Immune cell proliferation is suppressed by the interferon-gamma-induced indoleamine 2,3-dioxygenase expression of fibroblasts populated in collagen gel (FPCG)

Indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing enzyme, is an intracellular enzyme possessing various immunosuppressive properties. Here, we report the possible use of this enzyme to suppress proliferation of immune cells cocultured with IDO-expressing fibroblasts of an allogenic skin substitute. Fetal skin fibroblasts embedded within bovine collagen were treated with cytokine interferon-gamma (IFN-gamma) to induce expression of IDO mRNA and protein. Expression of IDO mRNA was evaluated by Northern analysis. IDO enzyme activity was evaluated by measurement of kynurenine and tryptophan levels in the IFN-gamma untreated and treated fibroblasts. The results of Northern analysis showed a dose-dependent increase in expression of IDO mRNA in response to various concentrations of IFN-gamma used. The levels of kynurenine and tryptophan measured, as the bioactivity of IDO, were significantly different in the IFN-gamma treated fibroblasts, compared to those of controls (P < 0.001). In a lasting effect experiment, the expression of IDO mRNA was gradually reduced to an undetectable level within 32 h of IFN-gamma removal. The results of Western blot analysis, however, revealed a significantly longer (192 h) lasting effect of IFN-gamma on IDO protein level, relative to that of mRNA expression. To demonstrate immunosuppressive effects of IDO on proliferation of immune cells, IDO-expressing fibroblasts were cocultured with peripheral blood mononuclear cells (PBMC) for a period of 5 days. The results of (3)H-thymidine incorporation showed a significant reduction in proliferation of PBMC when cocultured with IDO-expressing fibroblasts, compared to those cocultured with non-IDO-expressing fibroblasts (P < 0.001). Furthermore, addition of IDO-inhibitor (1-methyl-d-tryptophan) reversed the suppressive effects of IDO on PBMC proliferation in a dose-dependant fashion. To test the viability of immune cells cocultured with IDO-expressing fibroblasts, FACS analysis of the PI stained PBMC was conducted and no significant difference was found between these cells and the controls. In another set of experiments, we showed that migration rate and subsequent proliferation of IDO-expressing fibroblasts are also the same as those of control cells. In conclusion, IDO-expressing allogenic fibroblasts embedded within collagen gel suppress the proliferation of allogenic immune cells, while they still remain viable in this IDO-induced tryptophan-deficient culture environment.     

Immuno-Regulatory Function of Indoleamine 2,3 Dioxygenase through Modulation of Innate Immune Responses

Successful long-term treatment of type-1 diabetes mainly relies on replacement of β-cells via islet transplantation. Donor shortage is one of the main obstacles preventing transplantation from becoming the treatment of choice. Although animal organs could be an alternative source for transplantation, common immunosuppressive treatments demonstrate low efficacy in preventing xenorejection. Immunoprotective effects of indoleamine 2,3-dioxygenase (IDO) on T-cell mediated allorejection has been extensively studied. Our studies revealed that IDO expression by fibroblasts, induced apoptosis in T-cells while not affecting non-immune cell survival/function. Since macrophages play a pivotal role in xenograft rejection, herein we investigated the effect of IDO-induced tryptophan deficiency/kynurenine accumulation on macrophage function/survival. Moreover, we evaluated the local immunosuppressive effect of IDO on islet-xenograft protection. Our results indicated that IDO expression by bystander fibroblasts significantly reduced the viability of primary macrophages via apoptosis induction. Treatment of peritoneal macrophages by IDO-expressing fibroblast conditioned medium significantly reduced their proinflammatory activity through inhibition of iNOS expression. To determine whether IDO-induced tryptophan starvation or kynurenine accumulation is responsible for macrophage apoptosis and inhibition of their proinflammatory activity, Raw264.7 cell viability and proinflammatory responses were evaluated in tryptophan deficient medium or in the presence of kynurenine. Tryptophan deficiency, but not kynurenine accumulation, reduced Raw264.7 cell viability and suppressed their proinflammatory activity. Next a three-dimensional islet-xenograft was engineered by embedding rat islets within either control or IDO–expressing fibroblast-populated collagen matrix. Islets morphology and immune cell infiltration were then studied in the xenografts transplanted into the C57BL/6 mouse renal sub-capsular space. Local IDO significantly decreased the number of infiltrating macrophages (11±1.47 vs. 70.5±7.57 cells/HPF), T-cells (8.75±1.03 vs. 75.75±5.72 cells/HPF) and iNOS expression in IDO-expressing xenografts versus controls. Islet morphology remained intact in IDO-expressing grafts and islets were strongly stained for insulin/glucagon compared to control. These findings support the immunosuppressive role of IDO on macrophage-mediated xeno-rejection.     

Functional characterization of Cx43 based gap junctions during spermatogenesis

Indoleamine 2,3-dioxygenase (IDO) is an intracellular tryptophan-catabolizing enzyme possessing various immunosuppressive properties. Here, we report the use of this enzyme to suppress the proliferation of peripheral blood mononuclear cells (PBMC) co-cultured with IDO-expressing fibroblasts of an allogeneic skin substitute in vitro. Fetal foreskin fibroblasts populated within collagen gel (FPCG) were treated with interferon-gamma (IFN-gamma) conjugated with a temperature-sensitive polymer to induce the expression of IDO mRNA and protein. SDS-PAGE showed successful conjugation of IFN-gamma with the temperature-sensitive polymer. Expression of IDO mRNA was evaluated by Northern analysis. IDO enzyme activity was evaluated by the measurement of kynurenine levels. The results of Northern blot analysis showed an induction of IDO mRNA expression when treated with polymer-conjugated IFN-gamma. Kynurenine levels, as a measure of IDO bioactivity, were significantly higher in IFN-gamma-treated fibroblasts than in controls (P < 0.001). In a lasting effect experiment, the expression of IDO mRNA in FPCG treated with polymer-conjugated IFN-gamma was significantly longer than in those treated with free (non-conjugated) IFN-gamma (P < 0.001). IFN-gamma radiolabeling showed a prolonged retention of IFN-gamma within collagen gel in its polymer-conjugated form, compared to its free form. Presence of IDO protein in FPCG was demonstrated by Western analysis even 16 days after removal of the conditioned medium (containing released IFN-gamma). To demonstrate the immunosuppressive effects of IDO on the proliferation of PBMC, IDO-expressing FPCG treated with polymer-conjugated IFN-gamma were co-cultured with PBMC for a period of 5 days. The results showed a significant reduction in proliferation of PBMC co-cultured with IFN-gamma-treated IDO-expressing fibroblasts, compared to those co-cultured with non-IDO-expressing fibroblasts (P < 0.001). The addition of an IDO inhibitor (1-methyl-D-tryptophan) reversed the suppressive effects of IDO on PBMC proliferation. In conclusion, IDO expression in FPCG suppresses the proliferation of immune cells in vitro. The use of a temperature-sensitive polymer further prolongs the effect of IFN-gamma on the expression of IDO. Therefore, modulating IDO levels in situ might be an alternative for prolonging the survival of skin allografts.     

Suppression of islet allogeneic immune response by indoleamine 2,3 dioxygenase-expressing fibroblasts

Success of transplantation of pancreatic islets which is a promising way for restoring efficient insulin regulation in type 1 diabetes depends on lifelong use of immunosuppressive drugs. To eliminate the use of systemic immunosuppressive drugs for islet transplantation, we examined the potential use of a local immunosuppressive factor, indoleamine 2,3-dioxygenase (IDO). Thus, the aim of this study was to determine whether local expression of IDO in bystander syngeneic fibroblasts could prevent islet allogeneic immune response in vitro. C57BL/6 (B6) mouse fibroblasts were induced to express IDO by either IFN-gamma treatment or transduction with an adenoviral vector and were co-cultured with B6 mouse lymphocytes and BALB/c mouse pancreatic islets in the presence or absence of an IDO inhibitor. Proliferation of lymphocytes were then assessed using [(3)H]-thymidine incorporation assay. IDO-expression by co-cultured syngeneic fibroblasts resulted in a five-fold decrease in lymphocyte proliferation rate upon stimulation of lymphocytes by allogeneic mouse pancreatic islets (21.9% +/- 5.3 and 22.1% +/- 4.9 in the preparations with IFN-gamma treated and genetically modified IDO-expressing fibroblasts, respectively vs. 100% in control groups, P < 0.01). Allogeneic response was restored when IDO inhibitor was added to the culture indicating that suppression was due to IDO. In conclusion, this study shows that local expression of IDO by syngeneic bystander fibroblasts can suppress in vitro proliferation of lymphocytes in response to stimulation with allogeneic pancreatic islets. This local immunosuppressive function of IDO may be employed for development of a novel alternative strategy for preventing allogeneic islet graft rejection.     

IDO induces expression of a novel tryptophan transporter in mouse and human tumor cells

IDO is the rate-limiting enzyme in the kynurenine pathway, catabolizing tryptophan to kynurenine. Tryptophan depletion by IDO-expressing tumors is a common mechanism of immune evasion inducing regulatory T cells and inhibiting effector T cells. Because mammalian cells cannot synthesize tryptophan, it remains unclear how IDO(+) tumor cells overcome the detrimental effects of local tryptophan depletion. We demonstrate that IDO(+) tumor cells express a novel amino acid transporter, which accounts for ～50% of the tryptophan uptake. The induced transporter is biochemically distinguished from the constitutively expressed tryptophan transporter System L by increased resistance to inhibitors of System L, resistance to inhibition by high concentrations of most amino acids tested, and high substrate specificity for tryptophan. Under conditions of low extracellular tryptophan, expression of this novel transporter significantly increases tryptophan entry into IDO(+) tumors relative to tryptophan uptake through the low-affinity System L alone, and further decreases tryptophan levels in the microenvironment. Targeting this additional tryptophan transporter could be a way of pharmacological inhibition of IDO-mediated tumor escape. These findings highlight the ability of IDO-expressing tumor cells to thrive in a tryptophan-depleted microenvironment by expressing a novel, highly tryptophan-specific transporter, which is resistant to inhibition by most other amino acids. The additional transporter allows tumor cells to strike the ideal balance between supply of tryptophan essential for their own proliferation and survival, and depleting the extracellular milieu of tryptophan to inhibit T cell proliferation.     

Establishment of 3D organotypic cultures using human neonatal epidermal cells

This protocol describes an ex vivo three-dimensional coculture system optimized to study the skin regenerative ability of primary human keratinocytes grown at the air-liquid interface on collagen matrices embedded with human dermal fibroblasts. An option for enrichment of keratinocyte stem cells and their progeny using fluorescence-activated cell sorting is also provided. Initially, dermal equivalents, comprising human passaged fibroblasts seeded in a collagen matrix, are grown on porous filters (3 mum) placed in transwells. After 1 week, primary human keratinocytes are seeded on this base. One week later, an air-lift transition is performed, leading to the differentiation of the keratinocytes, which are macroscopically visible as artificial skin after a couple of days. The cultures can be harvested 1 week after the air-lift and processed for immunohistochemistry or gene expression analysis. The overall procedure can be completed in 3 weeks, including the preparation of the dermal equivalent and the seeding of the primary keratinocytes.     

Indoleamine 2,3-dioxygenase is regulated by IFN-gamma in the mouse placenta during Listeria monocytogenes infection

The tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) is expressed in macrophages that have been differentiated in the presence of CSF-1 and is important in the containment of intracellular pathogens. IDO also appears to play a role in suppression of T cell responses in a variety of contexts. In the placenta, its enzymatic activity is believed to establish a chemical barrier that protects the fetal allograft from T cell-mediated immune aggression. We have studied the regulation of IDO in the utero-placental unit of mice following infection with the Gram-positive, intracellular bacterium Listeria monocytogenes that has a predilection for replication in the decidua basalis. IDO mRNA and protein expression is enhanced in the utero-placental unit following infection with L. monocytogenes. However, in contrast to the human where IDO is expressed by the CSF-1R-positive syncytial trophoblast, IDO is not expressed in murine trophoblastic tissue but instead is found in stromal cells of the decidua basalis and metrial gland and following infection, in endothelial cells. Using mice carrying null mutations in cytokine/growth factor genes, we explored the regulation of IDO in the placenta. Consistent with the absence of CSF-1R expression in the IDO-expressing cells of mice, neither the basal levels of IDO nor its induction following infection is affected by the absence of CSF-1. However, although the basal level of IDO is normal, the enhanced expression during Listeriosis is completely abrogated in the absence of IFN-gamma, a cytokine required for the resolution of this infection. These data suggest that IDO plays a role in resolving bacterial infection in the placenta while at the same time maintaining a barrier to T cells whose presence might result in fetal rejection.     

High expression of IMPACT protein promotes resistance to indoleamine 2,3‐dioxygenase‐induced cell death

Indoleamine 2,3‐dioxygenase (IDO), a tryptophan degrading enzyme, is a potent immunomodulatory factor. IDO expression in fibroblasts selectively induces apoptosis in immune cells but not in primary skin cells. However, the mechanism(s) of this selective effect of IDO‐induced low tryptophan environment is not elucidated. The aim of present study was to investigate whether the activity of general control non‐derepressible‐2(GCN2) kinase stress‐responsive pathway and its known inhibitor, protein IMPACT homolog, in immune and skin cells are differentially regulated in response to IDO‐induced low tryptophan environment. IDO‐expressing human fibroblasts were co‐cultured with Jurkat cells, human T cells, fibroblasts, or keratinocytes. Activation of GCN2 pathway was significantly higher in immune cells exposed to IDO‐expressing environment relative to that of skin cells. In contrast, IMPACT was highly and constitutively expressed in skin cells while its expression was very low in stimulated T cells and undetectable in Jurkat cells. A significant IDO‐induced suppressive as well as apoptotic effect was demonstrated in IMPACT knocked down fibroblasts co‐cultured with IDO‐expressing fibroblasts. Proliferation of Jurkat cells, stably transduced with IMPACT‐expressing vector, was rescued significantly in tryptophan‐deficient but not IDO‐expressing environment. This may be due to the ability of IMPACT to recover the effects of IDO‐mediated tryptophan depletion (GCN2 dependent) but not the effects of IDO‐generated cytotoxic metabolites. These findings collectively suggest for the first time that high expression of protein IMPACT homolog in non‐immune cells such as skin cells acts as a protective mechanism against IDO‐induced GCN2 activation, therefore, makes them resistant to the amino acid‐deprived environment caused by IDO. J. Cell. Physiol. 225: 196–205, 2010. © 2010 Wiley‐Liss, Inc.     

The specific targeting of immune regulation: T-cell responses against Indoleamine 2,3-dioxygenase

Indoleamine 2,3-dioxygenase (IDO) is an immunoregulatory enzyme that is implicated in suppressing T-cell immunity in many settings including cancer. In recent years, we have described spontaneous CD8(+) as well as CD4(+) T-cell reactivity against IDO in the tumor microenvironment of different cancer patients as well as in the peripheral blood of both cancer patients and to a lesser extent in healthy donors. We have demonstrated that IDO-reactive CD8(+) T cells were peptide-specific, cytotoxic effector cells, which are able to recognize and kill IDO-expressing cells including tumor cells as well as dendritic cells. Consequently, IDO may serve as a widely applicable target for immunotherapeutic strategies with a completely different function as well as expression pattern compared to previously described antigens. IDO constitutes a significant counter-regulatory mechanism induced by pro-inflammatory signals, and IDO-based immunotherapy may consequently be synergistic with additional immunotherapy. In this regard, we have shown that the presence of IDO-specific T cells boosted immunity against CMV and tumor antigens by eliminating IDO(+) suppressive cells and changing the regulatory microenvironment. The current review summarizes current knowledge of IDO as a T-cell antigen, reports the initial results that are suggesting a general function of IDO-specific T cells in immunoregulation, and discusses future opportunities.     

Indoleamine 2,3-dioxygenase mediates the antiviral effect of gamma interferon against hepatitis B virus in human hepatocyte-derived cells

Alpha interferon (IFN-α) is an approved medication for chronic hepatitis B. Gamma interferon (IFN-γ) is a key mediator of host innate and adaptive antiviral immunity against hepatitis B virus (HBV) infection in vivo. In an effort to elucidate the antiviral mechanism of these cytokines, 37 IFN-stimulated genes (ISGs), which are highly inducible in hepatocytes, were tested for their ability to inhibit HBV replication upon overexpression in human hepatoma cells. One ISG candidate, indoleamine 2,3-dioxygenase (IDO), an IFN-γ-induced enzyme catalyzing tryptophan degradation, efficiently reduced the level of intracellular HBV DNA without altering the steady-state level of viral RNA. Furthermore, expression of an enzymatically inactive IDO mutant did not inhibit HBV replication, and tryptophan supplementation in culture completely restored HBV replication in IDO-expressing cells, indicating that the antiviral effect elicited by IDO is mediated by tryptophan deprivation. Interestingly, IDO-mediated tryptophan deprivation preferentially inhibited viral protein translation and genome replication but did not significantly alter global cellular protein synthesis. Finally, tryptophan supplementation was able to completely restore HBV replication in IFN-γ- but not IFN-α-treated cells, which strongly argues that IDO is the primary mediator of IFN-γ-elicited antiviral response against HBV in human hepatocyte-derived cells.     

Preparation and characterization of calibration beads for sorting cells expressing a beta-lactamase gene reporter.

BACKGROUND: Modern drug discovery has been based on high-throughput screening using whole-cell assays. A prominent role has been assigned to the reporter gene technology based on a beta-lactamase and the fluorogenic substrate CCF2. Successful application of this technology requires fluorescence-activated cell sorting. We describe the preparation and characterization of calibration beads for sorting cells expressing the beta-lactamase gene using the CCF2 substrate. METHODS: To model Forster resonance energy transfer (FRET) between the coumarin donor and the fluorescein acceptor of the CCF2 reporting dye, we used activated polystyrene beads with primary amino groups. Donor and acceptor fluorophores were attached to the beads at different ratios via succinimidyl esters. The beads were characterized with a fluorescence plate reader and a flow cytometer. RESULTS: We prepared polystyrene beads with five different ratios of donor and acceptor fluorophores and beads that carried a donor or a receptor fluorophore alone. Fluorescence measurements demonstrated that the prepared beads well represent the FRET of CCF2 substrate. CONCLUSION: We have demonstrated that the prepared beads can be successfully used for the setup of fluorescence-activated cell sorting to sort cells with CCF2 reporter substrate and the beta-lactamase reporter gene.     

Inhibition of allogeneic T-cell responses by dendritic cells expressing transduced indoleamine 2,3-dioxygenase

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO) is an enzyme involved in the catabolism of tryptophan and has been shown to prevent rejection of the fetus during pregnancy by inhibiting alloreactive T cells. METHODS: In this study we investigated dendritic cells (DCs) that are transfected with IDO cDNA in the inhibition of T-cell proliferation after antigen-specific interaction. XS106 DCs, derived from A/J mice (H-2k), were transduced with IDO with a gene-delivery system using a recombinant adenoviral vector. RESULTS: Western blotting and immune staining revealed IDO expression in XS106 DCs transduced with IDO (XS106-IDO DCs), and its catabolic effect was confirmed by an increase in kynurenine concentration. Fluorescence-activated cell sorting revealed that XS106-IDO DCs were not changeable for Ia, CD80, and CD86 expression. After XS106-IDO DCs were co-cultured with C57BL/6 allogeneic splenic T cells, the proliferation of the T cell was significantly inhibited. The co-cultured T cells with XS106-IDO DCs exhibited cell-cycle arrest. Furthermore, injection of XS160-IDO DCs into the footpads of C57BL/6 (H-2b) mice demonstrated a reduced T-cell response against allo-antigen. CONCLUSIONS: These results suggest that overexpression of IDO in the DCs effectively inhibited T-cell proliferation, and may expand a new immunomodulatory strategy for the prevention of allo-rejection of organ transplantation.     

利用流式细胞仪分选拟南芥根尖发育早期非根毛细胞

建立了应用流式细胞仪分选植物特定类型细胞的方法。以拟南芥（Arabidopsis thaliana）Wer：：GFP转基因株系为材料,用激光共聚焦显微镜鉴定GFP的表达位置,采用酶解法制备拟南芥根尖原生质体,应用流式细胞仪荧光激活细胞分选技术（FACS）分选收集GFP阳性细胞,并提取细胞的RNA。结果表明,Wer：：GFP转基因株系仅在根表皮发育早期的非根毛细胞中表达GFP;利用酶解法制备的根尖原生质体数目较多;从FACS分选收集的细胞中提取的RNA质量较好,可用于研究特定类型细胞的基因表达谱。应用流式细胞仪分选拟南芥非根毛细胞的方法为研究植物特定类型细胞的基因表达谱及基因功能奠定了技术基础。     

Molecular cloning, sequencing and expression of human interferon-gamma-inducible indoleamine 2,3-dioxygenase cDNA

The antiproliferative action of human interferon (HuIFN)-gamma on human cells and the inhibition of intracellular pathogens, e.g. Toxoplasma gondii and Chlamydia psittaci, is at least in part due to an induction of indoleamine 2,3-dioxygenase (IDO) enzyme which degrades tryptophan, an essential amino acid. A cDNA clone (called C42) was isolated from a cDNA library made from poly(A)+ RNA obtained from HuIFN-gamma-treated human fibroblasts. Its nucleotide sequence revealed an open reading frame coding for a polypeptide of 403 amino acids, but no homology with any known gene in GenBank database was found. Evidence was obtained indicating that this cDNA codes for IDO: (i) Hybrid selected C42 specific poly(A)+ RNA from IFN-gamma-treated human cells coded for a polypeptide in vitro of approximately 42 kD (reported size of IDO, approximately 40 kD) which was immunoprecipitated by monoclonal anti-IDO antibody but not by a control antibody; and (ii) transfection of human fibroblasts with an expression plasmid containing C42 cDNA transcribed from chicken beta-actin promoter led to constitutive expression of C42 specific RNA as well as IDO activity. This cDNA clone will be useful in studying the role of IDO in the biological effects of IFN-gamma, and the regulation of IDO gene by IFN-gamma.     

Enrichment and characterization of mouse putative epidermal stem cells

Epidermis, a continuously renewing tissue, is maintained by stem cells that proliferate and replenish worn out or damaged cells in the tissue during life. Cultured epidermal stem cells have great potential in scientific research and clinical application. However, isolating a pure and viable population of epidermal stem cells and culturing them has been challenging. In this study, putative epidermal stem cells of mouse were isolated by combining Hoechst 33342 and propidium iodide staining with fluorescence-activated cell sorting. Molecular markers expression pattern analysis showed that cytokeratin 14, integrin beta1 and p63 are expressed in the sorted putative stem cells, but not active beta-catenin, nestin and involucrin. Our results provide further supporting data that mouse putative epidermal stem cells could be successfully isolated by combining Hoechst dye staining with fluorescence-activated cell sorting and cultured in vitro. The cultured mouse putative epidermal stem cells could be used as a potent tool for studying stem cell biology and testing stem cell therapy.     

Watching osteogenesis: life monitoring of osteogenic differentiation using an osteocalcin reporter

Human bone marrow-derived mesenchymal stem cells have the potential to differentiate into several cell types such as osteoblasts, chondrocytes, and adipocytes. When cultured under appropriate medium conditions stem cells can be directed toward the osteoblast lineage in vitro. Progression of osteogenic differentiation is accompanied by changes in the expression pattern of several marker proteins including bone-specific alkaline phosphatase (bALP), collagen I (Col I), and osteocalcin (OC) and can be analyzed by well-established methods like immunohistochemical staining and quantitative RT-PCR. Furthermore, expression of fluorescent protein driven by an osteogenesis promoter facilitates online monitoring of proceeding osteogenic differentiation in transiently transfected human bone marrow-derived cells. In the present study we established a new double reporter gene construct comprising OC promoter-driven expression of green fluorescent protein and constitutive expression of red fluorescent protein-tagged histone H2B for transient transfection of primary human bone cells (HBCs). Osteogenic differentiation of transiently transfected cells was visualized by fluorescence microscopy. Immunohistochemical analysis and RT-PCR confirmed the progression into the osteo-specific lineage of transfected cells. Transfection efficiency was determined by fluorescence-activated cell sorting (FACS).     

Fluorescence-activated cell sorting of specific affibody-displaying staphylococci

Efficient enrichment of staphylococcal cells displaying specific heterologous affinity ligands on their cell surfaces was demonstrated by using fluorescence-activated cell sorting. Using bacterial surface display of peptide or protein libraries for the purpose of combinatorial protein engineering has previously been investigated by using gram-negative bacteria. Here, the potential for using a gram-positive bacterium was evaluated by employing the well-established surface expression system for Staphylococcus carnosus. Staphylococcus aureus protein A domains with binding specificity to immunoglobulin G or engineered specificity for the G protein of human respiratory syncytial virus were expressed as surface display on S. carnosus cells. The surface accessibility and retained binding specificity of expressed proteins were demonstrated in whole-cell enzyme and flow cytometry assays. Also, affibody-expressing target cells could be sorted essentially quantitatively from a moderate excess of background cells in a single step by using a high-stringency sorting mode. Furthermore, in a simulated library selection experiment, a more-than-25,000-fold enrichment of target cells could be achieved through only two rounds of cell sorting and regrowth. The results obtained indicate that staphylococcal surface display of affibody libraries combined with fluoresence-activated cell sorting might indeed constitute an attractive alternative to existing technology platforms for affinity-based selections.