Immune modulation in transplant medicine: a comprehensive review of cell therapy applications and future directions.

Balancing the immune response after solid organ transplantation (SOT) and vascularized composite allotransplantation (VCA) remains an ongoing clinical challenge. While immunosuppressants can effectively reduce acute rejection rates following transplant surgery, some patients still experience recurrent acute rejection episodes, which in turn may progress to chronic rejection. Furthermore, these immunosuppressive regimens are associated with an increased risk of malignancies and metabolic disorders. Despite significant advancements in the field, these IS related side effects persist as clinical hurdles, emphasizing the need for innovative therapeutic strategies to improve transplant survival and longevity. Cellular therapy, a novel therapeutic approach, has emerged as a potential pathway to promote immune tolerance while minimizing systemic side-effects of standard IS regiments. Various cell types, including chimeric antigen receptor T cells (CAR-T), mesenchymal stromal cells (MSCs), regulatory myeloid cells (RMCs) and regulatory T cells (Tregs), offer unique immunomodulatory properties that may help achieve improved outcomes in transplant patients. This review aims to elucidate the role of cellular therapies, particularly MSCs, T cells, Tregs, RMCs, macrophages, and dendritic cells in SOT and VCA. We explore the immunological features of each cell type, their capacity for immune regulation, and the prospective advantages and obstacles linked to their application in transplant patients. An in-depth outline of the current state of the technology may help SOT and VCA providers refine their perioperative treatment strategies while laying the foundation for further trials that investigate cellular therapeutics in transplantation surgery.

Advancing our understanding of bioreactors for industrial-sized cell culture: health care and cellular agriculture implications.

Bioreactors are advanced biomanufacturing tools that have been widely used to develop various applications in the fields of health care and cellular agriculture. In recent years, there has been a growing interest in the use of bioreactors to enhance the efficiency and scalability of these technologies. In cell therapy, bioreactors have been used to expand and differentiate cells into specialized cell types that can be used for transplantation or tissue regeneration. In cultured meat production, bioreactors offer a controlled and efficient means of producing meat without the need for animal farming. Bioreactors can support the growth of muscle cells by providing the necessary conditions for cell proliferation, differentiation, and maturation, including the provision of oxygen and nutrients. This review article aims to provide an overview of the current state of bioreactor technology in both cell therapy and cultured meat production. It will examine the various bioreactor types and their applications in these fields, highlighting their advantages and limitations. In addition, it will explore the future prospects and challenges of bioreactor technology in these emerging fields. Overall, this review will provide valuable insights for researchers and practitioners interested in using bioreactor technology to develop innovative solutions in the biomanufacturing of therapeutic cells and cultured meat.

Co-transplantation of autologous Treg cells in a cell therapy for Parkinson's disease.

The specific loss of midbrain dopamine neurons (mDANs) causes major motor dysfunction in Parkinson's disease, which makes cell replacement a promising therapeutic approach1-4. However, poor survival of grafted mDANs remains an obstacle to successful clinical outcomes5-8. Here we show that the surgical procedure itself (referred to here as 'needle trauma') triggers a profound host response that is characterized by acute neuroinflammation, robust infiltration of peripheral immune cells and brain cell death. When midbrain dopamine (mDA) cells derived from human induced pluripotent stem (iPS) cells were transplanted into the rodent striatum, less than 10% of implanted tyrosine hydroxylase (TH)+ mDANs survived at two weeks after transplantation. By contrast, TH- grafted cells mostly survived. Notably, transplantation of autologous regulatory T (Treg) cells greatly modified the response to needle trauma, suppressing acute neuroinflammation and immune cell infiltration. Furthermore, intra-striatal co-transplantation of Treg cells and human-iPS-cell-derived mDA cells significantly protected grafted mDANs from needle-trauma-associated death and improved therapeutic outcomes in rodent models of Parkinson's disease with 6-hydroxydopamine lesions. Co-transplantation with Treg cells also suppressed the undesirable proliferation of TH- grafted cells, resulting in more compact grafts with a higher proportion and higher absolute numbers of TH+ neurons. Together, these data emphasize the importance of the initial inflammatory response to surgical injury in the differential survival of cellular components of the graft, and suggest that co-transplanting autologous Treg cells effectively reduces the needle-trauma-induced death of mDANs, providing a potential strategy to achieve better clinical outcomes for cell therapy in Parkinson's disease.

CAR immune cells: design principles, resistance and the next generation.

The remarkable clinical activity of chimeric antigen receptor (CAR) therapies in B cell and plasma cell malignancies has validated the use of this therapeutic class for liquid cancers, but resistance and limited access remain as barriers to broader application. Here we review the immunobiology and design principles of current prototype CARs and present emerging platforms that are anticipated to drive future clinical advances. The field is witnessing a rapid expansion of next-generation CAR immune cell technologies designed to enhance efficacy, safety and access. Substantial progress has been made in augmenting immune cell fitness, activating endogenous immunity, arming cells to resist suppression via the tumour microenvironment and developing approaches to modulate antigen density thresholds. Increasingly sophisticated multispecific, logic-gated and regulatable CARs display the potential to overcome resistance and increase safety. Early signs of progress with stealth, virus-free and in vivo gene delivery platforms provide potential paths for reduced costs and increased access of cell therapies in the future. The continuing clinical success of CAR T cells in liquid cancers is driving the development of increasingly sophisticated immune cell therapies that are poised to translate to treatments for solid cancers and non-malignant diseases in the coming years.

High-Throughput and Efficient Intracellular Delivery Method via a Vibration-Assisted Nanoneedle/Microfluidic Composite System.

Intracellular delivery and genetic modification have brought a significant revolutionary to tumor immunotherapy, yet existing methods are still limited by low delivery efficiency, poor throughput, excessive cell damage, or unsuitability for suspension immune cells, specifically the natural killer cell, which is highly resistant to transfection. Here, we proposed a vibration-assisted nanoneedle/microfluidic composite system that uses large-area nanoneedles to rapidly puncture and detach the fast-moving suspension cells in the microchannel under vibration to achieve continuous high-throughput intracellular delivery. The nanoneedle arrays fabricated based on the large-area self-assembly technique and microchannels can maximize the delivery efficiency. Cas9 ribonucleoprotein complexes (Cas9/RNPs) can be delivered directly into cells due to the sufficient cellular membrane nanoperforation size; for difficult-to-transfect immune cells, the delivery efficiency can be up to 98%, while the cell viability remains at about 80%. Moreover, the throughput is demonstrated to maintain a mL/min level, which is significantly higher than that of conventional delivery techniques. Further, we prepared CD96 knockout NK-92 cells via this platform, and the gene-edited NK-92 cells possessed higher immunity by reversing exhaustion. The high-throughput, high-efficiency, and low-damage performance of our intracellular delivery strategy has great potential for cellular immunotherapy in clinical applications.

Electrochemical Single-Cell Protein Therapeutics Using a Double-Barrel Nanopipette.

Single-cell protein therapeutics is expected to promote our in-depth understanding of how a specific protein with a therapeutic dosage treats the cell without population averaging. However, it has not yet been tackled by current single-cell nanotools. We address this challenge by the use of a double-barrel nanopipette, in which one lumen was used for electroosmotic cytosolic protein delivery and the other was customized for ionic evaluation of the consequence. Upon injection of protein DJ-1 through the delivery lumen, upregulation of the antioxidant protein could protect neural PC-12 cells against oxidative stress from phorbol myristate acetate exposure, as deduced by targeting of the cytosolic hydrogen peroxide by the detecting lumen. The nanotool developed in this study for single-cell protein therapeutics provides a perspective for future single-cell therapeutics involving different therapeutic modalities, such as peptides, enzymes and nucleic acids.

Engineering an inhibitor-resistant human CSF1R variant for microglia replacement.

Hematopoietic stem cell transplantation (HSCT) can replace endogenous microglia with circulation-derived macrophages but has high mortality. To mitigate the risks of HSCT and expand the potential for microglia replacement, we engineered an inhibitor-resistant CSF1R that enables robust microglia replacement. A glycine to alanine substitution at position 795 of human CSF1R (G795A) confers resistance to multiple CSF1R inhibitors, including PLX3397 and PLX5622. Biochemical and cell-based assays show no discernable gain or loss of function. G795A- but not wildtype-CSF1R expressing macrophages efficiently engraft the brain of PLX3397-treated mice and persist after cessation of inhibitor treatment. To gauge translational potential, we CRISPR engineered human-induced pluripotent stem cell-derived microglia (iMG) to express G795A. Xenotransplantation studies demonstrate that G795A-iMG exhibit nearly identical gene expression to wildtype iMG, respond to inflammatory stimuli, and progressively expand in the presence of PLX3397, replacing endogenous microglia to fully occupy the brain. In sum, we engineered a human CSF1R variant that enables nontoxic, cell type, and tissue-specific replacement of microglia.

Non-viral precision T cell receptor replacement for personalized cell therapy.

T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells1-3. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRß). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.

Multidimensional control of therapeutic human cell function with synthetic gene circuits.

Synthetic gene circuits that precisely control human cell function could expand the capabilities of gene- and cell-based therapies. However, platforms for developing circuits in primary human cells that drive robust functional changes in vivo and have compositions suitable for clinical use are lacking. Here, we developed synthetic zinc finger transcription regulators (synZiFTRs), which are compact and based largely on human-derived proteins. As a proof of principle, we engineered gene switches and circuits that allow precise, user-defined control over therapeutically relevant genes in primary T cells using orthogonal, US Food and Drug Administration-approved small-molecule inducers. Our circuits can instruct T cells to sequentially activate multiple cellular programs such as proliferation and antitumor activity to drive synergistic therapeutic responses. This platform should accelerate the development and clinical translation of synthetic gene circuits in diverse human cell types and contexts.

The emerging era of cell engineering: Harnessing the modularity of cells to program complex biological function.

A new era of biological engineering is emerging in which living cells are used as building blocks to address therapeutic challenges. These efforts are distinct from traditional molecular engineering-their focus is not on optimizing individual genes and proteins as therapeutics, but rather on using molecular components as modules to reprogram how cells make decisions and communicate to achieve higher-order physiological functions in vivo. This cell-centric approach is enabled by a growing tool kit of components that can synthetically control core cell-level functional outputs, such as where in the body a cell should go, what other cells it should interact with, and what messages it should transmit or receive. The power of cell engineering has been clinically validated by the development of immune cells designed to kill cancer. This same tool kit for rewiring cell connectivity is beginning to be used to engineer cell therapies for a host of other diseases and to program the self-organization of tissues and organs. By forcing the conceptual distillation of complex biological functions into a finite set of instructions that operate at the cell level, these efforts also shed light on the fundamental hierarchical logic that links molecular components to higher-order physiological function.

Understanding the Molecular Basis of iPSC Reprogrammed Cells to Fulfil Their Expectations in Future Clinical Applications.

IPSC-based disease modelling and pluripotency studies have sparked widespread enthusiasm for more than 16 years of research [...].

Two-year efficacy of SNK01 plus pembrolizumab for non-small cell lung cancer: Expanded observations from a phase I/IIa randomized controlled trial.

BACKGROUND: A previous trial showed that autologous ex-vivo expanded NK cell (SNK01) treatment combined with pembrolizumab showed better efficacy than pembrolizumab monotherapy in advanced non-small cell lung cancer (NSCLC). This study was a 2-year follow-up of that previous study to determine the long-term efficacy of the combination treatment. METHODS: This trial included 20 patients with advanced NSCLC with a PD-L1 tumor proportion score of 1% or greater who failed prior to front-line platinum-based therapy. The patients received pembrolizumab with low-dose SNK01 (2 × 109 cells/dose) or high-dose SNK01 (4 × 109 cells/dose), or pembrolizumab monotherapy. The primary study endpoint was overall survival (OS), and the secondary endpoint was progression-free survival (PFS). RESULTS: Two patients were excluded following serious adverse events. Among the 11 patients who died, five were from the NK groups (41.6%, n = 5/12), and six received pembrolizumab monotherapy (100%, n = 6/6). The estimated 2-year survival rate was 58.3% versus 16.7% (pembrolizumab plus SNK01 vs. pembrolizumab monotherapy). The hazard ratio of pembrolizumab plus SNK01 compared with pembrolizumab monotherapy was 0.32 (95% CI: 0.1, 1.08, p-value: 0.066). Although the median PFS was significantly higher in the pembrolizumab plus SNK01 group than in the pembrolizumab alone group, OS and PFS did not differ statistically between patients who received low doses of NK cells and those who received high doses of NK cells. CONCLUSIONS: Autologous NK cells can enhance the long-term OS and PFS for NSCLC. A larger study is needed to confirm this result. Clinical Research Information Service number: KCT0003463.

Potentiating adoptive cell therapy using synthetic IL-9 receptors.

Synthetic receptor signalling has the potential to endow adoptively transferred T cells with new functions that overcome major barriers in the treatment of solid tumours, including the need for conditioning chemotherapy1,2. Here we designed chimeric receptors that have an orthogonal IL-2 receptor extracellular domain (ECD) fused with the intracellular domain (ICD) of receptors for common γ-chain (γc) cytokines IL-4, IL-7, IL-9 and IL-21 such that the orthogonal IL-2 cytokine elicits the corresponding γc cytokine signal. Of these, T cells that signal through the chimeric orthogonal IL-2Rß-ECD-IL-9R-ICD (o9R) are distinguished by the concomitant activation of STAT1, STAT3 and STAT5 and assume characteristics of stem cell memory and effector T cells. Compared to o2R T cells, o9R T cells have superior anti-tumour efficacy in two recalcitrant syngeneic mouse solid tumour models of melanoma and pancreatic cancer and are effective even in the absence of conditioning lymphodepletion. Therefore, by repurposing IL-9R signalling using a chimeric orthogonal cytokine receptor, T cells gain new functions, and this results in improved anti-tumour activity for hard-to-treat solid tumours.

Mesenchymal Stem Cells and Extracellular Vesicles Derived from Canine Adipose Tissue Ameliorates Inflammation, Skin Barrier Function and Pruritus by Reducing JAK/STAT Signaling in Atopic Dermatitis.

Canine atopic dermatitis (AD) is a common chronic inflammatory skin disorder resulting from imbalance between T lymphocytes. Current canine AD treatments use immunomodulatory drugs, but some of the dogs have limitations that do not respond to standard treatment, or relapse after a period of time. Thus, the purpose of this study was to evaluate the immunomodulatory effect of mesenchymal stem cells derived from canine adipose tissue (cASCs) and cASCs-derived extracellular vesicles (cASC-EVs) on AD. First, we isolated and characterized cASCs and cASCs-EVs to use for the improvement of canine atopic dermatitis. Here, we investigated the effect of cASCs or cASC-EVs on DNCB-induced AD in mice, before using for canine AD. Interestingly, we found that cASCs and cASC-EVs improved AD-like dermatitis, and markedly decreased levels of serum IgE, (49.6%, p = 0.002 and 32.1%, p = 0.016 respectively) epidermal inflammatory cytokines and chemokines, such as IL-4 (32%, p = 0.197 and 44%, p = 0.094 respectively), IL-13 (47.4%, p = 0.163, and 50.0%, p = 0.039 respectively), IL-31 (64.3%, p = 0.030 and 76.2%, p = 0.016 respectively), RANTES (66.7%, p = 0.002 and 55.6%, p = 0.007) and TARC (64%, p = 0.016 and 86%, p = 0.010 respectively). In addition, cASCs or cASC-EVs promoted skin barrier repair by restoring transepidermal water loss, enhancing stratum corneum hydration and upregulating the expression levels of epidermal differentiation proteins. Moreover, cASCs or cASC-EVs reduced IL-31/TRPA1-mediated pruritus and activation of JAK/STAT signaling pathway. Taken together, these results suggest the potential of cASCs or cASC-EVs for the treatment of chronic inflammation and damaged skin barrier in AD or canine AD.

Adverse effects of cell-free and concentrated ascites reinfusion therapy for malignant ascites: a single-institute experience.

BACKGROUND: Cell-free and concentrated ascites reinfusion therapy (CART) is a strategy for improving various intractable symptoms due to refractory ascites, including hypoalbuminemia. CART has recently been applied in the treatment of cancer patients. This study was performed to assess the safety of CART in a single cancer institute. METHODS: We retrospectively reviewed 233 CART procedures that were performed for 132 cancer patients in our institute. RESULTS: The median weight of ascites before and after concentration was 4,720 g and 490 g (median concentration rate, 10.0-fold), The median amounts of total protein and albumin were 64.0 g and 32.6 g (median recovery rates, 44.9% and 49.0%), respectively. Thirty-three adverse events (AEs) were observed in 22 (9.4%) of 233 procedures; 30 of these events occurred after reinfusion. The most common reinfusion-related AEs were fever (13 events) and chills (10 events). Univariate analyses revealed no significant relationships between the frequency of AEs and age, sex, appearance of ascites, weight of harvested and concentrated ascites, the ascites processing rate (filtration and concentration), weight of saline used for membrane cleaning, amount of calculated total protein for infusion, or prophylaxis against AEs; the reinfusion rate of ≥ 125 mL/h or ≥ 10.9 g/h of total protein affected the frequency of AEs, regardless of the prophylactic use of steroids. CONCLUSIONS: The observed AEs were mainly mild reactions after reinfusion, which were related to a reinfusion rate of volume ≥ 125 mL/h, a simple indicator in practice, or total protein ≥ 10.9 g/h. Although our study was retrospective in nature and undertaken in a single institute, this information may be helpful for the management of cancer patients with refractory malignant ascites using CART.