RESUMEN
Glycans, chains of sugar molecules found conjugated to cell proteins and lipids, contribute to their growth, movement and differentiation. Aberrant glycosylation is a hallmark of several medical conditions including tumorigenesis. Glycosphingolipids (GSLs), consisting of glycans conjugated to a lipid (ceramide) core, are found in the lipid bilayer of eukaryotic cell membranes. GSLs, play an active role in cell processes. Several GSLs are expressed by human embryonic stem cells and have been found to be overexpressed in several types of cancer. In this review, we discuss the data, hypotheses and perspectives related to the GSLs Globo H and SSEA-4.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/fisiología , Neoplasias/etiología , Antígenos Embrionarios Específico de Estadio/fisiología , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Vacunas contra el Cáncer/uso terapéutico , Desarrollo Embrionario , Glicoconjugados/fisiología , Glicoesfingolípidos/antagonistas & inhibidores , Glicoesfingolípidos/fisiología , Humanos , Neoplasias/inmunología , Neoplasias/terapia , Antígenos Embrionarios Específico de Estadio/inmunologíaRESUMEN
Pancreatic cancer is usually asymptomatic in the early stages; the 5-y survival rate is around 9%; and there is a lack of effective treatment. Here we show that SSEA-4 is more expressed in all pancreatic cancer cell lines examined but not detectable in normal pancreatic cells; and high expression of SSEA-4 or the key enzymes B3GALT5 + ST3GAL2 associated with SSEA-4 biosynthesis significantly lowers the overall survival rate. To evaluate potential new treatments for pancreatic cancer, homogeneous antibodies with a well-defined Fc glycan for optimal effector functions and CAR-T cells with scFv construct designed to target SSEA-4 were shown highly effective against pancreatic cancer in vitro and in vivo. This was further supported by the finding that a subpopulation of natural killer (NK) cells isolated by the homogeneous antibody exhibited enhancement in cancer-cell killing activity compared to the unseparated NK cells. These results indicate that targeting SSEA-4 by homologous antibodies or CAR-T strategies can effectively inhibit cancer growth, suggesting SSEA-4 as a potential immunotherapy target for treating pancreatic disease.
Asunto(s)
Anticuerpos/inmunología , Neoplasias Pancreáticas/tratamiento farmacológico , Antígenos Embrionarios Específico de Estadio/inmunología , Animales , Línea Celular Tumoral , Tratamiento Basado en Trasplante de Células y Tejidos , Regulación de la Expresión Génica , Humanos , Inmunoterapia , Inmunoterapia Adoptiva , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The globo-series glycosphingolipids (SSEA3, SSEA4, and Globo H) were shown to express in many cancers selectively, and a combination of anti-SSEA4 and anti-Globo H antibodies was able to suppress tumor growth in mice inoculated with breast cancer cell lines. To further understand the effect, we focused on the combined effect of the two antibodies in target binding and antibody-dependent cellular cytotoxicity (ADCC) in vitro. Here, we report that the binding of anti-Globo H antibody (VK9) to MDA-MB231 breast cancer cells was influenced by anti-SSEA4 antibody (MC813-70), and a combination of both antibodies induced a similar effect as did anti-SSEA4 antibodies alone in a reporter-based ADCC assay, indicating that SSEA4 is a major target in breast cancer due to its higher expression than Globo H. Furthermore, we showed that a homogeneous anti-SSEA4 antibody (chMC813-70-SCT) designed to maximize the ADCC activity can be used to isolate a subpopulation of natural killer (NK) cells that exhibit an â¼23% increase in killing the target cells as compared to the unseparated NK cells. These findings can be used to predict a therapy outcome based on the expression levels of antigens and evaluate therapeutic antibody development.
Asunto(s)
Anticuerpos/inmunología , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Neoplasias de la Mama/metabolismo , Antígenos Embrionarios Específico de Estadio/inmunología , Animales , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Línea Celular Tumoral , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Ratones , Receptores de IgG/metabolismo , Antígenos Embrionarios Específico de Estadio/metabolismoRESUMEN
The stage-specific embryonic antigen-4 (SSEA-4) is a cell surface glycosphingolipid antigen expressed in early stages of human development. This surface marker is downregulated during the differentiation process but is found re-expressed in several types of tumors, including breast cancer. This feature makes SSEA-4 an attractive target for the development of therapeutic antibodies against tumors. In this work, we first studied the binding and intracellular fate of the monoclonal antibody MC-813-70 directed against SSEA-4. MC-813-70 was found to be rapidly internalized into triple-negative breast cancer cells following binding to its target at the plasma membrane, and to accumulate in acidic organelles, most likely lysosomes. Given the internalization feature of MC-813-70, we next tested whether the antibody was able to selectively deliver the saporin toxin inside SSEA-4-expressing cells. Results show that the immunotoxin complex was properly endocytosed and able to reduce cell viability of breast cancer cells in vitro, either alone or in combination with chemotherapeutic drugs. Our findings indicate that the MC-813-70 antibody has the potential to be developed as an alternative targeted therapeutic agent for cancer cells expressing the SSEA-4 glycolipid.
Asunto(s)
Inmunotoxinas/farmacología , Saporinas/farmacología , Antígenos Embrionarios Específico de Estadio/inmunología , Neoplasias de la Mama Triple Negativas/inmunología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/inmunología , Adenocarcinoma/metabolismo , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Endocitosis/efectos de los fármacos , Femenino , Humanos , Inmunotoxinas/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Saporinas/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
One of the important questions when studying established cancer cell lines is whether such cells contain a subpopulation of primitive cancer stem cells that maintains the expansion of the cell line. To address this issue, we performed studies on the established human embryonal carcinoma cell line NTera2 by evaluating the potential stemness of cells sorted according to their expression of the cell surface stem cell markers CD133 and SSEA4. By performing in vitro and in vivo assays, we observed different properties of cells expressing both, one, or neither of these antigens. While sorted SSEA4+ subpopulations exhibited the greatest propensity for migration toward normal serum and the highest seeding efficiency in the lungs of immunodeficient mice, CD133-SSEA4- cells displayed high seeding efficiency to the bone marrow after injection in vivo. It is worth noting that these properties did not depend on the size of the evaluated cells. To address the question of whether cancer stem cell phenotypes in cell lines are fixed or fluctuating, we sorted single cells according to their expression of CD133 and SSEA4 antigens and observed that cells which did not express these cancer stem cell markers gave rise to cells that express these markers after expansion in vitro. Therefore, our results support the idea that within established cancer cell lines, the phenotype of the cell subpopulation expressing cancer stem cell markers is not fixed but fluctuates during cell line expansion, and cells negative for these markers may acquire their expression.
Asunto(s)
Antígeno AC133/genética , Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión Génica , Células Madre Neoplásicas/metabolismo , Fenotipo , Células Madre Pluripotentes/metabolismo , Antígenos Embrionarios Específico de Estadio/genética , Antígeno AC133/inmunología , Antígeno AC133/metabolismo , Animales , Biomarcadores de Tumor/inmunología , Biomarcadores de Tumor/metabolismo , Médula Ósea/inmunología , Médula Ósea/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Embrión de Mamíferos , Citometría de Flujo , Perfilación de la Expresión Génica , Impresión Genómica , Humanos , Pulmón/inmunología , Pulmón/patología , Ratones , Ratones SCID , Células Madre Neoplásicas/inmunología , Células Madre Neoplásicas/patología , Células Madre Pluripotentes/inmunología , Células Madre Pluripotentes/patología , Antígenos Embrionarios Específico de Estadio/inmunología , Antígenos Embrionarios Específico de Estadio/metabolismoRESUMEN
Human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide a powerful model system for studies of cellular identity and early mammalian development, which hold great promise for regenerative medicine. It is necessary to develop a convenient method to discriminate hPSCs from other cells in clinics and basic research. Herein, a simple and reliable biosensor for stem cell detection was established. In this biosensor system, stage-specific embryonic antigen-3 (SSEA-3) and stage-specific embryonic antigen-4 (SSEA-4) were used to mark human pluripotent stem cells (hPSCs). Antibody specific for SSEA-3 was coated onto magnetic beads for hPSCs enrichment, and antibody specific for SSEA-4 was conjugated with carboxyl-modified tDNA sequence which was used as template for strand displacement amplification (SDA). The amplified single strand DNA (ssDNA) was detected with a lateral flow biosensor (LFB). This biosensor is capable of detecting a minimum of 19 human embryonic stem cells by a strip reader and 100 human embryonic stem cells by the naked eye within 80min. This approach has also shown excellent specificity to distinguish hPSCs from other types of cells, showing that it is promising for specific and handy detection of human pluripotent stem cells.
Asunto(s)
Técnicas Biosensibles/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Células Madre Pluripotentes/citología , Anticuerpos Inmovilizados/química , Anticuerpos Inmovilizados/inmunología , Antígenos de Carbohidratos Asociados a Tumores/química , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Técnicas Biosensibles/instrumentación , Técnicas de Cultivo de Célula , ADN de Cadena Simple/análisis , Diseño de Equipo , Oro/química , Humanos , Nanopartículas del Metal/química , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Células Madre Pluripotentes/inmunología , Sensibilidad y Especificidad , Antígenos Embrionarios Específico de Estadio/química , Antígenos Embrionarios Específico de Estadio/inmunologíaRESUMEN
BACKGROUND: "Weak P" is a rare red blood cell (RBC) phenotype, characterized by a global decrease in P(k) and P antigens. We now describe a second weak P individual who also typed LKE-negative (LKE-N) and possessed a clinically significant anti-LKE. STUDY DESIGN AND METHODS: Patient RBCs and plasma were examined by standard serology and flow cytometry. Glycosphingolipids (GSLs) from patient, P(k) , and LKE-strong (LKE-S) RBCs were isolated and analyzed by high-performance thin-layer chromatography (HPTLC). To confirm antibody specificity, patient serum and 30 human polyclonal controls, including alloanti-P and anti-PP1 P(k) , were tested against a panel of GSLs by HPTLC immunostaining. RESULTS: The patient typed P1 +, P+, and LKE-N and possessed a "P-like" panagglutinin. In a two-stage indirect antiglobulin test, the patient's plasma caused hemolysis of LKE-S cells but not p, P(k) , or LKE-N cells. Clinically, transfusion of P+ RBCs compatible by a prewarmed technique had shortened RBC survival with laboratory evidence of hemolysis. Analysis of the patient's isolated RBC GSLs showed a 30% relative decrease in Gb3 (P(k) ) and Gb4 (P) and a 90% decrease in monosialogalactosylgloboside (MSGG, LKE), accompanied by increased lactosylceramide (CDH), paragloboside, and GM3. On HPTLC immunostaining, the patient's plasma strongly bound MSSG with weak binding to galactosylgloboside (Gb5). Binding to MSGG, Gb5, and Gb4 was also observed with some examples of alloanti-P from P(k) individuals, but not anti-PP1 P(k) , autoanti-P, or normal controls. CONCLUSIONS: We describe the first example of a clinically significant anti-LKE in the setting of a rare weak P background. Human alloanti-LKE and some alloanti-P recognized Gb5 and MSGG.
Asunto(s)
Anemia Hemolítica Autoinmune/sangre , Globósidos/inmunología , Glicoesfingolípidos/inmunología , Isoanticuerpos/inmunología , Sistema del Grupo Sanguíneo P/inmunología , Antígenos Embrionarios Específico de Estadio/inmunología , Anemia Hemolítica Autoinmune/diagnóstico , Anemia Hemolítica Autoinmune/genética , Anemia Hemolítica Autoinmune/inmunología , Especificidad de Anticuerpos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Tipificación y Pruebas Cruzadas Sanguíneas/métodos , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Prueba de Coombs , Transfusión de Eritrocitos , Resultado Fatal , Humanos , Linfoma de Células B Grandes Difuso/complicaciones , Linfoma de Células B Grandes Difuso/diagnóstico , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Fenotipo , Antígenos Embrionarios Específico de Estadio/química , Reacción a la Transfusión/etiologíaRESUMEN
OBJECTIVES: It is well accepted that stage-specific embryonic antigen (SSEA)-4 is an antigen that is useful to isolate adult stem cells analogous to embryonic stem cells. Therefore, in the present study, we investigated whether SSEA-4 can also be used as a marker to identify human deciduous dental pulp (D-DP) stem cells. MATERIALS AND METHODS: Intact deciduous teeth were collected from healthy patients who were undergoing orthodontic treatment at Okayama University Hospital. Immunofluorescence analysis, flow cytometric analysis, and multilineage differentiation assay were performed to characterize SSEA-4+ D-DP cells. RESULTS: The D-DP cells had the characteristics of mesenchymal stem cells (MSCs), namely plastic adherence, specific surface antigen expression, and multipotent differentiation potential. SSEA-4 expression was detected in D-DP cells in vitro and ex vivo samples. A flow cytometric analysis demonstrated that 21.2 % of the D-DP cells were positive for SSEA-4. The SSEA-4+ clonal D-DP cells showed multilineage differentiation potential toward adipocytes, osteoblasts, and chondrocytes in vitro. In fact, 26.1 % (6/23) of the SSEA-4+ clonal D-DP cells showed adipogenic potential, 91.3 % (21/23) showed osteogenic potential, 91.3 % (21/23) showed chondrogenic potential, and 87.0 % (20/23) showed both osteogenic and chondrogenic potential. CONCLUSIONS: Thus, the majority of SSEA-4+ D-DP cells had the potential for multilineage differentiation. Hence, SSEA-4 appears to be a specific marker that can be used to identify D-DP stem cells. CLINICAL RELEVANCE: SSEA-4+ D-DP cells appear to be a promising source of stem cells for regenerative therapy.
Asunto(s)
Pulpa Dental/inmunología , Antígenos Embrionarios Específico de Estadio/inmunología , Diente Primario/inmunología , Adolescente , Niño , Pulpa Dental/citología , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/inmunología , Diente Primario/citologíaRESUMEN
Globo-series glycans are human cell-surface carbohydrates that include stem-cell marker SSEA-4 and cancer-cell antigen Globo H. These two hexasaccharides differ only in their terminal saccharide: N-acetylneuraminic acid in SSEA-4 and L-fucose in Globo H. Herein, we evaluated the affinity of the monoclonal antibodies α-SSEA-4 and α-GH for the glycans SSEA-4 and Globo H. Using fluorescence polarization, we find that the two monoclonal antibodies have affinity for their cognate glycan in the low nanomolar range, and have negligible affinity for the non-cognate glycan. Using surface plasmon resonance, we find that each cognate affinity is â¼20-fold greater if the glycan is immobilized on a surface rather than free in solution. We conclude that the terminal saccharide plays a dominant role in the ability of monoclonal antibodies to recognize these Globo-series glycans and that the extraordinary specificity of these antibodies supports their use for identifying and sorting stem-cells (α-SSEA-4) and as an agent in cancer immunotherapy (α-GH).
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Antígenos Embrionarios Específico de Estadio/inmunología , Especificidad de Anticuerpos , Antígenos de Carbohidratos Asociados a Tumores/química , Compuestos de Boro/química , Polarización de Fluorescencia , Humanos , Soluciones , Antígenos Embrionarios Específico de Estadio/química , Resonancia por Plasmón de SuperficieRESUMEN
Glioblastoma multiforme (GBM), the grade IV astrocytoma, is the most common and aggressive brain tumor in adults. Despite advances in medical management, the survival rate of GBM patients remains poor, suggesting that identification of GBM-specific targets for therapeutic development is urgently needed. Analysis of several glycan antigens on GBM cell lines revealed that eight of 11 GBM cell lines are positive for stage-specific embryonic antigen-4 (SSEA-4), and immunohistochemical staining confirmed that 38/55 (69%) of human GBM specimens, but not normal brain tissue, were SSEA-4(+) and correlated with high-grade astrocytoma. In addition, an SSEA-4-specific mAb was found to induce complement-dependent cytotoxicity against SSEA-4(hi) GBM cell lines in vitro and suppressed GBM tumor growth in mice. Because SSEA-4 is expressed on GBM and many other types of cancers, but not on normal cells, it could be a target for development of therapeutic antibodies and vaccines.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Glioblastoma/metabolismo , Antígenos Embrionarios Específico de Estadio/inmunología , Antígenos Embrionarios Específico de Estadio/metabolismo , Animales , Anticuerpos Monoclonales/administración & dosificación , Línea Celular Tumoral , Cromatografía en Capa Delgada , Citometría de Flujo , Glioblastoma/tratamiento farmacológico , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB CRESUMEN
Somatic cell nuclear reprogramming is opening new doors for the modeling of human disease phenotypes in vitro, the identification of novel therapeutic compounds and diagnostic factors as well as future autologous cell replacement therapies. Despite the potential that reprogramming technologies bring, there are remaining concerns preventing their broad application in the short-term. One of them is the safety concern associated with the use of stem cell derivatives, those generated by reprogramming or even when embryonic stem cells are employed. Here we summarize the current knowledge in the field of stem cells and reprogramming with a particular focus on the pitfalls preventing rapid translation of stem cell technologies into the clinic. We discuss the most recent findings on immunogenicity and tumorigenicity of reprogrammed cells. We additionally provide an overview on the potential applications that reprogramming approaches might bring to the immunological field and elaborate on the use of induced pluripotent stem cells (iPSCs) with pre-arranged immune receptors for the development of future immunotherapeutic approaches. The use of reprogramming approaches can represent and provide groundbreaking strategies previously unachievable for stem cell engineering aimed at modulating immune responses. In summary, we provide an overview on the different topics related to the use of stem cells and highlight the most provocative, yet perhaps currently underappreciated, aspect of combining immunological and reprogramming strategies for the treatment of human disease.
Asunto(s)
Células Madre Embrionarias/inmunología , Células Madre Pluripotentes Inducidas/inmunología , Neoplasias/inmunología , Complicaciones Posoperatorias/inmunología , Trasplante de Células Madre , Animales , Carcinogénesis/genética , Reprogramación Celular/inmunología , Terapia Genética , Humanos , Inmunidad/genética , Neoplasias/etiología , Neoplasias/prevención & control , Complicaciones Posoperatorias/prevención & control , Ingeniería de Proteínas , Receptores de Antígenos/genética , Receptores de Antígenos/metabolismo , Receptores de Reconocimiento de Patrones/genética , Receptores de Reconocimiento de Patrones/metabolismo , Antígenos Embrionarios Específico de Estadio/inmunologíaRESUMEN
A lateral flow biosensor based on immunoassay has been developed for the detection of human stem cells for the first time. Antibody specific for a human stem cell surface antigen, SSEA-4, is coated onto gold nanoparticles, whereas antibody against another human pluripotent stem cell surface antigen, SSEA-3, is immobilized on the test zone of the NC membrane. Target cells bind to the antibody coated on the gold nanoparticles to form nanoparticles-stem cell complexes, and the complexes are then captured by another antibody immobilized on the test zone to form a red line for visual detection. This biosensor has been successfully applied to human embryonic stem cells and induced pluripotent stem cells. It is capable of detecting a minimum of 10,000 human embryonic stem cells by the naked eye and 7000 cells with a portable strip reader within 20 min. This approach has also shown excellent specificity to distinguish other types of cells. The biosensor shows great promise for specific and handy detection of human pluripotent stem cells.
Asunto(s)
Técnicas Biosensibles/métodos , Células Madre Pluripotentes , Anticuerpos Inmovilizados/química , Anticuerpos Inmovilizados/inmunología , Antígenos de Carbohidratos Asociados a Tumores/química , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Técnicas Biosensibles/instrumentación , Células Madre Embrionarias/citología , Diseño de Equipo , Oro , Humanos , Inmunoglobulina G/química , Nanopartículas , Sensibilidad y Especificidad , Antígenos Embrionarios Específico de Estadio/química , Antígenos Embrionarios Específico de Estadio/inmunologíaRESUMEN
Globo H (GH) is a hexasaccharide specifically overexpressed on a variety of cancer cells and therefore, a good candidate for cancer vaccine development. To identify the optimal carrier and adjuvant combination, we chemically synthesized and linked GH to a carrier protein, including keyhole limpet hemocyanion, diphtheria toxoid cross-reactive material (CRM) 197 (DT), tetanus toxoid, and BSA, and combined with an adjuvant, and it was administered to mice for the study of immune response. Glycan microarray analysis of the antiserum obtained indicated that the combination of GH-DT adjuvanted with the α-galactosylceramide C34 has the highest enhancement of anti-GH IgG. Compared with the phase III clinical trial vaccine, GH-keyhole limpet hemocyanion/QS21, the GH-DT/C34 vaccine elicited more IgG antibodies, which are more selective for GH and the GH-related epitopes, stage-specific embryonic antigen 3 (SSEA3) and SSEA4, all of which were specifically overexpressed on breast cancer cells and breast cancer stem cells with SSEA4 at the highest level (>90%). We, therefore, further developed SSEA4-DT/C34 as a vaccine candidate, and after immunization, it was found that the elicited antibodies are also IgG-dominant and very specific for SSEA4.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/farmacología , Proteínas Bacterianas/inmunología , Neoplasias de la Mama/prevención & control , Vacunas contra el Cáncer/química , Antígenos Embrionarios Específico de Estadio/inmunología , Animales , Antígenos de Carbohidratos Asociados a Tumores/administración & dosificación , Antígenos de Carbohidratos Asociados a Tumores/química , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Neoplasias de la Mama/inmunología , Femenino , Citometría de Flujo , Hemocianinas , Sueros Inmunes/análisis , Inmunoglobulina G/inmunología , Ratones , Análisis por Micromatrices , Estructura Molecular , Células Madre Neoplásicas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Toxoide TetánicoRESUMEN
The potential to develop into any tissue makes pluripotent stem cells (PSCs) one of the most promising sources for cellular therapeutics. However, numerous hurdles exist to their clinical applications, three of the most concerning include the inability to separate therapeutic population from heterogeneously differentiated cultures, the risk of teratoma formation from residual pluripotent cells, and immunologic rejection of engrafted cells. The recent development of induced PSCs has been proposed as a solution to the histocompatibility barrier. Theoretically, creation of patient-specific induced PSC lines would exhibit a complete histocompatibility antigen match. However, regardless of the PSC source, in vitro propagation and nonphysiologic differentiation may result in other, likely less powerful, mechanisms of immune rejection. In light of recent progress towards clinical application, this review focuses on two such potential immunologic mechanisms applicable to isogenic PSC derivates: namely, the immunogenicity of aberrant antigens resulting from long-term in vitro maintenance and alterations in immunologic properties due to rapid in vitro differentiation. These issues will be considered with attention to their relation to effector cells in the adult immune system. In addition, we highlight immunosuppressive approaches that could potentially address the immunogenicity of these proposed mechanisms.