Cell Therapy for Parkinson's Disease: A Translational Approach to Assess the Role of Local and Systemic Immunosuppression.

Neural transplantation is a promising therapeutic approach for neurodegenerative diseases; however, many patients receiving intracerebral fetal allografts exhibit signs of immunization to donor antigens that could compromise the graft. In this context, we intracerebrally transplanted mesencephalic pig xenografts into primates to identify a suitable strategy to enable long-term cell survival, maturation, and differentiation. Parkinsonian primates received WT or CTLA4-Ig transgenic porcine xenografts and different durations of peripheral immunosuppression to test whether systemic plus graft-mediated local immunosuppression might avoid rejection. A striking recovery of spontaneous locomotion was observed in primates receiving systemic plus local immunosuppression for 6 mo. Recovery was associated with restoration of dopaminergic activity detected both by positron emission tomography imaging and histological examination. Local infiltration by T cells and CD80/86+ microglial cells expressing indoleamine 2,3-dioxigenase were observed only in CTLA4-Ig recipients. Results suggest that in this primate neurotransplantation model, peripheral immunosuppression is indispensable to achieve the long-term survival of porcine neuronal xenografts that is required to study the beneficial immunomodulatory effect of local blockade of T cell costimulation.

Clinical efficacy of a new CD28-targeting antagonist of T cell co-stimulation in a non-human primate model of collagen-induced arthritis.

T cells have a central pathogenic role in the aetiopathogenesis of rheumatoid arthritis (RA), and are therefore a favoured target of immunotherapy aiming at physical or functional elimination. Here we report an efficacy test of FR104, a new co-stimulation inhibitor directly targeting CD28 on T cells, in a translationally relevant model, the rhesus monkey model of collagen-induced arthritis (CIA). As a relevant comparator we used abatacept [cytotoxic T lymphocyte antigen immunoglobulin (CTLA Ig)], an antagonist of CTLA-4 binding to CD80/86 clinically approved for treatment of RA. Treatment with either compound was started at the day of CIA induction. Although FR104 previously demonstrated a higher control of T cell responses in vitro than abatacept, both compounds were equally potent in the suppression of CIA symptoms and biomarkers, such as the production of C-reactive protein (CRP) and interleukin (IL)-6 and anti-collagen type II (CII) serum antibody (IgM/IgG). However, in contrast to abatacept, FR104 showed effective suppression of CII-induced peripheral blood mononuclear cell (PBMC) proliferation. The current study demonstrates a strong potential of the new selective CD28 antagonist FR104 for treatment of RA.

FR104, an antagonist anti-CD28 monovalent fab' antibody, prevents alloimmunization and allows calcineurin inhibitor minimization in nonhuman primate renal allograft.

Selective targeting of CD28 might represent an effective immunomodulation strategy by preventing T cell costimulation, while favoring coinhibition since inhibitory signals transmitted through CTLA-4; PD-L1 and B7 would not be affected. We previously showed in vitro and in vivo that anti-CD28 antagonists suppress effector T cells while enhancing regulatory T cell (Treg) suppression and immune tolerance. Here, we evaluate FR104, a novel antagonist pegylated anti-CD28 Fab' antibody fragment, in nonhuman primate renal allotransplantation. FR104, in association with low doses of tacrolimus or with rapamycin in a steroid-free therapy, prevents acute rejection and alloantibody development and prolongs allograft survival. However, when FR104 was associated with mycophenolate mofetil and steroids, half of the recipients rejected their grafts prematurely. Finally, we observed an accumulation of Helios-negative Tregs in the blood and within the graft after FR104 therapy, confirmed by Treg-specific demethylated region DNA analysis. In conclusion, FR104 reinforces immunosuppression in calcineurin inhibitor (CNI)-low or CNI-free protocols, without the need of steroids. Accumulation of intragraft Tregs suggested the promotion of immunoregulatory mechanisms. Selective CD28 antagonists might become an alternative CNI-sparing strategy to B7 antagonists for kidney transplant recipients.

Bortezomib, C1-inhibitor and plasma exchange do not prolong the survival of multi-transgenic GalT-KO pig kidney xenografts in baboons.

Galactosyl-transferase KO (GalT-KO) pigs represent a potential solution to xenograft rejection, particularly in the context of additional genetic modifications. We have performed life supporting kidney xenotransplantation into baboons utilizing GalT-KO pigs transgenic for human CD55/CD59/CD39/HT. Baboons received tacrolimus, mycophenolate mofetil, corticosteroids and recombinant human C1 inhibitor combined with cyclophosphamide or bortezomib with or without 2-3 plasma exchanges. One baboon received a control GalT-KO xenograft with the latter immunosuppression. All immunosuppressed baboons rejected the xenografts between days 9 and 15 with signs of acute humoral rejection, in contrast to untreated controls (n = 2) that lost their grafts on days 3 and 4. Immunofluorescence analyses showed deposition of IgM, C3, C5b-9 in rejected grafts, without C4d staining, indicating classical complement pathway blockade but alternate pathway activation. Moreover, rejected organs exhibited predominantly monocyte/macrophage infiltration with minimal lymphocyte representation. None of the recipients showed any signs of porcine endogenous retrovirus transmission but some showed evidence of porcine cytomegalovirus (PCMV) replication within the xenografts. Our work indicates that the addition of bortezomib and plasma exchange to the immunosuppressive regimen did not significantly prolong the survival of multi-transgenic GalT-KO renal xenografts. Non-Gal antibodies, the alternative complement pathway, innate mechanisms with monocyte activation and PCMV replication may have contributed to rejection.

CD28-specific immunomodulating antibodies: what can be learned from experimental models?

Tolerance induction to alloantigens remains a major challenge in transplant immunology. Progress in the last decade of our understanding of T-cell activation has led to the development of new immunotherapeutic strategies to replace conventional immunosuppression which inhibits the immune system in a nonspecific way. In particular, positive and negative costimulatory molecules of the CD28 family have been consistently demonstrated to be critical for the development of productive immune responses as well as the establishment and maintenance of peripheral tolerance. However, recent discoveries of novel costimulatory interactions confer a novel dimension to the immunoregulatory interactions within the B7:CD28 family and compels a revised view within a "quintet" of costimulatory molecules: CD28/B7/CTLA-4/PD-L1/ICOSL. Complexity introduced in this more detailed costimulatory pathway has important implications in therapeutic interventions against human immunological diseases and, especially, highlight the fundamental differences in selectively targeting CD28 molecules instead of B7 counterparts. In this review, we discuss these differences and emphasize different CD28-specific immunomodulating strategies evaluated in experimental models of transplantation and autoimmune diseases.

Preclinical efficacy and immunological safety of FR104, an antagonist anti-CD28 monovalent Fab' antibody.

Antagonist anti-CD28 antibodies prevent T cell costimulation and differentiate from CTLA4Ig since they cannot block CTLA-4 and PDL-1 coinhibitory signals. They demonstrated efficacy in suppressing effector T cells while enhancing regulatory T cells function and immune tolerance. However, anti-CD28 antibodies devoid of immunotoxicity and with a good pharmacokinetic profile have not yet been developed. Here, we describe FR104, a novel humanized pegylated anti-CD28 Fab' antibody fragment presenting a long elimination half-life in monkeys. In vitro, FR104 failed to induce human T cell proliferation and cytokines secretion, even in the presence of anti-CD3 antibodies or when cross-linked with secondary antibodies. Furthermore, in humanized NOD/SCID mice adoptively transferred with human PBMC, whereas superagonist and divalent antibodies elicited rapid cytokines secretion and human T cell activation, FR104 did not. These humanized mice developed a florid graft-versus-host disease, which was prevented by administration of FR104 in a CTLA4-dependent manner. Interestingly, administration of high doses of CTLA4-Ig was ineffective to prevent GVHD, whereas administration of low doses was partially effective. In conclusion, we demonstrated that FR104 is devoid of agonist activity on human T cells and thus compatible with a clinical development that might lead to higher therapeutic indexes, by sparing CTLA-4, as compared to CD80/CD86 antagonists.

Antibody-mediated depletion of lymphocyte-activation gene-3 (LAG-3(+) )-activated T lymphocytes prevents delayed-type hypersensitivity in non-human primates.

Lymphocyte-activation gene-3 (LAG-3, CD223) is a marker for recently activated effector T cells. Activated T lymphocytes are of major importance in many autoimmune diseases and organ transplant rejection. Therefore, specifically depleting LAG-3(+) T cells might lead to targeted immunosuppression that would spare resting T cells while eliminating pathogenic activated T cells. We have shown previously that anti-LAG-3 antibodies sharing depleting as well as modulating activities inhibit heart allograft rejection in rats. Here, we have developed and characterized a cytotoxic LAG-3 chimeric antibody (chimeric A9H12), and evaluated its potential as a selective therapeutic depleting agent in a non-human primate model of delayed-type hypersensitivity (DTH). Chimeric A9H12 showed a high affinity to its antigen and depleted both cytomegalovirus (CMV)-activated CD4(+) and CD8(+) human T lymphocytes in vitro. In vivo, a single intravenous injection at either 1 or 0·1 mg/kg was sufficient to deplete LAG-3(+) -activated T cells in lymph nodes and to prevent the T helper type 1 (Th1)-driven skin inflammation in a tuberculin-induced DTH model in baboons. T lymphocyte and macrophage infiltration into the skin was also reduced. The in vivo effect was long-lasting, as several weeks to months were required after injection to restore a positive reaction after antigen challenge. Our data confirm that LAG-3 is a promising therapeutic target for depleting antibodies that might lead to higher therapeutic indexes compared to traditional immunosuppressive agents in autoimmune diseases and transplantation.

Agonist anti-ChemR23 mAb reduces tissue neutrophil accumulation and triggers chronic inflammation resolution.

Resolution of inflammation is elicited by proresolving lipids, which activate GPCRs to induce neutrophil apoptosis, reduce neutrophil tissue recruitment, and promote macrophage efferocytosis. Transcriptional analyses in up to 300 patients with Inflammatory Bowel Disease (IBD) identified potential therapeutic targets mediating chronic inflammation. We found that ChemR23, a GPCR targeted by resolvin E1, is overexpressed in inflamed colon tissues of severe IBD patients unresponsive to anti-TNFα or anti-α4ß7 therapies and associated with significant mucosal neutrophil accumulation. We also identified an anti-ChemR23 agonist antibody that induces receptor signaling, promotes macrophage efferocytosis, and reduces neutrophil apoptosis at the site of inflammation. This ChemR23 mAb accelerated acute inflammation resolution and triggered resolution in ongoing chronic colitis models, with a significant decrease in tissue lesions, fibrosis and inflammation-driven tumors. Our findings suggest that failure of current IBD therapies may be associated with neutrophil infiltration and that ChemR23 is a promising therapeutic target for chronic inflammation.

Immune regulation by non-lymphoid cells in transplantation.

Regulatory cells play a crucial role in the induction and maintenance of tolerance by controlling T cell as well as B and natural killer (NK) cell-mediated immunity. In transplantation, CD4+CD25+forkhead box P3+ T regulatory cells are instrumental in the maintenance of immunological tolerance, as are several other T cell subsets such as NK T cells, double negative CD3+ T cells, gammadelta T cells, interleukin-10-producing regulatory type 1 cells, transforming growth factor-beta-producing T helper type 3 cells and CD8+CD28(-) cells. However, not only T cells have immunosuppressive properties, as it is becoming increasingly clear that both T and non-T regulatory cells co-operate and form a network of cellular interactions controlling immune responses. Non-T regulatory cells include tolerogenic dendritic cells, plasmacytoid dendritic cells, mesenchymal stem cells, different types of stem cells, various types of alternatively activated macrophages and myeloid-derived suppressor cells. Here, we review the mechanism of action of these non-lymphoid regulatory cells as they relate to the induction or maintenance of tolerance in organ transplantation.

Porcine alpha1,3-galactosyltransferase: tissue-specific and regulated expression of splicing isoforms.

Expression of the Gal alpha1,3 Gal epitope on membrane glycolipids and glycoproteins is known to vary widely from one tissue to another. In the course of studying the mechanisms underlying this variability, we have isolated from pig cDNA four sequences corresponding to four isoforms of alpha1,3-galactosyltransferase (alpha1,3GT), the Golgi enzyme that links galactose in alpha1,3 on the galactose residue of N-acetyllactosamine. The isoforms differ from each other in the alternative presence of two nucleotide stretches of 36 and 63 base pairs in a segment encoding the stem region of the protein. Stable expression experiments show that all four isoenzymes can confer alpha-galactosyltransferase activity to HeLa cells, and that they are all located within the Golgi compartment, indicating that variations in length in the stem region do not affect enzyme activity or cellular localization. Analysis of RNA from different pig organs and cells shows quantitative differences between tissues in levels of alpha1,3GT, as well as qualitative differences, the four isoforms being unequally represented in different tissues.

IgG2b or IgE molecules can be co-expressed with those of the IgM and IgD isotypes of the membrane of normal rat B cells.

Expression of Ig isotypes other than IgD together with IgM on the membranes of single B cells has been reported in different experimental models. This paper describes the co-expression of IgG2b or IgE with IgM-IgD on the surface of single B cell subpopulations from normal rats. Their expression was demonstrated with anti-IgE or IgG2b monoclonal antibodies and their F(ab')2 fragments. After pronase digestion, the re-expression of these isotypes together with IgM-IgD was observed in vitro and was inhibited by cycloheximide. These observations imply that mechanisms other than class switching may participate in the expression of membrane isotypes in vivo. The role of these membrane isotypes is still to be established, but could be important as IgG2b molecules are found on a large B cell subpopulation.

Intracellular expression in pig cells of anti-alpha1,3galactosyltransferase single-chain FV antibodies reduces Gal alpha1,3Gal expression and inhibits cytotoxicity mediated by anti-Gal xenoantibodies.

BACKGROUND: The carbohydrate structure Gal alpha1,3Gal expressed on pig cells is the major antigen recognized by xenoreactive natural antibodies in the higher primates. In xenotransplantation, natural antibodies binding to that structure initiate hyperacute rejection, and the anti-Gal alpha1,3Gal antibodies that are elicited probably take part in later phases of vascularized graft rejection. This epitope also appears to be involved in innate cellular responses. Inactivation of alpha1,3 galactosyltransferase in transgenic pigs would certainly lead to the success of xenotransplantation, but gene knockout in pigs is not feasible yet. METHODS: As a novel strategy to inhibit alpha1,3 galactosylation, we generated recombinant single-chain Fv (ScFv) antibodies directed against pig alpha1,3-galactosyltransferase and evaluated the effect of their intracellular expression on enzyme activity and Gal alpha1,3Gal expression. RESULTS: After in vitro transfection in pig cells, the scFv antibody anti-pig alpha1,3-galactosyltransferase reduced the amount or function of enzyme by up to 70% as evidenced by immunofluorescence and measurement of cell-associated activity. Consequently, Gal alpha1,3Gal on cell membranes was reduced to the same extent. This led to a profound (more than 90%) reduction in the cytotoxicity involving anti-Gal antibodies and complement. CONCLUSION: Although not sufficient to knock out the overall human anti-pig natural xenoreactivity, intracellular expression of the scFv antibody anti-alpha1,3-galactosyltransferase in pig cells significantly decreases the amount of Gal alpha1,3Gal and could be important to protect cells from elicited antibodies as well as from innate effectors.

Induction of anti-Forssman antibodies in the hamster-to-rat xenotransplantation model.

BACKGROUND: In the hamster-to-rat heart xenotransplantation model, the serum response of the host contributes to determine whether the xenograft is accommodated or rejected. METHODS: To further characterize the serum response in this model, we compared anti-hamster antibodies found in naive LEW-1A rats, or in LEW-1A rats rejecting or accommodating a hamster heart, using a combination of cobra venom factor (CVF) and cyclosporin A (CsA) given for 10 days, and then CsA alone. RESULTS: Hamster hearts grafted into rat recipients contained IgG and IgA deposits to the same extent whether the xenograft was rejected or accommodated. Only immunoglobulins of the IgM isotype were found to be more abundant in recipients rejecting their graft. A significant part of this IgM response was directed toward the Forssman antigen, a sphingolipid present in the hamster but not in the rat. However, although anti-Forssman antibodies bind in situ to hamster tissues, this binding was not able to induce hyperacute rejection after antibody transfer. Furthermore, depletion of anti-Forssman antibodies from a rejecting serum did not modify its rejection properties. CONCLUSION: Unlike the pig-to-primate discordant xenotransplantation model, in which preexisting anti-carbohydrate antibodies are directly responsible for hyperacute rejection, in the concordant hamster-to-rat situation, the evoked IgM anti-Forssman carbohydrate antibodies do not appear to be the main cause of the vascular rejection.

Gem, a GTP-binding protein from mitogen-stimulated T cells, is induced in endothelial cells upon activation by inflammatory cytokines.

Using differential screening of cytokine-activated versus resting porcine aortic endothelial cells (PAEC), we have isolated a member of the family of Ras/GTP-binding proteins. The cDNA encodes a 34-kilodalton protein showing 97% homology to Gem, a gene recently isolated from activated T cells, likely representing its porcine homologue. The amino acid sequence differs from the Ras consensus by the absence of a C-terminal isoprenylation site and a glycine to glutamic acid substitution in the third GTP-binding domain. We report here, that pigGem mRNA is strongly inducible in PAEC upon activation by either IL-1 alpha, TNF alpha or lipopolysaccharide (LPS). Low constitutive expression is found in several organs. Epitope-tagged pigGem transfected into endothelial cells (EC) localizes to the cytoplasm and to the inner side of the plasma membrane. Structural features of Gem and its inducibility apparently restricted to T cells and endothelial cells, together with Rad, a GTPase overexpressed in skeletal muscle cells of type II diabetic individuals, define a new branch within the superfamily of GTP-binding proteins.

Genetic engineering in the pig. Gene knockout and alternative techniques.

Since endothelial cells (EC) are the major target cells during hyperacute rejection and are likely in delayed graft rejection, most of the genetic engineering of the xenotransplant donor is aimed at modifying their properties. Among the various strategies that are reviewed are the genotypic or phenotypic knockout of the alpha 1,3Gal antigen, which is a major target of xenoantibodies and is also probably involved in innate cellular response. In addition, the success of the transgeny of complement regulatory proteins is well established. In vitro data from analyses of the mechanisms of endothelial cell activation also suggest that other molecules could be used to regulate apoptosis or thrombotic microenvironment or to minimize recipient T-cell activation by inhibiting costimulatory proteins such as CD40 or B7. Alternative to usual knockout techniques (thus far not available in pigs, where no ES cells have been derived) will be presented.

Xenotransplantation of galactosyl-transferase knockout, CD55, CD59, CD39, and fucosyl-transferase transgenic pig kidneys into baboons.

Galactosyl-transferase knockout (GT-KO) pigs represent the latest major progress to reduce immune reactions in xenotransplantation. However, their organs are still subject to rapid humoral rejection involving complement activation requiring the ongoing development of further genetic modifications in the pig. In a pig-to-baboon renal transplantation setting, we have used donor pigs that are not only GT-KO, but also transgenic for human CD55 (hCD55), hCD59, hCD39, and fucosyl-transferase (hHT). We studied kidney xenograft survival, physiological and immunologic parameters, xenogeneic rejection characteristics, as well as viral transmission aspects among two groups of baboons: control animals (n = 2), versus those (n = 4) treated with a cocktail of cyclophosphamide, tacrolimus, mycophenolate mofetil, steroids, and a recombinant human C1 inhibitor. Whereas control animals showed clear acute humoral rejection at around day 4, the treated animals showed moderately improved graft survival with rejection at around 2 weeks posttransplantation. Biopsies showed signs of acute vascular rejection (interstitial hemorrhage, glomerular thrombi, and acute tubular necrosis) as well as immunoglobulin (Ig)M and complement deposition in the glomerular and peritubular capillaries. The low level of preformed non-Gal-α1.3Gal IgM detected prior to transplantation increased at 6 days posttransplantation, whereas induced IgG appeared after day 6. No porcine endogenous retrovirus (PERV) transmission was detected in any transplanted baboon. Thus, surprisingly, organs from the GT-KO, hCD55, hCD59, hCD39, and hHT transgenic donors did not appear to convey significant protection against baboon anti-pig antibodies and complement activation, which obviously continue to be significant factors under a suboptimal immunosuppression regimen. The association, timing, and doses of immunosuppressive drugs remain critical. They will have to be optimized to achieve longer graft survivals.