Association of HLA-DRB1 locus with treatment response to abatacept or TNF inhibitors in patients with seropositive rheumatoid arthritis.

The strongest genetic risk factor for rheumatoid arthritis (RA) has been known as HLA-DRB1 based on amino acid positions 11, 71, and 74. This study analyzed the association between specific HLA-DRB1 locus and treatment response to abatacept or TNF inhibitors (TNFi) in patients with seropositive RA. A total of 374 Korean RA patients were treated with abatacept (n = 110) or TNFi (n = 264). Associations between HLA-DRB1 and treatment response after 6 months were analyzed using multivariable logistic regression. Seropositive RA patients with HLA-DRB1 shared epitope (SE) had a favorable response to abatacept (OR = 3.67, P = 0.067) and an inversely associated response to TNFi (OR 0.57, P = 0.058) based on EULAR response criteria, but the difference was not statistically significant in comparison to those without SE. In analyses using amino acid positions of HLA-DRB1, a significant association was found between valine at amino acid position 11 of SE and good response to abatacept (OR = 6.46, P = 5.4 × 10-3). The VRA haplotype also showed a good response to abatacept (OR = 4.56, P = 0.013), but not to TNFi. Our results suggest that treatment response to abatacept or TNFi may differ depending on HLA-DRB1 locus in seropositive RA, providing valuable insights for selecting optimal therapy.

CTLA-4 Insufficiency due to a Novel CTLA-4 Deletion, Identified through Copy Number Variation Analysis.

BACKGROUND: The diagnostic yield of next-generation sequencing (NGS) technologies in the diagnosis of monogenic inborn errors of immunity (IEI) remains limited, rarely exceeding 30%. Monoallelic pathogenic germline variants in cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) result in variable immunodeficiency and immune dysregulation. The genetic diagnosis of CTLA-4 insufficiency can affect follow-up procedures and may lead to consideration of treatment with CTLA-4-Ig. OBJECTIVES: The aim of the study was to identify the genetic cause of familial immunodeficiency and immune dysregulation in cases where single nucleotide variant analysis of short-read NGS data yielded no diagnostic result. METHODS: Analysis of copy number variants (CNVs) was applied on short-read NGS data. RESULTS: We identified a novel monoallelic deletion-insertion variant in CTLA-4 (c.445_568-544delinsTTTGCGATTG) resulting in familial autoimmunity. This is the second larger scale variant in CTLA-4, which despite consistently reduced expression of CTLA-4 displayed variable expressivity, ranging from typical juvenile idiopathic arthritis to common variable immunodeficiency-like immunodeficiency. CONCLUSIONS: Our report suggests the significance of integration of CNV analysis in routine evaluation of NGS, which may increase its diagnostic yield in IEI.

CTLA-4 gene mutation and multiple sclerosis: A case report and literature review.

We reported a patient with autoimmunity (multiple sclerosis), immunodeficiency (hypogammaglobulinemia with severe infections), enteropathy (diarrhea with intestinal inflammation), splenomegaly, lymphadenopathy and lymphocytic infiltration of non-lymphoid organs (lung, gut and brain). The patient was found to have a heterozygous mutation in cytotoxic T lymphocyte antigen-4, and had excellent response to abatacept.

The preliminary efficacy evaluation of the CTLA-4-Ig treatment against Lupus nephritis through in-silico analyses.

The humanized cytotoxic T lymphocyte-associated antigen 4 immunoglobulin (CTLA-4-Ig) has been used to treat Lupus nephritis (LN) based on CTLA-4s negative regulation of T-cell activation through competent to binding with CD80/CD86, the inherent genetic factors influencing the CTLA-4-Ig treatment efficacy are widely unknown. Here, 62 nonsynonymous single nucleotide variants (nsSNVs) of CTLA-4 gene, 184 of CD80 and 201 of CD86 were identified and validated within both EMBL-EBI and dbSNP databases. Next, the nsSNVs rs1466152724 in CTLA-4, rs1196816748, rs765515058, rs1157880125, rs1022857991, and rs142547094 in CD80 and rs1203132714 in CD86 were consistently suggested to be deleterious by SIFT, PolyPhen-2, PROVEAN and meta LR. Based on the 3D structure stability analysis, the variant rs765515058 causing G167V in CD80 was found to reduce the protein's stability through changing the characters of constructed structure of complete CD80 apo form and stabilizing amino acid residues of CD80 holo form in a great degree. Furthermore, the interaction energy analysis results suggested that rs1022857991 causing C50F may reduce the binding energy of CTLA-4 with CD80. Along with the increasing variants, these nsSNVs' effects on the interaction of CTLA-4 with CD80/CD86 will increase, and thus influence the CTLA-4-Ig treatment efficacy against LN.

Conserved HA-peptide NG34 formulated in pCMV-CTLA4-Ig reduces viral shedding in pigs after a heterosubtypic influenza virus SwH3N2 challenge.

Swine influenza viruses (SIVs), the causal agents of swine influenza, are not only important to control due to the economic losses in the swine industry, but also can be pandemic pathogens. Vaccination is one of the most relevant strategies to control and prevent influenza infection. Current human vaccines against influenza induce strain-specific immunity and annual update is required due to the virus antigenic shift phenomena. Previously, our group has reported the use of conserved hemagglutinin peptides (HA-peptides) derived from H1-influenza virus as a potential multivalent vaccine candidate. Immunization of swine with these HA-peptides elicited antibodies that recognized and neutralized heterologous influenza viruses in vitro and demonstrated strong hemagglutination-inhibiting activity. In the present work, we cloned one HA-peptide (named NG34) into a plasmid fused with cytotoxic T lymphocyte-associated antigen (CTLA4) which is a molecule that modifies T cell activation and with an adjuvant activity interfering with the adaptive immune response. The resulting plasmid, named pCMV-CTLA4-Ig-NG34, was administered twice to animals employing a needle-free delivery approach. Two studies were carried out to test the efficacy of pCMV-CTLA4-Ig-NG34 as a potential swine influenza vaccine, one in seronegative and another in seropositive pigs against SIV. The second one was aimed to evaluate whether pCMV-CTLA4-Ig-NG34 vaccination would overcome maternally derived antibodies (MDA). After immunization, all animals were intranasally challenged with an H3N2 influenza strain. A complete elimination or significant reduction in the viral shedding was observed within the first week after the challenge in the vaccinated animals from both studies. In addition, no challenged heterologous virus load was detected in the airways of vaccinated pigs. Overall, it is suggested that the pCMV-CTLA4-Ig-NG34 vaccine formulation could potentially be used as a multivalent vaccine against influenza viruses.

[Role of bone marrow mesenchymal stem cells with CTLA4Ig and CD40LIg gene modification in rejection reaction after liver transplantation].

Objective: To investigate the role of bone marrow mesenchymal stem cells (BMSCs) with CTLA4Ig and CD40LIg gene modification in rejection reaction after liver transplantation in rats and possible mechanisms. Methods: The modified Kamada's two-cuff technique was used to establish a Lewis-BN rat model of orthotopic liver transplantation, and a total of 75 rats were randomly divided into groups A, B, C, D, and E, with 15 rats in each group. The rats in group A (control group) were given infusion of isotonic saline via the portal vein during liver transplantation, those in group B (BMSC group) were given infusion of BMSCs via the portal vein during liver transplantation, those in group C (BMSCs with CTLA4Ig gene modification) were given infusion of BMSCs carrying the CTLA4Ig gene via the portal vein during liver transplantation, those in group D (BMSCs with CD40LIg gene modification) were given infusion of BMSCs carrying the CD40LIg gene via the portal vein during liver transplantation, and those in group E (BMSCs with CTLA4Ig and CD40LIg gene modification) were given infusion of BMSCs carrying CTLA4Ig and CD40LIg gene modification via the portal vein during liver transplantation. Postoperative survival and change in liver function were observed. HE staining was used to observe the pathomorphological changes of the graft liver, and ELISA was used to measure the levels of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10), and interferon-γ (IFN-γ) in peripheral blood. A one-way analysis of variance was used for comparison of means of multiple samples, and the Kaplan-Meier survival curve analysis was used for comparison of survival rates between multiple groups. Results: Group E had a significantly longer survival time after surgery than groups A, B, C, and D (P < 0.05), groups C and D had a significantly longer survival time than groups A and B (P < 0.05), and there was no significant difference between groups C and D (P > 0.05). On day 10 after surgery, group A had significantly higher levels of alanine aminotransferase and total bilirubin than the other four groups (P < 0.05). HE staining showed severe rejection reaction in group A, moderate rejection reaction in group B, and mild rejection reaction in groups C and D; pathological examination showed no marked rejection reaction in group E. Group A had significant increases in the levels of IL-2 and IFN-γ and significant reductions in the levels of IL-4 and IL-10 after surgery compared with the other four groups (all P < 0.05). Conclusion: Infusion of BMSCs with modification of both CTLA4Ig and CD40LIg genes can significantly inhibit acute rejection reaction after liver transplantation in rats and effectively prolong the survival time of the graft liver, with a better effect than infusion of BMSCs alone or BMSCs with modification of CTLA4Ig or CD40LIg gene.

LEA29Y expression in transgenic neonatal porcine islet-like cluster promotes long-lasting xenograft survival in humanized mice without immunosuppressive therapy.

Genetically engineered pigs are a promising source for islet cell transplantation in type 1 diabetes, but the strong human anti-pig immune response prevents its successful clinical application. Here we studied the efficacy of neonatal porcine islet-like cell clusters (NPICCs) overexpressing LEA29Y, a high-affinity variant of the T cell co-stimulation inhibitor CTLA-4Ig, to engraft and restore normoglycemia after transplantation into streptozotocin-diabetic NOD-SCID IL2rγ-/- (NSG) mice stably reconstituted with a human immune system. Transplantation of INSLEA29Y expressing NPICCs resulted in development of normal glucose tolerance (70.4%) and long-term maintenance of normoglycemia without administration of immunosuppressive drugs. All animals transplanted with wild-type NPICCs remained diabetic. Immunohistological examinations revealed a strong peri- and intragraft infiltration of wild-type NPICCs with human CD45+ immune cells consisting of predominantly CD4+ and CD8+ lymphocytes and some CD68+ macrophages and FoxP3+ regulatory T cells. Significantly less infiltrating lymphocytes and only few macrophages were observed in animals transplanted with INSLEA29Y transgenic NPICCs. This is the first study providing evidence that beta cell-specific LEA29Y expression is effective for NPICC engraftment in the presence of a humanized immune system and it has a long-lasting protective effect on inhibition of human anti-pig xenoimmunity. Our findings may have important implications for the development of a low-toxic protocol for porcine islet transplantation in patients with type 1 diabetes.

AAV9-mediated engineering of autotransplanted kidney of non-human primates.

Ex vivo gene transfer to the graft before transplantation is an attractive option for circumventing systemic side effects of chronic antirejection therapy. Gene delivery of the immunomodulatory protein cytotoxic T-lymphocyte-associated protein 4-immunoglobulin (CTLA4-Ig) prevented chronic kidney rejection in a rat model of allotransplantation without the need for systemic immunosuppression. Here we generated adeno-associated virus type 2 (AAV2) and AAV9 vectors encoding for LEA29Y, an optimized version of CTLA4-Ig. Both LEA29Y vectors were equally efficient for reducing T-cell proliferation in vitro. Serotype 9 was chosen for in vivo experiments owing to a lower frequency of preformed antibodies against the AAV9 capsid in 16 non-human primate tested sera. AAV9-LEA29Y was able to transduce the kidney of non-human primates in an autotransplantation model. Expression of LEA29Y mRNA by renal cells translated into the production of the corresponding protein, which was confined to the graft but not detected in serum. Results in non-human primates represent a step forward in maintaining the portability of this strategy into clinics.

Effects of combined genes of CTLA4Ig and IDO in post-liver transplantation immune tolerance of rats.

UNLABELLED:  Background and rationale for the study. Previous studies showed that CTLA4Ig and indoleamine 2,3-dioxygenase (IDO) genes played regulatory role in organ transplantation but failed to reach satisfactory effects. In this study, we constructed an adenovirus- mediated gene expressing CTLA4Ig-IDO and established rat liver transplantation models. Recipients were randomly divided into four groups of 10 rats each. During the operation, CTLA4Ig, IDO, and CTLA4Ig-IDO genes, as well as a blank plasmid, were infused into different rat groups via portal vein to determine their effects on inducing immune tolerance. Survival rate of recipients, histological changes of graft liver, post-transplantation liver function, and cytokine levels were observed at day 14 after operation. RESULTS: Serum levels of alanine aminotransferase (ALT), aspartate transaminase (AST), and total bilirubin level (TBIL) in the CTLA4Ig-IDO group were lower than those in the other three groups at 14 days post-transplantation (P < 0.05); mRNA and protein expressions of IL-2 and IFN-γ were higher in the control group, but lower in the CTLA4Ig-IDO group (P < 0.05). By contrast, expressions of IL-4, TGF-b, IL-10, and T lymphocyte apoptosis were higher in the CTLA4Ig-IDO group than those in the other three groups (P < 0.05). The CTLA4Ig-IDO group exhibited mild acute rejection and higher survival rate compared with the other groups (P < 0.05). CONCLUSION: Compared with using CTLA4Ig or IDO alone, combined transfection of CTLA4Ig-IDO was more effective in inducing immune tolerance after liver transplantation.

ASP2408 and ASP2409, novel CTLA4-Ig variants with CD86-selective ligand binding activity and improved immunosuppressive potency, created by directed evolution.

The CTLA4-Ig therapeutics abatacept and belatacept inhibit CD28-mediated T cell activation by binding CD80 (B7-1) and CD86 (B7-2) co-stimulatory ligands. Both compounds preferentially bind CD80, yet CD86 has been implicated as the dominant co-stimulatory ligand. Using directed evolution methods, novel CTLA4-Ig variants were created with selective CD86 binding affinity, a property that confers increased immunosuppressive potency and potentially improved efficacy and safety profiles. Relative to abatacept (wild-type CTLA4-Ig), ASP2408 and ASP2409 have 83-fold and 220-fold enhanced binding affinity to CD86 while retaining 1.5-fold and 5.6-fold enhanced binding affinity to CD80, respectively. Improvements in CD86 binding affinity correlates with increased immunosuppressive potencyin vitroandin vivo Our results highlight the power of directed evolution methods to obtain non-intuitive protein engineering solutions and represent the first examples of CD86-selective CTLA4-Ig compounds that have entered clinical trials.

CTLA-4 Ig as an effective treatment in a patient with type I diabetes mellitus and seropositive rheumatoid arthritis.

We describe a patient suffering from seropositive rheumatoid arthritis (RA) and type I diabetes mellitus (T1DM), who achieved a good EULAR response together with an improvement of the glycemic profile under treatment with CTLA-4 Ig. A close association is known to exist between T1DM and RA, and CTLA-4 exon 1 polymorphism has been associated to RA with coexisting autoimmune endocrinopathies. The possible common genetic background and the potential role of CTLA-4 Ig in the early phases of T1DM, could be considered in the therapeutic interventions in RA patients with type 1 diabetes.

Targeting the CD80/CD86 costimulatory pathway with CTLA4-Ig directs microglia toward a repair phenotype and promotes axonal outgrowth.

Among the costimulatory factors widely studied in the immune system is the CD28/cytotoxic T-lymphocyte antigen-4 (CTLA4)-CD80/CD86 pathway, which critically controls the nature and duration of the T-cell response. In the brain, up-regulated expression of CD80/CD86 during inflammation has consistently been reported in microglia. However, the role of CD80/CD86 molecules has mainly been studied in a context of microglia-T cell interactions in pathological conditions, while the function of CD80/CD86 in the regulation of intrinsic brain cells remains largely unknown. In this study, we used a transgenic pig line in which neurons express releasable CTLA4-Ig, a synthetic molecule mimicking CTLA4 and binding to CD80/CD86. The effects of CTLA4-Ig on brain cells were analyzed after intracerebral transplantation of CTLA4-Ig-expressing neurons or wild-type neurons as control. This model provided in vivo evidence that CTLA4-Ig stimulated axonal outgrowth, in correlation with a shift of the nearby microglia from a compact to a ramified morphology. In a culture system, we found that the CTLA4-Ig-induced morphological change of microglia was mediated through CD86, but not CD80. This was accompanied by microglial up-regulated expression of the anti-inflammatory molecule Arginase 1 and the neurotrophic factor BDNF, in an astrocyte-dependent manner through the purinergic P2Y1 receptor pathway. Our study identifies for the first time CD86 as a key player in the modulation of microglia phenotype and suggests that CTLA4-Ig-derived compounds might represent new tools to manipulate CNS microglia.

Transgenic expression of human cytoxic T-lymphocyte associated antigen4-immunoglobulin (hCTLA4Ig) by porcine skin for xenogeneic skin grafting.

Porcine skin is frequently used as a substitute of human skin to cover large wounds in clinic practice of wound care. In our previous work, we found that transgenic expression of human cytoxicT-lymphocyte associated antigen4-immunoglobulin (hCTLA4Ig) in murine skin graft remarkably prolonged its survival in xenogeneic wounds without extensive immunosuppression in recipients, suggesting that transgenic hCTLA4Ig expression in skin graft may be an effective and safe method to prolong xenogeneic skin graft survival. In this work, using a transgene construct containing hCTLA4Ig coding sequence under the drive of human Keratine 14 (k14) promoter, hCTLA4Ig transgenic pigs were generated by somatic nuclear transfer. The derived transgenic pigs were healthy and exhibited no signs of susceptibility to infection. The hCTLA4Ig transgene was stably transmitted through germline over generations, and thereby a transgenic pig colony was established. In the derived transgenic pigs, hCTLA4Ig expression in skin was shown to be genetically stable over generations, and detected in heart, kidney and corneal as well as in skin. Transgenic hCTLA4Ig protein in pigs exhibited expected biological activity as it suppressed human lymphocyte proliferation in human mixed lymphocyte culture to extents comparable to those of commercially purchased purified hCTLA4Ig protein. In skin grafting from pigs to rats, transgenic porcine skin grafts exhibited remarkably prolonged survival compared to the wild-type skin grafts derived from the same pig strain (13.33 ± 3.64 vs. 6.25 ± 2.49 days, P < 0.01), further indicating that the transgenic hCTLA4Ig protein was biologically active and capable of extending porcine skin graft survival in xenogeneic wounds. The transgenic pigs generated in this work can be used as a reproducible resource to provide porcine skin grafts with extended survival for wound coverage, and also as donors to investigate the impacts of hCTLA4Ig on xenotransplantation of other organs (heart, kidney and corneal) due to the ectopic transgenic hCTLA4Ig expression.

Glucose metabolism in pigs expressing human genes under an insulin promoter.

BACKGROUND: Xenotransplantation of porcine islets can reverse diabetes in non-human primates. The remaining hurdles for clinical application include safe and effective T-cell-directed immunosuppression, but protection against the innate immune system and coagulation dysfunction may be more difficult to achieve. Islet-targeted genetic manipulation of islet-source pigs represents a powerful tool to protect against graft loss. However, whether these genetic alterations would impair islet function is unknown. METHODS: On a background of α1,3-galactosyltransferase gene-knockout (GTKO)/human (h)CD46, additional genes (hCD39, human tissue factor pathway inhibitor, porcine CTLA4-Ig) were inserted in different combinations under an insulin promoter to promote expression in islets (confirmed by immunofluorescence). Seven pigs were tested for baseline and glucose/arginine-challenged levels of glucose, insulin, C-peptide, and glucagon. RESULTS: This preliminary study did not show definite evidence of ß-cell deficiencies, even when three transgenes were expressed under the insulin promoter. Of seven animals, all were normoglycemic at fasting, and five of seven had normal glucose disposal rates after challenge. All animals exhibited insulin, C-peptide, and glucagon responses to both glucose and arginine challenge; however, significant interindividual variation was observed. CONCLUSIONS: Multiple islet-targeted transgenic expression was not associated with an overtly detrimental effect on islet function, suggesting that complex genetic constructs designed for islet protection warrants further testing in islet xenotransplantation models.

Therapeutic effects of CTLA4Ig gene-transduced adipose tissue-derived mesenchymal stem cell transplantation on established autoimmune thyroiditis.

This study aimed to identify the beneficial effects of adipose tissue-derived mesenchymal stem cells (ASCs) and ASCs that overexpress the CTLA4Ig gene (CTLA4Ig-ASCs) on established autoimmune thyroiditis and to examine changes in clinical chemistry parameters and the presence of humoral responses upon repeated long-term administration of autologous ASCs. This study also aimed to acquire desirable results in a preclinical study by using large-sized lab animals and applying ASCs that overexpress therapeutic genes. Experimental autoimmune thyroiditis was induced by immunization with thyroglobulin. Experimental dogs were divided into five groups: (i) ASC IT + IV, (ii) ASC IV, (iii) CTLA4Ig-ASC IT + IV, (iv) CTLA4Ig-ASC IV, and (v) control IT + IV (saline only), and they received intrathyroidal (IT; 10 million cells/250 µl saline per thyroid) administration one time or intravenous (IV; 20 million cells/5 ml) administration seven times within a 101-day period. Blood samples were collected every week, and thyroids were harvested on days 104-106. After serial ASC or CTLA4Ig transplantation, the levels of canine thyroglobulin autoantibodies (TgAA) in serum and the infiltration of T-lymphocytes between the follicles of the thyroid glands were decreased. The expression of FoxP3 in submandibular lymph nodes was significantly increased. Repeated long-term administration of autologous ASCs or CTLA4Ig-ASCs did not generate changes in clinical chemistry parameters or humoral responses.The TgAA test can detect autoimmune thyroiditis years before clinical signs of hypothyroidism occur. Thus, ASC and CTLA4Ig-ASC transplantation in that period can be attractive candidates to ameliorate autoimmune thyroiditis and prevent the development of hypothyroidism.