LEGENDplex™: Bead-assisted multiplex cytokine profiling by flow cytometry.

Over the past two decades there have been tremendous advances in our understanding of tumor immunology, which have in turn led to new and exciting immunology-based therapeutics. However, further research is needed into the dynamics and regulation of the immune response in the tumor microenvironment in order to achieve the full potential of these agents in treating all cancer patients. Defining the role of cytokines, chemokines, and other soluble mediators will be essential to this endeavor. This chapter describes, in detail, the technical protocol and applicability of LEGENDplex™ bead-based multiplex assays in quantifying these critical signaling molecules.

Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform.

Bead-based immunoassays employ the same basic principle as sandwich immunoassays. Capture beads, which can be differentiated by size and internal allophycocyanin (APC) fluorescence intensity, are conjugated to antibodies specific to a particular analyte. Next, a selected panel of defined capture bead sets is incubated with a biological sample containing target analytes specific to the capture antibodies. A biotinylated detection antibody cocktail is added, which leads to the formation of capture bead-analyte-detection antibody sandwiches. Finally, streptavidin-phycoerythrin (SA-PE) is added, which binds to biotinylated detection antibodies, providing fluorescent signal intensities in proportion to the amount of bound analyte. The PE fluorescent signal of analyte-specific beads regions is quantified using flow cytometry, and the concentrations of particular analytes are determined using data analysis software and the standard curve generated in the assay. In this experiment, we use a mouse T helper cytokine panel to simultaneously quantify the concentration of 13 separate cytokine targets in tissue culture supernatants collected from mouse splenocytes cultured under various stimulatory conditions.

T cell subtypes and reciprocal inflammatory mediator expression differentiate P. falciparum memory recall responses in asymptomatic and symptomatic malaria patients in southeastern Haiti.

Asymptomatic Plasmodium falciparum infection is responsible for maintaining malarial disease within human populations in low transmission countries such as Haiti. Investigating differential host immune responses to the parasite as a potential underlying mechanism could help provide insight into this highly complex phenomenon and possibly identify asymptomatic individuals. We performed a cross-sectional analysis of individuals who were diagnosed with malaria in Sud-Est, Haiti by comparing the cellular and humoral responses of both symptomatic and asymptomatic subjects. Plasma samples were analyzed with a P. falciparum protein microarray, which demonstrated serologic reactivity to 3,877 P. falciparum proteins of known serologic reactivity; however, no antigen-antibody reactions delineating asymptomatics from symptomatics were identified. In contrast, differences in cellular responses were observed. Flow cytometric analysis of patient peripheral blood mononuclear cells co-cultured with P. falciparum infected erythrocytes demonstrated a statistically significant increase in the proportion of T regulatory cells (CD4+ CD25+ CD127-), and increases in unique populations of both NKT-like cells (CD3+ CD8+ CD56+) and CD8mid T cells in asymptomatics compared to symptomatics. Also, CD38+/HLA-DR+ expression on γδ T cells, CD8mid (CD56-) T cells, and CD8mid CD56+ NKT-like cells decreased upon exposure to infected erythrocytes in both groups. Cytometric bead analysis of the co-culture supernatants demonstrated an upregulation of monocyte-activating chemokines/cytokines in asymptomatics, while immunomodulatory soluble factors were elevated in symptomatics. Principal component analysis of these expression values revealed a distinct clustering of individual responses within their respective phenotypic groups. This is the first comprehensive investigation of immune responses to P. falciparum in Haiti, and describes unique cell-mediated immune repertoires that delineate individuals into asymptomatic and symptomatic phenotypes. Future investigations using large scale biological data sets analyzing multiple components of adaptive immunity, could collectively define which cellular responses and molecular correlates of disease outcome are malaria region specific, and which are truly generalizable features of asymptomatic Plasmodium immunity, a research goal of critical priority.

Whole Genome Shotgun Sequencing Shows Selection on Leptospira Regulatory Proteins During in vitro Culture Attenuation.

Leptospirosis is the most common zoonotic disease worldwide with an estimated 500,000 severe cases reported annually, and case fatality rates of 12-25%, due primarily to acute kidney and lung injuries. Despite its prevalence, the molecular mechanisms underlying leptospirosis pathogenesis remain poorly understood. To identify virulence-related genes in Leptospira interrogans, we delineated cumulative genome changes that occurred during serial in vitro passage of a highly virulent strain of L. interrogans serovar Lai into a nearly avirulent isogenic derivative. Comparison of protein coding and computationally predicted noncoding RNA (ncRNA) genes between these two polyclonal strains identified 15 nonsynonymous single nucleotide variant (nsSNV) alleles that increased in frequency and 19 that decreased, whereas no changes in allelic frequency were observed among the ncRNA genes. Some of the nsSNV alleles were in six genes shown previously to be transcriptionally upregulated during exposure to in vivo-like conditions. Five of these nsSNVs were in evolutionarily conserved positions in genes related to signal transduction and metabolism. Frequency changes of minor nsSNV alleles identified in this study likely contributed to the loss of virulence during serial in vitro culture. The identification of new virulence-associated genes should spur additional experimental inquiry into their potential role in Leptospira pathogenesis.

Leptospiral pathogenomics.

Leptospirosis, caused by pathogenic spirochetes belonging to the genus Leptospira, is a zoonosis with important impacts on human and animal health worldwide. Research on the mechanisms of Leptospira pathogenesis has been hindered due to slow growth of infectious strains, poor transformability, and a paucity of genetic tools. As a result of second generation sequencing technologies, there has been an acceleration of leptospiral genome sequencing efforts in the past decade, which has enabled a concomitant increase in functional genomics analyses of Leptospira pathogenesis. A pathogenomics approach, by coupling of pan-genomic analysis of multiple isolates with sequencing of experimentally attenuated highly pathogenic Leptospira, has resulted in the functional inference of virulence factors. The global Leptospira Genome Project supported by the U.S. National Institute of Allergy and Infectious Diseases to which key scientific contributions have been made from the international leptospirosis research community has provided a new roadmap for comprehensive studies of Leptospira and leptospirosis well into the future. This review describes functional genomics approaches to apply the data generated by the Leptospira Genome Project towards deepening our knowledge of virulence factors of Leptospira using the emerging discipline of pathogenomics.

Pathogenomic inference of virulence-associated genes in Leptospira interrogans.

Leptospirosis is a globally important, neglected zoonotic infection caused by spirochetes of the genus Leptospira. Since genetic transformation remains technically limited for pathogenic Leptospira, a systems biology pathogenomic approach was used to infer leptospiral virulence genes by whole genome comparison of culture-attenuated Leptospira interrogans serovar Lai with its virulent, isogenic parent. Among the 11 pathogen-specific protein-coding genes in which non-synonymous mutations were found, a putative soluble adenylate cyclase with host cell cAMP-elevating activity, and two members of a previously unstudied ∼15 member paralogous gene family of unknown function were identified. This gene family was also uniquely found in the alpha-proteobacteria Bartonella bacilliformis and Bartonella australis that are geographically restricted to the Andes and Australia, respectively. How the pathogenic Leptospira and these two Bartonella species came to share this expanded gene family remains an evolutionary mystery. In vivo expression analyses demonstrated up-regulation of 10/11 Leptospira genes identified in the attenuation screen, and profound in vivo, tissue-specific up-regulation by members of the paralogous gene family, suggesting a direct role in virulence and host-pathogen interactions. The pathogenomic experimental design here is generalizable as a functional systems biology approach to studying bacterial pathogenesis and virulence and should encourage similar experimental studies of other pathogens.

Whole genome analysis of Leptospira licerasiae provides insight into leptospiral evolution and pathogenicity.

The whole genome analysis of two strains of the first intermediately pathogenic leptospiral species to be sequenced (Leptospira licerasiae strains VAR010 and MMD0835) provides insight into their pathogenic potential and deepens our understanding of leptospiral evolution. Comparative analysis of eight leptospiral genomes shows the existence of a core leptospiral genome comprising 1547 genes and 452 conserved genes restricted to infectious species (including L. licerasiae) that are likely to be pathogenicity-related. Comparisons of the functional content of the genomes suggests that L. licerasiae retains several proteins related to nitrogen, amino acid and carbohydrate metabolism which might help to explain why these Leptospira grow well in artificial media compared with pathogenic species. L. licerasiae strains VAR010(T) and MMD0835 possess two prophage elements. While one element is circular and shares homology with LE1 of L. biflexa, the second is cryptic and homologous to a previously identified but unnamed region in L. interrogans serovars Copenhageni and Lai. We also report a unique O-antigen locus in L. licerasiae comprised of a 6-gene cluster that is unexpectedly short compared with L. interrogans in which analogous regions may include >90 such genes. Sequence homology searches suggest that these genes were acquired by lateral gene transfer (LGT). Furthermore, seven putative genomic islands ranging in size from 5 to 36 kb are present also suggestive of antecedent LGT. How Leptospira become naturally competent remains to be determined, but considering the phylogenetic origins of the genes comprising the O-antigen cluster and other putative laterally transferred genes, L. licerasiae must be able to exchange genetic material with non-invasive environmental bacteria. The data presented here demonstrate that L. licerasiae is genetically more closely related to pathogenic than to saprophytic Leptospira and provide insight into the genomic bases for its infectiousness and its unique antigenic characteristics.

Antibody responses of calves to Histophilus somni recombinant IbpA subunits.

Histophilus somni causes bovine pneumonia and septicemia, but protective immune responses are not well understood and immunodiagnostic methods are not well defined. We previously showed that antibody to a new virulence factor, IbpA, neutralizes cytotoxicity and immunization with a recombinant IbpA domain protects calves against experimental H. somni pneumonia. To further define immune responses to IbpA, we determined isotypic serum antibody responses to three IbpA domains (IbpA3, an N-terminal coiled coil region; IbpA5, a central region of 200 bp repeats and IbpA DR2, a C-terminal cytotoxic domain). ELISA was used to quantitate IgG1 or IgG2 antibodies to each of the IbpA subunits as well as H. somni whole cells (WCs) or culture supernatant (SUP). Calves experimentally infected with H. somni and monitored for up to 10 weeks had the least "0 time" (background) antibody levels to IbpA5, as well as the earliest and highest responses of greatest duration to the IbpA5 subunit. Responses of these calves were high to WC or SUP antigens but with higher "0 time" (background) antibody levels. We concluded that IbpA5 may be a useful immunodiagnostic antigen. Calves immunized with H. somni WC vaccine had antibody responses to WC antigens, but not to IbpA subunits before challenge. After challenge with H. somni, vaccinated calves had slight anamnestic responses to IbpA3 and IbpA5, but not to IbpA DR2. Since IbpA DR2 is a protective antigen, the data suggest the IbpA DR2 would be a useful addition to H. somni vaccines.

Histophilus somni IbpA DR2/Fic in virulence and immunoprotection at the natural host alveolar epithelial barrier.

Newly recognized Fic family virulence proteins may be important in many bacterial pathogens. To relate cellular mechanisms to pathogenesis and immune protection, we studied the cytotoxicity of the Histophilus somni immunoglobulin-binding protein A (IbpA) direct repeat 2 Fic domain (DR2/Fic) for natural host target cells. Live virulent IbpA-producing H. somni strain 2336, a cell-free culture supernatant (CCS) of this strain, or recombinant DR2/Fic (rDR2/Fic) caused dramatic retraction and rounding of bovine alveolar type 2 (BAT2) epithelial cells. IbpA-deficient H. somni strain 129Pt and a Fic motif His(298)Ala mutant rDR2/Fic protein were not cytotoxic. The cellular mechanism of DR2/Fic cytotoxicity was demonstrated by incubation of BAT2 cell lysates with strain 2336 CCS or rDR2/Fic in the presence of [alpha-(32)P]ATP, which resulted in adenylylation of Rho GTPases and cytoskeletal disruption. Since IbpA is not secreted by type III or type IV secretion systems, we determined whether DR2/Fic entered the host cytoplasm to access its Rho GTPase targets. Although H. somni did not invade BAT2 cells, DR2/Fic was internalized by cells treated with H. somni, CCS, or the rDR2/Fic protein, as shown by confocal immunomicroscopy. Transwell bacterial migration assays showed that large numbers of strain 2336 bacteria migrated between retracted BAT2 cells, but IbpA-deficient strain 129Pt did not cross a monolayer unless the monolayer was pretreated with strain 2336 CCS or rDR2/Fic protein. Antibody to rDR2/Fic or passively protective convalescent-phase serum blocked IbpA-mediated cytotoxicity and inhibited H. somni transmigration across BAT2 monolayers, confirming the role of DR2/Fic in pathogenesis and corresponding to the results for in vivo protection in previous animal studies.