The Immunogenicity of the Tumor-Associated Antigen α-Fetoprotein Is Enhanced by a Fusion with a Transmembrane Domain

Aim. To investigate the ability of recombinant modified vaccinia virus Ankara (rMVA) vector to induce an immune response against a well-tolerated self-antigen. Methods. rMVA vectors expressing different form of α-fetoprotein (AFP) were produced and characterized. Naïve mice were vaccinated with MVA vectors expressing the AFP antigen in either a secreted, or a membrane-bound, or an intracellular form. The immune response was monitored by an IFNΓ ELISpot assay and antibody detection. Results. Vaccination with the membrane-associated form of AFP induced a stronger CD8+ T-cell response compared to the ones obtained with the MVA encoding the secreted or the intracellular forms of AFP. Moreover, the vaccination with the membrane-bound AFP elicited the production of AFP-specific antibodies. Conclusions. The AFP transmembrane form is more immunogenic. Expressing a membrane-bound form in the context of an MVA vaccination could enhance the immunogenicity of a self-antigen

Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens

Two well-characterized carbohydrate epitopes are absent in humans but present in other mammals. These are galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc) which are introduced by the activities of two enzymes including α(1,3) galactosyltransferase (encoded by the GGTA1 gene) and CMP-Neu5Gc hydroxylase (encoded by the CMAH gene) that are inactive in humans but present in cattle. Hence, bovine-derived products are antigenic in humans who receive bioprosthetic heart valves (BHVs) or those that suffer from red meat syndrome. Using programmable nucleases, we disrupted (knockout, KO) GGTA1 and CMAH genes encoding for the enzymes that catalyse the synthesis of αGal and Neu5Gc, respectively, in both male and female bovine fibroblasts. The KO in clonally selected fibroblasts was detected by polymerase chain reaction (PCR) and confirmed by Sanger sequencing. Selected fibroblasts colonies were used for somatic cell nuclear transfer (SCNT) to produce cloned embryos that were implanted in surrogate recipient heifers. Fifty-three embryos were implanted in 33 recipients heifers; 3 pregnancies were carried to term and delivered 3 live calves. Primary cell cultures were established from the 3 calves and following molecular analyses confirmed the genetic deletions. FACS analysis showed the double-KO phenotype for both antigens confirming the mutated genotypes. Availability of such cattle double-KO model lacking both αGal and Neu5Gc offers a unique opportunity to study the functionality of BHV manufactured with tissues of potentially lower immunogenicity, as well as a possible new clinical approaches to help patients with red meat allergy syndrome due to the presence of these xenoantigens in the diet

Alloantigen gene transfer to hepatocytes promotes tolerance to pancreatic islet graft by inducing CD8 + regulatory T cells

International audienceBACKGROUND & AIMS: Induction of donor-specific immune tolerance is a good alternative to chronic life-long immunosuppression for transplant patients. Donor major histocompatibility complex (MHC) molecules represent the main targets of the allogeneic immune response of transplant recipients. Liver targeted gene transfer with viral vectors induces tolerance toward the encoded antigen. The aim of this work was to determine whether alloantigen gene transfer to hepatocytes induces tolerance and promotes graft acceptance.METHODS: C57BL/6 (H-2b) mice were treated with adeno-associated viral (AAV) vector targeting the expression of the MHC class I molecule H-2Kd to hepatocytes, before transplantation with fully allogeneic pancreatic islet from BALB/c mice (H-2d).RESULTS: AAV H-2Kd treated mice were tolerant to the alloantigen, as demonstrated by its long-term expression by the hepatocytes, even after a highly immunogenic challenge with an adenoviral vector. After chemical induction of diabetes, the AAV treated mice had significantly delayed rejection of fully allogeneic pancreatic islet grafts, with more than 40% of recipients tolerant (>100days). AAV-mediated expression of H-2Kd in the liver induced the local expansion of CD8+ T lymphocytes with allo-specific suppressive properties. The adoptive transfer of these liver-generated CD8+ Tregs into naive diabetic mice promoted the long-term survival of allogeneic pancreatic islet grafts.CONCLUSION: AAV-mediated long-term expression of a single MHC class I molecule in the liver induces the generation of a subset of allo-specific CD8+ Treg cells, which promote tolerance toward fully allogeneic graft. Liver gene transfer represents a promising strategy for in vivo induction of donor-specific tolerance.LAY SUMMARY: The liver has a special immune system, biased toward tolerance. In this study, we investigated the possibility of harnessing this property of the liver to induce tolerance to an allogeneic transplantation. We demonstrate for the first time that the in vivo gene transfer of an allogeneic antigen with an adeno-associated viral vector to mouse hepatocytes induces the expansion of a population of CD8+ regulatory T lymphocytes. These Tregs are then instrumental in preventing the rejection of allogeneic pancreatic islets transplanted in these animals. Allogeneic transplantation is the main treatment for the end-stage diseases of a number of organs. Life-long immunosuppressive treatments are still required to limit graft rejection, and these treatments exhibit serious side effects. Our present findings open a new avenue for promoting allo-specific tolerance via in vivo induction of CD8+ Treg expansion

Immunotherapy of hepatocellular carcinoma: is there a place for regulatory T-lymphocyte depletion?

Item does not contain fulltextImmunotherapy represents a potential therapeutic option for patients with hepatocellular carcinoma (HCC), especially as secondary treatment to prevent recurrence. It has been shown that a patient's survival is directly correlated to the type and number of tumor-infiltrating immune cells, indicating that immune responses have a direct effect on the clinical course of the disease. We have assessed the potential of immunotherapy against HCC in preclinical models of low tumor burden. An antigen-specific strategy targeting alpha-fetoprotein, and consisting of immunization with a DNA-based synthetic vector (DNAmAFP/704), was tested on an autochthonous model of chemical hepatocarcinogenesis and led to an important (65%) reduction of the tumor burden. A nonspecific approach of CD25(+) T-cell depletion by injection of PC61 antibody was also tested on an orthotopic HCC model and led to a significant protection against tumor development. Antigen-specific immunotherapy and Treg depletion are promising strategies in physiologically relevant HCC preclinical models. Future clinical trials will demonstrate if a combination of Treg depletion with an antigen-specific immunotherapy will also translate into clinical responses in HCC patients.1 april 201

Primary immune cell tracking with a new generation of multimodal far red emitting polymer probe

Primary immune cell tracking with a new generation of multimodal far red emitting polymer probe. 52ème Annual meeting of French Society for immunolog

Multiscale fluorescent tracking of immune cells in the liver with a highly biocompatible far-red emitting polymer probe

International audienceThe development of innovative immune cell therapies relies on efficient cell tracking strategies. For this, multiscale fluorescence-based analyses of transferred cells into the host with complementary techniques, including flow cytometry for high-throughput cell analysis and two-photon microscopy for deep tissue imaging would be highly beneficial. Ideally, cells should be labelled with a single fluorescent probe combining all the properties required for these different techniques. Due to the intrinsic autofluorescence of most tissues and especially the liver, far-red emission is also an important asset. However, the development of far-red emitting probes suitable for two-photon microscopy and compatible with clearing methods to track labelled immune cells in thick samples, remains challenging. A newly-designed water-soluble far-red emitting polymer probe, 19K-6H, with a large Stokes shift, was thus evaluated for the tracking of primary immune CD8 T cells. These cells, prepared from mouse spleen, were efficiently labelled with the 19K-6H probe, which was internalized via endocytosis and was highly biocompatible at concentrations up to 20 μM. Labelled primary CD8 T cells were detectable in culture by both confocal and two-photon microscopy as well as flow cytometry, even after 3 days of active proliferation. Finally, 19K-6H-labelled primary CD8 T cells were injected to mice in a classical model of immune mediated hepatitis. The efficient tracking of the transferred cells in the liver by flow cytometry (on purified non-parenchymal cells) and by two-photon microscopy on 800 μm thick cleared sections, demonstrated the versatility of the 19K-6H probe

Immune Alterations in Patients With Type 1 Autoimmune Hepatitis Persist Upon Standard Immunosuppressive Treatment

International audienceAutoimmune hepatitis (AIH) is a rare disease characterized by an immune attack of the liver. This study consists of a comprehensive analysis of immune alterations related to AIH at diagnosis, and during remission phase under treatment. A total of 37 major lymphocyte populations were analyzed from the peripheral blood of new-onset AIH patients (AIHn; n = 14), AIH patients with controlled disease (n = 11), and healthy subjects (n = 14). Liver biopsy analyses were performed to complete the blood phenotypic analysis. Four blood lymphocyte populations were significantly altered in AIHn patients at diagnosis compared with healthy subjects. Levels of mucosal-associated invariant T cells (MAIT), Type 1/Type 17 helper (Th1/ Th17) cells, clusters of differentiation (CD4) T cells, and invariant natural killer T cells were decreased, whereas MAIT granzyme B+ (GrB) cells were increased. A trend toward an increase of CD8+CD161+GrB+ cells was also observed. These alterations were not restored with standard immunosuppressive treatments. In the liver of AIHn patients, CD4, forkhead box P3 (Foxp3), and MAIT cell markers were enriched in the portal tract, and CD8, CD161, and GrB markers were enriched in the hepatic lobule. During remission, the hepatic lobule was clear of infiltrating T cells, but residual CD4 and MAIT cells were found in the portal tract, where Foxp3 was decreased, as previously described. In vitro, MAIT cells were functionally altered in AIH patients. Ex vivo MAIT cell activity (GrB) was linked to severe fibrosis. Conclusion: Our work proposes a global view of the lymphocyte alterations from diagnosis to remission phase in AIH patients. The absence of blood immune homeostasis restoration and the persistence of a CD4 infiltrate in the liver under standard immunosuppres-sion could form the basis of the high risk of relapse observed in AIH

Broad Impairment of Natural Killer Cells From Operationally Tolerant Kidney Transplanted Patients

The role of natural killer (NK) cells in organ transplantation is controversial. This study aims to decipher their role in kidney transplant tolerance in humans. Previous studies highlighted several modulated genes involved in NK cell biology in blood from spontaneously operationally tolerant patients (TOLs; drug-free kidney-transplanted recipients with stable graft function). We performed a phenotypic, functional, and genetic characterization of NK cells from these patients compared to kidney-transplanted patients with stable graft function under immunosuppression and healthy volunteers (HVs). Both operationally TOLs and stable patients harbored defective expression of the NKp46 activator receptor and lytic molecules perforin and granzyme compared to HVs. Surprisingly, NK cells from operationally TOLs also displayed decreased expression of the CD16 activating marker (in the CD56Dim NK cell subset). This decrease was associated with impairment of their functional capacities upon stimulation, as shown by lower interferon gamma (IFNγ) production and CD107a membranous expression in a reverse antibody-dependent cellular cytotoxicity (ADCC) assay, spontaneous lysis assays, and lower target cell lysis in the 51Cr release assay compared to HVs. Conversely, despite impaired K562 cell lysis in the 51Cr release assay, patients with stable graft function harbored a normal reverse ADCC and even increased amounts of IFNγ+ NK cells in the spontaneous lysis assay. Altogether, the strong impairment of the phenotype and functional cytotoxic capacities of NK cells in operationally TOLs may accord with the establishment of a pro-tolerogenic environment, despite remaining highly activated after transplantation in patients with stable graft function