Light Gradient-Based Screening of Arabidopsis thaliana on a 384-Well Type Plant Array Chip.

Arabidopsis thaliana (Arabidopsis), as a model for plant research, is widely used for various aspects of plant science. To provide a more sophisticated and microscopic environment for the germination and growth of Arabidopsis, we report a 384-well type plant array chip in which each Arabidopsis seed is independently seeded in a solid medium. The plant array chip is made of a poly(methyl methacrylate) (PMMA) acrylic material and is assembled with a home-made light gradient module to investigate the light effects that significantly affect the germination and growth of Arabidopsis. The light gradient module was used to observe the growth pattern of seedlings according to the intensity of the white light and to efficiently screen for the influence of the white light. To investigate the response to red light (600 nm), which stimulates seed germination, the light gradient module was also applied to the germination test. As a result, the germination results showed that the plant array chip can be used to simultaneously screen wild type seeds and phytochrome B mutant seeds on a single array chip according to the eight red light intensities.

Successful kidney transplantation across a positive complement-dependent cytotoxicity crossmatch by using C1q assay-directed, bortezomib-assisted desensitization: A case report.

RATIONALE: Human leukocyte antigen (HLA) is the major immunologic barrier in kidney transplantation (KT). Various desensitization protocols to overcome the HLA barrier have increased the opportunity for transplantation in sensitized patients. In addition, technological advances in solid-phase assays have permitted more comprehensive assessment of donor-specific antibodies. Although various desensitization therapies and immunologic techniques have been developed, the final transplantation decision is still based on the classic complement-dependent cytotoxicity (CDC) crossmatch (XM) technique. Some patients who fail to achieve negative XM have lost their transplant opportunities, even after receiving sufficient desensitization therapies. PATIENT CONCERNS: A 57-year-old male with end-stage renal disease secondary to chronic glomerulonephritis was scheduled to have a second transplant from his son, but CDC XM was positive. DIAGNOSES: Initial CDC XM (Initial T-AHG 1:32) and flow-cytometry XM were positive. Anti-HLA-B59 donor specific antibody was detected by Luminex single antigen assay. INTERVENTIONS: Herein, we report a successful case of KT across a positive CDC XM (T-AHG 1:8 at the time of transplantation) by using C1q assay-directed, bortezomib-assisted desensitization. After confirming a negative conversion in the C1q donor-specific antibody, we decided to perform KT accepting a positive AHG-CDC XM of 1:8 at the time of transplantation. OUTCOMES: The posttransplant course was uneventful and a protocol biopsy at 3 months showed no evidence of rejection. The patient had excellent graft function at 12 months posttransplant. LESSONS: The results of XM test and solid-phase assay should be interpreted in the context of the individual patient.

Plant array chip for the germination and growth screening of Arabidopsis thaliana.

A screening process for the germination and growth of seed is generally required for plant research. Such a repetitive screening process is costly and time-consuming, and its bulky setup requires a lot of space. In particular, the control of the variables, such as light, nutrients, hormones and temperature, is difficult due to the limited space for incubation. In addition, small seeds such as Arabidopsis thaliana are difficult to handle as they are hundreds of microns in diameter and require a more precisely controllable screening environment. However, conventional screening methods involve the seeding of multiple seeds on a single agarose plate without physical partitions. Such methods need to be improved because they lack control over the growth environment and the results are highly dependent on the researchers. To overcome the above-mentioned limitations, a novel seeding array chip has been developed which can be filled with conventional solid agarose while enabling more efficient screening. Individual seeds can be partitioned from each other and a number of different agarose conditions can be tested in a single plant array chip. As a demonstration, we tested the effect of various concentrations of Murashige and Skoog medium and a plant hormone (e.g., abscisic acid) on the growth of Arabidopsis. The chip can efficiently save the space required for screening by providing different conditions for ∼400 seeds in a 59 × 55 mm chip, and it also provides easy observation and analysis of seed growth. The proposed plant array chip is expected to contribute to more efficient screening of essential phenotypes such as germination and growth for both academic and industrial purposes.

Activation of Epidermal Growth Factor Receptor/p38/Hypoxia-inducible Factor-1α Is Pivotal for Angiogenesis and Tumorigenesis of Malignantly Transformed Cells Induced by Hexavalent Chromium.

Hexavalent chromium (Cr(VI))-containing compounds are well established environmental carcinogens. Most mechanistic investigations of Cr(VI)-induced carcinogenesis focus on oxidative stress and various cellular responses, leading to malignant cell transformation or the first stage of metal-induced carcinogenesis. The development of malignantly transformed cells into tumors that require angiogenesis is the second stage. This study focuses on the second stage, in particular, the role of EGF receptor (EGFR) signaling in angiogenesis and tumorigenesis of Cr(VI)-transformed cells. Our preliminary studies have shown that EGFR is constitutively activated in Cr(VI)-transformed cells, in lung tissue from Cr(VI)-exposed animals, and in lung tumor tissue from a non-smoking worker occupationally exposed to Cr(VI) for 19 years. Using in vitro and in vivo models, the present study has investigated the role of EGFR in angiogenesis of Cr(VI)-transformed cells. The results show that Cr(VI)-transformed cells are angiogenic. Hypoxia-inducible factor-1α, pro-angiogenic protein matrix metalloproteinase 1, and VEGF are all highly expressed in Cr(VI)-transformed cells, in lung tissue from animals exposed to Cr(VI), and in lung tumor tissue from a non-smoking worker occupationally exposed to Cr(VI) for 19 years. p38 MAPK is also activated in Cr(VI)-transformed cells and in human lung tumor tissue. Inhibition of EGFR reduces p38 MAPK, resulting in decreased expression of hypoxia-inducible factor-1α, metalloproteinase 1, and VEGF, leading to suppressions of angiogenesis and tumorigenesis. Overall, the present study has demonstrated that EGFR plays an important role in angiogenesis and tumorigenesis of Cr(VI)-transformed cells.

On-chip generation of monodisperse giant unilamellar lipid vesicles containing quantum dots.

Monodispersed lipid vesicles have been used as a drug delivery vehicle and a biochemical reactor. To generate monodispersed lipid vesicles in the nano- to micrometer size range, an extrusion step should be included in conventional hand-shaking method of lipid vesicle synthesis. In addition, lipid vesicles as a drug carrier still need to be improved to effectively encapsulate concentrated biomolecules such as cells, proteins, and target drugs. To overcome these limitations, this paper reports a new microfluidic platform for continuous synthesis of small-sized (∼10 µm) giant unilamellar vesicles (GUVs) containing quantum dots (QDs) as a nanosized model drug. To generate GUVs, we introduced an additional cross-flow to break vesicles into small size. 1,2 - dimyristoyl-sn-glycero - 3 - phosphocholine (DMPC) in an octanol-chloroform mixture was used in the construction of self-assembled membrane. Consequently, we have successfully demonstrated the fabrication of monodispersed GUVs with 7-12 µm diameter containing QDs. The proposed synthesis method of cell-sized GUVs would be highly desirable for applications such as multipurpose drug encapsulation and delivery.

Human bronchial epithelial BEAS-2B cells, an appropriate in vitro model to study heavy metals induced carcinogenesis.

Occupational and environmental exposure to arsenic (III) and chromium VI (Cr(VI)) have been confirmed to cause lung cancer. Mechanisms of these metals carcinogenesis are still under investigation. Selection of cell lines to be used is essential for the studies. Human bronchial epithelial BEAS-2B cells are the cells to be utilized by most of scientists. However, due to p53 missense mutation (CCG→TCG) at codon 47 and the codon 72 polymorphism (CGC→CCC) in BEAS-2B cells, its usage has frequently been questioned. The present study has examined activity and expression of 53 and its downstream target protein p21 upon acute or chronic exposure of BEAS-2B cells to arsenic and Cr(VI). The results show that short-term exposure of BEAS-2B cells to arsenic or Cr(VI) was able to activate both p53 and p21. Chronic exposure of BEAS-2B cells to these two metals caused malignant cell transformation and tumorigenesis. In arsenic-transformed BEAS-2B cells reductions in p53 promoter activity, mRNA expression, and phosphorylation of p53 at Ser392 were observed, while the total p53 protein level remained the same compared to those in passage-matched parent ones. p21 promoter activity and expression were decreased in arsenic-transformed cells. Cr(VI)-transformed cells exhibit elevated p53 promoter activity, mRNA expression, and phosphorylation at Ser15, but reduced phosphorylation at Ser392 and total p53 protein level compared to passage-matched parent ones. p21 promoter activity and expression were elevated in Cr(VI)-transformed cells. These results demonstrate that p53 is able to respond to exposure of arsenic or Cr(VI), suggesting that BEAS-2B cells are an appropriate in vitro model to investigate arsenic or Cr(VI) induced lung cancer.

Diagnostic value of tolerance-related gene expression measured in the recipient alloantigen-reactive T cell fraction.

The efficient development of tolerance-inducing therapies and safe reduction of immunosuppression should be supported by early diagnosis and prediction of tolerance in transplantation. Using mouse models of donor-specific tolerance to allogeneic skin and islet grafts we tested whether measurement of tolerance-related gene expression in their alloantigen-reactive peripheral T cell fraction efficiently reflected the tolerance status of recipients. We found that Foxp3, Nrn1, and Klrg1 were preferentially expressed in conditions of tolerance compared with rejection or unmanipulated controls if their expression is measured in CD69(+) T cells prepared from coculture of recipient peripheral T cells and donor antigen-presenting cells. The same pattern of gene expression was observed in recipients grafted with either skin or islets, recipients of different genetic origins, and even those taking immunosuppressive drugs. These findings suggest that the expression of tolerance-related genes in the alloantigen-reactive T cell fraction could be used to detect tolerance in the clinic.

Hypothermia enhances induction of protective protein metallothionein under ischemia.

BACKGROUND: Hypothermic protection against ischemic stroke has been reported by many studies. Hypothermia is supposed to mitigate the effects of deleterious genes and proteins and promote the activity of protective genes and proteins in the ischemic brain. Metallothionein (MT)-1/2 is thought to be a crucial factor for metal homeostasis, immune function, and apoptosis. This protein was found to exert protective effects in models of brain injury as well. In the present study, we investigated the effect of hypothermia on MT expression and the underlying mechanisms. METHODS: Cultured bEnd.3 brain endothelial cells were exposed to oxygen glucose deprivation and reperfusion (OGD+R). Reverse transcription PCR and western blot analyses were performed to measure the expression of MT, transcription factors, and methylation regulating factors. Transcription factor binding assays were also performed. Methylation profiles of the promoter area were obtained with pyrosequencing. RESULTS: Hypothermia protected bEnd.3 cells from OGD+R. When the cells were exposed to OGD+R, MT expression was induced. Hypothermia augmented MT levels. While OGD+R-induced MT expression was mainly associated with metal regulatory transcription factor 1 (MTF-1), MT expression promoted by hypothermia was primarily mediated by the signal transducer and activator of transcription 3 (STAT3). Significantly increased STAT3 phosphorylation at Ser727 was observed with hypothermia, and JSI-124, a STAT-3 inhibitor, suppressed MT expression. The DNA demethylating drug 5-aza-2'-deoxycytidine (5-Aza) enhanced MT expression. Some of the CpG sites in the promoter MT=> it should be "the CpG sites in the MT promoter" showed different methylation profiles and some methylation regulating factors had different expressional profiles in the presence of OGD+R and hypothermia. CONCLUSIONS: We demonstrated that hypothermia is a potent inducer of MT gene transcription in brain endothelial cells, and enhanced MT expression might contribute to protection against ischemia. MT gene expression is induced by hypothermia mainly through the STAT3 pathway. DNA methylation may contribute to MT gene regulation under ischemic or hypothermic conditions.

An effective immune-monitoring protocol based on gene expression profiles in the peripheral T-cell fraction reactive to graft antigens.

BACKGROUND: The ability to induce tolerance, or at least minimize the need for immunosuppressive therapy, is a high priority in organ transplantation. Accomplishing this goal requires a novel method for determining when a patient has become tolerant to or is rejecting their graft. Here, we sought to develop an efficient monitoring protocol based on gene expression profiles of recipient T cells in murine skin and islet allograft models. METHODS: Unlike previous studies, here, gene expression analysis was focused on donor antigen-reactive T cells, which were prepared by collecting CD69(+) T cells from cocultures of recipient peripheral T cells and donor antigen-presenting cells. Candidate tolerance and rejection biomarker genes were selected from a CD69(+) T-cell microarray analysis, and their expression levels were measured in the recipient CD69(+) T-cell fraction using quantitative reverse transcription polymerase chain reaction. RESULTS: Our new monitoring protocol was capable of precisely detecting the immune status of recipients relative to their graft regardless of the organ received, whether they were taking immunosuppressive drugs, or different strains of origin. CONCLUSIONS: Gene expression analysis focusing on recipient CD69(+) T cells as the donor antigen-reactive T-cell population could be used as an effective and sensitive method for monitoring transplant patients.

Aurintricarboxylic acid promotes the conversion of naive CD4+CD25- T cells into Foxp3-expressing regulatory T cells.

Naive peripheral CD4(+)CD25(-) T cells can be converted into Foxp3-expressing regulatory T cells under appropriate stimulation conditions. Considering that continuous exposure to antigens is one of the prerequisites for the differentiation and maintenance of Treg cells, we investigated whether preventing activation-induced cell death while providing continuous TCR stimulation could promote the expression of Foxp3 in murine naive CD4(+) T cells. Among the several anti-apoptotic agents tested, aurintricarboxylic acid (ATA) was found to induce the in vitro conversion of naive CD4(+) T cells into Foxp3(+) Treg cells with suppressive activity. Neutralizing studies with an antibody against transforming growth factor (TGF)-ß revealed that ATA requires the presence of TGF-ß to induce Foxp3 expression in naive CD4(+)CD25(-) T cells. Although ATA itself did not activate the Smad signaling pathway, it down-regulated the extracellular signal-regulated kinase and mammalian target of rapamycin signaling cascade in activated T cells. Lastly, combined exposure to ATA and TGF-ß had a synergistic effect on the rate of induction and maintenance of Foxp3 expression. These results indicate that ATA could be exploited to efficiently prepare inducible regulatory T cells in vitro and may aid in more precisely identifying the specific signaling pathways that drive Foxp3 expression in T cells.

Impact of immunosuppressants on the therapeutic efficacy of in vitro-expanded CD4+CD25+Foxp3+ regulatory T cells in allotransplantation.

BACKGROUND: Although the therapeutic potential of regulatory T lymphocytes (Tregs) in preventing allograft rejection has been well documented, accumulating evidence indicates that supplemental measures, such as concomitant use of immunosuppressive agents, are essential for effective application of Treg cell therapy in clinical transplantation. Thus, it is important to know the effect of immunosuppressive agents on Treg cell therapy. METHODS: We examined the impact of various immunosuppressive agents on the in vivo proliferation and therapeutic efficacy of in vitro-expanded Tregs using the murine graft-versus-host reaction and skin allograft model (BDF1 [H-2] to C57BL/6 [H-2]), respectively. RESULTS: All six immunosuppressive agents tested inhibited the alloantigen-stimulated proliferation of Tregs as efficiently as they inhibited the proliferative response of conventional CD3 T cells. We further show that blockade of the CD40-CD40L interaction by treatment with a MR-1 antibody significantly increased the therapeutic efficacy of Tregs, a synergistic effect that seemed to be related to the strong regulatory activity of adoptively transferred Tregs together with effector T-cell hyporesponsiveness. Although concomitant use of rapamycin marginally augmented the therapeutic effectiveness of Tregs, mycophenolate mofetil and cyclosporine A at their full therapeutic doses exerted an antagonistic effect on Treg cell therapy. CONCLUSION: These results demonstrate that inhibition of CD40-CD40L interaction or treatment with rapamycin could be successfully combined with in vitro-expanded Treg cell therapy, but the concomitant use of mycophenolate mofetil or cyclosporine A in this type of Treg cell therapy should be carefully considered.

Effect of in vitroexpanded CD4(+)CD25(+)Foxp3(+) regulatory T cell therapy combined with lymphodepletion in murine skin allotransplantation.

A promising approach for preventing allograft rejection involves shifting the balance between cytopathic and regulatory T cells to dominance of the latter cell type. Nonspecific lymphodepletion was conducted by administration of depleting anti-CD4 and anti-CD8 antibodies to reduce effector T cells and adoptive transfer of ex vivo-expanded host Treg cells by stimulation with donor dendritic cells to augment the Treg cell compartment. Evaluation of an MHC-mismatched skin allograft model revealed that combined therapy with these two protocols consistently induced modest prolongation of allograft survival, although all skin grafts were eventually rejected. The administration of IL-2/anti-IL-2 complexes significantly improved the efficacy of combination therapy via promoting the expansion of adoptively transferred Treg cells as well as endogenous recipient Treg cells. We conclude that Treg cell therapy combined with lymphodepletion is of practical benefit for the control of allograft rejection, but requires supplementary measures to promote immune tolerance.

Anti-inflammatory effects of Artemisia princeps in antigen-stimulated T cells and regulatory T cells.

OBJECTIVES: The aim was to investigate the anti-inflammatory effects of Artemisia princeps extract on the activity of anti-CD3/CD28-stimulated CD4(+)CD25(-) T cells and antigen-expanded regulatory T cells. METHODS: CD4(+)CD25(-) T cells were activated with coated anti-CD3 and anti-CD28 and cultured in the presence or absence of various concentrations of A. princeps extract. The cultures were pulsed on Day 6 with [(3)H]thymidine and, after harvesting the cells, [(3)H]thymidine incorporation was measured. For analysis of interleukin-2 and interferon-gamma secreted from CD4(+)CD25(-) T cells, culture supernatants were collected on Days 2 and 6. For the analysis of interleukin-10 secreted from the CD4(+)CD25(-) T cells and expanded regulatory T cells, supernatants were collected after 2 and 7 days, respectively. Cytokine levels were determined using an enzyme-linked immunosorbent assay. Potential medicinal components of the A. princeps extract were determined using gas chromatography-mass spectrometry. KEY FINDINGS: A. princeps (30 microg/ml) effectively suppressed proliferation of CD4(+)CD25(-) T cells that were stimulated with anti-CD3/CD28 without causing cytotoxicity in spleen cells incubated under conditions lacking antigen stimulation. A. princeps inhibited production of the pro-inflammatory cytokines interleukin-2 and interferon-gamma in anti-CD3/CD28-stimulated CD4(+)CD25(-) T cells. Also, the extract slightly increased production of the anti-inflammatory cytokine interleukin-10 in these cells. In regulatory T cells expanded by anti-CD3/CD28, A. princeps increased production of interleukin-10 and Foxp3. CONCLUSIONS: The results suggest that A. princeps may be useful in the treatment of autoimmune diseases and organ transplantation rejection by inhibiting proliferation of inflammatory T cells, suppressing inflammatory processes in antigen-stimulated CD4(+)CD25(-) T cells and increasing activity of expanded regulatory T cells.

Optoelectrofluidic control of colloidal assembly in an optically induced electric field.

This letter reports a method for controlling the organization of colloidal particles using optoelectrofluidic mechanisms. This optoelectrofluidic colloidal assembly (OCA) can be an efficient and simple way of preparing 2D colloidal crystals. Here, we present the first investigation of 2D colloidal assembly due to the electrohydrodynamic flows in an optoelectrofluidic device. The normalized distance among the assembled particles and the assembly rate according to the applied ac signal were analyzed. This OCA allows the control of both the colloidal crystal pattern over a large area and the distance between the assembled particles by adjusting the projected light pattern and the applied ac signal.

Clinical performance evaluation of four automated chemiluminescence immunoassays for hepatitis C virus antibody detection.

Various automated chemiluminescence immunoassay (CLIA) analyzers for the detection of antibodies to hepatitis C virus (HCV) are now commercially available in clinical laboratories and are replacing conventional enzyme immunoassays. We investigated the performance of four anti-HCV CLIAs (the Architect Anti-HCV assay on the Architect i2000 system, the Vitros Anti-HCV assay on the Vitros ECiQ Immunodiagnostic System, the Access HCV Ab PLUS assay on the UniCel DxI 800 analyzer, and the newly developed Elecsys Anti-HCV assay on the Cobas e 411 analyzer). The total percent coefficient of variation values of imprecision were 3.5 to 5.7% with positive control materials and 7.2 to 10.2% with negative control materials. The agreement between the results of the Elecsys, Architect, Vitros, and Access CLIAs ranged from 94.5 to 98.1%. The clinical sensitivity of all CLIAs was 100%. Each CLIA showed excellent reproducibility and clinical sensitivity. The Elecsys, Architect, Vitros, and Access CLIAs showed clinical specificities of 98.2, 98.8, 96.5, and 98.2%.

Viral IL-10 gene transfer prolongs rat islet allograft survival.

Islet transplantation is a potential cure for diabetes. However, allotransplant rejection severely limits its clinical application. In this study, we sought to transfect rat islets with an adenoviral vector containing the viral IL-10 (vIL-10) gene and examine its efficacy in preventing graft rejection. The immunosuppressive effect of vIL-10 is reported but its efficacy is somehow debatable in transplantation model. vIL-10 transfected islets were transplanted into streptozotocin-induced diabetic rats. Blood glucose, serum vIL-10 concentration, graft histology, and graft cytokine expression were used to monitor graft function up to day 21 after transplantation. Transfected islets released a large amount of vIL-10 protein without affecting their viability and functional integrity. When we transplanted the transfected islets into allogeneic hosts, the survival of grafted islets was not significantly increased. However, the combined use of vIL-10 and subtherapeutic doses of CsA (cyclosporine) significantly prolonged graft survival beyond that achieved with either agent alone (p < 0.001). vIL-10 and CsA-treated rats contain high level of vIL-10 in serum, which is evidenced by the inhibition of allogeneic mixed lymphocyte reaction (MLR). Histological analysis additionally revealed the presence of viable islets up to 21 days. IL-10 mRNA expression in grafted liver was higher and IFN-gamma mRNA was lower in vIL-10 and CsA-treated animals, compared with other groups. The synergistic effect of this combination therapy is potentially correlated with the induction of inhibitory cytokine secretion and downregulation of proinflammatory cytokine secretion from host cells.

Anti-inflammatory action of alpha-melanocyte stimulating hormone (alpha-MSH) in anti-CD3/CD28-mediated spleen and CD4(+)CD25(-) T cells and a partial participation of IL-10.

alpha-Melanocyte stimulating hormone (alpha-MSH) has been shown to inhibit the production and the effects of pro-inflammatory cytokine by inflammatory cells in innate immunity. We have determined whether alpha-MSH inhibits anti-CD3/CD28-mediated spleen cells and CD4(+)CD25(-) T cells proliferation and its mechanism of action. The proliferation of anti-CD3/CD28-mediated spleen cells and CD4(+)CD25(-) T cells markedly were suppressed by 50-100 nM and 5-100 nM alpha-MSH, respectively. alpha-MSH (100 nM) increased the production of the anti-inflammatory cytokine IL-10 and decreased the production of the pro-inflammatory cytokine IL-2 and IFN-gamma from CD4(+)CD25(-) T cells. Moreover, anti-IL-10 blocking Ab decreased the inhibitory effects of anti-CD3/CD28-mediated spleen cells and CD4(+)CD25(-) T cells proliferation by alpha-MSH, indicating a partial participation of IL-10 in its mechanism of inhibitory action. These results suggest that alpha-MSH may be useful for treatment of autoimmune diseases and transplantation involving innate and adaptive immunity.

Cell surface modification by activated polyethylene glycol prevents allosensitization after islet transplantation.

The necessity to transplant islet tissue without the need for immunosuppressant therapy has led to the development of materials for immune modulation. Pegylation makes islets antigenically silent, protecting them from the adsorption of foreign protein and thus avoiding immune injury. The aim of this study is to determine whether pegylation of islets prolongs islet survival and function both during tissue culture and posttransplantation. We used cyanuric chloride-activated methoxy-polyethylene glycol for cell surface modification. To detect the pegylation effect on splenocytes, we measured antibody binding inhibition and abrogation of lymphocyte proliferation. To detect the pegylation effect on islet grafts, we performed rodent islet transplantation. Islet viability and function were maintained after pegylation. Pegylated islets showed a 90% decrease in antibody binding and decreased lymphocyte proliferation in a mixed lymphocyte culture. However, when pegylated islets were transplanted, no prolongation of graft survival was observed. When a subtherapeutic dose of immunosuppressant was given at the time of transplantation of pegylated islets, islet graft survival was significantly prolonged. In addition, when rats were sensitized with donor splenocytes, graft survival was prolonged by pegylation. We observed that pegylation of islets, combined with a subtherapeutic dose of immunosuppressant, protects the graft from rejection. Prolonged graft survival in sensitized recipients showed that pegylation of islets shifted the pattern of rejection from an acute humoral response to a less aggressive cellular alloresponse.

Effect of immunosuppressants on the expansion and function of naturally occurring regulatory T cells.

The induction of immune tolerance is one of the final therapeutic goals in clinical transplantation. Regulatory T lymphocytes are important for the induction and maintenance of immune tolerance to grafts. If immunosuppressive drugs used clinically to prevent immune rejection also inhibit regulatory T lymphocytes, tolerance would not be achieved. We therefore tested the effect of several immunosuppressants with different mechanisms of action on the proliferation and suppressive activity of CD4(+)CD25(+) regulatory T cells. Highly purified CD4(+)CD25h(+) T cells from C57BL/6 (H-2(b)) mice were stimulated with allogeneic T-depleted splenocytes (BALB/c; H-2(d)) in the presence of various immunosuppressants. After one week in culture, viable T cells were recovered, their regulatory capacity was assessed by their ability to inhibit responder T cell proliferation in MLR, and their cytokine production profile was measured by ELISA. The immunosuppressants rapamycin, cyclosporine A, and methylprednisolone significantly inhibited the expansion of regulatory T cells upon stimulation with alloantigen, whereas mycophenolic acid and the costimulatory blockers, anti-CD40L and CTLA4Ig, did not. None of these immunosuppressants, however, reduced the suppressive capacity of regulatory T cells. Pretreatment with immunosuppressants did not induce significant changes in the cytokine production profile of regulatory T cells. Our results suggest that costimulatory blockers and mycophenolate mofetil can be utilized therapeutically in the induction of immune tolerance. In contrast, the use of rapamycin, cyclosporine A, and methylprednisolone should be reconsidered, due to their deleterious effects on the expansion of naturally occurring regulatory T cells.