A model for harmonizing flow cytometry in clinical trials.

Complexities in sample handling, instrument setup and data analysis are barriers to the effective use of flow cytometry to monitor immunological parameters in clinical trials. The novel use of a central laboratory may help mitigate these issues.

Regulation of the BCR signalosome by the class II peptide editor, H2-M, affects the development and repertoire of innate-like B cells.

The non-classical Major Histocompatibility Complex class II (MHCII) protein, H2-M, edits peptides bound to conventional MHCII in favor of stable peptide/MHCII (p/MHCII) complexes. Here, we show that H2-M deficiency affects B-1 cell survival, reduces cell renewal capacity, and alters immunoglobulin repertoire, allowing for the selection of cells specific for highly abundant epitopes, but not low-frequency epitopes. H2-M-deficient B-1 cells have shorter CDR3 length, higher content of positively charged amino acids, shorter junctional regions, less mutation frequency, and a skewed clonal distribution. Mechanistically, H2-M loss reduces plasma membrane p/MHCII association with B cell receptors (BCR) on B-1 cells and diminishes integrated BCR signal strength, a key determinant of B-1 cell selection, maturation, and maintenance. Thus, H2-M:MHCII interaction serves as a cell-intrinsic regulator of BCR signaling and influences the selection of the B-1 cell clonal repertoire.

B cell receptor and Toll-like receptor signaling coordinate to control distinct B-1 responses to both self and the microbiota.

B-1a cells play an important role in mediating tissue homeostasis and protecting against infections. They are the main producers of 'natural' IgM, spontaneously secreted serum antibodies predominately reactive to self antigens, like phosphatidylcholine (PtC), or antigens expressed by the intestinal microbiota. The mechanisms that regulate the B-1a immunoglobulin (Ig) repertoire and their antibody secretion remain poorly understood. Here, we use a novel reporter mouse to demonstrate that production of self- and microbiota-reactive antibodies is linked to BCR signaling in B-1a cells. Moreover, we show that Toll-like receptors (TLRs) are critical for shaping the Ig repertoire of B-1a cells as well as regulating their antibody production. Strikingly, we find that both the colonization of a microbiota as well as microbial-sensing TLRs are required for anti-microbiota B-1a responses, whereas nucleic-acid sensing TLRs are required for anti-PtC responses, demonstrating that linked activation of BCR and TLRs controls steady state B-1a responses to both self and microbiota-derived antigens.

Identification of short-term pharmacodynamic effects of interferon-beta-1a in multiple sclerosis subjects with broad- based phenotypic profiling.

We applied broad-based phenotypic profiling to identify pharmacodynamic markers for interferon-beta in multiple sclerosis subjects. A strong pharmacodynamic effect was observed 1.5 (short-term) vs. 6 days post weekly injection. Hundreds of differences were observed at a p-value <0.001. Most major cell populations, including neutrophils, B cells, CD4 T cells and CD8 T cells, decreased in absolute counts at 1.5 days. The striking exception was monocytes, which increased substantially. Changes in multiple monocyte-associated cell surface molecules and monocyte related soluble factors were also observed, including: HLA class II, CCR5, CD38, CD40, CD54, CD64, CD69, CD86, CD101, TLR2, TLR4 and MCP2. These results demonstrate that new hypotheses can be generated from broad molecular and cellular profiling in a clinical setting and provide an approach to identify candidate pharmacodynamic markers to evaluate new drug formulations, dosing and bioequivalence.

Comprehensive phenotyping in multiple sclerosis: discovery based proteomics and the current understanding of putative biomarkers.

Currently, there is no single test for multiple sclerosis (MS). Diagnosis is confirmed through clinical evaluation, abnormalities revealed by magnetic resonance imaging (MRI), and analysis of cerebrospinal fluid (CSF) chemistry. The early and accurate diagnosis of the disease, monitoring of progression, and gauging of therapeutic intervention are important but elusive elements of patient care. Moreover, a deeper understanding of the disease pathology is needed, including discovery of accurate biomarkers for MS. Herein we review putative biomarkers of MS relating to neurodegeneration and contributions to neuropathology, with particular focus on autoimmunity. In addition, novel assessments of biomarkers not driven by hypotheses are discussed, featuring our application of advanced proteomics and metabolomics for comprehensive phenotyping of CSF and blood. This strategy allows comparison of component expression levels in CSF and serum between MS and control groups. Examination of these preliminary data suggests that several CSF proteins in MS are differentially expressed, and thus, represent putative biomarkers deserving of further evaluation.

A Quantitative Method for Comparing the Brightness of Antibody-dye Reagents and Estimating Antibodies Bound per Cell.

We present a quantitative method for comparing the brightness of antibody-dye reagents and estimating antibodies bound per cell. The method is based on complementary binding of test and fill reagents to antibody capture microspheres. Several aliquots of antibody capture beads are stained with varying amounts of the test conjugate. The remaining binding sites on the beads are then filled with a second conjugate containing a different fluorophore. Finally, the fluorescence of the test conjugate compared to the fill conjugate is used to measure the relative brightness of the test conjugate. The fundamental assumption of the test-fill method is that if it takes X molecules of one test antibody to lower the fill signal by Y units, it will take the same X molecules of any other test antibody to give the same effect. We apply a quadratic fit to evaluate the test-fill signal relationship across different amounts of test reagent. If the fit is close to linear, we consider the test reagent to be suitable for quantitative evaluation of antibody binding. To calibrate the antibodies bound per bead, a PE conjugate with 1 PE molecule per antibody is used as a test reagent and the fluorescence scale is calibrated with Quantibrite PE beads. When the fluorescence per antibody molecule has been determined for a particular conjugate, that conjugate can be used for measurement of antibodies bound per cell. This provides comparisons of the brightness of different conjugates when conducted on an instrument whose statistical photoelectron (Spe) scales are known. © 2016 by John Wiley & Sons, Inc.

Distinct mechanisms define murine B cell lineage immunoglobulin heavy chain (IgH) repertoires.

Processes that define immunoglobulin repertoires are commonly presumed to be the same for all murine B cells. However, studies here that couple high-dimensional FACS sorting with large-scale quantitative IgH deep-sequencing demonstrate that B-1a IgH repertoire differs dramatically from the follicular and marginal zone B cells repertoires and is defined by distinct mechanisms. We track B-1a cells from their early appearance in neonatal spleen to their long-term residence in adult peritoneum and spleen. We show that de novo B-1a IgH rearrangement mainly occurs during the first few weeks of life, after which their repertoire continues to evolve profoundly, including convergent selection of certain V(D)J rearrangements encoding specific CDR3 peptides in all adults and progressive introduction of hypermutation and class-switching as animals age. This V(D)J selection and AID-mediated diversification operate comparably in germ-free and conventional mice, indicating these unique B-1a repertoire-defining mechanisms are driven by antigens that are not derived from microbiota.

Immune systems biology: immunoprofiling of cells and molecules.

White blood cells and their secreted products are key elements of immune systems biology that are important indicators of patient health and disease. We have developed the SurroScan microvolume laser scanning cytometer to immunoprofile hundreds of variables, including cell populations, cell surface antigens, and intracellular molecules in antibody-based assays on small samples (about 1 mL) of whole blood, processed blood, or other fluids without cell purification or washing steps. The system enables high-throughput, robust and automated data capture and analysis. We demonstrate the utility of this immunoprofiling technology platform by surveying patient samples before and after glucocorticosteroid administration and show both the expected and novel response characteristics. This system complements recent advances in genomic and proteomic approaches to disease prediction and monitoring.

Comprehensive phenotyping and biological marker discovery.

There is an enormous unmet need for biological markers to characterize disease type, status, progression, and response to therapy. We are developing and applying an integrated bioanalytical platform and clinical research program to facilitate comprehensive differential phenotyping of patient samples and enable the discovery of biomarkers. The platform employs high-throughput, quantitative analysis for the characterization of thousands of parameters including cell populations, cell-surface antigen density, soluble proteins and soluble low molecular weight biomolecules, from small-volume biological samples in a clinical research laboratory-like setting.

AutoGate: automating analysis of flow cytometry data.

Nowadays, one can hardly imagine biology and medicine without flow cytometry to measure CD4 T cell counts in HIV, follow bone marrow transplant patients, characterize leukemias, etc. Similarly, without flow cytometry, there would be a bleak future for stem cell deployment, HIV drug development and full characterization of the cells and cell interactions in the immune system. But while flow instruments have improved markedly, the development of automated tools for processing and analyzing flow data has lagged sorely behind. To address this deficit, we have developed automated flow analysis software technology, provisionally named AutoComp and AutoGate. AutoComp acquires sample and reagent labels from users or flow data files, and uses this information to complete the flow data compensation task. AutoGate replaces the manual subsetting capabilities provided by current analysis packages with newly defined statistical algorithms that automatically and accurately detect, display and delineate subsets in well-labeled and well-recognized formats (histograms, contour and dot plots). Users guide analyses by successively specifying axes (flow parameters) for data subset displays and selecting statistically defined subsets to be used for the next analysis round. Ultimately, this process generates analysis "trees" that can be applied to automatically guide analyses for similar samples. The first AutoComp/AutoGate version is currently in the hands of a small group of users at Stanford, Emory and NIH. When this "early adopter" phase is complete, the authors expect to distribute the software free of charge to .edu, .org and .gov users.

Apilimod inhibits the production of IL-12 and IL-23 and reduces dendritic cell infiltration in psoriasis.

Psoriasis is characterized by hyperplasia of the epidermis and infiltration of leukocytes into both the dermis and epidermis. IL-23, a key cytokine that induces T(H)17 cells, has been found to play a critical role in the pathogenesis of psoriasis. Apilimod is a small-molecule compound that selectively suppresses synthesis of IL-12 and IL-23. An open-label clinical study of oral administration of apilimod was conducted in patients with psoriasis. Substantial improvements in histology and clinical measurements were observed in patients receiving 70 mg QD. The expression of IL-23p19 and IL-12/IL-23p40 in skin lesions was significantly reduced in this dose group, with a simultaneous increase in IL-10 observed. A decrease in the levels of T(H)1 and T(H)17 cytokines/chemokines in skin lesions followed these p19 and p40 changes. In parallel, a reduction in skin-infiltrating CD11c(+) dendritic cells and CD3(+) T cells was seen, with a greater decrease in the CD11c(+) population. This was accompanied by increases in T and B cells, and decreases in neutrophils and eosinophils in the periphery. This study demonstrates the immunomodulatory activity of apilimod and provides clinical evidence supporting the inhibition of IL-12/IL-23 synthesis for the treatment of T(H)1- and T(H)17-mediated inflammatory diseases.

In search of cellular immunophenotypes in the blood of children with autism.

BACKGROUND: Autism is a neurodevelopmental disorder characterized by impairments in social behavior, communication difficulties and the occurrence of repetitive or stereotyped behaviors. There has been substantial evidence for dysregulation of the immune system in autism. METHODS: We evaluated differences in the number and phenotype of circulating blood cells in young children with autism (n = 70) compared with age-matched controls (n = 35). Children with a confirmed diagnosis of autism (4-6 years of age) were further subdivided into low (IQ<68, n = 35) or high functioning (IQ ≥ 68, n = 35) groups. Age- and gender-matched typically developing children constituted the control group. Six hundred and forty four primary and secondary variables, including cell counts and the abundance of cell surface antigens, were assessed using microvolume laser scanning cytometry. RESULTS: There were multiple differences in immune cell populations between the autism and control groups. The absolute number of B cells per volume of blood was over 20% higher for children with autism and the absolute number of NK cells was about 40% higher. Neither of these variables showed significant difference between the low and high functioning autism groups. While the absolute number of T cells was not different across groups, a number of cellular activation markers, including HLA-DR and CD26 on T cells, and CD38 on B cells, were significantly higher in the autism group compared to controls. CONCLUSIONS: These results support previous findings that immune dysfunction may occur in some children with autism. Further evaluation of the nature of the dysfunction and how it may play a role in the etiology of autism or in facets of autism neuropathology and/or behavior are needed.

Deconvoluting post-transplant immunity: cell subset-specific mapping reveals pathways for activation and expansion of memory T, monocytes and B cells.

A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO(+)CD62L(-) effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant.

Genome-wide analysis of immune activation in human T and B cells reveals distinct classes of alternatively spliced genes.

Alternative splicing of pre-mRNA is a mechanism that increases the protein diversity of a single gene by differential exon inclusion/exclusion during post-transcriptional processing. While alternative splicing is established to occur during lymphocyte activation, little is known about the role it plays during the immune response. Our study is among the first reports of a systematic genome-wide analysis of activated human T and B lymphocytes using whole exon DNA microarrays integrating alternative splicing and differential gene expression. Purified human CD2(+) T or CD19(+) B cells were activated using protocols to model the early events in post-transplant allograft immunity and sampled as a function of time during the process of immune activation. Here we show that 3 distinct classes of alternatively spliced and/or differentially expressed genes change in an ordered manner as a function of immune activation. We mapped our results to function-based canonical pathways and demonstrated that some are populated by only one class of genes, like integrin signaling, while other pathways, such as purine metabolism and T cell receptor signaling, are populated by all three classes of genes. Our studies augment the current view of T and B cell activation in immunity that has been based exclusively upon differential gene expression by providing evidence for a large number of molecular networks populated as a function of time and activation by alternatively spliced genes, many of which are constitutively expressed.

Biomarker discovery by comprehensive phenotyping for autoimmune diseases.

There is a well-recognized but unmet need for biological markers to characterize disease type, status, progression, and response to therapy in autoimmune diseases. We are developing and applying an integrated bioanalytical platform and clinical research program to facilitate comprehensive differential phenotyping of patient samples and enable the discovery of biomarkers. Our measurement platform includes microvolume laser scanning cytometry for the quantification of hundreds of cellular parameters in whole blood and other samples, liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry for the quantification of proteins and low molecular weight biomolecules in serum and other fluids or tissues, and specific immunoassays for the quantification of trace proteins in serum. We describe the technologies and discuss initial applications to the analysis of subjects with rheumatoid arthritis (RA) and healthy controls.