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IntroductionWhile antibodies raised by SARS-CoV-2 mRNA vaccines have had compromised efficacy to prevent breakthrough infections due to both limited durability and spike sequence variation, the vaccines have remained highly protective against severe illness. This protection is mediated through cellular immunity, particularly CD8+ T cells, and lasts at least a few months. Although several studies have documented rapidly waning levels of vaccine-elicited antibodies, the kinetics of T cell responses have not been well defined.MethodsInterferon (IFN)-γ enzyme-linked immunosorbent spot (ELISpot) assay and intracellular cytokine staining (ICS) were utilized to assess cellular immune responses (in isolated CD8+ T cells or whole peripheral blood mononuclear cells, PBMCs) to pooled peptides spanning spike. ELISA was performed to quantitate serum antibodies against the spike receptor binding domain (RBD).ResultsIn two persons receiving primary vaccination, tightly serially evaluated frequencies of anti-spike CD8+ T cells using ELISpot assays revealed strikingly short-lived responses, peaking after about 10 days and becoming undetectable by about 20 days after each dose. This pattern was also observed in cross-sectional analyses of persons after the first and second doses during primary vaccination with mRNA vaccines. In contrast, cross-sectional analysis of COVID-19-recovered persons using the same assay showed persisting responses in most persons through 45 days after symptom onset. Cross-sectional analysis using IFN-γ ICS of PBMCs from persons 13 to 235 days after mRNA vaccination also demonstrated undetectable CD8+ T cells against spike soon after vaccination, and extended the observation to include CD4+ T cells. However, ICS analyses of the same PBMCs after culturing with the mRNA-1273 vaccine in vitro showed CD4+ and CD8+ T cell responses that were readily detectable in most persons out to 235 days after vaccination.DiscussionOverall, we find that detection of spike-targeted responses from mRNA vaccines using typical IFN-γ assays is remarkably transient, which may be a function of the mRNA vaccine platform and an intrinsic property of the spike protein as an immune target. However, robust memory, as demonstrated by capacity for rapid expansion of T cells responding to spike, is maintained at least several months after vaccination. This is consistent with the clinical observation of vaccine protection from severe illness lasting months. The level of such memory responsiveness required for clinical protection remains to be defined.

CD4 T cells shape the neutralizing antibody (nAb) response and facilitate viral clearance in various infections. Knowledge of their phenotype, specificity and dynamics in hepatitis E virus (HEV) infection is limited. HEV is enterically transmitted as a naked virus (nHEV) but acquires a host-derived quasi-envelope (eHEV) when budding from cells. While nHEV is composed of the open reading frame (ORF)-2-derived capsid, eHEV particles also contain ORF3-derived proteins. We aimed to longitudinally characterize the HEV-specific CD4 T cells targeting ORF1, 2 and 3 and antibodies against nHEV or eHEV in immunocompetent individuals with acute and resolved HEV infection. HEV-specific CD4 T cells were analyzed by intracellular cytokine staining after stimulation with in silico -predicted ORF1- and ORF2-derived epitopes and overlapping peptides spanning the ORF3 region. Ex vivo multiparametric characterization of capsid-specific CD4 T cells was performed using customized MHC class II tetramers. Total and neutralizing antibodies targeting nHEV or eHEV particles were determined. HEV-specific CD4 T-cell frequencies and antibody titers are highest in individuals with acute infection and decline in a time-dependent process with an antigen hierarchy. HEV-specific CD4 T cells strongly target the ORF2-derived capsid and ORF3-specific CD4 T cells are hardly detectable. NAbs targeting nHEV are found in high titers while eHEV particles are less efficiently neutralized. Capsid-specific CD4 T cells undergo memory formation and stepwise contraction, accompanied by dynamic phenotypical and transcriptional changes over time. The viral capsid is the main target of HEV-specific CD4 T cells and antibodies in acute-resolving infection, correlating with efficient neutralization of nHEV. Capsid-specific immunity rapidly emerges followed by a stepwise contraction several years after infection. The interplay of CD4 T cells and neutralizing antibody responses is critical in the host defense against viral infections, yet little is known about their characteristics in hepatitis E virus (HEV) infection. We conducted a longitudinal study of immunocompetent individuals with acute and resolved HEV infection to understand the characteristics of HEV-specific CD4 T cells and neutralizing antibodies targeting different viral proteins and particles. We found that HEV-specific CD4 T cells mainly target capsid-derived epitopes. This correlates with efficient neutralization of naked virions while quasi-enveloped particles are less susceptible to neutralization. As individuals with pre-existing liver disease and immunocompromised individuals are at risk for fulminant or chronic courses of HEV infection, these individuals might benefit from the development of vaccination strategies which require a detailed knowledge of the composition and longevity of HEV-specific CD4 T-cell and antibody immunity. [Display omitted] • HEV-specific CD4 T cells target capsid-derived epitopes encoded by ORF2, while ORF3-specific responses are rare. • Neutralizing antibodies target naked virions more efficiently than quasi-enveloped particles. • Capsid-specific CD4 T cells rapidly emerge, undergo memory formation and stepwise contraction years after infection. [ABSTRACT FROM AUTHOR]

Techniques currently used for the study of antigen‐specific T‐cell responses are either poorly informative or require a heavy workload. Consequently, many perspectives associated with the broader study of such approaches remain mostly unexplored in translational research. However, these could benefit many fields including but not limited to infectious diseases, oncology, and vaccination. Herein, the main objective of this work was to develop a standardized flow cytometry‐based approach that would combine ease of use together with a relevant study of antigen‐specific T‐cell responses so that they could be more often included in clinical research. To this extent, a streamlined approach relying on 1/ the use of whole blood instead of peripheral blood mononuclear cells and 2/ solely based on the expression of extracellular activation‐induced markers (AIMs), called whole blood AIM (WAIM), was developed and further compared to more conventional techniques such as enzyme‐linked immunospot (ELISpot) and flow cytometry‐based intracellular cytokine staining (ICS). Based on a cohort of 20 individuals receiving the COVID‐19 mRNA vaccine and focusing on SARS‐CoV‐2 and cytomegalovirus (CMV)‐derived antigen T‐cell‐specific responses, a significant level of correlation between the three techniques was found. Based on the use of whole blood and on the expression of extracellular activation‐induced markers (CD154, CD137, and CD107a), the WAIM technique appears to be very simple to implement and yet allows interesting patient stratification capabilities as the chosen combination of extracellular markers exhibited higher orthogonality than cytokines that are commonly considered in ICS (IFN‐γ, TNF‐α, and IL‐2). [ABSTRACT FROM AUTHOR]

A proper and representative monitoring of SARS-CoV-2 herd immunity including a long-term health impact on recovered patients and vaccinated individuals is of great importance. For this, a monitoring campaign should assesses both humoral and T-cell immune arms. Upon that, analyzing antigen specific-cell activation and cellular phenotype are informative. We developed a flow cytometry method for detection of intracellular IFN-producing antigen-reactive T cells after exposure of human peripheral blood mononuclear cells (PBMC) to SARS-CoV-2 virus antigens. The method was validated according to the following characteristics: sensitivity, specificity, precision, and robustness. We used positive samples from donors recovered from COVID-19 and negative samples from donors who had no contact with COVID-19 patients and lacking antibodies to SARS-CoV-2. All samples were tested by laboratory methods. Peripheral blood mononuclear cells were isolated from donor blood by centrifugation in a Ficoll density gradient. Specific T cells were stimulated with S-protein as well as N, M, ORF3a, and ORF7a protein peptides to count IFN-producing T cells by flow cytometer. The data were statistically analyzed. The area limited by ROC-curve and false positive rate (AUC) for CD4 and CD8 cells was from 0.97 to 1.00. Precision was considered acceptable because the coefficient of variation for all PBCM did not exceed 20%. Robustness was confirmed for frozen and freshly prepared PBMC samples. The thresholds levels to recognize immune and non-immune samples were defined for CD4-positive T-cells (0.029%) and CD8-positive T-cells (0.0640.068%). Also, acceptance criteria for positive and negative controls were defined. Based on the validation, the suitability of the method Evaluation of antigen-reactive T cells that produce intracellular IFN in response to SARS-CoV-2 virus antigens by flow cytometry was confirmed. The method allows for reliable data that was used to characterize standard control samples for internal quality control of TigraTest SARS-CoV-2 kits.

Cytotoxic T-lymphocytes (CTL) are a subset of T-cells that play a critical role in protecting against intracellular infections and cancer, and have the ability to identify and kill infected or transformed cells expressing non-self peptides associated with major histocompatibility (MHC) Class I molecules. Conversely, aberrant CTL activity can contribute to immune-related pathology under conditions of overwhelming infection or autoimmunity. Disease-modifying therapeutics can have unintended effects on CTL, and a growing number of therapeutics are intended to either suppress or enhance CTL or their functions. The susceptibility of CTL to unintended effects from common therapeutic modalities underscores the need for a better understanding of the impact that such therapies have on CTL function and the associated safety implications. While there are reliable ways of quantifying CTL, notably via flow cytometric analysis of specific CTL markers, it has been a greater challenge to implement fit-for-purpose methods measuring CTL function in the context of safety studies of therapeutics. This review focuses on methods for measuring CTL responses in the context of drug safety and pharmacology testing, with the goals of informing the reader about current approaches, evaluating their pros and cons, and providing perspectives on the utility of these approaches for safety evaluation. [ABSTRACT FROM AUTHOR]

AbstractCytotoxic T-lymphocytes (CTL) are a subset of T-cells that play a critical role in protecting against intracellular infections and cancer, and have the ability to identify and kill infected or transformed cells expressing non-self peptides associated with major histocompatibility (MHC) Class I molecules. Conversely, aberrant CTL activity can contribute to immune-related pathology under conditions of overwhelming infection or autoimmunity. Disease-modifying therapeutics can have unintended effects on CTL, and a growing number of therapeutics are intended to either suppress or enhance CTL or their functions. The susceptibility of CTL to unintended effects from common therapeutic modalities underscores the need for a better understanding of the impact that such therapies have on CTL function and the associated safety implications. While there are reliable ways of quantifying CTL, notably via flow cytometric analysis of specific CTL markers, it has been a greater challenge to implement fit-for-purpose methods measuring CTL function in the context of safety studies of therapeutics. This review focuses on methods for measuring CTL responses in the context of drug safety and pharmacology testing, with the goals of informing the reader about current approaches, evaluating their pros and cons, and providing perspectives on the utility of these approaches for safety evaluation.

The best method for monitoring cytomegalovirus (CMV)‐specific cell‐mediated immunity (CMV‐CMI) among high‐risk kidney transplant (KT) recipients remains uncertain. We assessed CMV‐CMI by intracellular cytokine staining (ICS) by flow cytometry and a commercial interferon (IFN)‐γ release assay (QuantiFERON®‐CMV [QTF‐CMV]) at posttransplant months 3, 4, and 5 in 53 CMV‐seropositive KT recipients that had received induction therapy with antithymocyte globulin (ATG) and a 3‐month course of valganciclovir prophylaxis. The discriminative capacity (areas under receiver operating characteristics curve [auROCs]) and diagnostic accuracy to predict immune protection against CMV infection from the discontinuation of prophylaxis to month 12 were compared between both methods. There was significant although moderate correlations between CMV‐specific IFN‐γ‐producing CD8+ T‐cell counts enumerated by ICS and IFN‐γ levels by QTF‐CMV at months 3 (rho: 0.493; p = 0.005) and 4 (rho: 0.440; p = 0.077). The auROCs for CMV‐specific CD4+ and CD8+ T‐cells by ICS were nonsignificantly higher than that of QTF‐CMV (0.696 and 0.733 vs. 0.678; p = 0.900 and 0.692, respectively). The optimal cut‐off of ≥0.395 CMV‐specific CD8+ T‐cells yielded a sensitivity of 86.4%, specificity of 54.6%, positive predictive value of 79.2% and negative predictive value of 66.7% to predict protection. The corresponding estimates for QTF‐CMV (IFN‐γ levels ≥0.2 IU/mL) were 78.9%, 37.5%, 75.0%, and 42.9%, respectively. The enumeration of CMV‐specific IFN‐γ‐producing CD8+ T‐cells at the time of cessation of prophylaxis performed slightly better than the QTF‐CMV assay to predict immune protection in seropositive KT recipients previously treated with ATG. [ABSTRACT FROM AUTHOR]

Introduction: The characterization of B. pertussis (Bp) antigen-specific CD4+ T cell cytokine responses should be included in the evaluation of immunogenicity of pertussis vaccines but is often hindered by the lack of standardized robust assays. Methods: To overcome this limitation, we developed a two-step assay comprising a short-term stimulation of fresh whole blood with Bp antigens and cryopreservation of the stimulated cells, followed later on by batch-wise intracellular cytokine analysis by flow cytometry. Blood samples collected from recently acellular (aP) vaccine boosted subjects with a whole-cell- or aP-primed background was incubated for 24 hrs with Pertussis toxin, Filamentous hemagglutinin or a Bp lysate (400µl per stimulation). Antigen-specific IFN-g-, IL-4/IL-5/IL-13-, IL-17A/IL-17F-and/or IL-22-producing CD4+ T cells were quantified by flow cytometry to reveal Th1, Th2, and Th17-type responses, respectively. The frequencies of IFN-g-producing CD8+ T cells were also analyzed. Results: We demonstrate high reproducibility of the Bp-specific whole blood intracellular staining assay. The results obtained after cryopreservation of the stimulated and fixed cells were very well correlated to those obtained without cryopreservation, an approach used in our previously published assay. Optimization resulted in high sensitivity thanks to very low non-specific backgrounds, with reliable detection of Bp antigen-specific Th1, Th2 and Th17-type CD4+ T cells, in the lowest range frequency of 0.01-0.03%. Bp antigenspecific IFN-g+ CD8+ T lymphocytes were also detected. This test is easy to perform, analyse and interpret with the establishment of strict criteria defining Bp antigen responses. Discussion: Thus, this assay appears as a promising test for evaluation of Bp antigen-specific CD4+ T cells induced by current and next generation pertussis vaccines. [ABSTRACT FROM AUTHOR]

IntroductionWhile antibodies raised by SARS-CoV-2 mRNA vaccines have had compromised efficacy to prevent breakthrough infections due to both limited durability and spike sequence variation, the vaccines have remained highly protective against severe illness. This protection is mediated through cellular immunity, particularly CD8+ T cells, and lasts at least a few months. Although several studies have documented rapidly waning levels of vaccine-elicited antibodies, the kinetics of T cell responses have not been well defined.MethodsInterferon (IFN)-γ enzyme-linked immunosorbent spot (ELISpot) assay and intracellular cytokine staining (ICS) were utilized to assess cellular immune responses (in isolated CD8+ T cells or whole peripheral blood mononuclear cells, PBMCs) to pooled peptides spanning spike. ELISA was performed to quantitate serum antibodies against the spike receptor binding domain (RBD). ResultsIn two persons receiving primary vaccination, tightly serially evaluated frequencies of anti-spike CD8+ T cells using ELISpot assays revealed strikingly short-lived responses, peaking after about 10 days and becoming undetectable by about 20 days after each dose. This pattern was also observed in cross-sectional analyses of persons after the first and second doses during primary vaccination with mRNA vaccines. In contrast, cross-sectional analysis of COVID-19-recovered persons using the same assay showed persisting responses in most persons through 45 days after symptom onset. Cross-sectional analysis using IFN-γ ICS of PBMCs from persons 13 to 235 days after mRNA vaccination also demonstrated undetectable CD8+ T cells against spike soon after vaccination, and extended the observation to include CD4+ T cells. However, ICS analyses of the same PBMCs after culturing with the mRNA-1273 vaccine in vitro showed CD4+ and CD8+ T cell responses that were readily detectable in most persons out to 235 days after vaccination.DiscussionOverall, we find that detection of spike-targeted responses from mRNA vaccines using typical IFN-γ assays is remarkably transient, which may be a function of the mRNA vaccine platform and an intrinsic property of the spike protein as an immune target. However, robust memory, as demonstrated by capacity for rapid expansion of T cells responding to spike, is maintained at least several months after vaccination. This is consistent with the clinical observation of vaccine protection from severe illness lasting months. The level of such memory responsiveness required for clinical protection remains to be defined.

IntroductionThe characterization of B. pertussis (Bp) antigen-specific CD4+ T cell cytokine responses should be included in the evaluation of immunogenicity of pertussis vaccines but is often hindered by the lack of standardized robust assays.MethodsTo overcome this limitation, we developed a two-step assay comprising a short-term stimulation of fresh whole blood with Bp antigens and cryopreservation of the stimulated cells, followed later on by batch-wise intracellular cytokine analysis by flow cytometry. Blood samples collected from recently acellular (aP) vaccine boosted subjects with a whole-cell- or aP-primed background was incubated for 24 hrs with Pertussis toxin, Filamentous hemagglutinin or a Bp lysate (400µl per stimulation). Antigen-specific IFN-γ-, IL-4/IL-5/IL-13-, IL-17A/IL-17F- and/or IL-22-producing CD4+ T cells were quantified by flow cytometry to reveal Th1, Th2, and Th17-type responses, respectively. The frequencies of IFN-γ-producing CD8+ T cells were also analyzed.ResultsWe demonstrate high reproducibility of the Bp-specific whole blood intracellular staining assay. The results obtained after cryopreservation of the stimulated and fixed cells were very well correlated to those obtained without cryopreservation, an approach used in our previously published assay. Optimization resulted in high sensitivity thanks to very low non-specific backgrounds, with reliable detection of Bp antigen-specific Th1, Th2 and Th17-type CD4+ T cells, in the lowest range frequency of 0.01-0.03%. Bp antigen-specific IFN-γ+ CD8+ T lymphocytes were also detected. This test is easy to perform, analyse and interpret with the establishment of strict criteria defining Bp antigen responses.DiscussionThus, this assay appears as a promising test for evaluation of Bp antigen-specific CD4+ T cells induced by current and next generation pertussis vaccines.

Elongated peptides (EPs), containing possibly one or multiple epitope/s, are increasingly used for the screening of antigen-specific CD8+ and CD4+ cell responses. Here, we present an in vitro protocol that allows the amplification of antigen-specific cells and the subsequent functional analysis of both T cell types using EPs. Known viral-derived epitopes were elongated to 20 mer EPs on the N-, C-, and both termini for HLA class I binders, or on the N- and C- termini for HLA class II binders. With EP stimulation only, the percentage of responding CD8+ T cells was dependent on the elongation site of the EP, whereas CD4+ T cell responses were completely lost in 22% of the tests performed ex vivo. A short-term amplification step plus the addition of a TLR3 agonist (Poly-ICLC) together with an increased EP concentration improved markedly the detection of CD8+ and CD4+ T cell reactivities. [ABSTRACT FROM AUTHOR]

Despite the knowledge that cell-mediated immunity (CMI) contributes to the reduction of severe influenza infection, transmission, and disease outcome, the correlates of protection for cell-mediated immunity remain still unclear. Therefore, measuring the magnitude and quality of influenza-specific T cell responses in a harmonized way is of utmost importance to improve characterisation of vaccine-induced immunity across different clinical trials. The present study, conducted as part of the FLUCOP project, describes the development of a consensus protocol for the intracellular cytokine staining (ICS) assay, in order to reduce inter-laboratory variability, and its qualification. In order to develop a consensus protocol, the study was divided into different stages. Firstly, two pilot studies evaluated critical parameters in the analytical (read-outs) and post-analytical (gating strategies and data analysis) methods applied by eight different laboratories within the FLUCOP consortium. The methods were then harmonized by fixing the critical parameters and the subsequent consensus protocol was then qualified by one FLUCOP member. The antigen-specific cell population was defined as polypositive CD4+ T cells (i.e. positive for at least two markers among CD40L/IFNγ/IL2/TNFα), which was shown to be the most sensitive and specific read-out. The qualification of this consensus protocol showed that the quantification of polypositive CD4+ T cells was precise, linear and accurate, and sensitive with a lower limit of quantification of 0.0335% antigen-specific polypositive CD4+ T cells. In conclusion, we provide the description of a harmonized ICS assay, which permits quantitative and qualitative evaluation of influenza vaccine-induced T cell responses. Application of this harmonized assay may allow for future comparisons of T cell responses to different influenza vaccines. It may facilitate future assessments of potential correlates of protection with the promise of application across other pathogens.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most relevant porcine pathogens worldwide. Active control of the disease relies on modified live virus vaccines (MLVs), as most inactivated vaccines provide very limited protection. Neutralizing antibodies occur late in infection; therefore, CD8+ T cells are considered important correlates of protection and are a frequent focus of investigation. Our aim was to identify viral peptides naturally bound by the class I major histocompatibility complex (MHC-I) and to confirm their ability to stimulate CD8+ T cells. For this purpose, we immunoprecipitated MHC-I/peptide complexes of PRRSV (strain AUT15-33) -infected cells (SLA-I Lr-Hp 35.0/24 mod) to isolate the viral epitopes and analyzed them with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Furthermore, we employed these identified peptides to stimulate peripheral blood mononuclear cells (PBMCs) of previously PRRSV-infected pigs and measured the PRRSV-specific CD8+ T-cell response with an intracellular cytokine staining (ICS). Our data revealed that PRRSV non-structural proteins (NSPs), encoded in open reading frame 1a and 1b (ORF1), present the major source of MHC-I-presented peptides. Additionally, we show that our identified epitopes are able to trigger IFNγ responses in vitro. These findings are a basis for understanding the proteasomal degradation of PRRSV proteins, the cellular ability to display them via MHC-I, and their potential to restimulate CD8+ T cells. [ABSTRACT FROM AUTHOR]

Background: The ALVAC/gp120 + MF59 vaccines in the HIV Vaccine Trials Network (HVTN) 702 efficacy trial did not prevent human immunodeficiency virus-1 (HIV-1) acquisition. Vaccine-matched immunological endpoints that were correlates of HIV-1 acquisition risk in RV144 were measured in HVTN 702 and evaluated as correlates of HIV-1 acquisition.Methods: Among 1893 HVTN 702 female vaccinees, 60 HIV-1-seropositive cases and 60 matched seronegative noncases were sampled. HIV-specific CD4+ T-cell and binding antibody responses were measured 2 weeks after fourth and fifth immunizations. Cox proportional hazards models assessed prespecified responses as predictors of HIV-1 acquisition.Results: The HVTN 702 Env-specific CD4+ T-cell response rate was significantly higher than in RV144 (63% vs 40%, P = .03) with significantly lower IgG binding antibody response rate and magnitude to 1086.C V1V2 (67% vs 100%, P < .001; Pmag < .001). Although no significant univariate associations were observed between any T-cell or binding antibody response and HIV-1 acquisition, significant interactions were observed (multiplicity-adjusted P ≤.03). Among vaccinees with high IgG A244 V1V2 binding antibody responses, vaccine-matched CD4+ T-cell endpoints associated with decreased HIV-1 acquisition (estimated hazard ratios = 0.40-0.49 per 1-SD increase in CD4+ T-cell endpoint).Conclusions: HVTN 702 and RV144 had distinct immunogenicity profiles. However, both identified significant correlations (univariate or interaction) for IgG V1V2 and polyfunctional CD4+ T cells with HIV-1 acquisition. Clinical Trials Registration . NCT02968849. [ABSTRACT FROM AUTHOR]

Intracellular cytokine staining (ICS) is a widely employed ex vivo method for quantitative determination of the activation status of immune cells, most often applied to T cells. ICS test samples are commonly prepared from animal or human tissues as unpurified cell mixtures, and cell-specific cytokine signals are subsequently discriminated by gating strategies using flow cytometry. Here, we show that when ICS samples contain Ly6G+ neutrophils, neutrophils are ex vivo activated by an ICS reagent – phorbol myristate acetate (PMA) – which leads to hydrogen peroxide (H2O2) release and death of cytokine-expressing T cells. This artifact is likely to result in overinterpretation of the degree of T cell suppression, misleading immunological research related to cancer, infection, and inflammation. We accordingly devised easily implementable improvements to the ICS method and propose alternative methods for assessing or confirming cellular cytokine expression. [ABSTRACT FROM AUTHOR]

Objectives: We previously described the Phase I‐II evaluation of SARS‐CoV‐2 recombinant protein candidate vaccine, CoV2‐PreS‐dTM, with AF03‐ or AS03‐adjuvant systems (ClinicalTrials.gov, NCT04537208). Here, we further characterise the cellular immunogenicity profile of this vaccine candidate using a whole‐blood secretion assay in parallel to intracellular cytokine staining (ICS) of cryopreserved peripheral blood mononuclear cells (PBMCs). Methods: A randomly allocated subset of 90 healthy, SARS‐CoV‐2‐seronegative adults aged ≥ 18 years who had received (random allocation) one or two separate injections (on study day [D]1 and D22) of saline placebo or CoV2‐PreS‐dTM formulated with AS03 or AF03 were included. Cytokine secretion was assessed using a TruCulture® whole‐blood stimulation system in combination with multiplex bead array, and intracellular cytokine profiles were evaluated on thawed PBMCs following ex vivo stimulation with recombinant S protein at pre‐vaccination (D1), post‐dose 1 (D22) and post‐dose 2 (D36). Results: Both methods detected similar vaccine‐induced responses after the first and second doses. We observed a Th1 bias (Th1/Th2 ratio > 1.0) for most treatment groups when analysed in whole blood, mainly characterised by increased IFN‐γ, IL‐2 and TNF‐α secretion. Among participants aged ≥ 50 years, the Th1/Th2 ratio was higher for those who received vaccine candidate with AS03 versus AF03 adjuvant. ICS revealed that this higher Th1/Th2 ratio resulted from higher levels of IFN‐γ expression and that the vaccine induced polyfunctional CD4+ T cells. Conclusions: The whole‐blood cytokine secretion assay is a high‐throughput alternative for assessing the quantity and character of vaccine‐induced cellular responses. [ABSTRACT FROM AUTHOR]

Intracellular cytokine staining (ICS) is a widely employed ex vivo method for quantitative determination of the activation status of immune cells, most often applied to T cells. ICS test samples are commonly prepared from animal or human tissues as unpurified cell mixtures, and cell-specific cytokine signals are subsequently discriminated by gating strategies using flow cytometry. Here, we show that when ICS samples contain Ly6G+ neutrophils, neutrophils are ex vivo activated by an ICS reagent – phorbol myristate acetate (PMA) – which leads to hydrogen peroxide (H2O2) release and death of cytokine-expressing T cells. This artifact is likely to result in overinterpretation of the degree of T cell suppression, misleading immunological research related to cancer, infection, and inflammation. We accordingly devised easily implementable improvements to the ICS method and propose alternative methods for assessing or confirming cellular cytokine expression.

Abstract Objectives We previously described the Phase I‐II evaluation of SARS‐CoV‐2 recombinant protein candidate vaccine, CoV2‐PreS‐dTM, with AF03‐ or AS03‐adjuvant systems (ClinicalTrials.gov, NCT04537208). Here, we further characterise the cellular immunogenicity profile of this vaccine candidate using a whole‐blood secretion assay in parallel to intracellular cytokine staining (ICS) of cryopreserved peripheral blood mononuclear cells (PBMCs). Methods A randomly allocated subset of 90 healthy, SARS‐CoV‐2‐seronegative adults aged ≥ 18 years who had received (random allocation) one or two separate injections (on study day [D]1 and D22) of saline placebo or CoV2‐PreS‐dTM formulated with AS03 or AF03 were included. Cytokine secretion was assessed using a TruCulture® whole‐blood stimulation system in combination with multiplex bead array, and intracellular cytokine profiles were evaluated on thawed PBMCs following ex vivo stimulation with recombinant S protein at pre‐vaccination (D1), post‐dose 1 (D22) and post‐dose 2 (D36). Results Both methods detected similar vaccine‐induced responses after the first and second doses. We observed a Th1 bias (Th1/Th2 ratio > 1.0) for most treatment groups when analysed in whole blood, mainly characterised by increased IFN‐γ, IL‐2 and TNF‐α secretion. Among participants aged ≥ 50 years, the Th1/Th2 ratio was higher for those who received vaccine candidate with AS03 versus AF03 adjuvant. ICS revealed that this higher Th1/Th2 ratio resulted from higher levels of IFN‐γ expression and that the vaccine induced polyfunctional CD4+ T cells. Conclusions The whole‐blood cytokine secretion assay is a high‐throughput alternative for assessing the quantity and character of vaccine‐induced cellular responses.

Elongated peptides (EPs), containing possibly one or multiple epitope/s, are increasingly used for the screening of antigen-specific CD8+ and CD4+ cell responses. Here, we present an in vitro protocol that allows the amplification of antigen-specific cells and the subsequent functional analysis of both T cell types using EPs. Known viral-derived epitopes were elongated to 20 mer EPs on the N-, C-, and both termini for HLA class I binders, or on the N- and C- termini for HLA class II binders. With EP stimulation only, the percentage of responding CD8+ T cells was dependent on the elongation site of the EP, whereas CD4+ T cell responses were completely lost in 22% of the tests performed ex vivo. A short-term amplification step plus the addition of a TLR3 agonist (Poly-ICLC) together with an increased EP concentration improved markedly the detection of CD8+ and CD4+ T cell reactivities.

Anaphylaxis is a life-threatening allergic reaction caused by cross-linking of high-affinity IgE antibodies on the surface of mast cells and basophils. Understanding the cellular mechanisms that lead to high-affinity IgE production is required to develop better therapeutics for preventing this severe reaction. A recently discovered population of T follicular helper T fh 13 cells regulates the production of high-affinity IgE in mouse models of allergy and can also be found in patients with allergies with IgE antibodies against food or aeroallergens. Here we describe optimized protocols for identifying T fh 13 cells in both mice and humans. [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]

Intracellular cytokine staining is a versatile technique used to analyze cytokine production in individual cells by flow cytometry. This methodology has the specific advantage of enabling the simultaneous assessment of multiple phenotypic, differentiation, and functional parameters pertaining to responding T cells. This methodology applied after short-term culture of cells, followed by fixation and permeabilization make this technique ideal for the assessment of T-cell immune responses induced by different challenges. Here we describe an intracellular staining method followed by flow cytometry after cell stimulation with immune-relevant antigens for Lyme disease., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The encounter of T cells with the antigen through the interaction of T cell receptors with peptides and major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs) can generate effector response and memory T cells. Memory T cells developed following infections or vaccination may persist, leading to the generation of a specific immune response upon reexposure to the same pathogen through rapid clonal proliferation and activation of effector functions. T cell memory subsets can be identified based on the expression of several membrane markers such as CCR7, CD27, and CD45RA. Using fluorescent antibodies against these markers and a flow cytometer, it is possible to perform immunophenotyping via the analysis of cell surface expression of proteins by different subpopulations such as the subsets of naïve, effector, and memory T cells as well as via the analysis of functional markers that further characterize each sample. Intracellular cytokine staining allows for the evaluation of intracellular proteins expressed in T cells in response to antigenic stimulation. This chapter presents the phenotypic and functional characterization of memory T cells after antigenic stimulation, detailing the procedures for identifying intracellular and surface protein markers. Herein, we review and present a reproducible standardized protocol using antibodies for specific markers and applying flow cytometry., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

We developed this comprehensive 28‐color flow cytometry panel with the aim to measure a variety of T cell effector functions in combination with T cell differentiation markers (CCR7, CD27, CD28, CD45RO, CD95) in γδ T cells and CD4+ and CD8+ αβ T cells (Table 1). The effector functions measured in this panel include activation and co‐stimulatory molecules (CD69, CD137, and CD154), cytokines (IL‐2, IL‐13, IL‐17A, IL‐21, IL‐22, TNF, and IFNγ), the chemokine IL‐8, cytotoxic molecules (perforin and granzyme B), and the degranulation marker CD107a. In addition, Ki67 enables the identification and analysis of recently activated T cells. To characterize regulatory T cells (Tregs), we included CD25, CD39, and the canonical Tregs transcription factor FoxP3. We developed and optimized this panel for cryopreserved human peripheral blood mononuclear cells (PBMC) and stimulation with phorbol 12‐myristate 13‐acetate (PMA) and ionomycin. However, we successfully tested other types of stimulation such as staphylococcus enterotoxin B (SEB) or a mix of immunodominant peptides (CEF peptide pool) from cytomegalovirus (CMV), Epstein–Barr virus (EBV) and influenza. Published 2019. This article is a U.S. Government work and is in the public domain in the USA. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

IL-17A as important cytokine in host defense has been analysed intensively and various homologous have been identified. To further gain insight into the functional properties of chicken (gg) IL-17A its expression profile was analysed by intracellular cytokine staining. In splenocytes and peripheral blood mononuclear cells gg IL-17A was detected in subsets of CD4 + T cells and γδ T cells. In contrast the gg IL-17A producing populations in intestinal intraepithelial lymphocytes were characterized as either CD3 + CD25 + cells or γδ T cells. Furthermore, using FLAG tagged gg IL-17A, binding to its receptor was demonstrated on the macrophage cell line HD11. In peripheral blood IL-17A binding activity was found on αβ and γδ T cell subsets, monocytes and a distinct population of CD25 high cells. Treatment of HD11 cells with gg IL-17A induced IL-6 mRNA expression and nitric oxide production. These results demonstrate the presence of a αβ T helper 17 cell subset and IL-17 producing γδ T cells in the chicken. [ABSTRACT FROM AUTHOR]

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-α, and CD40L and HBsAg-specific CD4+ T producing IFN-γ 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 aim of this study was to optimise and evaluate an intracellular cytokine staining (ICS) assay for assessment of T cell IFN-γ responses in chickens vaccinated against Newcastle disease (ND). We aimed to validate currently available antibodies to chicken IFN-γ using transfected CHO cells. Moreover, this ICS assay was evaluated for use to detect mitogen and antigen induced IFN-γ production in chicken peripheral blood leucocytes. Chickens from an inbred white leghorn line containing two MHC haplotypes, B19 and B21, were divided into three experimental groups; one group was kept as naive controls, one group was vaccinated intramuscularly twice with a commercial inactivated ND virus (NDV) vaccine, and the last group was vaccinated orally twice with a commercial live attenuated NDV vaccine. PBMC were ex vivo stimulated with ConA or with NDV antigen. The ICS assay was used to determine the phenotype and frequency of IFN-γ positive cells. ConA stimulation induced extensive IFN-γ production in both CD3 + TCRγδ + (γδ T cells) cells and CD3 + TCRγδ − cells (αβ T cells), but no significant differences were observed between the experimental groups. Furthermore, a large proportion of the IFN-γ producing cells were CD3 − indicating that other cells than classic T cells, secreted this cytokine. NDV antigen stimulation induced IFN-γ production but to a lower extent than ConA and with a large variation between individuals. The CD3 + TCR1γδ − CD8α + (CTL) population produced the highest NDV specific IFN-γ responses, with significantly elevated levels of IFN-γ producing cells in the B19 chickens vaccinated orally with live attenuated NDV vaccine. This was not the case in the B21 animals, indicating a haplotype restricted variation. In contrast, the CD3 + TCR1γδ − CD4 + (Th) population did not show a significant increase in IFN-γ production in NDV stimulated samples which was in part due to a high number of IFN-γ producing cells after incubation with medium alone. In conclusion, an ICS assay for phenotyping of IFN-γ producing chicken leukocytes was set up that proved useful in identifying cytokine producing cells upon either mitogen or antigen-specific stimulation. [ABSTRACT FROM AUTHOR]

Baboons naturally infected with simian T lymphotropic virus (STLV) are a potentially useful model system for the study of vaccination against human T lymphotropic virus (HTLV). Here we expanded the number of available full-length baboon STLV-1 sequences from one to three and related the T cell responses that recognize the immunodominant Tax protein to the tax sequences present in two individual baboons. Continuously growing T cell lines were established from two baboons, animals 12141 and 12752. Next-generation sequencing (NGS) of complete STLV genome sequences from these T cell lines revealed them to be closely related but distinct from each other and from the baboon STLV-1 sequence in the NCBI sequence database. Overlapping peptides corresponding to each unique Tax sequence and to the reference baboon Tax sequence were used to analyze recognition by T cells from each baboon using intracellular cytokine staining (ICS). Individual baboons expressed more gamma interferon and tumor necrosis factor alpha in response to Tax peptides corresponding to their own STLV-1 sequence than in response to Tax peptides corresponding to the reference baboon STLV-1 sequence. Thus, our analyses revealed distinct but closely related STLV-1 genome sequences in two baboons, extremely low heterogeneity of STLV sequences within each baboon, no evidence for superinfection within each baboon, and a ready ability of T cells in each baboon to recognize circulating Tax sequences. While amino acid substitutions that result in escape from CD8+ T cell recognition were not observed, premature stop codons were observed in 7% and 56% of tax sequences from peripheral blood mononuclear cells from animals 12141 and 12752, respectively. [ABSTRACT FROM AUTHOR]

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-α, and CD40L and HBsAg-specific CD4+ T producing IFN-γ 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 HBsAgspecific 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. [ABSTRACT FROM AUTHOR]

Interleukin 17 (IL-17) cytokines play a crucial role in host defense and inflammatory diseases. Of the six mammalian IL-17 members five are described in the chicken (gg) genome. A novel method that attached cytokines to the surface of cells via a GPI linker was established to generate two chicken IL-17A and one chicken IL-17F specific mab. Recombinant gg IL-17A and gg IL-17F that showed dimerization in Western blot were used to verify the antibodies specificity. The mab could detect gg IL-17 by intracellular cytokine staining as demonstrated on cells expressing recombinant IL-17. Furthermore IL-17A and lower amounts of IL-17F were detectable in CD4 positive T cells of stimulated splenocytes. In conclusion, we have generated novel tools to analyze chicken IL-17 in more detail and demonstrated that the surface expression of cytokines is a reliable method to generate specific mab applicable for intracellular cytokine staining. [ABSTRACT FROM AUTHOR]

Background: It is important to identify vaccine-induced immune responses that predict the preventative efficacy of a human immunodeficiency virus (HIV)-1 vaccine. We assessed T-cell response markers as correlates of risk in the HIV Vaccine Trials Network (HVTN) 505 HIV-1 vaccine efficacy trial.Methods: 2504 participants were randomized to DNA/rAd5 vaccine or placebo, administered at weeks 0, 4, 8, and 24. Peripheral blood mononuclear cells were obtained at week 26 from all 25 primary endpoint vaccine cases and 125 matched vaccine controls, and stimulated with vaccine-insert-matched peptides. Primary variables were total HIV-1-specific CD4+ T-cell magnitude and Env-specific CD4+ polyfunctionality. Four secondary variables were also assessed. Immune responses were evaluated as predictors of HIV-1 infection among vaccinees using Cox proportional hazards models. Machine learning analyses identified immune response combinations best predicting HIV-1 infection.Results: We observed an unexpectedly strong inverse correlation between Env-specific CD8+ immune response magnitude and HIV-1 infection risk (hazard ratio [HR] = 0.18 per SD increment; P = .04) and between Env-specific CD8+ polyfunctionality and infection risk (HR = 0.34 per SD increment; P < .01).Conclusions: Further research is needed to determine if these immune responses are predictors of vaccine efficacy or markers of natural resistance to HIV-1 infection. [ABSTRACT FROM AUTHOR]

Background The evaluation of durable immune responses is important in HIV vaccine research and development. The efficiency of such evaluation could be increased by incorporating predictors of the responses in the statistical analysis. In this paper, we investigated whether and how baseline demographic variables and immune responses measured two weeks after vaccination predicted durable immune responses measured six months later. Methods We included data from seven preventive HIV vaccine regimens evaluated in three clinical trials: a Phase 1 study of four DNA, NYVAC and/or AIDSVAX vaccine regimens (HVTN096), a Phase 2 study of two DNA and/or MVA vaccine regimens (HVTN205), and a Phase 3 study of a single ALVAC/AIDSVAX regimen (RV144). Regularized random forests and linear regression models were used to identify and evaluate predictors of the positivity and magnitude of durable immune responses. Results We analyzed 201 vaccine recipients with data from 10 to 127 immune response biomarkers, and 3–5 demographic variables. The best prediction of participants’ durable response positivity based on two-week responses rendered up to close-to-perfect accuracy; the best prediction of participants’ durable response magnitude rendered correlation coefficients between the observed and predicted responses ranging up to 0.91. Though prediction performances differed among biomarkers, durable immune responses were best predicted by the two-week response level of the same biomarker. Adding demographic information and two-week response levels of different biomarkers provided little or no improvement in the predictions. Conclusions For some biomarkers and for the vaccines we studied, two-week post-vaccination responses can well predict durable responses six months later. Therefore, if immune response durability is only assessed in a sub-sample of vaccine recipients, statistical analyses of durable responses will have increased efficiency by incorporating two-week response data. Further research is needed to generalize the findings to other vaccine regimens and biomarkers. Clinicaltrials.gov identifiers: NCT01799954 , NCT00820846 , NCT00223080 . [ABSTRACT FROM AUTHOR]

In recent years, mRNA-based vaccines have emerged to be a great alternative to DNA-based vaccines due to the safety of not inserting into host genome. However, mRNA molecules are single-stranded nucleic acids that are vulnerable under RNase existing in human skin and tissues. In this study, a self-assembled cationic nanomicelles based on polyethyleneimine-stearic acid (PSA) copolymer were developed to delivery HIV-1 gag encoding mRNA to dendritic cells and BALB/c mice. We evaluated the transfection efficiency and cell uptake efficiency of naked EGFP mRNA, PSA, PEI-2k and PEI-25k nanoparticles format on DC2.4 cell lines. Immune responses after sub-cutaneous administration of gag mRNA to BALB/c mice were notably induced by PSA as compared with naked gag mRNA. We found the PSA/mRNA nanomicelles were potent systems that can effectively deliver mRNA and induce antigen-specific immune response, stimulating various new vaccine strategies using mRNA. [ABSTRACT FROM AUTHOR]