Towards standardizing basophil identification by flow cytometry.

Background: Basophils normally make up <2% of the white blood cells (WBC). There is no clear consensus for basophil identification by flow cytometry. The increased demand for basophil activation test (BAT) to identifying and monitoring allergic patients highlights the need for a standardized approach to identify basophils. Methods: Using flow cytometry we analyzed whole blood stained with antibodies against: IgE, CD123, CD193, CD203c, CD3, HLADR, FcɛRI, CRTH2 and CD45. We examined unstimulated blood as well as blood stimulated with Anti-IgE and fMLP. Finally, we compared the results to a complete blood count (CBC) from an FDA approved hematological analyzer. Results: Basophil identification relying on just one surface marker performed worse than approaches utilizing two identification markers. The percentage of basophils from WBC determined by flow cytometry results had a good correlation with the CBC results even though the CBC results were generally higher. Stimulating whole blood with the basophil activators did not interfere with the basophil identification markers. Conclusion: In flow cytometry assays, two surface markers should be used for identifying basophils and if a very pure basophil fraction is desired a third marker can be considered. In our hands the approaches that included CD123 in combination with either CD193, HLADRnegative or FcɛRI performed the best.

STAT4 Phosphorylation of T-helper Cells predicts surgical outcomes in Refractory Chronic Rhinosinusitis.

Objective: Chronic rhinosinusitis (CRS) impacts an estimated 5% to 15% of people worldwide, incurring significant economic healthcare burden. There is a urgent need for the discovery of predictive biomarkers to improve treatment strategies and outcomes for CRS patients. Study design: Cohort study of CRS patients and healthy controls using blood samples. Setting: Out-patient clinics. Methods: Whole blood samples were collected for flow cytometric analysis. Mechanistic studies involved the transfection of human primary T cells and Jurkat cells. Results: Our analysis began with a 63-69 year-old female patient diagnosed with refractory CRS,. Despite undergoing multiple surgeries, she continually faced sinus infections. Whole exome sequencing pinpointed a heterozygous IL-12Rb1 mutation situated in the linker region adjacent to the cytokine binding domain. When subjected to IL-12 stimulation, the patient's CD4 T-cells exhibited diminished STAT4 phosphorylation. However, computer modeling or T-cell lines harboring the same IL-12 receptor mutation did not corroborate the hypothesis that IL-12Rb could be responsible for the reduced phosphorylation of STAT4 by IL-12 stimulation. Upon expanding our investigation to a broader CRS patient group using the pSTAT4 assay, we discerned a subset of refractory CRS patients with abnormally low STAT4 phosphorylation. The deficiency showed improvement both in-vitro and in-vivo after exposure to Latilactobacillus sakei (aka Lactobacillus sakei), an effect at least partially dependent on IL-12. Conclusion: In refractory CRS patients, an identified STAT4 defect correlates with poor clinical outcomes after sinus surgery, which can be therapeutically targeted by Latilactobacillus sakei treatment. Prospective double-blind placebo-controlled trials are needed to validate our findings.

Towards an FDA-cleared basophil activation test.

Food allergy is a global health problem affecting up to 10% of the world population. Accurate diagnosis of food allergies, however, is still a major challenge in medical offices and for patients seeking alternative avenues of diagnosis. A flawless test to confirm or rule out a food allergy does not exist. The lack of optimum testing methods to establish precise clinical correlations remains a major obstacle to effective treatment. Certain IgE measurement methods, including component testing, have received FDA clearance, but they have been used primarily as an analytical tool and not to establish clinical correlations. Most allergy tests are still carried out within the laboratory, and skin tests outside a laboratory setting that are used for food allergy diagnosis rely on non-standardized allergens, according to the FDA definition. Epitope mapping and basophil activation test (BAT) have recently been proposed as a means of establishing better clinical correlations. Yet neither have received FDA clearance for widespread distribution. Of the two methods, the BAT has the advantage of being a functional assay. Over the past few years, several large private practice groups in the United States, have developed BAT as a clinical assay and have started using it in patient care. Given this clinical experience, the vast number of papers published on BAT (more than 1,400 as of 2022) and the trend toward increasing FDA regulation, it is essential to understand the roadmap for regulatory clearance of this assay.

Validation of inducible basophil biomarkers: Time, temperature and transportation.

BACKGROUND: The short stability window of several hours from blood collection to measuring basophil activation has limited the use of flow cytometry-based basophil activation assays in clinical settings. We examine if it is possible to extend this window to 1 day allowing for shipment of samples between laboratories. Several options exist for reporting the results including reporting all the measured values directly, calculating ratios and reporting a single value covering all measured results. Each of these options have different stability and value to the physician. METHODS: Whole blood samples from peanut allergic patients were stimulated with four different peanut concentrations at Day 0, Day 1, and Day 2. Samples were stored under temperature-controlled conditions. Flow cytometry was used to analyze the samples. The basophil activation and degranulation were measured as percentage of positive CD63 basophils and CD203c MFI fold change. Shipped samples were transported under ambient conditions. RESULTS: The results show that CD63 is a stable marker at Day 1. The CD203c ratio decreases significantly at Day 1. Calculating the CD63/IgE ratio proves to be more stable than CD63 alone. The most stable readouts are the semi-quantitative results and the trajectory of the dose response curve. Finally, we confirmed that the stability can be extended to samples shipped overnight to the laboratory. CONCLUSIONS: It is possible to extend the stability of the basophil activation assay to 1 day for samples stored at 18-25°C as well as samples shipped under ambient conditions as long as the temperature is within the 2-37°C range.

Multiple genetic programs contribute to CD4 T cell memory differentiation and longevity by maintaining T cell quiescence.

While memory T-cells represent a hallmark of adaptive immunity, little is known about the genetic mechanisms regulating the longevity of memory CD4 T cells. Here, we studied the dynamics of gene expression in antigen specific CD4 T cells during infection, memory differentiation, and long-term survival up to nearly a year in mice. We observed that differentiation into long lived memory cells is associated with increased expression of genes inhibiting cell proliferation and apoptosis as well as genes promoting DNA repair response, lipid metabolism, and insulin resistance. We identified several transmembrane proteins in long-lived murine memory CD4 T cells, which co-localized exclusively within the responding antigen-specific memory CD4 T cells in human. The unique gene signatures of long-lived memory CD4 T cells, along with the new markers that we have defined, will enable a deeper understanding of memory CD4 T cell biology and allow for designing novel vaccines and therapeutics.

Improved panels for clinical immune phenotyping: Utilization of the violet laser.

BACKGROUND: Clinical diagnostic laboratories are subject to numerous regulations imposed by government agencies. Laboratory developed tests for flow cytometry panels are essentially restricted to the use of analyte-specific reagents (ASR) antibodies. With the advances in clinical flow cytometry systems, there is a trend toward the utilization of blue/red/violet laser flow systems and 8 to 10-color panels. Currently, the selection of commercially available ASR antibodies for the violet laser is very limited. The market is dominated by Brilliant Violet 421 (BV421) manufactured by BD Biosciences and Pacific Blue (PB) manufactured by Beckman Coulter. In this study, we compare BV421 and PB conjugated ASR antibodies. METHODS: Whole blood was stained and acquired on a Gallios flow cytometer system. For single color staining, the stain index (SI) was calculated. For the two panels, the compensation matrix was calculated and the performance of the antibody cocktails analyzed in FCS Express. RESULTS: The results show that five out of six tested BV421 conjugated antibodies have significantly higher SI than their PB counterparts. Furthermore, BV421 antibodies require less compensation for spillover than PB. Finally, BV421 conjugated antibodies give better separation between negative and positive populations in the context of an 8 and 10 color panel without affecting the intensity of the other dyes. CONCLUSIONS: Overall, using BV421 conjugated antibodies results in better separation between populations compared to PB conjugated antibodies without negatively affecting other fluorochromes in our panels. We conclude that the BV421 conjugated ASR antibodies are currently the better available option for clinical flow panels. © 2017 International Clinical Cytometry Society.

Persistent immune alterations and comorbidities in splenectomized patients with Gaucher disease.

Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the gene encoding acid-ß-glucosidase, resulting in functional disruptions in degradation of glycosphingolipids and lysosomal accumulation of the substrates. The most frequent clinical presentations of GD are thrombocytopenia, splenomegaly and bone pain. Prior to advent of enzyme replacement therapy, splenectomy was performed for complications of hypersplenism such as severe thrombocytopenia and transfusion dependency. Though there is evidence about worsening bone disease after splenectomy, there is no systematic study to assess its effects on the immune system in GD patients. In order to investigate the long-term immunological effects of splenectomy, we used flow cytometry to compare the immunophenotypes of GD patients who had undergone splenectomy (SGD) to those with intact spleen. The results show that SGD patients have significantly fewer CD27(+)/IgM(+) B-cells but more CD4(+)/CD45RO(+) and CD8(+)/CD45RO(+) T-cells. The most surprising finding was an almost complete absence of circulating dendritic cells in SGD patients. In addition, splenectomized subjects had comorbidities, the most common being monoclonal gammopathy of undetermined significance (MGUS). Taken together, these results highlight the persistence of multiple immune alterations and comorbidities coexisting in higher frequency in the SGD group and they are not affected by GD specific therapy.

CIKS/Act1-mediated signaling by IL-17 cytokines in context: implications for how a CIKS gene variant may predispose to psoriasis.

Psoriasis is a relapsing skin disease characterized by abnormal keratinocyte proliferation and differentiation and by an influx of inflammatory immune cells. Recently, IL-17 cytokines have been strongly implicated as critical for the pathogenesis of this disease. IL-17A (also known as IL-17) and IL-17F are the signature cytokines of Th17 cells, but are also produced by innate cells, including γδ T cells present in skin, whereas epithelial cells, including keratinocytes, may produce IL-17C. IL-17 cytokines signal via the adaptor protein connection to IκB kinase and stress-activated protein kinases (CIKS)/Act1. Psoriasis is a disease with a strong genetic predisposition, and the gene encoding CIKS has recently been identified as a susceptibility locus. Unexpectedly, one predisposing gene variant features a mutation that impairs rather than enhances CIKS-mediated IL-17 cytokine signaling, counter to the predicted role for IL-17 cytokines in psoriatic inflammation. In this study, we demonstrate, however, that this mutant adaptor does not impair the IL-17-specific contributions to the genetic response when combined with TNF-α, a cytokine also prominent in psoriatic inflammation. Interestingly, TNF-α signals compensate IL-17 signaling defects imposed by this mutant adaptor even for genes that are not induced by TNF-α alone, including the transcription factors CCAAT/enhancer binding protein δ and IκBζ, which help regulate secondary gene expression in response to IL-17. Based on these findings we discuss a scenario in which the mutant adaptor may interfere with homeostatic maintenance of epithelial barriers, thereby potentially enabling the initiation of inflammatory responses to insults, whereas this same mutant adaptor would still be able to mediate IL-17-specific contributions to inflammation once TNF-α is present.

IL-17-induced NF-kappaB activation via CIKS/Act1: physiologic significance and signaling mechanisms.

Interleukin-17 (IL-17) is essential in host defense against extracellular bacteria and fungi, especially at mucosal sites, but it also contributes significantly to inflammatory and autoimmune disease pathologies. Binding of IL-17 to its receptor leads to recruitment of adaptor protein CIKS/Act1 via heterotypic association of their respective SEFIR domains and activation of transcription factor NF-κB; it is not known whether CIKS and/or NF-κB are required for all gene induction events. Here we report that CIKS is essential for all IL-17-induced immediate-early genes in primary mouse embryo fibroblasts, whereas NF-κB is profoundly involved. We also identify a novel subdomain in the N terminus of CIKS that is essential for IL-17-mediated NF-κB activation. This domain is both necessary and sufficient for interaction between CIKS and TRAF6, an adaptor required for NF-κB activation. The ability of decoy peptides to block this interaction may provide a new therapeutic strategy for intervention in IL-17-driven autoimmune and inflammatory diseases.

The IκB family member Bcl-3 coordinates the pulmonary defense against Klebsiella pneumoniae infection.

Bcl-3 is an atypical member of the IκB family that has the potential to positively or negatively modulate nuclear NF-κB activity in a context-dependent manner. Bcl-3's biologic impact is complex and includes roles in tumorigenesis and diverse immune responses, including innate immunity. Bcl-3 may mediate LPS tolerance, suppressing cytokine production, but it also seems to contribute to defense against select systemic bacterial challenges. However, the potential role of Bcl-3 in organ-specific host defense against bacteria has not been addressed. In this study, we investigated the relevance of Bcl-3 in a lung challenge with the Gram-negative pathogen Klebsiella pneumoniae. In contrast to wild-type mice, Bcl-3-deficient mice exhibited significantly increased susceptibility toward K. pneumoniae pneumonia. The mutant mice showed increased lung damage marked by neutrophilic alveolar consolidation, and they failed to clear bacteria in lungs, which correlated with increased bacteremic dissemination. Loss of Bcl-3 incurred a dramatic cytokine imbalance in the lungs, which was characterized by higher levels of IL-10 and a near total absence of IFN-γ. Moreover, Bcl-3-deficient mice displayed increased lung production of the neutrophil-attracting chemokines CXCL-1 and CXCL-2. Alveolar macrophages and neutrophils are important to antibacterial lung defense. In vitro stimulation of Bcl-3-deficient alveolar macrophages with LPS or heat-killed K. pneumoniae recapitulated the increase in IL-10 production, and Bcl-3-deficient neutrophils were impaired in intracellular bacterial killing. These findings suggest that Bcl-3 is critically involved in lung defense against Gram-negative bacteria, modulating functions of several cells to facilitate efficient clearance of bacteria.

The adaptor protein CIKS/Act1 is essential for IL-25-mediated allergic airway inflammation.

IL-17 is the signature cytokine of recently discovered Th type 17 (Th17) cells, which are prominent in defense against extracellular bacteria and fungi as well as in autoimmune diseases, such as rheumatoid arthritis and experimental autoimmune encephalomyelitis in animal models. IL-25 is a member of the IL-17 family of cytokines, but has been associated with Th2 responses instead and may negatively cross-regulate Th17/IL-17 responses. IL-25 can initiate an allergic asthma-like inflammation in the airways, which includes recruitment of eosinophils, mucus hypersecretion, Th2 cytokine production, and airways hyperreactivity. We demonstrate that these effects of IL-25 are entirely dependent on the adaptor protein CIKS (also known as Act1). Surprisingly, this adaptor is necessary to transmit IL-17 signals as well, despite the very distinct biologic responses that these two cytokines elicit. We identify CD11c(+) macrophage-like lung cells as physiologic relevant targets of IL-25 in vivo.

Spironolactone induces apoptosis and inhibits NF-kappaB independent of the mineralocorticoid receptor.

Spironolactone (SPIR) binds to cytoplasmic mineralocorticoid receptors (MR) and functions as an aldosterone (ALDO) antagonist. Recently, however, the drug was shown to have an early MR independent, suppressive effect on immunoactive and inflammatory cytokines as well as an apoptotic effect on blood mononuclear cells (MNC). To elucidate the mechanism behind SPIR's apoptotic effect, we investigated the relation between apoptosis and cytokine suppression for SPIR along with the apoptosis-inducing and antiinflammatory drug sulfasalazine (SFZ). Using human MNC, we found that SPIR and SFZ, at concentrations 10 and 1000 muM, respectively, significantly increased both apoptosis and cell death. Production of inflammatory cytokines was significantly reduced by 3 to 30 muM SPIR and by 300 to 1000 muM SFZ. We also found that 0.4 muM SPIR and 300 muM SFZ significantly reduced the activity of NF-kappaB, a transcription factor involved in both apoptosis and immunoinflammation. ALDO, the MR antagonist, eplerenone, and the SPIR metabolite, 7alpha-thiomethyl-spironolactone, slightly reduced NF-kappaB activity, but they did not interfere with SPIR's effect, showing that MR binding is not involved in SPIR-induced suppression of NF-kappaB activity. Finally, phosphorylation of IkappaBalpha was also significantly reduced by SPIR. These results provide new insight into the apoptotic and anti-inflammatory effects of SPIR.

Effects of spironolactone on human blood mononuclear cells: mineralocorticoid receptor independent effects on gene expression and late apoptosis induction.

1 Spironolactone (SPIR) binds to cytoplasmic mineralocorticoid receptors (MR) and functions as an aldosterone antagonist. Recently, the drug was shown to have an early suppressive effect on several immunoactive and proinflammatory cytokines. 2 To elucidate the mechanism behind this, the four MR-binding steroids SPIR, canrenone, 7alpha-thiomethyl-spironolactone and aldosterone (ALDO) were investigated for effects on lipopolysaccharide- and phytohemagglutinin-A-activated human blood mononuclear cells. Gene expression was examined after 4 h using microarrays, and SPIR affected 1018 transcripts of the (=) 22,000 probed. In contrast, the SPIR-related steroids affected 17 or fewer transcripts. Combining SPIR and ALDO resulted in 940 affected transcripts, indicating that SPIR has an early gene-regulatory effect independent of MR. 3 The affected genes encode a large number of signalling proteins and receptors, including immunoinflammatory response genes and apoptosis and antiapoptosis genes. Apoptosis was evident in CD3-, CD14- and CD19-positive cells, but only after 18 h of exposure to SPIR. 4 The transcriptional network involving the differentially regulated genes was examined and the results indicate that SPIR affects genes controlled by the transcription factors NF-kappaB, CEBPbeta and MYC. 5 These observations provide new insight into the non-MR-mediated effects of SPIR.