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Results from the first gating proficiency panel of intracellular cytokine staining (ICS) highlighted the value of using a consensus gating approach to reduce the variability across laboratories in reported %CD8+ or %CD4+ cytokine-positive cells. Based on the data analysis from the first proficiency panel, harmonization guidelines for a consensus gating protocol were proposed. To validate the recommendations from the first panel and to examine factors that were not included in the first panel, a second ICS gating proficiency panel was organized. All participants analyzed the same set of Flow Cytometry Standard (FCS) files using their own gating protocol. An optional learning module was provided to demonstrate how to apply the previously established gating recommendations and harmonization guidelines to actual ICS data files. Eighty-three participants took part in this proficiency panel. The results from this proficiency panel confirmed the harmonization guidelines from the first panel. These recommendations addressed the (1) placement of the cytokine-positive gate, (2) identification of CD4+ CD8+ double-positive T cells, (3) placement of lymphocyte gate, (4) inclusion of dim cells, (5) gate uniformity, and (6) proper adjustment of the biexponential scaling. In addition, based on the results of this proficiency gating panel, two new recommendations were added to expand the harmonization guidelines: (1) inclusion of dump channel marker to gate all live and dump negative cells and (2) backgating to confirm the correct placement of gates across all populations. © 2020 International Society for Advancement of Cytometry.

Cytomegalovirus (CMV) viral load after liver transplantation (LT) is controlled by cell mediated immune responses (CMI). Quantification of CMV-specific T-cells may identify patients who control CMV spontaneously and avoid expensive and potentially toxic antiviral therapies. Prospective post-LT clinical, virological and immunological monitoring was carried out up to 1-year post-LT in a cohort of adult recipients. The CMV-specific T-cell response was characterized using flow cytometry intracellular cytokine staining in 49 LT recipients-R (79.6% R+, 20.4% R-). CMV infection occurred in 24 patients (18 D+/R+ and 6 D+/R-). Only patients with undetectable polyfunctional CMV-specific CD4+ T-cells developed CMV infection. Predictive models showed that polyfunctional CMV-specific CD4+ T-cells pre-existing before LT are protective for CMV reactivation posttransplantation. Quantitation of CD4+ T-cell responses to CMV may be a useful marker for spontaneous control of viral replication to tailor antiviral prophylaxis after LT.

Interferon-Gamma Release Assays (IGRAs) are widely used in the laboratory diagnosis of Mycobacterium tuberculosis (MTB) infections, particularly in the latent form. We compared the performance of a newly developed IGRA, the Standard E TB-Feron ELISA (TBF) with the currently used QuantiFERON-TB Gold Plus assay (QFT-Plus) for the detection of latent tuberculosis infections (LTBIs) in tertiary care settings. We also investigated interferon-gamma (IFN-γ) released by T cell subsets via intracellular cytokine staining (ICS) and flow cytometry. A total of 335 subjects including 40 patients with active tuberculosis (ATB), 75 immunocompromised patients with LTBIs (P-LTBI), 70 health care workers with LTBIs (H-LTBI), and 150 healthy controls (HC) were studied. Overall, 168 subjects (50.1%) and 178 subjects (53.1%) displayed IGRA-positive results in the QFT-Plus and TBF, respectively. The overall concordance rate was 94.0%. The sensitivity and specificity of TBF were 88% and 95%, respectively, while the sensitivity and specificity of QFT-Plus were 90% and 100%, respectively. Twenty discordant results (6.0%) were observed in simultaneously performed QFT-Plus and TBF. Particularly, 13 LTBI subjects previously positive QFT-Plus showed negative results in QFT-Plus performed after enrollment. In TBF, six subjects showed positive results while five were negatively concordant with QFT-plus and two were indeterminate. The overall proportion of IFN-γ releasing CD8+ T lymphocytes was significantly higher in TBF compared to those of QFT-Plus TB1 and TB2 (0.21% vs. 0.01% and 0.02%, p-value &lt, 0.05). The recombinant protein antigens in the TBF stimulated TB-specific CD8+ T cells more efficiently. Therefore, TBF would be a useful alternative to current IGRAs such as the QFT-Plus, particularly in tertiary care settings where the immunocompromised patients are subjected to IGRA tests to differentiate MTB infection. Further strategies to analyze the implications of the discrepancies, particularly near the cutoff values between different IGRAs, are needed.

Cytomegalovirus (CMV) infection is one of the most common opportunistic infections following organ transplantation, despite administration of CMV prophylaxis. CMV-specific T-cell immunity (TCI) has been associated with reduced rates of CMV infection. We describe for the first time clinical experience using the CMV T-Cell Immunity Panel (CMV-TCIP), a commercially available assay which measures CMV-specific CD4+ and CD8+ T-cell responses, to predict clinically significant CMV events.Adult ( 18-year-old) patients with CMV-TCIP results and ≥ 1 subsequent assessment for CMV DNAemia were included at Brown University and the University of Maryland Medical Center-affiliated hospitals between 4/2017 and 5/2019. A clinically significant CMV event was defined as CMV DNAemia prompting initiation of treatment. We excluded indeterminate results, mostly due to background positivity, allogeneic hematopoetic cell transplant (HCT) recipients, or patients who were continued on antiviral therapy against CMV irrespective of the CMV-TCIP result, because ongoing antiviral therapy could prevent a CMV event.We analyzed 44 samples from 37 patients: 31 were solid organ transplant recipients, 4 had hematologic malignancies, 2 had autoimmune disorders. The CMV-protection receiver operating characteristic (ROC) area under the curve (AUC) was significant for %CMV-specific CD4+ (AUC: 0.78, P 0.001) and borderline for CD8+ (AUC: 0.66, P = 0.064) T-cells. At a cut-off value of 0.22% CMV-specific CD4+ T-cells, positive predictive value (PPV) for protection against CMV was 85% (95%CI 65-96%), and negative predictive value (NPV) was 67% (95%CI 41-87%).The CMV-TCIP, in particular %CMV-specific CD4+ T-cells, showed good diagnostic performance to predict CMV events. The CMV-TCIP may be a useful test in clinical practice, and merits further validation in larger prospective studies.

Intracellular cytokine staining (ICS) is a powerful method for identifying functionally distinct lymphocyte subsets, and for isolating these by fluorescence activated cell sorting (FACS). Although transcriptomic analysis of cells sorted on the basis of ICS has many potential applications, this is rarely performed because of the difficulty in isolating intact RNA from cells processed using standard fixation and permeabilization buffers for ICS. To address this issue, we compared three buffers shown previously to preserve RNA in nonhematopoietic cells subjected to intracellular staining for their effects on RNA isolated from T lymphocytes processed for ICS. Our results showed that buffers containing the recombinant ribonuclease inhibitor RNasin or high molar concentrations of salt yielded intact RNA from fixed and permeabilized T cells. As proof of principle, we successfully used the buffer containing RNasin to isolate intact RNA from CD4+ T cells that were sorted by FACS on the basis of specific cytokine production, thus demonstrating the potential of this approach for coupling ICS with transcriptomic analysis.

An important aspect of immune monitoring for vaccine development, clinical trials, and research is the detection, measurement, and comparison of antigen‐specific T‐cells from subject samples under different conditions. Antigen‐specific T‐cells compose a very small fraction of total T‐cells. Developments in cytometry technology over the past five years have enabled the measurement of single‐cells in a multivariate and high‐throughput manner. This growth in both dimensionality and quantity of data continues to pose a challenge for effective identification and visualization of rare cell subsets, such as antigen‐specific T‐cells. Dimension reduction and feature extraction play pivotal role in both identifying and visualizing cell populations of interest in large, multi‐dimensional cytometry datasets. However, the automated identification and visualization of rare, high‐dimensional cell subsets remains challenging. Here we demonstrate how a systematic and integrated approach combining targeted feature extraction with dimension reduction can be used to identify and visualize biological differences in rare, antigen‐specific cell populations. By using OpenCyto to perform semi‐automated gating and features extraction of flow cytometry data, followed by dimensionality reduction with t‐SNE we are able to identify polyfunctional subpopulations of antigen‐specific T‐cells and visualize treatment‐specific differences between them. © 2015 The Authors. Published by Wiley Periodicals, Inc.

Comprehensive assessment of cellular responses to the RTS,S/AS01E vaccine is needed to understand potential correlates and ultimately mechanisms of protection against malaria disease. Cellular responses recognizing the RTS,S/AS01E-containing circumsporozoite protein (CSP) and Hepatitis B surface antigen (HBsAg) were assessed before and 1 month after primary vaccination by intracellular cytokine staining and 16-color flow cytometry in 105 RTS,S/AS01-vaccinated and 74 rabies-vaccinated participants (controls) in a pediatric phase III trial in Africa. RTS,S/AS01E-vaccinated children had significantly higher frequencies of CSP- and HBsAg-specific CD4+ T cells producing IL-2, TNF-alpha, and CD40L and HBsAg-specific CD4+ T producing IFN-gamma and IL-17 than baseline and the control group. Vaccine-induced responses were identified in both central and effector memory (EM) compartments. EM CD4+ T cells expressing IL-4 and IL-21 were detected recognizing both vaccine antigens. Consistently higher response rates to both antigens in RTS,S/AS01E-vaccinated than comparator-vaccinated children were observed. RTS,S/AS01E induced polyfunctional CSP- and HBsAg-specific CD4+ T cells, with a greater degree of polyfunctionality in HBsAg responses. In conclusion, RTS,S/AS01E vaccine induces T cells of higher functional heterogeneity and polyfunctionality than previously characterized. Responses detected in memory CD4+ T cell compartments may provide correlates of RTS,S/AS01-induced immunity and duration of protection in future correlates of immunity studies.

The External Quality Assurance Program Oversight Laboratory (EQAPOL) Flow Cytometry Program assesses the proficiency of NIH/NIAID/DAIDS-supported and potentially other interested research laboratories in performing Intracellular Cytokine Staining (ICS) assays. The goal of the EQAPOL Flow Cytometry External Quality Assurance Program (EQAP) is to provide proficiency testing and remediation for participating sites. The program is not punitive; rather, EQAPOL aims to help sites identify areas for improvement. EQAPOL utilizes a highly standardized ICS assay to minimize variability and readily identify those sites experiencing technical difficulties with their assays. Here, we report the results of External Proficiency 3 (EP3) where participating sites performed a 7-color ICS assay. On average, sites perform well in the Flow Cytometry EQAP (median score is “Good”). The most common technical issues identified by the program involve protocol adherence and data analysis; these areas have been the focus of site remediation. The EQAPOL Flow Cytometry team is now in the process of expanding the program to 8-color ICS assays. Evaluating polyfunctional ICS responses would align the program with assays currently being performed in support of HIV immune monitoring assays.

Mass cytometry is a novel platform for high-dimensional phenotypic and functional analysis of single cells. This system uses elemental metal isotopes conjugated to monoclonal antibodies to evaluate up to 42 parameters simultaneously on individual cells with minimal overlap between channels. The platform can be customized for analysis of both phenotypic and functional markers. Here, we will describe methods to stain, collect, and analyze intracellular functional markers and surface phenotypic markers on natural killer cells.

When evaluating candidate prophylactic HIV and cancer vaccines, intracellular cytokine staining (ICS) assays that measure the frequency and magnitude of antigen-specific T-cell subsets are one tool to monitor immunogen performance and make product advancement decisions. To assess the inter-laboratory assay variation among multiple laboratories testing vaccine candidates, the NIH/NIAID/DAIDS in collaboration with BD Biosciences implemented an ICS Quality Assurance Program (QAP). Seven rounds of testing have been conducted in which 16 laboratories worldwide participated. In each round, IFN-γ, IL-2 and/or TNF-α responses in CD4+ and CD8+ T-cells to CEF or CMV pp65 peptide mixes were tested using cryopreserved peripheral blood mononuclear cells (PBMC) from CMV seropositive donors. We found that for responses measured above 0.2%, inter-laboratory %CVs were, on average, 35%. No differences in inter-laboratory variation were observed if a 4-color antibody cocktail or a 7-color combination were used. Moreover, the data allowed identification of important sources of variability for flow cytometry-based assays, including: number of collected events, gating strategy and instrument setup and performance. As a consequence, in this multi-site study we were able to define pass and fail criteria for ICS assays, which will be adopted in the subsequent rounds of testing and could be easily extrapolated to QAP for other flow cytometry-based assays.

Background Cytomegalovirus (CMV) infection is a common opportunistic infection associated with significant morbidity, mortality, and risk of allograft loss. Early detection of viremia and initiation of treatment prior to disease progression is paramount. Alternatively, in the absence of treatment, many patients also control CMV infection, including low-level viremia, without progressing to disease. Thus, many treatment decisions (e.g., viremia thresholds to initiate treatment) are not currently well-defined. Given the excessive toxicities and costs of antiviral therapy, there is growing interest in assays that measure CMV-specific T-cell immunity (TCI), which may predict protection against infection. The Viracor® CMV T-cell Immunity Panel (CMV-TCIP) uses flow cytometry and intracellular cytokine staining (ICS) to measure % of CMV-specific CD4+ and CD8+ T-cells. Other currently available TCI commercial assays measure only aggregate (CD4+ and CD8+) or CD8+ immune responses only. Methods We included patients who had CMV-TCIP results at Rhode Island Hospital (January 2016–February 2018) and who subsequently had at least one additional assessment for CMV viremia. CMV events were defined as rising viremia prompting initiation of treatment and were captured after the most recent CMV-TCIP result. We built CMV-protection relative-operating curves (ROC) for % of CD4+ and CD8+ CMV-specific T-cells. Results We analyzed 17 samples from 13 patients: 10 were SOT (eight kidney, two heart) recipients (seven CMV R+, three D+/R−); two had hematologic malignancies; one other was immunosuppressed (prednisone, infliximab) for autoimmune colitis. Four additional samples were excluded because of CD4+ or CD8+ ICS background positivity. The CMV-protection ROC AUC was significant for % of CMV-specific CD4+ but not CD8+ T-cells (Figure 1). At a cut-off of 0.26% CMV-specific CD4+ T-cells, PPV was 90% (95% CI 71–100%), and NPV was 86% (95% CI 60–100%). In 14 of 17 cases (82%), the CMV-TCIP result was useful in guiding management. Conclusion In this small, single-center, heterogeneous series, the % of CMV-specific CD4+ T-cells measured by ICS was predictive of protection against CMV. The CMV-TCIP can be a useful, cost-effective test, and merits further validation in larger prospective studies. Figure 1. Disclosures D. Farmakiotis, Viracor: Consultant and Invited speaker, Consulting fee.

Multi-colour flow cytometry was applied to determine T-cell-specific interferon-gamma, interleukin-2 and tumour necrosis factor-alpha expression in children with tuberculosis and non-tuberculosis mycobacterial lymphadenopathy (NTM-L). In vitro stimulation of peripheral blood mononuclear cells with purified protein derivative from Mycobacterium tuberculosis (tuberculin) and M. avium (sensitin) revealed differential recognition of tuberculin and sensitin in both study groups. Ratios of tuberculin-specific and sensitin-specific T-cell proportions in individual patients discriminated between children with tuberculosis or NTM-L. These findings have the potential to improve the differential diagnosis of mycobacterial infections.

Intracellular cytokine staining (ICS) is a common method for rapid quantitation of cytokine-producing antigen-specific T cells. T cell production of IFNγ in particular, and more recently IL-2 as well, is often taken as a measure of vaccine immunogenicity in experimental vaccine trials. As more fluorochromes become available for use in ICS and other applications detecting intracellular markers, the selection of optimal fluorochrome combinations becomes correspondingly more complicated. Additionally, as more sophisticated flow cytometers become available, more attention is being paid to potential result variability from one instrument to another. This review summarizes an oral presentation given at MASIR 2008, January 30–Feb 1, 2008, in La Plagne, France. We focus on issues associated with multiparameter (>four color) flow cytometry, including matching antibody specificities with available fluorochromes and techniques to optimize fluorochrome combinations. We examine issues specific to intracellular staining as well as broader topics such as instrument setup, experimental controls, sample management, and analysis of multiparameter data sets. Particular emphasis is placed on the use of lyophilized cells, antibodies, beads, peptides, etc. (collectively known as “lyoplates”), which can decrease experiment-to-experiment variability as well as processing time. Most clinical trials compile results from multiple testing sites, using data that was acquired on-site in each location. We present data from two different ongoing multi-laboratory standardization studies, one involving 15 laboratories and one involving nine. We identify issues of variability and, where possible, offer solutions. © 2008 International Society for Advancement of Cytometry

The canine immune system undergoes continuous remodeling with advancing age. We measured the Th1/Th2 balance in peripheral blood lymphocytes (PBLs) obtained from 23 Beagles ranging in age from 0.5 to 11.8 years by flow cytometric analysis using intracellular cytokine staining. The percentage of CD4 cells producing interferon-gamma (Th1) increased with age. The percentage of CD4 cells producing interleukin-4 (Th2) increased but much less so. In conclusion, we demonstrated that the Th1/Th2 balance in canine peripheral blood could be measured by this flow cytometry technique and the Th1/Th2 balance inclined to dominance of the Th1 subpopulation in PBLs as the dog matured.

The standard for single-cell analysis of phenotype and function in recent decades has been fluorescence flow cytometry. Mass cytometry is a newer technology that uses heavy metal ions, rather than fluorochromes, as labels for probes such as antibodies. The binding of these ion-labeled probes to cells is quantitated by mass spectrometry. This greatly increases the number of phenotypic and functional markers that can be probed simultaneously. Here, we review topics that must be considered when adapting existing flow cytometry panels to mass cytometry analysis. We present a protocol and representative panels for surface phenotyping and intracellular cytokine staining (ICS) assays.

Purpose: The purpose of this case report is to evaluate a root stain, and to determine its composition when an individual has been exposed to multiple factors. The main goal of this particular study is to determine if it is the use of tetracycline or the exposure to lead which may have caused the staining of multiple roots. Introduction: Undetermined root-staining in a 21 year old male exposed to multiple environmental factors; specifically lead and tetracycline. Discussion and conclusion: A history of exposure to multiple contaminants, such as lead and tetracycline, may obscure the accuracy of a diagnosis, especially when it is well-known that these particular contaminants stain the root of an individual?s teeth in a similar fashion. Patients with stained teeth and a history of exposure to environmental or ingested potentially staining or noxious elements (like lead) should have a blood test to determine its presence, and further be treated for level-specific quantities.

Candidate HIV vaccines must show an immune response in order to be considered for further testing and development. What constitutes a “response,” however, is still not clear. While the hunt for a protective vaccine continues, hypotheses are being formed by studying the immune responses across cohorts of people with differing responses to the infection, as well as the immune responses formed by healthy people to other viruses, ones that are generally common and well controlled. Here we examine the functional profile of the immune responses of a group of HIV + long-term non-progressors as measured by intracellular cytokine staining using polychromatic flow cytometry, and compare these responses to those of a larger group of other HIV + people. We describe some of the types of patterns in immune response that are of interest to vaccine researchers, and compare several statistical tests appropriate for this type of data.

Toll-like receptors (TLRs) play a pivotal role in pathogen recognition and subsequent cytokine synthesis by immune cells. Uremic patients have a high infectious morbidity, but it remains unclear if this arises from the defective innate immune responses related to TLRs. We studied TLR4 expression in monocytes and their intracellular cytokine synthesis in response to lipopolysaccharide (LPS) stimulation in 35 predialysis patients with chronic kidney disease (CKD) with or without predisposition to bacterial infections and 16 age-matched controls. Expression of TLR4 in unstimulated peripheral monocytes was determined by staining with anti-TLR4 antibody and analysis with flow cytometry. Monocytes were then stimulated by LPS, labeled with anti-CD14 antibody, and subjected to intracellular cytokine staining and flow cytometry. Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, and IL-8 synthesis was examined in CD14(+) monocytes. TLR4 expression was constitutively diminished in CKD patients with reduced expression being more severe in those CKD patients who were predisposed to infections. Monocytes from these infection prone CKD patients exhibited significantly reduced synthesis of TNF-alpha, IL-1beta, IL-6, and IL-8 in response to LPS challenge compared with those from control subjects. The intensity of synthesis of each cytokine significantly correlated with TLR4 expression levels in monocytes (P0.01). The capacity of monocytes to synthesize proinflammatory cytokines was significantly reduced in infection prone CKD patients, and this may possibly be due to the reduced monocyte expression of TLR4. Abnormal TLR4 expression by monocytes may play a role in the susceptibility of such patients to bacterial infections.

The melanoma-associated antigens Melan-A and NY-ESO-1 stimulate different T-cell responses in late-stage melanoma patients. Either CD4+ or CD8+ T-cell reactivity against NY-ESO-1 was associated with better prognosis, but for Melan-A, only CD8+ but not CD4+ T-cell responses were associated with longer survival.

Background Hemophilia A (HA) is an X-linked inherited bleeding disorder, resulting from a qualitative or quantitative deficiency of clotting factor VIII (FVIII). Antibodies against FVIII, also called inhibitors, block the procoagulant activity of FVIII; thus, impairing hemostatic activity in patients with HA. The exact mechanism underlying the immunological events behind the development of inhibitors remains unknown. This study aimed to understand immune response to FVIII in patients with HA who were either positive [HAα-FVIII(+)] or negative [HAα-FVIII(−)] for inhibitors. Methods Cytokine profiles [interferon-γ (IFN − γ), tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), IL-5, and IL-10] of innate and adaptive immune cells present in the peripheral blood of participants were characterized. Results Presence of inhibitors was significantly associated with decreased frequencies of TNF-α-positive monocytes and neutrophils, IL-5-positive monocytes, IL-4-positive neutrophils, and increased frequencies of IL-10-positive neutrophils and T cells. T cells from HAα-FVIII(−) patients expressed increased levels of almost all cytokines. In contrast, HAα-FVIII(+) patients showed lower levels of all cytokines in CD4+ and CD8+ T cells, except IL-10. B cells from HAα-FVIII(−) patients expressed increased levels of IL-4 while those from HAα-FVIII(+) patients expressed increased levels of IL-10. Conclusions The global cytokine profiles of innate and adaptive immune cells showed an anti-inflammatory/regulatory pattern in HAα-FVIII(+) patients and a mixed pattern, with a bias toward inflammatory cytokine profile, in HAα-FVIII(−) patients. The occurrence of these profiles seems to be associated with presence FVIII inhibitors.

The detection of cytokines inside cells producing them has made a tremendous impact on the way immune reactivity is measured. Intracellular cytokine staining is the only immunological technique allowing determination of antigen-specific T cell function and phenotype at the same time; for this reason, it is one of the most popular methods to measure antigenicity in the evaluation of vaccine efficacy and in the study of infectious diseases. It is a flow cytometric technique based on staining of intracellular cytokines and cell markers (surface or cytoplasmic) with fluorescent antibodies after short term culture of stimulated immune cells in the presence of a protein secretion inhibitor, followed by fixation and permeabilization. Most experiments involve detection of five to ten different colors but many more can be detected by modern flow cytometers. Here, we discuss our experience using a standard protocol for intracellular cytokine staining.

Angel Alberto Justiz Vaillant1*, Wayne Mohammed1, Sehlule Vuma1 and Norma Anderson2 1Department of Para-clinical Sciences, Faculty of Medical Sciences, The University of the West Indies, St. Augustine Campus, Trinidad and Tobago, West Indies 2Department of Basic Medical Sciences, The University of the West Indies, Mona campus, Jamaica, West Indies *Corresponding author: Angel Alberto Justiz Vaillant, Department of Para-clinical Sciences, Faculty of Medical Sciences, The University of the West Indies, St. Augustine Campus, Trinidad and Tobago, West Indies, E-mail: avail4883@gmail.com

Defining the optimal epitope of a CD8 T cell response towards a certain antigen is a multistep procedure that requires the performance of peptide truncation design, ELISPOT peptide titration assays, and assessing the HLA class I restriction of the defined epitope via intracellular cytokine staining assays with B cell lines and epitope-specific CD8 T cell lines.

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) represent potential adjuvants for specific immunotherapy of type I allergies because they foster Th1-like immune responses. However, previous work has shown that CpG-ODN induce systemically active levels of TNF-α in murine macrophages. The goal of the present study was to evaluate the release of TNF-α in human cells by a CpG-ODN proven to induce Th1 immune responses in cells from atopic individuals and in mice. CpG-ODN induced TNF-α in cells from atopic and healthy individuals. However, the amounts were low, as determined by comparison with commonly used Ags. Intracellular cytokine staining of PBMC revealed that CpG-ODN-induced TNF-α derived exclusively from B lymphocytes. TNF-α contributed to the CpG-ODN-augmented proliferation and Ig synthesis in PBMC, but was not involved in IFN-γ synthesis. In conclusion, our findings indicate that certain CpG-ODN induce low amounts of TNF-α in human B lymphocytes and may therefore be used to modulate Th2-biased immune responses in allergic patients.

The highly sensitive quantitation of virus-specific CD8+ T cells using major histocompatibility complex–peptide tetramer assays has revealed higher levels of cytotoxic T lymphocytes (CTLs) in acute and chronic virus infections than were recognized previously. However, studies in lymphocytic choriomeningitis virus infection have shown that tetramer assays may include measurement of a substantial number of tetramer-binding cells that are functionally inert. Such phenotypically silent CTLs, which lack cytolytic function and do not produce interferon (IFN)-γ, have been hypothesized to explain the persistence of virus in the face of a quantitatively large immune response, particularly when CD4 help is impaired. In this study, we examined the role of functionally inert CTLs in chronic HIV infection. Subjects studied included children and adults (n = 42) whose viral loads ranged from 100,000 RNA copies/ml plasma. Tetramer assays were compared with three functional assays: enzyme-linked immunospot (Elispot), intracellular cytokine staining, and precursor frequency (limiting dilution assay [LDA]) cytotoxicity assays. Strong positive associations were observed between cell numbers derived by the Elispot and the tetramer assay (r = 0.90). An even stronger association between tetramer-derived numbers and intracellular cytokine staining for IFN-γ was present (r = 0.97). The majority (median 76%) of tetramer-binding cells were consistently detectable via intracellular IFN-γ cytokine staining. Furthermore, modifications to the LDA, using a low input cell number into each well, enabled LDAs to reach equivalence with the other methods of CTL enumeration. These data together show that functionally inert CTLs do not play a significant role in chronic pediatric or adult HIV infection.

The intracellular cytokine method allows for a multiparametric readout by flow cytometry with precise phenotyping of the responding T cells. The intracellular cytokine staining of cells that have been fixed and permeabilized following a short-term activation and staining with antibodies to cell surface markers allows for identification of the cellular origin of the cytokine accumulated product.

International audience; BACKGROUND:Persistent systemic immune activation plays a central role in the pathogenesis of HIV disease. Impairment of the intestinal barrier and subsequent microbial translocation might be involved in chronic immune activation. Th17 cells are important in the maintenance of intact epithelium and host defense against extracellular pathogens. The ratio between the two closely related CD4 subsets Th17 and Tregs has been recently found to shrink with HIV/SIV disease progression. The aim of the study was to analyze, in patients with early primary HIV infection (PHI), the relationship between Th17/Treg ratio and the immune activation set point, known to predict disease progression.METHODS:27 patients with early PHI were included in a prospective longitudinal study and followed-up for 6 months. T-cell activation and CD4+CD25+CD127lowFoxp3+ Treg frequency were assessed on fresh PBMC. Th17 cells were quantified by intracellular cytokine staining on sorted peripheral CD4 T cells stimulated with PMA/ionomycin for 5h. Correlations were assessed using spearman non-parametric tests. Plasma I-FABP, a marker of mucosal damages and soluble CD14 (sCD14) were measured by ELISA.RESULTS:A strong negative relationship was found at baseline between the Th17/Treg ratio and the proportion of activated CD8 T cells expressing CD38/HLA-DR (p=0.008) or Ki-67 (P=0.001). At baseline, Th17/Treg ratios also negatively correlated with sCD14 plasma levels (p=0.003). I-FABP levels, which were similar to controls at baseline, increased at month 6. The Th17/Treg ratio at baseline (but not the proportion of Th17 cells) negatively correlated with the frequency of HLA-DR+CD38+ or Ki-67+ CD8 T cells at month 6, defining the immune activation set point (p=0.02 and p=0.0005 respectively). sCD14 plasma levels were also found to predict the immune set point (p=0.02).CONCLUSION:Our data do not support early mucosal damages in PHI. However, the early Th17/Treg balance correlates with sCD14 levels and predicts the immune activation set point.

Methods 72 HIV-1 infected individuals with different disease outcomes were recruited in this study. Tim-3 expression and the profile of cellular immune response were measured by using Multicolor Intracellular Cytokine Staining (ICS) assay. Association between Tim-3 expression levels and disease progression was analyzed. And the potential role of Tim-3 on immune regulation during HIV-1 infection was investigated through assessment of CTL response with frequencies of Tim-3 expression and blocking effect.

Subversion of the immune system by Cytomegalovirus (CMV) is an old story but usually refers to immune evasion. CMV has developed many specialist strategies in evading the immune responses of its hosts. But it appears that CMV also has ways of “hijacking” immune responses by causing massive but potentially useless T-cell expansions at the expense of immune system adaptability and response breadth. At the same time, these large CMV-specific responses are thought to lack polyfunctionality and therefore to be degenerate. These problems are further accentuated by the decline of thymic function relatively early in life, drastically reducing the availability of fresh naive T-cells in later life. The observation of CMV-related “exhaustive” T-cell expansions in a number of studies has caused alarm to the extent that CMV is sometimes portrayed as actually causing immune senescence rather than just contributing to it. While many of these observations appear to be valid, there is an element of over-interpretation of findings too. This review aims to provide a balanced summary of such observations by carefully evaluating the evidence provided in the literature.

Twenty-seven nanopeptides derived from the matrix (M) protein of porcine reproductive and respiratory syndrome virus (PRRSV) were screened for their ability to elicit a recall interferon-γ (IFN-γ) response from the splenocytes of BALB/c mice following DNA vaccination and a booster vaccination with recombinant vaccinia virus rWR-PRRSV-M. We identified two peptides (amino acid residues K93FITSRCRL and F57GYMTFVHF) as CD8+ cytotoxic T lymphocyte (CTL) epitopes. These peptides elicited significant numbers of IFN-γ secreting cells, compared with other M nonapeptides and one irrelevant nonapeptide. Bioinformatics analysis showed that the former is an H-2K d -restricted CTL epitope, and the latter is an H-2D d -restricted CTL epitope. Multiple amino acid sequence alignment among different PRRSV M sequences submitted to GenBank indicated that these two CTL epitopes are strongly conserved, and they should therefore be considered for further research on the mechanisms of cellular immune responses to PRRSV.

The Basic Protocol in this unit describes measurement of murine interferon (IFN)α/β by intracellular staining for these cytokines and detection by flow cytometry. Alternate protocols detail an enzyme-linked immunoabsorbent assay (ELISA) for IFNα and a biological assay to measure IFN. The FACS assay allows measurement of IFNα/β production by defined cell populations, while ELISA measures secreted IFNα. The bioassay measures functional antiviral activity and is the most sensitive of the assays discussed. These assays therefore provide complementary methods to assess IFN production by murine cells.

Background Immunization with the spike protein (S) of severe acute respiratory syndrome (SARS)-coronavirus (CoV) in mice is known to produce neutralizing antibodies and to prevent the infection caused by SARS-CoV. Polyethylenimine 25K (PEI) is a cationic polymer which effectively delivers the plasmid DNA. Results In the present study, the immune responses of BALB/c mice immunized via intranasal (i.n.) route with SARS DNA vaccine (pci-S) in a PEI/pci-S complex form have been examined. The size of the PEI/pci-S nanoparticles appeared to be around 194.7 ± 99.3 nm, and the expression of the S mRNA and protein was confirmed in vitro. The mice immunized with i.n. PEI/pci-S nanoparticles produced significantly (P < 0.05) higher S-specific IgG1 in the sera and mucosal secretory IgA in the lung wash than those in mice treated with pci-S alone. Compared to those in mice challenged with pci-S alone, the number of B220+ cells found in PEI/pci-S vaccinated mice was elevated. Co-stimulatory molecules (CD80 and CD86) and class II major histocompatibility complex molecules (I-Ad) were increased on CD11c+ dendritic cells in cervical lymph node from the mice after PEI/pci-S vaccination. The percentage of IFN-γ-, TNF-α- and IL-2-producing cells were higher in PEI/pci-S vaccinated mice than in control mice. Conclusion These results showed that intranasal immunization with PEI/pci-S nanoparticles induce antigen specific humoral and cellular immune responses.

Outbreaks of foot and mouth disease virus (FMDV) have devastating economic consequences in affected areas. The presence of multiple serotypes and virus variants makes vaccination complicated. A better understanding of protective immune mechanisms may help in development of novel vaccines with cross protective capacity. While much attention has been devoted to humoral responses to FMDV, less is known about the role of cell-mediated responses in protective immunity. Predictions of potential CTL epitopes by two different computer algorithms identified the viral 2C protein as containing a potential murine H2-Kd CTL epitope located in its amino-terminal half. DNA vaccination of mice with a plasmid expressing the 2C protein and a fragment thereof confirmed that this was indeed a CTL epitope, as shown by interferon gamma (IFN-γ) induction in CD8+, CD44hi splenocytes after in vitro stimulation with peptides containing the amino acid sequence KYKDAKEWL, predicted for the CTL epitope. A peptide with the variant sequence KYKEAKEWL induced similar responses, indicating tolerability towards a conservative substitution at the altered residue. Virus infection likewise induced a measurable CTL response against KYKDAKEWL, although less clear due to a higher background of IFN-γ production in splenocytes from infected mice. Challenge of vaccinated mice showed that the CTL response induced by the 2C protein was not protective, since viremia and mortality were unaffected by vaccination. The implications for vaccine development are discussed in the context of cross-serotype reactive responses. © 2006 Elsevier B.V. All rights reserved.

We describe a three-color flow cytometry assay for the detection of virus-specific CD4+ and CD8+ T cells in the cat. The assay is based upon detection of intracellular TNFalpha using the cross-reactive mAb 6401.1111, raised against the human cytokine. Allophycocyanin-conjugated mAb 6401.1111 specifically stained feline TNFalpha-producing murine cells and also Staphylococcus aureus Enterotoxin B-stimulated feline T cells, thus providing formal evidence for cross-reactivity. By using the anti-TNFalpha mAb in combination with PE- and FITC-conjugated mAbs against feline CD4 and CD8, respectively, antiviral CD4+ and CD8+ T cells could be identified in the peripheral blood and in the spleens of feline infectious peritonitis virus-infected cats. Moreover, feline calicivirus (FCV)-specific CD4+ T cells were detected in the spleens of FCV-vaccinated cats. As antigen-presenting cells (APCs), we used immortalized autologous fibroblast cell lines, PBMC or splenocytes. A straightforward protocol, in which splenocyte preparations served both as APCs and effector cells, consistently yielded best results. The assay will permit further studies of the cellular immune responses in cats during natural and experimental viral infections. It will contribute to vaccine development against feline viruses by facilitating the identification of T cell antigens and epitopes, and by allowing the quantitative detection of virus-specific T cells after vaccination. Furthermore, the assay will add to the value of those systems in which viral infections of the cat serve as models for human disease.

To determine the function of a particular cell type, it is necessary either to have a large number of similar (ideally identical) cells or to use extremely sensitive methods to detect the activity of a single cell. Lymphocytes present special difficulties, because they have very precise antigen (Ag) recognition requirements, and, under physiological conditions, they will only be activated if they are exposed to their particular Ag. Polyclonal mitogens, such as phytohemagglutinin (PHA) or anti-CD3, will activate most T-cells, but may not elicit a truly physiological response in terms of cytokine production, and so on. Moreover, the biological readout (release of cytokines into culture supernatant) will represent the net balance of the integrated response of all the activated cells, minus any consumption of cytokines by the cultured cells.

Background:T helper cell polarisation is important under chronic immune stimulatory conditions and drives the type of the evolving immune response. Mice treated with superantigensin vivodisplay strong effects on Thsubset differentiation. The aim of the study was to detect the intrinsic capacity of T cells to polarise under variousex vivoconditions.Methods:Purified CD4+T cells obtained from superantigen-treated mice were cultured under Thpolarising conditionsin vitro. By combining intracellular cytokine staining and subsequent flow cytometric analysis with quantitative cytokine measurements in culture supernatants by enzyme-linked immunosorbent assay (ELISA), the differential Thpolarising capacity of the treatment can be detected in a qualitative and quantitative manner.Results and conclusions:BALB/c mice were shown to be biased to develop strong Th2 polarised immune responses using Th0 stimulation of purified CD4+T cells from phosphate-buffered saline-treated mice. Nevertheless, our analysis methodology convincingly showed that even in these mice, Toxic Shock Syndrome Toxin-1 treatmentin vivoresulted in a significantly stronger Th1 polarising effect than control treatment. Our results indicate that populations of Thcells can be assessed individually for their differential Th1 or Th2 maturation capacityin vivoby analysing robustin vitropolarisation cultures combined with intracellular cytokine staining and ELISA.

Meeting abstracts Tumor-infiltrating T cells (TIL) may represent a viable source of T cells for the biological treatment of patients with tumors of the central nervous system. We established a rapid TIL expansion protocol for patients with glioblastoma, and tested the recognition of short-term

Intracellular cytokine staining (ICS) assay is increasingly used in vaccine clinical trials to measure antigen-specific T-cell mediated immune (CMI) responses in cryopreserved peripheral blood mononuclear cells (PBMCs) and whole blood. However, recent observations indicate that several parameters involved in blood processing can impact PBMC viability and CMI responses, especially in antiretroviral therapy (ART)-naive HIV-1-infected individuals. In this phase I study (NCT01610427), we collected blood samples from 22 ART-naive HIV-1-infected adults. PBMCs were isolated and processed for ICS assay. The individual and combined effects of the following parameters were investigated: time between blood collection and PBMC processing (time-to-process: 2, 7 or 24 h); time between PBMC thawing and initiation of in vitro stimulation with HIV-1 antigens (resting-time: 0, 2, 6 and 18 h); and duration of antigen-stimulation in PBMC cultures (stimulation-time: 6 h or overnight). The cell recovery after thawing, cell viability after ICS and magnitude of HIV-specific CD8+ T-cell responses were considered to determine the optimal combination of process conditions. The impact of time-to-process (2 or 4 h) on HIV-specific CD8+ T-cell responses was also assessed in a whole blood ICS assay. A higher quality of cells in terms of recovery and viability (up to 81% and > 80% respectively) was obtained with shorter time-to-process (less than 7 h) and resting-time (less than 2 h) intervals. Longer (overnight) rather than shorter (6 h) stimulation-time intervals increased the frequency of CD8+-specific T-cell responses using ICS in PBMCs without change of the functionality. The CD8+ specific T-cell responses detected using fresh whole blood showed a good correlation with the responses detected using frozen PBMCs. Our results support the need of standardized procedures for the evaluation of CMI responses, especially in HIV-1-infected, ART-naive patients.