MMP inhibition as a novel strategy for extracellular matrix preservation during whole liver decellularization.

As the only reliable treatment option for end-stage liver diseases, conventional liver transplantation confronts major supply limitations. Accordingly, the decellularization of discarded livers to produce bioscaffolds that support recellularization with progenitor/stem cells has emerged as a promising translational medicine approach. The success of this approach will substantially be determined by the extent of extracellular matrix (ECM) preservation during the decellularization process. Here, we assumed that the matrix metalloproteinase (MMP) inhibition could reduce the ECM damage during the whole liver decellularization of an animal model using a perfusion-based system. We demonstrated that the application of doxycycline as an MMP inhibitor led to significantly higher preservation of collagen, glycosaminoglycans, and hepatic growth factor (HGF) contents, as well as mechanical and structural features, including tensile strength, fiber integrity, and porosity. Notably, produced bioscaffolds were biocompatible and efficiently supported cell viability and proliferation in vitro. We also indicated that produced bioscaffolds efficiently supported HepG2 cell function upon seeding onto liver ECM discs using albumin and urea assay. Additionally, MMP inhibitor pretreated decellularized livers were more durable in contact with collagenase digestion compared to control bioscaffolds in vitro. Using zymography, we confirmed the underlying mechanism that results in these promising effects is through the inhibition of MMP2 and MMP9. Overall, we demonstrated a novel method based on MMP inhibition to ameliorate the ECM structure and composition preservation during liver decellularization as a critical step in fabricating transplantable bioengineered livers.

Exosomal Cargo: Pro-angiogeneic, anti-inflammatory, and regenerative effects in ischemic and non-ischemic heart diseases - A comprehensive review.

Heart diseases are the primary cause of mortality and morbidity worldwide which inflict a heavy social and economic burden. Among heart diseases, most deaths are due to myocardial infarction (MI) or heart attack, which occurs when a decrement in blood flow to the heart causes injury to cardiac tissue. Despite several available diagnostic, therapeutic, and prognostic approaches, heart disease remains a significant concern. Exosomes are a kind of small extracellular vesicles released by different types of cells that play a part in intercellular communication by transferring bioactive molecules important in regenerative medicine. Many studies have reported the diagnostic, therapeutic, and prognostic role of exosomes in various heart diseases. Herein, we reviewed the roles of exosomes as new emerging agents in various types of heart diseases, including ischemic heart disease, cardiomyopathy, arrhythmia, and valvular disease, focusing on pathogenesis, therapeutic, diagnostic, and prognostic roles in different areas. We have also mentioned different routes of exosome delivery to target tissues, the effects of preconditioning and modification on exosome's capability, exosome production in compliance with good manufacturing practice (GMP), and their ongoing clinical applications in various medical contexts to shed light on possible clinical translation.

Randomized crossover clinical trial of coenzyme Q10 and nicotinamide riboside in chronic kidney disease.

BackgroundCurrent studies suggest mitochondrial dysfunction is a major contributor to impaired physical performance and exercise intolerance in chronic kidney disease (CKD). We conducted a clinical trial of coenzyme Q10 (CoQ10) and nicotinamide riboside (NR) to determine their impact on exercise tolerance and metabolic profile in patients with CKD.MethodsWe conducted a randomized, placebo-controlled, double-blind, crossover trial comparing CoQ10, NR, and placebo in 25 patients with an estimated glomerular filtration rate (eGFR) of less than 60mL/min/1.73 m2. Participants received NR (1,000 mg/day), CoQ10 (1,200 mg/day), or placebo for 6 weeks each. The primary outcomes were aerobic capacity measured by peak rate of oxygen consumption (VO2 peak) and work efficiency measured using graded cycle ergometry testing. We performed semitargeted plasma metabolomics and lipidomics.ResultsParticipant mean age was 61.0 ± 11.6 years and mean eGFR was 36.9 ± 9.2 mL/min/1.73 m2. Compared with placebo, we found no differences in VO2 peak (P = 0.30, 0.17), total work (P = 0.47, 0.77), and total work efficiency (P = 0.46, 0.55) after NR or CoQ10 supplementation. NR decreased submaximal VO2 at 30 W (P = 0.03) and VO2 at 60 W (P = 0.07) compared with placebo. No changes in eGFR were observed after NR or CoQ10 treatment (P = 0.14, 0.88). CoQ10 increased free fatty acids and decreased complex medium- and long-chain triglycerides. NR supplementation significantly altered TCA cycle intermediates and glutamate that were involved in reactions that exclusively use NAD+ and NADP+ as cofactors. NR decreased a broad range of lipid groups including triglycerides and ceramides.ConclusionsSix weeks of treatment with NR or CoQ10 improved markers of systemic mitochondrial metabolism and lipid profiles but did not improve VO2 peak or total work efficiency.Trial registrationClinicalTrials.gov NCT03579693.FundingNational Institutes of Diabetes and Digestive and Kidney Diseases (grants R01 DK101509, R03 DK114502, R01 DK125794, and R01 DK101509).

Wet-spinnability and crosslinked Fiber properties of alginate/hydroxyethyl cellulose with varied proportion for potential use in tendon tissue engineering.

Researchers have examined different bio-inspired materials in tissue engineering and regenerative medicine to fabricate scaffolds to address tendon regeneration requirements. We developed fibers based on alginate (Alg) and hydroxyethyl cellulose (HEC) by wet-spinning technique to mimic the fibrous sheath of ECM. Various proportions (25:75, 50:50, 75:25) of 1 % Alg and 4 % HEC were blended to this aim. Two steps of crosslinking with different concentrations of CaCl2 (2.5 and 5 %) and glutaraldehyde (2.5 %) were used to improve physical and mechanical properties. The fibers were characterized by FTIR, SEM, swelling, degradation, and tensile tests. The in vitro proliferation, viability, and migration of tenocytes on the fibers were also evaluated. Moreover, the biocompatibility of implanted fibers was investigated in an animal model. The results showed ionic and covalent molecular interactions between the components. In addition, by properly maintaining surface morphology, fiber alignment, and swelling, lower concentrations of HEC in the blending provided good degradability and mechanical features. The mechanical strength of fibers was in the range of collagenous fibers. Increasing the crosslinking led to significantly different mechanical behaviors in terms of tensile strength and elongation at break. Because of good in vitro and in vivo biocompatibility, tenocyte proliferation, and migration, the biological macromolecular fibers could serve as desirable tendon substitutes. This study provides more practical insight into tendon tissue engineering in translational medicine.

Angiogenesis and Re-endothelialization in decellularized scaffolds: Recent advances and current challenges in tissue engineering.

Decellularization of tissues and organs has recently become a promising approach in tissue engineering and regenerative medicine to circumvent the challenges of organ donation and complications of transplantations. However, one main obstacle to reaching this goal is acellular vasculature angiogenesis and endothelialization. Achieving an intact and functional vascular structure as a vital pathway for supplying oxygen and nutrients remains the decisive challenge in the decellularization/re-endothelialization procedure. In order to better understand and overcome this issue, complete and appropriate knowledge of endothelialization and its determining variables is required. Decellularization methods and their effectiveness, biological and mechanical characteristics of acellular scaffolds, artificial and biological bioreactors, and their possible applications, extracellular matrix surface modification, and different types of utilized cells are factors affecting endothelialization consequences. This review focuses on the characteristics of endothelialization and how to optimize them, as well as discussing recent developments in the process of re-endothelialization.

Immunogenicity of decellularized extracellular matrix scaffolds: a bottleneck in tissue engineering and regenerative medicine.

Tissue-engineered decellularized extracellular matrix (ECM) scaffolds hold great potential to address the donor shortage as well as immunologic rejection attributed to cells in conventional tissue/organ transplantation. Decellularization, as the key process in manufacturing ECM scaffolds, removes immunogen cell materials and significantly alleviates the immunogenicity and biocompatibility of derived scaffolds. However, the application of these bioscaffolds still confronts major immunologic challenges. This review discusses the interplay between damage-associated molecular patterns (DAMPs) and antigens as the main inducers of innate and adaptive immunity to aid in manufacturing biocompatible grafts with desirable immunogenicity. It also appraises the impact of various decellularization methodologies (i.e., apoptosis-assisted techniques) on provoking immune responses that participate in rejecting allogenic and xenogeneic decellularized scaffolds. In addition, the key research findings regarding the contribution of ECM alterations, cytotoxicity issues, graft sourcing, and implantation site to the immunogenicity of decellularized tissues/organs are comprehensively considered. Finally, it discusses practical solutions to overcome immunogenicity, including antigen masking by crosslinking, sterilization optimization, and antigen removal techniques such as selective antigen removal and sequential antigen solubilization.

Chronic Kidney Disease is Associated With Attenuated Plasma Metabolome Response to Oral Glucose Tolerance Testing.

OBJECTIVE: Chronic kidney disease (CKD) is associated with decreased anabolic response to insulin contributing to protein-energy wasting. Targeted metabolic profiling of oral glucose tolerance testing (OGTT) may help identify metabolic pathways contributing to disruptions to insulin response in CKD. METHODS: Using targeted metabolic profiling, we studied the plasma metabolome response in 41 moderate-to-severe nondiabetic CKD patients and 20 healthy controls at fasting and 2 hours after an oral glucose load. We used linear mixed modeling with random intercepts, adjusting for age, gender, race/ethnicity, body weight, and batch to assess heterogeneity in response to OGTT by CKD status. RESULTS: Mean estimated glomerular filtration rate among CKD participants was 38.9 ± 12.7 mL/min per 1.73 m2 compared to 87.2 ± 17.7 mL/min per 1.73 m2 among controls. Glucose ingestion induced an anabolic response resulting in increased glycolysis products and a reduction in a wide range of metabolites including amino acids, tricarboxylic acid cycle intermediates, and purine nucleotides compared to fasting. Participants with CKD demonstrated a blunted anabolic response to OGTT evidenced by significant changes in 13 metabolites compared to controls. The attenuated metabolome response predominant involved mitochondrial energy metabolism, vitamin B family, and purine nucleotides. Compared to controls, CKD participants had elevated lactate:pyruvate (L:P) ratio and decreased guanosine diphosphate:guanosine triphosphate ratio during OGTT. CONCLUSION: Metabolic profiling of OGTT response suggests a broad disruption of mitochondrial energy metabolism in CKD patients. These findings motivate further investigation into the impact of insulin sensitizers and mitochondrial targeted therapeutics on energy metabolism in patients with nondiabetic CKD.

Impaired incretin homeostasis in non-diabetic moderate-severe CKD.

Background: Incretins are regulators of insulin secretion and glucose homeostasis that are metabolized by dipeptidyl peptidase-4 (DPP-4). Moderate-severe CKD may modify incretin release, metabolism, or response. Methods: We performed 2-hour oral glucose tolerance testing (OGTT) in 59 people with non-diabetic CKD (eGFR<60 ml/min per 1.73 m2) and 39 matched controls. We measured total (tAUC) and incremental (iAUC) area under the curve of plasma total glucagon-like peptide-1 (GLP-1) and total glucose-dependent insulinotropic polypeptide (GIP). Fasting DPP-4 levels and activity were measured. Linear regression was used to adjust for demographic, body composition, and lifestyle factors. Results: Mean eGFR was 38 ±13 and 89 ±17ml/min per 1.73 m2 in CKD and controls. GLP-1 iAUC and GIP iAUC were higher in CKD than controls with a mean of 1531 ±1452 versus 1364 ±1484 pMxmin, and 62370 ±33453 versus 42365 ±25061 pgxmin/ml, respectively. After adjustment, CKD was associated with 15271 pMxmin/ml greater GIP iAUC (95% CI 387, 30154) compared to controls. Adjustment for covariates attenuated associations of CKD with higher GLP-1 iAUC (adjusted difference, 122, 95% CI -619, 864). Plasma glucagon levels were higher at 30 minutes (mean difference, 1.6, 95% CI 0.3, 2.8 mg/dl) and 120 minutes (mean difference, 0.84, 95% CI 0.2, 1.5 mg/dl) in CKD compared to controls. There were no differences in insulin levels or plasma DPP-4 activity or levels between groups. Conclusion: Incretin response to oral glucose is preserved or augmented in moderate-severe CKD, without apparent differences in circulating DPP-4 concentration or activity. However, neither insulin secretion nor glucagon suppression are enhanced.

Targeting the CTLA-4/B7 axes in glioblastoma: preclinical evidence and clinical interventions.

INTRODUCTION: Glioblastoma Multiforme (GBM) is one of the fatal cancers of the Central Nervous System (CNS). A variety of reasons exist for why previous immunotherapy strategies, especially Immune Checkpoint Blockers (ICBs), did not work in treating GBM patients. The cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a key immune checkpoint receptor. Its overexpression in cancer and immune cells causes tumor cell progression. CTLA-4 suppresses anti-tumor responses inside the GBM tumor-immune microenvironment. AREAS COVERED: It has been attempted to explain the immunobiology of CTLA-4 as well as its interaction with different immune cells and cancer cells that lead to GBM progression. Additionally, CTLA-4 targeting studies have been reviewed and CTLA-4 combination therapy, as a promising therapeutic target and strategy for GBM immunotherapy, is recommended. EXPERT OPINION: CTLA-4 could be a possible supplement for future cancer immunotherapies of GBM. However, many challenges remain such as the high toxicity of CTLA-4 blockers, and the unresponsiveness of most patients to immunotherapy. For the future clinical success of CTLA-4 blocker therapy, combination approaches with other targeted treatments would be a potentially effective strategy. Going forward, predictive biomarkers can be used to reduce trial timelines and increase the chance of success.

Osteogenic Differentiation Effect of BMP-9 with Phenamil and Simvastatin on Intact Human Amniotic Epithelial Stem Cells

Background: Background: Bone tissue engineering has shown to be a promising strategy for repairing bone defects without causing harmful side effects to the patient. Three main building blocks of tissue engineering, including seeding cells, scaffold, and signaling molecules, are required for adequate bone regeneration. The human amniotic membrane (hAM) is the innermost of the placental membranes. In addition to providing a source of stem cells and growth factors, hAM has several features that make it an appropriate scaffold containing stem cells for use in tissue engineering purposes. The present investigation aimed to assess the effect of bone morphogenetic protein-9 (BMP-9) combined with phenamil and simvastatin on osteogenic induction of hAM with its human amniotic membrane epithelial cells (hAECs). Method: Methods: Using six different osteogenic medium (OMs), we cultured hAM for 14 days. The basic OMs were chosen as the first group and other media were made by adding BMP-9, phenamil, simvastatin, BMP-9 alongside phenamil, and BMP-9 alongside simvastatin to the basic OMs. Finally, viability assay, tissue mineralization, calcium and phosphate content determination, and measurement of lactic acid dehydrogenase (LDH), and alkaline phosphatase (ALP) activity were performed. Results: Results: Among all study groups, groups containing simvastatin showed a significantly lower level of viability. Although all media could induce osteogenic features, the hAECs cultured in media containing BMP-9 and phenamil demonstrated a wider area of mineralization and a significantly higher level of calcium and phosphate content, LDH, and ALP activity. Conclusion: Conclusion: Our findings indicated that the use of phenamil together with BMP-9 could synergistically show in situ osteogenic induction in hAECs, which could be a new insight into translational medicine.

Recent advances on small molecules in osteogenic differentiation of stem cells and the underlying signaling pathways.

Bone-related diseases are major contributors to morbidity and mortality in elderly people and the current treatments result in insufficient healing and several complications. One of the promising areas of research for healing bone fractures and skeletal defects is regenerative medicine using stem cells. Differentiating stem cells using agents that shift cell development towards the preferred lineage requires activation of certain intracellular signaling pathways, many of which are known to induce osteogenesis during embryological stages. Imitating embryological bone formation through activation of these signaling pathways has been the focus of many osteogenic studies. Activation of osteogenic signaling can be done by using small molecules. Several of these agents, e.g., statins, metformin, adenosine, and dexamethasone have other clinical uses but have also shown osteogenic capacities. On the other hand, some other molecules such as T63 and tetrahydroquinolines are not as well recognized in the clinic. Osteogenic small molecules exert their effects through the activation of signaling pathways known to be related to osteogenesis. These pathways include more well-known pathways including BMP/Smad, Wnt, and Hedgehog as well as ancillary pathways including estrogen signaling and neuropeptide signaling. In this paper, we review the recent data on small molecule-mediated osteogenic differentiation, possible adjunctive agents with these molecules, and the signaling pathways through which each small molecule exerts its effects.

Recent advances in mRNA-LNP therapeutics: immunological and pharmacological aspects.

In the last decade, the development of messenger RNA (mRNA) therapeutics by lipid nanoparticles (LNP) leads to facilitate clinical trial recruitment, which improves the efficacy of treatment modality to a large extent. Although mRNA-LNP vaccine platforms for the COVID-19 pandemic demonstrated high efficiency, safety and adverse effects challenges due to the uncontrolled immune responses and inappropriate pharmacological interventions could limit this tremendous efficacy. The current study reveals the interplay of immune responses with LNP compositions and characterization and clarifies the interaction of mRNA-LNP therapeutics with dendritic, macrophages, neutrophile cells, and complement. Then, pharmacological profiles for mRNA-LNP delivery, including pharmacokinetics and cellular trafficking, were discussed in detail in cancer types and infectious diseases. This review study opens a new and vital landscape to improve multidisciplinary therapeutics on mRNA-LNP through modulation of immunopharmacological responses in clinical trials.

The prognostic and therapeutic potentials of CTLA-4 in hematological malignancies.

INTRODUCTION: Hematological Malignancies (HMs) are a group of progressive, difficult-to-treat, and highly recurrent diseases. A suppressed phenotype of the immune system is present in HMs and growing evidence indicates the role of Cytotoxic T lymphocyte-Associated protein 4 (CTLA-4) in the course of HMs. AREAS COVERED: This article reviews the recent literature on the role of CTLA-4 in different subtypes of HMs. Here, the studies on the expression pattern, its effect on the prognosis of different HMs, and polymorphisms of CTLA-4 have been elaborated. Finally, the effect of targeting CTLA-4 in vitro and in vivo, as well as in clinical trials, is discussed. EXPERT OPINION: According to the recent literature, CTLA-4 is overexpressed in different HMs, which is correlated with poor survival, while it is associated with better a prognosis in Chronic Lymphocytic Leukemia (CLL). Targeting CTLA-4 in Acute Myeloid Leukemia (AML), Sezary Syndrome (SS), Hodgkin's Lymphoma (HL), and so on, is helpful. While this is not recommended and may even be harmful in multiple myeloma (MM) and CLL. Also, it seems that certain CTLA-4 gene polymorphisms are efficient factors in the course of HMs. Future studies may broaden our knowledge regarding the role of CTLA-4 in HMs.

Evaluation of immunomodulatory effects of co-culture or supernatant of dexamethasone or IFN-γ-treated adipose-derived mesenchymal stem cells on spleen mononuclear cells

Although mesenchymal stem cells (MSCs) have exhibited promising immunomodulatory potential in preclinical studies, clinical studies have revealed variable results. These results often depend on environmental cues. Pre-conditioning MSCs with cytokines is one of the methods used to enhance their immunomodulatory effects. In this study, we harvested adipose-derived MSCs from mice and cultured them with different doses of the cytokine, IFN-γ, and the corticosteroid drug, dexamethasone, in order to investigate their effects on MSC immunosuppressive function. We found the co-culture or supernatant of MSCs, pre-conditioned with IFN-γ, together with spleen mononuclear cells resulted in a significant reduction of mononuclear cell proliferation. Although the supernatant of MSCs, pre-conditioned with dexamethasone, showed similar results, dexamethasone pre-conditioning of co-cultured MSCs increased mononuclear cell proliferation. The results further our understanding of immune-related effects of MSCs which may provide a basis for further in vivo studies to achieve better clinical results. We propose that pre-conditioning with cytokines might be an effective method to boost the immunomodulatory effects of MSCs.

Targeted Association and Intracellular Delivery of Nanocargoes into Primary T Lymphocytes via Interleukin-2 Receptor-Mediated Endocytosis.

Despite the tremendous progress in immunotherapy regimens using T cells, efforts to modulate the functions of T cells are still significantly hampered by the lack of reliable methods to deliver various cargoes into the T cells. This ongoing challenge originates from the intrinsic resistance of T cells in taking up exogenous materials. Here, we strategically aimed to hijack the natural endocytosis of Interleukin-2 (IL2) by the activated T cells for the targeted association and intracellular delivery of cargoes in varying sizes. First, we carefully characterized the fluctuations in the expression levels of IL2 receptor (IL2R) subunits (CD25, CD122, and CD132) during the murine primary T cell cultures over 12 days. We identified the highest fraction of T cells that would express the high-affinity trimeric IL2R on Day 3. By examining the association and uptake efficiencies of IL2 molecules that are biotinylated via either random lysine-targeting chemical reaction (using NHS-PEG4-Biotin) or site-specific enzymatic modification (using Avitag sequence), we demonstrated that the most efficient delivery of cargo can be achieved by C-terminal conjugation. Upon confirmation of successful delivery of a small model cargo, streptavidin, we employed superparamagnetic iron oxide nanoparticles (SPIONs) as bigger model cargoes having core diameters of 50, 100, and 200 nm. We examined the association and intracellular delivery of the IL2-conjugated nanocargoes using flow cytometry, confocal laser scanning microscopy, and transmission electron microscopy. While cargoes of all tested sizes were successfully associated with the IL2R-expressing T cells in comparable efficiencies, the uptake efficiencies were inversely proportional to the sizes of the cargoes. Nevertheless, our current definitive report confirms that nanocargoes with a practical maximum size limit around 100-200 nm can be intracellularly delivered into activated primary T cells using IL2R-mediated endocytosis, which opens a new horizon for engineering and manufacturing of various T cell immunotherapeutics.

Nanoparticle-mediated synergistic chemoimmunotherapy for tailoring cancer therapy: recent advances and perspectives.

Nowadays, a potent challenge in cancer treatment is considered the lack of efficacious strategy, which has not been able to significantly reduce mortality. Chemoimmunotherapy (CIT) as a promising approach in both for the first-line and relapsed therapy demonstrated particular benefit from two key gating strategies, including chemotherapy and immunotherapy to cancer therapy; therefore, the discernment of their participation and role of potential synergies in CIT approach is determinant. In this study, in addition to balancing the pros and cons of CIT with the challenges of each of two main strategies, the recent advances in the cancer CIT have been discussed. Additionally, immunotherapeutic strategies and the immunomodulation effect induced by chemotherapy, which boosts CIT have been brought up. Finally, harnessing and development of the nanoparticles, which mediated CIT have expatiated in detail.

Blockade of HIF-1α and STAT3 by hyaluronate-conjugated TAT-chitosan-SPION nanoparticles loaded with siRNA molecules prevents tumor growth.

HIF-1α and STAT3 are two of the critical factors in the growth, proliferation, and metastasis of cancer cells and play a crucial role in inhibiting anti-cancer immune responses. Therefore, we used superparamagnetic iron oxide (SPION) nanoparticles (NPs) coated with thiolated chitosan (ChT) and trimethyl chitosan (TMC) and functionalized with hyaluronate (H) and TAT peptide for delivery of siRNA molecules against STAT3 and HIF-1α to cancer cells both in vivo and in vitro. The results indicated that tumor cell transfection with siRNA-encapsulated NPs robustly inhibited proliferation and migration and induced apoptosis in tumor cells. Furthermore, simultaneous silencing of HIF-1α and STAT3 significantly repressed cancer development in two different tumor types (4T1 breast cancer and CT26 colon cancer) which were associated with upregulation of cytotoxic T lymphocytes and IFN-γ secretion. The findings suggest inhibiting the HIF-1α/STAT3 axis by SPION-TMC-ChT-TAT-H NPs as an effective way to treat cancer.

Calcium signaling on Jurkat T cells induced by microbeads coated with novel peptide ligands specific to human CD3ε.

CD3ε is expressed on T lymphocytes as a part of the T cell receptor (TCR)-CD3 complex. Together with other CD3 molecules, CD3ε is responsible for the activation of T cells via transducing the event of antigen recognition by the TCR into intracellular signaling cascades. The present study first aims to identify a novel peptide ligand that binds to human CD3ε in a specific manner and to perform an initial evaluation of its biological efficacy on the human T cell line, Jurkat cells. We screened a phage-display peptide library against human CD3ε using a subtractive biopanning process, from which we identified 13 phage clones displaying unique peptide sequences. One dominant phage clone displaying the 7 amino acid sequence of WSLGYTG, which occupied 90% of tested plaques (18 out of 20) after the 5th round of biopanning, demonstrated a superior binding behavior to other clones in the binding assays against recombinant CD3ε on microbeads or Jurkat cells. The synthesized peptide also showed specific binding to Jurkat cells in a dose-dependent manner but not to B cell lymphoma line, 2PK3 cells. Molecular modeling and docking simulation confirmed that the selected peptide ligand in an energetically stable conformation binds to a pocket of CD3ε that is not hidden by either CD3γ or CD3δ. Lastly, magnetic microbeads conjugated with the synthesized peptide ligands showed a weak but specific association with Jurkat cells and induced the calcium flux, a hallmark indication of proximal T cell receptor signaling, which gave rise to an enhancement of IL-2 section and cell proliferation. The novel peptide ligand and its various multivalent forms have a great potential in applications related to T cell biology and T cell immunotherapy.