Cytotoxic T-lymphocyte Antigen 4-immunoglobulin Research Articles

형질전환 벼 현탁세포 배양에서 세포 사멸 억제를 통한 hCTLA4Ig 생산성 증대

Transgenic plant cell cultures are an attractive expression system for the production of industrial and pharmaceutical proteins because of their advantages in safety and low production cost. Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) was produced and secreted when sugar was depleted in culture medium by transgenic rice cell lines (Oryza sativa L.) using RAmy3D promoter. Due to the production of the target protein by sugar depletion, concomitant occurrence of cell death is inevitable. For that reason, inhibition of cell death for enhancing productivity was necessary for the production period without energy sources. Supplementation of 0.1 mM sodium nitroprusside improved cell viability by 1.4-fold and maximum hCTLA4Ig production by 1.3-fold compared to those of control. Addition of 1 and 10 mM glutathione, N-acetylcysteine (NAC), and nicotinamide inhibited apoptotic-like programmed cell death by decreasing the activity of reactive oxygen species. Production hCTLA4Ig was enhanced 1.4-, 1.25-, and 1.15-fold with 10 mM NAC, 1 mM NAC, and 1 mM glutathione, respectively. In addition, it was found that the supplementation of NAC enhanced the cell viability.

Assessment of long-term cryopreservation for production of hCTLA4Ig in transgenic rice cell suspension cultures

For the commercialization of plant-made pharmaceuticals (PMPs) using transgenic plant cell cultures, the establishment of a cell-banking system has been known to be an essential process. Plant cells are traditionally maintained by repeated subcultures. However, this method has several problems including genetic instability of transformed cell lines, time- and cost-consuming. In this study, long-term cryopreserved rice suspension cells were firstly investigated for the production of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig). The cryopreserved cells for 5 years were regrowed to callus successfully and then suspended into the liquid medium. Consequently, the maximum cell mass and the hCTLA4Ig production were similar levels compared to those of the non-cryopreserved cells (control) even though hCTLA4Ig productivity was 1.7-fold higher than that of control. To further assess the level of improvements in hCTLA4Ig productivity in cryopreserved cells, hCTLA4Ig production profiles were statistically assessed between data of the cryopreserved cells for 5 years and annual data of non-cryopreserved cells maintained by subculture for 5 years. These results also indicate that hCTLA4Ig productivity in cryopreserved cells for 5 years was significantly increased (p-value: <0.001, 95% confidence interval) and it could be related to cell lysis resulting in release of hCTLA4Ig which was confirmed by the measurement of electrolyte leakage. In conclusion, we show that the long-term cryopreservation of transgenic rice cells was possible to support stable cell lines for the production of PMPs.

Bioreactor engineering using disposable technology for enhanced production of hCTLA4Ig in transgenic rice cell cultures

Two kinds of disposable bioreactors, air-lift disposable bioreactors (ADB) and wave disposable bioreactors (WDB) were compared with stirred-tank reactors (5-L STR). These bioreactors were successfully applied to transgenic rice cell cultures for the production of recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig). In both systems, a fed-batch culture method was used to produce hCTLA4Ig efficiently by feeding concentrated amino acids and production levels were enhanced when dissolved oxygen (DO) level was regulated at 30% using pure oxygen sparging. Agitation and aeration rate during cultivation in ADB and WDB were determined by the same mixing time. The results in both disposable bioreactors showed similar values in maximum cell density (11.9 gDCW/L and 12.6 gDCW/L), doubling time (4.8- and 5.0-day), and maximum hCTLA4Ig concentration (43.7 and 43.3 mg/L). Relatively higher cell viability was sustained in the ADB whereas hCTLA4Ig productivity was 1.2-fold higher than that in WDB. The productivity was improved by increasing aeration rate (0.2 vvm). Overall, our experiments demonstrate pneumatically driven disposable bioreactors are applicable for the production of recombinant proteins in plant cell cultures. These results will be useful for development and scale-up studies of disposable bioreactor systems for transgenic plant cell cultures.

Process Characterization of hCTLA4Ig Production in Transgenic Rice Cell Cultures Using a 3-L Bioreactor

Most of the technical know-how and experience of bioreactor engineering is applicable to plant cell cultures. In this study, transgenic rice cell cultures using RAmy3D promoter were used for the production of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig). In process aspect, the rice cells during production phase are strongly influenced by hydrodynamic stresses, such as shear stress and bubble burst. Therefore, the effects of agitation and aeration rates on cell growth and hCTLA4Ig production were investigated in a 3-L multi-bioreactor. By increasing over 240 rpm, the detrimental effects on cell growth and hCTLA4Ig production were observed. At an aeration rate of 0.3 vvm, relative cell viability sharply decreased 2 days earlier than those of lower aeration rates. In addition, it was confirmed that the specific yields and the specific productivity at 0.3 vvm were superior to those values at 0.05 vvm. Overall, higher aeration rate showed the improved hCTLA4Ig production in combination experiment. High aeration rates in general, however, have an undesired effect as excessive aeration was found to negatively affect the quality of hCTLA4Ig. Consequently, the hydrodynamic conditions must be tightly regulated during bioreactor operation in order to enhance hCTLA4Ig productivity and quality in transgenic rice cell cultures.

Ex Vivo Alloanergization with Belatacept: A Strategy to Selectively Modulate Alloresponses after Transplantation

Ex vivo alloanergization of human immune cells, via allostimulation in the presence of costimulatory blockade with either a combination of anti-B7.1 and anti-B7.2 antibodies or first-generation cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4-Ig), induces alloantigen-specific hyporesponsiveness and expands alloantigen-specific regulatory T cells (Treg). We have successfully used this approach in the clinical setting of haploidentical hematopoietic stem cell transplantation. Recently, the in vivo use of a new second-generation CTLA4-Ig, belatacept, has shown promise in controlling alloresponses after transplantation of both human kidneys and islet cells. We therefore compared the efficiency of first- and second-generation CTLA4-Ig in alloanergizing human peripheral blood mononuclear cells (PBMCs) and investigated whether ex vivo alloanergization with belatacept could be used to engineer an alloantigen-specific immunoregulatory population of autologous cells suitable for administration to recipients of cellular or solid organ transplant recipients. Alloanergization of HLA-mismatched human PBMCs with belatacept resulted in a greater reduction in subsequent alloresponses than alloanergization with first generation CTLA4-Ig. Moreover, subsequent ex vivo re-exposure of alloanergized cells to alloantigen in the absence of belatacept resulted in a significant expansion of Tregs with enhanced alloantigen-specific suppressive function. Alloanergized PBMCs retained functional Epstein-Barr virus (EBV)-specific T-cell responses, and expanded Tregs did not suppress EBV-specific proliferation of autologous cells. These results suggest that ex vivo alloanergization with belatacept provides a platform to engineer populations of recipient Treg with specificity for donor alloantigens but without nonspecific suppressive capacity. The potential advantages of such cells for solid organ transplantation include (1) reduction of the need for nonspecific immunosuppression, (2) retention of pathogen-specific immunity, and (3) control of graft rejection, if used as an intervention.

Interleukin-6 Receptor Signaling Disruption Prevents Cardiac Allograft Deterioration in Mice

Interleukin-6, a pleiotropic cytokine that functions in both innate and adaptive immune responses, has been implicated in allograft rejection. We analyzed the efficacy of anti interleukin-6 receptor monoclonal antibody in delaying allograft rejection in a murine model of a heart. To investigate the role of interleukin-6 receptor signal transduction in acute and chronic allograft rejection, we blocked interleukin-6 receptor signaling to suppress the alloimmune response in C57BL/6 recipients of BALB/c cardiac allografts. Administration of a high-dose α-interleukin-6 receptor monoclonal antibody prevented the intragraft infiltration of inflammatory cells and lymphocytes and prolonged allograft survival during the peritransplant period. However, all allografts were rejected by 23.5 days after transplant. In contrast, cardiac allograft recipients treated with a cytotoxic T-lymphocyte antigen 4-immunoglobulin plus continued administration of low-dose α-interleukin-6 receptor monoclonal antibody showed long-term graft survival compared with cytotoxic T-lymphocyte antigen 4-immunoglobulin monotherapy. A histologic analysis revealed that graft fibrosis was prevented in cytotoxic T-lymphocyte antigen 4-immunoglobulin plus high-dose α-interleukin-6 receptor monoclonal antibody group, but not in the cytotoxic T-lymphocyte antigen 4-immunoglobulin alone group. This suggests that deterioration of graft function associated with chronic rejection could be prevented by blocking interleukin-6 receptor signaling. Disruption of interleukin-6 receptor signaling is an effective strategy for modulating proinflammatory immune responses and preventing chronic rejection.

Adsorptive loss of secreted recombinant proteins in transgenic rice cell suspension cultures

Adsorptive loss of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) in transgenic rice cell suspension cultures was investigated using glass flasks, plastic flasks, disposable vessels, and stainless steel vessels. When hCTLA4Ig was added to the glass flasks containing sterile AA medium, a rapid decrease in the concentration of hCTLA4Ig, independent on pH, was observed resulting in more than 90% of the protein loss within 1 h due to the surface adsorption. When the same experiments were performed on four different types of culture equipments mentioned above, the lowest adsorption level was observed in the plastic flasks and the highest level was observed in the glass flasks. The use of the plastic flasks retarded the adsorptive loss of hCTLA4Ig at the early stage of the protein production. There was a significant increase in the production of hCTLA4Ig when the flasks were coated with bovine serum albumin. However, the spike test of purified hCTLA4Ig at two different concentrations of 15 and 100 mg L(-1) in 500-mL spinner flasks confirmed that the amount of hCTLA4Ig adsorbed was dependent on the surface area of the flasks but not on the concentrations. In conclusion, although the protein adsorption affected the total amount of the protein yielded to some extent, it could be regarded as a minor factor in transgenic plant cell cultures with higher titer.

Biologics and Donor Bone Marrow Cells for Targeted Immunomodulation in Vascularized Composite Allotransplantation: A Translational Trial in Swine

Introduction Bone marrow (BM) infusion following organ transplantation is a prerequisite for potential donor-antigen-specific tolerance induction. We developed a preclinical swine model to determine the optimal dose of BM cells to achieve microchimerism. Furthermore, induction therapy was optimized by augmenting the BM infusion with biologics in the form of costimulatory blockade: cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA4-Ig). Materials and Methods Yucatan miniature swine ( n = 12) underwent total body and thymic irradiation for cytodepletion. Animal groups received 15, 30, or 60 million cells per kilogram of whole unmodified BM. The optimal dose of BM cell infusion (BMT) was then applied to subsequent experiments evaluating the addition of CTLA4lg. Group 1 (control) received no treatment. Group 2 received FK506 only; group 3 received irradiation, BMT, and FK506; group 4 received FK506 and CTLA4-lg. Results Microchimerism was established in all animals after BM cell infusion; at postoperative day 9, it was significantly increased for 60 million cells per kilogram ( P = .0001). Transplanted animals in group 1 rejected the allograft 5 to 8 days after transplantation. Group 2 rejected the allograft (skin and muscle) 30 to 32 days after transplantation (2 days after cessation of immunosuppression). Group 3 rejected the skin portion of the allograft at 50, 52, and 53 days posttransplant. Remaining allograft components (muscle, bone, nerve, vessel) survived indefinitely. Group 4 animals demonstrated significantly prolonged muscle survival beyond 150 days posttransplant; the skin component survived past 150 days in two of three animals. Skin and muscle histology in all long-term surviving animals were normal. Conclusions BM cell infusion with 60 million cells per kilogram results in stable levels of microchimerism. The addition of costimulatory blockade (CTLA4lg) prolonged allograft skin survival and overall graft survival. Such targeted immunomodulatory protocols might facilitate immune tolerance and eliminate the need for multidrug immunosuppression to maintain graft survival after vascularized composite allotransplantation.

Sodium butyrate와 sodium pyruvate 첨가에 의한 hCTLA4Ig 생산성 증대

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig), an immunosuppressive agent, was expressed in transgenic rice cells using RAmy3D promoter and RAmy1A signal peptide for the inducible production and secretion into culture media by sugar depletion. In this study, sodium butyrate was used as a small molecular enhancer (SME) to enhance the production of hCTLA4Ig in transgenic rice cell suspension cultures. When 1 mM sodium butyrate was added in sugar-free media, relative viability was not reduced, while the productivity was improved 1.3-fold. In addition, by supplementing 87 mM sodium pyruvate as an alternative energy source during the production phase, death rate of the cells was decreased. When sodium pyruvate was not added, most cells became dead at day 6. However, by adding sodium pyruvate, 18% of viability can be maintained until day 10 and the production of hCTLA4Ig was enhanced 1.4-fold. When the combination of sodium pyruvate and sodium butyrate at optimum concentrations was added, the highest viability and hCTLA4Ig production could be obtained. The highest level of hCTLA4Ig reached up to 35 mg/L at day 10.

Cytotoxic T lymphocyte antigen 4 immunoglobulin modified dendritic cells attenuate allergic airway inflammation and hyperresponsiveness by regulating the development of T helper type 1 (Th1)/Th2 and Th2/regulatory T cell subsets in a murine model of asthma

T helper type 2 (Th2) and regulatory T cells (T(reg) ) have been postulated to have critical roles in the pathogenesis of allergic asthma. Cytotoxic T lymphocyte antigen 4 immunoglobulin (CTLA4Ig) gene-modified dendritic cells (DC-CTLA4Ig) have the potential to reduce Th2 cells and induce T(reg) cells. In the present study, we evaluated the therapeutic effects and potential mechanisms of the adoptive transfer of DC-CTLA4Ig into mice in an experimental model of asthma. BALB/c mice were sensitized with ovalbumin (OVA) and challenged with aerosolized OVA for 7 days. Just prior to the first challenge, DC-CTLA4Ig, DCs or DCs infected with DC-green fluorescent protein (GFP) were injected intravenously into mice. The administration of DC-CTLA4Ig reduced airway hyperresponsiveness, relieved asthmatic airway inflammation and decreased the numbers of esosinophils in the BALF in OVA-sensitized/challenged mice. In addition, DC-CTLA4Ig altered the balance of Th1/Th2 cytokine production in the lungs with increased interferon (IFN)-γ levels and decreased interleukin (IL)-4 levels, decreased the percentage of Th2 and increased both the percentage of Th1 and T(reg) cells in the lungs of OVA-sensitized/challenged mice. This research demonstrates that DC-CTL4Ig reduces airway hyperresponsiveness effectively and prevents airway inflammation in OVA-sensitized/challenged mice, which is due most probably to attenuated secretion of Th2 cytokines and increased secretion of Th1 cytokines in the local airway, and the correction of the pulmonary imbalance between Th1/Th2 cells and Th2/T(reg) cells.

Production and characterization of LEA29Y, a variant of cytotoxic T-lymphocyte antigen 4-immunoglobulin, in Pichia pastoris

Blocking the CD28/B7 costimulatory pathway is a promising strategy in the treatment of graft rejection, graft-versus-host disease and autoimmune diseases. LEA29Y, a high-affinity variant of cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4Ig), is a more potent inhibitor of the interaction between CD28 and B7 than is CTLA4Ig. In a previous study, LEA29Y was produced in a mammalian cell system, which is time-consuming and expensive. To obtain LEA29Y more efficiently and cost effectively, we attempted to produce LEA29Y using a Pichia pastoris expression system. The gene encoding LEA29Y, with an additional 6-His tag at the N-terminus, was cloned into the yeast vector pPIC9K and expressed in the P. pastoris strain GS115. Under the optimized induction conditions for protein expression (inoculum density, OD(600) = 80; methanol concentration added daily, 1.0-3.0%; induction time point, 72-96 h; culture medium pH = 6.0), the yield of purified LEA29Y was approximately 30 mg l(-1) by one-step Ni-agarose affinity chromatography. PNGase F treatment showed the purified LEA29Y to be post-translational modified by N-linked glycosylation. In biological function assays, LEA29Y expressed in P. pastoris demonstrated specific binding to B7-1/B7-2-positive Raji cells and also suppressed lymphocyte proliferation in a dose-dependent manner. These results suggest that LEA29Y produced in P. pastoris is biologically active and will be useful for experimental therapy on immunotherapy for transplant rejection and autoimmune diseases.

Effect of Triple Costimulation Blockade on Islet Allograft Survival in Sensitized Mice

Background Islet allograft rejection in sensitized recipients is difficult to control by costimulation blockade using anti-CD154 and cytotoxic T-lymphocyte antigen-4 immunoglobulin (CTLA4Ig). Because leukocyte function antigen (LFA) 1 is highly expressed on memory T cells, adding an LFA-1 blockade may inhibit memory T-cell activities. We examined the effects on islet allograft survival of triple costimulation blockade in presensitized recipient mice. Methods C57BL/6 mice were sensitized by transplantation under the kidney capsule or intraperitoneal injection of Balb/c islets. Four weeks after transplantation, sensitization was confirmed by flow-cytometric detection of alloreactive antibodies. Diabetes was induced by a single intravenous injection of streptozotocin. Recipients were transplanted with 200 Balb/c islets under the right kidney capsule. Graft function was assessed by daily blood glucose and body weight records. Transplanted animals were divided into 3 treatment groups: group 1, control antibody; group 2, anti-CD154 and CTLA-4 Ig double therapy; group 3, anti-CD154, CTLA4Ig, and anti–LFA-1 triple therapy. Injections were administered every second day from day −2 to day 8. Results Naïve mice rejected islet allografts between days 7 and 29 (mean 16 ± 6 d; n = 5), sensitized mice in group 1 between days 0 and 14 (mean 7 ± 5 d; n = 8), in group 2 between days 4 and 16 (mean 8 ± 4 d; n = 7), and in group 3 between days 4 and 26 (mean 11 ± 7 d; n = 10). Conclusion Triple costimulation blockade with anti-CD154, CTLA4Ig, and anti–LFA-1 was not sufficient to improve islet allograft survival in sensitized recipients.

Fed-batch cultivation of transgenic rice cells for the production of hCTLA4Ig using concentrated amino acids

RAmy3D promoter is capable of expressing high levels of recombinant proteins in response to the depletion of sugar in transgenic rice cell suspension cultures. For this reason, it is necessary to change the growth medium into sugar-free production medium to produce the target protein, human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig), using the inducible RAmy3D promoter. Since the two-stage culture is a complex process to perform in large-scale, a fed-batch method was evaluated with the addition of concentrated amino acids before the depletion of sugar to induce hCTLA4Ig production. This fed-batch culture was found to be effective and the production of hCTLA4Ig was enhanced up to 1.2-fold compared to that of two-stage cultures with medium exchange. In addition, when this fed-batch culture was performed in a 15-l stirred-tank bioreactor, maximum hCTLA4Ig level was 76.5 mg l −1 at day 10.

Effects of culture media on hCTLA4Ig production and protein expression patterns in transgenic rice cell suspension cultures

The effects of culture media on the production of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) and intracellular protein expression patterns were investigated in transgenic rice cell suspension cultures. Using comparative proteomic analysis, changes in the intracellular proteome in different culture media were identified. Culture media were found to be an important factor for the production of the recombinant target protein in this expression system, which was under the control of the rice α-amylase 3D (RAmy3D) promoter. In terms of hCTLA4Ig production, the N6 medium produced a 3.7-fold higher level of protein than the AA medium. In addition, the N6 medium provided better protein stability and cell viability. In the intracellular proteome analysis, we identified eight proteomes that were differentially expressed. These results could provide valuable information for the improvement of cell growth and target protein production.

Therapeutic effects of antibodies to tumor necrosis factor-α, interleukin-6 and cytotoxic T-lymphocyte antigen 4 immunoglobulin in mice with glucose-6-phosphate isomerase induced arthritis

IntroductionImmunization with glucose-6-phosphate isomerase (GPI) induces severe arthritis in DBA/1 mice. The present study was designed to identify the cytokines and co-stimulatory molecules involved in the development of GPI-induced arthritis.MethodsArthritis was induced in DBA/1 mice with 300 μg human recombinant GPI. CD4+ T cells and antigen-presenting cells from splenocytes of arthritic mice were cultured in the presence of GPI. Tumor necrosis factor (TNF)-α, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-12 levels were assessed using cytometric bead array. Monoclonal antibodies to TNF-α, IFN-γ, IL-12, CD40L, inducible co-stimulator (ICOS), and cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA-4Ig) were used to block TNF-α and IFN-γ production, examine clinical index in mice with GPI-induced arthritis, and determine anti-GPI antibody production.ResultsLarge amounts of TNF-α and IFN-γ and small amounts of IL-2 and IL-6 were produced by splenocytes from mice with GPI-induced arthritis. Anti-TNF-α mAbs and CTLA-4Ig suppressed TNF-α production, whereas anti-IFN-γ mAbs, anti-IL-12 mAbs, and CTLA-4 Ig inhibited IFN-γ production. A single injection of anti-TNF-α and anti-IL-6 mAbs and two injections of CTLA-4Ig reduced the severity of arthritis in mice, whereas injections of anti-IFN-γ and anti-IL-12 mAbs tended to exacerbate arthritis. Therapeutic efficacy tended to correlate with reduction in anti-GPI antibodies.ConclusionTNF-α and IL-6 play an important role in GPI-induced arthritis, whereas IFN-γ appears to function as a regulator of arthritis. Because the therapeutic effects of the tested molecules used in this study are similar to those in patients with rheumatoid arthritis, GPI-induced arthritis appears to be a suitable tool with which to examine the effect of various therapies on rheumatoid arthritis.

Negative and Positive Co-Signaling With Anti-BTLA (PJ196) and CTLA4Ig Prolongs Islet Allograft Survival

The novel coinhibitory receptor B and T lymphocyte attenuator (BTLA) has been implicated in the regulation of autoimmune and may potentially play a role in alloimmune responses. An anti-BTLA monoclonal antibody has been reported to prolong fully major histocompatibility complex-mismatched cardiac allograft survival, and we test the hypothesis that anti-BTLA monoclonal antibody PJ196 may synergize with cytotoxic T lymphocyte antigen-4 immunoglobulin (CTLA4Ig) costimulatory blockade in islet transplantation. We investigated the potential of PJ196, and show that it did not deplete BTLA expressing cells, but it caused down-regulation of BTLA on the surface of lymphocytes and accumulation of cells with regulatory phenotype at the graft site, promoting islet allograft acceptance together with CTLA4Ig. The combination of BTLA coinhibitory modulation and CTLA4Ig costimulatory blockade may be an effective adjunctive strategy for inducing long-term allograft survival.

Donor-Derived T Cells Can Be Rendered Hyporesponsive to Alloantigen without Loss of Pathogen or Tumor Immune Responses.

The prevention of severe acute Graft-versus-Host Disease (GvHD) without impairment of immune reconstitution is the major challenge in HLA-mismatched hematopoietic stem cell transplantation (HSCT). One alternative to experimental strategies to selectively destroy or remove alloreactive T cells from the donor T cell pool is to induce hyporesponsiveness (anergy) in alloreactive T cells within the donor T cell pool and thus preserve the vast majority of T cell repertoire. We previously reported early clinical data of HLA-mismatched HSCT after alloanergization of donor bone marrow via ex vivo allostimulation in the presence of co-stimulatory blockade (CSB) with Cytotoxic T Lymphocyte Antigen-4 Immunoglobulin (CTLA4-Ig). Analysis of a larger cohort of such patients revealed a low rate of severe acute GvHD and very few clinically significant viral infections, with over 30% of patients (pts) surviving long-term without disease relapse. This suggested that CSB might indeed be controlling alloreactivity with preservation of pathogen-specific immunity and a graft-versus-leukemia (GvL) effect. We therefore sought to directly determine the effect of alloanergization of human donor T cells on alloreactivity, pathogen- and leukemia-antigen-specific immunity. After alloanergization via blockade of CD28-mediated co-stimulation with clinical-grade humanized anti-B7.1 and anti B7.2 antibodies, HLA-mismatched alloproliferative responses were reduced by 2 logs, a more efficient reduction in alloreactivity than previously reported with the use of CTLA4 Ig. Using CFSE-based labeling of human responder T cells we have demonstrated directly for the first time that alloanergization efficiently abrogates stimulator-specific alloproliferation in both CD4 and CD8 donor T cells, whereas third party responses are retained (Figure 1). Importantly, the strategy does not diminish the capacity of donor CD4 and CD8 T cells to mount a range of functional immune responses, including proliferation, cytokine production and cytotoxic responses, in response to stimulation with several human herpes viruses. We have also demonstrated that frequencies of WT1-specific IFN-g+ CD4 and CD8 T cells are not diminished after the process of alloanergization, showing that a T cell mediated GvL effect may be retained. Importantly we demonstrated retention of pathogen and leukemia antigen-specific responses to both MHC Class I- and II-restricted antigens and in both HLA-A2+ and non-HLA-A2+ responders. These data confirm that the technique of alloanergization can be used to provide non-alloreactive donor T cells without loss of beneficial CD4 and CD8 donor immunity. The optimal dose of HLA-mismatched alloanergized donor T cells that will improve immune reconstitution whilst controlling acute GvHD after HLA-mismatched HSCT remains to be defined. To answer this question, we have embarked on a dose-escalating clinical study of delayed alloanergized donor T cell infusion to improve immune reconstitution after haploidentical HSCT. [Display omitted]

Immunomodulatory and osteogenic differentiation effects of mesenchymal stem cells by adenovirus‐mediated coexpression of CTLA4Ig and BMP2

Many reports have previously utilized a human bone morphogenetic protein 2 (BMP2)-expressing recombinant adenoviral vector (AdBMP2) and mesenchymal stem cells (MSCs) for osteoinductive gene therapy. However, immunosuppression is essential for osteoinduction by AdBMP2, and this is one of the major impediments to its clinical use. In the present study, in vitro propagated MSCs were transduced using an adenoviral (Ad) vector to express the gene encoding cytotoxic T lymphocyte antigen 4-immunoglobulin (CTLA4Ig). Lymphocyte response was induced by allogeneic-irradiated MSCs as stimulators. We also examined the effects of cotransfection with a combination of the CTLA4Ig and the BMP2 gene on osteoblastic cell differentiation. The results showed that BMP2 gene transfected MSC elicited significant stimulatory responses, and one-way MLR reactions were significantly blunted by CTLA4Ig. Further study demonstrates that cotransfection of MSCs with the combination of the CTLA4Ig and the BMP2 gene stimulates osteoblastic cell differentiation in vitro. The findings suggest that genetic engineering of MSCs to express an immunosuppressive molecule in combination with an osteogenic protein gene may have potential application in the treatment of several genetic diseases and in bone reconstruction.

The Glycosylation and Pharmacokinetics of CTLA4Ig Produced in Rice Cells

Cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4Ig) has immunosuppressive activity and the ability to induce immune tolerance. There has been no report of its glycosylation ratio or of the role of its glycans. We investigated the terminal sialylation of rice cell-derived recombinant human CTLA4Ig (rrhCTLA4Ig) using lectins. The glycosylation ratios of rrhCTLA4Ig and Chinese hamster ovary (CHO) cell-derived recombinant human CTLA4Ig (crhCTLA4Ig) were evaluated by chemical deglycosylation. After intravenous (i.v.) or subcutaneous (s.c.) administration to rats, the pharmacokinetics of rrhCTLA4Ig and crhCTLA4Ig as well as of their deglycosylated forms were evaluated. rrhCTLA4Ig does not have terminal sialic acids and its glycosylation ratio was slightly lower than that of crhCTLA4Ig. Its terminal elimination half-life (T(1/2)) was shorter than that of crhCTLA4Ig following i.v. administration. However, the half-life was significantly prolonged and was similar with that of crhCTLA4Ig following s.c. administration. Moreover, the deglycosylated forms of both preparations were cleared from the circulation faster than the native forms. These results suggest that the presence of glycans on rrhCTLA4Ig and crhCTA4Ig are important for their in vivo stability. In addition, the glycan structure of rrhCTLA4Ig is more effective in maintaining in vivo stability after s.c. administration than after i.v. administration although the glycans on rrhCTLA4Ig lack terminal sialic acids, suggesting that its glycans have the potential for in vivo stability.

Cryopreservation of transgenic rice suspension cells producing recombinant hCTLA4Ig

Transgenic suspension cells of Oryza sativa L. cv. Dongjin utilized as a host for producing recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) were preserved in liquid nitrogen (-196 degrees C) after slow prefreezing in a deep freezer (-70 degrees C). The development of an optimal procedure for long-term storage was investigated by the addition of various concentrations of cryoprotectant mixture and osmoticum in preculture media before cooling. A pre-deep-freezing time of 120 min was the most effective for maintaining cell viability. Compared with mannitol, sorbitol, trehalose, and NaCl under the same osmotic conditions, 0.5 M sucrose was found to be the best osmoticum for preculture media. The cryoprotectant comprising sucrose, glycerol, and dimethylsulfoxide (DMSO) was applied to the precultured cells, and a combination of 1 M sucrose, 1 M glycerol, and 1 M DMSO provided the best result. The viability with this optimized condition was 88% after cryocell-banking for 1 day. The expression of hCTLA4Ig in recovered callus from cryopreservation was also kept stable, and the production level was similar to that observed in noncryopreserved cultures.