Tissue Immunogenicity Research Articles

Whole-eye transplantation: Current challenges and future perspectives.

Whole-eye transplantation emerges as a frontier in ophthalmology, promising a transformative approach to irreversible blindness. Despite advancements, formidable challenges persist. Preservation of donor eye viability post-enucleation necessitates meticulous surgical techniques to optimize retinal integrity and ganglion cell survival. Overcoming the inhibitory milieu of the central nervous system for successful optic nerve regeneration remains elusive, prompting the exploration of neurotrophic support and immunomodulatory interventions. Immunological tolerance, paramount for graft acceptance, confronts the distinctive immunogenicity of ocular tissues, driving research into targeted immunosuppression strategies. Ethical and legal considerations underscore the necessity for stringent standards and ethical frameworks. Interdisciplinary collaboration and ongoing research endeavors are imperative to navigate these complexities. Biomaterials, stem cell therapies, and precision immunomodulation represent promising avenues in this pursuit. Ultimately, the aim of this review is to critically assess the current landscape of whole-eye transplantation, elucidating the challenges and advancements while delineating future directions for research and clinical practice. Through concerted efforts, whole-eye transplantation stands to revolutionize ophthalmic care, offering hope for restored vision and enhanced quality of life for those afflicted with blindness.

In vitro оценка иммуногенности хондроцитов, полученных из индуцированных плюрипотентных стволовых клеток с нокаутом B2M

Today, the cell-based technologies are one of the instruments used for the cartilage tissue repair. Creation of a universal hypoimmunogenic cartilage tissue graft from the differentiated derivatives of induced pluripotent stem cells (iPSCs) might solve the problem of the lack of the cartilage cell product. However, currently there is little data on immunogenicity of such tissue-engineered preparations. The study was aimed to create a cartilage implant from the differentiated derivatives of the B2M-deficient iPSCs and assess its immunogenicity. The previously developed protocol was used to ensure differentiation of both wild-type and B2M knockout iPSCs into chondrocyte-like cells. After quality control of the resulting cell lines by conducting polymerase chain reaction and immunocytochemical assessment, the resulting cell lines were co-cultured with the peripheral blood mononuclear cells of a healthy donor. When co-cultivation was over, activation and degranulation of CD8+ T cells was assessed by flow cytometry analysis based on the CD69 and CD107a expression on the cell surface, respectively. The iPSC-derived chondrocytes expressed the cartilage tissue markers. Flow cytometry analysis revealed no substantial differences in immunogenicity between the derivatives of wild-type and B2M knockout iPSCs, as well as from the cartilage tissue cells of a healthy donor. Immunogenicity of chondrocyte-like cells was higher than that of hypoimmunogenic non-edited iPSCs. The B2M knockout iPSCs demonstrated a trend towards greater activation of CD8+ T cells. Thus, the B2M knockout in the iPSC-derived chondrocytes had no significant effect on the tissue immunogenicity. It is necessary to further edit the genes encoding MHC II and CD47 to obtain a less immunogenic product.

Biomimetic-modified bioprosthetic heart valves with controlled release of glycyrrhizin acid mediated by the inflammatory microenvironment for anti-thrombotic, anti-inflammatory, and anti-calcification

Artificial heart valve replacement is optimum for treating severe valvular heart disease (VHD). Bioprosthetic heart valves (BHVs) have received widespread attention from patients and doctors due to their good hydrodynamics, hemocompatibility, and minimally invasive implantation. However, the inflammatory microenvironment caused by cytotoxicity of residual aldehyde groups, the immunogenicity of heterogeneous tissue, and implantation damage, have led to problems such as endothelialization difficulties, leaflet thrombosis, and calcification of BHVs, limiting service life and applicability to the population of BHVs. In this study, we introduced chondroitin sulfate (CS) and p-aminophenyl boronic acid-glycyrrhizin acid (PBA-GA) into glutaraldehyde (Glut) cross-linked BHV material, thereby preparing a bionic-modified BHV material (CS-GA-PP), which can remould the inflammatory microenvironment at the injured site. CS can form an extracellular matrix (ECM)-like structure, reducing the immunogenicity and thrombosis of BHVs, at the same time, GA can be released under the inflammatory microenvironment, exerting an anti-inflammatory effect, reducing inflammation and calcification. CS-GA-PP maintained the great structural stability and mechanical properties brought about by cross-linking with Glut. In addition, the strategy significantly improved the anti-thrombotic, anti-inflammatory, endothelialization, and anti-calcification properties of the BHV material. It is expected to provide a new idea and a solution for the intelligent biomedical design of Glut cross-linked BHV material.

Genetic knockout of porcine GGTA1 or CMAH/GGTA1 is associated with the emergence of neo-glycans.

Pig-derived tissues could overcome the shortage of human donor organs in transplantation. However, the glycans with terminal α-Gal and Neu5Gc, which are synthesized by enzymes, encoded by the genes GGTA1 and CMAH, are known to play a major role in immunogenicity of porcine tissue, ultimately leading to xenograft rejection. The N-glycome and glycosphingolipidome of native and decellularized porcine pericardia from wildtype (WT), GGTA1-KO and GGTA1/CMAH-KO pigs were analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection. We identified biantennary and core-fucosylated N-glycans terminating with immunogenic α-Gal- and α-Gal-/Neu5Gc-epitopes on pericardium of WT pigs that were absent in GGTA1 and GGTA1/CMAH-KO pigs, respectively. Levels of N-glycans terminating with galactose bound in β(1-4)-linkage to N-acetylglucosamine and their derivatives elongated by Neu5Ac were increased in both KO groups. N-glycans capped with Neu5Gc were increased in GGTA1-KO pigs compared to WT, but were not detected in GGTA1/CMAH-KO pigs. Similarly, the ganglioside Neu5Gc-GM3 was found in WT and GGTA1-KO but not in GGTA1/CMAH-KO pigs. The applied detergent based decellularization efficiently removed GSL glycans. Genetic deletion of GGTA1 or GGTA1/CMAH removes specific epitopes providing a more human-like glycosylation pattern, but at the same time changes distribution and levels of other porcine glycans that are potentially immunogenic.

FABP6 Expression Correlates with Immune Infiltration and Immunogenicity in Colorectal Cancer Cells.

Background Immune checkpoint inhibitors (ICIs) have rapidly revolutionized colorectal cancer (CRC) treatment, but resistance caused by the heterogeneous tumor microenvironment (TME) still presents a challenge. Therefore, it is necessary to characterize TME immune infiltration and explore new targets to improve immunotherapy. Methods The compositions of 64 types of infiltrating immune cells and their relationships with CRC patient clinical characteristics were assessed. Differentially expressed genes (DEGs) between “hot” and “cold” tumors were used for functional analysis. A prediction model was constructed to explore the survival of CRC patients treated with and without immunotherapy. Finally, fatty acid-binding protein (FABP6) was selected for in vitro experiments, which revealed its roles in the proliferation, apoptosis, migration, and immunogenicity of CRC tissues and cell lines. Results The infiltration levels of several immune cells were associated with CRC tumor stage and prognosis. Different cell types showed the synergistic or antagonism infiltration patterns. Enrichment analysis of DEGs revealed that immune-related signaling was significantly activated in hot tumors, while metabolic process pathways were altered in cold tumors. In addition, the constructed model effectively predicted the survival of CRC patients treated with and without immunotherapy. FABP6 knockdown did not significantly alter tumor cell proliferation, apoptosis, and migration. FABP6 was negatively correlated with immune infiltration, and knockdown of FABP6 increased major histocompatibility complex (MHC) class 1 expression and promoted immune-related chemokine secretion, indicating the immunogenicity enhancement of tumor cells. Finally, knockdown of FABP6 could promote the recruitment of CD8+ T cells. Conclusion Collectively, we described the landscape of immune infiltration in CRC and identified FABP6 as a potential immunotherapeutic target for treatment.

Split bullets loaded nanoparticles for amplified immunotherapy

Dendritic cells (DCs) play central role in adaptive antitumor immunity, while their function is often hampered by low immunogenicity of tumor tissues and surrounding hostile microenvironment. Herein, a "split bullets" loaded nanoplatform that can bidirectionally injure mitochondria (MT) and endoplasmic reticulum (ER) of tumor cells is developed. After cellular uptake, the released "split bullets" separately target to different subcellular destinations and exert distinct effects on DCs: (1) MT-targeted "bullet" recruits peripheral DCs into tumor sites, due to its capability to trigger adenosine triphosphate release from tumor cells; (2) ER-targeted "bullet" activates tumor-infiltrating DCs, which is attributed to its ability to evoke calreticulin exposure on tumor cells. These effects collectively improve the tropism and reactivity of DCs to tumor-specific antigen in a two-pronged way. As a result of enhanced function of DCs in antigen capture, treatment of the "split bullets" loaded nanoplatform ignites robust immune response to suppress primary melanoma, and establishes systemic immune memory against post-surgical tumor recurrence. Overall, this nanoplatform offers a generalizable approach to boost DCs and augment immunotherapy.

Subcutaneous surgical rat models for the evaluation of decellularised heart valve immunogenicity: A systematic review

BackgroundTissue engineered heart valves are being designed as alternative valvular conduits to standard bioprostheses with the potential to meet the requirements of an ideal valvular prosthesis: non-thrombogenicity, non-immunogenicity, long-lasting, and capability of growth, repair, and remodelling upon implantation. Assessments of immunogenicity are required before in vivo testing in large animals may be undertaken. Rats are a cost-effective model that are widely used for such assessments. However, it is unclear whether the surgical rat models used for examining tissue immunogenicity are homogenous in their design. MethodsSystematic searches were conducted in the PubMed, Scopus, and Web of Science databases for articles in which the immunogenicity of decellularised heart valves was examined using subcutaneous surgical rat models. Assessments were made of the animal, surgical, and donor scaffold characteristics used in such models. The heterogeneity of the various characteristics was then evaluated qualitatively. ResultsIn total, 54 articles were qualitatively assessed in this systematic review. The donor scaffold characteristics were homogenous. Whereas, the animal and surgical characteristics were heterogenous and infrequently reported. The collective data suggest that an agreed subcutaneous rat model might consist of implanting four, 1 cm2, decellularised heart valve samples in the dorsal aspect of 6-week-old, Sprague Dawley, rats. ConclusionsRat subcutaneous implantation may be undertaken to assess the immunogenicity of decellularised heart valves before proceeding to in vivo studies in large animal models. There is significant heterogeneity in the characteristics of the subcutaneous rat models that have been used, to date. Most studies insufficiently report the techniques used.

Recovery of Recombinant Avian Paramyxovirus Type-3 Strain Wisconsin by Reverse Genetics and Its Evaluation as a Vaccine Vector for Chickens.

A reverse genetic system for avian paramyxovirus type-3 (APMV-3) strain Wisconsin was created and the infectious virus was recovered from a plasmid-based viral antigenomic cDNA. Green fluorescent protein (GFP) gene was cloned into the recombinant APMV-3 genome as a foreign gene. Stable expression of GFP by the recovered virus was confirmed for at least 10 consecutive passages. APMV-3 strain Wisconsin was evaluated against APMV-3 strain Netherlands and APMV-1 strain LaSota as a vaccine vector. The three viral vectors expressing GFP as a foreign protein were compared for level of GFP expression level, growth rate in chicken embryo fibroblast (DF-1) cells, and tissue distribution and immunogenicity in specific pathogen-free (SPF) day-old chickens. APMV-3 strain Netherlands showed highest growth rate and GFP expression level among the three APMV vectors in vitro. APMV-3 strain Wisconsin and APMV-1 strain LaSota vectors were mainly confined to the trachea after vaccination of day-old SPF chickens without any observable pathogenicity, whereas APMV-3 strain Netherlands showed wide tissue distribution in different body organs (brain, lungs, trachea, and spleen) with mild observable pathogenicity. In terms of immunogenicity, both APMV-3 strain-vaccinated groups showed HI titers two to three fold higher than that induced by APMV-1 strain LaSota vaccinated group. This study offers a novel paramyxovirus vector (APMV-3 strain Wisconsin) which can be used safely for vaccination of young chickens as an alternative for APMV-1 strain LaSota vector.

Models of Immunogenicity in Preclinical Assessment of Tissue Engineered Heart Valves

Tissue engineered heart valves offer an exciting alternative to traditional valve prostheses. Methods of construction vary, from decellularised animal tissue to synthetic hydrogels, but the goal is the same: the creation of a ‘living valve’ populated with autologous cells that may persist indefinitely upon implantation. Previous failed attempts in humans have highlighted the difficulty in predicting how a novel heart valve will perform in vivo. The final hurdle in bringing these prostheses to market is understanding the immune reaction in the short and long term. In particular, the chronic remodelling of a tissue engineered implant by macrophages remains poorly understood. No silver bullet exists, rather researchers must draw upon a number of in vitro and in vivo models to fully elucidate the effect a host will exert on the graft. This review details the methods by which the immunogenicity of tissue engineered heart valves may be investigated and reveals areas that would benefit from more research.

Subcutaneous Surgical Rat Models for the Evaluation of Tissue-Engineered Heart Valve Immunogenicity: A Systematic Review

Background: Tissue engineered heart valves are being designed as alternative valvular conduits to standard bioprostheses with the potential to meet the requirements of an ideal valvular prosthesis: non-thrombogenicity, non-immunogenicity, long-lasting, and capability of growth, repair, and remodelling upon implantation. Assessments of immunogenicity are required before in vivo testing in large animals may be undertaken. Rats are a cost-effective model that are widely used for such assessments. However, it is unclear whether the surgical rat models used for examining tissue immunogenicity are homogenous in their design. Methods: Systematic searches were conducted in the PubMed, Scopus, and Web of Science databases for articles in which the immunogenicity of tissue engineered heart valves was examined using subcutaneous surgical rat models. Assessments were made of the animal, surgical, and donor scaffold characteristics used in such models. The heterogeneity of the various characteristics was then evaluated qualitatively. Results: In total, 54 articles were qualitatively assessed in this systematic review. The donor scaffold characteristics were homogenous. Whereas, the animal and surgical characteristics were heterogenous and infrequently reported. The collective data suggest that an agreed subcutaneous rat model might consist of implanting four, 1 cm2, tissue engineered heart valve samples in the dorsal aspect of 6-week-old, Sprague Dawley, rats. Conclusions: Rat subcutaneous implantation may be undertaken to assess the immunogenicity of tissue engineered heart valves before proceeding to in vivo studies in large animal models. There is significant heterogeneity in the characteristics of the subcutaneous rat models that have been used, to date. Most studies insufficiently report the techniques used. Funding Information: This work was supported by funding gratefully received from the Green Lane Research and Education Fund (Greenlane Research and Education Fund, Grafton, Auckland, New Zealand), and the National Heart Foundation of New Zealand (National Heart Foundation of New Zealand, Ellerslie, Auckland, New Zealand). S.W. received a postgraduate scholarship from the National Heart Foundation of New Zealand, M.G. received and Green Lane Research and Education Fund Postgraduate Scholarship, and C.A. received a University of Auckland summer research scholarship. Declaration of Interests: The authors declare that they have no conflicts of interest related to this work.

A histological study of mouse tissues and water loss following lyophilization

ABSTRACTLyophilization is a practical method for product storage and transportation; it commonly is used in the food and pharmaceutical industries. Lyophilization also is used for preserving biological samples such as serum, plasma and animal tissues. We found that lyophilization does not affect the stability of RNAs and proteins in tissue samples. To investigate histological characteristics, we prepared lyophilized tissues for paraffin sectioning and hematoxylin and eosin (H & E) staining. We also measured water loss from organs during lyophilization. We used immunohistochemistry of frozen brain sections to identify potential protective effects of three concentrations of sucrose, glucose and trehalose against the effects of lyophilization. H & E staining revealed vacuoles in heart, lung, liver, kidney, spleen and brain after lyophilization without pretreatments, especially heart and kidney. We found that 10% solutions of sucrose, glucose and trehalose helped preserve tissue morphology. Immunohistochemistry of frozen brain tissue showed that 10% glucose and 30% sucrose preserved cellular characteristics and immunogenicity following lyophilization. Lyophilization removed > 70% of the water from organs, and lyophilized tissues without protectants were not suitable for histological study. Overall, we found that 10% glucose helped preserve both optimal tissue morphology and immunogenicity of freeze-dried tissue.

γδ T Cell Update: Adaptate Orchestrators of Immune Surveillance.

As interest in γδ T cells grows rapidly, what key points are emerging, and where is caution warranted? γδ T cells fulfill critical functions, as reflected in associations with vaccine responsiveness and cancer survival in humans and ever more phenotypes of γδ T cell-deficient mice, including basic physiological deficiencies. Such phenotypes reflect activities of distinct γδ T cell subsets, whose origins offer interesting insights into lymphocyte development but whose variable evolutionary conservation can obfuscate translation of knowledge from mice to humans. By contrast, an emerging and conserved feature of γδ T cells is their "adaptate" biology: an integration of adaptive clonally-restricted specificities, innate tissue-sensing, and unconventional recall responses that collectively strengthen host resistance to myriad challenges. Central to adaptate biology are butyrophilins and other γδ cell regulators, the study of which should greatly enhance our understanding of tissue immunogenicity and immunosurveillance and guide intensifying clinical interest in γδ cells and other unconventional lymphocytes.

Composite tissue allotransplantation: opportunities and challenges.

Vascularized composite allotransplants (VCAs) have unique properties because of diverse tissue components transplanted en mass as a single unit. In addition to surgery, this type of transplant also faces enormous immunological challenges that demand a detailed analysis of all aspects of alloimmune responses, organ preservation, and injury, as well as the immunogenicity of various tissues within the VCA grafts to further improve graft and patient outcomes. Moreover, the side effects of long-term immunosuppression for VCA patients need to be carefully balanced with the potential benefit of a non-life-saving procedure. In this review article, we provide a comprehensive update on limb and face transplantation, with a specific emphasis on the alloimmune responses to VCA, established and novel immunosuppressive treatments, and patient outcomes.

Generation of GGTA1 <sup>-/-</sup>β2M <sup>-/-</sup>CIITA <sup>-/-</sup> Pigs Using CRISPR/Cas9 Technology to Alleviate Xenogeneic Immune Reactions

Background: The generation of glycoprotein galactosyltransferase alpha 1, 3 (GGTA1) knockout pigs has greatly contributed to the reduction of hyperacute xenograft rejection. However, severe xenograft rejection can still be induced by xenoimmune responses to the porcine major histocompatibility complex (MHC) antigens SLA-I and SLA-II. Methods: We simultaneously depleted GGTA1, β 2M, and CIITA genes using CRISPR-Cas9 technology to generate GGTA1-/-β2M-/-CIITA-/- triple knockout (GBC-3KO) pigs. Mixed lymphocyte reaction (MLR) and BrdU incorporating assays were utilized to quantify the potency of pig cells to stimulate human T cell responses. Skin grafts were determined the immunogenicity of pig tissues in xenogeneic hosts in vivo. Findings: The genotype of GBC-3KO pigs was confirmed by PCR and Sanger sequencing, and the loss of expression of α-Gal, SLA-I, and SLA-II was demonstrated by flow cytometric analysis. Mixed lymphocyte reaction (MLR) assay revealed that splenocytes from GBC-3KO pigs were significantly less effective than WT pig splenocytes in inducing human CD3+CD4+ and CD3+CD8+ T cell activation (measured by CD25 expression) and proliferation. Furthermore, GBC-3KO pig skin grafts showed a significantly prolonged survival (with a median survival time (MST) of 16 days) in immunocompetent C57BL/6 mice, when compared to WT pig skin grafts (MST = 13.5 days). Interpretation: These results demonstrate that elimination of GGTA1, β2M, and CIITA genes in pigs can effectively alleviate xenogeneic immune responses. Funding Statement: This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16030000 and XDA16030303), the National Key Research and Development Program (2017YFA0104400, 2016YFA0100202 and 2017YFA0104402), the National Natural Science Foundation of China (31621004, 81570145, 81870091), and the Key Research Projects of the Frontier Science of the Chinese Academy of Sciences (QYZDB-SSW-SMC022). Declaration of Interests: The authors stated: None. Ethics Approval Statement: Bama miniature pigs were raised and cloned at the Beijing Farm Animal Research Center. Human blood was obtained from healthy volunteers. Protocols related to the use of animals and human tissues were approved by the Institutional Review Board and Institutional Animal Care and Use Committee of the First Hospital of Jilin University and the Institute of Zoology of Chinese Academy of Sciences. All the experiments were performed in accordance with the protocols.

Impact of VS83 and its components upon tissue immunogenicity

Impact of VS83 and its components upon tissue immunogenicity

Histological and Mechanical Assessment of Decellularized Porcine Biografts, and Its Biological Evaluation following Aortic Implantation during Mid-Term Follow-Up

Aims: Prosthetic graft infection frequently requires graft replacement. Among other options, a biological graft could serve as an alternative choice. Decellularization reduces tissue immunogenicity. Our aim was to determine an efficient decellularization method and to evaluate the decellularized porcine biografts’ adaptability. Methods: Four different protocols were implemented to decellularize porcine aortic segments (n = 4). Cell removal effectiveness and matrix structure preservation were histologically examined. Mechanical tests were performed. Decellularized porcine grafts were interpositioned in a porcine aorta. After a 6-month period, implanted samples were removed and evaluated using light and electron microscopy. Results: Histological results showed complete removal of cells and preserved connective tissue fiber structure following decellularization, using sodium dodecyl sulfate and sodium azide. Pressure tests demonstrated similar compliance to fresh vessels. In 9 out of 10 cases, pigs survived the follow-up period. Graft rejection, intimal hyperplasia, reocclusion and/or aneurysm formation were not observed. Presence of host cells and neoendothelialization were microscopically confirmed. Conclusions: This decellularization protocol enables a cost-effective preparation of biological grafts featuring reduced immunogenicity. The implanted grafts did not degenerate during the 6-month follow-up period, the lack of graft rejection suggests acceptable immunological tolerance, while recipient cells migrate into, proliferate and differentiate, thus creating the possibility for further use as an optional vascular graft.

The Neglected Villain of Bioprosthetic Degeneration: Inflammatory and Immune Processes.

In an attempt to avoid the destructive process of bioprosthetic heart-valve calcification associated with the use of glutaraldehyde, valves are today prepared using low concentrations of the crosslinking reagent. In this review, we summarize our findings and those of others that confirm that the immunogenicity of such tissue is not sufficiently masked and that a defined humoral response is indeed mounted against a repertoire of antigens unrelated to those associated with vascularized and non—cross-linked xenograft organs. We demonstrate the need for increased cross-linking of tissue to satisfactorily mitigate that response; furthermore, we examine the impact of increased cross-link density on the macrophage as antigen presenting cell with respect to its involvement in both tissue erosion and pannus overgrowth. Finally we present evidence for a role of circulating antibodies in bioprosthesis calcification.

Beneath the sword of Damocles: regenerative medicine and the shadow of immunogenicity.

Few topics in regenerative medicine have inspired such impassioned debate as the immunogenicity of cell types and tissues differentiated from pluripotent stem cells. While early predictions suggested that tissues derived from allogeneic sources may evade immune surveillance altogether, the pendulum has since swung to the opposite extreme, with reports that the ectopic expression of a few developmental antigens may prompt rejection, even of tissues differentiated from autologous cell lines. Here we review the evidence on which these contradictory claims are based in order to reach an objective assessment of the likely magnitude of the immunological challenges ahead. Furthermore, we discuss how the inherent properties of pluripotent stem cells may inform strategies for reducing the impact of immunogenicity on the future ambitions of regenerative medicine.

Taming the lion: the challenge of immunity in regenerative medicine.

Taming the lion: the challenge of immunity in regenerative medicine.

A future with less HLA: potential clinical applications of HLA-universal cells.

The high variability of the human leukocyte antigen (HLA) remains a major obstacle to the application of allogeneic products in cell-based therapies. We have developed a strategy to decrease the immunogenicity of cell and tissues to improve their survival after allogeneic transplantation in the absence of immunosuppression. Using RNA interference technology, the expression of HLA class I and II was stably downregulated. HLA-silenced cells demonstrated to prevent a de novo and escape a pre-formed alloimmune response in vitro and in vivo. Also, they demonstrated to be capable of engraft and survive after allogeneic transplantation independently of the donor's and recipient's genetic background. The generation of HLA-universal cells has may open new horizons in the field of regenerative medicine. Some of the potential clinical applications of HLA universal cells will be discussed in this review.