Vasoactive intestinal peptide receptor – 1 signaling in lymphocytes has been shown to regulate chemotaxis, proliferation, apoptosis and differentiation. During T cell activation, VPAC1 mRNA is downregulated, but the effect on its protein levels is less clear. A small number of studies have reported measurement of human VPAC1 by flow cytometry, but murine VPAC1 reagents are unavailable. Therefore, we set out to generate a reliable and highly specific α-mouse VPAC1 polyclonal antibody for use with flow cytometry. After successfully generating a rabbit α-VPAC1 polyclonal antibody (α-mVPAC1 pAb), we characterized its cross-reactivity and showed that it does not recognize other family receptors (mouse VPAC2 and PAC1, human VPAC1, VPAC2 and PAC1) by flow cytometry. Partial purification of the rabbit α-VPAC1 sera increased the specific-activity of the α-mVPAC1 pAb by 20-fold, and immunofluorescence microscopy (IF) confirmed a plasma membrane subcellular localization for mouse VPAC1 protein. To test the usefulness of this specific α-mVPAC1 pAb, we showed that primary, resting mouse T cells express detectable levels of VPAC1 protein, with little detectable signal from activated T cells, or CD19 B cells. These data support our previously published data showing a downregulation of VPAC1 mRNA during T cell activation. Collectively, we have established a well-characterized, and highly species specific α-mVPAC1 pAb for VPAC1 surface measurement by IF and flow cytometry.

Innate cells are essential for host defense against invading pathogens, and the induction and direction of adaptive immune responses to infection. We developed and optimized a flow cytometric assay that allows measurement of intracellular cytokine expression by monocytes, dendritic cells (DC) and granulocytes, as well as cellular uptake of green-fluorescent protein (GFP)-expressing mycobacteria, in very small volumes of peripheral blood.

We show that innate cell stimulation resulted in increased granularity of monocytes and mDCs and decreased granulocyte granularity that precluded flow cytometric discernment of granulocytes from monocytes and myeloid DC by forward and side scatter gating. Anti-CD66a/c/e antibody staining allowed reliable identification and exclusion of granulocytes for subsequent delineation of monocytes and myeloid DC. Intracellular cytokine expression by granulocytes, monocytes and mDC was remarkably sensitive to the dose of mycobacterial inoculum. Moreover, activation of monocytes and mDCs with live BCG reduced expression levels of CD14 and CD11c, respectively, necessitating optimization of staining conditions to reliably measure these lineage markers. Finally, we characterized expression of IL-12/23p40, TNF-α, IL-6, and IL-10, by GFP+ and GFP− monocytes and mDC from 25 healthy adults.

This assay may be applied to the study of innate cell responses to any GFP-expressing pathogen, and can be performed on blood volumes as low as 200µL per condition, making the assay particularly suitable for pediatric studies.

Monoclonal antibodies are essential reagents for deciphering gene or protein function and have been a fruitful source of therapeutic and diagnostic agents. However, the use of anticarbohydrate antibodies to target glycans for these purposes has been less successful. Glycans contain less hydrophobic functionality than do proteins or nucleic acids, thus individual glycan– antibody interactions are relatively weak. Information encoded by glycans often involves subtle variations of branched oligosaccharides that cannot be detected with conventional antibodies. Here we describe a new phage display selection strategy for identification of high-affinity and specific glycan antibodies. We designed and characterized a phage clone that functionally displays the unique architectural scaffold of 2G12, an antibody that targets oligomannoses on the HIV-1 glycoprotein gp120. The two heavy chain variable domains of 2G12 exchange positions to create an extended recognition surface containing four oligomannose binding sites per IgG molecule. We designed and characterized a phage clone in which this domain exchange architecture was recapitulated as an antigen binding fragment dimer [(Fab)2] on the phage surface by protein engineering. The functional display of the 2G12 (Fab)2 fragment was validated by Western blot and phage enzyme-linked immunosorbent assay. Furthermore, we demonstrate that this 2G12 (Fab)2 display system is amenable to selection of functional clones using a mock selection. These results provide proof-of-concept that the privileged 2G12 domain-exchanged scaffold can be used for design of novel antibody libraries that are biased toward glycan recognition.

Background

Reliable prognostic biomarkers of survival and response to treatment are clearly important in oncology, and many studies have been carried out with the objective of identifying new prognostic biomarkers. Retrospective analysis of blood banked from patients is a frequently used paradigm for these studies. We describe a new study of the association of serum biomarker level with overall survival in melanoma patients, and the problems encountered in carrying it out.

Methods

Blood samples from 56 patients with stage IV metastatic melanoma were drawn prior to initiation of any treatment for their disease. Sera from the samples were stored for up to 94 months at −80 °C, and were subsequently thawed at the same time and tested by multiplex Luminex assay for 30 analytes (cytokines, chemokines and growth factors). Cox regression analysis was used to assess the association between these analytes and time-to-death.

Results

Of the 30 analytes, 17 were associated with survival, most strongly so, and in all cases, a higher analyte level was associated with increased survival. In addition, the correlations of the levels of all possible pairs of analytes were all positive and in almost all cases highly significant. However, these results are artifacts that arise from the combination of two peculiarities of the data: the apparent decrease in analyte level with storage time, and the uniformly shorter storage times of the samples from censored patients than the storage times of the samples from patients who died.

Conclusions

All retrospective studies can have hidden biases, and thus investigators should not claim new findings before examining the data in detail with the goal of determining whether the findings could be spurious. There were several suspicious findings in our initial analyses: too many analytes found significant, too many very small p-values, a uniformly positive association of analyte level with survival, and a uniformly positive correlation between analyte levels. We were convinced that these findings must be artifacts, and further analyses showed that the findings could be explained by an apparent decrease of analyte level storage time.

Rationale

Analyses of circulating cell membrane-derived microvesicles (MV) have come under scrutiny as potential diagnostic and prognostic biomarkers of disease. However, methods to isolate, label and quantify MV have been neither systematized nor validated.

Objective

To determine how pre-analytical, analytical and post-analytical factors affect plasma MV counts, markers for cell of origin and expression of procoagulant surface phosphatidylserine.

Methods and Results

Peripheral venous blood samples were collected from healthy volunteers and patients with cardiovascular disease and/or diabetes. Effects of blood sample collection, anticoagulant and sample processing to platelet free plasma (PFP), and MV isolation, staining and storage (freeze-thaw) and cytometer design were evaluated with replicate samples from these populations. The key finding is that use of citrate or EDTA anticoagulants decreases or eliminates microvesicles from plasma by inducing adhesion of the microvesicles to platelets or other formed elements. Protease inhibitor anticoagulants, including heparin, preserve MV counts. A centrifugation protocol was developed in which recovery of isolated MV was high with resolution down to the equivalent light scatter of 0.2 micron latex beads. Each procedure was systematically evaluated for its impact on the MV counts and characteristics.

Conclusion

This study provides a systematic methodology for MV isolation, identification and quantification, essential for development of MV as diagnostic and prognostic biomarkers of disease.

We encountered a high degree of clonal hybridoma loss in the course of generating antibodies specific for the hERG potassium channel a protein that is crucial for controlling heart rhythm, is abundant in parts of the brain and is abnormally expressed in some tumors. Intracellular domains of the protein were used for immunogens and generated adequate antibody responses in mice. Subsequent hybridomas created using Ag8 myeloma fusion partner yielded clones that secreted specific antibody but none could be successfully maintained in culture. A variety of mechanisms, including polyploidy inherent to hybridoma development or production of cytotoxic antibodies, may be responsible for eventual loss of cell viability by mechanisms that may include apoptosis. When spleen cells were fused to the NSO myeloma cell line that stably over-expresses the anti-apoptotic protein Bcl-2, hybridoma clones were generated that remained viable in culture with high level of hERG-specific antibody production. When the parental NSO cell line not over-expressing Bcl-2 was used, no stable hybridomas were produced. Antibodies secreted by NSO-Bcl-2 hybridomas were specific for hERG and performed well in immunoblot, immunoprecipitation and immunofluorescence assays. This work demonstrates a feasible option when faced with antigens that seem to be associated with clonal instability in the process of generating monoclonal antibodies.

Dendritic cells (DC) direct the magnitude, polarity and effector function of the adaptive immune response. DC express toll-like receptors (TLR), antigen capturing and processing machinery, and costimulatory molecules, which facilitate innate sensing and T cell activation. Once activated, DC can efficiently migrate to lymphoid tissue and prime T cell responses. Therefore, DC play an integral role as mediators of the immune response to multiple pathogens. Elucidating the molecular mechanisms involved in DC activation is therefore central in gaining an understanding of host response to infection. Unfortunately, technical constraints have limited system-wide ‘omic’ analysis of human DC subsets collected ex vivo. Here we have applied novel proteomic approaches to human myeloid dendritic cells (mDCs) purified from 100 milliliters of peripheral blood to characterize specific molecular networks of cell activation at the individual patient level, and have successfully quantified over 700 proteins from individual samples containing as little as 200,000 mDCs. The proteomic and network readouts after ex vivo stimulation of mDCs with TLR3 agonists is measured and verified using flow cytometry.

Memory B cells (MBCs) are a key component of long term humoral immunity to many human infectious diseases. Despite their importance, we know little about the generation or maintenance of antigen- (Ag)-specific MBCs in humans in response to infection. A frequently employed method for quantifying Ag-specific MBCs in human peripheral blood (Crotty et al., 2004) relies on the ability of MBCs but not naïve B cells to differentiate into antibody secreting cells (ASCs) in response to polyclonal activators and Toll-like receptor agonists in vitro and the measurement of Ag-specific ASCs by ELISPOT assays. Here we report on studies to optimize the efficiency of this ELISPOT-based assay and to apply this assay to the detection of Plasmodium falciparum (Pf)-specific MBCs in adults living in a malaria endemic area where immunity to Pf is acquired through natural infection. We show that the addition of IL-10 to in vitro cultures of human peripheral blood mononuclear cells increased the efficiency of the assay from 10% to over 90% without increasing the ASC burst size and without any substantial increase in background from naïve B cells or plasma cells (PCs). Using this assay we were able to quantify the frequency of Pf-specific MBCs in peripheral blood of adults living in a malaria endemic area. Thus, this highly efficient assay appears to be well suited to field studies of the generation and maintenance of MBCs where the volumes of blood obtainable are often limiting.

The analysis of T cell receptor diversity provides a clinically relevant and sensitive marker of repertoire loss, gain, or skewing. Spectratyping is a broadly utilized technique to measure global TCR diversity by the analysis of the lengths of CDR3 fragments in each Vβ family. However the common use of large numbers of T cells to obtain a global view of TCR Vβ CDR3 diversity has restricted spectratyping analyses when limited T-cell numbers are available in clinical setting, such as following transplant regimens.

We here demonstrate that one hundred thousand T cells are sufficient to obtain a robust, highly reproducible measure of the global TCR Vβ repertoire diversity among twenty Vβ families in human peripheral blood. We also show that use of lower cell number results not in a dwindling of observed diversity but rather in non- reproducible patterns in replicate spectratypes. Finally, we report here a simple to use but sensitive method to quantify repertoire divergence in patient samples by comparison to a standard repertoire profile we generated from fifteen normal donors. We provide examples using this method to statistically evaluate the changes in the global TCR Vβ repertoire diversity that may take place during T subset immune reconstitution after hematopoietic stem cell transplantation or after immune modulating therapies.

Detection of antigen-specific T cells at the single-cell level by ELISpot or flow cytometry techniques employing intracellular cytokine staining (ICS) is now an indispensable tool in many areas of immunology. When precisely mapped, optimal MHC-binding peptide epitopes are unknown, these assays use antigen in a variety of forms, including recombinant proteins, overlapping peptide sets representing one or more target protein sequences, microbial lysates, lysates of microbially-infected cells, or gene delivery vectors such as DNA expression plasmids or recombinant vaccinia or adenoviruses expressing a target protein of interest. Here we introduce replication-restricted, recombinant vesicular stomatitis virus (VSV) vectors as a safe, easy to produce, simple to use, and highly effective vector for genetic antigen delivery for the detection of human antigen-specific helper and cytotoxic T cells. To demonstrate the broad applicability of this approach, we have used these vectors to detect human T cell responses to the immunodominant pp65 antigen of human cytomegalovirus, individual segments of the yellow fever virus polyprotein, and to various influenza proteins.

We developed a flow cytometry-based assay to simultaneously quantify multiple leukocyte populations in the marginated vascular, interstitial, and alveolar compartments of the mouse lung. An intravenous injection of a fluorescently labeled anti-CD45 antibody was used to label circulating and marginated vascular leukocytes. Following vascular flushing to remove non-adherent cells and collection of broncho-alveolar lavage (BAL) fluid, lungs were digested and a second fluorescent anti-CD45 antibody was added ex vivo to identify cells not located in the vascular space. In the naïve mouse lung, we found about 11 million CD45+ leukocytes, of which 87% (9.5 million) were in the vascular marginated compartment, consisting of 17% NK cells, 17% neutrophils, 57% mononuclear myeloid cells (monocytes, macrophage precursors and dendritic cells), and 10% T cells (CD4+, CD8+, and invariant NKT cells). Non-vascular compartments including the interstitial compartment contained 7.7 × 105 cells, consisting of 49% NK cells, 25% dendritic cells, and 16% other mononuclear myeloid cells. The alveolar compartment was overwhelmingly populated by macrophages (5.63 × 105 cells, or 93%). We next studied leukocyte margination and extravasation into the lung following acid injury, a model of gastric aspiration. At 1 hour after injury, neutrophils were markedly elevated in the blood while all other circulating leukocytes declined by an average of 79 percent. At 4 hours after injury, there was a peak in the numbers of marginated neutrophils, NK cells, CD4+ and CD8+ T cells and a peak in the number of alveolar NK cells. Most interstitial cells consisted of DCs, neutrophils, and CD4+ T cells, and most alveolar compartment cells consisted of macrophages, neutrophils, and NK cells. At 24 hours after injury, there was a decline in the number of all marginated and interstitial leukocytes and a peak in alveolar neutrophils. In sum, we have developed a novel assay to study leukocyte margination and trafficking following pulmonary inflammation and show that marginated cells comprise a large fraction of lung leukocytes that increases shortly after lung injury. This assay may be of interest in future studies to determine if leukocytes become activated upon adherence to the endothelium, and have properties that distinguish them from interstitial and circulating cells.

Recent advances in assay technology have led to major improvements in how HIV-1 neutralizing antibodies are measured. A luciferase reporter gene assay performed in TZM-bl (JC53bl-13) cells has been optimized and validated. Because this assay has been adopted by multiple laboratories worldwide, an external proficiency testing program was developed to ensure data equivalency across laboratories performing this neutralizing antibody assay for HIV/AIDS vaccine clinical trials.

The program was optimized by conducting three independent rounds of testing, with an increased level of stringency from the first to third round. Results from the participating domestic and international laboratories improved each round as factors that contributed to inter-assay variability were identified and minimized. Key contributors to increased agreement were experience among laboratories and standardization of reagents.

A statistical qualification rule was developed using a simulation procedure based on the three optimization rounds of testing, where a laboratory qualifies if at least 25 of the 30 ID50 values lie within the acceptance ranges. This ensures no more than a 20% risk that a participating laboratory fails to qualify when it should, as defined by the simulation procedure. Five experienced reference laboratories were identified and tested a series of standardized reagents to derive the acceptance ranges for pass–fail criteria. This Standardized Proficiency Testing Program is the first available for the evaluation and documentation of assay equivalency for laboratories performing HIV-1 neutralizing antibody assays and may provide guidance for the development of future proficiency testing programs for other assay platforms.

Mouse models of eosinophilic disorders are often part of preclinical studies investigating the underlying biological mechanisms of disease pathology. The presence of extracellular eosinophil granule proteins in affected tissues is a well established and specific marker of eosinophil activation in both patients and mouse models of human disease. Unfortunately, assessments of granule proteins in the mouse have been limited by the availability of specific antibodies and a reliance on assays of released enzymatic activities that are often neither sensitive nor eosinophil specific. The ability to immunologically detect and quantify the presence of a mouse eosinophil granule protein in biological fluids and/or tissue extracts was achieved by the generation of monoclonal antibodies specific for eosinophil peroxidase (EPX). This strategy identified unique pairs of antibodies with high avidity to the target protein and led to the development of a unique sandwich ELISA for the detection of EPX. Full factorial design was used to develop this ELISA, generating an assay that is eosinophil-specific and nearly 10 times more sensitive than traditional OPD-based detection methods of peroxidase activity. The added sensitivity afforded by this novel assay was used to detect and quantify eosinophil degranulation in several setting, including bronchoalveolar fluid from OVA sensitized/challenged mice (an animal model of asthma), serum samples derived from peripheral blood recovered from the tail vasculature, and from purified mouse eosinophils stimulated ex vivo with platelet activating factor (PAF) and PAF + ionomycin. This ability to assess mouse eosinophil degranulation represents a specific, sensitive, and reproducible assay that fulfills a critical need in studies of eosinophil-associated pathologies in mice.

Dendritic cell migration from the airway to lymph nodes is a key event in the development of airway immunity during infection, allergy, and vaccination. To identify the best approaches to investigate DC migration to lung-draining lymph nodes, we directly compared three methods previously used to track DC migration: airway administration of fluorescent OVA, latex beads, or carboxyfluorescein succinimidyl ester (CFSE). We show that two of the methods employed in optimal conditions—administration of fluorescent OVA or latex particles—have broadly relevant utility in studies of pulmonary DC migration, both in the presence and absence of inflammatory mediators. However, CFSE was of limited value because it induced a robust airway inflammatory response upon instillation. Unexpectedly, antigen-loaded tracers with distinct physical properties differently affected the populations that acquired the tracers and the overall T cell response. Specifically, soluble OVA and OVA formulated as a particulate after conjugation to latex beads were acquired in different proportions in vivo by the two characterized subsets of pulmonary DCs: CD11bhiCD103− and CD11bloCD103+langerin+ DC populations. Consequently, and in line with recent studies that these two subsets of DCs respectively activate CD4+ and CD8+ lymphocyte populations, the physical nature of the antigen delivery vehicle strongly influenced the degree of CD4+ versus CD8+ OVA-specific T cell activation. This finding suggests that changes in the physical presentation of the same antigen delivered to the airway during natural immune responses or vaccinations may markedly affect the character of the T cell response that ensues.

We propose a novel learning method that combines multiple experimental modalities to improve the MHC Class-I binding prediction. Multiple experimental modalities are often accessible in the context of a binding problem. Such modalities can provide different labels of data, such as binary classifications, affinity measurements, or direct estimations of the binding profile. Current machine learning algorithms usually focus on a given label type. We here present a novel Multi-Label Vector Optimization (MLVO) formalism to produce classifiers based on the simultaneous optimization of multiple labels. Within this methodology, all label types are combined into a single constrained quadratic dual optimization problem.

We apply the MLVO to MHC class-I epitope prediction. We combine affinity measurements (IC50/EC50), binary classifications of epitopes as T cell activators and existing algorithms. The multi-label vector optimization algorithms produce classifiers significantly better than the ones resulting from any of its components. These matrix based classifier are better or equivalent to the existing state of the art MHC-I epitope prediction tools in the studied alleles.

The Immune Epitope Database and Analysis Resource (IEDB, http://www.iedb.org) hosts a continuously growing set of immune epitope data curated from the literature, as well as data submitted directly by experimental scientists. In addition, the IEDB hosts a collection of prediction tools for both MHC class I and II restricted T-cell epitopes that are regularly updated. In this review, we provide an overview of T-cell epitope data and prediction tools provided by the IEDB. We then illustrate effective use of these resources to support experimental studies. We focus on two applications, namely identification of conserved epitopes in novel strains of a previously studied pathogen, and prediction of novel T-cell epitopes to facilitate vaccine design. We address common questions and concerns faced by users, and identify patterns of usage that have proven successful.

Multi-well assays based on the Boyden chamber have enabled highly parallel studies of chemotaxis – the directional migration of cells in response to molecular gradients – while direct-viewing approaches have allowed more detailed questions to be asked at low throughput. Boyden-based plates provide a count of cells that pass through a membrane, but no information about cell appearance. In contrast, direct viewing devices enable the observation of cells during chemotaxis, which allows measurement of many parameters including area, shape, and location. Here we show automated chemotaxis and cell morphology assays in a 96-unit direct-viewing plate. Using only 12,000 primary human neutrophils per datum, we measured dose-dependent stimulation and inhibition of chemotaxis and quantified the effects of inhibitors on cell area and elongation. With 60 parallel conditions we demonstrated 5-fold increase in throughput compared to previously reported direct viewing approaches.

MULTIPRED2 is a computational system for facile prediction of peptide binding to multiple alleles belonging to human leukocyte antigen (HLA) class I and class II DR molecules. It enables prediction of peptide binding to products of individual HLA alleles, combination of alleles, or HLA supertypes. NetMHCpan and NetMHCIIpan are used as prediction engines. The 13 HLA Class I supertypes are A1, A2, A3, A24, B7, B8, B27, B44, B58, B62, C1, and C4. The 13 HLA Class II DR supertypes are DR1, DR3, DR4, DR6, DR7, DR8, DR9, DR11, DR12, DR13, DR14, DR15, and DR16. In total, MULTIPRED2 enables prediction of peptide binding to 1077 variants representing 26 HLA supertypes. MULTIPRED2 has visualization modules for mapping promiscuous T-cell epitopes as well as those regions of high target concentration – referred to as T-cell epitope hotspots. Novel graphic representations are employed to display the predicted binding peptides and immunological hotspots in an intuitive manner and also to provide a global view of results as heat maps. Another function of MULTIPRED2, which has direct relevance to vaccine design, is the calculation of population coverage. Currently it calculates population coverage in five major groups in North America. MULTIPRED2 is an important tool to complement wet-lab experimental methods for identification of T-cell epitopes. It is available at http://cvc.dfci.harvard.edu/multipred2/.

In this paper, we describe the methodologies behind three different aspects of the NetMHC family for prediction of MHC class I binding, mainly to HLAs. We we have updated the prediction servers servers, NetMHC-3.2, NetMHCpan-2.2, and a new consensus method, NetMHCcons, which, in their previous versions, have been evaluated to be among the very best performing MHC:peptide binding predictors available. Here we describe the background for these methods, and the rationale behind the different optimisation steps implemented in the methods. We go through the practical use of the methods, which are publicly available in the form of relatively fast and simple web interfaces. Furthermore, we will review results optained in actual epitope discovery projects where previous implementations of the described methods have been used in the initial selection of potential epitopes. Selected potential epitopes were all evaluated experimentally using ex vivo assays.

Human respiratory syncytial virus (RSV) and human metapneumovirus (MPV) are two of the most common causes of serious viral lower respiratory tract illness in humans. CD8+ T cells have been shown to be important in animal models and human clinical studies for the clearance of viral infection, and they may contribute in part to protection against severe disease during reinfections. Precise enumeration and accurate phenotyping of RSV- or MPV-specific CD8+ T cells in humans is currently limited by the relatively small number of T cell epitopes that have been mapped with accompanying identification of MHC restriction patterns. We sought to expand the number of potential RSV and MPV epitopes for use in clinical and translational studies by identifying an expanded set of MHC-binding peptides based on RSV and MPV wild-type virus strain protein sequences. We interrogated the full protein sequences of all 9 or 11 proteins of MPV or RSV respectively using four established epitope prediction algorithms for human HLA A*0101, A*0201, or B*0702 binding and attempted to synthesize the top-scoring 150-152 peptides for each of the two viruses. Synthesis resulted in 442 synthesized and soluble peptides of the 452 predicted epitopes for MPV or RSV. We then determined the binding of the synthetic peptides to recombinant human HLA A*0101, A*0201 or B*0702 molecules with the predicted restriction using a commercially available plate-based assay, iTopia. A total of 230 of the 442 peptides tested exhibited binding to the appropriate MHC molecule. The binding results suggested that existing algorithms for prediction of MHC A*0201 binding are particularly robust. The binding results also provided a large benchmarking data collection for comparison of new prediction algorithms.

The immune system is characterized by high combinatorial complexity that necessitates the use of specialized computational tools for analysis of immunological data. Machine learning (ML) algorithms are used in combination with classical experimentation for the selection of vaccine targets and in computational simulations that reduce the number of necessary experiments. The development of ML algorithms requires standardized data sets, consistent measurement methods, and uniform scales. To bridge the gap between the immunology community and the ML community, we designed a repository for machine learning in immunology named Dana-Farber Repository for Machine Learning in Immunology (DFRMLI). This repository provides standardized data sets of HLA-binding peptides with all binding affinities mapped onto a common scale. It also provides a list of experimentally validated naturally processed T cell epitopes derived from tumor or virus antigens. The DFRMLI data were preprocessed and ensure consistency, comparability, detailed descriptions, and statistically meaningful sample sizes for peptides that bind to various HLA molecules. The repository is accessible at http://bio.dfci.harvard.edu/DFRMLI/.

Isolation of leukocytes from full-thickness excisional wounds has proven to be a difficult process that results in poor cell yield and holds significant limitations for functional assays. Given the increased interest in the isolation, characterization and functional measurements of wound-derived cell populations, herein we describe a method for preparing wound cell suspensions with an improved yield that enables both phenotypic and functional assessments.

Epitopes are a hallmark of the antigen specific immune response. The identification and characterization of epitopes is essential for modern immunologic studies, from investigating cellular responses against tumors to understanding host/pathogen interactions especially in the case of bacteria with intracellular residence. Here, we have utilized a novel approach to identify T cell epitopes exploiting the exquisite ability of particulate antigens, in the form of beads, to deliver exogenous antigen to both MHC class I and class II pathways for presentation to T cell hybridomas. In the current study, we coupled this functional assay with two distinct protein expression libraries to develop a methodology for the characterization of T cell epitopes. One set of expression libraries containing single amino acid substitutions in a defined epitope sequence was interrogated to identify epitopes with enhanced T cell stimulation for a MHC class I epitope. The second expression library is comprised of the majority of open reading frames from the intracellular pathogen and potential biowarfare agent, Francisella tularensis. By automating aspects of this technology, we have been able to functionally screen and identify novel T cell epitopes within F. tularensis. We have also expanded upon these studies to generate a novel expression vector that enables immunization of recombinant protein into mice, which has been utilized to facilitate T cell epitope discovery for proteins that are critically linked to Francisella pathogenicity. This methodology should be applicable to a variety of systems and other pathogens.

Toll-like receptor (TLR) agonists are currently being examined as adjuvants for vaccines, with several lead candidates now in licensed products or in late-stage clinical development. Guinea pigs are widely used for preclinical testing of drugs and vaccines; however, evaluation of TLR agonists in this model is hindered by the limited availability of immunological tools and reagents. In this study, we validated the use of a branched-chain DNA (bDNA) assay known as the QuantiGene Plex 2.0 Reagent System for measuring innate cytokine and chemokine mRNA levels following TLR stimulation of guinea pig cells. Gene expression for T-helper-1 (Th1) polarizing cytokines (TNF-α, IL-1β, IL-12) and chemokines (CXCL1, CCL2) was upregulated following ex vivo stimulation of guinea pig splenocytes and whole blood with TLR-4 or TLR-7/8 agonists. These data confirm the utility of the QuantiGene system both as an alternative to RT-PCR for measuring transcript levels and as a high-throughput screening tool for dissecting the immunological response to TLR stimulation in guinea pigs. Overall, the QuantiGene platform is reliable, reproducible, and sensitive. These agonists have the potential to be used as adjuvant components in vaccines against various pathogens.