |
| Paul Nana Kwame Sagoe |
Syracuse Universsity |
"INTRODUCTION: The role of synovial inflammation in the progression and pain associated with osteoarthritis (OA) has garnered significant attention. Upon activation a sub-population of macrophage secretes pro-inflammatory and catabolic mediators that exacerbate OA severity. Thus, specifically targeting macrophages offers promise for disease modification in OA therapy. While bisphosphonates (BPs) exhibit high affinity and toxicity against macrophages; their rapid clearance upon delivery renders them ineffective when injected intraarticularly. Thus, we developed a novel therapeutic system using nanoparticles in a microparticle formulation (NiM) to deliver zoledronate (Zol), a potent third-generation BP, to activated macrophages for OA treatment. METHODS: The NiM formulation composed of polymeric microparticles (PEG-PLGA MP) and calcium-zoledronate nanoparticles (CaZol NP) was achieved via a coaxial flow phase separation technique by adding the nanoparticles to the organic phase used for the microparticles synthesis. Nanoparticle and microparticle sizes were characterized using Dynamic light scattering (DLS), transmission electron microscopy (TEM), and inverted microscopy. CaZol NiM was confirmed using Fourier transform infrared (FTIR) and energy-dispersive x-ray (EDAX). Zol release from microparticles was quantified via inductively coupled plasma–optical emission spectroscopy (ICP-OES). Cellular uptake studies were conducted with Raw 264.7 macrophage cells using confocal microscopy and flow cytometry. RESULTS: The average sizes of microparticles and nanoparticles were determined as 6.7±3.1 μm and 44.6±3.1 nm respectively. Successful loading of Zol into NiM was confirmed by the presence of phosphate group using FTIR and EDAX. Cell uptake was evidenced by nanoparticle co-localization with lysosomes through confocal microscopy. CaZol NP displayed a burst release profile and higher uptake rates by RAW 264.7 cells compared to CaZol NiM, as observed through ICP-OES and flow cytometry. CONCLUSION: Altogether, PEG-PLGA microparticles provide a suitable platform for sustained and targeted delivery of Zol by minimizing burst release, thus delivering Zol as nanoparticles in microparticles promises to enhance Zol efficacy for OA treatment. |
| Erin M. O'Brien |
Drexel University |
Cell therapy is a quickly growing field in biomedical engineering with a wide range of applications in immunotherapy and tissue regeneration. Due to their role as high-level regulators of healing and angiogenesis, macrophages are an attractive target for cell therapy platforms. Studies have shown that the delivery of macrophages to sites of injury can improve healing outcomes, such as biomaterial integration and blood vessel regrowth. Pro-regenerative IL-4-polarized macrophages in particular have demonstrated some success in promoting angiogenesis. However, macrophage plasticity and rapid response to environmental stimuli can make it difficult to maintain macrophage phenotype in the highly inflammatory injury milieu. In this study, we sought to overcome this problem by designing a biomaterials-based strategy to control macrophage phenotype intracellularly.Both human and murine primary monocytes were isolated from peripheral blood and bone marrow, respectively, and differentiated into macrophages with macrophage colony-stimulating factor. Macrophages were either maintained in an unactivated phenotype (M0) or M1-activated with interferon-gamma and lipopolysaccharide (inflammatory media). Lipid nanoparticles carrying IL-4 mRNA (IL4-LNPs) were cultured with macrophages for 12 hours to facilitate phagocytosis. After co-culture, macrophages were cultured in normal or inflammatory media. Culture media was collected from days 1-7 for ELISA, and cell lysates were collected on day 2 for gene expression analysis. ELISA results showed that macrophages treated with IL4-LNPs were successfully transfected and secreted IL-4, a cytokine not typically produced by macrophages, at detectable levels for at least 7 days. Gene expression data demonstrated that this secreted IL-4 successfully polarized M0 macrophages and re-polarized M1 macrophages to a pro-angiogenic M2 phenotype. M1 macrophages significantly downregulated typical M1 markers, indicating that IL4-LNPs were able to overcome the inflammatory culture conditions of this group. Future work will administer IL4-LNP-carrying murine macrophages to volumetric muscle loss injuries to determine their effects on tissue regeneration and angiogenesis in vivo. |
| Eliana Du |
West Windsor-Plainsboro High School South |
Major Depressive Disorder (MDD, i.e. “depression”) has only recently begun to be recognized as a biological affliction that stems from the degeneration of key neurological tissues. Given its ubiquity and full-body reach, its treatment is an important point of discussion. However, the diagnosis of MDD has long relied on subjective measures, such as patient self-evaluation and doctor analysis. With few quantitative factors, this leaves depressed patients liable to be misdiagnosed and to not receive the necessary help, should they require it. To improve depression diagnosis, more quantitative methods must be explored. One such method is the analysis of electroencephalographic (EEG) data, which has a long history of usage in large-scale neurological assessment and, when taken from a resting-state patient, has potential applications in clinical settings. However, there are many different signal processing methods that can be used to extract features from EEG data. Thus, it is important to choose the most accurate method for depression-specific features. In this paper, two signal processing methods, the Fourier transform (FT) and the continuous wavelet transform (CWT), are used to analyze actual continuous, resting-state EEG data at well-established frequency bands to create similar topographic maps of a subject’s brain. The transforms and topographic maps are created using the most recently updated MATLAB software as of September 9th, 2023, the 2023.0 version of the EEGLAB software, and the most recently updated FieldTrip toolbox software as of August 17th, 2023. The transforms’ respective topographic maps are then assessed qualitatively to detect depression-specific outputs to ultimately determine the strengths of each transform in potential quantitative depression-diagnosis. |
| Iris Baurceanu |
Cancer Innovation Laboratory, Center for Cancer Research, National Cancer Institute |
Biologic scaffold materials derived from decellularized extracellular matrix (ECM) are used to promote wound healing after tumor resection by initiating a Type 2 inflammatory response, which has been recently leveraged to enhance efficacy as a therapeutic cancer vaccine. We investigated the mechanisms of activation in an ECM scaffold-assisted cancer vaccine. Decellularized small intestinal submucosa (SIS) ECM was milled into particles to create an injectable scaffold delivery platform with the immune adjuvant cyclic di-AMP (CDA), a stimulator of interferon genes (STING) agonist, and the model antigen ovalbumin (OVA) for subcutaneous injection in C57Bl/6 mice. After 7 days, spectral cytometry showed that F4/80+ macrophages represented 51.7% of the immune population in SIS and 32.9% in SIS+CDA, and both maintained high eosinophil proportions. Although SIS recruited more macrophages, SIS+CDA increased CD86 expression. Histologic staining showed increases in CD86+ cells within the dermis adjacent to the vaccination site. Next, we explored the potential of SIS+CDA to induce antigen specific cytotoxic T cell responses. CD103+ migratory dendritic cells internalized fluorescently labeled OVA and trafficked to draining lymph nodes, which increased 2.5 fold with CDA. Subsequently, CDA induced a greater number of OVA-specific CD8+ T cells via tetramer staining. CDA delivery did not diminish Type 2 signatures; SIS+CDA recruited cells exhibiting both STAT1 phosphorylation (interferon-gamma signaling) and STAT6 phosphorylation (induced by interleukin 4 (IL-4) signaling). In summary, we found that SIS scaffold-assisted vaccines using the adjuvant CDA increased local immune activation, leading to antigen trafficking to lymph nodes, and antigen-specific cytotoxic T cell priming while maintaining ECM immune features such as IL-4 signaling and eosinophil infiltration. In conclusion, the STING agonist CDA can induce antigen specific cytotoxic T cell response while retaining a SIS scaffold associated Type 2 immune response needed for wound repair, and eventually be utilized for a therapeutic cancer vaccine formulation. |
| Matthew M. C. Kwan |
University of Colorado Boulder |
Cell-mediated drug delivery systems are emerging as a new approach to deliver bioactive drugs packaged in nanoparticles to specific, pathologic sites. Circulating immune cells like macrophages have the capacity to enhance nanoparticle delivery in vivo through chemotaxis, and once delivered, the cells can participate in the intended therapeutic effect as well. However, nanoparticles are known to exert stresses on macrophages, causing them to polarize between M1-like (proinflammatory) to M2-like (anti-inflammatory) phenotypes. Despite this understanding, little is known about the underlying relationship between nanoparticle shape and macrophage polarization. It has been demonstrated that isotropic particle geometries are rapidly phagocytosed within the phagosome within minutes to hours; however, anisotropic particle geometries are capable of frustrating phagocytosis by macrophages for days, resulting in cell spreading along the particle surface due to incomplete formation of actin structures. Herein, we study the interaction of poly(D,L lactic-co-glycolic acid) discoidal (major axis 7.7 ± 0.7 µm, thickness 261.3 ± 50.4 nm) and spherical (diameter 1 – 3 µm) particles on C57BL6-derived primary macrophages to respectively promote or suppress phagocytosis. Discoidal and spherical particles of a fixed volume were fabricated using microcontact printing and homogenization techniques, respectively. Macrophage-particle complexes were manufactured by incubating particles in vitro. Upon particle association, macrophages were profiled with assay for transposase-accessible chromatin with sequencing (ATAC-seq), RNA-seq, flow cytometry, and ELISA. Relative to control macrophages (i.e., no particles associated), macrophages coupled with discs assumed a strong M2-like phenotype, while spheres induce only subtle M2-like characteristics. Specifically, macrophage-disc complexes presented enriched signaling by VEGF and TLR9 as well as significant upregulation of M2 biomarker Arg1. These findings highlight the importance of physical cues on macrophage behavior and will largely improve upon engineered cell-based therapies for disease targeting. |
| Jordan Seibold |
University of Cincinnati |
The neuroimmune system is complex and is regulated by small chemical messengers released from resident immune and neuronal cells. Catecholamines like dopamine and norepinephrine are important chemical messengers responsible for initiating and propagating messages between neurons and immune cells; however, the mechanism and dynamics of this signaling is relatively unknown. The presence of the intracellular machinery to synthesize and interact with catecholamines in T lymphocytes has been known for over a decade; however, direct measurement of catecholamine secretion in real time from immune cells has not been possible. Here, we have discovered that Naïve CD4+ T cells are capable of releasing catecholamines on a subsecond time scale. We have used a revolutionary technique, often used in the neuroscience field, called fast-scan cyclic voltammetry (FSCV) to measure subsecond fluctuations of catecholamines from isolated CD4+ T cells. This work provides direct evidence that T cells are capable of releasing catecholamines; potentially as a feedback communicator to resident sympathetic neurons in host immune organs. These results also prove that immune cells can signal on the same time scale as neurons. This work will hopefully open the door to enable more sophisticated studies at the neuron-immune synapse in the future and the understanding of neuro-immune based diseases. |
| Deepa Reddy |
Drexel University College of Medicine |
Surgeries induce post-operative pain that must be alleviated effectively to reduce suffering, and to promote healing and recovery. Depending on the surgery and the specific conditions, post-operative pain is not effectively managed in >80% of the patients in US. Traditional opioids, such as morphine, remain the standard of care for perioperative pain regulation. Limitations of opioid use include a narrow therapeutic window, undesirable adverse events, and toxicity. Better treatment strategies are needed during and immediately after surgery to prevent the progression of acute pain to long term persistent pain. Small extracellular vesicles (sEVs), including exosomes, mediate intercellular communication by carrying biomolecular cargo to recipient cells. Our previous studies have shown that sEVs isolated from macrophage derived RAW 264.7 cells attenuate pain and inflammation in a mouse model of inflammatory pain. We hypothesize that macrophage-derived sEVs can attenuate post-operative pain therapeutically (administered immediately after surgery) or prophylactically (administered 2 weeks prior to surgery), decreasing the dose and duration of analgesic needed for pain relief. We generated and validated the model and our preliminary data reveal sEVs given after surgery results in faster attenuation of hypersensitivity compared to control. Future studies will determine the synergistic effect of therapeutic and prophylactic sEVs, to assess whether sEVs can a) lower the efficacious dose of commonly prescribed analgesic drugs or b) increase the duration of pain relief in a post-operative pain model. Studies are ongoing to elucidate the mechanisms underlying efficacy of sEVs, including immune cell trafficking in paw tissue. |
| Kenneth M. Kim |
Drexel University College of Medicine |
Autoimmune diseases and chronic tissue injury are associated with deficient regulatory T cell (Treg) populations and an increase in autoreactive or pro-inflammatory T cells. Moreover, Tregs in pro-inflammatory environments are dysfunctional, limiting their capacity for expansion and effector phenotype changes. Treg therapies have focused on adoptive cell transfer and in vivo induction via co-delivery of soluble factors (e.g., IL-2, TGF-β, and rapamycin). However, the timespan and relative delivery rate for these factors are difficult to control. Furthermore, their use remains limited by Treg phenotype instability and loss of function. To address these challenges, we have developed an injectable granular hydrogel for the local presentation of an immune checkpoint, which is associated with Treg abundance in disease (e.g., cancer). Hydrogels were formed from hyaluronic acid, modified by methacrylates (for photopolymerization) and DBCO (to anchor azide-terminated checkpoint ligands via copper-free click chemistry). Hydrogels were subsequently processed into granules (155±53µm) by extrusion fragmentation (18-22G needles) to yield an injectable and self-healing material for local Treg induction. Soluble checkpoint ligands increased Treg populations in a dose-dependent fashion ex vivo. Subcutaneous injection of the bioconjugated granular hydrogel in C57BL/6J mice was performed and T cell populations were profiled after 7 days from the spleen, inguinal lymph nodes, and injected hydrogels. The system elicited desirable immunomodulatory function, including an increase in Treg populations. Interestingly, these changes were observed not only at the injection site, but also in secondary lymphoid organs by two-fold, relative to soluble TGF-β. To our knowledge, this is the first reported use of the specific checkpoint ligand as an immunomodulatory biotherapeutic. The innovative strategy is a promising approach to treat autoimmune dysfunction and tissue injury pathologies, such as autoimmune hypothyroidism and organ injury. |
| Emily Konopka |
Drexel College of Medicine |
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 further understand the age-associated impairments in immune response to C. difficile for development of effective therapeutics, aging mouse models of CDI are critical. We hypothesized that immune dysfunction in the elderly will make them more susceptible to infection with C. difficile. To test this, we first established the lethal CD196 spore dose in aged (72-80 weeks) mice. After identification of this lethal dose, vaccination followed by challenge was performed to determine vaccine efficacy and impairments in immune response associated with elevated mortality in young versus aged mice. Mice were immunized twice over 30 days with RBDA/RBDB-expressing plasmids (5ug/plasmid/mouse) followed by orogastric challenge with 200 CD196 spores at thirty days post boost. Serum analyses showed that while young and aged mice developed similar anti-toxin IgG responses following two doses of the RBDA/RBDB vaccine, aged mice that succumbed to CD196 infection trended to lower anti-toxin B IgG responses. Additionally, post-challenge, aged mice developed significantly less toxin A-specific memory B cells compared to young unvaccinated mice and significantly lower toxin B-specific memory B cells compared to young RBD vaccinated mice. Together, these results indicate that increased susceptibility is associated with reduced vaccine-specific anti-toxin B IgG in sera and lower frequencies of toxin-specific memory B cells suggesting the necessity for adjuvanted vaccine formulations for improvement of aged immune responses to C. difficile infection. |
| Asheley Chapman, PhD |
MIT |
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. One such bnAb, VRC01, 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 co-immunize with an “on-target” antigen (germline targeting trimer MD39 VGT3.1) bearing the neutralizing epitope of interest along with an “off-target” competitor antigen lacking said epitope (“MD39-ko”) carrying a chemotherapeutic drug to elicit cell death of the off-target B cells specific for the competitor. Antigen-drug conjugates were designed using FDA-approved chemotherapeutic small molecules tesirine, a DNA alkylator, as our warhead and validated in vitro. B cell-directed tesirine toxicity was tested in the Alt lab VH1-2 human Ig mouse model of VRC01-class B cell priming. Administration of epitope-KO-tesirine conjugates after priming suppressed the overall GC response, substantially reduced “off-target” KO-specific B cells, and conferred an early transient advantage to VRC01-class clones in the germinal center. We are hopeful that this strategy will prove effective at reducing the distracting off-target response against HIV antigens and provide a path to broadly neutralizing vaccinations. |
| Tyler Blanch |
University of Pennsylvania |
Introduction: In conventional injury responses, inflammatory signaling is critical in tendon repair, but aged tissues often struggle to resolve these cues. However, the mechanisms by which inflammation impacts tendon resident cells (tenocytes) and how aging influences these responses remain enigmatic. Thus, this study aims to elucidate disparities in cellular reactions to inflammatory cues (e.g., tumor necrosis factor-α, TNFα) between young and mature tenocytes to understand how inflammation affects age-related histone reorganization.Methods: Mouse tenocytes were isolated from young (<5 weeks) and mature (>45 weeks) tail tendons and cultured in TNFα-containing media. Fixed cells were immunostained for either super-resolution imaging (for Histone-H2B, TNFαR1, or TNFaR2) for their density quantifications or immunofluorescence (for histone modification markers). Results: Super-resolution H2B localizations revealed a global elevation of H2B density within mature nuclei, which continued to increase with TNFα treatment. Examining histone modification status, the marker associated with gene repression (H3K27me3) increased in intensity with increasing treatment duration, with more substantial changes in mature cells. Interestingly, the marker associated with gene activation (H3K9ac) exhibited an increase in young cells but a decrease in mature cells in response to inflammation. Quantification of TNFα receptor densities revealed no difference in TNFαR1, but mature tenocytes exhibited an elevation in TNFαR2 surface density. Conclusions: This study reveals age-related differences in tenocyte responses to inflammation. TNFα induces global chromatin compaction in both groups, with mature cells showing a substantial increase after 6-hours of treatment. The interplay between changes in H3K27me3 and H3K9ac between young and mature cells likely contributes to the observed chromatin condensation changes, suggesting that the inflammatory environment in aged tendons may drive abnormal tenocyte behavior. Elevated TNFαR2 levels in mature tenocytes may be a factor in age-associated inflammatory responsiveness. Ongoing studies investigate the downstream effects of these chromatin changes in the context of tendon aging and disease. |
| Nahtalee Lomeli |
University of California Berkeley and University of California San Francisco |
Introduction: Tumor-associated macrophages (TAMs) play a pivotal role in solid tumors where higher TAM density in the tumor microenvironment (TME) often indicates poor prognosis across a range of cancers. This connection is primarily due to TAMs' immunosuppressive behavior, dampening inflammatory signals and limiting the effectiveness of cancer immunotherapies designed to activate robust immune-inflammatory responses against solid tumors. Studies highlight the benefits of shifting TAMs from the M2 immunosuppressive phenotype to M1 inflammatory phenotype to enhance tumor clearance. Inspired by these findings, our work focuses on reprogramming TAMs towards an M1 inflammatory state by transfecting them with immune-stimulatory lipid nanoparticles (LNP) coated with lipopolysaccharide (LPS) and loaded with interferon-gamma (IFNγ) mRNA. Methods/Results: In our in-vitro model, CD14+ isolated peripheral blood mononuclear cells (PBMCs) are co-cultured with human A375 melanoma cells to recapitulate recruited monocytes in the tumor niche. Our preliminary results reveal that stimulation of these co-cultures with IFNγ and LPS leads to a significant increase in the expression of the immunostimulatory marker CD86, accompanied by a decrease in the immunosuppressive marker CD206. Furthermore, this reprogramming corroborates with elevated interleukin-6 (IL-6) secretion and diminished interleukin-10 (IL-10) production, reinforcing the shift towards an immunostimulatory phenotype. Conclusion: Our results highlight the important biochemical cues provided by LPS and IFNγ in re-polarizing TAMs towards an immunostimulatory phenotype within the TME and motivate their use in cancer immunotherapy. We plan to validate these findings further through a functional assay assessing the phagocytic and killing activity of IFNγ/LPS-stimulated CD14+ macrophages using CFSE-labeled A375 cells in a co-culture setup. These investigations hold promise for innovative approaches to enhance antitumor immunity through TAM reprogramming. |
| Joshua Montano |
Johns Hopkins University |
"Triple Negative Breast Cancer (TNBC) is categorized as a uniquely aggressive breast cancer for its ability to be unresponsive to hormonal therapies with its lack of estrogen receptor (ER-), progesterone receptor (PR-), and human epidermal growth factor receptor 2 (EGFR2-/HER2-). 13% of women in the US are diagnosed with breast cancer, and 50-72% of women who possess the heritable Breast Cancer gene (BRCA1) mutation will develop breast cancer. Drug X is an anti-metabolite (AMeb) chemotherapeutic which acts as an active nucleoside analog to cause “masked termination” in DNA, causing single strand breaks (SSBs). However, many studies have implicated toxicity risk of Drug X upon combination with other chemotherapeutics such as Docetaxel, Doxorubicin, and Cyclophosphamide in advanced breast cancer. Drug Y is a PARP inhibitor (PARPi) that can inhibit PARP1, a protein responsible for recognizing SSBs and recruiting proteins for DNA repair through the base-exclusion repair (BER) pathway. PARP inhibition of the BER pathway forces BRCA-mutated cancer cells to convert SSBs to double strand breaks for subsequent repair through homologous recombination (HR). Because BRCA-mutated cancer cells have issues using the HR DNA repair pathway, BRCA-mutated cancer cells are forced to opt for the error-prone non-homologous end joining (NHEJ) DNA repair pathway. However, Drug Y is only soluble in organic solvents that are toxic upon administration. Our formulation of Drug Y is stable in aqueous solutions and continuously slow-releases Drug Y, sensitizing TNBC cells to Drug X while keeping within the therapeutic index. Using crystal violet viability assays and qPCR, we find that: 1) Alone, Drug X did not cause a significant decrease of TNBC 4T1 cell viability, but Drug Y with Drug X decreased 4T1s cell viability significantly and 2) Gene markers TNFa and GM-CSF are significantly upregulated in 4T1 cells in vitro. |
| Serena Omo-Lamai |
University of Pennsylvania |
Lipid nanoparticles (LNPs) have become the dominant drug delivery technology in industry, holding the promise to deliver RNA to up- or down-regulate any protein of interest. LNPs have been targeted to specific cell types or organs by physicochemical targeting, in which LNP lipid compositions are adjusted to find mixtures with the desired tropism. In a popular approach, physicochemical targeting is accomplished by formulating LNPs with charged lipids. Negatively charged lipids localize LNPs to the spleen and positively charged lipids to the lungs. Here we found that lung-tropic LNPs employing cationic lipids induce massive thrombosis. We demonstrate that thrombosis is induced in the lungs and other organs, and greatly exacerbated by pre-existing inflammation. This clotting is induced by a variety of LNP formulations with cationic lipids including non-LNP nanoparticles. We isolate the mechanism of clotting induction which depends on the LNPs binding to the coagulation protein fibrinogen as part of the protein corona and causing platelet and thrombin activation. Based on these mechanisms, we engineered multiple solutions which enable positively charged LNPs to target the lungs without inducing thrombosis. We determined the optimal anticoagulants to prevent clotting while maintaining LNP lung-tropism and mRNA expression. We also demonstrated the efficacy of cationic LNPs functionalized with the direct thrombin inhibitor PPACK. Finally, we found that the size of positively charged LNPs has a positive correlation with side-effects and we showed that decreasing LNP size to <80nm ameliorates these side effects while preserving lung-tropism and mRNA expression. Our findings implicate thrombosis as a major barrier that blood erects against LNPs with cationic components and illustrate how physicochemical targeting approaches must be investigated early for risks and re-engineered with a careful understanding of biological mechanisms. |
| Jason DaCunza |
Drexel University College of Medicine |
Small extracellular vesicles (sEVs) are bilipid membrane particles between 30-150 nm in diameter. These vesicles are an integral means of intercellular communication by transporting a variety of cargo such as nucleic acids, proteins, and lipids. sEVs perform many regulatory functions, and their uptake can induce immune system activation and alterations in gene expression. Recent studies have also shown that sEVs released from allogeneic tissues and organs after transplantation play a crucial role in immune recognition and response, leading to rejection or tolerance in mice. Our recent findings show that allogeneic sEVs from RAW 264.7 mouse macrophage cells are immunomodulatory and act as a pain therapeutic and prophylactic in a complete Freud adjuvant (CFA) mouse (C57BL/6J) model of inflammatory pain. Differential expression of relevant anti-inflammatory miRNA under lipopolysaccharide stimulation provides a possible mechanism for pain attenuation. However, it is unclear if these effects are dependent on an allogeneic immune response in the recipient. Additionally, we have yet to characterize sEVs derived from primary cells for cargo and efficacy. To better understand the impact of sEV allogenicity in pain attenuation, we will utilize primary macrophage-derived sEVs of different donors. We have successfully established cultures of bone marrow derived macrophages (BMDMs) from male and female C57BL/6J mice, confirming purity using flow cytometry. sEVs released from primary BMDMs were characterized for size, surface marker expression, and miRNA cargo using nanoparticle tracking analysis, flow cytometry, and qPCR respectively. Our preliminary findings suggest that primary BMDM sEVs contain similar cargo to those of cell line macrophages. Future studies will determine if primary sEVs are as efficacious in attenuating inflammatory pain as cell line-derived sEVs, and if allogeneic (BALB/c) and syngeneic (C57BL/6J) BMDM sEVs differ in efficacy in the CFA model. |
| Rimsha Bhatta |
University of Illinois at Urbana Champaign |
Dendritic cells (DCs) play a critical role in orchestrating immune responses and are pivotal in the field of immunotherapy. Harnessing the potential of DCs for targeted immunotherapy requires the development of biomaterials that can selectively recruit and modulate these specialized immune cells. Notably, macroporous hydrogels loaded with granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor antigens have demonstrated the ability to recruit and modulate dendritic cells (DCs) in the local microenvironment, thereby priming tumor-specific T cells. However, the mechanism underlying immune cell recruitment and enrichment within macroporous materials remains largely unknown, posing a challenge to the rational design of materials that can selectively enrich and modulate specific immune cell populations. Here, we designed a macroporous hydrogel that can independently tune the pore size, viscosity, and stiffness, thereby elucidating their impact on immune cell recruitment behaviors within the hydrogels. We show that by tuning these parameters, we can enhance the immune cell recruitment behaviors and overall recruitment profiles of DC. Further, we incorporated this macroporous hydrogel with tumor antigen and adjuvant to function as a DC-homing hydrogel for cancer immunotherapy. |
| Manthan Patel |
University of Pennsylvania |
RNA-containing lipid nanoparticle [LNPs] not only enclose and ferry delicate RNA to target cells, but also allow the release of enclosed RNA into the cytosol, often termed endosomal escape. This process requires the action of an ionizable lipid, the primary component in all LNP formulations, which becomes positively charged within the acidic endosome environment and damages the endosome membrane. As a result, RNA is released into the cytosol and subsequently translated into protein of interest. Nonetheless, here we show that LNPs are inflammatory in mice, regardless of route of administration (intravenous and intratracheal). Although the inflammatory nature of LNPs is advantageous for vaccine applications, its implications within contexts of other diseases, particularly those with underlying inflammation, are less favorable. We screened various ionizable lipids, observing a positive correlation between mRNA expression levels and inflammation, quantified by cytokine levels. Moreover, differences in mRNA expression also correlate with the extent of endosomal disruption. While screening for more potent ionizable lipids remains a crucial objective, it is equally important to consider the potential escalation of inflammation. In response to this concern, we identified various LNP formulation changes (independent of ionizable lipid) that enhance mRNA expression without the consequent increase in inflammation, thus allowing for application of LNPs for inflammatory diseases and enabling increased dosing capacity. |
| Jacqueline Li |
University of Pennsylvania |
Glioblastoma (GBM) is a primary brain cancer with a median survival of 15 months post-diagnosis. Conventional treatments have limited efficacy and are plagued with off-target toxicities. Chimeric Antigen Receptor (CAR) T-cell therapy has emerged as a beacon of hope in cancer therapy, offering a targeted approach. However, its potential in GBM treatment is compromised by the immunosuppressive tumor microenvironment (TME). This suppression is largely driven by M2-phenotype tumor-associated macrophages (TAMs) that promote an immunologically cold site, thereby limiting the effectiveness of immune responses. Our strategy aims to counteract this by delivering CAR mRNA to TAMs, potentially converting them into a pro-inflammatory M1 phenotype and creating an 'immunological hot spot'. A major challenge, however, is the non-invasive delivery of CAR mRNA across the blood-brain barrier (BBB). Our approach leverages lipid nanoparticles (LNPs) for this purpose, especially with the promise of intranasal administration. GBM mouse models were developed using GFP-labeled U87 cells. Two LNPs, one formulated with the C14i-494 ionizable lipid and the other with C12-200, were equipped with mCherry mRNA to monitor translation. These LNPs were introduced via various routes: intranasal, intravenous, and intraperitoneal. IVIS system monitoring indicated a specific targeting of the GBM TME, further corroborated by confocal microscopy showing mCherry translation predominantly near GFP-labeled tumor cells. Notably, the C14i-494 LNP demonstrated a superior fluorescence signal compared to the C12-200 LNP. Intranasal delivery emerged as the most promising, potentially offering a way around the BBB. Reduced fluorescence in non-target organs further augments the specificity of our approach. In sum, our findings indicate that LNPs, particularly those formulated with the C14i-494 lipid, may present an effective method for delivering mRNA to TAMs within the GBM TME. As we progress, our research will further delve into biodistribution validation and therapeutic efficacy, with the overarching aim of pioneering a more efficacious GBM therapy. |
| Breana Channer |
Drexel University College of Medicine |
Macrophages are central to the innate immune response and defining mechanisms by which these cells mediate inflammation is critical to managing disease. Distinct culture conditions influence the macrophage inflammatory response, and our prior data show that dopamine, classically considered a neurotransmitter, can increase NF-kB activity and cytokine production in macrophages. As human monocyte derived macrophages (MDM), are a widely used tool to study macrophage function and inflammation, we examined the baseline activity, morphology, and dopaminergic inflammatory response of MDM grown in distinct culture media. We examined Dulbecco’s Modified Eagle Medium (DMEM) +/- serum, Rosewell Park Memorial Institute 1640 (RPMI) +/- serum and Macrophage Serum Free Media (M-SFM), as these are commonly used to culture macrophages. MDM cultured in these distinct media were treated with vehicle (H2O), dopamine or lipopolysaccharide (LPS) as a positive control. High content imaging was used to examine changes in cell morphology and NF-kB nuclear translocation as a read out for inflammatory profile. Inflammatory profiles were also evaluated via changes in cytokine secretion, measured by AlphaLISA, and dopamine receptor expression was evaluated by RT-qPCR. Our results showed that dopamine increases NF-kB nuclear translocation and inflammatory cytokine production, but the magnitude of the results varied between media, with no serum cultures showing less robust responses than serum cultures. Cells cultured in M-SFM showed lower cytokine secretion levels and less dopamine receptor expression compared to other medias. Cultures in DMEM or RPMI without serum showed lower inflammatory response than similar cultures with serum. These findings demonstrate the importance of microenvironment conditions on macrophage response to inflammatory stimulation and the importance of optimizing experimental conditions when examining highly plastic cells like human macrophages. |
| Laura C. Van Eyndhoven |
University of Pennsylvania |
Single-cell analyses of innate immune responses have revealed that only a small subset of cells is able to initiate type I interferon (IFN-I) production upon infection, serving a crucial role in establishing antiviral immunity in a dynamic paracrine fashion. Despite this new understanding, the spatiotemporal role of these so-called “first responders” remains elusive. Herpes Simplex virus type I (HSV-1) infection manifests at extremely local regions in the epithelial mucosa, and therefore serves as a good model to further characterize the molecular determinants that drive first responders and study their spatiotemporal role in establishing antiviral immunity in tissues. Monolayers of human primary keratinocytes and dermal fibroblasts were infected with genetically modified HSV-1 expressing fluorescently tagged ICP4 to monitor spatiotemporal virus replication and infection progression. IFN-I production and subsequent upregulation of interferon stimulated genes (ISGs) were assessed and quantified using single-molecule RNA fluorescence in situ hybridization (smFISH). Cell lineages were traced using a nuclear labelling method based on the stochastic expression of three fluorescent proteins. Our results confirmed that only a small (~1-5%) fraction of the total population of infected cells can initiate antiviral immunity by mass-producing IFN-I, thereby upregulating ISGs in both the infected and yet uninfected cells. These cells are only abortively infected and originate at the borders of infection. Lineage tracing revealed that IFN-I production quantities correlate between sister cells, indicating that transiently heritable host intrinsic factors dictate IFN-I production. Moreover, different clonal lineages showed heterogeneous viral susceptibility, with some clones being able to fully stop viral replication upon infection. In conclusion, cellular heterogeneity is a key feature of antiviral immunology, driving both viral susceptibility and IFN-I production, thereby dictating the spatiotemporal dynamics of viral replication and progression. |
| Oluwatofunmi Oteju |
Drexel University College of Medicine |
The neurological sequelae of HIV infection of the CNS persist in people living with HIV (PLWH) despite antiretroviral therapy. Within PLWH, stimulant use is highly comorbid, worsening clinical outcomes. Despite the prevalence of stimulant use in HIV-infected populations, the mechanism(s) by which stimulants impact HIV infection are undefined. Our data show that dopamine, which is increased in the CNS in response to stimulant use, can increase HIV replication in CNS myeloid cells. However, it is not clear if stimulants act only via changes in dopamine or if they have distinct effects on HIV replication. To examine this, we inoculated human primary monocyte-derived macrophages (hMDM) and induced pluripotent stem cell-derived microglia (iMicroglia) with an R5-tropic strain of HIV (HIVADA) in the presence of dopamine, methamphetamine (Meth), or cocaine (Coc). High-content image analysis and p24 AlphaLISA were used to define infection dynamics by evaluating percentage of cells infected, amount of secreted p24 protein, and the infectivity of the secreted virions. Our data show that DA and Coc, but not Meth, increases p24 secretion in hMDM and iMicroglia. Inhibition of endogenous myeloid dopamine receptors did not attenuate the impact of cocaine, but the effect of Coc was blocked by inhibition of sigma-1, a chaperone protein targeted by Coc. A sigma-1 agonist also increased p24 secretion, suggesting that the effects of cocaine are mediated, at least in part, via sigma-1 and are distinct from those of dopamine. Future studies using mixed culture systems combining syngeneic iMicroglia or iMac with iPSC-derived neurons or astrocytes will examine the transcriptional changes and downstream proteins driving the actions of Coc in CNS myeloid cells using single-cell RNA/ATACseq. These will enable more comprehensive analyses of changes in HIV infection dynamics, inflammation, and neuronal/glial health and function in response to HIV + substances of misuse. |
| Magdalena Maria Samojlik |
University of Florida |
Background: Robust in vitro platforms for the co-culture of immune cells and pancreatic islets that allow for the stable spatial distribution of the cells, migration, and dynamic cell-cell interactions are needed to bridge the knowledge gap in our understanding of Type 1 Diabetes (T1D) immunopathogenesis. Herein, we describe an extracellular matrix (ECM) hydrogel in vitro platform, which permits for the interrogation of the role of antigen-specific cell-cell interactions in T-cell migration. Methods: OTI-GFP cytotoxic CD8+ T-cells (CTLs) were co-cultured with mOVA (antigen-specific) or B6 (negative control) islets in ECM hydrogel. Confocal timelapse imaging was performed at 90 second intervals for 16 hours and Fiji TrackMate was used to track the migration of T-cells. Outputs were processed in MATLAB for visualization, temporal analysis, and trajectory classification. Results: Compared to the negative control group, the antigen-specific group exhibited significantly decreased displacement and speed, but increased mean directional change rate upon interactions with the target islets. Additionally, significant accumulation of the CTLs on the mOVA islet surface over time was observed, in contrast to the B6 islet surface. Temporal analysis of total distance traveled by the CTLs revealed that mOVA islet-interacting CTLs exhibited overall significantly lower total distance traveled than the negative control. Trajectory classification of the migration tracks, based on their mean squared displacement, revealed that CTLs responding to mOVA islets exhibited less Lévy walk (superdiffusive) and more subdiffusive migration than those responding to non-antigen-specific islets. Conclusions: This data illustrates unique insights provided by this 3-D ECM T-cell-islet co-culture platform for the study of T cell migration characteristics. Our data indicates that the interactions between antigen-specific T-cells and their cognate antigen targets result in significant temporal changes in general migration parameters and in the type of migration pattern exhibited, with switching from the exploratory Lévy walk to subdiffusive migration upon target interaction. |
| Carolann Espy |
University of Pennsylvania |
Treatment with lipid nanoparticles (LNPs) causes an inflammatory response leading to rapid LNP clearance and negative side effects. These side effects are exacerbated when there is pre-existing inflammation, which limits LNPs therapeutic use. Therefore, it is important to create LNPs capable of avoiding/ preventing inflammatory responses. Cytokines are some of the major regulators and signalers for inflammatory pathways. Two cytokines that we have shown to be upregulated after LNP administration are Interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFα). Therefore, these cytokine pathways could be potential targets to intervene and ameliorate LNP based inflammation. We hypothesize that interrupting IL-6 or TNFα signaling will decrease the inflammatory response caused by LNP administration. siRNA LNPs for both IL-6 and TNFα along with an antibody for IL-6 were investigated as potential candidates for decreasing LNP based inflammation. Inflammatory responses were measured using cytokine ELISAs or Multiplex assays on in vitro and in vivo samples. RAW 264.7 cells, a macrophage cell line, were used as an in vitro model for inflammation; justified by the lab’s previous demonstration that macrophages are required for LNP based inflammation. An in vitro model developed by the lab for pre-existing inflammation with LPS treated C57BL/6 mice was also used. Using the aforementioned techniques, IL-6 and TNFα siRNA LNPs were shown to decrease cytokine production and IL-6 antibodies decreased inflammatory markers and mice appeared healthier. Therefore, supporting our hypothesis that abrogation in cytokine signaling can decrease LNP based inflammation. Fine tuning of these techniques can alleviate the immunological consequences of LNPs, thereby improving them as a drug delivery method and increasing the demographic able to receive this medication. |
| Anna Romanov |
MIT |
Nanoparticulate display of vaccine antigens is a well-established method of enhancing humoral responses to vaccines. Here, we leveraged wireframe DNA origami virus-like nanoparticles (DNA-VLPs) as a vaccine scaffold, which enables fully programmable and addressable nanoparticle assembly on the 10-100 nm scale with nanometer precision of immunogen conjugation. Unlike protein scaffolds which may generate scaffold-specific antibodies, DNA origami does not generate adaptive immune responses or memory but can be tuned to be inert or highly stimulatory via innate toll-like receptor recognition, which may be leveraged as a vaccine adjuvant. Previously, the Bathe and Irvine labs used wireframe DNA origami VLPs (DNA-VLPs) displaying an engineered HIV immunogen to reveal how antigen valency, spacing, and affinity impact B cell receptor activation in vitro and discovered that B cell activation plateaus after reaching a certain minimal valency and that larger antigen spacing leads to stronger B cell activation in vitro. Learning if these rules translate in vivo is of high interest to inform next-generation vaccine designs. We have demonstrated with two different vaccine antigens that prime-boost immunization in mice with DNA-VLPs leads to a valency-dependent elevation in antigen-specific IgG titers compared to soluble protein monomer; furthermore, these mice do not generate anti-DNA or anti-origami antibodies. In ongoing work, we are investigating the fate of DNA-VLP vaccines in lymph, including biodegradation, interactions with immune cells, and methods of follicular trafficking. Future designs of DNA origami vaccines will co-deliver antigen and therapeutic modalities to lymph, such as adjuvants, siRNAs, and/or mRNAs. |
| Marco E Zamora |
Drexel University/University of Pennsylvania |
"Delivery of RNA cargo to the lung holds enormous potential for treatment of acute lung illnesses like acute respiratory distress syndrome [ARDS]. Currently, there are two methods to achieve extrahepatic delivery: 1) antibody-mediated targeting, where antibodies are covalently conjugated to the surface of an LNP; and 2) physico-chemical tropism, where modification of a lipid component or addition of a helper lipid, bias expression to other sites of interest. However, these two methods have never been combined or rigorously tested head-to-head. Here, we test three modalities for extrahepatic delivery to enhance lung delivery: Conjugation of antibodies against platelet endothelial cell adhesion molecule [PECAM], targeting the vascular endothelium, LNPs generated with a permanently cationic helper lipid, DOTAP, and an LNP combining these technologies. These three formulations were then assessed for organ localization via biodistribution, the contribution of first-pass circulation to localization, expression capacity, and cell type uptake via flow cytometry. We further assessed how these parameters changed in the context of acute lung injury using our model of nebulized LPS to simulate an ARDS phenotype. Using these parameters, we found that combined antibody-plus-physicochemical targeting dramatically increased both localization and expression capacity in the lung, while also enhancing uptake by epithelial and mesenchymal cells. Taken together, we show that a combined antibody-plus-physicochemical approach markedly enhances the overall delivery, expression, and cell-type-targeting, and has the potential to provide therapeutic benefit for lung diseases. |
| Ann Metzloff |
University of Pennsylvania |
Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable clinical success. However, producing these bespoke cancer-killing cells is a complicated ex vivo process of leukapheresis, artificial T cell activation, and CAR construct introduction. Activation is vital for CAR construct uptake and differentiation into effector T cell phenotype. In the body, native T cells are activated when CD3/TCR and CD28 (two T cell surface molecules) engage with antigen presenting cells (APCs). Ex vivo, this process is mimicked with antibodies against CD3 and CD28, often conjugated to magnetic beads. While effective, removal of beads is cumbersome and results in loss of engineered cells. To overcome this challenge, we have developed APC-mimetic, T cell activating lipid nanoparticles (aLNPs) for one-step activation of and CAR mRNA delivery to human T cells. To generate aLNPs, human CD3 and CD28 antibodies (αCD3/αCD28) are conjugated to the LNP surface via thiol-maleimide chemistry. We demonstrate that aLNPs efficiently transfect primary human T cells with luciferase mRNA in the absence of activating beads. We further show that anti-CD19 CAR T cells generated with aLNPs perform potent cancer cell killing in vitro and express cytokines and cell-surface activation markers at levels similar to those for bead activated T cells. Finally, we show that the adoptive transfer of anti-CD19 CAR T cells generated with aLNPs reduces tumor burden in a murine xenograft model of leukemia. Overall, our APC-mimetic aLNPs with surface-bound CD3 and CD28 antibody fragments rapidly activate and deliver CAR mRNA to primary human T cells ex vivo, which may eliminate the need for T cell activating beads. This simplifies the complex CAR T cell engineering process, potentially improving cell yields and decreasing engineering cost and time. In the future, this platform could be used to formulate LNPs for the delivery of other cargoes to T cells. |
| Russell Urie |
University of Michigan |
Introduction: As there is no assay to predict alloimmunity in transplant or fetal rejection, clinicians rely on invasive tissue biopsy, non-specific blood-based assays, and, in transplant, aggressive immunosuppression. Immunosuppression protects transplants but increases systemic toxicities. Primary tissue histology is a flawed standard for alloimmunity surveillance and diagnostics, as histological evidence of rejection inherently lags behind molecular biomarkers and suffers from variability. Noninvasive alternatives, including gene profiling and cell-free DNA, also measure lagging indicators of rejection. A minimally invasive surveillance method is urgently needed to identify early risk of rejection for minimizing invasive procedures and personalizing interventions. We have developed porous biomaterial scaffolds for minimally-invasive sampling. These scaffolds amass immune cells producing biomarkers of disease as an engineered niche which can predict rejection onset. In this work, we employ microporous scaffolds to capture the longitudinal immune domain of healthy and rejecting transplants and healthy and miscarriage-prone rodent pregnancies without disrupting the primary tissue and with greater specificity than blood. Methods: We implanted subcutaneous poly-caprolactone scaffolds in murine heart transplant recipients and miscarriage-prone pregnancies. Scaffolds were biopsied and analyzed for differential gene expression by RNA sequencing using elastic net regularization across mice, tissue, and day to generate a biomarker signature of rejection. We performed singular value decomposition and supervised machine learning (Random Forest) to derive single-metric scores and a predictive model for graft or fetus rejection. Results and Conclusions: Gene expression in the cell-capture scaffold identified biomarker signatures of early rejection in heart transplant and miscarriage prone pregnancies, without invasive biopsy. We have developed an implant to remotely derive and monitor early immunological markers of alloimmunity in transplantation and pregnancy. Scaffold gene expression differentiates immune state across time and disease progression prior to the onset of symptoms, creating an early, novel therapeutic window to prevent transplant or fetal injury. |
| Eric Ginter |
University of Colorado Boulder |
Endothelial cells (ECs) compose the interior wall of blood vasculature and are key to maintaining vessel barrier function. In addition, ECs are now broadly recognized as active participants in the immune response, with ECs secreting and responding to various chemokines to mediate inflammation. Previous work has shown vessel origination within the body leads to heterogeneity in EC phenotype, but implications of these differences are not understood in context of the inflammatory response. Other work demonstrates that arteries and veins differ in neutrophil recruitment, with neutrophils preferentially extravasating through veins. This project will reveal how endothelial heterogeneity causes differences in cytokine secretion, and how those differences impact neutrophil-EC interactions. To accomplish this, we are using an infection-on-a-chip in vitro system that integrates a model blood vessel surrounded by ECM, and allows modular addition of ECs, neutrophils, and immune stimulation via cytokines or live pathogens. Others have shown that 3D culture constructs support native EC behavior better than 2D plates, and our system harnesses this advantage. In addition to providing growth conditions more relevant to in vivo, our device allows for live imaging of neutrophil extravasation – a traditionally difficult process to observe. We have successfully integrated multiple EC sources into our device from sites including microvascular lung and dermis, and umbilical vein and artery. Each cell source was separately optimized for growth in the device by varying cell seeding density and incubation time. We have characterized the vessels by confirming barrier integrity and function through staining and FITC-dextran diffusion assays. Neutrophils have been added to the system, and we have observed differences in extravasation from different EC vessels. We’re currently collecting samples to determine protein and RNA expression levels across cell types with and without inflammatory stimulus. With the completion of this research, we will unveil how endothelial heterogeneity impacts inflammatory mediation. |
| Emily Boltcreed |
Stevens Institute of Technology |
Neural stimulating electrodes can remain implanted for over 10 years to treat symptoms of Parkinson’s Disease (PD). However, long-term effectiveness of probes is hindered by the foreign body response. The gamma band (30-60Hz) has been indicated in modulating glial recruitment using unique inflammation pathways to the electrode-tissue interface in visual and ultrasound applications. We explore here the potential for chronic microelectrode stimulation at 40Hz to change the long-term glial response to implanted electrodes. Male Long Evans rats (n = 12) received 6-OHDA lesioning and were fitted with an 4x4 Pl-Ir microelectrode array in one randomized hemisphere. One week post-implant, stimulation was applied on a single experimental electrode shank randomized out of the four at 40Hz for 1hr at a constant current of 10uA using 60us-100us, charged balance biphasic pulses. Acute animals were sacrificed post-stimulation and chronic animals were sacrificed 2 weeks post-initial-stimulation. Passive recordings were taken before and after stimulation. Fixed samples were stained for ED1 (activated microglia), GFAP (activated astrocytes), TH (dopaminergic neurons) and DAP (nonspecific nuclei). Both acutely and chronically, microglial cells were at their densest in the 100𝜇m around the stimulated probe, although the percentage difference between stimulated and non-stimulated probes was 3x acutely and less than 1.5x chronically. Acutely and chronically, in the first 100𝜇m away from the implant, astrocytic density around the stimulated probe was significantly lower than that of adjacent probes but higher than that around opposite probes. This trend reversed past 100𝜇m into the tissue. The mechanism for this is not clear and likely due to a molecular reaction to the gamma stimulation. These results suggest that gamma stimulation is changing the immune response in ways that could be used to minimize glial scarring long term. |
| Bumjun Kim |
Princeton University |
Introduction: The success of mRNA COVID LNP vaccines has revolutionized nanoparticle- based drug delivery. LNPs target the liver due to PEG-lipid desorption and ApoE binding, leading to LDLR-mediated uptake by hepatocytes. Alnylam's Onpattro employs this to deliver TTR-silencing RNA to hepatocytes. However, targeting LNPs to non-liver cells remains a challenge. Recent attempts to guide LNPs using antibodies often face premature separation due to weak lipid-antibody binding. Thus, there's a need for a next-generation LNP that reduces liver targeting and improves T cell modulation in vivo. Methods: Here, we present the development of ngLNPs that replace the lipid-PEG with block copolymer (BCP), specifically poly(ε-caprolactone)-block-poly(ethylene glycol) (PLC-b-PEG). The incorporation of PCL-b-PEG, possessing a more substantial hydrophobic domain than lipid tails in PEG-lipids, reduces the likelihood of "shedding" post-Ab attachment. To craft and refine these pioneering LNP blueprints, we employed a design of experiments (DOE) approach and synthesized LNPs through a scalable Flash NanoPrecipitation (FNP) technique. Results: Our research identified a design space wherein the LNPs maintain a diameter below 140 nm, encapsulate efficiently (>80%), and outperform standard LNPs (sLNPs) in terms of transfection within benchmark cell lines. Notably, sucrose emerged as the superior cryoprotectant for both ngLNPs and sLNPs during freeze-thaw cycles.. Conclusions: The optimized LNP formulations demonstrate superior in vitro transfection capabilities without any trade-off in their physicochemical attributes. The enhanced stability offered by the larger BCP suggests that ngLNPs can be effectively directed towards extrahepatic sites using Abs tethered to the BCP. Upcoming research will focus on conjugating anti-CD5 Abs to ngLNPs to investigate their biodistribution and to gauge their efficacy in in situ CAR-T production. |
| Sabrina N. VandenHeuvel |
Texas A&M University |
The 5-year survival rate of metastasized ovarian cancer (OvCa) is <30%. Clinical biospecimen link poor prognosis to increased macrophage immune checkpoint signaling. OvCa cells express CD47 which binds macrophage checkpoint SIRP (signal regulatory protein-) to suppress anti-tumor immunity and enhance OvCa chemoresistance and metastasis. We developed a short interfering RNA (siRNA) nano-immunotherapy (siSIRP) to disrupt macrophage checkpoint signaling by reducing SIRP expression. Microfluidic techniques were used to prepare stable and reproducible lipid nanoparticles encapsulating SIRP siRNA. OvCa/macrophage co-culture heterospheroids were generated on a hanging drop array and maintained for up to 6 days with 25 nM siSIRP administration on day 2. Resulting SIRP gene (RT-qPCR) and protein (flow cytometry) expressions as well as spheroid proliferation and carboplatin chemotherapy response (MTS viability assay) were evaluated. Invasive potential was quantified by seeding compact spheroids onto culture dishes and monitoring spheroid area expansion over 5 days. Owing to the terminal differentiation state of macrophages, heterospheroids were smaller and less proliferative than monospheroids (OvCa cells alone). Despite smaller spheroid size, macrophages contributed to carboplatin chemoresistance making heterospheroids 4.7-fold more resistant. OvCa cells in heterospheroids also showed significantly increased invasive potential (compare 11.1-fold to 4.4-fold area increase in monospheroids, ***p<0.001). siSIRP therapy reduced SIRP gene and protein expression in heterospheroids by 42 and 59%, respectively (****p<0.0001) and improved chemosensitivity by 60%. Notably, when siSIRP was administered to monospheroids, neither chemoresistance nor invasive potential were significantly altered, supporting that our therapy directly affects macrophages in the system, as desired. Our work underscores that CD47-SIRP signaling inhibitors are a novel therapeutic agent against OvCa progression and chemotherapy resistance. Optimizing nano-therapies is advantageous to control transport, timing and specificity of RNA therapies. An additional benefit is the ability to design these nanoparticles for uptake by intrinsically phagocytic macrophages to treat and curb metastatic OvCa and positively impact survival. |
| Christopher Ashdown |
Stony Brook University |
Chimeric antigen receptor T cell (CAR-T) therapy is an incredibly effective cancer therapeutic. Nevertheless, autologous CAR-T therapy is a relatively inefficient process that remains confounded by a time-intensive ex vivo expansion process. Current strategies to reduce this manufacturing time involve adding various cocktails of cytokines or expansion beads. However, these chemical additives can often have deleterious effects on CAR-T therapy outcomes, as the increased expansion they offer comes at the expense of higher levels of T cell exhaustion, which are associated with less functional CAR-T cells. Mechanical signals represent a previously underutilized strategy for improving expansion of suspension cells and may be able to influence cell growth without the side effects generated by traditional chemical manipulation. We tested the hypothesis that mechanical simulation, delivered non-invasively using low intensity vibration (LIV, at: 30 Hz, 0.7g, 2 bouts of 1h/d, 2-hour refractory period) could be used to improve T cell expansion without disrupting phenotype. We found that LIV was able to drive a significant increase in T cell proliferation (30% in 5 days). This increase in proliferation was driven by roughly 20% increase in the number of activated T cells (CD62L-/CD69+), showing that we can mechanically and noninvasively activate T cells with vibration. We also show that LIV may have beneficial effects on T cell exhaustion. Our LIV stimulation caused a 25% decrease in the expression of the inhibitory receptors PD-1 and LAG-3, suggesting that LIV may be used in the future to improve functionality of T cells in addition to proliferation. This proof-of-concept work provides evidence that mechanical stimulation in the form of LIV, can not only be used to significantly improve the speed of CAR-T manufacturing, but that it can do so while generating beneficial phenotypes related to T cell exhaustion, that are often sacrificed during traditional pharmacologic expansion processes. |
| Samantha Hall |
The University of Memphis |
Guided bone regeneration (GBR) is an alveolar bone loss procedure that utilizes barrier membranes to occlude soft tissue infiltration. Electrospun chitosan membranes (ESCMs) have shown promise for enhanced GBR due to drug loading capabilities, biocompatibility, and pro-healing properties. Raspberry Ketone (RK) is a phenolic compound of red raspberry that has shown significant potential in the promotion of macrophage polarization toward the M2, pro-healing, phenotype. The aim of this study was to use an in vitro macrophage polarization model and microarray analysis to evaluate the immunomodulatory effects of RK when released from ESCMs. RAW cells were seeded in 24 well plates at 100,00 cell/ml in DMEM + 10% FBS-1% PSN media. After 24 hours, M1, pro-inflammatory, activation was induced using lipopolysaccharide (LPS). After 24 hours of LPS incubation, medium was replaced with complete DMEM, DMEM containing 300 ng/ml PGE2, or 10mm diameter ESCMs loaded with 0, 50, 100, 250, or 500 µg RK /membrane. Positive controls only received 1 µg/mL LPS, and negative controls did not receive any treatment. Groups (n=3) were assayed on days 1, 3, and 5 for 20 pro and anti-inflammatory cytokines present in the culture medium using microarray analyses (RayBiotech Mouse Cytokine Array Q1, GA). Results showed that RK treated groups had a decreased production of the immune cell recruitment chemokine, RANTES. RK groups also saw an increase in M2 associated, pro-inflammatory inhibiting factors IL-10, IL-9, and IL-4 in comparison to non-treated groups. Additionally, RK groups saw an increased production in the angiogenic factor VEGF-A at day 3. RK groups also saw an increased production of the osteogenic cytokine IFNϒ at days 1 and 3 in comparison to RK treated groups. This indicates that RK has potential for uses in GBR and other biomaterials-based applications for the facilitation of wound healing. |
| Benjamin S Haslund-Gourley |
Drexel College of Medicine |
A major factor contributing to severe COVID-19 infection is the extensive deposition of complement in the pulmonary and renal systems. IgM is a potent initiator of complement deposition, containing glycans that can participate in C1q binding. These glycans are sugar structures post-translationally attached to IgM in the Golgi by transferases. However, the glycans on IgM have not been studied during any infectious disease. For the first time, we report alterations in IgM glycosylation significantly correlate with COVID-19 severity and promote higher levels of complement deposition. These findings were confirmed within total IgM and SARS-CoV-2-specific IgM glycan profiles. In contrast to the IgG glycans from severe patients, IgM glycans present more negatively charged sialic acids and unprocessed branching mannose structures. We link the changes of IgM glycosylation with the mRNA expression of Golgi transferases quantified from circulating immune cells. These IgM glycan changes significantly correlate with other markers of disease severity including D-dimer, creatinine, potassium, and blood urea nitrogen. To further explore the role of IgM glycans during severe COVID-19, we developed an antigen-specific complement deposition assay. Using this assay, we observe that IgM-dependent complement deposition is elevated in severe COVID-19 patients. Moreover, we modulated the amount of IgM-dependent complement deposition by enzymatically removing sialic acid glycans from IgM. Taken together, this work identifies the glycans on IgM as biomarkers of COVID-19 severity and highlights a mechanism promoting COVID-19 pathogenesis through complement deposition. |