Cell Sorter Cleaning Practices and Their Impact on Instrument Sterility.

Cells isolated using electrostatic cell sorters are subsequently evaluated in a variety of in vitro and in vivo applications. Thus, manipulations to the cells during the pre- and post-sort processing as well as when the cells are being analyzed by and passing through the sorter fluidics has the potential to affect the experimental results. There are many variables to consider when seeking to preserve cellular integrity and function during the cell-sorting process. A previous study by the Association of Biomolecular Resource Facilities Flow Cytometry Research Group (FCRG) investigated downstream effects on different cell types as a function of sorting variables such as pressure, nozzle size, and temperature. This multisite study revealed site-to-site variability based on differential gene expression when the same cell type and sort conditions were used. These results indicated the possibility that environmental factors such as the presence of contaminants in the sorter fluidics could exhibit effects on downstream molecular assays (ie, endotoxins or RNases). In the study described here, the FCRG sought to better understand how sorters are maintained and evaluated for contaminants such as bacteria, endotoxin, and RNases. In addition, the efficacy of an endotoxin decontamination method was evaluated. The results demonstrated that the majority of sorters in shared resource laboratories are free of RNase activity and bacteria; however, many are contaminated with endotoxin. The efficacy of a hydrogen peroxide cleaning procedure was tested and found to exhibit only a short-term effectiveness in eliminating endotoxin contamination.

Evaluating the Effects of Cell Sorting on Gene Expression.

Cell sorting is a commonly used technology to isolate highly purified cell populations for downstream applications. Because the sorted cells are destined for further analysis, i.e., gene expression assays or functional assays, ensuring that the sorting process itself has little effect on the cells is of utmost importance. Previous studies examining the effects of sorting on cellular function have primarily focused on a specific cell type or condition. One of the goals of the Flow Cytometry Research Group of the Association of Biomolecular Resource Facilities is to establish best practice guidelines for cell sorting conditions that minimize cell stress, perturbation, or injury to the sorted cell population. In this study, the effects of nozzle size, sample pressure, UV exposure, and instrument type were evaluated for their effects on gene expression and cell cycle using both established cell lines and primary cells across several flow cytometry shared facilities. Results indicate that nozzle size and pressure, as well as UV exposure and instrument type, have only minor effects on gene expression, which were diminished by subsequent culturing of the sorted cells. In this assessment, these data demonstrate that cell sorting itself, regardless of instrumentation used, has minimal effects on downstream cellular applications.

Evaluating the Effects of Cell Sorting on Gene Expression.

Cell sorting is a commonly used technology to isolate highly purified cell populations for downstream applications. Because the sorted cells are destined for further analysis, i.e., gene expression assays or functional assays, ensuring that the sorting process itself has little effect on the cells is of utmost importance. Previous studies examining the effects of sorting on cellular function have primarily focused on a specific cell type or condition. One of the goals of the Flow Cytometry Research Group of the Association of Biomolecular Resource Facilities is to establish best practice guidelines for cell sorting conditions that minimize cell stress, perturbation, or injury to the sorted cell population. In this study, the effects of nozzle size, sample pressure, UV exposure, and instrument type were evaluated for their effects on gene expression and cell cycle using both established cell lines and primary cells across several flow cytometry shared facilities. Results indicate that nozzle size and pressure, as well as UV exposure and instrument type, have only minor effects on gene expression, which were diminished by subsequent culturing of the sorted cells. In this assessment, these data demonstrate that cell sorting itself, regardless of instrumentation used, has minimal effects on downstream cellular applications.

Phenotypic Analysis of the Mouse Hematopoietic Hierarchy Using Spectral Cytometry: From Stem Cell Subsets to Early Progenitor Compartments.

Phenotypic analysis by flow cytometry is one of the most utilized primary tools to study the hematopoietic system. Here, we present a complex panel designed for spectral flow cytometry that allows for the in-depth analysis of the mouse hematopoietic stem and progenitor compartments. The developed panel encompasses the hematopoietic stem cell (HSC) compartment, an array of multipotent progenitors with early marks of lineage specification and a series of progenitors associated with lymphoid, granulo-macrophagic, megakaryocytic and erythroid lineage commitment. It has a built-in redundancy for key markers known to decipher the fine architecture of the HSC compartment by segregating subsets with different functional potential. As a resource, we used this panel to provide a snapshot view of the evolution of these phenotypically defined hematopoietic compartments during the life of the animals. We show that by using a spectral cytometer, this panel is compatible with the analysis of GFP-expressing gene-reporter mice across the hematopoietic system. We leverage this tool to determine how previously described markers such as CD150, CD34, CD105, CD41, ECPR, and CD49b define specific HSC subsets and confirm that high expression of the transcription factor Gfi1 is a hallmark of the most primitive HSC compartment. Altogether, our results provide a convenient protocol to obtain in one analysis a more extensive view of the hematopoietic architecture in mouse models. Our results could also serve as a base for further development of high-end panels leveraging spectral flow cytometry beyond the 15-fluorochrome panel presented in this report. © 2020 International Society for Advancement of Cytometry.

A Multiparameter Flow Cytometry Analysis Panel to Assess CD163 mRNA and Protein in Monocyte and Macrophage Populations in Hyperinflammatory Diseases.

CD163 facilitates regulation and resolution of inflammation and removal of free hemoglobin and is highly expressed in myeloid cells from patients with inflammatory disorders, such as systemic juvenile idiopathic arthritis (SJIA) and macrophage activation syndrome (MAS). Our recent studies indicate that regulation of CD163 mRNA expression is a key functional property of polarized monocytes and macrophages and is mediated at the transcriptional and posttranscriptional level, including via microRNAs. The goal of the current study is to develop a multiparameter flow cytometry panel incorporating detection of CD163 mRNA for polarized monocyte and macrophage populations in disorders such as SJIA and MAS. THP-1 cells and CD14+ human monocytes were stained using fluorochrome-conjugated Abs to myeloid surface markers, along with CD163 mRNA. Staining for mRNA could reliably detect CD163 expression while simultaneously detecting different macrophage populations using Abs targeting CD14, CD64, CD80, CD163, and CD209. This approach was found to be highly sensitive for increased mRNA expression when macrophages were polarized with IL-10 [M(IL-10)], with a strong signal over a broad range of IL-10 concentrations, and showed distinct kinetics of CD163 mRNA and protein induction upon IL-10 stimulation. Finally, this panel demonstrated clear changes in polarization markers in unstimulated monocytes from patients with SJIA and MAS, including upregulated CD163 mRNA and increased CD64 expression. This approach represents a robust and sensitive system for RNA flow cytometry, useful for studying CD163 expression as part of a multimarker panel for human monocytes and macrophages, with broad applicability to the pathogenesis of hyperinflammatory diseases.

A guide to choosing fluorescent protein combinations for flow cytometric analysis based on spectral overlap.

The advent of facile genome engineering technologies has made the generation of knock-in gene-expression or fusion-protein reporters more tractable. Fluorescent protein labeling of specific genes combined with surface marker profiling can more specifically identify a cell population. However, the question of which fluorescent proteins to utilize to generate reporter constructs is made difficult by the number of candidate proteins and the lack of updated experimental data on newer fluorescent proteins. Compounding this problem, most fluorescent proteins are designed and tested for use in microscopy. To address this, we cloned and characterized the detection sensitivity, spectral overlap, and spillover spreading of 13 monomeric fluorescent proteins to determine utility in multicolor panels. We identified a group of five fluorescent proteins with high signal to noise ratio, minimal spectral overlap, and low spillover spreading making them compatible for multicolor experiments. Specifically, generating reporters with combinations of three of these proteins would allow efficient measurements even at low-level expression. Because the proteins are monomeric, they could function either as gene-expression or as fusion-protein reporters. Additionally, this approach can be generalized as new fluorescent proteins are developed to determine their usefulness in multicolor panels. © 2018 International Society for Advancement of Cytometry.

Neutrophils From Children With Systemic Juvenile Idiopathic Arthritis Exhibit Persistent Proinflammatory Activation Despite Long-Standing Clinically Inactive Disease.

Background: Systemic juvenile idiopathic arthritis (SJIA) is a chronic childhood arthropathy with features of autoinflammation. Early inflammatory SJIA is associated with expansion and activation of neutrophils with a sepsis-like phenotype, but neutrophil phenotypes present in longstanding and clinically inactive disease (CID) are unknown. The objective of this study was to examine activated neutrophil subsets, S100 alarmin release, and gene expression signatures in children with a spectrum of SJIA disease activity. Methods: Highly-purified neutrophils were isolated using a two-step procedure of density-gradient centrifugation followed by magnetic-bead based negative selection prior to flow cytometry or cell culture to quantify S100 protein release. Whole transcriptome gene expression profiles were compared in neutrophils from children with both active SJIA and CID. Results: Patients with SJIA and active systemic features demonstrated a higher proportion of CD16+CD62Llo neutrophil population compared to controls. This neutrophil subset was not seen in patients with CID or patients with active arthritis not exhibiting systemic features. Using imaging flow cytometry, CD16+CD62Llo neutrophils from patients with active SJIA and features of macrophage activation syndrome (MAS) had increased nuclear hypersegmentation compared to CD16+CD62L+ neutrophils. Serum levels of S100A8/A9 and S100A12 were strongly correlated with peripheral blood neutrophil counts. Neutrophils from active SJIA patients did not show enhanced resting S100 protein release; however, regardless of disease activity, neutrophils from SJIA patients did show enhanced S100A8/A9 release upon PMA stimulation compared to control neutrophils. Furthermore, whole transcriptome analysis of highly purified neutrophils from children with active SJIA identified 214 differentially expressed genes (DEG) compared to neutrophils from healthy controls. The most significantly upregulated gene pathway was Immune System Process, including AIM2, IL18RAP, and NLRC4. Interestingly, this gene set showed intermediate levels of expression in neutrophils from patients with long-standing CID yet persistent serum IL-18 elevation. Indeed, all patient samples regardless of disease activity demonstrated elevated inflammatory gene expression, including inflammasome components and S100A8. Conclusion: We identify features of neutrophil activation in SJIA patients with both active disease and CID, including a proinflammatory gene expression signature, reflecting persistent innate immune activation. Taken together, these studies expand understanding of neutrophil function in chronic autoinflammatory disorders such as SJIA.

International Society for Advancement of Cytometry (ISAC) flow cytometry shared resource laboratory (SRL) best practices.

The purpose of this document is to define minimal standards for a flow cytometry shared resource laboratory (SRL) and provide guidance for best practices in several important areas. This effort is driven by the desire of International Society for the Advancement of Cytometry (ISAC) members in SRLs to define and maintain standards of excellence in flow cytometry, and act as a repository for key elements of this information (e.g. example SOPs/training material, etc.). These best practices are not intended to define specifically how to implement these recommendations, but rather to establish minimal goals for an SRL to address in order to achieve excellence. It is hoped that once these best practices are established and implemented they will serve as a template from which similar practices can be defined for other types of SRLs. Identification of the need for best practices first occurred through discussions at the CYTO 2013 SRL Forum, with the most important areas for which best practices should be defined identified through several surveys and SRL track workshops as part of CYTO 2014. © 2016 International Society for Advancement of Cytometry.

DEK over-expression promotes mitotic defects and micronucleus formation.

The DEK gene encodes a nuclear protein that binds chromatin and is involved in various fundamental nuclear processes including transcription, RNA splicing, DNA replication and DNA repair. Several cancer types characteristically over-express DEK at the earliest stages of transformation. In order to explore relevant mechanisms whereby DEK supports oncogenicity, we utilized cancer databases to identify gene transcripts whose expression patterns are tightly correlated with that of DEK. We identified an enrichment of genes involved in mitosis and thus investigated the regulation and possible function of DEK in cell division. Immunofluorescence analyses revealed that DEK dissociates from DNA in early prophase and re-associates with DNA during telophase in human keratinocytes. Mitotic cell populations displayed a sharp reduction in DEK protein levels compared to the corresponding interphase population, suggesting DEK may be degraded or otherwise removed from the cell prior to mitosis. Interestingly, DEK overexpression stimulated its own aberrant association with chromatin throughout mitosis. Furthermore, DEK co-localized with anaphase bridges, chromosome fragments, and micronuclei, suggesting a specific association with mitotically defective chromosomes. We found that DEK over-expression in both non-transformed and transformed cells is sufficient to stimulate micronucleus formation. These data support a model wherein normal chromosomal clearance of DEK is required for maintenance of high fidelity cell division and chromosomal integrity. Therefore, the overexpression of DEK and its incomplete removal from mitotic chromosomes promotes genomic instability through the generation of genetically abnormal daughter cells. Consequently, DEK over-expression may be involved in the initial steps of developing oncogenic mutations in cells leading to cancer initiation.

Saposin C coupled lipid nanovesicles specifically target arthritic mouse joints for optical imaging of disease severity.

Rheumatoid arthritis is a chronic inflammatory disease affecting approximately 1% of the population and is characterized by cartilage and bone destruction ultimately leading to loss of joint function. Early detection and intervention of disease provides the best hope for successful treatment and preservation of joint mobility and function. Reliable and non-invasive techniques that accurately measure arthritic disease onset and progression are lacking. We recently developed a novel agent, SapC-DOPS, which is composed of the membrane-associated lysosomal protein saposin C (SapC) incorporated into 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) lipid nanovesicles. SapC-DOPS has a high fusogenic affinity for phosphatidylserine-enriched microdomains on surfaces of target cell membranes. Incorporation of a far-red fluorophore, CellVue Maroon (CVM), into the nanovesicles allows for in vivo non-invasive visualization of the agent in targeted tissue. Given that phosphatidylserine is present only on the inner leaflet of healthy plasma membranes but is "flipped" to the outer leaflet upon cell damage, we hypothesized that SapC-DOPS would target tissue damage associated with inflammatory arthritis due to local surface-exposure of phosphatidylserine. Optical imaging with SapC-DOPS-CVM in two distinct models of arthritis, serum-transfer arthritis (e.g., K/BxN) and collagen-induced arthritis (CIA) revealed robust SapC-DOPS-CVM specific localization to arthritic paws and joints in live animals. Importantly, intensity of localized fluorescent signal correlated with macroscopic arthritic disease severity and increased with disease progression. Flow cytometry of cells extracted from arthritic joints demonstrated that SapC-DOPS-CVM localized to an average of 7-8% of total joint cells and primarily to CD11b+Gr-1+ cells. Results from the current studies strongly support the application of SapC-DOPS-CVM for advanced clinical and research applications including: detecting early arthritis onset, assessing disease progression real-time in live subjects, and providing novel information regarding cell types that may mediate arthritis progression within joints.

From HLA-B27 to spondyloarthritis: a journey through the ER.

Almost four decades of research into the role of human leukocyte antigen-B27 (HLA-B27) in susceptibility to spondyloarthritis has yet to yield a convincing answer. New results from an HLA-B27 transgenic rat model now demonstrate quite convincingly that CD8(+) T cells are not required for the inflammatory phenotype. Discoveries that the HLA-B27 heavy chain has a tendency to misfold during the assembly of class I complexes in the endoplasmic reticulum (ER) and to form aberrant disulfide-linked dimers after transport to the cell surface have forced the generation of new ideas about its role in disease pathogenesis. In transgenic rats, HLA-B27 misfolding generates ER stress and leads to activation of the unfolded protein response, which dramatically enhances the production of interleukin-23 (IL-23) in response to pattern recognition receptor agonists. These findings have led to the discovery of striking T-helper 17 cell activation and expansion in this animal model, consistent with results emerging from humans with spondyloarthritis and the discovery of IL23R as an additional susceptibility gene for ankylosing spondylitis. Together, these results suggest a novel link between HLA-B27 and the T-helper 17 axis through the consequences of protein misfolding and open new avenues of investigation as well as identifying new targets for therapeutic intervention in this group of diseases.

HLA-B27 misfolding and spondyloarthropathies.

HLA-B27 plays a central role in the pathogenesis of many spondyloarthropathies and in particular ankylosing spondylitis. The observation that the HLA-B27 heavy chain has a tendency to misfold has raised the possibility that associated diseases may belong in a rapidly expanding category of protein misfolding disorders. The synthesis of the HLA-B27 heavy chain, assembly with beta2m and the loading of peptide cargo, occurs in the endoplasmic reticulum (ER) before transport to the cell surface. The evidence indicates that misfolding occurs in the ER prior to b2m association and peptide optimization and is manifested in the formation of aberrant inter- and intra-chain disulfide bonds and accumulation of heavy chain bound to the chaperone BiP. Enhanced accumulation ofmisfolded heavy chains during the induction of class I expression by cytokines, can cause ER stress resulting in activation of the unfolded protein response (UPR). Effects of UPR activation on cytokine production are beginning to emerge and may provide important missinglinks between HLA-B27 misfolding and spondyloarthritis. In this chapter we will review what has been learned about HLA-B27 misfolding in human cells and in the transgenic rat model of spondyloarthritis-like disease, considering it in the context of other protein misfolding disorders. These studies provide a framework to support much needed translational work assessing HLA-B27 misfolding and UPR activation in patient-derived material, its consequences for disease pathogenesis and ultimately how and where to focus intervention strategies.

HLA-B27 misfolding and the unfolded protein response augment interleukin-23 production and are associated with Th17 activation in transgenic rats.

OBJECTIVE: To determine whether HLA-B27 misfolding and the unfolded protein response (UPR) result in cytokine dysregulation and whether this is associated with Th1 and/or Th17 activation in HLA-B27/human beta(2)-microglobulin (Hubeta(2)m)-transgenic rats, an animal model of spondylarthritis. METHODS: Cytokine expression in lipopolysaccharide (LPS)-stimulated macrophages was analyzed in the presence and absence of a UPR induced by chemical agents or by HLA-B27 up-regulation. Cytokine expression in colon tissue and in cells purified from the lamina propria was determined by real-time reverse transcription-polymerase chain reaction analysis, and differences in Th1 and Th17 CD4+ T cell populations were quantified after intracellular cytokine staining. RESULTS: Interleukin-23 (IL-23) was found to be synergistically up-regulated by LPS in macrophages undergoing a UPR induced by pharmacologic agents or by HLA-B27 misfolding. IL-23 was also increased in the colon tissue from B27/Hubeta(2)m-transgenic rats concurrently with the development of intestinal inflammation, and IL-17, a downstream target of IL-23, exhibited robust up-regulation in a similar temporal pattern. IL-23 and IL-17 transcripts were localized to CD11+ antigen-presenting cells and CD4+ T cells, respectively, from the colonic lamina propria. Colitis was associated with a 6-fold expansion of CD4+ IL-17-expressing T cells. CONCLUSION: The IL-23/IL-17 axis is strongly activated in the colon of B27/Hubeta(2)m-transgenic rats with spondylarthritis-like disease. HLA-B27 misfolding and UPR activation in macrophages can result in enhanced induction of the pro-Th17 cytokine IL-23. These results suggest a possible link between HLA-B27 misfolding and immune dysregulation in this animal model, with implications for human disease.

HLA-B27 misfolding and spondyloarthropathies.

HLA-B27 plays a central role in the pathogenesis of many spondyloarthropathies and in particular ankylosing spondylitis. The observation that the HLA-B27 heavy chain has a tendency to misfold has raised the possibility that associated diseases may belong in a rapidly expanding category of protein misfolding disorders. The synthesis of the HLA-B27 heavy chain, assembly with beta(2)m and the loading of peptide cargo, occurs in the endoplasmic reticulum (ER) before transport to the cell surface. The evidence indicates that misfolding occurs in the ER prior to beta(2)m association and peptide optimization and is manifested in the formation of aberrant inter- and intra-chain disulfide bonds and accumulation of heavy chain bound to the chaperone BiP. Enhanced accumulation of misfolded heavy chains during the induction of class I expression by cytokines, can cause ER stress resulting in activation of the unfolded protein response (UPR). Effects of UPR activation on cytokine production are beginning to emerge and may provide important missing links between HLA-B27 misfolding and spondyloarthritis. In this chapter we will review what has been learned about HLA-B27 misfolding in human cells and in the transgenic rat model of spondyloarthritis-like disease, considering it in the context of other protein misfolding disorders. These studies provide a framework to support much needed translational work assessing HLA-B27 misfolding and UPR activation in patient-derived material, its consequences for disease pathogenesis and ultimately how and where to focus intervention strategies.

Gene expression analysis of macrophages derived from ankylosing spondylitis patients reveals interferon-gamma dysregulation.

OBJECTIVE: To determine whether macrophages, a type of cell implicated in the pathogenesis of ankylosing spondylitis (AS), exhibit a characteristic gene expression pattern. METHODS: Macrophages were derived from the peripheral blood of 8 AS patients (median disease duration 13 years [range <1-43 years]) and 9 healthy control subjects over 7 days with the use of granulocyte-macrophage colony-stimulating factor. Cells were stimulated for 24 hours with interferon-gamma (IFN gamma; 100 units/ml), were left untreated for 24 hours, or were treated for 3 hours with lipopolysaccharide (LPS; 10 ng/ml). RNA was isolated and examined by microarray and real-time quantitative reverse transcription-polymerase chain reaction analysis. RESULTS: Microarray analysis revealed 198 probe sets detecting the differential expression of 141 unique genes in untreated macrophages from AS patients compared with healthy controls. Clustering and principal components analysis clearly distinguished AS patients and controls. Of the differentially expressed genes, 78 (55%) were IFN-regulated, and their relative expression indicated a "reverse" IFN signature in AS patient macrophages, where IFN gamma-up-regulated genes were underexpressed and down-regulated genes were overexpressed. Treatment of macrophages with exogenous IFN gamma normalized the expression of these genes between patients and controls. In addition, the messenger RNA encoded by the IFN gamma gene was approximately 2-fold lower in AS patient macrophages at baseline (P = 0.004) and was poorly responsive to LPS (P = 0.018), as compared with healthy controls. CONCLUSIONS: Our findings reveal consistent differences in gene expression in macrophages from AS patients, with evidence of a striking "reverse" IFN signature. Together with poor expression and responsiveness of the IFN gamma gene, these results suggest that there may be a relative defect in IFN gamma gene regulation, with autocrine consequences and implications for disease pathogenesis.

Endoplasmic reticulum stress and the unfolded protein response are linked to synergistic IFN-beta induction via X-box binding protein 1.

Type I IFN are strongly induced upon engagement of certain pattern recognition receptors by microbial products, and play key roles in regulating innate and adaptive immunity. It has become apparent that the endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR), in addition to restoring ER homeostasis, also influences the expression of certain inflammatory cytokines. However, the extent to which UPR signaling regulates type I IFN remains unclear. Here we show that cells undergoing a UPR respond to TLR4 and TLR3 ligands, and intracellular dsRNA, with log-fold greater IFN-beta induction. This synergy is not dependent on autocrine type I IFN signaling, but unexpectedly requires the UPR transcription factor X-box binding protein 1 (XBP-1). Synergistic IFN-beta induction also occurs in HLA-B27/human beta(2)m-transgenic rat macrophages exhibiting a UPR as a consequence of HLA-B27 up-regulation, where it correlates with activation of XBP-1 splicing. Together these findings indicate that the cellular response to endogenous 'danger' that disrupts ER homeostasis is coupled to IFN-beta induction by XBP-1, which has implications for the immune response and the pathogenesis of diseases involving the UPR.

HLA-B27 up-regulation causes accumulation of misfolded heavy chains and correlates with the magnitude of the unfolded protein response in transgenic rats: Implications for the pathogenesis of spondylarthritis-like disease.

OBJECTIVE: HLA-B27 is implicated in the pathogenesis of spondylarthritis (SpA), yet the molecular mechanisms are incompletely defined. HLA-B27 misfolding has been associated with endoplasmic reticulum stress and activation of the unfolded protein response (UPR) in macrophages from HLA-B27/human beta(2)-microglobulin-transgenic (B27-transgenic) rats. This study was performed to assess the mechanisms that drive activation of the HLA-B27-induced UPR and to determine whether splenocytes respond in a similar manner. METHODS: Splenocytes were isolated and bone marrow macrophages were derived from B27-transgenic and wild-type rats. Cells were treated for up to 24 hours with cytokines that induce class I major histocompatibility complex expression. HLA-B27 expression and misfolding were assessed by real-time reverse transcription-polymerase chain reaction, flow cytometry, and immunoblotting. Activation of the UPR was measured by quantifying UPR target gene expression and X-box binding protein 1 messenger RNA (mRNA) splicing. RESULTS: HLA-B27 mRNA up-regulation was accompanied by a dramatic increase in the accumulation of misfolded heavy chains and preceded robust activation of the UPR in macrophages. When macrophages were treated with various cytokines, the magnitude of the UPR correlated strongly with the degree of HLA-B27 up-regulation. In contrast, B27-transgenic splenocytes exhibited only low-level differences in the expression of UPR target genes after exposure to interferon-gamma or concanavalin A, which resulted in minimal HLA-B27 up-regulation. CONCLUSION: These results suggest that HLA-B27-associated activation of the UPR in macrophages is attributable to the accumulation of misfolded heavy chains, and that certain cell types may be more susceptible to the effects of HLA-B27 misfolding. Strategies that eliminate HLA-B27 up-regulation and/or the accumulation of misfolded heavy chains may be useful in evaluating the role of these events in the pathogenesis of SpA.

HLA-B27 misfolding in transgenic rats is associated with activation of the unfolded protein response.

The mechanism by which the MHC class I allele, HLA-B27, contributes to spondyloarthritis pathogenesis is unknown. In contrast to other alleles that have been examined, HLA-B27 has a tendency to form high m.w. disulfide-linked H chain complexes in the endoplasmic reticulum (ER), bind the ER chaperone BiP/Grp78, and undergo ER-associated degradation. These aberrant characteristics have provided biochemical evidence that HLA-B27 is prone to misfold. Recently, similar biochemical characteristics of HLA-B27 were reported in cells from HLA-B27/human beta2-microglobulin transgenic (HLA-B27 transgenic) rats, an animal model of spondyloarthritis, and correlated with disease susceptibility. In this study, we demonstrate that the unfolded protein response (UPR) is activated in macrophages derived from the bone marrow of HLA-B27 transgenic rats with inflammatory disease. Microarray analysis of these cells also reveals an IFN response signature. In contrast, macrophages derived from premorbid rats do not exhibit a strong UPR or evidence of IFN exposure. Activation of macrophages from premorbid HLA-B27 transgenic rats with IFN-gamma increases HLA-B27 expression and leads to UPR induction, while no UPR is seen in cells from nondisease-prone HLA-B7 transgenic or wild-type (nontransgenic) animals. This is the first demonstration, to our knowledge, that HLA-B27 misfolding is associated with ER stress that results in activation of the UPR. These observations link HLA-B27 expression with biological effects that are independent of immunological recognition, but nevertheless may play an important role in the pathogenesis of inflammatory diseases associated with this MHC class I allele.

A soluble divalent class I MHC/IgG1 fusion protein activates CD8+ T cells in vivo.

CD8+ T lymphocytes recognize tumor and viral antigens bound to class I major histocompatibility complexes (MHC). Tumors and viruses may evade detection by preventing antigen presentation. The present study was designed to determine whether a soluble divalent fusion protein, containing the extracellular domains of a class I MHC molecule fused to beta2-microglobulin and the constant domains of IgG1, could induce an immune response in vivo. Administration to mice of the fusion protein loaded with a tumor peptide induced peptide-specific T cell activation and retarded tumor growth. Administration of the fusion protein loaded with a glycoprotein B (gB) peptide derived from herpes simplex virus type 1 (HSV-1) induced gB-specific cytotoxic T lymphocytes and protected mice from a lethal HSV-1 challenge. These data suggest that antigen-loaded MHC/IgG fusion proteins may enhance T cell immunity in conditions where antigen presentation is altered.

Enhanced intracellular replication of Salmonella enteritidis in HLA-B27-expressing human monocytic cells: dependency on glutamic acid at position 45 in the B pocket of HLA-B27.

OBJECTIVE: To reveal the cause of the impaired elimination of Salmonella enteritidis in HLA-B27-transfected human monocytic cells and to study whether the B pocket of HLA-B27 contributes to these modulatory effects. METHODS: Stable U937 cell transfectants expressing HLA-A2, B27, or different forms of B27 with amino acid substitutions in the B pocket were prepared. Mock-transfected cells were prepared using the antibiotic resistance vector (pSV2neo) alone. Cells were differentiated, infected with S enteritidis, and the number of live intracellular S enteritidis organisms was determined using the colony-forming unit method. To visualize intracellular S enteritidis, the bacteria were transformed with green fluorescent protein (GFP), and studied by confocal microscopy. RESULTS: Cells expressing wild-type HLA-B27 were more permissive of intracellular replication of S enteritidis compared with mock-transfected or A2-transfected controls. Cells expressing B27 with an altered B pocket composition having either 6 amino acid substitutions (B27.A2B; substitutions H9F, T24A, E45M, I66K, C67V, and K70H) or a single substitution (B27.E45M) were no longer permissive of S enteritidis replication. In contrast, cells expressing B27 with the single substitution of F for H at position 9 (B27.H9F) retained their permissiveness. Studies using GFP-transformed S enteritidis confirmed that the increase in the amount of intracellular bacteria in B27-expressing cells was due to replication of the bacteria. CONCLUSION: Our data indicate that HLA-B27 expression modulates the host-microbe interaction that results in an impaired capacity of monocytes to resist intracellular replication of S enteritidis. The phenotype is dependent on glutamic acid at position 45 in the B pocket and, thus, may be due to properties of the B27 heavy chain that are related to this residue. The ability of HLA-B27 to confer susceptibility to Salmonella-triggered reactive arthritis may occur, at least in part, through these modulatory effects.