Cytotoxic T Lymphocyte-associated Antigen 4 Immunoglobulin Research Articles

Low dose post-transplant cyclophosphamide and sirolimus induce mixed chimerism with CTLA4-Ig or lymphocyte depletion in an MHC-mismatched murine allotransplantation model

Allogeneic hematopoietic cell transplantation (allo-HCT) offers a curative option for patients with certain non-malignant hematological diseases. High-dose post-transplant cyclophosphamide (PT-Cy) (200 mg/kg) and sirolimus (3 mg/kg), (HiC) synergistically induce stable mixed chimerism. Further, sirolimus and cytotoxic T lymphocyte-associated antigen-4 immunoglobulin (CTLA4-Ig), also known as Abatacept (Aba), promote immune tolerance and allograft survival. Here, in a major histocompatibility complex (MHC)-mismatched allo-HCT murine model, we combined Aba and/or T-cell depleting anti-Thy1.2 (Thy) with a lower dose of PT-Cy (50 mg/kg) and Sirolimus (3 mg/kg), (LoC). While mice in the LoC group showed graft rejection, the addition of Thy to LoC induced similar donor chimerism levels when compared to the HiC group. However, the addition of Aba to LoC led to graft acceptance only in younger mice. When Thy was added to the LoC+Aba setting, graft acceptance was restored in both age groups. Engrafted groups displayed significantly reduced frequencies of recipient-specific interferon-γ-producing T cells as well as an increased frequency in regulatory T cells (Tregs) except in the LoC+Aba group. Splenocytes from engrafted mice showed no proliferation upon restimulation with Balb/c stimulators. Collectively, in combination with Aba or Thy, LoC may be considered to reduce graft rejection in patients who undergo allo-HCT.

Comparison of retention of biologics in Japanese patients with elderly-onset rheumatoid arthritis-the ANSWER cohort study.

This multicentre, retrospective study aimed to compare retention and reasons for discontinuation between Janus kinase inhibitors (JAKi) and biologic disease-modifying antirheumatic drugs in patients with elderly-onset rheumatoid arthritis (EORA). Patients with RA enrolled in a Japanese multicentre observational registry between 2015 and 2022 were included. EORA was defined as RA with onset at 60 or over. To adjust confounding factors by indication for initiation of tumor necrosis factor inhibitors (TNFi), interleukin-6 inhibitors (IL-6i), cytotoxic T-lymphocyte associated antigen 4 immunoglobulin (CTLA4-Ig) blockers, or JAKi, a propensity score based on baseline characteristics was used to compare drug retention. To assess the reasons for discontinuation, retention rates for ineffectiveness, adverse events, and remission were analyzed as secondary outcomes. A total of 572 patients with 835 treatment courses were identified (314 TNFi, 175 IL-6i, 228 CTLA4-Ig, and 118 JAKi). After adjusting for differences in baseline characteristics, drug retention was significantly higher for IL-6i (HR = 0.38, 95%CI = 0.27-0.55, p< 0.01) as compared with TNFi. Discontinuation due to lack of effectiveness was lower with the JAKi (HR = 0.38, 95%CI = 0.22-0.66, p< 0.01) and the IL-6i (HR = 0.29, 95%CI = 0.19-0.46, p< 0.01) as compared with the TNFi although the CTLA4-Ig had a similar HR to TNFi. The adjusted incidence of discontinuation due to adverse event was higher in the JAKi (HR = 2.86, 95%CI = 1.46-5.59, p< 0.01) than the TNFi. In EORA patients, IL-6i and JAKi had longer retention and less discontinuation due to ineffectiveness than TNFi. The potential risks of JAKi should be approached with an individualized perspective.

CTLA4-Ig protects tacrolimus-induced oxidative stress via inhibiting the AKT/FOXO3 signaling pathway in rats

Although the conversion from tacrolimus (TAC) to cytotoxic T-lymphocyte-associated antigen 4-immunoglobulin (CTLA4-Ig) is effective in reducing TAC-induced nephrotoxicity, it remains unclear whether CTLA4-Ig has a direct effect on TAC-induced renal injury. In this study, we evaluated the effects of CTLA4-Ig on TAC-induced renal injury in terms of oxidative stress. In vitro study was performed to assess the effect of CTLA4-Ig on TAC-induced cell death, reactive oxygen species (ROS), apoptosis, and the protein kinase B (AKT)/forkhead transcription factor (FOXO) 3 pathway in human kidney 2 cells. In the in vivo study, the effect of CTLA4-Ig on TAC-induced renal injury was evaluated using renal function, histopathology, markers of oxidative stress (8-hydroxy-2'-deoxyguanosine) and metabolites (4-hydroxy-2-hexenal, catalase, glutathione S-transferase, and glutathione reductase), and activation of the AKT/FOXO3 pathway with insulin-like growth factor 1 (IGF-1). CTLA4-Ig significantly decreased cell death, ROS, and apoptosis caused by TAC. TAC treatment increased apoptotic cell death and apoptosis-related proteins (increased Bcl-2-associated X protein and caspase-3 and decreased Bcl-2), but it was reversed by CTLA4-Ig treatment. The activation of p-AKT and p-FOXO3 by TAC decreased with CTLA4-Ig treatment. TAC-induced renal dysfunction and oxidative marker levels were significantly improved by CTLA4-Ig in vivo. Concomitant IGF-1 treatment abolished the effects of CTLA4-Ig. CTLA4-Ig has a direct protective effect on TAC-induced renal injury via the inhibition of AKT/FOXO3 pathway.

Fragmentation stability and retention time-shift obtained by LC-MS/MS to distinguish sialylated N-glycan linkage isomers in therapeutic glycoproteins

Sialylated N-glycan isomers with α2-3 or α2-6 linkage(s) have distinctive roles in glycoproteins, but are difficult to distinguish. Wild-type (WT) and glycoengineered (mutant) therapeutic glycoproteins, cytotoxic T lymphocyte-associated antigen-4-immunoglobulin (CTLA4-Ig), were produced in Chinese hamster ovary cell lines; however, their linkage isomers have not been reported. In this study, N-glycans of CTLA4-Igs were released, labeled with procainamide, and analyzed by liquid chromatography-tandem mass spectrometry (MS/MS) to identify and quantify sialylated N-glycan linkage isomers. The linkage isomers were distinguished by comparison of 1) intensity of the N-acetylglucosamine ion to the sialic acid ion (Ln/Nn) using different fragmentation stability in MS/MS spectra and 2) retention time-shift for a selective m/z value in the extracted ion chromatogram. Each isomer was distinctively identified, and each quantity (>0.1%) was obtained relative to the total N-glycans (100%) for all observed ionization states. Twenty sialylated N-glycan isomers with only α2-3 linkage(s) in WT were identified, and each isomer's sum of quantities was 50.4%. Furthermore, 39 sialylated N-glycan isomers (58.8%) in mono- (3 N-glycans; 0.9%), bi- (18; 48.3%), tri- (14; 8.9%), and tetra- (4; 0.7%) antennary structures of mutant were obtained, which comprised mono- (15 N-glycans; 25.4%), di- (15; 28.4%), tri- (8; 4.8%), and tetra- (1; 0.2%) sialylation, respectively, with only α2-3 (10 N-glycans; 4.8%), both α2-3 and α2-6 (14; 18.4%), and only α2-6 (15; 35.6%) linkage(s). These results are consistent with those for α2-3 neuraminidase-treated N-glycans. This study generated a novel plot of Ln/Nn versus retention time to distinguish sialylated N-glycan linkage isomers in glycoprotein.

Usage of fingolimod combined with polyacetone system in the treatment of mouse kidney transplantation model

A kidney transplantation model was established with mouse subjects, which was treated with the fingolimod (FTY720) combined with polyacetone (PCL) membrane controlled release system to analyze its therapeutic effect on kidney transplantation. 45 C57BL/6 (H-2b) mice were randomly enrolled into 3 groups, 9 in the Sham group did not receive any treatment, 18 in the model group and FTY720-PCL group, with 9 donors and recipients, respectively. The mice in model group were treated with kidney transplantation, and those in the FTY720-PCL group were treated with FTY720-PCL based on the model group. The three groups of mice were analyzed for serum creatinine (Scr), urine output, survival time of transplanted kidney (SToTK), overall survival time (OST), and the spleen tissue of the recipient mice. RT-PCR was used to detect the expression of IL-2 and ICAM-1 mRNA, and Western blot was used to detect the expression of CTLA4Ig and Fas/FasL protein. The pathological changes of the transplanted kidney were graded and evaluated according to the BanffSchema pathological diagnostic criteria. The results showed that the Scr level of the recipient mice in the FTY720-PCL group was greatly reduced (P &lt; 0.05). Compared with the model group, urine output, SToTK, and OST were greatly increased (P &lt; 0.05). The levels of interleukin 2 (IL-2) and intercellular adhesion molecule 1 (ICAM-1) mRNA in the spleen (P &lt; 0.05) of the mice in the FTY720-PCL group were obviously reduced, the level of cytotoxic T lymphocyte-associated antigen 4 immunoglobulin (CTLA4Ig) in the kidney was increased obviously, and factor related apoptosis (Fas) and factor related apoptosis ligand (FasL) (P &lt; 0.05) levels dropped dramatically compared with the model group. The results of hematoxylin-eosin staining (HE staining) showed that the rejection rate of transplanted kidneys in the FTY720-PCL group was greatly reduced. In summary, FTY720-PCL membrane system can reduce the rejection rate of mouse kidney transplantation and prolong SToTK.

Systemic administration of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)-Ig abrogates alveolar bone resorption in induced periodontitis through inhibition of osteoclast differentiation and activation: An experimental investigation.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a critical immunoregulatory molecule expressed on T cells. CTLA-4 also binds to the surfaces of monocytes and macrophages, precursors of osteoclasts. Research on rheumatoid arthritis demonstrated that CTLA-4suppresses inflammation and bone resorption. However, its effects on alveolar bone have yet to be understood. The purpose of this study was to investigate the role and potential mechanism of CTLA-4 in bone resorption in periodontitis. In vivo, the effects of systemic administration of CTLA-4 immunoglobulin fusion protein (CTLA-4-Ig) on alveolar bone resorption were investigated using a periodontitis mouse model. A total of 20 C57BL/6J mice were randomly assigned to two groups according to the administration modes. Periodontitis was induced by placing a ligature around the left maxillary second molar. The contralateral tooth was left un-ligated. In the CTLA-4-Ig (+) group, CTLA-4-Ig was administered by intraperitoneal injection at 1 and 3days after ligature placement. Animals in the CTLA-4-Ig (-) group were given only phosphate-buffered saline each time. At 5days after ligature placement, bone resorption was assessed by micro-computed tomography and histological examination, and the prevalence of osteoclast-like cells was assessed by tartrate-resistant acid phosphatase (TRAP) staining. In vitro, the effects of CTLA-4-Ig on osteoclasts were evaluated. Viability of RAW 264.7 cells treated with receptor activator of nuclear factor-κB ligand (RANKL) and CTLA-4-Ig was tested by WST-1 assay. Osteoclast-like cells were enumerated by TRAP staining, and osteoclast activity was evaluated by resorption pit assay. Gene expression levels of osteoclast differentiation markers (macrophage-colony stimulating factor receptor, carbonic anhydrase II, cathepsin K, and Trap) and protein phosphatase 2A (PP2A), a major serine-threonine phosphatase, were assessed by quantitative real-time polymerase chain reaction. The effect of CTLA-4-Ig on the nuclear factor-κB (NF-κB) activation was assessed by enzyme-linked immunosorbent assay. In vivo, ligature-induced bone resorption and the numbers of osteoclast-like cells were significantly decreased by the administration of CTLA-4-Ig. In vitro, treatment with RANKL and CTLA-4-Ig had no significant effect on cell viability. CTLA-4-Ig significantly reduced the prevalence and activation of osteoclast-like cells and decreased the expressions of osteoclast differentiation markers, compared with the RANKL-treated control. CTLA-4-Ig significantly suppressed RANKL-induced phosphorylation of NF-κB p65 but increased PP2A expression. These results suggest that CTLA-4-Ig abrogates bone resorption in induced periodontitis, possibly via inhibition of osteoclast differentiation and activation. The regulation of the NF-κB pathway and PP2A expression may be one mechanism by which CTLA-4-Ig suppresses osteoclast behavior.

Effects of Cytotoxic T Lymphocyte-Associated Antigen 4 Immunoglobulin Combined with Microbubble-Mediated Irradiation on Hemodynamics of the Renal Artery in Rats with Diabetic Nephropathy

Cytotoxic T lymphocyte-associated antigen 4 immunoglobulin (CTLA-4-Ig) can inhibit the effect of B7-1 and improve renal hemodynamics in rats with diabetic nephropathy (DN). Nevertheless, a strategy that could increase the permeation of CTLA-4-Ig through endothelial cells and basement membrane remains to be discovered. We investigated the effect of CTLA-4-Ig combined with microbubble-mediated irradiation on the hemodynamics of renal arteries in DN rats. Rats were treated with CTLA-4-Ig and/or microbubble exposure. After 8 wk of intervention, color Doppler ultrasonography was used to detect peak systolic velocity (PSV), end-diastolic velocity (EDV), mean velocity (MV), systolic acceleration (SAC), pulsatility index (PI) and resistance index (RI) of the renal artery trunk. The CTLA-4-Ig + microbubble exposure group exhibited significantly higher PSV, EDV and MV than the CTLA-4-Ig group, which had significantly higher values than the non-intervention group. The CTLA-4-Ig + microbubble exposure group exhibited significantly lower SAC, PI and RI than the CTLA-4-Ig group, which had significantly lower values than the non-intervention group. Our results indicate that both CTLA-4-Ig and CTLA-4-Ig + microbubble exposure can reduce the blood flow resistance and improve the blood flow velocity of renal arteries in rats. Moreover, the effect of CTLA-4-Ig + microbubble exposure is better than that of CTLA-4-Ig alone. Our study provides a new, effective and non-invasive strategy for the treatment of DN.

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.

Costimulation blockade by combining CTLA4Ig with anti-CD40L mAb markedly inhibits the inflammatory response of experimental autoimmune myocarditis

The aim of this study was to investigate the effect of costimulation blockade with cytotoxic T-lymphocyte-associated-antigen 4-immunoglobulin (CTLA4Ig) and anti-CD40L monoclonal antibody (anti-CD40L mAb) on an experimental autoimmune myocarditis (EAM) mouse model. Characteristics of myocardial tissue were observed by hematoxylin and eosin (H&amp;E) staining. The messenger RNA (mRNA) levels of CTLA4, CD40L, IFN-γ, and IL-4 were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Serum concentrations of IFN-γ and IL-4 were determined by ELISA. After immune intervention, the inflammatory score, mRNA levels of CTLA4 and CD40L, and IFN-γ level were decreased. Furthermore, these parameters in the combinational intervention group (blockade by CTLA4Ig and anti-CD40L mAb) were significantly decreased, compared to the single intervention group (blockade by CTLA4Ig or anti-CD40L mAb). However, after costimulation, blockade serum IL-4 levels were increased. Therefore, costimulation blockade by combination CTLA4Ig and anti-CD40L mAb could more effectively inhibit the inflammatory response of EAM than single use of CTLA4Ig or anti-CD40L mAb.

Immunosuppression in early postnatal days induces persistent and allergen-specific immune tolerance to asthma in adult mice.

Bronchial asthma is a chronic airway inflammatory condition with high morbidity, and effective treatments for asthma are limited. Allergen-specific immunotherapy can only induce peripheral immune tolerance and is not sustainable. Exploring new therapeutic strategies is of great clinical importance. Recombinant adenovirus (rAdV) was used as a vector to make cells expressing cytotoxic T lymphocyte-associated antigen-4-immunoglobulin (CTLA4Ig) a soluble CTLA4 immunoglobulin fusion protein. Dendritic cells (DCs) were modified using the rAdVs together with allergens. Then these modified DCs were transplanted to mice before allergen sensitization. The persistence and specificity of immune tolerance were evaluated in mice challenged with asthma allergens at 3 and 7 months. DCs modified by CTLA4Ig showed increased IL-10 secretion, decreased IL-12 secretion, and T cell stimulation in vitro. Mice treated with these DCs in the early neonatal period developed tolerance against the allergens that were used to induce asthma in the adult stage. Asthma symptoms, lung damage, airway reactivity, and inflammatory response all improved. Humoral immunity indices showed that this therapeutic strategy strongly suppressed mice immune responses and was maintained for as long as 7 months. Furthermore, allergen cross-sensitization and challenge experiments demonstrated that this immune tolerance was allergen-specific. Treatment with CTLA4Ig modified DCs in the early neonatal period, inducing persistent and allergen-specific immune tolerance to asthma in adult mice. Our results suggest that it may be possible to develop a vaccine for asthma.

Local Cytotoxic T-lymphocyte–associated Antigen-4 Immunoglobulin Inhibition of Rejection Response Is Dependent on Indoleamine 2,3-dioxygenase Activities in the Allograft

A previous study showed that local gene transfer of cytotoxic T-lymphocyte–associated antigen-4 immunoglobulin (CTLA4Ig) significantly prolonged the survival time of rat flap allografts. However, the underlying mechanism is not fully understood. Indoleamine 2,3-dioxygenase (IDO) is considered to be able to modulate the unresponsiveness state of allografts. In this study, we tested the expression of the CD80 molecule, IDO mRNA, and the level of the tryptophan metabolite kynurenine with or without the application of the IDO blocker 1-methyl-tryptophan (1-MT) in a rat composite tissue allotransplantation model. CD80 expression could be detected in the allograft. The ration of IDO mRNA/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA and the level of kynurenine were both enhanced (46.3 ± 8.8 versus 4.6 ± 1.8 and 18.9 ± 1.3 μmol/L versus 2.1 ± 0.2 μmol/L separately) after adenovirus-mediated CTLA4Ig (AdCTLA4Ig) transduction. When 1-MT was applied to the AdCTLA4Ig perfusion recipients, the ration of IDO mRNA/GAPDH mRNA (5.2 ± 2.9) and the level of kynurenine (0.8 ± 0.5μmol/L) were significantly reduced. Moreover, the allograft survival time was greatly reduced when 1-MT was applied to AdCTLA4Ig perfusion recipients compared to single AdCTLA4Ig perfusion therapy recipients (7.2 days versus 13.6 days). We showed that the inhibitory effect of locally delivered CTLA4Ig is dependent on IDO activities within the allograft.

Tartrate-resistant acid phosphatase 5b is a potential biomarker for rheumatoid arthritis: a pilot study in Han Chinese

Background Bone damage around the joints is one of the major pathophysiological mechanisms that leads to rheumatoid arthritis (RA) chronic disability. Serum tartrate-resistant acid phosphatase 5b (TRACP-5b) is secreted by osteoclasts, its activity can be used as a clinically relevant bone resorption marker. The aim of this study was to test whether the measurement of serum levels of TRACP-5b in patients with RA would correlate with measures of disease activity and with responses to therapy. Methods Fifty-six patients were randomly assigned to receive recombinant human cytotoxic tlymphocyte-associated antigen-4 immunoglobulin (RhCTLA4-Ig), infliximab or methotrexate (MTX). The clinical and serologic indicators of RA activity were evaluated at baseline and at 24 weeks. Serum TRACP-5b was measured by Enzyme-linked Immunosorbent Assay (ELISA) at 0, 12 and 24 weeks. Hand X-rays were obtained at baseline. Results At baseline, the levels of TRACP-5b correlated with the severity of X-ray damage, disease duration (r=0.332, P=0.012), and tender joint count (r=0.408, P=0.002). The 24 weeks values of TRACP-5b for RhCTLA4-Ig group and infliximab group differed significantly from the baseline values in each group (P &lt;0.05; P &lt;0.05), whereas only the value for RhCTLA4-Ig group differed significantly from the 24 weeks value for the MTX group (P &lt;0.01). Considering the two biologics-treated groups together, the TRACP-5b levels at 24 weeks differed significantly from the baseline values only in those patients who reached an ACR70 level (P &lt;0.05). Conclusions Measurement of serum TRACP-5b in RA patients reflects clinical and radiological measures of disease activity, treatment with certain biologics, and degree of response to therapy. TRACP-5b should be investigated further as a potential biomarker to predict response to therapy, including slowing of radiographic progression.

Belatacept: A New Biologic and Its Role in Kidney Transplantation

To review the pharmacology, efficacy, safety, and role of belatacept in maintenance immunosuppression in adult kidney transplant recipients (KTR). PubMed, EMBASE, International Pharmaceutical Abstracts, Web of Knowledge (1990-November 2011), and Google were searched using the terms belatacept, kidney or renal, and transplant. Relevant articles (English language and human subjects) were reviewed. Selected studies included 3 Phase 2 and 2 Phase 3 trials. Data were compared with Food and Drug Administration (FDA) briefing documents and belatacept full prescribing information. Belatacept, a cytotoxic T-lymphocyte-associated antigen 4-immunoglobulin, is the first marketed intravenous maintenance immunosuppressant. It is approved for use in combination with basiliximab induction, mycophenolate mofetil, and corticosteroids to prevent rejection in adult KTR. Belatacept exhibits linear pharmacokinetics and first-order elimination. The less intensive regimen used in Phase 3 trials is approved by the FDA. In low-moderate immunologic risk KTR, short-term patient and allograft survival appear comparable with that seen with cyclosporine, with improved renal function despite more frequent and severe early acute rejection. Preliminary data from Phase 2 corticosteroid-avoidance and conversion trials suggest that better renal function, acceptable rejection rates, and comparable patient and allograft survival may be achieved with belatacept compared with calcineurin inhibitors (CNIs). Common adverse effects of belatacept include anemia, neutropenia, urinary tract infection, headache, and peripheral edema. While a more favorable cardiovascular and metabolic profile and lack of requirement for therapeutic drug monitoring are attractive, a higher frequency of posttransplant lymphoproliferative disorder is concerning. Belatacept drug costs are significantly higher than those of standard CNI- or sirolimus-based regimens. Belatacept provides a new option for maintenance immunosuppression in adult KTR. Further research is needed to compare its efficacy and safety with standard tacrolimus-based regimens, to evaluate whether increased drug costs are offset by long-term improvements in patient and allograft survival, and to establish its role in the immunosuppression armamentarium.

Simultaneous Blockade of Costimulatory Signals CD28-CD80 and CD40-CD154 Combined with Monoclonal Antibody against CD25 Induced a Stable Chimerism and Tolerance without Graft-versus-Host Disease in Rat

Background: The conditioning regimen to induce chimerism for immune tolerance is usually accompanied by high toxicity and graft-versus-host disease (GVHD). Our aim was to explore a nontoxic strategy for the induction of mixed chimerism by pretreatment with anti-CD25 monoclonal antibody (mAb), cytotoxic T-lymphocyte-associated antigen 4 immunoglobulin (CTLA4Ig) and anti-CD154 mAb. Methods: A total of 30 Lewis recipient rats (RT1l) were randomly divided into control (A and B) and treated (C, D and E) groups. Recipients serving as controls were without any pretreatment (group A) or pretreated with anti-CD25 mAb, CTLA4Ig and anti-CD154 mAb on days 0, 2, 4, 6 and 8 without bone marrow transplantation (BMT; group B). In the treated groups, the recipients were pretreated with anti-CD25 mAb and CTLA4Ig (group C), with anti-CD25 mAb and anti-CD154 mAb (group D) or with anti-CD25 mAb, CTLA4Ig and anti-CD154 mAb (group E) on days 0, 2, 4, 6 and 8 plus BMT [2 × 10⁸ unmodified whole bone marrow cells from Brown Norway donor rats (RT1n)] on day 0. Full-thickness skin grafts from donor-specific Brown Norway rats were grafted to the dorsal thoracic wall of Lewis recipients on day 8. GVHD was assessed after BMT, and chimerism and T cell apoptosis on days 7, 21, 35 and 49 were detected by flow cytometry. Results: GVHD was not observed in any groups. On days 7, 21 and 35, hematopoietic chimerism was present and maintained in the recipients of the 3 treated groups (groups C, D and E), and thereafter disappeared on day 49. The rate of chimerism in group E was significantly higher compared to that in group C on day 7 and that in group C or D on day 21, but there was no significant difference on day 35 among the 3 groups. The rate of T cell apoptosis in group C, D or E was significantly higher than in group A or B on days 14, 21 and 35. The grafted skin survival in group C, D or E was longer than in group A or B, and survival was significantly longer in group E than in group C or D. Conclusion: Preconditioning with anti-CD25 mAb, CTLA4Ig and anti-CD154 mAb could effectively induce chimerism and immune tolerance without GVHD in a major histocompatibility complex-disparate rat model. This strategy may be attractive for induction of transplantation tolerance. T cell apoptosis is one of the important considerations in tolerance induction.

Co-Stimulation Blockade as a New Strategy in Kidney Transplantation

New immunosuppressive drugs have greatly decreased the frequency of graft failure due to acute rejection but have had little impact on long-term graft survival. This is due, at least in part, to the broad non-immune effects of the current immunosuppressive drugs, which are involved in the death of patients and in chronic allograft dysfunction, particularly due to their nephrotoxicity. Recent progress in the development of biologicals, i.e. antibodies and fusion proteins, allows precise targeting of the immune system, preventing the non-immune side effects encountered with current protocols. In particular, targeting of the two most important co-stimulation pathways critical for T-cell activation, i.e. B7/CD28 and CD40/CD40L, has provided excellent results in many experimental models of organ transplantation. This has led to the clinical development of belatacept, a cytotoxic T-lymphocyte-associated antigen 4 immunoglobulin (CTLA4-Ig) fusion protein, which has proved to be efficient in preventing acute rejection in kidney transplant recipients. Its use is associated with improved renal function and a better metabolic profile than calcineurin inhibitors. However, because belatacept does not selectively target alloreactive T lymphocytes and must be combined with classical immunosuppressive drugs, infectious and neoplastic complications may occur, particularly post-transplantation lymphoproliferative disorders. We also address the current development of molecules targeting other co-stimulatory pathways (CD40/CD40L, leukocyte function-associated antigen [LFA]-1/intercellular adhesion molecule [ICAM], CD2/LFA-3). Many unresolved issues regarding the use of co-stimulation blocking agents are also discussed, e.g. their long half-life, which can be problematic in cases of serious adverse events, their long-term safety and efficacy, and the lack of monitoring tools to allow modulation of their use over time.

Effects of Gene Transfer CTLA4Ig and Anti-CD40L Monoclonal Antibody on Islet Xenograft Rejection in Mice

Blockade of a costimulatory pathway by adenovirus-mediated cytotoxic T lymphocyte associated antigen 4 immunoglobulin (CTLA4-Ig) gene transfer and anti-CD40L mAb(MR1) have been reported to enhance graft survival in several experimental transplantation models. In this study, we investigated the effects of gene transfer of CTLA4Ig and MR1 on islet xenograft rejection in mice. Recombinant adenovirus AdCTLA4Ig was constructed to express CTLA4Ig. Islet grafts from adult male DA rats transferred with AdCTLA4Ig were transplanted to streptozocin-induced diabetic Balb/c mice. The diabetic mice were treated with MR1 after transplantation. We evaluated the islet xenograft mean survival time as well as changes in interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-α) levels in transplanted mice. The mean survival of islet xenografts in the MR1 treatment group was 34.9 ± 5.62 days, in the AdCTLA4Ig treatment group it was 56.5 ± 10.64 days, and in the AdCTLA4Ig plus MR1 treatment group it was 112.9 ± 19.26 days, all significantly prolonged compared with an untreated group (8.1 ± 0.83 days). Within 1 week after transplantation the levels of IL-2 and TNF-α showed sharp increases in the untreated group, being significantly higher than those observed prior to transplantation. In conclusion, using both AdCTLA4Ig and MR1 can improve the islet xenograft survival. The beneficial effects of the combined use of the 2 reagents were superior to either 1 alone, possibly related to down-regulated expression of Th1 cell–related cytokines.

CTLA4-Ig interacts with cultured synovial macrophages from rheumatoid arthritis patients and downregulates cytokine production

IntroductionCo-stimulatory signal B7(CD80/CD86):CD28 is needed in order to activate T cells in immune response. Cytotoxic T lymphocyte-associated antigen-4-immunoglobulin (CTLA4-Ig) binding to the B7 molecules on antigen-presenting cells downregulates this activation and represents a recent biological treatment in rheumatoid arthritis (RA). Objectives of the study were to investigate the presence of the B7.2 (CD86) molecule and its masking by CTLA4-Ig on cultures of both RA synovial macrophages (RA SM), and of macrophages differentiated from THP-1 cells (M). In addition, the anti-inflammatory effects of CTLA4-Ig on co-cultures of RA SM and M with activated T cells were tested.MethodsAll macrophages were co-cultured for 24 hours with activated T cells, without or with CTLA4-Ig (10, 100, 500 μg/ml for 1 hour, 3 hours and overnight, respectively). Immunofluorescence (IF) staining for B7.2, and an analysis of inflammatory cytokine expression (interleukin (IL) -6, tumor necrosis factor (TNF) α, IL-1β, transforming growth factor (TGF) β) by immunocytochemistry (ICC), western blot (WB) and reverse transcriptase-polymerase chain reaction (RT-PCR) were performed.ResultsMacrophages showed intense B7.2 expression. CTLA4-Ig/B7.2 masking was evident for all macrophages, even after only 1 hour of cell culture (range from 10 to 100 μg/ml). ICC of co-cultures showed a dose-dependent decrease in inflammatory cytokines (P < 0.001 for IL-6, TNFα, IL-1β and TGFβ). Data were confirmed by WB and RT-PCR analysis.ConclusionsOptimal concentrations of CTLA4-Ig for the CTLA4-Ig/B7.2 masking on activated macrophages were identified and were found to induce significant downregulation in the cell production of IL-6, TNFα, IL1-β and TGFβ. In conclusion, macrophages would appear to be a sensitive target for CTLA4-Ig treatment in RA.

Transgenic Expression of Cytotoxic T-Lymphocyte-Associated Antigen 4-Immunoglobulin Prolongs Xenogeneic Skin Graft Survival Without Extensive Immunosuppression in Rat Burn Wounds

We sought to establish a transgenic animal line skin-specifically overexpressing cytotoxic T-lymphocyte-associated antigen 4-immunoglobulin (CTLA4Ig) as a reproducible source of xenogeneic skin grafts with extended survival for wound coverage. We tested this strategy in mice based on a previously established transgenic mouse line that stably and skin-specifically expresses CTLA4Ig for lifetimes and generations. CTLA4Ig expression was examined by immunohistochemical assay, and its bio-activity was tested by mixed lymphocyte reaction. The survival of transgenic mouse skin grafted onto rat burn wounds was observed. The impact of transgenic skin grafting on recipient immunity was evaluated by inspecting the survival of the wild-type skin grafted along with transgenic skin onto a separate wound on the same rat. The circulatory CTLA4Ig protein in recipient was detected by sandwich enzyme-linked immunosorbent assay, and its impact on recipient lymphocyte response against donor antigen was tested by mixed lymphocyte reaction. The transgenic CTLA4Ig protein suppressed lymphocyte proliferation in vitro, and the transgenic skin graft survival was remarkably prolonged compared with the wild-type skin derived from the same mouse strain. The survival of the wild-type skin grafted along with transgenic skin exhibited no significant difference from that grafted alone. Circulatory CTLA4Ig protein was detected in recipients, however, no significantly reduced recipient lymphocyte response against donor antigen was observed. transgenic expression of CTLA4Ig may be a potential and safe method to prolong xenogenic skin graft survival in burn wounds, and transgenic animal lines can be established as a reproducible source of skin grafts with extended survival for wound coverage.

Rapamycin, Mycophenolate Mofetil, Methylprednisolone, and Cytotoxic T-Lymphocyte–Associated Antigen 4 Immunoglobulin–Based Conditioning Regimen to Induce Partial Tolerance to Hind Limb Allografts Without Cytoreductive Conditioning

Background Composite tissue allograft transplantation may represent the next frontier in the field of reconstructive surgery. However, the main obstacles precluding the routine use of composite tissue allotransplants are rejection and toxicity associated with life-long immunosuppressive therapy. In this study, we investigated a nontoxic immunosuppressant and cytotoxic T-lymphocyte–associated antigen 4 immunoglobulin (CTLA4-Ig)–based protocol to induce donor-specific tolerance to hind limb allografts in rats. Methods Fully mismatched, 4- to 10-week-old Brown Norway (BN, RT1 n) and Lewis (RT 1) rats were used as cell/organ donors and recipients, respectively. Recipients were treated with CTLA4-Ig (2 mg/kg/d) on days -30, -28, -26, -24, and -22, rapamycin, mycophenolate mofetil, and methylprednisolone (RAPA/MMF/MP) combined therapy (from days -30 to day 100), a single dose of anti-lymphocyte serum (10 mg, on day -30), and donor bone marrow (10 × 10 7 T-cell–depleted cells) transplantation (BMT, on day -30). Thirty days after BMT, chimeric animals received hind limb allotransplantations (on day 0). The RAPA/MMF/MP combined therapy was changed to Cyclosporine (CsA, 8 mg/kg/d) on day 100 and maintained thereafter at this level. Results Hematopoietic chimerism of 17.6 ± 9.5% at day 0, was stable (15.2 ± 5.6%) at 230 days post-BMT; there was no sign of graft-versus-host disease. Chimeric recipients (Lewis) permanently accepted (>200 days) donor (BN)–specific (RT1 1, n = 6) hind limbs, yet rapidly rejected (20 ± 2 days) third-party hind limbs (Wistar Furth [WF]). Lymphocytes of graft-tolerant animals demonstrated hyporesponsiveness in mixed lymphocyte cultures in a donor-specific manner. Tolerant graft histology showed no signs of acute and chronic rejection. Conclusions The immunosuppressant and CTLA4-Ig–based conditioning regimen with donor BMT produced mixed chimerism and induced partial donor-specific tolerance to hind limb allografts.

Nitric Oxide and Indoleamine 2,3-Dioxygenase Mediate CTLA4Ig-Induced Survival in Heart Allografts in Rats

Cytotoxic T lymphocyte-associated antigen 4 immunoglobulin (CTLA4Ig) leads to transplantation tolerance in mice depending on indoleamine 2,3-dioxygenase (IDO). We have shown that CTLA4Ig induces indefinite heart allograft survival in rats and that nitric oxide (NO) was implicated in the in vitro active tolerogenic mechanisms mediated by dendritic cells (DCs). Here we studied the in vivo tolerogenic mechanisms by which CTLA4Ig induces graft survival in rats receiving a cardiac allograft. Treatment of recipients with the IDO inhibitor 1-methyltryptophan (1-MT) did not abrogate the indefinite graft survival observed with CTLA4Ig alone. This was also the case after administration of the inducible nitric oxide synthase inhibitor aminoguanidine when again, indefinite allograft survival was maintained. However, administration of both inhibitors led to acute rejection. We show that IDO and NO are responsible for the impaired capacity of DCs from CTLA4Ig-treated rats to stimulate allogeneic T cells. In conclusion, we show that NO and IDO mediate CTLA4Ig-induced tolerance in rat allograft recipients.