Antigen-stimulated Peripheral Blood Mononuclear Cells Research Articles

Zinc aspartate induces proliferation of resting and antigen-stimulated human PBMC under high-density cell culture condition

BackgroundZinc, one of the most important essential trace elements in the human body, regulates a wide range of cellular functions of immune cells, such as proliferation, differentiation and survival. Zinc deficiency affects both the innate and adaptive immune system. Zinc supplementation was discussed as possible therapy for infectious diseases and T cell-mediated autoimmune diseases. However, the influence of commercial zinc preparations on proliferation and cytokine production of resting and antigen-stimulated peripheral blood mononuclear cells (PBMC) has not yet been completely investigated. MethodsHere, we examined whether zinc aspartate (Unizink®), an approved drug to treat zinc deficiency in patients, induces proliferation, cytokine production, and induction of apoptosis/caspase 3/7 activity of resting PBMC under high-density cell culture condition. In addition, we performed antigen-specific proliferation experiments, where PBMCs of healthy donors vaccinated against Influenza A (H1N1) and/or SARS-CoV-2 were stimulated with Influenza A (H1N1) peptides or SARS-CoV-2 peptides as well as the Mixed Lymphocyte Culture (MLC) in the presence of increasing concentrations of zinc aspartate. ResultsWe observed a dose-dependent enhancement of proliferation and induction of cytokine production (IFN-γ, IL-5, GM-CSF and CXCL10) of resting PBMC in presence of zinc aspartate. The number of cells with active caspase 3/7 and, consecutively, the amount of cells undergoing apoptosis steadily decreased in presence of zinc aspartate. Moreover, zinc aspartate was capable of stimulating antigen-specific PBMC proliferation using MLC or influenza A (H1N1) and SARS-CoV-2 peptides in both a dose-dependent and a donor-specific manner. In the absence of zinc aspartate, we clearly could discriminate two groups of responders: low and high responders to antigenic stimulation. The addition of increasing concentration of zinc aspartate significantly stimulated the proliferation of PBMC from low responders, but not from high responders. ConclusionTaken together, our results suggest that zinc aspartate induces the proliferation of resting and antigen-stimulated PBMCs under high-density cell culture conditions. Thus, zinc might represent a supportive treatment in patients suffering from infectious diseases.

Evaluation of IFIT3 and ORM1 as Biomarkers for Discriminating Active Tuberculosis from Latent Infection.

Current commercially available immunological tests cannot be used for discriminating active tuberculosis (TB) from latent TB infection. To evaluate the value of biomarker candidates in the diagnosis of active TB, this study aimed to identify differentially expressed genes in peripheral blood mononuclear cells (PBMCs) between patients with active TB and individuals with latent TB infection by transcriptome sequencing. The differentially expressed genes in unstimulated PBMCs and in Mycobacterium tuberculosis (Mtb) antigen-stimulated PBMCs from patients with active TB and individuals with latent TB infection were identified by transcriptome sequencing. Selected candidate genes were evaluated in cohorts consisting of 110 patients with TB, 30 individuals with latent TB infections, and 50 healthy controls by quantitative real-time RT-PCR. Receiver operating characteristic (ROC) curve analysis was performed to calculate the diagnostic value of the biomarker candidates. Among the differentially expressed genes in PBMCs without Mtb antigen stimulation, interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) had the highest area under curve (AUC) value (0.918, 95% CI: 0.852-0.984, P<0.0001) in discriminating patients with active TB from individuals with latent TB infection, with a sensitivity of 91.86% and a specificity of 84.00%. In Mtb antigen-stimulated PBMCs, orosomucoid 1 (ORM1) had a high AUC value (0.833, 95% CI: 0.752-0.915, P<0.0001), with a sensitivity of 81.94% and a specificity of 70.00%. IFIT3 and ORM1 might be potential biomarkers for discriminating active TB from latent TB infection.

Transcriptional correlates of malaria in RTS,S/AS01-vaccinated African children: a matched case-control study.

In a phase 3 trial in African infants and children, the RTS,S/AS01 vaccine (GSK) showed moderate efficacy against clinical malaria. We sought to further understand RTS,S/AS01-induced immune responses associated with vaccine protection. Applying the blood transcriptional module (BTM) framework, we characterized the transcriptomic response to RTS,S/AS01 vaccination in antigen-stimulated (and vehicle control) peripheral blood mononuclear cells sampled from a subset of trial participants at baseline and month 3 (1-month post-third dose). Using a matched case-control study design, we evaluated which of these 'RTS,S/AS01 signature BTMs' associated with malaria case status in RTS,S/AS01 vaccinees. Antigen-specific T-cell responses were analyzed by flow cytometry. We also performed a cross-study correlates analysis where we assessed the generalizability of our findings across three controlled human malaria infection studies of healthy, malaria-naive adult RTS,S/AS01 recipients. RTS,S/AS01 vaccination was associated with downregulation of B-cell and monocyte-related BTMs and upregulation of T-cell-related BTMs, as well as higher month 3 (vs. baseline) circumsporozoite protein-specific CD4+ T-cell responses. There were few RTS,S/AS01-associated BTMs whose month 3 levels correlated with malaria risk. In contrast, baseline levels of BTMs associated with dendritic cells and with monocytes (among others) correlated with malaria risk. The baseline dendritic cell- and monocyte-related BTM correlations with malaria risk appeared to generalize to healthy, malaria-naive adults. A prevaccination transcriptomic signature associates with malaria in RTS,S/AS01-vaccinated African children, and elements of this signature may be broadly generalizable. The consistent presence of monocyte-related modules suggests that certain monocyte subsets may inhibit protective RTS,S/AS01-induced responses. Funding was obtained from the NIH-NIAID (R01AI095789), NIH-NIAID (U19AI128914), PATH Malaria Vaccine Initiative (MVI), and Ministerio de Economía y Competitividad (Instituto de Salud Carlos III, PI11/00423 and PI14/01422). The RNA-seq project has been funded in whole or in part with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under grant number U19AI110818 to the Broad Institute. This study was also supported by the Vaccine Statistical Support (Bill and Melinda Gates Foundation award INV-008576/OPP1154739 to R.G.). C.D. was the recipient of a Ramon y Cajal Contract from the Ministerio de Economía y Competitividad (RYC-2008-02631). G.M. was the recipient of a Sara Borrell-ISCIII fellowship (CD010/00156) and work was performed with the support of Department of Health, Catalan Government grant (SLT006/17/00109). This research is part of the ISGlobal's Program on the Molecular Mechanisms of Malaria which is partially supported by the Fundación Ramón Areces and we acknowledge support from the Spanish Ministry of Science and Innovation through the 'Centro de Excelencia Severo Ochoa 2019-2023' Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program.

An allergen-fused dendritic cell-binding peptide enhances in vitro proliferation of equine T-cells and cytokine production

Allergen-specific immunotherapy (AIT) constitutes the only curative approach for allergy treatment. There is need for improvement of AIT in veterinary medicine, such as in horses suffering from insect bite hypersensitivity, an IgE-mediated dermatitis to Culicoides. Dendritic cell (DC)-targeting represents an efficient method to increase antigen immunogenicity. It is studied primarily for its use in improvement of cancer therapy and vaccines, but may also be useful for improving AIT efficacy. Immunomodulators, like the Toll-like receptor 4 (TLR-4) agonist monophosphoryl lipid-A (MPLA) has been shown to enhance the IL-10 response in horses, while CpG-rich oligonucleotides (CpG-ODN), acting as TLR-9 agonists, have been shown to induce Th1 or regulatory responses in horses with equine asthma. Our aim was to evaluate in vitro effects of antigen-targeting to equine DC with an antigen-fused peptide known to target human and mouse DC and investigate whether addition of MPLA or CpG-ODN would further improve the induced immune response with regard to finding optimal conditions for equine AIT. For this purpose, DC-binding peptides were fused to the model antigen ovalbumin (OVA) and to the recombinant Culicoides allergen Cul o3. Effects of DC-binding peptides on cellular antigen uptake and induction of T cell proliferation were assessed. Polarity of the immune response was analysed by quantifying IFN-γ, IL-4, IL-10, IL-17 and IFN-α in supernatants of antigen-stimulated peripheral blood mononuclear cells (PBMC) in presence or absence of adjuvants. Fusion of DC-binding peptides to OVA significantly enhanced antigen-uptake by equine DC. DC primed with DC-binding peptides coupled to OVA or Cul o3 induced a significantly higher T-cell proliferation compared to the corresponding control antigens. PBMC stimulation with DC-binding peptides coupled to Cul o3 elicited a significant increase in the pro-inflammatory cytokines IFN-γ, IL-4, IL-17, as well as the anti-inflammatory IL-10, but not of IFN-α. Adjuvant addition further enhanced the effect of the DC-binding peptides by significantly increasing the production of IFN-γ, IL-4, IL-10 and IFN-α (CpG-ODN) and IL-10 (MPLA), while simultaneously suppressing IFN-γ, IL-4 and IL-17 production (MPLA). Targeting equine DC with allergens fused to DC-binding peptides enhances antigen-uptake and T-cell activation and may be useful in increasing the equine immune response against recombinant antigens. Combination of DC-binding peptide protein fusions with adjuvants is necessary to appropriately skew the resulting immune response, depending on intended use. Combination with MPLA is a promising option for improvement of AIT efficacy in horses, while combination with CpG-ODN increases the effector immune response to recombinant antigens.

Impact of immunosuppressive and antifungal drugs on PBMC- and whole blood-based flow cytometric CD154+ Aspergillus fumigatus specific T-cell quantification.

Flow cytometric quantification of CD154+ mould specific T-cells in antigen-stimulated peripheral blood mononuclear cells (PBMCs) or whole blood has been described as a supportive biomarker to diagnose invasive mould infections and to monitor therapeutic outcomes. As patients at risk frequently receive immunosuppressive and antifungal medication, this study compared the matrix-dependent impact of representative drugs on CD154+ T-cell detection rates. PBMCs and whole blood samples from healthy adults were pre-treated with therapeutic concentrations of liposomal amphotericin B, voriconazole, posaconazole, cyclosporine A (CsA) or prednisolone. Samples were then stimulated with an Aspergillus fumigatus lysate or a viral antigen cocktail (CPI) and assessed for CD154+ T-helper cell frequencies. Specific T-cell detection rates and technical assay properties remained largely unaffected by exposure of both matrices to the studied antifungals. By contrast, CsA and prednisolone pre-treatment of isolated PBMCs and whole blood adversely impacted specific T-cell detection rates and caused elevated inter-replicate variation. Unexpectedly, the whole blood-based protocol that uses additional α-CD49d co-stimulation was less susceptible to CsA and prednisolone despite prolonged drug exposure in the test tube. Accordingly, addition of α-CD49d during PBMC stimulation partially attenuated the impact of immunosuppressive drugs on test performance. Translating these results into the clinical setting, false-negative results of CD154+ antigen-specific T-cell quantification need to be considered in patients receiving T-cell-active immunosuppressive medication. Optimized co-stimulation regimes with α-CD49d could contribute to an improved feasibility of functional T-cell assays in immunocompromised patient populations.

2888. STAT4 Mutation in Three Generations with Disseminated Coccidioidomycosis (DCM) also Exhibits Increased Susceptibility to Coccidioidal Infection in Transfected Mice

BackgroundReported coccidioidomycosis has increased with case rates of 198/100,000 in Arizona (2012). In California alone, 2000–2011 hospitalizations were $2.2B. Dissemination occurs in 8% of reports with significant morbidity and occasional deaths. DCM was found in 3 generations: grandmother (skin), mother (skin), and son (bone). Whole exome sequencing identified a heterozygous (het) STAT4 mutation (p.E626G) in all three. This mutation alters the phosphotyrosine binding pocket and is predicted to impair STAT4 function, interfering with (i) receptor binding and phosphorylation, (ii) nuclear localization, and/or (iii) transcription. Expression profiling of antigen-stimulated peripheral blood mononuclear cells from one patient showed dampening of known STAT4 targets compared with controls.MethodsSTAT4 p.E626G was generated and confirmed in C57BL/6NJ (WT) mice using CRISPR-Cas9. With continued breeding, neither homozygous (hom) nor het mice had gross abnormalities. There were normal spleen and lung lymphoid cell numbers. Thymus and bone marrow had normal development of lymphoid subsets. We performed intranasal infection with reduced virulence C. posadasii strain 1038 or with F. tularensis live vaccine- strain (LVS). Naïve or Δcps1-vaccinated mice were tested for resistance to C. posadasii strain Silveira.ResultsAt day 21 post Cp 1038 infection, hom, het, and WT mice had similar lung fungal burdens (~104.7 cfu). All p.E626G mice died between days 31 and 39 with lung burden significantly higher (~9 × 106 cfu) than WT sacrificed on day 44 (7 × 105 cfu, P = 0.015). After LVS infection, p.E626G mice had increased lung bacterial cfu and all had dissemination to the spleen compared with WT lung bacterial burden and no splenic dissemination. Immunized het and WT mice all had significantly reduced lung cfu 14 days following C. posadasii infection compared with unvaccinated WT mice.ConclusionThe STAT4 p.E626G mutated mouse recapitulated patients’ increased susceptibility to coccidioidal infection. The decreased fungal burdens seen in Δcps1-vaccinated mice suggest that vaccination may be effective in those persons genetically susceptible to DCM. Given the increasing frequency and economic burdens of coccidioidomycosis, pursuit of vaccination strategies should continue. Disclosures All Authors: No reported Disclosures.

A DNA priming and protein boosting immunization scheme to augment immune responses against parvovirus in ducks.

To evaluate the effect of a DNA priming and protein boosting immunization scheme in ducks. Pekin ducks were immunized with pTCY/VP2 DNA vaccine; on day 14 (D14) after primary immunization, the ducks were boosted with either the same vaccine (DNA+DNA) or the rVP2 vaccine (DNA+rVP2). CpG oligodeoxynucleotides containing three copies of GACGTT motifs were used as the adjuvant in the vaccines. Compared with unimmunized controls, both immunization schemes significantly increased the titre of antigen-specific antibodies, lymphocyte proliferation index, percentage of CD4+ and CD8+ cells in peripheral blood mononuclear cells (PBMCs) and mRNA expression of interferon (IFN)-α, IFN-γ, interleukin (IL)-6 and IL-12 in antigen-stimulated PBMCs. Furthermore, compared with the DNA+DNA homologous scheme, the DNA+rVP2 heterologous scheme significantly increased lymphocyte proliferation, percentage of CD4+ and CD8+ cells in PBMCs and upregulation of mRNA expression of cytokines 2weeks after the boost (D28). The DNA+rVP2 immunization scheme enhanced immune responses, mainly Th1 type, against parvovirus in ducks. The DNA priming and protein boosting heterologous immunization strategy can be applied to develop vaccines against viral infections in ducks. It can potentially be used in breeding ducks because of long-term immunity may confer protection for ducklings.

An RORγt Oral Inhibitor Modulates IL-17 Responses in Peripheral Blood and Intestinal Mucosa of Crohn's Disease Patients.

Background and Aims: Despite the negative results of blocking IL-17 in Crohn's disease (CD) patients, selective modulation of Th17-dependent responses warrants further study. Inhibition of retinoic acid-related orphan receptor gamma (RORγt), the master regulator of the Th17 signature, is currently being explored in inflammatory diseases. Our aim was to determine the effect of a novel oral RORγt antagonist (BI119) in human CD and on an experimental model of intestinal inflammation.Methods: 51 CD patients and 11 healthy subjects were included. The effects of BI119 were tested on microbial-stimulated peripheral blood mononuclear cells (PBMCs), intestinal crypts and biopsies from CD patients. The ability of BI119 to prevent colitis in vivo was assessed in the CD4+CD45RBhigh T cell transfer model.Results: In bacterial antigen-stimulated PBMCs from CD patients, BI119 inhibits Th17-related genes and proteins, while upregulating Treg and preserving Th1 and Th2 signatures. Intestinal crypts cultured with supernatants from BI119-treated commensal-specific CD4+ T cells showed decreased expression of CXCL1, CXCL8 and CCL20. BI119 significantly reduced IL17 and IL26 transcription in colonic and ileal CD biopsies and did not affect IL22. BI119 has a more profound effect in ileal CD with additional significant downregulation of IL23R, CSF2, CXCL1, CXCL8, and S100A8, and upregulation of DEFA5. BI119 significantly prevented development of clinical, macroscopic and molecular markers of colitis in the T-cell transfer model.Conclusions: BI119 modulated CD-relevant Th17 signatures, including downregulation of IL23R while preserving mucosa-associated IL-22 responses, and abrogated experimental colitis. Our results provide support to the use of RORγt antagonists as a novel therapy to CD treatment.

INTERFERON GAMMA EXPRESSION CD8+-T LYMPHOCYTE WITH ESAT-6-CFP-10 FUSION ANTIGEN STIMULATION BETWEEN ACTIVE TUBERCULOSIS, LATENT TUBERCULOSIS AND HEALTHY PEOPLE

Tuberculosis (TB) is an infectious disease in the world causing a global problem. Vaccination with Purified Protein Derivative (PPD) still cannot prevent tuberculosis in Indonesia. Interferon-gamma (IFN-γ) produced by CD8+-T lymphocyte has an important role in eliminating Mycobacterium tuberculosis. The vaccine candidate antigenic test was done to observe the inducible ability of IFN-γ as a main protection cytokine. This study aim was to research the difference of IFN-γ expression CD8+-T lymphocyte percentage with ESAT-6-CFP-10 fusion antigen stimulations as a TB vaccine candidate. This research is a quasi-experimental study design in the laboratory by ESAT-6-CFP-10 fusion antigen-stimulated Peripheral Blood Mononuclear Cells (PBMC) culture in vitro in TB patients, latent TB, and healthy subjects' groups. IFN-γ expression CD8+-T lymphocyte percentage were examined by flow cytometry BDFACSCalibur with results: without antigen fusion stimulation IFN-γ expression CD8+-T lymphocyte percentage mean (TB patients 2,560, latent TB 2,173, and healthy people 2,153) and with antigen fusion (TB patients 3,039, latent TB 2,471, and healthy people 2,405). There was no significant difference in fusion antigen stimulation PBMC between TB patients, latent TB, and healthy subjects' group, and also within groups.

IL-1β Production by Intermediate Monocytes Is Associated with Immunopathology in Cutaneous Leishmaniasis

Cutaneous leishmaniasis due to Leishmania braziliensis infection is an inflammatory disease in which skin ulcer development is associated with mononuclear cell infiltrate and high levels of inflammatory cytokine production. Recently, NLRP3 inflammasome activation and IL-1β production have been associated with increased pathology in murine cutaneous leishmaniasis. We hypothesized that cutaneous leishmaniasis patients have increased expression of NLRP3, leading to high levels of IL-1β production. In this article we show high production of IL-1β in biopsy samples and Leishmania antigen-stimulated peripheral blood mononuclear cells from patients infected with L.braziliensis and reduced IL-1β levels after cure. IL-1β production positively correlated with the area of necrosis in lesions and duration of the lesions. The main source of IL-1β was intermediate monocytes (CD14++CD16+). Furthermore, our murine experiments show that IL-1β production in response to L.braziliensis was dependent on NLRP3, caspase-1, and caspase-recruiting domain (ASC). Additionally, we observed an increased expression of the NLRP3 gene in macrophages and the NLRP3 protein in intermediate monocytes from cutaneous leishmaniasis patients. These results identify an important role for human intermediate monocytes in the production of IL-1β, which contributes to the immunopathology observed in cutaneous leishmaniasis patients.

Combined Expression of IFN-γ, IL-17, and IL-4 mRNA by Recall PBMCs Moderately Discriminates Active Tuberculosis from Latent Mycobacterium tuberculosis Infection in Patients with Miscellaneous Inflammatory Underlying Conditions.

New biomarkers are needed for discriminating active tuberculosis (TB) from latent TB infection (LTBI), especially in vulnerable groups representing the major diagnostic challenge. This pilot study was carried out to explore the diagnostic potential of selected genes, IFN-γ, IL-17, IL-4, and FoxP3, associated with TB immunity and immunopathology. IFN-γ, IL-17, IL-4, and FoxP3 mRNA expression levels were measured by quantitative reverse transcription PCR (RT-qPCR) from antigen-stimulated peripheral blood mononuclear cells of patients with active TB (n = 25); patients with miscellaneous inflammatory disorders and concomitant LTBI (n = 20), rheumatoid arthritis (RA) being the most predominant in the group (n = 11); and in healthy Bacillus Calmette–Guérin (BCG) vaccinees (n = 8). While the levels of FoxP3 mRNA did not differ between the tested groups, the cumulative expression levels of purified protein derivative-stimulated IFN-γ, IL-17, and IL-4 mRNAs were found to distinguish active TB from the whole group of LTBI with 48% sensitivity and 85% specificity. When restricting the LTBI group to RA cases only, the sensitivity was 56% and specificity 100%. When interpreting the result as positive in at least one of the mRNAs IFN-γ, IL-17, or IL-4, sensitivity of 64% and specificities of 75% (heterogeneous group of LTBI) or 100% (LTBI with RA) were achieved. Moderate discrimination of active TB from LTBI with miscellaneous inflammatory underlying conditions by using combined quantitative expression of IFN-γ, IL-17, and IL-4 mRNA seems not to be of high diagnostic potential.

Effects of aspirin-triggered resolvin D1 on peripheral blood mononuclear cells from patients with Chagas' heart disease

Chagas disease is caused by Trypanosoma cruzi (T. cruzi). In some patients with Chagas disease, symptoms progress to chronic chagasic cardiomyopathy. Endogenously, inflammation is resolved in the presence of lipid mediators such as aspirin-triggered RvD1 (AT-RvD1) which has anti-inflammatory and pro-resolution effects. Here, we demonstrated, for the first time, the effects of AT-RvD1 on T. cruzi antigen-stimulated peripheral blood mononuclear cells (PBMCs) from patients with Chagas heart disease. The levels of IFN-γ, TNF-α, IL-10, and IL-13 increased in PBMCs from cardiac-form Chagas patients in stage B1 (patients with fewer heart abnormalities) stimulated with T. cruzi antigen compared to those in non-stimulated PBMCs. AT-RvD1 reduced the IFN-γ concentrations in PBMCs from patients with Chagas disease stimulated with T. cruzi antigen compared to stimulated with T. cruzi antigen cells. AT-RvD1 treatment resulted in no observable changes in TNF-α, IL-10, and IL-13 levels. AT-RvD1 significantly decreased the percentage of necrotic cells and caused a significant reduction in the proliferation rate of T. cruzi antigen-stimulated PBMCs from patients with Chagas disease. These findings demonstrate that AT-RvD1 modulates the immune response in Chagas disease patients and might have potential to be used as an alternative approach for slowing the development of further heart damage.

Evaluation of immunogenicity and protective efficacy of adjuvanted Salmonella Typhimurium ghost vaccine against salmonellosis in chickens

ABSTRACTBackground: Salmonella Typhimurium, a non-host-adapted Gram-negative intracellular pathogen, is capable of infecting a variety of animal hosts and humans.Objective: This study utilized the prime-booster immunization strategy using Salmonella Typhimurium–LTB (S. Typhimurium–LTB) ghost with the aim of inducing a robust immune response for the prevention of avian salmonellosis. In addition, the effect of MontanideTM ISA 70VG adjuvant on S. Typhimurium–LTB ghost vaccination was investigated.Animals and methods: A total of 75 chickens were divided into three groups (n=25) for intramuscular immunization: group A (non-immunized control injected with sterile PBS), group B (immunized with S. Typhimurium–LTB ghost), and group C (immunized with S. Typhimurium–LTB ghost plus MontanideTM ISA70VG adjuvant).Results: Compared with group A, the immunized chickens (groups B and C) exhibited increased titers of antigen specific plasma IgG and intestinal secretory IgA antibodies. In addition, group C showed enhanced induction of the humoral immune response compared to group B. The populations of splenic CD3+CD4+ and CD3+CD8+ T-cells increased significantly in both immunized groups. In addition, increased mRNA expression of the Th1 cytokines, IFN-γ, and IL-2 were observed in S. Typhimurium antigen-stimulated peripheral blood mononuclear cells from groups B and C chickens. Chickens from both vaccinated groups showed significant protection against virulent S. Typhimurium oral challenge compared to non-vaccinated chickens and a lower challenge strain count was recovered from the internal organs of group C.Conclusions: Injection of S. Typhimurium-LTB ghost with or without MontanideTM ISA70VG adjuvant is capable of inducing protective immunity against the virulent S. Typhimurium infection in chickens.

A benchmark for evaluation of algorithms for identification of cellular correlates of clinical outcomes.

The Flow Cytometry: Critical Assessment of Population Identification Methods (FlowCAP) challenges were established to compare the performance of computational methods for identifying cell populations in multidimensional flow cytometry data. Here we report the results of FlowCAP-IV where algorithms from seven different research groups predicted the time to progression to AIDS among a cohort of 384 HIV+ subjects, using antigen-stimulated peripheral blood mononuclear cell (PBMC) samples analyzed with a 14-color staining panel. Two approaches (FlowReMi.1 and flowDensity-flowType-RchyOptimyx) provided statistically significant predictive value in the blinded test set. Manual validation of submitted results indicated that unbiased analysis of single cell phenotypes could reveal unexpected cell types that correlated with outcomes of interest in high dimensional flow cytometry datasets.

Superior antigen-specific CD4+ T-cell response with AS03-adjuvantation of a trivalent influenza vaccine in a randomised trial of adults aged 65 and older.

BackgroundThe effectiveness of trivalent influenza vaccines may be reduced in older versus younger adults because of age-related immunosenescence. The use of an adjuvant in such a vaccine is one strategy that may combat immunosenescence, potentially by bolstering T-cell mediated responses.MethodsThis observer-blind study, conducted in the United States (US) and Spain during the 2008–2009 influenza season, evaluated the effect of Adjuvant System AS03 on specific T-cell responses to a seasonal trivalent influenza vaccine (TIV) in ≥65 year-old adults.Medically-stable adults aged ≥65 years were randomly allocated to receive a single dose of AS03-adjuvanted TIV (TIV/AS03) or TIV. Healthy adults aged 18–40 years received only TIV. Blood samples were collected on Day 0, Day 21, Day 42 and Day 180. Influenza-specific CD4+ T cells, defined by the induction of the immune markers CD40L, IL-2, IFN-γ, or TNF-α, were measured in ex vivo cultures of antigen-stimulated peripheral blood mononuclear cells.ResultsA total of 192 adults were vaccinated: sixty nine and seventy three ≥65 year olds received TIV/AS03 and TIV, respectively; and fifty 18 - 40 year olds received TIV. In the ≥65 year-old group on Day 21, the frequency of CD4+ T cells specific to the three vaccine strains was superior in the TIV/AS03 recipients to the frequency in TIV (p < 0.001). On Days 42 and 180, the adjusted-geometric mean specific CD4+ T-cell frequencies were also higher in the TIV/AS03 recipients than in the TIV recipients (p < 0.001). Furthermore, the adjusted-geometric mean specific CD4+ T-cell frequencies were higher in the ≥65 year-old recipients of TIV/AS03 than in the18 - 40 year old recipients of TIV on Days 21 (p = 0.006) and 42 (p = 0.011).ConclusionThis positive effect of AS03 Adjuvant System on the CD4+ T-cell response to influenza vaccine strains in older adults could confer benefit in protection against clinical influenza disease in this population.Trial registration(Clinicaltrials.gov.). NCT00765076.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2334-14-425) contains supplementary material, which is available to authorized users.

Mycobacterium Tuberculosis-Specific TNF-α Is a Potential Biomarker for the Rapid Diagnosis of Active Tuberculosis Disease in Chinese Population

Interferon-gamma release assays (IGRAs) have proven to be useful to accurately detect Mycobacterium tuberculosis (Mtb) infection, but they cannot reliably discriminate between active tuberculosis (TB) and latent tuberculosis infection (LTBI). This study aims to test whether Mtb-specific tumor necrosis factor-alpha (TNF-α) could be used as a new tool for the rapid diagnosis of active TB disease. The secretion of TNF-α by Mtb-specific antigen-stimulated peripheral blood mononuclear cells (PBMCs) of sixty seven participants was investigated in the study. Our results showed that the total measurement of TNF-α secretion by Mtb-specific antigen-stimulated PBMCs is not a good biomarker for active TB diagnosis. However, we found that calculation of Mtb-specific TNF-α not only distinguish between active and latent TB infection, but also can differentiate active TB from non-TB patients. Using the cutoff value of 136.9 pg/ml for Mtb-specific TNF-α, we were able to differentiate active TB from LTBI. Sensitivity and specificity were 72% and 90.91%. These data suggest that Mtb-specific TNF-α could be a potential biomarker for the diagnosis of active TB disease.

Immune responses associated with progression and control of infection in calves experimentally challenged with Mycobacterium avium subsp. paratuberculosis

This study examined the immune responses related to the infection, progression and control of Mycobacterium avium subsp. paratuberculosis (MAP) infection in calves. Twenty calves were challenged orally with MAP and 11 non-challenged calves served as controls. Approximately half the calves from each group were sacrificed at either 7 or 15 months post-challenge (PC). The majority of the challenged calves (19/20) shed MAP in feces 2–4 months PC, but thereafter fecal shedding reduced markedly. The severity of infection was reduced at 15 months PC compared to that at 7 months PC as seen from a significantly lower isolation of MAP from tissues and lower lesion scores (P<0.05). In addition, there was a reduction in the upregulation of gene expression of gamma interferon, interleukin-10 (IL-10) and inducible nitric oxide synthase from the antigen-stimulated mesenteric lymph node (MLN) cultures of the challenged calves. No evidence of infection was detected in the control calves. The severity of the infection in individual calves at 15 months PC as indicated from the number of tissue culture positive sites, was negatively related to IL-10 released from antigen-stimulated peripheral blood mononuclear cells (P<0.05). Collectively the data indicated that the severity of the MAP infection was reduced in the calves at 15 months PC and in a specific time period during infection, IL-10 may play a role in reducing the severity of this disease.

Altered patterns of toll-like receptor gene expression in cull cows infected with Mycobacterium avium subsp. paratuberculosis

Johne's disease caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic enteric disease of cattle. The mechanism how MAP can co-exist in the gastro-intestinal tract despite a massive infiltration of immune cells is not known. Toll-like receptors (TLRs) are known to play an important role in both innate and acquired immune responses but it is unclear what role different TLRs play in response to MAP. In this study, 38 cull cows from herds infected with MAP were classified into four groups, based on MAP culture from gut tissues and histopathological lesion scores. The expression of TLR1, 2 and 4 mRNA from MAP antigen-stimulated mesenteric lymph node (MLN) cultures and peripheral blood mononuclear cells (PBMCs) and in the MLN and ileum tissues of these animals was determined. MAP antigen-specific expression of TLR1 in MLN and PBMC was significantly lower in the MAP-infected groups than the non-infected control group, suggesting that in MAP-infected animals there is impairment in the up-regulation of TLR1 in response to MAP antigen. TLR4 expression in MLN tissues was significantly higher in the severely infected group than the control group suggesting up-regulation of endogenous TLR4 expression at a site of MAP infection in animals severely affected with Johne's disease. A preliminary screening of TLR1, 2 and 4 in the cull cows revealed the presence of polymorphisms in TLR1 and TLR2. In summary, one mechanism how MAP may subvert the immune system is that there is an apparent lack of recognition of MAP antigens as foreign by TLR1 in MAP-infected cows.

Lower levels of interleukin-12 precede the development of tuberculosis among HIV-infected women

Tuberculosis (TB) is the worldwide leading cause of death among HIV-infected individuals, accounting for more than half of AIDS-related deaths. A high risk of tuberculosis (TB) has been shown in early stages of the HIV disease, even in the presence of normal CD4+ cell counts. Moreover, the factors that determine protective immunity vs. susceptibility to Mycobacterium tuberculosis cannot be fully explained by simple changes in IFNγ levels or a shift from Th1 to Th2 cytokines. This work investigated the relationship between cytokine expression profiles in peripheral blood mononuclear cells (PBMC) and susceptibility to M. tuberculosis in 10 HIV+ women who went onto develop TB. RNA transcripts for IL-4, IL-4δ2, IL-10, IL-12(p35), IL-13, IL-17A, IFNγ and TNFα were measured by real-time quantitative PCR in unstimulated or TB peptide antigen-stimulated PBMCs from 10 HIV+ women with positive tuberculin skin tests (TST) and compared with HIV-seropositive and seronegative women without previous TB and negative TST. Stimulated PBMC cultures showed significantly lower expression of IL-12p35 (p=0.004) and IL-10 (p=0.026) in the HIV+TB+ group 6–12months before onset of TB compared to HIV+TB− women. Unstimulated PBMC from HIV+TB+ women also had lower expression of Th2 cytokines [IL-4 (p=0.056) and IL-13 (p=0.050)] compared to HIV+TB− women. These results suggest that lower IL-12 production by PBMC in response to TB antigens and lower levels of both Th1 and Th2 cytokines by PBMC correlate with future development of TB in HIV-infected women and may be responsible for their increased susceptibility.

Plasma biomarkers in the prediction and diagnosis of tuberculosis-associated immune reconstitution inflammatory syndrome

Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is the most common form of immune restoration disease (IRD) complicating early antiretroviral therapy (ART) in patients from resource-limited countries with HIV infection [1]. Increasing attention is being paid to the definition of plasma biomarkers that might assist in the prediction and diagnosis of TB-IRIS, as well as other forms of IRD [2,3]. The findings of the study by Haddow et al.[4] are, therefore, an important contribution to the literature on IRD but might have been more informative if considered in the context of data from other studies of plasma biomarkers. We described a prospective study of the immunopathogenesis of TB-IRIS and ART-associated TB (ART-TB; equivalent to ‘unmasking’ TB-IRIS [1] reported by Haddow et al.) undertaken using plasma from whole blood interferon (IFN)-gamma release assays (IGRAs) [5,6]. The advantage of using whole blood IGRAs in this context is that plasma from antigen-stimulated and antigen-unstimulated tubes can be analyzed simultaneously. However, this advantage must be balanced against the uncertainty of knowing whether biomarkers in plasma from unstimulated tubes reflect the in-vivo state or the production or consumption of biomarkers during the 24-h culture. The demonstration by Haddow et al.[4] that plasma CCL2 (monocyte chemotactic protein-1) levels are low in patients with ‘paradoxical’ TB-IRIS is supported by our finding of low CCL2 levels in unstimulated IGRA plasma from patients with TB-IRIS [6]. In addition, we demonstrated low levels of CCL2 before ART. In contrast to the findings of Haddow et al.[4], we found similar levels of interleukin (IL)-10 in unstimulated IGRA plasma from TB-IRIS cases and controls [6]. However, low production of IL-10 by antigen-stimulated peripheral blood mononuclear cells from patients with IRD associated with infection by nontuberculous mycobacteria has been demonstrated previously [7,8]. In the study by Seddiki et al.[8], low IL-10 production was associated with increased numbers of circulating regulatory T (Treg) cells, so the authors suggested that low IL-10 production might reflect dysfunctional Treg cells. As CCL2 and IL-10 are both produced by monocytes and macrophages, an alternative explanation is that CCL2 and IL-10 deficiency reflect monocyte/macrophage dysfunction. We agree with Haddow et al.[4] that there may be differences in the immunopathogenesis of TB-IRIS and ART-TB [5,6]. They reported that pre-ART plasma IFN-gamma levels were approximately 10-fold higher in patients who subsequently developed ‘unmasking’ TB IRIS compared with controls, but that there was no difference in pre-ART IFN-gamma levels when ‘paradoxical’ TB-IRIS cases were compared with controls [4]. We reported that pre-ART IFN-gamma responses to both region of difference 1 and purified protein derivative antigens of Mycobacterium tuberculosis in whole blood IGRAs, corrected for CD4+ T cell counts, were increased in patients who developed ART-TB and had strong performance characteristics in the prediction of ART-TB by receiver operator curve (ROC) analysis [5]. We also reported that levels of CXCL10 (IP-10) in unstimulated IGRA plasmas were higher in TB-IRIS patients than ART-TB patients during 24 weeks of ART and, furthermore, that pre-ART levels of CXCL10 in combination with CCL2 and IL-18 levels predicted the occurrence of TB-IRIS by ROC analysis. In contrast, Haddow et al.[4] did not demonstrate a difference in plasma levels of CXCL10 levels between ‘paradoxical’ TB-IRIS or ‘unmasking’ TB-IRIS cases and their controls [4], perhaps suggesting that in our study, these biomarkers were produced in cell culture. Unlike previous studies of various forms of IRD [2], Haddow et al.[4] did not demonstrate increased plasma levels of IL-6 in patients with ‘paradoxical’ TB-IRIS. A possible explanation for this comprises differences in the assay method. In most previous studies of IRD, plasma IL-6 levels have been assayed using enzyme-linked immunosorbent assays (ELISAs), whereas Haddow et al.[4] used a multiplex bead array assay (MBAA). Plasma IL-6 levels in HIV patients may differ significantly when assayed by ELISA or MBAA [9]. It is now clear that assays of selected biomarkers can assist in both the prediction and diagnosis of TB-IRIS and ART-TB. Large-scale studies are now needed to delineate the value of these assays as diagnostic tests. In doing this, consideration should be given to the use of both plasma and IGRA plasma from antigen-stimulated and antigen-unstimulated tubes and also to the method used to assay cytokines and chemokines. Acknowledgements Conflicts of interest There are no conflicts of interest.