Delivery Of Cytokines Research Articles

Tertiary Lymphoid Structures and Immunotherapy: Challenges and Opportunities.

Tertiary lymphoid structures (TLS)are accumulations of lymphoid cells that arise in ectopic sites through the process of lymphoid neogenesis in chronic inflammation in autoimmunity, microbial infections, organ rejection, aging, and cancer. Their cellular composition and function and regulation via members of the lymphotoxin (LT)/tumor necrosis factor (TNF) family resemble that of secondary lymphoid organs (SLOs). Tumor-associated (TA)-TLS can be associated with favorable clinical outcomes. Immunotherapy in the form of immune checkpoint inhibitors (ICI) has contributed to tremendous advances in cancer therapy. However, ICI are effective in only some tumors, can give rise to resistance, and can precipitate immune-related adverse events (irAEs), many of which appear to have hallmarks of autoimmunity and can resemble TLS. TA-TLS correlate with a positive response to immunotherapy, but they can also be associated with susceptibility to irAEs, suggesting that TA-TLS in combination with ICI could lead to uncontrolled autoimmunity. The tumor environment can be manipulated to ensure that, not only the number of TLS, but also their cellular composition and appropriate function allow for judicious combinations of TLS and immunotherapy that can synergize and contribute to better outcomes with a minimum of destructive irAEs. Strategies include directed delivery of lymphoneogenic cytokines and chemokines or vascular growth factors directly, via transgenes or via adenovirus vectors.

CAR T-cells for pediatric solid tumors: where to go from here?

Despite the great success that chimeric antigen receptor (CAR) T-cells have had in patients with B-cell malignancies and multiple myeloma, they continue to have limited efficacy against most solid tumors. Especially in the pediatric population, pre- and post-treatment biopsies are rarely performed due to ethical reasons, and thus, our understanding is still very limited regarding the mechanisms in the tumor microenvironment by which tumor cells exclude effectors and attract immune-suppressive cells. Nevertheless, based on the principles that are known, current T-cell engineering has leveraged some of these processes and created more potent CAR T-cells. The recent discovery of new oncofetal antigens and progress made in CAR design have expanded the potential pool of candidate antigens for therapeutic development. The most promising approaches to enhance CAR T-cells are novel CAR gating strategies, creative ways of cytokine delivery to the TME without enhancing systemic toxicity, and hijacking the chemokine axis of tumors for migratory purposes. With these new modifications, the next step in the era of CAR T-cell development will be the clinical validation of these promising preclinical findings.

Open-Source Throttling of CD8+ T Cells in Brain with Low-Intensity Focused Ultrasound-Guided Sequential Delivery of CXCL10, IL-2, and aPD-L1 for Glioblastoma Immunotherapy.

Improving clinical immunotherapy for glioblastoma (GBM) relies on addressing the immunosuppressive tumor microenvironment (TME). Enhancing CD8+ T cell infiltration and preventing its exhaustion holds promise for effective GBM immunotherapy. Here, a low-intensity focused ultrasound (LIFU)-guided sequential delivery strategy is developed to enhance CD8+ T cells infiltration and activity in the GBM region. The sequential delivery of CXC chemokine ligand 10 (CXCL10) to recruit CD8+ T cells and interleukin-2 (IL-2) to reduce their exhaustion is termed an "open-source throttling" strategy. Consequently, up to 3.39-fold of CD8+ T cells are observed with LIFU-guided sequential delivery of CXCL10, IL-2, and anti-programmed cell death 1 ligand 1 (aPD-L1), compared to the free aPD-L1 group. The immune checkpoint inhibitors (ICIs) therapeutic efficacy is substantially enhanced by the reversed immunosuppressive TME due to the expansion of CD8+ T cells, resulting in the elimination of tumor, prolonged survival time, and long-term immune memory establishment in orthotopic GBM mice. Overall, LIFU-guided sequential cytokine and ICIs delivery offers an "open-source throttling" strategy of CD8+ T cells, which may present a promising strategy for brain-tumor immunotherapy.

An IL-7 fusion protein targeting EDA fibronectin upregulates TCF1 on CD8+ T-cells, preferentially accumulates to neoplastic lesions, and boosts PD-1 blockade

BackgroundAnti-PD-1 antibodies have revolutionized cancer immunotherapy due to their ability to induce long-lasting complete remissions in a proportion of patients. Current research efforts are attempting to identify biomarkers and suitable...

Delayed administration of interleukin-4 coacervate alleviates the neurotoxic phenotype of astrocytes and promotes functional recovery after a contusion spinal cord injury

Objective. Macrophages and astrocytes play a crucial role in the aftermath of a traumatic spinal cord injury (SCI). Infiltrating macrophages adopt a pro-inflammatory phenotype while resident astrocytes adopt a neurotoxic phenotype at the injury site, both of which contribute to neuronal death and inhibit axonal regeneration. The cytokine interleukin-4 (IL-4) has shown significant promise in preclinical models of SCI by alleviating the macrophage-mediated inflammation and promoting functional recovery. However, its effect on neurotoxic reactive astrocytes remains to be elucidated, which we explored in this study. We also studied the beneficial effects of a sustained release of IL-4 from an injectable biomaterial compared to bolus administration of IL-4.Approach. We fabricated a heparin-based coacervate capable of anchoring and releasing bioactive IL-4 and tested its efficacyin vitroandin vivo. Main results. We show that IL-4 coacervate is biocompatible and drives a robust anti-inflammatory macrophage phenotype in culture. We also show that IL-4 and IL-4 coacervate can alleviate the reactive neurotoxic phenotype of astrocytes in culture. Finally, using a murine model of contusion SCI, we show that IL-4 and IL-4 coacervate, injected intraspinally 2 d post-injury, can reduce macrophage-mediated inflammation, and alleviate neurotoxic astrocyte phenotype, acutely and chronically, while also promoting neuroprotection with significant improvements in hindlimb locomotor recovery. We observed that IL-4 coacervate can promote a more robust regenerative macrophage phenotypein vitro, as well as match its efficacyin vivo,compared to bolus IL-4.Significance. Our work shows the promise of coacervate as a great choice for local and prolonged delivery of cytokines like IL-4. We support this by showing that the coacervate can release bioactive IL-4, which acts on macrophages and astrocytes to promote a pro-regenerative environment following a SCI leading to robust neuroprotective and functional outcomes.

Tumor micro-environment induced TRAIL secretion from engineered macrophages for anti-tumor therapy

The high plasticity and long-term persistency make macrophages excellent vehicles for delivering anti-tumor cytokines. Macrophage delivery of chemokines and cytokines shows potential in tumor therapy. TRAIL, a promising anti-tumor cytokine, induces apoptosis in tumor cells with low toxicity to normal cells. However, its off-target toxicity and limited stability have limited its clinical progress. Here, we engineered macrophages with Mono-TRAIL and Tri-TRAIL and found that Tri-TRAIL had higher cytotoxic activity against tumor cells than Mono-TRAIL in vitro. To target the tumor microenvironment (TME), we generated macrophages secreting trimeric TRAIL (Tri-TRAIL-iM) induced by the TME-specific promoter Arg1. The Tri-TRAIL-iM cells displayed high specific activatable activity in cell-based co-culture assay and tumor-baring mice models. In addition, we demonstrated that compared to macrophages over-expressing TRAIL under a non-inducible promoter, Tri-TRAIL-iM could more effectively induce apoptosis in cancer cells, inhibit tumor growth, and reduce systemic side effects. This strategy of inducing TRAIL delivery holds great potential for cancer therapy. It is promising to be combined with other engineering methods to maximize the therapeutic effects of solid tumors.

Agent-based model demonstrates the impact of nonlinear, complex interactions between cytokinces on muscle regeneration.

Muscle regeneration is a complex process due to dynamic and multiscale biochemical and cellular interactions, making it difficult to identify microenvironmental conditions that are beneficial to muscle recovery from injury using experimental approaches alone. To understand the degree to which individual cellular behaviors impact endogenous mechanisms of muscle recovery, we developed an agent-based model (ABM) using the Cellular-Potts framework to simulate the dynamic microenvironment of a cross-section of murine skeletal muscle tissue. We referenced more than 100 published studies to define over 100 parameters and rules that dictate the behavior of muscle fibers, satellite stem cells (SSCs), fibroblasts, neutrophils, macrophages, microvessels, and lymphatic vessels, as well as their interactions with each other and the microenvironment. We utilized parameter density estimation to calibrate the model to temporal biological datasets describing cross-sectional area (CSA) recovery, SSC, and fibroblast cell counts at multiple timepoints following injury. The calibrated model was validated by comparison of other model outputs (macrophage, neutrophil, and capillaries counts) to experimental observations. Predictions for eight model perturbations that varied cell or cytokine input conditions were compared to published experimental studies to validate model predictive capabilities. We used Latin hypercube sampling and partial rank correlation coefficient to identify in silico perturbations of cytokine diffusion coefficients and decay rates to enhance CSA recovery. This analysis suggests that combined alterations of specific cytokine decay and diffusion parameters result in greater fibroblast and SSC proliferation compared to individual perturbations with a 13% increase in CSA recovery compared to unaltered regeneration at 28 days. These results enable guided development of therapeutic strategies that similarly alter muscle physiology (i.e. converting extracellular matrix [ECM]-bound cytokines into freely diffusible forms as studied in cancer therapeutics or delivery of exogenous cytokines) during regeneration to enhance muscle recovery after injury.

Agent-based model demonstrates the impact of nonlinear, complex interactions between cytokines on muscle regeneration

Muscle regeneration is a complex process due to dynamic and multiscale biochemical and cellular interactions, making it difficult to identify microenvironmental conditions that are beneficial to muscle recovery from injury using experimental approaches alone. To understand the degree to which individual cellular behaviors impact endogenous mechanisms of muscle recovery, we developed an agent-based model (ABM) using the Cellular-Potts framework to simulate the dynamic microenvironment of a cross-section of murine skeletal muscle tissue. We referenced more than 100 published studies to define over 100 parameters and rules that dictate the behavior of muscle fibers, satellite stem cells (SSCs), fibroblasts, neutrophils, macrophages, microvessels, and lymphatic vessels, as well as their interactions with each other and the microenvironment. We utilized parameter density estimation to calibrate the model to temporal biological datasets describing cross-sectional area (CSA) recovery, SSC, and fibroblast cell counts at multiple timepoints following injury. The calibrated model was validated by comparison of other model outputs (macrophage, neutrophil, and capillaries counts) to experimental observations. Predictions for eight model perturbations that varied cell or cytokine input conditions were compared to published experimental studies to validate model predictive capabilities. We used Latin hypercube sampling and partial rank correlation coefficient to identify in silico perturbations of cytokine diffusion coefficients and decay rates to enhance CSA recovery. This analysis suggests that combined alterations of specific cytokine decay and diffusion parameters result in greater fibroblast and SSC proliferation compared to individual perturbations with a 13% increase in CSA recovery compared to unaltered regeneration at 28 days. These results enable guided development of therapeutic strategies that similarly alter muscle physiology (i.e. converting extracellular matrix [ECM]-bound cytokines into freely diffusible forms as studied in cancer therapeutics or delivery of exogenous cytokines) during regeneration to enhance muscle recovery after injury.

A phase 1 dose-escalation and expansion study of CUE-101, given as monotherapy and in combination with pembrolizumab, in patients with recurrent/metastatic HPV16+ head and neck squamous cell cancer (R/M HNSCC).

6004 Background: Immuno-STATs are modular T cell engagers engineered to selectively activate tumor-antigen specific CD8+ T cells via targeted delivery of cytokines. CUE-101, the first Immuno-STAT in clinical trials, is composed of a human leukocyte antigen (HLA) complex, HLA-A*0201, a peptide epitope derived from the HPV16 E7 protein and 4 molecules of attenuated human interleukin-2 (IL-2), to bind, expand, and activate HPV16-specific CD8+ T cells for the treatment of HPV16+ HNSCC. Methods: CUE-101-01 is an ongoing first-in-human study in HLA-A*0201 patients with HPV16+ R/M HNSCC. Escalating doses of CUE-101 monotherapy (0.06 mg/kg to 8 mg/kg) were evaluated in R/M HNSCC refractory to ≥ 1 platinum or checkpoint inhibitor (CPI) based therapy, alone or combined with pembrolizumab (1 mg/kg to 4 mg/kg + 200 mg pembrolizumab) in the first line treatment of PD-L1+ R/M HNSCCs. Enrollment at the recommended phase 2 dose (RP2D) was expanded. Therapy was administered every 3 weeks (Q3W) until disease progression or intolerable toxicity. Safety, PK/PD, and antitumor activity were assessed. Results: Enrollment in both monotherapy and combination cohorts is now completed (N=80 patients, 49 in monotherapy and 31 CUE-101 plus pembrolizumab). Following dose escalation, 4 mg/kg Q3W of CUE-101 was selected for RP2D for both monotherapy and pembrolizumab combination cohorts. At data cut-off, adverse events (AEs) have been manageable and 92% grade ≤2. The most frequent grade 3 AEs reported include lymphocyte count decreased (7.6%), anemia (6.3%), decreased appetite (5.1%) and infusion-related reactions (5.1%). In combination with pembrolizumab no unanticipated significant safety concerns have emerged. Exposure-dependent PD effects of CUE-101 are consistent with IL-2 pharmacology and indicate preferential expansion of CUE-101 on E7-specific T cells. Among the 19 evaluable patients treated with the RP2D of CUE-101 plus pembrolizumab, an ORR of 47% (1 CR, 8 PRs), a Disease Control Rate (ORR + durable SDs) of 74%, and mPFS of 5.8 months [95% CI 2.56; NA] were observed. A median OS has not been reached. Of the 9 patients with confirmed objective responses, all achieved >99% reduction in HPV16 cfDNA in plasma during their treatment course. Among the 19 evaluable monotherapy RP2D patients, 1 PR and 6 durable SD (SD ≥ 12 weeks) and mOS of 20.8 months [95% CI 11.0; NA] were observed. Conclusions: An ORR of 47% and a mPFS of 5.8 months were observed in R/M HNSCC patients treated with CUE-101 4 mg/kg + pembrolizumab as 1L therapy. A median OS of 20.8 was observed in patients treated with CUE-101 monotherapy as post-platinum/CPI therapy. CUE-101 continues to demonstrate safety, tolerability and meaningful clinical benefit in patients with HPV16+ R/M HNSCC. Clinical trial information: NCT03978689 .

Abstract 58: Immunocytokines with specificity for invariant region of CAR molecules enhance effector function of multiple CAR T cell therapies

Abstract The effector functions of CAR T cells can be enhanced through delivery of immunostimulatory cytokines, such as IL-12, IL-18, or IL-23. However, systemic delivery of recombinant cytokines or paracrine secretion by CAR T cells can elicit toxicities due to non-specific targeting of the cytokines. Here, we designed immunocytokines with antibodies specific for the invariant linker peptides that join variable domains in the CAR scFv and immunostimulatory cytokines. The immunocytokines were capable of targeting CAR molecules agnostic of target antigen, demonstrating broad applicability without the need for further genetic engineering of CAR therapies, such as co-expression of a surrogate molecule (tEGFR, etc.). The addition of immunocytokines enhanced CAR T cell cytolytic activity and secretion of type I cytokines, even at low concentrations of immunocytokine. In a stringent Nalm6 xenograft model, the addition of immunocytokines delivering IL-12 enhanced the anti-tumor activity of anti-CD19 CAR T cells and overall survival compared to controls of antibody alone or PBS. This technological development presents the opportunity to specifically enhance the activity of multiple CAR T cell therapies in the absence of non-specific toxicities. Citation Format: J T. Keane, Oula K. Dagher, Fang Liu, Avery D. Posey. Immunocytokines with specificity for invariant region of CAR molecules enhance effector function of multiple CAR T cell therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 58.

Abstract 3999: Augmentation of antitumor effects by CAR-T cells secreting anti-PD-1 single-chain antibody fused with IL-21

Abstract Background: Chimeric antigen receptor (CAR) T cell therapy has shown remarkable responses in hematologic malignancies. However, many patients suffer from limited response and tumor relapse due to lack of persisting CAR-T cells and immune escape. Delivery of immunomodulatory cytokines is one of the strategies to improve immune function; however, the systemic effect of cytokines can be detrimental. Methods: To prevent the toxic effects of cytokines, we proposed the concept of targeting cytokines to tumor-specific T cells in vivo. To this end, we developed CAR-T cells secreting a fusion protein (PD-1Ab21) composed of anti-PD-1 single chain antibody and IL-21, which target IL-21 to CAR-T cells to promote their proliferation and differentiation into memory cells while blocking the PD-1 signal of CAR-T cells. The secretion and functions of PD-1Ab21 were identified. The proliferation and differentiation of CAR-T cells secreting PD-1Ab21 (PD-1Ab21-CAR-T) were compared in vitro with that of the conventional CAR-T cells. The therapeutic effects of CAR-T cells secreting PD-1Ab21 were studied in immunodeficient mice engrafted with human Burkitt lymphoma Daudi, multiple myeloma cell U266 and Her2/neu+ breast cancer cell BT474. Results: The results showed that the culture supernatant of PD-1Ab21-CAR-T cells, like anti-PD-1 antibody, blocked the binding of PD-1 to PD-L1 and stimulated STAT3 phosphorylation in human T lymphocytic leukemia cell Hut78 with the same activity as IL-21. In the conventional culture milieu with high dose of IL-2, the proliferation of the PD-1Ab21-CAR-T cells was not different from that of conventional CAR-T cells. While cultured with low dose of IL-2 that could not support the proliferation of conventional CAR-T cells, the PD-1Ab21-CAR-T cells still showed significant proliferation. A major fraction of the PD-1Ab21-CAR-T products was shown to co-express CCR7, CD62L and CD45RA, and express high level of TCF-1, indicating a memory stem-like phenotype. While the most of conventional CAR-T cells showed effector phenotype defined as CD45RA+CCR7-. Furthermore, adoptive transfer of PD-1Ab21-CAR-T cells exhibited significantly improved CAR-T cell expansion and resulted in superior tumor eradication in the xenograft models of human B-cell malignancy, multiple myeloma and Her2/neu+ breast cancer. The enhanced cellular function by secreting PD-1Ab21 was mediated through an autocrine and/or paracrine manners. Conclusion: We developed an enhanced CAR-T cells. Our data provide preclinical evidence to support the translation of this design for an improved CAR-T cell-mediated anti-tumor response. Citation Format: Ying Li, Jitao Zhao, Xiangrui Cheng, Hang Li, Ran Tian, Qianqian He, Shengdian Wang. Augmentation of antitumor effects by CAR-T cells secreting anti-PD-1 single-chain antibody fused with IL-21 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3999.

Abstract 4070: CIS-demasking cytokine linker technology allows a selective cytokine delivery to activated immune cells while sparing others and peripheral toxicity

Abstract Immunocytokines are promising new therapeutic approaches to enhance T-cell response by delivering interleukins into the tumoral site. Fusion of wild-type or attenuated interleukins to anti-PD-1 antibody has shown some efficacy to preferentially cis-activate PD1-expressing T-cells but on-target/off-tumor activity on PD-1-negative cells is still observed due to the high affinity of the cytokine to its receptor leading a broad systemic effect, toxicity, high clearance and low tumor biodistribution limiting the potential of some immunocytokines (e.g. anti-PD-1/IL-2/IL-15/IL-21). Various strategies have emerged to achieve localized cytokine activation using conditional strategies (e.g. allosteric modulators, MMP-cleavable linkers or pH-dependent binding) that remain challenging due to high dependency to specific TME composition (MMP, acidosis...). We developed the OSE-Cytomask® Platform, a universal and innovative linker technology allowing exclusive cytokine CIS-demasking upon binding of the fused antibody to its target without TRANS-activation associated with undesired effects (e.g. Toxicity). A series of different linkers were screened for optimal cis-activation of cytokine on PD1-expressing vs PD1-negative T-cells using different cytokines fused to a high-affinity anti-PD1. OSE-Cytomask® linker technology decreases IL-2 or IL-15 cytokine activity (pSTAT5 signaling) on PD1-negative cells while maintaining high activation of PD1-transduced T-cells even in co-culture. We confirmed activity of OSE-Cytomask® Immunocytokine on (PD1+) human T-cells while very low activation has been observed in naïve T-cells. Importantly, Cytomask® linker technology does not induce TRANS-activation on PD1-negative T-cells illustrating an innovative and strict CIS-demasking mechanism of action. In vivo, Anti-PD-1/IL-15 and/or anti-PD1/IL-2 OSE-Cytomask® molecules illustrated reduced toxicity after single or multiple injections (2mg/kg). High T-cell proliferation has been observed in tumor-micro-environment where PD1-expressing T cells are located. Low T-cell proliferation in periphery was seen, illustrating the potential of CIS-demasking technology to target the right T-cells at the right place. Altogether, OSE-Cytomask® linker technology illustrates specific intrinsic property to mask cytokine on naïve peripheral immune cells not expressing the target of the antibody while allowing selective CIS-demasking of the cytokine and CIS-activation of activated immune cells expressing (e.g. PD-1). This linker technology could be used with a broad range of cytokine to abrogate OFF-tumor cytokine activity associated with toxicity while selectively CIS-activating activated immune cells in the TME. Citation Format: Aurore Morello, Caroline Mary, Virginie Thepenier, Margaux Seité, Justine Durand, Cécile Batty, Kévin Biteau, Julien Taurelle, Géraldine Teppaz, Amandine Georges, Nicolas Poirier. CIS-demasking cytokine linker technology allows a selective cytokine delivery to activated immune cells while sparing others and peripheral toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4070.

A novel strategy to generate immunocytokines with activity-on-demand using small molecule inhibitors

Cytokine-based therapeutics have been shown to mediate objective responses in certain tumor entities but suffer from insufficient selectivity, causing limiting toxicity which prevents dose escalation to therapeutically active regimens. The antibody-based delivery of cytokines significantly increases the therapeutic index of the corresponding payload but still suffers from side effects associated with peak concentrations of the product in blood upon intravenous administration. Here we devise a general strategy (named “Intra-Cork”) to mask systemic cytokine activity without impacting anti-cancer efficacy. Our technology features the use of antibody-cytokine fusions, capable of selective localization at the neoplastic site, in combination with pathway-selective inhibitors of the cytokine signaling, which rapidly clear from the body. This strategy, exemplified with a tumor-targeted IL12 in combination with a JAK2 inhibitor, allowed to abrogate cytokine-driven toxicity without affecting therapeutic activity in a preclinical model of cancer. This approach is readily applicable in clinical practice.

Development of Recombinant Oncolytic rVSV-mIL12-mGMCSF for Cancer Immunotherapy.

Anti-cancer therapy based on oncolytic viruses (OVs) is a targeted approach that takes advantage of OVs' ability to selectively infect and replicate in tumor cells, activate the host immune response, and destroy malignant cells over healthy ones. Vesicular stomatitis virus (VSV) is known for its wide range of advantages: a lack of pre-existing immunity, a genome that is easily amenable to manipulation, and rapid growth to high titers in a broad range of cell lines, to name a few. VSV-induced tumor immunity can be enhanced by the delivery of immunostimulatory cytokines. The targeted cytokine delivery to tumors avoids the significant toxicity associated with systemic delivery while also boosting the immune response. To demonstrate this enhanced effect on both tumor growth and survival, a novel recombinant VSV (rVSV)-mIL12-mGMCSF, co-expressing mouse IL-12 (interleukin-12) and GM-CSF (granulocyte-macrophage colony-stimulating factor), was tested alongside rVSV-dM51-GFP (rVSV-GFP) that was injected intratumorally in a syngeneic in vivo C57BL/6 mouse model infused subcutaneously with B16-F10 melanoma cells. The pilot study tested the effect of two viral injections 4 days apart and demonstrated that treatment with the two rVSVs resulted in partial inhibition of tumor growth (TGII of around 40%) and an increased survival rate in animals from the treatment groups. The effect of the two VSVs on immune cell populations will be investigated in future in vivo studies with an optimized experimental design with multiple higher viral doses, as a lack of this information presents a limitation of this study.

Variant rs4986790 of toll-like receptor 4 affects the signaling and induces cell dysfunction in patients with severe COVID-19

ObjectivesWe investigated the expression of toll-like receptor (TLR)-4 on the cell surface of innate and adaptive cells from patients with COVID-19 carrying the rs4986790 GG genotype in the TLR4 gene and the functional profile of these cells. MethodsWe included 1169 hospitalized patients with COVID-19. The rs4986790 in TLR4 was identified by real-time polymerase chain reaction. Peripheral blood mononuclear cells were isolated and cultured to evaluate TLR-4 expression on immune cells. Supernatants recovered culture assays were stored, and we measured cytokines and cytotoxic molecules. ResultsWe showed that the rs4986790 (GG) was significantly associated (P = 0.0310) with severe COVID-19. Cells of patients with COVID-19 carrying the GG genotype have increased the frequency of monocytes and activated naïve and non-switched B cells positive to TLR-4 when cells are stimulated with lipopolysaccharide and with spike protein of SARS-CoV-2. Also, cells from patients with GG COVID-19 cannot produce pro-inflammatory cytokines after lipopolysaccharide stimulus, but they are high producers of cytotoxic molecules at baseline. ConclusionsThe rs4986790 GG genotype of the TLR4 is associated with the risk of COVID-19 and acute respiratory distress syndrome. Peripheral blood mononuclear cells of patients carrying the rs4986790 (TLR4) GG genotype had a limited delivery of pro-inflammatory cytokines compared to the AA and AG genotypes in which TLR-4 stimulation induces IL-10, IL-6, tumor necrosis factor-α, and Fas ligand production.

Application of Hydrogels in Cardiac Regeneration.

Myocardial infarction (MI) is a leading cause of death globally. Due to limited cardiac regeneration, infarcted myocardial tissue is gradually replaced by cardiac fibrosis, causing cardiac dysfunction, arrhythmia, aneurysm, free wall rupture, and sudden cardiac death. Thus, the development of effective methods to promote cardiac regeneration is extremely important for MI treatment. In recent years, hydrogels have shown promise in various methods for cardiac regeneration. Hydrogels can be divided into natural and synthetic types. Different hydrogels have different features and can be cross-linked in various ways. Hydrogels are low in toxicity and highly stable. Since they have good biocompatibility, biodegradability, and transformability, moderate mechanical properties, and proper elasticity, hydrogels are promising biomaterials for promoting cardiac regeneration. They can be used not only as scaffolds for migration of stem cells, but also as ideal carriers for delivery of drugs, genetic materials, stem cells, growth factors, cytokines, and small molecules. In this review, the application of hydrogels in cardiac regeneration during or post-MI is discussed in detail. Hydrogels open a promising new area in cardiac regeneration for treating MI.

Adipose-derived stem cells enriched with therapeutic mRNA TGF-β3 and IL-10 synergistically promote scar-less wound healing in preclinical models.

Skin wound healing often leads to scar formation, presenting physical and psychological challenges for patients. Advancements in messenger RNA (mRNA) modifications offer a potential solution for pulsatile cytokine delivery to create a favorable wound-healing microenvironment, thereby preventing cutaneous fibrosis. This study aimed to investigate the effectiveness of human adipose-derived stem cells (hADSCs) enriched with N 1-methylpseudouridine (m1ψ) modified transforming growth factor-β3 (TGF-β3) and interleukin-10 (IL-10) mRNA in promoting scar-free healing in preclinical models. The results demonstrated that the modified mRNA (modRNA)-loaded hADSCs efficiently and temporarily secreted TGF-β3 and IL-10 proteins. In a dorsal injury model, hADSCs loaded with modRNA TGF-β3 and IL-10 exhibited multidimensional therapeutic effects, including improved collagen deposition, extracellular matrix organization, and neovascularization. In vitro experiments confirmed the ability of these cells to markedly inhibit the proliferation and migration of keloid fibroblasts, and reverse the myofibroblast phenotype. Finally, collagen degradation mediated by matrix metalloproteinase upregulation was observed in an ex vivo keloid explant culture model. In conclusion, the synergistic effects of the modRNA TGF-β3, IL-10, and hADSCs hold promise for establishing a scar-free wound-healing microenvironment, representing a robust foundation for the management of wounds in populations susceptible to scar formation.

In vitro modelling of local gene therapy with IL-15/IL-15Rα and a PD-L1 antagonist in melanoma reveals an interplay between NK cells and CD4+ T cells

Blockade of the immune checkpoint axis consisting of programmed death-1 (PD-1) and its ligand PD-L1 alleviates the functional inhibition of tumor-infiltrating lymphoid cells yet weakly induces their expansion. Exogenous cytokines could further expand lymphoid cells and thus synergize with αPD-L1 therapy. However, systemic delivery of most cytokines causes severe toxicity due to unspecific expansion of immune cells in the periphery. Here, we modelled local delivery of cytokines and αPD-L1 therapeutics to immune cell-containing in vitro melanoma tumors. Three-dimensional tumor models consisting of 624-MEL cells were co-cultured with human peripheral blood lymphoid cells (PBLs) in presence of the cytokines IL-2, IL-7, IL-15, IL-21 and IFN-γ. To model local gene therapy, melanoma tumors were modified with lentiviral vectors encoding IL-15 fused to IL-15Rα (IL-15/IL-15Rα) and K2-Fc, a fusion of a human PD-L1 specific single domain antibody to immunoglobulin (Ig)G1 Fc. To evaluate the interplay between PBL fractions, NK cells, CD4+ T cells or CD8+ T cells were depleted. Tumor cell killing was followed up using real time imaging and immune cell expansion and activation was evaluated with flow cytometry. Among the tested cytokines, IL-15 was the most potent cytokine in stimulating tumor cell killing and expanding both natural killer (NK) cells and CD8+ T cells. Gene-based delivery of IL-15/IL-15Rα to tumor cells, shows expansion of NK cells, activation of NK cells, CD4+ and CD8+ T cells, and killing of tumor spheroids. Both NK cells and CD8+ T cells are necessary for tumor cell killing and CD4+ T-cell activation was reduced without NK cells. Co-delivery of K2-Fc improved tumor cell killing coinciding with increased activation of NK cells, which was independent of bystander T cells. CD4+ or CD8+ T cells were not affected by the co-delivery of K2-Fc even though NK-cell activation impacted CD4+ T-cell activation. This study demonstrates that gene-based delivery of IL-15/IL-15Rα to tumor cells effectively mediates anti-tumor activity and sensitizes the tumor microenvironment for therapy with αPD-L1 therapeutics mainly by impacting NK cells. These findings warrant further investigation of gene-based IL-15 and K2-Fc delivery in vivo.

Charged Fibrous Dressing to Promote Diabetic Chronic Wound Healing.

Diabetic chronic wounds cause a significant amount of pain to patients because of their low cure rates and high recurrence rates. Traditional approaches to treating diabetic chronic wounds often involve the delivery of drugs or cytokines that regulate the microenvironment and eliminate bacterial infection in the wound area, but they are passive in controlling cell behaviors and may lead to drug resistance. Emerging drug-free wound treatments are important for convenient, effective, and safe treatment strategies. However, the current approaches cannot fully promote tissue regeneration or prevent bacterial infections. Here, the efficacy of a negatively charged fiber dressing in promoting diabetic chronic wound healing is investigated. The negatively charged fiber dressing can generate reactive oxygen species to inhibit bacterial reproduction with the assistance of ultrasound during the inflammatory phase. Furthermore, the dressing provides an electrostatic field that regulates cellular behavior during the inflammatory and proliferative phases. In particular, the dressing can promote fibroblast migration and induce macrophage polarization and neovascularization without any additional drugs. It is demonstrated that this strategy enables the healing of diabetic chronic wounds in a mouse model, achieving effective wound closure over a 12-day treatment cycle and providing a drug-free therapeutic strategy for diabetic chronic wound care.

Triply Enhanced Immunotherapy via Dual Glycan Reforming Integrated with Perforation.

The enhancement of immunotherapy is an emerging direction to develop highly effective and practical cancer therapeutic methods. Here a triply enhanced immunotherapy drug (TEID) is designed for ingeniously integrating in situ dual glycan reforming with perforation on cell membrane. The TEID is composed of galactose and neuraminidase conjugated streptolysin O (SLO-Gal and SLO-NEU), which are encapsulated in a hyaluronic acid (HA) shell for targeted recognition to tumor tissue via cell surface CD44. After targeted delivery and HAase-mediated degradation in the tumor region, the TEID releases SLO-Gal and SLO-NEU, which can easily anchor Gal and NEU on the tumor cell membrane via the perforation of SLO to perform dual glycan reforming for the introduction of Gal and the cleavage of sialic acid. The former can activate immune cells to secret cytokines for immune-killing, and the latter can weaken the immune inhibition to improve the immunotherapeutic efficacy. Meanwhile, the perforation of SLO can promote the delivery of cytokines into the tumor cells to further enhance the efficacy. The designed triply enhanced immunotherapy strategy opens a significant and promising route to promote clinical immunotherapy of cancer.