| Shreya Soni |
Drexel University |
Small molecule immunotherapeutics are readily available to treat inflammatory diseases, ranging from cancer to heart disease. Their efficacy, however, remains limited by poor solubility, rapid clearance, and off-target effects in other tissues. Injectable hydrogels enable minimally-invasive and localized delivery to overcome these challenges. Here, we develop an injectable hydrogel for small molecule drug delivery, composed of cyclodextrin nanoparticles (CDNPs) dynamically crosslinked by adamantane-modified hyaluronic acid (Ad-HA). Individual components, CDNP and Ad-HA, are viscous solutions that form a viscoelastic hydrogel when mixed. The hydrogel exhibits shear-thinning under high strain for ease of injection and rapid self-healing for retention in the tissue. Hydrogel properties are dependent on crosslinking density, controlled via Ad-HA modification (25% or 50% of HA units), component concentrations (2.5 to 10 wt%), the ratio of guest to host components, and molecular weight of Ad-HA. We found that a high Ad-HA modification (50%) and a 1:1.5 ratio of Ad-HA:CDNP formed gels with greatest viscoelastic moduli and the slowest rate of degradation. Celastrol was encapsulated as a model anti-inflammatory drug (IC50 <100 nM for NF-κB inhibition); drug affinity for CD enables controlled release (Keq = 0.474mM, determined by SPR). Celastrol-loaded CDNPs were released in bioactive concentrations for >14 days in vitro, rapidly uptaken by macrophages (MF), and knocked down inflammatory pathway activity (>80%). In sum, the polymer-particle hydrogels developed exhibit sustained release of immunomodulatory drugs and injectable properties that enable their application towards localized and MF-targeted immunotherapy. |
| Stephen Linderman |
Georgia Institute of Technology |
Surgical interventions are frequently complicated by local inflammation and swelling, leading to postoperative pain, scarring, and foreign body responses. While hydrogels are effective vehicles for local, immunomodulatory drug delivery, extreme swelling upon degradation limits clinical applications. When implanted in tight surgical spaces (e.g., around the spine or airway), hydrogel swelling creates pressure on surrounding tissue that can cause pain and tissue necrosis. Amphiphilic hydrogel networks can minimize swelling. Here, we evaluate the effects of thiolated PEG-PPG-PEG (Plu-DT) amphiphilic crosslinkers versus hydrophilic thiolated PEG controls (PEG-DT) in PEG-4 arm maleimide (PEG-4Mal) networks on swelling and hydrogel stiffness with degradation. Compared to control hydrogels with similarly sized hydrophilic crosslinkers, amphiphilic gels exhibited lower swelling following casting. This effect was more pronounced in gels with lower PEG-4Mal molecular weight, i.e., higher hydrophobic content. Furthermore, amphiphilic hydrogels exhibited 2- to 3.5-fold lower swelling weights following lyophilization compared to before lyophilization, indicating significant fiber annealing beyond that seen in control hydrophilic hydrogels, possibly from clustering of hydrophobic segments. Within 7 hours of accelerated ester hydrolysis at pH 10, amphiphilic hydrogels (PEG-4-ester-Mal 20 kDa, 10% w/v, crosslinked with Plu-DT) swelled 21.3% ± 1.7% by weight, while hydrophilic hydrogels crosslinked with PEG-DT swelled 87.1% ± 3.4%, or 4.1-times more. Finally, these amphiphilic hydrogels were loaded with dexamethasone-containing PLGA microparticles for sustained drug release. Dexamethasone has been shown to counter inflammation via reducing vascular permeability, inhibiting T-cell based inflammation via increasing CTLA4 checkpoints and blocking CD28-mediated T cell proliferation, inhibiting neutrophil and macrophage infiltration, and inhibiting IL1 production. This approach allows local, postoperative drug delivery for immune modulation in tight surgical spaces, with significantly reduced hydrogel swelling and reduced pressure on surrounding tissues. By reducing local tissue swelling and inflammation, these immunomodulatory drug delivery gels promise to reduce postoperative pain, scarring, and associated clinical complications. |
| Serena Omo-Lamai |
University of Pennsylvania |
The COVID-19 vaccine has demonstrated the power of RNA-containing lipid nanoparticles (RNA-LNPs). While there is great potential for RNA-LNPs to be applied in a variety of diseases, their use is very limited in inflammatory conditions. This is due to a phenomenon elucidated in this work termed inflammation exacerbation (IE), in which LNPs dramatically worsen pre-existing inflammation, whether in the diseased organ or in a remote organ (present due to comorbidities). We have proven that IE is caused by the escape of RNA from the endosome and is, therefore, intrinsic to any RNA delivery system - even nucleoside-modified RNAs. We have also shown that the IE effect decreases the biodistribution and translation of the LNP-delivered RNA. The IE phenomenon therefore limits the use of RNA-LNP therapeutics in any condition with inflammation such as ARDS, heart attack, and stroke. In this study, we examine and characterize the signaling pathways that propagate IE and show that this effect is likely due to the NLRP3 inflammasome. We then test the efficacy of MCC950- a selective and potent NLRP3 inhibitor. We also ameliorate the IE phenomenon using anti-inflammatory agents that act up- and downstream of NLRP3 – dexamethasone and IL-1Ra. The IE effect poses a significant problem for nanomedicine and perhaps gene therapy at large. This proposal will therefore be the first to elucidate the mechanisms of IE and develop multiple therapeutic interventions. |
| Marian Ackun-Farmmer |
University of Maryland |
Multiple sclerosis (MS) develops when autoreactive lymphocytes mistakenly attack myelin in the central nervous system, resulting in reduced quality of life for patients. Treatments for MS are non-curative, require life-long compliance, and due to lack of specificity can increase patient susceptibility to infection. A therapeutic approach to treat MS involves co-delivery of self-antigens and immunomodulatory cues to induce tolerance by driving naïve T cell polarization away from inflammation and towards tolerogenic phenotypes such as regulatory T cells (TREGS). Toll-like receptors (TLRs), which are traditionally associated with pathogens, detect pathogen-associated patterns on antigen presenting cells (APCs) and are upregulated in MS1. Thus, to achieve the goal of promoting tolerance in MS, we developed self-assembled carriers built entirely from immune cues – termed immune polyelectrolyte multilayers (iPEMs). We showed that iPEMs assembled using myelin oligodendrocyte glycoprotein (MOG) and GpG blunt innate immunity while promoting TREGS to improve disease severity in a preclinical MS model known as experimental encephalomyelitis (EAE). MS impacts both female and male patients4, however, EAE is typically induced in female mice due to their susceptibility to the disease. Thus, to support translation of iPEMs and test if biological sex-differences impact its effectiveness, we developed iPEMs for male mice induced with EAE and female mice induced with relapsing-remitting EAE (RR-EAE). We found that male EAE mice treated before disease onset with MOG/GpG iPEMs showed reduced disease severity. Since RR-EAE represents a majority of human MS cases, we next tested if iPEMs assembled using PLP139-151/GpG would dampen relapses. Excitingly, we found that iPEM treated RR-EAE mice also exhibited reduced overall disease severity and lessened relapses. These important findings support our hypothesis that iPEMs motivate subsequent studies to interrogate the role of sex-differences in iPEM effectiveness. |
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| Rimsha Bhatta |
University of Illinois at Urbana Champaign |
Tumor cell-derived exosomes, nano-sized extracellular vesicles with a size of 30-150 nm, inherit various types of proteins, RNAs, polysaccharides, and lipids from the parent tumor cells, and are considered a good source of tumor antigens. These EVs have been widely explored as cancer vaccines due to their excellent safety profile, easy manufacturing, and capability to generate antitumor immune responses. However, the resultant antitumor efficacy of tumor exosome vaccines is still far from satisfactory, likely due to the poor cytotoxic T lymphocyte (CTL) response. Here we report a universal yet facile metabolic labeling approach to generate chemically tagged exosomes from tumor cells, which enable conjugation of immunomodulatory agents for the development of enhanced exosome vaccines. This exosome tagging and targeting technology provides an unprecedented and generalizable approach to generating chemically tagged exosomes from all types of tumor cells for the development of potent exosome vaccines. |
| Zain Clapacs |
Washington University in St. Louis |
Plasmacytoid dendritic cells (pDCs) play a unique role in responding to viral infection as the chief producers of interferon-a (IFNa), which is known to enhance development of cellular and humoral immunity. Recent studies have found that pDCs uniquely overexpress the endocytosing iron transporter CD71 compared to other leukocyte populations. By presenting gambogic acid (GA), a known noncompetitive ligand of CD71, on PEGylated liposomes we have selectively targeted pDCs for liposomal vaccine delivery in murine lymphoid organs. Our results demonstrate selective uptake of GA-liposomes by spleen and lymph node pDCs compared to other phagocyte populations. We also observed that pDC uptake occurs rapidly (~ 4h) compared to conventional dendritic cell (~ 18h) and no targeting effects were observed in bone marrow (consistent with poor CD71 expression). To confirm the robustness of the GA-targeting mechanism we also replicated the effect using alginate nanoparticles. To the best of our knowledge, this work is the first to report non-competitive active transport of vaccines to pDCs, which may confer protective immunity through IFNa stimulation, which is crucial for antiviral immunity. |
| Christine Hamadani |
The University of Mississippi |
Introduction: <1% of IV-injected nanoparticles (NPs) reach destined tumor sites in diseased tissue. By hitchhiking onto blood components post-injection, NPs can be selectively delivered to targeted tissue sites. Ionic liquids (ILs) are liquid salts <100℃ composed of bulky asymmetric cations and anions. We have shown that IL coated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) significantly extend circulation time in BALB/c mice post-IV injection. This occurs via hitchhiking on red blood cell (RBC) membranes, resulting in selective lung accumulation. We now investigate the impact of structurally engineering choline carboxylate ILs to selectively interface and hitchhike different blood components in whole blood to achieve targeted drug delivery. Methods: We synthesized & screened 60 choline carboxylic acid-based ILs to coat PLGA NPs (IL-PLGA NPs). The structural relationship between anion and biological membrane affinity was studied ex-vivo in whole pooled-gender BALB/c mouse blood [100 NP:RBC]. Major cell fractions (white blood cells (WBCs), platelets, RBCs) were separated & washed to eliminate unbound NPs (6). The degree of hitchhiking was screened qualitatively by fluorescence activated cell sorting (FACS). Identified leading candidates were measured quantitatively as % Injected Dose (%ID,n=4) by fluorescent plate reader, observed by cellular imaging/microscopy, and assayed for biosafety/biocompatibility (hemolysis(n=9), platelet activation(n=4), and WBC cytotoxicity(n=3)). Results: Choline carboxylate IL-PLGA NP lead candidates direct selective hitchhiking affinity towards lymphocytes(40.5 ± 0.69%), monocytes(30.1 ± 0.66%), granulocytes(32.7 ± 2.6%), platelets (19.8 ± 1.7%), and RBCs(50.9 ± 9.8%). Confocal microscopy showed IL-PLGA NPs on targeted cell populations. All leading candidates exhibited high biocompatibility (sub-10% toxicity) vs. PLGA across all cell biosafety assays. Conclusions: We have established a modular and transformative targeting technology to hitchhike onto blood components that may drive selective biodistribution with high efficacy and safety in the bloodstream post-IV administration. Further research will examine precise chemical mechanisms driving the hitchhiking phenomena, safety, and immune responses. |
| Grace Farbin |
University of Toronto |
The delivery of drugs to the central nervous system is grossly limited by the presence of the blood-brain and blood-spinal cord barriers. These barriers can be transiently permeabilized using low-intensity focused ultrasound combined with circulating microbubbles. The spectral content of acoustic emissions from cavitating microbubbles can be analyzed to control for and mitigate potential bioeffects. However, there is a large degree of uncertainty as to where these cavitation signals are originating. Passive beamforming of microbubble emissions recorded using multi-element arrays can enable spatial mapping of cavitation activity. Passive acoustic mapping (PAM) is challenging to implement in the presence of an intervening bone layer but has been successfully demonstrated through the skull. Here we present the first experimental demonstration of PAM through human vertebral bone. A tube containing flowing microbubbles was placed in the canal of ex vivo human thoracic vertebrae (stack of 3 levels) and excited (250 kHz) through the right laminae. A 64-element large aperture 2D array was used to receive the harmonic emissions through the left laminae. Reconstructed maps successfully localized the cavitation activity to the spinal canal. Future work will examine effects of phase/amplitude correction, receiver number, and location of the cavitation relative to the vertebral anatomy. |
| Kathryn Wofford |
University of Pennsylvania |
The central nervous system (CNS) regulates the peripheral immune system (PIS) and maintains a homeostatic balance. Indeed, during an infection, the brain counters mounting inflammatory cascades by inducing immunodepressive signals in the body, thereby helping the body return to a healthy homeostasis. However, following a traumatic brain injury (TBI), the brain becomes flooded with inflammatory signals, disrupting the balance between the CNS and PIS. For instance, CNS injury-induced immunodepression is a leading cause of death following severe TBI. However, specific immunological consequences of TBI have yet to be investigated in a clinically relevant model of TBI. Here, we employed a highly translational preclinical model of diffuse closed-head TBI in swine to characterize changes to the PIS over time and injury severity. We hypothesized that following a moderate-to-severe closed-head TBI, the PIS would exhibit hypersensitivity at acute timepoints and immunodepression at more chronic timepoints. To test this, we collected peripheral whole blood from female Yucatan mini pigs in sham (n=3), mild (n=4), or a moderate-to-severe closed-head diffuse TBI (n=7) conditions. Whole blood was drawn prior to injury procedure and over 14 days to collect peripheral blood mononuclear cells (PBMCs) and plasma as repeated measures. Relative to pre-injury baseline levels, select plasma cytokine expression levels were significantly diminished 14 days after TBI – interleukin (IL)-6 after moderate-to-severe injury and IL-10 and TNF after mild injury. PBMC numbers significantly increased 3- and 10-days post injury relative to pre-injury levels. The phagocytic activity of isolated PBMCs was increased at 7- and 10-days post moderate-to-severe TBI when cells were challenged with polystyrene particles or bacteria-laden nanoparticles, respectively. Together, these data suggest that significant changes to PIS demographics, phagocytic reactivity, and plasma proteins occur 1-2 weeks after TBI. Future studies aim to learn how the dysregulated PIS contributes to neuropathology and secondary injury after TBI. |
| Sabrina DeStefano |
NIBIB NIH |
Upon wounding and material implantation there is a disruption in tissue homeostasis. With analysis of antigen-presenting cells in biomaterial-treated muscle injury in a pro-fibrotic and pro-regenerative context, previous data completed has shown that pro-regenerative materials enrich Batf3-dependent CD103+ XCR1+CD301b+ dendritic cells associated with cross-presentation and self-tolerance. Trauma was accompanied by CD8+ iTregs and expansion of CD103+XCR1+CD62L- adaptive immune cells. Up-regulation of E-Cadherin and XCL-1 in injured tissue suggests a mechanism for cell recruitment to trauma. Without cross-presenting cells, there is an increase in T cell activation, a decrease in pro-regenerative macrophage polarization, and a defect in muscle healing. These data describe a communication network through CD103+XCR1+ antigen-presenting and adaptive immune cells resulting in downstream effects on tissue regeneration. With this knowledge in context of a volumetric muscle loss (VML) traumatic injury, the next question is if we see this cross presenting dendritic cell communication occur throughout different tissue context upon stimulus of an implanted pro-regenerative material. To evaluate immune responses in the context of trauma and tissue regeneration, we looked at the immune response of porcine derived small intestine submucosa (SIS) decellularized extracellular matrix (ECM) material in a C57BL/6J (B6) mouse model. To test the variable of this immune response in context of tissue location, an ECM paste material was injected intraperitoneally (IP), subcutaneous (subq), and in the quadricep muscle within a VML injury. To analyze this, we ethically sacrificed the mice and removed the quadricep muscle and both IP and subq implants to further run the specimen on a flow cytometer using a 22-color flow cytometry panel. This data has shown a varied set of innate immune cells recruited to three different tissue locations which received the ECM material by 7- and 21-days post-injury. Data was analyzed using FlowJo and spectral unmixing was done after acquisition on SpectroFlo software. |
| Ishan Sinha |
Wayne Hills High School |
The COVID-19 pandemic brought unprecedented challenges for healthcare. In particular, surgeries were delayed due to resource constraints and concerns about hospital spread of COVID-19. This study assesses the impact of the pandemic on surgery, by examining malfunctions and injuries attributed to surgical sutures. The Food and Drug Administration (FDA) Manufacturer and User Facility Device Experience (MAUDE) database was mined for reported adverse events for non-absorbable and absorbable sutures, from January 2018 through June 2022. The results reveal that injuries attributed to non-absorbable sutures rose significantly from 2019 through 2021. Between 2019 and 2020, monthly injuries attributed to non-absorbable sutures rose 85.5%, from 40.9 per month in 2019, to 75.9 per month in 2020 (p<0.03). Between 2020 and 2021, monthly injuries attributed to non-absorbable sutures further increased 69.5%, from 75.9 per month in 2020 to 128.7 per month in 2021 (p<0.02). Overall, injuries attributed to non-absorbable sutures rose 214.5% from 2019 to 2021 (p<0.0002). The rise in injuries was specific to the pandemic; there was no significant increase in injuries from 2018 to 2019 (p>0.24). In addition, malfunctions attributed to absorbable sutures rose 30.6% between 2020 and 2021, from 255.9 per month in 2020, to 334.2 per month in 2021 (p<0.003). Delays in diagnosis and surgery during the COVID-19 pandemic may have raised the risk of complications; further research is required to determine long-term outcomes of surgeries performed during the pandemic. |
| Yixiang Deng |
Ragon Institute |
The COVID-19 pandemic catalyzed a revolution in vaccine development, leading to the testing and approval of several global vaccine platforms that have shown tremendous promise in curbing the pandemic. Yet, despite these successes, waning immunity, and the emergence of variants of concern linked to rising breakthrough infections among vaccinees, have begun to highlight opportunities to improve vaccine platforms and deployment. Real-world vaccine efficacy has highlighted the reduced risk of breakthrough infection and disease among individuals infected and vaccinated, otherwise referred to as hybrid immunity. The hybrid immunity points to the potential for more vigorous or distinct immunity primed by the infection and may confer enhanced protection from COVID-19. Beyond augmented hybrid induced neutralizing antibody and T cell immune responses, here we sought to define whether hybrid immunity may shape the functional humoral immune response to SARS-CoV-2 following Pfizer/BNT162b2 and Moderna mRNA1273 mRNA-based, and ChadOx1/AZ1222 and Ad26.COV2.S vector-based SARS-CoV-2 vaccination. Each vaccine exhibited a unique functional humoral immune profile in the setting of naïve or hybrid immunity. However, hybrid immunity showed a unique augmentation in S2-domain specific functional humoral immunity that was poorly induced in the setting of naïve immune response. These data highlight the immunodominant effect of the S1-domain in the setting of natural immunity, which is highly variable during viral evolution, and the importance of natural infection in breaking this immunodominance in driving immunity to the S2 region of the SARS-CoV-2 S2 domain that is more conserved across variants of concern. |
| Niamh Ward |
University of Galway, Ireland |
TITLE: Immunomodulation of the host foreign body response. INTRODUCTION: The long-term performance of medical implants is hindered by the foreign body response (FBR). This protective mechanism is intended to isolate potentially harmful objects from the body. Ultimately, inflammatory cytokines such as TGF-β1 activate myofibroblasts, which deposit a hypo-permeable, collagen-rich fibrotic capsule (FC). This is problematic for implants designed for therapy delivery as the FC impedes molecular exchange. We previously reported that intermittent pneumatic actuation (IA) of an implanted reservoir can significantly reduce FC thickness in a 14-day rat model1. Recently, we impressively demonstrated that insulin diffusion kinetics were maintained at baseline levels for the entire eight-week study in a mouse model when implanted reservoirs received IA2. We hypothesise that actuation-induced tissue strain can modulate local inflammatory pathways: to investigate this, we examine cellular interactions that modulate collagen production under IA in vitro. METHODS: Myofibroblasts (WPMY-1) were seeded onto reservoirs and cultured for 14 days under IA. Cell viability was evaluated using metabolic activity and cytotoxicity assays. Collagen (Sircol Soluble Collagen assay) and TGF-β1 (ELISA) production were compared to non-actuated controls. Analytical models of device strain were developed based on Von Kármán plate theory. RESULTS: We observed that IA did not negatively affect cell viability. Interestingly, IA induced a significant reduction (p=0.0463) in TGF-β1 production, followed by a significant reduction in collagen production after 9 (p=0.0132) and 14 (p=0.0038) days actuation compared to non-actuated controls. Models predict maximum radial and tangential strains of 3.9% during IA. CONCLUSION: We have demonstrated the compelling potential of IA to modulate the FBR, optimising the local implant environment for therapy diffusion1,2. In vitro, we have uncovered alterations in the TGF-β1 pathway leading to a reduction in collagen production by WPMY-1 cells exposed to IA. Ongoing work is investigating a wider range of inflammatory markers, and correlating strain predictions to cell and tissue responses to fully elucidate the mechanism of IA mediated FBR modulation. (REFERENCES: 1. Dolan et al. Sci Rob 2019. 2. Whyte, Goswami, & Wang et al. Nat Comms 2022) |
| Tina Tylek |
Drexel University |
Phagocytosable Dexamethasone Loaded PLGA-Microparticles Control Macrophage Phenotype Intracellularly in Vitro and in Vivo Tina Tylek*1, Joanna Wong2, Andrew Vaughan2, Kara Spiller1 1: Drexel University, Philadelphia; 2: University of Pennsylvania, Philadelphia 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 stages of wound healing and tissue repair, making them an attractive choice for cell therapy applications. However, macrophage cell therapy has been limited by their tendency to rapidly change phenotype in response to local cues. Therefore, control over macrophage phenotype is critical for their use in cell therapy applications. Hence, in this study, we investigated the efficacy of phagocytosable PLGA microparticles (MP) encapsulating dexamethasone (Dex), an anti-inflammatory and antifibrotic drug, to control the macrophage phenotype intracellularly. Murine macrophages that phagocytosed Dex-MPs exhibited an anti-inflammatory, antifibrotic macrophage phenotype characterized by downregulation of inflammatory markers and cytokines under pro-inflammatory conditions in vitro and an expression pattern of MMPs and TIMPs (markers involved in the inhibition/promotion of fibrosis) previously shown to be effective in downregulating fibrosis in lungs or kidneys. Upon administration directly to inflamed or fibrotic lungs of mice, macrophages containing Dex-loaded microparticles exhibited a phenotypic profile that was distinct from those containing blank particles as well as those containing Dex-loaded microparticles that were cultured in vitro. In particular, expression of the M2 markers CD206 and CD301b was maintained. These results show that the intracellular release of Dex did affect macrophage phenotype but that it was also affected by the microenvironment. Furthermore, analysis of bystander macrophages (in vivo and in vitro via direct coculture) revealed phenotypical differences depending on administered macrophages. Thus, intracellular control of macrophages for cell therapy with Dex is a promising strategy as it induced an antifibrotic, anti-inflammatory phenotype in vitro, which could be partly maintained in vivo. As the host environment strongly influenced macrophage phenotypes, further studies will include optimization of dosing and timing of drug and cell administration. |
| Gina Cusimano |
Drexel University |
Dendritic cells (DCs) are critical mediators of antigen-specific immunity through their ability to present antigen and drive T cell differentiation into different effector subsets such as follicular helper T cells (TFH). TFH cells provide both physical and cytokine-mediated stimuli to B cells resulting in somatic hypermutation and class-switching of B cell receptors. Molecules that target TFH cells, such as adenosine deaminase-1 (ADA-1), would improve humoral immunity. We have previously demonstrated that co-delivery of plasmid-encoded adenosine deaminase with an HIV-1 envelope and SARS-CoV-2 DNA vaccines in vivo, enhanced both humoral and cellular responses. However, the mechanism by which ADA-1 is acting as an adjuvant remains to be elucidated. To this end, we treated monocyte-derived DCs from healthy human donors in vitro with recombinant ADA-1 protein and evaluated the expression of maturation markers, cytokines and chemokines. ADA-1-treated DCs had a significantly increased expression of CD40 and CD86 as well as HLA-DR compared to their unstimulated, immature counterparts. The level of co-stimulatory marker and HLA-DR expression on ADA-1-treated DCs was similar to that on LPS/IFN-γ-treated DCs, indicating ADA-1-mediated DC maturation. ADA-1-treated DCs also exhibited a significant increase in IL-6, IL-1ß and CXCL13 expression. IL-6 is a key pro-TFH cytokine and IL-1ß and CXCL13 may play a role in TFH cell differentiation, function, and proliferation. Ongoing studies are aimed to drive ADA-1 overexpression in DCs and evaluate effects on antigen presentation. Overall, elucidating the mechanism of ADA-1’s adjuvanticity, will allow for its progression as a clinical adjuvant. |
| Leonor Teles |
University of Miami |
Type 1 Diabetes (T1D) results in β cell destruction from impaired T cell tolerance. Exploiting therapeutic tolerance induction mechanisms using fibroblastic reticular cells (FRC) could protect against the disease. FRCs contribute to peripheral tolerance in lymph nodes (LN) and create supportive, interconnected reticula and allow expansion during inflammation. FRCs also express and present self-antigens, including β cell antigens, to autoreactive T cells. However, unlike professional antigen-presenting cells, FRCs do so with limited co-stimulation, thereby inducing T cell tolerance. In T1D LNs, the FRC frequency, expression of T1D-relevant antigens, and reticular organization decrease, likely reducing FRC tolerogenic engagement of autoreactive T cells. Our goal is to tissue-engineer FRC reticula that recapitulate these organizational changes in inflamed and healthy LNs to determine tolerance mechanisms and develop T1D FRC-based therapies. We previously showed that genetically-engineered FRCs overexpressing T1D-specific autoantigens seeded in collagen scaffolds with 400μm-diameter pores generated reticula with pores recapitulating inflamed LNs (100-200 μm) and engaged antigen-specific T cells. We then fabricated freeze-dried gelatin scaffolds with tunable pore sizes to generate FRC reticula resembling non-inflamed LN pores (15-20µm). The reticular pores of scaffolds frozen in liquid nitrogen had smaller pore sizes (<50µm) compared to -80ºC (<150µm) when characterized by SEM. We confirmed that FRCs seeded in each scaffold form reticula with pore sizes mimicking healthy (37um) and inflamed (89um) LNs via confocal microscopy and remain viable for at least 21 days via CellTiter-Glow assay. We are currently investigating the effects of FRC reticula pore sizes on the engagement of T1D antigen-specific T cells with T1D antigen-expressing FRCs from a T1D mouse model. Our tissue-engineering approach will allow us to study FRC-T cell interactions in an environment that recapitulates the T1D FRC reticula. We ultimately aim to test FRC tolerogenic mechanisms and develop novel FRC-based therapies for peripheral tolerance induction in T1D. |
| Lauren M. DeLong |
University of Cincinnati |
The oxygen gradient in the intestine influences intestinal physiology, and the microbial environment. Neurons that innervate the intestine are in constant communication with the local microbiome and this communication is regulated by the physiological health of the tissue. Measuring the communication between neurons and resident gastrointestinal cells could provide essential information about gut health. Despite this, a method to simultaneously culture intestinal tissue slices with a physiologically-relevant oxygen gradient with the capability of monitoring neural-gut communication in real-time has not been explored. Here, we designed and fabricated a 3D printed microfluidic device with precisely placed 500 μm delivery ports to maintain the oxygen gradient. The gradient is maintained from outlets below while allowing access to the slice from above for neurochemical detection with fast scan cyclic voltammetry and carbon-fiber microelectrodes. Outlets are placed in an oval where deoxygenated media is delivered to the middle of the slice, then oxygenated media is delivered to the outside of the slice. An oxygen sensitive fluorescent dye, Tris(2,2’-bipyridyl)dichlororuthenium(II), is used to characterize the tunability of the gradient delivery on a slice. Then while maintaining the oxygen gradient tissue function and viability were examined with fast scan cyclic voltammetry paired with carbon fiber microelectrodes by measuring transient release of neurotransmitters in the intestine. This chip allows direct access to intestinal slices for real time measurements, and imaging while maintaining an oxygen gradient to recreate the physiological environment which has not previously been done giving new insights to neuro-immune communication. |
| Shuyue Zhan |
University of Georgia |
Bladder cancer is the tenth most common cause of cancer-related death. About 75% of newly diagnosed cases are non-muscle-invasive bladder cancer (NMIBC). Surgical transurethral resection of bladder tumor (TURBT) is the standard treatment for NMIBC but recurrence rates are high. To prevent tumor recurrence and progression, TURBT is often followed by intravesical chemotherapy or immunotherapy with Bacillus Calmette–Guérin (BCG). However, about 30% of the patients are not responsive to BCG and chemotherapy as a second-line treatment is not effective. We investigated sodium chloride nanoparticles (SCNPs) as a novel ajuvent therapy for NMIBC. SCNPs are expected to be delivered into the bladder through a urethral catheter for effective killing of cancer cells. After treatment, SCNPs will degrade into constituent ions, which are safely excreted in the urine. The project is based on the hypothesis that SCNPs can break the osmotic balance across the plasma membrane of cancer cells and interrupt essential cellular processes, eventually causing cancer cell death. While the plasma membrane is not permeable to ions, we hypothesize that SCNPs can enter the cell through endocytosis and degrade inside cells, releasing large quantities of ions into cancer cells therein. Our extensive in vitro studies showed that SCNPs are more toxict to cancer cells than normal cells. This is because cancer cells have higher intracellular sodium levels, making them more susceptible to SCNPs-induced ion disruption. We have tested SCNPs in vivo in tumor models established with bladder cancer cell lines. Our data confirmed that SCNPs can effectively suppress tumor growth without causing additional systemic toxicity. Besides, we observed that cancer cells succumbed to SCNPs released immunogenic cell death (ICD) signals including HMGB1, ATP, and CRT. Preliminary studies indicate that combination therapy with SCNPs and anti-PD-1 antibodies can trigger anti-cancer immune response, which contributes to inhibited tumor growth and metastasis. |
| Oluwatofunmi Oteju |
Drexel University College of Medicine |
Title: Modeling of HIV infection of human iPSC derived microglia In-Vitro using high content screening Oluwatofunmi Oteju1, Stephanie Matt Ph.D.1, Peter Gaskill Ph.D.1 1Department of Pharmacology and Physiology, Drexel University, Philadelphia, PA Abstract: HIV enters the central nervous system (CNS) after initial infection, and HIV persistence in the CNS can lead to a variety of neurological symptoms collectively known as neuroHIV. These neurological sequelae remain prevalent in people living with HIV despite antiretroviral therapy (ART). Myeloid cells, such as microglia and other CNS macrophages, are the main targets of HIV in the CNS and are the primary cells comprising the CNS reservoir. Microglia have long been difficult to access for study, but recent advances enable the differentiation of induced pluripotent stem cells (iPSCs) into iMicroglia that closely resemble primary human microglia. We have optimized the differentiation and plating density of iMicroglia and evaluated infection in response to multiple concentrations of an R5-tropic strain of HIV (HIVADA) +/- ART. We used high content image analysis of p24 staining coupled with Alphalisa analysis of p24 secretion as a surrogate for viral replication to evaluate viral kinetics and combined this with other high content analyses of cell size and morphology to assess changes in microglial health during infection and ART treatment. Our analyses validate our optimization and infection protocols, setting up more comprehensive analyses in upcoming studies that will use high content analyses to explore other myeloid cell models associated with neuroHIV such as iPSC-derived macrophages (iMac), as well as mixed culture systems combining iMicroglia or iMac with iPSC-derived neurons or astrocytes. These will better model how conditions seen in the human brain affect HIV infection dynamics, inflammation, and neuronal/glial health and function. |
| Lillian DeCostanza |
University of Virginia |
Introduction: Acellular Dermal Matrices (ADMs) have revolutionized post-mastectomy breast reconstructions by providing support to the silicone implant. ADM incorporation creates a host inflammatory response, implicating a significant macrophage response that is poorly understood. However, ADM has been shown to positively modulate inflammation. Many breast reconstruction patients have also undergone radiation treatment, affecting the tissue microenvironment. Here, we investigated the effects of radiation on ADM-initiated macrophage response in vivo. Methods: C57BL/6J mice were randomly assigned to receive 0 or 35Gy dorsal skin radiation. 12 weeks post radiation, animals received subcutaneous ADM implants. Implants were harvested at 1 and 3 weeks. Flow cytometry was performed using a robust panel of pan-, M1, and M2 macrophage markers. Fibroblast infiltration into the ADM was evaluated through immunohistochemical staining with vimentin. Hematoxylin and eosin staining of implant cross sections was also performed to evaluate cellularization. Results: The macrophage response to ADM integration was greatly impacted by skin irradiation. At 1 week, radiation led to the increased expression of CD9, Jag1, and CD86, and a decrease in CD163, Arg1, CD38, CD301b, and CCR2. By 3 weeks, differences in the expression profiles persisted, with radiation leading to a maintained decrease in CD163 and an increase in CCR2. When evaluating the temporal differences, the radiation expression profiles were also significantly altered. Discussion: The results provide the first investigation into the complex macrophage phenotypes over time in response to ADM with or without radiation. The macrophage response suggests that the effect of radiation is more complex than simply being more inflammatory. A comprehensive analysis of the macrophage response to ADM implantation will provide critical supporting evidence of the merits of clinical use and identify potential immunomodulatory strategies to improve ADM-associated healing. Differences in expression of CD163, Arg1, and CD301b support the use of IL-4 to promote pro-regenerative M2 phenotypes. Acknowledgements: This research was funded by the Plastic Surgery Foundation and the MTF Biologics Allograft Tissue Research Grant. |
| Xiaonan Ma |
University of Pennsylvania |
Idiopathic pulmonary fibrosis (IPF) is a type of interstitial lung disease that causes progressive decline in lung function as lung tissue becomes replaced by scar tissue. IPF has significant morbidity, exhibited by shortness of breath and oxygen dependence, and an average survival of only 5 years following diagnosis. The current standard of care is to treat IPF patients with anti-fibrotic drugs that slow the rate of declining lung function but do not reduce symptoms or prolong life. Additionally, these drugs have significant side effects that often necessitate dose reduction or limit patients’ ability to take them at all. To improve the pharmacokinetics of the anti-fibrotic drug nintedanib, we utilize two methods, nano-scale drug carriers and inhaled delivery directly to the lungs. Combined, these techniques are referred to as Pulmonary Inhaled Nanocarreirs (PINs). PINs deliver 24,000x more nintedanib to the lung and have a 10x longer lung half-life compared to conventional oral drug delivery in a murine model. Further, PINs reach multiple cell types in the distal lung, retain their shape after exposure to murine surfactant, and have no demonstrable toxicity. Ultimately we hypothesize that the improved pharmacokinetics attained with PINs will enable better efficacy and reduced side effects in the treatment of IPF and other diseases of the distal lung. |
| Ricardo Checchia Whitaker |
Drexel University |
Volumetric muscle loss (VML) is a debilitating condition resulting from the loss of a large portion soft muscle due to injury or disease. Macrophages play a crucial role in VML outcome; however, a lot is still unknown about its phenotype and interplay with other cells. We have identified systemic and local changes in immune cell trafficking, macrophage behavior and cytokine secretion in fibrotic and non-fibrotic VML injuries. Immune cell trafficking at main myeloid reservoirs (blood, bone marrow and spleen) show little differences between fibrotic and non-fibrotic injuries at the first 7 days post injury. Nonetheless, immune cell accumulation at the site of injury, mainly neutrophils at early timepoints, accompanied by an increase in systemic levels of G-CSF, and macrophages at later timepoints is significantly increased in fibrotic group compared to non-fibrotic. Macrophage phenotype is also drastically different between groups. Systemically, splenic macrophages in fibrotic show higher levels of M1 markers compared to non-fibrotic injuries. At the site of injury, fibrotic injuries presented a significantly higher expression of inflammatory markers CD9, CD38 and CXCR4 at earlier timepoints. Fibrotic injuries also presented a significantly lower expression of anti-inflammatory markers CD163 and CD301b. Nanostring analysis on FACS sorted macrophages at Day 3 post injury indicated a general downregulation of most genes in the fibrotic group compared to non-fibrotic. Many of the significantly downregulated genes were part of phagocytic pathways, while the few upregulated genes generally played a role in ECM deposition and adhesion. We have thoroughly characterized immune cell trafficking patterns and macrophage phenotype, systemically and locally, in fibrotic and non-fibrotic injuries. Due to the clear increase in neutrophil presence preceding changes in macrophage phenotype, alongside to high levels of G-CSF systemically and vast literature on neutrophil influence in macrophage phenotype, macrophage-neutrophil interactions will be studied next. |
| Breana Channer |
Drexel University College of Medicine |
Human immunodeficiency virus (HIV) is a global epidemic that can dysregulate immune cell function and survival in both the periphery and central nervous system (CNS), leading to the development of comorbid conditions. Many of these comorbidities are inflammatory and can be driven by activation of NF-B and inflammasome signaling in myeloid populations. Substance use disorders are disproportionately common in people living with HIV and are a major risk factor for CNS dysfunction, suggesting substances of misuse could exacerbate HIV-associated neuroinflammation. All addictive substances increase extracellular dopamine, and we have shown that dopamine promotes inflammation in human macrophages. However, the mechanisms by which dopamine affects HIV mediated inflammation are not clear. We hypothesize that dopamine concentrations induced by stimulant use will have more robust inflammatory effects in HIV-infected human macrophages than uninfected cells. To test this, uninfected and HIV-infected human monocyte derived macrophages were treated with dopamine. Changes in inflammatory profiles were assessed by measuring NF-kB nuclear translocation using high content imaging, inflammasome activation by western blot, and quantifying cytokine production by AlphaLISA analysis. Dopamine significantly increased the production of IL-6 and IL-1b, which were specifically mediated by dopamine receptor expression and the activation of NF-kB. Nod-like receptor (NLR) inflammasome transcripts and NF-kB baseline activation were altered in the presence of HIV. We also found that NF-kB and Akt may interact to drive dopaminergic inflammation in macrophages. Understanding the immunological effects of dopamine could usher initiatives to repurpose dopaminergic therapeutics to target immunological diseases. |
| Samuel Hofbauer |
Rowan University |
Tissue resident macrophages play a pivotal role in the local immune system of tissues in the body. There is a balance between pro-inflammatory M1 and anti-inflammatory M2 macrophages in tissues, and macrophage phenotype is controlled by the level of cytokine expression in the local microenvironment. Our goal is to induce cytokine expression in the placenta using mRNA to modulate the local immune activity as a potential treatment for preeclampsia, the most common disorder of pregnancy that has no therapeutic options. In preeclampsia, M1 macrophages predominate in the decidua contributing to the poor spiral artery invasion and remodeling that leads to the hallmark symptom of hypertension. We have developed ionizable lipid nanoparticles (LNPs) to deliver IL4 and IL13 mRNA to trophoblasts, the main cell type in the placenta. We hypothesize that LNPs will be taken up by trophoblasts to induce them to secrete IL4 and IL13 to polarize tissue-resident macrophages to an M2 phenotype. LNPs encapsulating IL4 mRNA (IL4-LNPs) or IL13 mRNA (IL13-LNPs) were delivered to two trophoblast cell lines, BeWo cells (a 3rd trimester choriocarcinoma line) and HTR8/SVneo cells (a 1st trimester line). Following LNP treatment, both cell lines secreted high levels of cytokine as evaluated by enzyme-linked immunosorbent assays. Macrophages collected from human donors were cultured in the conditioned media from LNP-treated trophoblasts and assessed for polarization by flow cytometry. Treatment with the IL4/IL13 conditioned media resulted in 97.5% M2 expression. By comparison, macrophages cultured on conditioned media from PBS-treated trophoblasts yielded 77.16% M2 expression. Further, our preliminary studies revealed that LNPs accumulate in the placenta following IV administration. This demonstrates that LNP delivery to trophoblasts in the placenta have the potential to locally modulate immune activity. More broadly, our work demonstrates that LNPs can induce local immune modulation important for treating many diseases. |
| George Fotakos |
Millennium Brooklyn High School |
The COVID-19 disease pandemic brought unprecedented challenges for healthcare, as hospitals struggled to balance inpatient and outpatient procedures with public health measures to limit the spread of infectious disease. Healthcare facilities suffered from limitations on personnel, supplies, resources, and capacity. Moreover, many elective surgeries were postponed, and hospital stays were minimized. Arthroscopy is a minimally invasive procedure used for the diagnosis and treatment of diseases of the joints, particularly autoimmune disorders and traumatic injuries. This research sought to determine possible impacts of the COVID-19 pandemic on arthroscopic procedures, and specifically examined trends in adverse events attributed to arthroscopes before and after the pandemic. The Food and Drug Administration (FDA) Manufacturer and User Facility Device Experience (MAUDE) database contains all adverse event reports for medical devices in the United States, and is a valuable resource for post-market surveillance of devices. The MAUDE database was searched for all reported adverse events for arthroscopes from 2018 to present. The results reveal a statistically significant rise in both malfunctions and injuries attributed to arthroscopes during the pandemic. Monthly reported malfunctions for arthroscopes rose by 51%, from 108.5 monthly malfunctions in June 2019-May 2020, to 163.75 monthly malfunctions in June 2020-May 2021 (p <0.0006). Monthly reported injuries for arthroscopes rose by 50%, from 9.5 monthly injuries in June 2019-May 2020, to 14.25 monthly injuries in June 2020-May 2021 (p <0.02). These results suggest that changes to arthroscopy practices during the pandemic, as well as changes in underlying health status of patients, may have contributed to increases in complications from arthroscopic surgery. Future research should examine the impacts of the pandemic on other surgical procedures, along with longer-term trends in reported adverse events for arthroscopy and other surgeries. The long-term impacts on patients undergoing arthroscopy should also be carefully followed. |
| Erin O'Brien |
Drexel University |
Macrophages play an active role in multiple phases of wound healing, transitioning from a predominantly pro-inflammatory (M1) population to less inflammatory, pro-regenerative phenotypes (M2). As high-level regulators of healing, macrophages pose a strong influence on other cell types through the secretion of cytokines and the expression of immunomodulatory ligands. Therefore, macrophages are an attractive target for cell therapies aiming to remedy dysfunctional wound healing characterized by chronic inflammation, such as that seen in aging patients. Previous work has shown that M2 macrophages that have previously been M1-activated, compared to those that have not, are phenotypically unique and exhibit enhanced M2 function. These “M1M2” macrophages are characterized by expression of the M2 marker CCL17, a chemokine known to attract skin-homing T cells, particularly IL-4-secreting Th2 cells and anti-inflammatory regulatory T cells (Tregs). This finding, along with the upregulation of immunosuppressive ligands PD-L1 and PD-L2 on M1 and M1M2 macrophages, respectively, begs the question of how macrophages may influence T cell differentiation (and vice versa) in the context of wound healing. In this study, we cultured human primary CD4+ T cells directly with autologous M0 or M1 macrophages, or with conditioned media generated by those phenotypes. After three days of co-culture, both macrophages and T cells were characterized via flow cytometry. Compared to T cells cultured alone, T cells co-cultured with macrophages exhibited increased differentiation into the Th2 and Treg phenotypes, and decreased differentiation into the inflammatory Th1 phenotype. These findings suggest that macrophages may direct the resolution of inflammation via crosstalk with T cells. Interestingly, similar results were seen in T cells cultured in macrophage-conditioned media, suggesting this crosstalk is not dependent on cell-cell interactions. Finally, macrophages co-cultured with T cells upregulated the expression of PD-L1, indicating that T cells may also promote immunosuppressive activity in macrophages. |
| Erica Jiang |
Horace Mann School |
Addiction involves functional changes to brain circuits that are involved with reward, stress, and self-control. Studies estimate that 20-60% of athletes suffer from stress due to excessive exercise and inadequate recovery. This research addresses certain neuroadaptations in the striatum of the brain that renders elite athletes more vulnerable to addictive behavior, particularly as a result of an increased tolerance to dopamine. Dopamine receptors in the striatum are crucial to the addiction process. Dopamine receptor type 2 (D2) receptors have a 10-to-100-fold greater affinity for dopamine (DA) than dopamine receptor type 1 (D1) receptors. The intense exercise that elite athletes must complete on a day-to-day basis can increase D2 receptor expression and binding in the striatum, until tolerance to the elevated levels of dopamine is eventually developed. In animal models, studies show that consistent, moderate aerobic exercise amplifies the availability and processing of dopamine neurotransmitters in the striatal region of the brain. In MPTP-induced mice models of Parkinson’s disease, intense daily exercise for 28 days (four weeks) resulted in higher levels of dopamine neurotransmission compared to non-exercise mice, through an increase in D2 receptor expression and binding in the dorsolateral striatum. Using a fast-scan cyclic voltammetry technique on mice, studies show that treadmill exercise increased stimulus-evoked DA release in MPTP-induced PD mice while also decreasing the decay of DA in the dorsal striatum. Additionally, positron emission tomography (PET) validated that treadmill exercise upregulates striatal D2 receptors. These increases in dopaminergic levels due to exercise can consequently increase motivation for rewards. Taken together, evidence supports the hypothesis that long-term, intense exercise modulates dopamine receptor expression. These changes may render elite athletes more vulnerable to addictive behavior, mainly due to an increased tolerance to dopamine. Tolerance leads to increased risk-taking behavior to compensate for the decrease in D2 receptor expression. |
| Jianwen Li |
University of Georgia |
Therapeutics that can be activated by radiation in situ to enhance the efficacy of radiotherapy are highly desirable. Herein, 7-Dehydrocholesterol (7-DHC), a biosynthetic precursor of cholesterol, as a radiosensitizer, exploiting its ability to propagate the free radical chain reaction is explored. The studies show that 7-DHC can react with radiation-induced reactive oxygen species and in turn promote lipid peroxidation, double-strand breaks, and mitochondrial damage in cancer cells. For efficient delivery, 7-DHC is encapsulated into nanoparticles, and been tested in tumor bearing mice, results showed a significantly enhanced the efficacy of radiotherapy, causing complete tumor eradication in 30% of the treated animals. After treatment, 7-DHC is converted to cholesterol, causing no detectable side effects or hypercalcemia. This new method represents a radiation-responsive sensitizer with great potential in clinical translation. |
| Magdalena Samojlik |
University of Florida |
Islet-Derived Chemokine Gradients Alter T cell Migration Characteristics Magdalena M. Samojlik a, Smit N. Patel a, Ying Li a,b, Aidan T. Fernandez a, Todd M. Brusko b, Clayton E. Mathews b, Edward A. Phelps a, and Cherie L. Stabler a a J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, USA b Department of Pathology, Immunology, and Laboratory Medicine, University of Florida Gainesville, FL, USA Robust in vitro models that provide a relevant 3-D microenvironment amenable to soluble gradient creation are needed to improve our understanding of the role of inflammation in the precipitation and progression of Type 1 Diabetes (T1D), as current animal models or 2-D cell culture methods cannot adequately fill this knowledge gap. Herein, we describe a decellularized extracellular matrix (ECM) 3-D hydrogel platform that allows for the study of the role of inflammation in the purposeful migration of immune cells to their target pancreatic islet cells. ECM hydrogel, procured using established methods, was adapted for the co-culture of human CD8+ T cells and pancreatic islets. To investigate inflammatory factors associated with T cell homing in T1D, donor human islets were pre-treated with pro-inflammatory cytokines (IFN, TNF, and IL-1), and the induced production of chemokine CXCL10 was quantified. Migration of T cells co-cultured with pro-inflammatory cytokine-treated or untreated islets within the hydrogel was tracked through real-time high-resolution, time-lapse confocal imaging and analyzed with Fiji TrackMate. The pre-treatment of human islets with the cocktail of pro-inflammatory cytokines resulted in production of the chemoattractant CXCL10 that significantly decreased the total distance traveled by the T cells while increasing their mean speed, compared to untreated islets. Extensive image analysis further provided key metrics regarding global T cell migratory behavior. Collectively, these results validate that this 3-D hydrogel platform provides a unique opportunity to co-culture human islets and immune cells, track the purposeful migration of these cells, and investigate factors involved in the chemotaxis of T cells in human T1D. |
| Sarah Y Neshat |
Johns Hopkins University |
T cell immunotherapies have demonstrated robust clinical success in treating some cancers but are not without their challenges. Engineering T cells, such as chimeric antigen receptor (CAR) strategies, has been shown to be a powerful approach to direct the adaptive immune response in cancer. However, these approaches are expensive, time consuming, and inefficient given the need for T cell extraction from the patient, ex vivo engineering, then reinfusion. Genetically modifying T cells in situ would significantly simplify the process and reduce costs. Non-viral polymeric nanoparticles offer a potential vehicle for in situ gene engineering for their well-established efficacy and safety in multiple cell types. Here, we developed a modular, targeted polymeric/lipid NP that can deliver mRNA cargo and effectively stimulate T cells in situ. Using synthetic poly(beta amino ester) (PBAE), mRNA nanoparticles were synthesized by self-assembly in buffer. Particles were subsequently conjugated with either anti-CD3 or both anti-CD3 and anti-CD28, a costimulatory signal, to transfect primary murine T cells isolated from C57BL/6. Our results suggest that both in vitro and in vivo, mRNA-NPs with anti-CD3 conjugated to the surface significantly outperformed unconjugated NPs. In vitro, transfection efficacy of NPs + anti-CD3 reached ~17%, a 3-fold increase over the unconjugated particles. Likewise, particles conjugated with both anti-CD3 and anti-CD28 were able to induce ~5-fold T cell proliferation with minimal toxicity. For in vivo studies, transgenic Ai9 mice were injected systemically with mRNA-NPs encoding a Cre mRNA molecule for tdTomato expression analysis. NPs + anti-CD3 achieved significantly higher transfection in the spleen and lymph nodes than unconjugated particles, and preferentially transfected CD4+ T cells. Unconjugated particles had no significant preference for transfection in CD8+ or CD4+ T cells. Studies are currently underway to further investigate antibody conjugation and apply this T lymphocyte targeted gene delivery platform in situ for immunoengineering applications. |
| Wei Yang |
University of Georgia |
Photodynamic therapy (PDT) is an emerging cancer treatment modality. PDT produces ROS under photo-irradiation that damage tumor cells. Moreover, it may also induce immunogenic cell death, eliciting an antitumor immunity. However, immunosuppressive tumor microenvironment may thwart the immune response. In particular, indoleamine 2,3-dioxygenase (IDO), an immune suppressor enzyme that metabolizes tryptophan (Trp) to kynurenine (Kyn), is frequently upregulated in tumors and induced after PDT. IDO overexpression leads to the depletion of Trp, inhibiting the activation of effector CTLs. Meanwhile, the production of Kyn promotes differentiation and activation of Tregs, as well as facilitates tumor-infiltrating myeloid-derived suppressor cells (MDSCs). It is envisioned that combining IDO inhibition with PDT may augment PDT-induced immune response, thereby enhancing tumor suppression. Herein, we report a composite, core/satellite nanoparticle, which allows for combination PDT and checkpoint blockage therapy. Specifically, ZnF16Pc, a photosensitizer, is loaded into ferritin protein cage, whereas NLG919, an IDO inhibitor, is encapsulated into PEG-PLGA nanoparticles. These two compartments are then covalently conjugated to form composite nanoparticles, referred to as PPF NPs. It is hypothesized that under photo-irradiation, ZnF16Pc produces ROS to kill cancer cells, while NLG919 is controlled released to inhibit IDO thus boosting the antitumor immunity post-PDT. We test the efficacy of this approach in B16F10-tumor bearing C57/BL6 mice. Our studies demonstrate that the treatment with PPF NPs plus photo-irradiation promotes infiltration of CD8+ T cells while reducing MDSCs and Tregs in tumors. Compared to PDT monotherapy which causes modest tumor suppression, PPF NPs plus photo-irradiation significantly inhibits tumor growth, with 30% of the animals remaining tumor-free after 6 months. Moreover, these surviving animals successfully reject a second tumor inoculation, indicating that a strong immunity is developed through the treatment. Overall, our composite nanoparticles simultaneously deliver photosensitizer and IDO inhibitor to tumors, allowing combination PDT and immunotherapy for sustained tumor control. |
| Nora Jean-Jacques |
Miss Porter's School |
Shifts in care delivery during the COVID-19 pandemic, particularly the shift from surgical aortic valve replacement to percutaneous aortic valve replacement, may have had adverse outcomes for patients. We analyzed data from the Food and Drug Administration (FDA) Manufacturer and User Facility Device Experience (MAUDE) database, to determine trends in reported malfunctions, injuries, and deaths for transcatheter aortic valve prostheses during the decade 2012 to 2021. We find that reported deaths attributed to transcatheter aortic valve prostheses more than doubled between the pre-pandemic year of 2019 to the pandemic year of 2020. Monthly reported deaths attributed to transcatheter aortic valve prostheses increased by 124%, from 25.0 monthly deaths in 2019, to 55.9 monthly deaths in 2020 (p<0.003). The increased level of deaths persisted in 2021, with 58.3 monthly reported deaths in 2021; the overall increase in deaths between 2019 and 2021 was 133%. We find that reported injuries attributed to transcatheter aortic valve prostheses increased by 25%, from 297.8 monthly injuries in 2019, to 371.4 monthly injuries in 2020 (p<0.03). Finally, we find that reported malfunctions attributed to transcatheter aortic valve prostheses increased by 39%, from 40.1 monthly malfunctions in 2019, to 55.7 monthly malfunctions in 2020 (p<0.007). These results suggest an overall increase in adverse events following transcatheter aortic valve prostheses during the pandemic, and indicate a pressing need for further research into short-term and long-term patient outcomes following transcatheter aortic valve prostheses. |
| Jascha Brettschneider |
1Center for Clinical and Translational Medicine, Institute for Molecular Medicine and Infectious Disease and Department of Microbiology and Immunology, College of Medicine, Drexel University |
Investigating non-thermal plasma induced calcium ion flux in an in vitro human keratinocyte wound closure model for the development of a closed loop control system to regulate plasma delivery Jascha Brettschneider1, Sara Mamchur2, John DiStefano1, Julia Sutter1, Francois Berthiaume3, Katharina Stapelmann4, Fred Krebs1, and Vandana Miller1 1Center for Clinical and Translational Medicine, Institute for Molecular Medicine and Infectious Disease and Department of Microbiology and Immunology, College of Medicine, Drexel University, Philadelphia, PA, USA 2Department of Biology, College of Arts and Sciences, Drexel University, Philadelphia, PA, USA 3Department of Biomedical Engineering, School of Engineering, Rutgers University, Piscataway, NJ, USA 4Department of Nuclear Engineering, College of Engineering, North Carolina State University, Raleigh, NC, USA Non-thermal plasma (NTP) generated through dielectric barrier discharge is an innovative treatment for wounds. An amalgam of short- and long-lived reactive oxygen and nitrogen species generated by NTP alter cellular redox processes that enhance wound closure. However, the establishment and application of appropriate NTP dosage for clinical use is challenging due to variation in devices used and biological responses. One such response is the flux of cytoplasmic calcium released from intracellular stores within minutes of treatment. As calcium acts as a second messenger during wound healing, it is expected that an NTP-induced calcium release will influence wound closure. Therefore, we anticipate that detection of calcium in real-time will provide feedback on cellular responses that enhance wound closure. Our research utilizes an in vitro wound healing model that incorporates the HaCaT human keratinocyte cell line. A linear gap (~0.75 mm wide) is created in a confluent cell layer using the Aglient BioTek Autoscratcher. Acute changes in calcium flux and subsequent wound closure are evaluated using the Aglient BioTek Cytation-5 cell imaging and multimode reader, a high content automated microscope that images cells (in visible and fluorescent wavelengths). Calcium flux is measured in conjunction with Fluo-4 NW, a calcium sensitive fluorescent dye, through imaging and fluorescence intensity. Wound closure is measured through kinetic imaging and automatic detection of the leading edge over a 24-hour period using the Aglient BioTek Gen5 software. This enables us to quantify changes in area and closure of the generated gap in real time. With our collaborators at Rutgers University and North Carolina State University, we will optimize this methodology in a wound healing Balb/c mouse model to develop a sensor-based, closed loop control system capable of regulating NTP dosage using real-time feedback from biological signals such as calcium flux. These studies are supported by NIH grant 1R01EB029705-01A1. |
| Theinmozhi Arulraj |
Johns Hopkins University |
Transcriptomics-aided in silico biomarker predictions for anti-PD-1 immunotherapy in metastatic triple negative breast cancer using a quantitative pharmacology model Theinmozhi Arulraj1, Hanwen Wang1, Cesar A. Santa-Maria2 and Aleksander S. Popel1,2 1 Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA 2 Department of Oncology, and the Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA Immune checkpoint inhibitors have demonstrated promising outcomes in the treatment of various cancer types but offer durable responses only in a limited number of patients. Thus, there is an unmet need in identifying predictive biomarkers for selecting patients most likely to derive clinical benefit. Triple negative breast cancer (TNBC), a subtype of breast cancer characterized by a high propensity for metastatic spread is considered an ideal target of immunotherapy due to the high immunogenicity compared to other breast cancer subtypes. However, treatment of metastatic TNBC is remarkably challenging due to the heterogeneity between metastatic tumors in different organs. We present a quantitative systems pharmacology (QSP) model, specifically developed to investigate tumor responses in the metastatic setting, by incorporating multiple metastatic tumors. The QSP model was calibrated using immune cell abundances estimated from transcriptomic and clinical data from KEYNOTE-119 trial, to investigate (anti-PD-1) pembrolizumab monotherapy as a second/third line treatment for metastatic TNBC. We performed an extensive search of cellular and molecular components of the QSP model to identify and rank potential biomarkers. We predict that the density of antigen presenting cells in lymph nodes, richness of cancer clones, fraction of cytotoxic T cells in lymph nodes or in the blood, could serve individually as potential biomarkers but have a higher predictive power as combinations of two biomarkers. We find that differences in immunogenicity is sufficient to confer higher resistance of metastatic tumors to pembrolizumab compared to primary TNBC tumors. Collectively, these findings suggest potential biomarkers that would enable a prospective identification of responders to pembrolizumab monotherapy and highlight the critical role of tumor immunogenicity in conferring resistance against immunotherapy. We expect that the developed QSP platform will be useful in the optimization of different treatment strategies in the metastatic setting of various cancer types. |
| Julia Sutter |
Drexel University College of Medicine |
Herpes simplex virus type 1 is a human pathogen known to cause cold sores around the mouth, eyes, and genitalia as clinical manifestations of acute infection. Infection with HSV-1 is lifelong, primarily due to its ability to travel into the nervous system and establish latency, which provides a reservoir for recurrent reactivation of acute infection in epithelial cells. While current antiviral therapies can alleviate the symptoms of acute infection, they are ineffective in preventing the establishment of latent infection and eliminating the virus. We investigated the use of nonthermal plasma (NTP), a partially ionized gas with demonstrated antiviral activity against many viruses, as a potential therapeutic against HSV-1 infection. Exposure of HSV-1 infected cells or cell-free virus to NTP resulted in reductions in infection (as measured 24 hours after infection). Additionally, an antiviral effect was observed in experiments in which uninfected cells exposed to NTP were less susceptible to HSV-1 infection. The antiviral activity of NTP is likely attributable to its ability to generate reactive oxygen and nitrogen species (RONS) that are known to react with macromolecular structures in viruses and are associated with immune responses in NTP-exposed cells. To establish a relationship between NTP-generated RONS and the antiviral effect of NTP, we measured markers of oxidative stress and immunogenicity in cells exposed to NTP. Overall, these initial studies are critical in developing NTP as an alternative therapy for HSV-1 infection that can target latent infection through the reduction of reactivation of virus replication in the periphery, minimizing the amount of virus that reaches the ganglia during primary infection. Over time, this control of HSV-1 infection can further be mediated by the anti-HSV-1 immune responses stimulated by NTP exposure. |
| Lars Howell |
University of Massachusetts Amherst |
Salmonella-mediated tumor immunotherapy by localized delivery of LPS. Immunotherapies are promising treatments for cancer; however, many tumors are non-responsive due to immunosuppression. Advancing immunotherapies requires treatments that convert the tumor microenvironment from cold to hot. Lipopolysaccharides (LPS) are strong adjuvants which increase cytokine production and innate immune cell activation; however, systemic delivery of LPS can induce sepsis, limiting their use. Salmonella typhimurium preferentially grow in the tumor microenvironment and can produce therapeutic compounds after delivery, making them an ideal vehicle for localized therapies. We engineered a therapeutic strain of Salmonella, which does not constitutively produce LPS, to produce LPS upon induction of msbB (Sal-msbB). We hypothesize delivery of Sal-msbB to tumors will decrease tumor growth by initiating a pro-inflammatory immune response and activating innate immune cells. We tested this hypothesis by culturing immune cells with Sal-msbB in vitro and delivering Sal-msbB to tumors in vivo. In vitro, induced Sal-msbB increased dendritic cell activation and doubled uptake of soluble antigen. Co-culture of induced Sal-msbB with immature monocytes produced 3.75 times as much pro-inflammatory TNFα as uninduced Sal-msbB, which remained consistent with the non-LPS producing control. In vivo, delivery of Sal-msbB decreased tumor growth. Sal-msbB treated tumors grew 0.67 times the rate of tumors treated with control bacteria and 0.35 times the rate of untreated tumors. Delivery of Sal-msbB increased overall immune infiltration to tumors and increased systemic dendritic cell activation compared to untreated mice. Finally, activation of Sal-msbB demonstrated low toxicity with all mice experiencing either no adverse effects or minor stress which resolved four hours after injection. Together, this data shows that delivery of immunostimulatory LPS by therapeutic Salmonella induces anti-tumor efficacy. This provides a promising new avenue for overcoming tumor immunosuppression, as therapeutic Salmonella localize to tumors prior to producing and delivering immunostimulatory molecules overcoming limitations of systemic treatments. |
| Anush Raghav Polamraju |
Rutgers Prep |
Brain aneurysms result from focal weakening of blood vessel walls within the cerebral vasculature; risk factors include age, sex, and hypertension. Neurovascular embolization coils are commonly used to halt growth and prevent rupture of brain aneurysms. These metallic coils are inserted via catheter into the center of the aneurysm, and function by inducing a clotting response, so that blood can no longer enter the aneurysm. While embolization coils enable minimally invasive treatment of brain aneurysms, the devices carry significant risks, including mechanical failure and patient interaction problems. This study quantitatively characterizes adverse events attributed to neurovascular embolization coils. The Food and Drug Administration (FDA) Manufacturer and User Facility Device Experience (MAUDE) database was mined using a computer algorithm created using the Pandas Python module. A total of 17,992 reports of deaths, injuries, and malfunctions were retrieved and analyzed using the algorithm. The results reveal that the most common device problems for embolization coils are unidentified problems, activation failures, separation/detachment, positioning failures, and advancing difficulties. The most frequent adverse patient outcomes were hemorrhage, stroke, rupture, thrombosis, and no known patient consequences. The delay between event date and report date was also characterized. Deaths exhibited an average reporting delay of 69.21 days, injuries exhibited an average reporting delay of 65.35 days, and malfunctions exhibited an average reporting delay of 35.52 days; all were above the FDA 30-day reporting delay mandate. Finally, injury and malfunction narratives were searched for terms indicative of patient deaths; this analysis revealed that 1.7% of injuries and 0.29% of malfunctions were misreported. These findings highlight the need for more rigorous monitoring of device reporting to ensure the safety of neurovascular embolization coils. |
| Katerina Morgaenko |
University of Virginia |
Lymph node metastasis impacts disease progression and contributes to mortality in breast cancer patients. However, the process of tumor invasion in tumor-draining lymph nodes (TDLN) is poorly understood. While it is established that tumor cell entry into TDLNs through the subcapsular sinuses is facilitated by CCL1 chemokine, information is lacking on the extent to which tumor cells invade the TDLN parenchyma and whether tumor cells are guided specifically to the cortex (B cell zones) or deep parenchyma (T cell zone) of the lymph node (LN) by hijacking chemokine gradients used for homing circulating lymphocytes. Elucidating the mechanisms underlying tumor cell traffic to TDLN and metastasis localization is important for identification of druggable targets and advancing therapy. This study presents the development of an ex-vivo model that replicates spread of tumor cells in the LN parenchyma and its use to identify regions of preferential tumor cell accumulation. Spread of syngeneic 4T1 and BRPKp110 breast cancer cell lines seeded onto live murine lymph tissue slices was quantified at multiple timepoints during ex vivo culture. We observed tumor cells infiltrating into the tissue, acquiring elongated morphology indicative of adhesion, and proliferating according to Ki-67 staining. In naïve slices, tumor cells invade into subcapsular sinus initially, reminiscent of in vivo behavior, and later distribute to deeper parenchyma, with high accumulation in B cell follicles. In ongoing work, we will i) quantify tumor cell distribution relative to chemokine gradients in live LN tissue slice (CCL1, CXCL13, CCL19, CCL21) and ii) test the impact of blocking chemokine function via neutralizing antibodies on tumor cell distribution in the LN. |
| Marco E Zamora |
Drexel University/University of Pennsylvania |
For the past 20 years, it has been assumed that vascular targeted antibody-mediated delivery to the lung directed delivery of nanocarriers exclusively to the pulmonary endothelium. To test this, we developed liposomal nanocarriers covalently conjugated with antibodies against intracellular cell adhesion molecule 1 (ICAM-1), which were then assessed by flow cytometry, biodistribution, and mass spectroscopy. Using flow cytometry techniques, we found not only that pulmonary endothelial cells, but also marginated neutrophils are important players in uptake of liposomes, especially in the context of acute lung injury (ALI). We were then interested in elucidating the kinetics of uptake and clearance for these liposomes and comparing them in healthy and ALI injured mouse models. For the ALI model, we performed all studies four hours after injury, modeling clinically relevant administration of liposomal drug carriers. In both healthy and nebulized mice, we found rapid uptake of liposomes as early as 10 minutes post injection by both neutrophils and endothelial cells reaching a peak uptake around 30 min post injection. After 1 hour of circulation, we found that in the nebulized model, a sharp decrease in uptake pointing to clearance of the liposomes, likely by marginated neutrophils now leaving the lung vasculature. Biodistribution studies over time further corroborate this finding showing reduced signal in the lung over time in the nebulized model and more prominent after 1 hour of circulation. Finally, mass spectroscopy tracking of drug liposomes showed similarly fast clearance of drugs away from the lung in vivo. In summary, these findings have major implications for these systems, as rapid loss of carrier signal implies that while therapies are arriving to the lung where they are needed, they are removed just as quickly, impairing therapeutic efficacy. However it also provides further ground for engineering solutions to reduce this rapid clearance. |
| Eno-Obong Essien |
University of Pennsylvania |
Title: Pulmonary Marginated Neutrophils: A Novel Target for Vascular Targeted Nanoparticles Authors: Eno-Obong Essien*1, Marco Zamora*1 Aditi Murthy1, Elizabeth Hood2, Laura Ferguson1, Oscar Marcos-Contreras2, Jacob S. Brenner1,2 Pulmonary disease is one of the leading causes of mortality in the world, and continues to remain in need of effective therapies. To address this need, nanomedicine is a promising strategy that achieves organ specific delivery of RNA and small molecular drugs. Antibody conjugated nanoparticles targeting vascular endothelial moieties such as ICAM, PLVAP and PECAM can concentrate drugs in the lungs up to 300 fold more than standard drug delivery. However, for decades, this organ specificity had been assumed to be solely due to endothelial cell specific uptake in the pulmonary vasculature. Thus, in our study, we sought to determine the cell type specificity of vascular targeted liposomes. We achieved this by conjugating antibodies targeting ICAM, PECAM and PLVAP, and delivered them intravenously in both healthy mice and mice with acute lung injury. Surprisingly, we found that in addition to endothelial cell uptake, there was avid uptake by marginated neutrophils in both healthy and diseased mice. Across the vascular targeting moieties, PLVAP had the most endothelial specific uptake though there was still some uptake by marginated neutrophils. To further understand the mechanisms of uptake, we showed that marginated neutrophil liposome uptake is not mediated by surface presentation of ICAM and PECAM as previously thought. Furthermore, we demonstrated that the mechanism of uptake is similar amongst other forms of vascular targeted conjugated nanoparticles. Finally, we showed that these mechanisms of liposomal uptake can be recapitulated in human lungs. In conclusion, from our data, cell specific targeting remains elusive and will require further engineering to improve specificity. However, neutrophil nanoparticle uptake does present a unique opportunity for innovative drug delivery strategies to ameliorate lung disease. |
| Michael Buckenmeyer |
National Cancer Institute |
Building a 3D Tumor Spheroid-induced Tumor-Associated Macrophage Model for Cancer Immunotherapy Testing-in-a-Dish Tumor associated macrophages (TAMs) play an integral role in tumor progression by mediating the transition from clustered cancer cells to a complex 3D tumor microenvironment. Macrophages are phenotypically plastic and are reprogrammed in cancer to support tumor growth. However, testing TAM responses to immunotherapy is complicated by the difficulty of culturing macrophages in high throughput 3D models. To overcome this challenge, we discovered that supplementing spheroid culture with decellularized intestine extracellular matrix (SI-ECM) enabled complex tumor spheroid formation with MC38 murine colorectal cancer cells, fibroblasts, and bone marrow macrophages. In the absence of ECM, macrophages remained excluded whereas with SI-ECM, macrophages were completely integrated within a single cohesive spheroid. Spheroid growth was monitored using a Celigo cytometer, reaching an average diameter of 698 +/- 15 um after 7 days. Picro-sirius red (PSR) histologic staining confirmed that ECM assembly and collagen distribution within the tumor spheroid mimicked colorectal tumor morphology. 3D tumor spheroids were dissociated to analyze spheroid-induced TAM (siTAM) phenotype by flow cytometry compared to 2D macrophages in M0 (unstimulated), M1 (LPS+IFNy), and M2 (IL4) conditions. siTAMs upregulated the immune checkpoint PD-L1 (3.4-fold) and co-stimulatory molecule CD86 (4.3-fold) compared to M0. M1 conditions enhanced major histocompatibility complex II (3.7-fold); however, siTAMs did not, suggesting defective antigen presentation. We added the immune adjuvant CDA (c-di-AMP, STING agonist) to 7-day spheroid cultures and analyzed the supernatant with cytokine arrays. siTAMs caused a 2-fold decrease in CCL5 secretion compared to macrophage-free spheroids. This inhibited secretory phenotype was reversed with CDA treatment and a 3.5-fold increase in CCL5 secretion. In conclusion, a decellularized ECM scaffold enables macrophage integration into 3D tumor spheroids and subsequent immunotherapy testing. This is the first study to demonstrate the integral role of ECM in tumor assembly in vitro creating a platform to study ECM interactions during TAM reprogramming. |
| Bumjun Kim |
Princeton University |
Current chimeric antigen receptor (CAR) T cell therapy employees the ex-vivo engineering of T cells, which require expensive and complicated CAR-T cell and viral vector manufacturing facilities. In-situ engineering of T cells via synthetic lipid nanoparticles (LNPs) may obviate the needs for the complex manufacturing processes and facilities. However, targeting non-hepatic cells and tissues remain the major challenge. Liver targeting of LNPs is a result of the shedding of poly(ethylene glycol) (PEG)-lipids and adsorption of apolipoprotein E (ApoE) on LNPs, leading to low-density lipoprotein receptor (LDLR)-mediated uptake by hepatocytes. Despite the recent promise of antibody (Abs)-directed targeting of LNPs toward T cells, anchoring the large Abs onto the LNP surface by the weak anchoring energy of lipid tails will result in partitioning the Ab-conjugate lipids off of the circulating LNPs even more rapidly than PEG-lipids. Here, we replaced lipid-PEG with poly(ε-caprolactone)-block-poly(ethylene glycol) (PLC-b-PEG) and optimized the formulation for T cell transfection. The larger PCL block copolymer will serve as a better Ab conjugation site due to stronger anchoring on the LNP surface. This polymer-lipid hybrid vesicles have a potential to unlock the full potential of targeted delivery of nucleic acids. |
| Victoria Nash |
Drexel University |
Immunomodulatory biomaterials aim to facilitate in situ tissue regeneration but leveraging biotin-avidin interactions as the drug delivery system to achieve this goal has not been thoroughly investigated. The goal of this study was to characterize how avidin variants and varying biotin densities on two different porous gelatin scaffolds can influence differentiation of primary human macrophages in vitro by delivering the immunomodulatory cytokine, interleukin 4 (IL4). Here, we show increasing the degree of scaffold biotinylation determined the amount of avidin variants, streptavidin and CaptAvidin, bound to the scaffolds. Interestingly, we found that protein release kinetics differed between the two materials, Gelfoam and Surgifoam, highlighting the influence of material choice on this binding system. Finally, we found that increasing scaffold biotinylation led to increased M2-like polarization of primary human macrophages in vitro over time, showcasing how a biotin-avidin delivery system can modulate macrophage phenotype. These results exhibit a robust and modular drug delivery system to impart immunomodulatory activity to biomaterials. |
| Sanjay Pal |
National Cancer Institute, NIH |
Inducing Antigen Specific Anti-Tumor Immunity in a Type 2 Biologic Scaffold Immune Environment Using a STING Agonist Sanjay Pal, Brenna J. Hill, Rohan B. Chaudhari, Matthew T. Wolf Cancer Innovation Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD-21702 Tissue engineering is intertwined with surgical oncology, where biomaterials are implanted to repair tissues post tumor resection. Every implanted biomaterial initiates a host immune response, which exist on a spectrum; Type 1 immunity is associated with cytotoxic anti-tumor responses and Type 2 with wound healing. Biologic scaffolds prepared by tissue decellularization initiate a Type 2-like response, but it is unknown if this host response is compatible with cancer immunotherapy. To test this question, we compared immune stimulating adjuvants delivered with a biologic scaffold (decellularized small intestine, SIS) in C57Bl/6 mice. SIS particulate was injected subcutaneously with the adjuvant MPLA (monophosphoryl lipid A), CDA (cyclic diAMP, STING agonist), GM-CSF (granulocyte-macrophage colony stimulating factor), or vehicle. Quantitative histology showed that MPLA induced the greatest cellularity (4906 cells/µm2) after 7-14 days, qualitatively mimicking infection. However, SIS+MPLA only achieved an antigen specific cell killing index of 60% using the ovalbumin (OVA) model antigen in a cytotoxic T cell assay. SIS+CDA led to greater than 95% specific killing, indicating that CDA can be delivered locally with SIS to induce antigen specific immunity. SIS+CDA was further evaluated via flow cytometry. CDA did not reverse key Type 2 features associated with SIS such as CD206+ macrophages or eosinophil recruitment. However, CDA increased the proportion of dendritic cells and CD86 expression in SIS scaffolds. Finally, SIS+CDA was delivered with OVA as a therapeutic vaccine to treat established (75mm3) OVA antigen expressing Eg.7 lymphoma tumors. SIS+CDA led to complete responses and protection on tumor rechallenge in 4/7 animals. No tumor regression was observed in control SIS or CDA groups. In conclusion, the STING agonist CDA can drive antigen specific tumor immunity while not compromising a SIS scaffold associated Type 2 immune response needed for wound repair. Likewise, a pro-healing immune environment does not impair cell mediated cytotoxicity. |
| Theresa Raimondo |
Massachusetts Institute of Technology |
Inhibiting adaptive immune checkpoints has proven therapeutic efficacy, however only a minority of patients respond. Myeloid cells, especially monocytes and macrophages, are the predominant immune cell in many solid tumors and are potent immunosuppressors. Here, we hypothesized that silencing a macrophage-checkpoint would promote their activation, enhance cancer phagocytosis and antigen presentation, to ultimately promote a therapeutic antitumor T cell response. Optimized short interfering RNA (siRNA) was encapsulated in lipid nanoparticles (LNPs) formulated with a novel, biodegradable, ionizable lipid which allowed high levels of silencing (~90%) in peritoneal macrophages following low-dose (0.01 mg/kg), intraperitoneal, administration. The novel siRNA-LNPs demonstrated macrophage-tropism in vivo, with uptake mediated by clathrin-mediated endocytosis and macropinocytosis, independent of ApoE and LDLR. siRNA-LNP driven silencing shifted murine and primary human macrophages towards an immunostimulatory (M1) phenotype, in vivo and ex vivo, respectively; and, compared to antibody blocking, minimized counterproductive mutual phagocytosis (macrophage-macrophage attack often triggered by antibody-opsonization). siRNA-driven macrophage-silencing was synergistic with cancer-opsonizing antibody therapy, in vitro, the combination yielding ~70% cancer phagocytosis vs ≤10% phagocytosis by cells lacking either opsonizing-treatment or siRNA-silencing. Following macrophage coculture with ovalbumin-expressing melanoma, siRNA-LNP lead to a 2-fold increase in MHCI cross-presentation of the ovalbumin antigen. Ultimately, in a murine model of peritoneal metastasized ovarian cancer, siRNA-LNP + a cancer opsonizing-antibody significantly prolonged survival vs treatment with control siRNA-LNP + antibody. The combination increased both the recruitment, and tumor-specific activation of cytotoxic T lymphocytes. Administration of siRNA-LNP with standard chemotherapy (3-rounds), significantly slowed tumor growth following treatment cessation. The slowed growth was concomitant with increases in effector memory CD4 and CD8 T cells. These data demonstrate that siRNA-LNP elicits potent checkpoint silencing in myeloid cells capable of driving antitumor adaptive immunity, and represents a promising platform for innate immune checkpoint inhibitors |
| Alexis Brantly |
Drexel University College of Medicine |
HIV and HBV cause chronic viral infections and coinfected individuals have a 5-6 fold increased risk for developing cirrhosis and hepatocellular carcinoma (HCC) over mono-infection. The basis for this difference is not clear, but HIV-infected T-cells and liver macrophages likely contribute to an altered liver microenvironment, exacerbating disease. This effect may be mediated by the effect of viral proteins that are still produced because of the integration of HIV-1 proviral DNA from infected cells. The heterogenous liver macrophage population, including Kupffer cells and human monocyte derived macrophages (hMDMs), are likely reservoirs for HIV as viral RNA and DNA have been detected in patient-derived livers. This project examined hMDMs and liver macrophages isolated from peripheral blood resected liver tissue, respectively. These cells were infected with HIVADA, a R5 tropic virus. The number of p24 containing cells and formation of multi-nucleated giant cells, a hallmark of HIV infection in tissues, particularly the brain, was evaluated using high content imaging on the CX7 High Content scanner. Changes in virion production were evaluated by measuring p24 levels in supernatant using AlphaLISAs. Our results show that an increasing number of infected cells correlates with increased viral production over time and production of multi-nucleated giant cells. We have also showed that liver macrophages derived from patient samples are susceptible to HIV-1ADA infection, and that treatment with anti-retroviral Biktarvy results in reduced p24 levels. Furthermore, treatment of hMDMs with HBV proteins known to be chronically present in infected individuals resulted in altered p24 production and percentage of p24 positive cells, which may occur in a serotype specific manner. This data is a critical step in establishing a baseline model of HIV infection dynamics to further evaluating changes in HIV infection in cells of a myeloid origin. |