Anti-C1GALT1 Autoantibody Is a Novel Prognostic Biomarker for Patients With Head and Neck Cancer.

OBJECTIVES: The objective of this study is to determine the value of the anti- glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase 1 (C1GALT1) autoantibody as a biomarker for distant metastasis and good response to immune checkpoint inhibitors in patients with head and neck squamous cell carcinoma (HNSCC). METHODS: In this retrospective study with a median follow-up of 55.7 months, 186 HNSCC patients were enrolled between July 2013 and August 2014. Data were analyzed between April 2018 and November 2019. Titers of autoantibody against the C1GALT1 peptide were measured by ELISA. Student t test, Kaplan-Meier analysis, and univariate and multivariate Cox proportional hazard models were used to evaluate the association of anti-C1GALT1 autoantibody titer with clinicopathologic factors, survival, and response to immunotherapy. RESULTS: Our results showed that high levels of the anti-C1GALT1 autoantibody is an independent marker for distant metastasis and poor disease-specific survivals in HNSCC patients. In 19 recurrent or metastatic (R/M) HNSCC patients who have received nivolumab or pembrolizumab, higher autoantibody titers are associated with a better treatment response. CONCLUSION: We propose that the anti-C1GALT1 autoantibody can serve as a novel biomarker for distant metastasis in HNSCC patients. It is also useful in individualized medicine for R/M HNSCC patients who are considering immunotherapy. LEVEL OF EVIDENCE: IV Laryngoscope, 131:E196-E202, 2021.

A New Humanized Mouse Model Mimics Humans in Lacking α-Gal Epitopes and Secreting Anti-Gal Antibodies.

Mice have been used as accepted tools for investigating complex human diseases and new drug therapies because of their shared genetics and anatomical characteristics with humans. However, the tissues in mice are different from humans in that human cells have a natural mutation in the α1,3 galactosyltransferase (α1,3GT) gene and lack α-Gal epitopes on glycosylated proteins, whereas mice and other nonprimate mammals express this epitope. The lack of α-Gal epitopes in humans results in the loss of immune tolerance to this epitope and production of abundant natural anti-Gal Abs. These natural anti-Gal Abs can be used as an adjuvant to enhance processing of vaccine epitopes to APCs. However, wild-type mice and all existing humanized mouse models cannot be used to test the efficacy of vaccines expressing α-Gal epitopes because they express α-Gal epitopes and lack anti-Gal Abs. Therefore, in an effort to bridge the gap between the mouse models and humans, we developed a new humanized mouse model that mimics humans in that it lacks α-Gal epitopes and secretes human anti-Gal Abs. The new humanized mouse model (Hu-NSG/α-Galnull) is designed to be used for preclinical evaluations of viral and tumor vaccines based on α-Gal epitopes, human-specific immune responses, xenotransplantation studies, and in vivo biomaterials evaluation. To our knowledge, our new Hu-NSG/α-Galnull is the first available humanized mouse model with such features.

Evaluation of the host immune response to decellularized lung scaffolds derived from α-Gal knockout pigs in a non-human primate model.

Whole organ tissue engineering is a promising approach to address organ shortages in many applications, including lung transplantation for patients with chronic pulmonary disease. Engineered lungs may be derived from animal sources after removing cellular content, exposing the extracellular matrix to serve as a scaffold for recellularization with human cells. However, the use of xenogeneic tissue sources in human transplantation raises concerns due to the presence of the antigenic Gal epitope. In the present study, lungs from wild type or α-Gal knockout pigs were harvested, decellularized, and implanted subcutaneously in a non-human primate model to evaluate the host immune response. The decellularized porcine implants were compared to a sham surgery control, as well as native porcine and decellularized macaque lung implants. The results demonstrated differential profiles of circulating and infiltrating immune cell subsets and histological outcomes depending on the implanted tissue source. Upon implantation, the decellularized α-Gal knockout lung constructs performed similarly to the decellularized wild type lung constructs. However, upon re-implantation into a chronic exposure model, the decellularized wild type lung constructs resulted in a greater proportion of infiltrating CD45+ cells, including CD3+ and CD8+ cytotoxic T-cells, likely mediated by an increase in production of Gal-specific antibodies. The results suggest that removal of the Gal epitope can potentially reduce adverse inflammatory reactions associated with chronic exposure to engineered organs containing xenogeneic components.

A practical approach to pancreatic cancer immunotherapy using resected tumor lysate vaccines processed to express α-gal epitopes.

OBJECTIVES: Single-agent immunotherapy is ineffective against poorly immunogenic cancers, including pancreatic ductal adenocarcinoma (PDAC). The aims of this study were to demonstrate the feasibility of production of novel autologous tumor lysate vaccines from resected PDAC tumors, and verify vaccine safety and efficacy. METHODS: Fresh surgically resected tumors obtained from human patients were processed to enzymatically synthesize α-gal epitopes on the carbohydrate chains of membrane glycoproteins. Processed membranes were analyzed for the expression of α-gal epitopes and the binding of anti-Gal, and vaccine efficacy was assessed in vitro and in vivo. RESULTS: Effective synthesis of α-gal epitopes was demonstrated after processing of PDAC tumor lysates from 10 different patients, and tumor lysates readily bound an anti-Gal monoclonal antibody. α-gal(+) PDAC tumor lysate vaccines elicited strong antibody production against multiple tumor-associated antigens and activated multiple tumor-specific T cells. The lysate vaccines stimulated a robust immune response in animal models, resulting in tumor suppression and a significant improvement in survival without any adverse events. CONCLUSIONS: Our data suggest that α-gal(+) PDAC tumor lysate vaccination may be a practical and effective new immunotherapeutic approach for treating pancreatic cancer.

Microbial, metabolomic, and immunologic dynamics in a relapsing genetic mouse model of colitis induced by T-synthase deficiency.

Intestinal dysbiosis is thought to confer susceptibility to inflammatory bowel disease (IBD), but it is unknown whether dynamic changes in the microbiome contribute to fluctuations in disease activity. We explored this question using mice with intestine-specific deletion of C1galt1 (also known as T-synthase) (Tsyn mice). These mice develop spontaneous microbiota-dependent colitis with a remitting/relapsing course due to loss of mucin core-1 derived O-glycans. 16S rRNA sequencing and untargeted metabolomics demonstrated age-specific perturbations in the intestinal microbiome and metabolome of Tsyn mice compare with littermate controls at weeks 3 (disease onset), 5 (during remission), and 9 (after relapse). Colitis remission corresponded to increased levels of FoxP3+RORγt+CD4+ T cells in the colonic lamina propria that were positively correlated with operational taxonomic units (OTUs) in the S24-7 family and negatively correlated with OTUs in the Clostridiales order. Relapse was characterized by marked expansion of FoxP3-RORγt+CD4+ T cells expressing IFNγ and IL17A, which were associated with Clostridiales OTUs distinct from those negatively correlated with FoxP3+RORγt+CD4+ T cells. Our findings suggest that colitis remission and relapse in the Tsyn model may reflect alterations in the microbiome due to reduced core-1 O-glycosylation that shift the balance of regulatory and pro-inflammatory T cell subsets. We investigated whether genetic variation in C1galt1 correlated with the microbiome in a cohort of 78 Crohn's disease patients and 101 healthy controls. Polymorphisms near C1galt1 (rs10486157) and its molecular chaperone, Cosmc (rs4825729), were associated with altered composition of the colonic mucosal microbiota, supporting the relevance of core-1 O-glycosylation to host regulation of the microbiome.

The Immune Responses and Calcification of Bioprostheses in the α1,3-Galactosyltransferase Knockout Mouse.

BACKGROUND: The study aim was to evaluate the immune reaction, difference of degenerative calcification, and anti-calcification effect of decellularization with or without α-galactosidase in bovine pericardium and porcine heart valves, using an α1,3-galactosyltransferase (α-Gal) knockout (KO) mouse model. METHODS: In order to elucidate the anti-calcification effect of decellularization with or without α-galactosidase, bovine pericardium and porcine heart valve tissues were assigned to four groups according to the tissue preparation method: (i) glutaraldehyde (GA) fixation only; (ii) decellularization + GA fixation (Decell); (iii) α-galactosidase + GA fixation (α-galactosidase); and (iv) decellularization +α-galactosidase + GA fixation (Decell + α-galactosidase). Each prepared tissue was implanted subcutaneously into α-Gal KO mice. Anti-α-Gal immunoglobulin (Ig) G and IgM antibody titers were monitored prior to implantation and at four, eight and 12 weeks after implantation using an enzyme-linked immunosorbent assay. Calcium contents of explanted tissues were measured at 12 weeks after implantation. RESULTS: There were no significant differences in the anti-α-Gal IgG antibody titers according to the type of bioprosthetic material or tissue preparation method (p >0.05). The calcium content was significantly lower in porcine heart valves than in bovine pericardium when implanted in α-Gal-KO mice (p <0.001). Calcium contents in bovine pericardium and porcine heart valves were significantly lower in the Decell, α-galactosidase and Decell + α-galactosidase groups than in the GA group (all p <0.05). CONCLUSIONS: The porcine heart valve induced lower levels of calcium deposition than did the bovine pericardium, but the anti-α-Gal IgG antibody titers did not differ significantly between the bioprosthetic tissues. Decellularization had significant anticalcification effects in both the bovine pericardium and porcine heart valves, though there was no significant difference in the anti-α-Gal IgG antibody titers among tissue preparation methods.

Enhanced anti-tumor immunity against breast cancer induced by whole tumor cell vaccines genetically modified expressing α-Gal epitopes.

Whole tumor cell vaccines have shown much promise, but demonstrated poor efficiency in phase III trials. In this study, we modified MDA-MB­231 tumor cells (MDA-MB­231Gal+) to express α-1, 3-galactosyltransferase (α-1, 3-GT) protein, to potentially enhance antitumor effect of whole tumor cell vaccines. MDA-MB­231 tumor cell vaccines were transfected with a reconstructed lentiviral containing α-1, 3-GT genes. Tumor growth, tumorigenesis and survival of Hu-NOD-SCID mice were observed when tumor-bearing mice were injected with tumor cell vaccines. Proliferation and apoptosis in MDA-MB­231 tumor xenografts were observed by immunohistochemistry. The levels of cytokine secretion in the serum of mice were tested by ELISA. CD8+ T cells infiltrating tumors were assessed by flow cytometry. MDA-MB­231Gal+ cells expressed active α-1, 3-GT and produced α-Gal in vitro. MDA-MB­231Gal+ cell vaccines suppressed tumor growth and tumorigenesis in immunized Hu-NOD-SCID mice. Additionally, decrease of TGF-ß, IL-10 and increase of INF-γ, IL-12 were observed in tumor cell vaccinated mice. Furthermore, the cell vaccines enhanced infiltration of cytotoxic CD8+ T cells in the tumor microenvironment of immunized mice. The MDA-MB­231Gal+ cell vaccines modified α-1, 3-GT genes improved the antitumor effect.

Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft.

Preventing xenograft rejection is one of the greatest challenges of transplantation medicine. Here, we describe a reproducible, long-term survival of cardiac xenografts from alpha 1-3 galactosyltransferase gene knockout pigs, which express human complement regulatory protein CD46 and human thrombomodulin (GTKO.hCD46.hTBM), that were transplanted into baboons. Our immunomodulatory drug regimen includes induction with anti-thymocyte globulin and αCD20 antibody, followed by maintenance with mycophenolate mofetil and an intensively dosed αCD40 (2C10R4) antibody. Median (298 days) and longest (945 days) graft survival in five consecutive recipients using this regimen is significantly prolonged over our recently established survival benchmarks (180 and 500 days, respectively). Remarkably, the reduction of αCD40 antibody dose on day 100 or after 1 year resulted in recrudescence of anti-pig antibody and graft failure. In conclusion, genetic modifications (GTKO.hCD46.hTBM) combined with the treatment regimen tested here consistently prevent humoral rejection and systemic coagulation pathway dysregulation, sustaining long-term cardiac xenograft survival beyond 900 days.

Characteristics of α-Gal epitope, anti-Gal antibody, α1,3 galactosyltransferase and its clinical exploitation (Review).

The α-Gal epitope (Galα1,3Galα1,4GlcNAc­R) is ubiquitously presented in non-primate mammals, marsupials and New World Monkeys, but it is absent in humans, apes and Old World monkeys. However, the anti-Gal antibody (~1% of immunoglobulins) is naturally generated in human, and is found as the immunoglobulin G (IgG), IgM and IgA isotypes. Owing to the specific binding of the anti­Gal antibody with the α­Gal epitope, humans have a distinct anti­α­gal reactivity, which is responsible for hyperacute rejection of organs transplanted from α­gal donors. In addition, the α1,3 galactosyltransferases (α1,3GT) can catalyze the synthesis of the α­Gal epitope. Therefore, the α1,3GT gene, which encodes the α1,3GT, is developed profoundly. The distributions of the α­Gal epitope and anti­Gal antibody, and the activation of α1,3GT, reveal that the enzyme of α1,3GT in ancestral primates is ineffective. Comparison of the nucleotide sequence of the human α1,3­GT pseudogene to the corresponding different species sequence, and according to the evolutionary tree of different species, the results of evolutionary inactivation of the α1,3GT gene in ancestral primates attribute to the mutations under a stronger selective pressure. However, on the basis of the structure, the mechanism and the specificity of the α­Gal epitope and anti­Gal antibody, they can be applied to clinical exploitation. Knocking out the α1,3GT gene will eliminate the xenoantigen, Gal(α1,3)Gal, so that the transplantation of α1,3GT gene knockout pig organ into human becomes a potential clinically acceptable treatment for solving the problem of organ shortage. By contrast, the α­Gal epitope expressed through the application of chemical, biochemical and genetic engineering can be exploited for the clinical use. Targeting anti­Gal­mediated autologous tumor vaccines, which express α­Gal epitope to antigen­presenting cells, would increase their immunogenicity and elicit an immune response, which will be potent enough to eradicate the residual tumor cells. For tumor vaccines, the way of increasing immunogenicity of certain viral vaccines, including flu vaccines and human immunodeficiency virus vaccines, can also be used in the elderly. Recently, α­Gal epitope nanoparticles have been applied to accelerate wound healing and further directions on regeneration of internally injured tissues.

Epithelial ovarian cancer stem-like cells expressing α-gal epitopes increase the immunogenicity of tumor associated antigens.

BACKGROUND: As ovarian cancer stem cells (CSCs) are responsible for tumor initiation, invasion, metastasis, and chemo-resistance, new stratagems that selectively target ovarian CSCs are critically significant. Our previous work have demonstrated that ovarian cancer spheroid cells are tumorigenic and chemo-resistant, and have the properties of ovarian CSCs. Herein, we hypothesized that expressing α-gal epitopes on ovarian spheroid cells may help eliminate CSCs and improve the outcome of therapeutic intervention for ovarian cancer patients. METHODS: Lentivirus-mediated transfer of a pig α(1,3)galactosyltransferase [α1,3GT] enzyme gene into human ovarian cell line SKOV3 cells formed α-gal epitope-expressing cells (SKOV3-gal cells), and then these cells were maintained in a serum-free culture system to form SKOV3-gal spheroid cells. Efficacy of this cell vaccine was demonstrated in α1,3GT knockout mice (α1,3GT KO mice). RESULTS: The antibody titers to α-gal epitopes measured by ELISA were significantly increased in α1,3GT KO mice after immunization with SKOV3-gal spheroid cells. Furthermore, compared with the non-immunized KO mice, the SKOV3 tumors grafted under renal capsules of KO mice immunized with SKOV3-gal spheroid cells grew slower and began to shrink on day 12. Western blot analysis also showed that immunized KO mice can produce effective antibody against certain tumor associated antigens (TAAs) derived from both SKOV3 cells and SKOV3 spheroid cells. The TAAs were further investigated by mass spectrometry and RNA interference (RNAi) technology. The results suggested that antibodies responding to protein c-erbB-2 may be raised in the sera of the mice after immunization with SKOV3-gal spheroid cells. Ultimately, vaccination with SKOV3-gal spheroid cells induced more CD3+CD4+T cells in the spleen of immunized mice than non-immunized KO mice. CONCLUSIONS: The results suggest that vaccination using ovarian cancer stem-like cells engineered to express α-gal epitopes may be a novel strategy for treatment of ovarian cancer.

The Role of Liver Sinusoidal Endothelial Cells in Induction of Carbohydrate Reactive B Cells Tolerance Through the Programmed Death 1/Programmed Death Ligand 1 Pathway.

BACKGROUND: A spontaneous tolerance of B cells responding to blood group antigens frequently develops in ABO-incompatible pediatric liver transplantation (LT). Liver sinusoidal endothelial cells (LSECs), which exclusively express blood group antigens in the liver, possess a capacity to induce alloantigen-specific tolerance. In this study, we elucidated the role of LSECs in the tolerance induction of blood group antigen-reactive B cells after ABO-incompatible LT using mice that lack galactose-α(1,3)galactose (Gal) epitopes resembling blood group carbohydrate antigens. METHODS: Using adoptive transfer of LSECs from wild type (WT) C57BL/6J mice to congenic α1,3-galactosyltransferase gene knockout (GalT) mice, we established orthotropic GalT → GalT LSEC chimera mice. Anti-Gal Ab (antibody) production was evaluated after immunization of GalT → GalT LSEC chimera mice with Gal rabbit RBC. RESULTS: Adoptive transfer of LSECs isolated from WT GalT mice via the portal vein resulted in persistent engraftment of Gal LSECs in congenic GalT mouse livers. Only when GalT mice were splenectomized before LSEC inoculation, the GalT → GalT LSEC chimera lost the ability to produce anti-Gal Abs. The administration of blocking monoclonal Abs (mAbs) against programmed death ligand 1 to the splenectomized GalT → GalT LSEC chimera resulted in the recovery of anti-Gal Ab production. CONCLUSIONS: These findings suggest that LSECs take a part in tolerization of immature but not mature B cells specifically for Gal. Furthermore, the programmed death 1/programmed death ligand 1 pathway likely plays a crucial role in the mechanisms underlying spontaneous tolerization of B cells responding to ABO-blood group antigens in LT.

Pig kidney graft survival in a baboon for 136 days: longest life-supporting organ graft survival to date.

The longest survival of a non-human primate with a life-supporting kidney graft to date has been 90 days, although graft survival > 30 days has been unusual. A baboon received a kidney graft from an α-1,3-galactosyltransferase gene-knockout pig transgenic for two human complement-regulatory proteins and three human coagulation-regulatory proteins (although only one was expressed in the kidney). Immunosuppressive therapy was with ATG+anti-CD20mAb (induction) and anti-CD40mAb+rapamycin+corticosteroids (maintenance). Anti-TNF-α and anti-IL-6R were administered. The baboon survived 136 days with a generally stable serum creatinine (0.6 to 1.6 mg/dl) until termination. No features of a consumptive coagulopathy (e.g., thrombocytopenia, decreased fibrinogen) or of a protein-losing nephropathy were observed. There was no evidence of an elicited anti-pig antibody response. Death was from septic shock (Myroides spp). Histology of a biopsy on day 103 was normal, but by day 136, the kidney showed features of glomerular enlargement, thrombi, and mesangial expansion. The combination of (i) a graft from a specific genetically engineered pig, (ii) an effective immunosuppressive regimen, and (iii) anti-inflammatory agents prevented immune injury and a protein-losing nephropathy, and delayed coagulation dysfunction. This outcome encourages us that clinical renal xenotransplantation may become a reality.

In vitro exposure of pig neonatal isletlike cell clusters to human blood.

BACKGROUND: Pig islet grafts have been successful in treating diabetes in animal models. One remaining question is whether neonatal pig isletlike cell clusters (NICC) are resistant to the early loss of islets from the instant blood-mediated inflammatory reaction (IBMIR). METHODS: Neonatal isletlike cell clusters were harvested from three groups of piglets-(i) wild-type (genetically unmodified), (ii) α1,3-galactosyltransferase gene-knockout (GTKO)/CD46, and (iii) GTKO/CD46/CD39. NICC samples were mixed with human blood in vitro, and the following measurements were made-antibody binding; complement activation; speed of islet-induced coagulation; C-peptide; glutamic acid decarboxylase (GAD65) release; viability. RESULTS: Time to coagulation and viability were both reduced in all groups compared to freshly drawn non-anticoagulated human blood and autologous combinations, respectively. Antibody binding to the NICC occurred in all groups. CONCLUSIONS: Neonatal isletlike cell clusters were subject to humoral injury with no difference associated to their genetic characteristics.

Immune modulation in xenotransplantation.

The use of animals as donors of tissues and organs for xenotransplantations may help in meeting the increasing demand for organs for human transplantations. Clinical studies indicate that the domestic pig best satisfies the criteria of organ suitability for xenotransplantation. However, the considerable phylogenetic distance between humans and the pig causes tremendous immunological problems after transplantation, thus genetic modifications need to be introduced to the porcine genome, with the aim of reducing xenotransplant immunogenicity. Advances in genetic engineering have facilitated the incorporation of human genes regulating the complement into the porcine genome, knockout of the gene encoding the formation of the Gal antigen (α1,3-galactosyltransferase) or modification of surface proteins in donor cells. The next step is two-fold. Firstly, to inhibit processes of cell-mediated xenograft rejection, involving natural killer cells and macrophages. Secondly, to inhibit rejection caused by the incompatibility of proteins participating in the regulation of the coagulation system, which leads to a disruption of the equilibrium in pro- and anti-coagulant activity. Only a simultaneous incorporation of several gene constructs will make it possible to produce multitransgenic animals whose organs, when transplanted to human recipients, would be resistant to hyperacute and delayed xenograft rejection.

Genetically engineered pigs and target-specific immunomodulation provide significant graft survival and hope for clinical cardiac xenotransplantation.

OBJECTIVES: Cardiac transplantation and available mechanical alternatives are the only possible solutions for end-stage cardiac disease. Unfortunately, because of the limited supply of human organs, xenotransplantation may be the ideal method to overcome this shortage. We have recently seen significant prolongation of heterotopic cardiac xenograft survival from 3 to 12 months and beyond. METHODS: Hearts from genetically engineered piglets that were alpha 1-3 galactosidase transferase knockout and expressed the human complement regulatory gene, CD46 (groups A-C), and the human thrombomodulin gene (group D) were heterotropically transplanted in baboons treated with antithymocyte globulin, cobra venom factor, anti-CD20 antibody, and costimulation blockade (anti-CD154 antibody [clone 5C8]) in group A, anti-CD40 antibody (clone 3A8; 20 mg/kg) in group B, clone 2C10R4 (25 mg/kg) in group C, or clone 2C10R4 (50 mg/kg) in group D, along with conventional nonspecific immunosuppressive agents. RESULTS: Group A grafts (n = 8) survived for an average of 70 days, with the longest survival of 236 days. Some animals in this group (n = 3) developed microvascular thrombosis due to platelet activation and consumption, which resulted in spontaneous hemorrhage. The median survival time was 21 days in group B (n = 3), 80 days in group C (n = 6), and more than 200 days in group D (n = 5). Three grafts in group D are still contracting well, with the longest ongoing graft survival surpassing the 1-year mark. CONCLUSIONS: Genetically engineered pig hearts (GTKOhTg.hCD46.hTBM) with modified targeted immunosuppression (anti-CD40 monoclonal antibody) achieved long-term cardiac xenograft survival. This potentially paves the way for clinical xenotransplantation if similar survival can be reproduced in an orthotopic transplantation model.

The characterization of IgG antibodies to GalNAc beta-terminated glycans of gastric cancer survivors.

UNLABELLED: The elevated anti-GalNAcß IgG level of serum was shown to be associated with the significantly better survival of patients with gastrointestinal cancer. AIM: To characterize the specificity of IgG antibodies to GalNAcß-terminated glycans of long-term gastric cancer survivors. METHODS: Serum antibodies and affinity-isolated antibodies were analysed by the indirect and competitive ELISA using glycan-polyacrylamide (PAA) conjugates as well as by isoelectric focusing and Western blotting. RESULTS: In the serum probes, a partial cross-reactivity of antibodies to GalNAcß, GalNAcß1-3Galß (X2di), GalNAcß1-3GalNAcß (PFdi) and GlcNAcß was observed. The isolated anti-GalNAcß IgGs demonstrated the cross-reactivity to the X2di glycan mainly. The affinity of the X2di-PAA to anti-GalNAcß IgGs was 11-21 times lower than that of the GalNAcß-PAA. Anti-X2di and anti-PFdi IgGs demonstrated monoreactivity to their key glycans-PAA used in isolation. The IC50 values of key glycoconjugates ranged from 1 to 5 · 10(-7) M. No polyreactivity of antibodies to the unrelated antigens (ferritin, casein and DNA) was found. The polyclonal or oligoclonal distribution of IgG bands was established and the monoreactivity of antibodies was not associated with the clonal distribution of bands. CONCLUSION: The cross-reactivity of anti-GalNAcß antibodies to X2di and related glycans deserves attention in the clarification of the role of antibodies in cancer progression and enhancement of the prognostic potential in the combined determination of antibody markers.

Human T cell activation results in extracellular signal-regulated kinase (ERK)-calcineurin-dependent exposure of Tn antigen on the cell surface and binding of the macrophage galactose-type lectin (MGL).

The C-type lectin macrophage galactose-type lectin (MGL) exerts an immunosuppressive role reflected by its interaction with terminal GalNAc moieties, such as the Tn antigen, on CD45 of effector T cells, thereby down-regulating T cell receptor signaling, cytokine responses, and induction of T cell death. Here, we provide evidence for the pathways that control the specific expression of GalNAc moieties on human CD4(+) T cells. GalNAc epitopes were readily detectable on the cell surface after T cell activation and required de novo protein synthesis. Expression of GalNAc-containing MGL ligands was completely dependent on PKC and did not involve NF-κB. Instead, activation of the downstream ERK MAPK pathway led to decreased mRNA levels and activity of the core 1 ß3GalT enzyme and its chaperone Cosmc, favoring the expression of Tn antigen. In conclusion, expression of GalNAc moieties mirrors the T cell activation status, and thus only highly stimulated T cells are prone to the suppressive action of MGL.

Discovery of the natural anti-Gal antibody and its past and future relevance to medicine.

This is a personal account of the discovery of the natural anti-Gal antibody, the most abundant natural antibody in humans, the reciprocal distribution of this antibody and its ligand the α-gal epitope in mammals and the immunological barrier this antibody has formed in porcine to human xenotransplantation. This barrier has been overcome in the recent decade with the generation of α1,3-galactosyltransferase gene-knockout pigs. However, anti-Gal continues to be relevant in medicine as it can be harnessed for various therapeutic effects. Anti-Gal converts tumor lesions injected with α-gal glycolipids into vaccines that elicit a protective anti-tumor immune response by in situ targeting of tumor cells for uptake by antigen-presenting cells. This antibody further accelerates wound and burn healing by interaction with α-gal nanoparticles applied to injured areas and induction of rapid recruitment and activation of macrophages. Anti-Gal/α-gal nanoparticle immune complexes may further induce rapid recruitment and activation of macrophages in ischemic myocardium and injured nerves, thereby inducing tissue regeneration and prevention of fibrosis.

Costimulation blockade in pig artery patch xenotransplantation - a simple model to monitor the adaptive immune response in nonhuman primates.

BACKGROUND: CD154 blockade-based immunosuppression successfully prevents both humoral and cellular adaptive immune responses in baboons receiving α1,3-galactosyltransferase gene-knockout (GTKO) pig organs. Using a GTKO pig artery transplantation model in baboons, we evaluated the efficacy of CD28/B7 costimulatory pathway blockade in comparison with CD154 blockade. METHODS: Baboons received artery patch grafts from GTKO pigs, with no (Group1), anti-CD154mAb-based (Group2), or CTLA4-Ig-based (Group3) immunosuppressive therapy. Anti-pig IgM and IgG antibody and cellular responses were monitored. Xenografts were immunohistologically evaluated for antibody and complement deposition, and cellular infiltration. RESULTS: Group1 baboons developed increased IgM and IgG antibody and cellular responses against GTKO antigens. In Group2, anti-CD154mAb alone prevented the development of both IgM and IgG antibody and cellular responses,but not cellular infiltration of the graft. In the single baboon that received anti-thymocyte globulin (ATG) + mycophenolate mofetil (MMF) + anti-CD154mAb, cellular infiltration of the graft was not seen. In Group3, CTLA4-Ig with ATG + MMF inhibited the cellular proliferative response to pig antigens but did not prevent the IgG response or cellular infiltration. CONCLUSIONS: (i) Artery patch transplantation is a simple model to monitor the adaptive immune response to xenografts; (ii) anti-CD154mAb prevents sensitization but not cellular infiltration (but, without anticoagulation, may result in early thrombosis of a pig xenograft); (iii) although in only one baboon, the addition of ATG and MMF prevents cellular infiltration and (iv) replacement of anti-CD154mAb by CTLA4-Ig (at the doses used), even in combination with ATG and MMF, prevents the cellular proliferative response to GTKO pig antigens but is insufficient to prevent the development of anti-pig antibodies.

The future is now: chimeric antigen receptors as new targeted therapies for childhood cancer.

Improved outcomes for children with cancer hinge on the development of new targeted therapies with acceptable short-term and long-term toxicity. Progress in basic, preclinical, and clinical arenas spanning cellular immunology, gene therapy, and cell-processing technologies have paved the way for clinical applications of chimeric antigen receptor-based therapies. This is a new form of targeted immunotherapy that merges the exquisite targeting specificity of monoclonal antibodies with the potent cytotoxicity, potential for expansion, and long-term persistence provided by cytotoxic T cells. Although this field is still in its infancy, clinical trials have already shown clinically significant antitumor activity in neuroblastoma, chronic lymphocytic leukemia, and B-cell lymphoma, and trials targeting a variety of other adult and pediatric malignancies are under way. Ongoing work is focused on identifying optimal tumor targets and elucidating and manipulating both cell- and host-associated factors to support expansion and persistence of the genetically engineered cells in vivo. In pediatric oncology, CD19 and GD2 are compelling antigens that have already been identified for targeting pre-B acute lymphoblastic leukemia and neuroblastoma, respectively, with this approach, but it is likely that other antigens expressed in a variety of childhood cancers will also soon be targeted using this therapy. The potential to target essentially any tumor-associated cell-surface antigen for which a monoclonal antibody can be made opens up an entirely new arena for targeted therapy of childhood cancer.