Respiratory Syncytial Virus Infects Regulatory B Cells in Human Neonates via Chemokine Receptor CX3CR1 and Promotes Lung Disease Severity.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in infants and is characterized by pulmonary infiltration of B cells in fatal cases. We analyzed the B cell compartment in human newborns and identified a population of neonatal regulatory B lymphocytes (nBreg cells) that produced interleukin 10 (IL-10) in response to RSV infection. The polyreactive B cell receptor of nBreg cells interacted with RSV protein F and induced upregulation of chemokine receptor CX3CR1. CX3CR1 interacted with RSV glycoprotein G, leading to nBreg cell infection and IL-10 production that dampened T helper 1 (Th1) cytokine production. In the respiratory tract of neonates with severe RSV-induced acute bronchiolitis, RSV-infected nBreg cell frequencies correlated with increased viral load and decreased blood memory Th1 cell frequencies. Thus, the frequency of nBreg cells is predictive of the severity of acute bronchiolitis disease and nBreg cell activity may constitute an early-life host response that favors microbial pathogenesis.

Impact of Maternal Pertussis Antibodies on the Infants' Cellular Immune Responses.

INTRODUCTION: Maternal antibody interference of the infant's humoral immune responses raises some concern to the strategy of maternal Tdap (tetanus, diphtheria, acellular pertussis [aP]) vaccination. This study assessed the impact of maternal Tdap antibodies on the infant's pertussis-specific T lymphocyte responses following infant vaccination with an aP containing vaccine, in a term and preterm born cohort. METHODS: Heparin samples (±0.5 mL) were conveniently drawn from infants of a Belgian prospective cohort study (N = 79, NCT02511327), including Tdap vaccinated (Boostrix®) and nonvaccinated women (no Tdap vaccine in the last 5 years) that delivered at term or prematurely. Sampling was performed before and 1 month after primary (8-12-16 weeks) and booster vaccination (13 or 15 months) with DTaP-IPV-HB-PRP~T vaccine (Hexyon®). Pertussis toxin (PT)-specific CD3+, CD3+ CD4+ and CD3+ CD8+ lymphoblasts and their cytokine secretions were measured using a flow cytometric assay on whole blood (FASCIA) and multiplex technology (Meso Scale Discovery), respectively. RESULTS: In total, 57% of all infants were considered PT-specific CD3+ CD4+ lymphoblasts responders after primary and booster vaccination, whereas 17% were CD3+ CD8+ lymphoblast responders. Interferon (IFN)-γ, interleukin (IL)-13, IL-17A, and IL-5 cytokine secretions after primary and booster vaccination were indicative of a mixed T helper (Th) 1/Th2/Th17 cell profile. Lymphoblast and cytokine levels were comparable between term and preterm infants. Nonresponders for IL-13 after booster vaccination had higher maternal PT immunoglobulin G (IgG) levels at birth when compared to responders. CONCLUSIONS: Term and preterm born infants are capable of inducing Th1, Th2, and Th17 responses after aP vaccination, yet maternal vaccination modulate these responses. Evaluation of this effect in larger trials is needed.

Natural T Cell Epitope Containing Methyl Lysines on Mycobacterial Heparin-Binding Hemagglutinin.

T cell epitopes are mostly nonmodified peptides, although posttranslationally modified peptide epitopes have been described, but they originated from viral or self-proteins. In this study, we provide evidence of a bacterial methylated T cell peptide epitope. The mycobacterial heparin-binding hemagglutinin (HBHA) is a protein Ag with a complex C-terminal methylation pattern and is recognized by T cells from humans latently infected with Mycobacterium tuberculosis By comparing native HBHA with recombinant HBHA produced in Mycobacterium smegmatis (rHBHA-Ms), we could link antigenic differences to differences in the methylation profile. Peptide scan analyses led to the discovery of a peptide containing methyl lysines recognized by a mAb that binds to native HBHA ∼100-fold better than to rHBHA-Ms This peptide was also recognized by T cells from latently infected humans, as evidenced by IFN-γ release upon peptide stimulation. The nonmethylated peptide did not induce IFN-γ, arguing that the methyl lysines are part of the T cell epitope.

HBHA-Induced Polycytotoxic CD4+ T Lymphocytes Are Associated with the Control of Mycobacterium tuberculosis Infection in Humans.

Heparin-binding hemagglutinin (HBHA), a surface protein of Mycobacterium tuberculosis, is an attractive vaccine candidate and marker of protective immunity against tuberculosis, although the mechanisms underlying this protective immunity are not fully understood. Comparisons of the immune responses of latently M. tuberculosis-infected (LTBI) subjects to those of patients with active tuberculosis (aTB) may help to identify surrogate markers of protection, as LTBI subjects are most often lifelong protected against the disease. HBHA was shown to induce strong Th1 responses and cytotoxic CD8+ responses in LTBI subjects, but additional mechanisms of control of M. tuberculosis infection remain to be identified. In this study, using HBHA-induced blast formation as a readout of specific T lymphocyte activation, we report the presence in M. tuberculosis-infected subjects of HBHA-induced CD4+ T cell blasts that degranulate, as measured by surface capture of CD107a. This suggests the induction by HBHA of a CD4+ T cell subset with cytolytic function, and as nearly half of these cells also contained IFN-γ, they had both Th1 and cytotoxic characteristics. We further identified a CD4+ T lymphocyte subset producing IFN-γ together with a combination of mediators of cytotoxicity, i.e., perforin, granzymes, and granulysin, and we called them polycytotoxic CD4+ T lymphocytes. Interestingly, whereas purified protein derivative induced such cells in both LTBI subjects and patients with aTB, HBHA-specific polycytotoxic CD4+ T lymphocytes were detected in LTBI subjects and not in patients with pulmonary aTB. To our knowledge, we thus identified a new HBHA-induced CD4+ T cell subset that may contribute to the control of M. tuberculosis infection.

Highlights of the 12th International Bordetella Symposium.

To commemorate the 100th anniversary of the Nobel prize being awarded to Jules Bordet, the discoverer of Bordetella pertussis, the 12th International Bordetella Symposium was held from 9 to 12 April 2019 at the Université Libre de Bruxelles, where Jules Bordet studied and was Professor of Microbiology. The symposium attracted more than 300 Bordetella experts from 34 countries. They discussed the latest epidemiologic data and clinical aspects of pertussis, Bordetella biology and pathogenesis, immunology and vaccine development, and genomics and evolution. Advanced technological and methodological tools provided novel insights into the genomic diversity of Bordetella and a better understanding of pertussis disease and vaccine performance. New molecular approaches revealed previously unrecognized complexity of virulence gene regulation. Innovative insights into the immune responses to infection by Bordetella resulted in the development of new vaccine candidates. Such discoveries will aid in the design of more effective approaches to control pertussis and other Bordetella-related diseases.

Early diagnosis of miliary tuberculosis in a hemodialysis patient by combining two interferon-γ-release assays: a case report.

BACKGROUND: Patients with end-stage renal disease undergoing chronic hemodialysis (HD) are at high risk to develop tuberculosis (TB) associated with a high mortality rate. TB diagnosis is often delayed due to non-specific symptoms, frequent extra-pulmonary manifestations, and rare microbiological confirmation. This case report illustrates the clear added value of combined interferon-γ -release assays (IGRA) in response to different mycobacterial antigens for an early diagnosis of TB in HD patients. CASE PRESENTATION: We report the case of an Egyptian patient under chronic HD treatment, who presented with recurrent episodes of fever and myalgia of unknown origin, associated with an important inflammatory syndrome. These episodes resolved partially or completely within less than 1 month without any treatment but recurred 10 times within 3 years. Chest Computed Tomography and 18F-fluorodeoxyglucose Positron Emission Tomography/Computed Tomography (18FDG PET-CT) demonstrated several active mediastinal lymphadenopathies. TB was the first suspected diagnosis but cultures and polymerase chain reaction (PCR) remained negative on a mediastinal lymph node aspiration. In contrast, the results from two different IGRA performed on blood were highly suggestive of TB disease. Several granulomas, some of them with central non-caseating necrosis, were demonstrated on a pulmonary nodule obtained by thoracoscopic resection, but PCR and culture remained negative for M. tuberculosis. Three years after the initial symptoms, a new PET-CT revealed a retro-clavicular lymphadenopathy in addition to the mediastinal lymphadenopathies, and the M. tuberculosis culture performed on the resected lymphadenopathy was positive. Antibiotic treatment for TB was started and resulted in a clear improvement of the patient's clinical condition, allowing him to successfully receive a renal graft. CONCLUSIONS: In view of the high frequency of TB in patients undergoing chronic HD and of the limitations of the classical diagnosis procedures, nephrologists have to diagnose TB mostly on clinical suspicion. We demonstrate here that the use of a combined IGRA to two different mycobacterial antigens may significantly raise the index of suspicion and help clinicians to decide starting anti-TB treatment in HD patients.

Human Immune Responses to Pertussis Vaccines.

Pertussis still represents a major cause of morbidity and mortality worldwide. Although vaccination is the most powerful tool in preventing pertussis and despite nearly 70 years of universal childhood vaccination, incidence of the disease has been rising in the last two decades in countries with high vaccination coverage. Two types of vaccines are commercially available against pertussis: whole-cell pertussis vaccines (wPVs) introduced in the 1940s and still in use especially in low and middle-income countries; less reactogenic acellular pertussis vaccines (aPVs), licensed since the mid-1990s.In the last years, studies on pertussis vaccination have highlighted significant gaps and major differences between the two types of vaccines in the induction of protective anti-pertussis immunity in humans. This chapter will discuss the responses of the immune system to wPVs and aPVs, with the aim to enlighten critical points needing further efforts to reach a good level of protection in vaccinated individuals.

Efficacy of treatment with belladonna in children with severe pallid breath-holding spells.

IntroductionPallid breath-holding spells are common and dramatic forms of recurrent syncope in infancy. They are very stressful despite their harmless nature and sometimes require treatment. OBJECTIVE: The objective of this study was to evaluate the efficacy of belladonna in severe breath-holding spells. METHODS: This is a multicentric, retrospective series involving 84 children with severe pallid breath-holding spells. Inclusion criteria were >1 pallid breath-holding spell with loss of consciousness, paediatric cardiology evaluation, and follow-up >6 months. In total, 45 patients received belladonna and 39 patients did not receive treatment, according to physician preference. RESULTS: Mean age was 11 months, ranging from 4 to 18 months, with 54% of males. Mean spell duration was 30 seconds (interquartile range 15, 60), and the frequency was four episodes per month (interquartile range 0.5, 6.5). Comparison of baseline characteristics between groups showed similar demographics, with the single difference in the severity of the spells, being more severe in the treated group. When comparing the treated and non-treated groups at 3 months, only two (5%) patients had a complete remission in the first group, whereas 20 (44%) had remission in the belladonna group (p<0.01). When considering the characteristics of the spells before and after the initiation of treatment with belladonna, 75% of the patients presented a positive response, with 44% of the patients presenting with complete resolution of the spells (p<0.01). No major adverse reaction was reported, with only 5% minor adverse events. CONCLUSIONS: Belladonna is highly effective to alleviate severe breath-holding spells in young children, without any major adverse effects.

Standardized whole blood stimulation improves immunomonitoring of induced immune responses in multi-center study.

Functional immune responses are increasingly important for clinical studies, providing in depth biomarker information to assess immunotherapy or vaccination. Incorporating functional immune assays into routine clinical practice has remained limited due to challenges in standardizing sample preparation. We recently described the use of a whole blood syringe-based system, TruCulture®, which permits point-of-care standardized immune stimulation. Here, we report on a multi-center clinical study in seven FOCIS Centers of Excellence to directly compare TruCulture to conventional PBMC methods. Whole blood and PBMCs from healthy donors were exposed to LPS, anti-CD3 anti-CD28 antibodies, or media alone. 55 protein analytes were analyzed centrally by Luminex multi-analyte profiling in a CLIA-certified laboratory. TruCulture responses showed greater reproducibility and improved the statistical power for monitoring differential immune response activation. The use of TruCulture addresses a major unmet need through a robust and flexible method for immunomonitoring that can be reproducibly applied in multi-center clinical studies. ONE SENTENCE SUMMARY: A multi-center study revealed greater reproducibility from whole blood stimulation systems as compared to PBMC stimulation for studying induced immune responses.

Persistence of T-cell immune response induced by two acellular pertussis vaccines in children five years after primary vaccination.

The resurgence of pertussis suggests the need for greater efforts to understand the long-lasting protective responses induced by vaccination. In this paper we dissect the persistence of T memory responses induced by primary vaccination with two different acellular pertussis (aP) vaccines, hexavalent Hexavac® vaccine (Hexavac) (Sanofi Pasteur MSD) and Infanrix hexa® (Infanrix) (Glaxo-SmithKline Biologicals). We evaluated magnitude and duration of T-cell responses to pertussis toxin (PT) by measuring T-cell proliferation, cytokines (IL-2 and IFNγ) production and memory subsets in two groups of children 5 years after primary vaccination. Some of the enrolled children received only primary vaccination, while others had the pre-school boost dose. Positive T-cell responses to PT were detected in 36% of children. Percentage of responsive children, T-cell proliferation and CD4IL-2+ cells were significantly higher in the children primed with Hexavac than in those who received Infanrix vaccine. No major effects of the boost on PT-specific proliferation were observed. Overall, our data documented a persistence of T-cell memory against PT in a minor fraction of children 5 years after primary vaccination. The different responses induced by Hexavac and Infanrix vaccine could rely on differences in PT inactivation process or excipients/adjuvants formulations.

Contribution of a heparin-binding haemagglutinin interferon-gamma release assay to the detection of Mycobacterium tuberculosis infection in HIV-infected patients: comparison with the tuberculin skin test and the QuantiFERON-TB Gold In-tube.

BACKGROUND: The screening and treatment of latent tuberculosis (TB) infection reduces the risk of progression to active disease and is currently recommended for HIV-infected patients. The aim of this study is to evaluate, in a low TB incidence setting, the potential contribution of an interferon-gamma release assay in response to the mycobacterial latency antigen Heparin-Binding Haemagglutinin (HBHA-IGRA), to the detection of Mycobacterium tuberculosis infection in HIV-infected patients. METHODS: Treatment-naïve HIV-infected adults were recruited from 4 Brussels-based hospitals. Subjects underwent screening for latent TB using the HBHA-IGRA in parallel to a classical method consisting of medical history, chest X-ray, tuberculin skin test (TST) and QuantiFERON-TB Gold In-Tube (QFT-GIT). Prospective clinical and biological follow-up ensued, with repeated testing with HBHA-IGRA. A group of HIV-infected patients with clinical suspicion of active TB was also recruited and tested with the HBHA-IGRA. Multiplex analysis was performed on the culture supernatants of this in-house assay to identify test read-outs alternative to interferon-gamma that could increase the sensitivity of the test. RESULTS: Among 48 candidates enrolled for screening, 9 were identified with latent TB by TST and/or QFT-GIT results. Four of these 9 patients and an additional 3 screened positive with the HBHA-IGRA. This in-house assay identified all the patients that were positive for the TST and showed the best concordance with the presence of a M. tuberculosis exposure risk. During follow-up (median 14 months) no case of active TB was reported and HBHA-IGRA results remained globally constant. Fourteen HIV-infected patients with clinical suspicion of active TB were recruited. Active TB was confirmed for 6 of them among which 3 were HBHA-IGRA positive, each with very high interferon-gamma concentrations. All patients for whom active TB was finally excluded, including 2 non-tubercular mycobacterial infections, had negative HBHA-IGRA results. Multiplex analysis confirmed interferon-gamma as the best read-out. CONCLUSIONS: The HBHA-IGRA appears complementary to the QuantiFERON-TB Gold In-Tube for the screening of latent TB in HIV-infected patients. Large-scale studies are necessary to determine whether this combination offers sufficient sensitivity to dismiss TST, as suggested by our results. Furthermore, HBHA-IGRA may help in the diagnosis work-up of clinical suspicions of active TB.

Human CD19 and CD40L deficiencies impair antibody selection and differentially affect somatic hypermutation.

BACKGROUND: Individuals with genetic defects in CD40 ligand (CD40L) or B-cell antigen receptor coreceptor molecules CD19 and CD81 suffer from an antibody deficiency. Still, these patients carry low levels of memory B cells and serum antibodies. OBJECTIVE: We sought to assess why the remaining memory B cells and antibodies in the blood of these patients do not provide functional immunity. METHODS: We included CD19-deficient patients (n = 8), CD40L-deficient patients (n = 8), and healthy controls (n = 50) to perform detailed flow cytometry on blood B cells, molecular analysis of IgA and IgG transcripts, as well as functional analysis of B-cell activation. RESULTS: CD19-deficient and CD40L-deficient patients carried reduced numbers of all memory B-cell subsets except CD27(-)IgA(+) B cells. Their immunoglobulin heavy chain class-switched transcripts contained less somatic mutations and reduced usage of IgM-distal IgG2 and IgA2 subclasses. The selection strength of mutations for antigen binding was significantly lower than in controls, whereas selection to maintain superantigen binding was normal. Furthermore, the patients showed impaired selection against inherently autoreactive properties of their immunoglobulins. Somatic hypermutation analysis revealed decreased activation-induced cytidine deaminase and uracil-DNA glycosylase 2 activity in CD40L deficiency and increased uracil-DNA glycosylase 2 but decreased mismatch repair in CD19 deficiency. B-cell activation studies revealed that this was at least in part due to transcriptional regulation of DNA repair genes. CONCLUSIONS: This study on CD19 and CD40L deficiencies illustrates that both the B-cell antigen receptor and CD40 signaling pathways are required for the selection of immunoglobulin reactivity. Still, they differentially mediate DNA repair pathways during somatic hypermutation, thereby together shaping the human in vivo antigen-experienced B-cell repertoire.

NF-κB activation and severity of gastritis in Helicobacter pylori-infected children and adults.

BACKGROUND: In contrast to adults, Helicobacter pylori gastritis in children is reported as milder and ulcer disease as uncommon, but unequivocal data are lacking. OBJECTIVES: To compare the frequency of gastro-duodenal ulcers in children and adults as well as the proportion of Helicobacter pylori infection in these patients and to study the effect of chronological age on NF-κB activation and on severity of gastritis. DESIGN: Patients referred in one pediatric and one adult facility for upper GI endoscopy were included. Gastric biopsies were obtained in consecutive Helicobacter pylori-infected patients and age-matched negative controls for immunohistochemistry and electrophoresis mobility shift assay. Three age groups were defined: younger than 8 years, 8-17 years, and adults. RESULTS: Peptic ulcer disease was less frequent in children and less frequently associated with Helicobacter pylori infection. When comparing infected subjects to controls, densities of neutrophils and CD20 cells in the lamina propria increased in all age groups, CD3 cells increasing only in patients older than 8 years and CD8 cells only in adults. NF-κB-p65-positive cells were also increased only in infected adults as well as NF-κB-binding activity. A positive correlation was found between age and densities of neutrophils and CD3, but not of CD8 or CD20 cells. CONCLUSION: Peptic ulcer disease was less frequent in children and less frequently caused by Helicobacter pylori infection. The different clinical outcome of the infection in children can be the consequence of the lower mucosal immune response.

Antibody deficiency due to a missense mutation in CD19 demonstrates the importance of the conserved tryptophan 41 in immunoglobulin superfamily domain formation.

Immunoglobulin superfamily (IgSF) domains are conserved structures present in many proteins in eukaryotes and prokaryotes. These domains are well-capable of facilitating sequence variation, which is most clearly illustrated by the variable regions in immunoglobulins (Igs) and T cell receptors (TRs). We studied an antibody-deficient patient suffering from recurrent respiratory infections and with impaired antibody responses to vaccinations. Patient's B cells showed impaired Ca(2+) influx upon stimulation with anti-IgM and lacked detectable CD19 membrane expression. CD19 sequence analysis revealed a homozygous missense mutation resulting in a tryptophan to cystein (W52C) amino acid change. The affected tryptophan is CONSERVED-TRP 41 located on the C-strand of the first extracellular IgSF domain of CD19 and was found to be highly conserved, not only in mammalian CD19 proteins, but in nearly all characterized IgSF domains. Furthermore, the tryptophan is present in all variable domains in Ig and TR and was not mutated in 117 Ig class-switched transcripts of B cells from controls, despite an overall 10% amino acid change frequency. In vitro complementation studies and CD19 western blotting of patient's B cells demonstrated that the mutated protein remained immaturely glycosylated. This first missense mutation resulting in a CD19 deficiency demonstrates the crucial role of a highly conserved tryptophan in proper folding or stability of IgSF domains.

IL-12Rß1 deficiency and disseminated Mycobacterium tilburgii disease.

Mycobacterium tilburgii rarely causes disseminated disease. We describe a case of M. tilburgii infection in an otherwise healthy 33-year-old woman, who was found to carry bi-allelic mutations of the gene encoding the ß1 chain of the IL-12 receptor.

Large decrease of anti-tetanus anatoxin and anti-pneumococcal antibodies at one year after renal transplantation.

AIMS: In kidney transplant recipients (KTR), antibody (Ab) synthesis is hampered by AZA and CsA. We here report in a prospective cohort study, the effects of mycophenolate mofetil (MMF) associated to a calcineurin inhibitor on plasma levels of anti-tetanus anatoxin Ab (TAnAb) and anti-pneumococcal Ab (PnPsAb). METHODS: Serum titers of the TAnAb and the PnPsAb against serotypes 14, 19F and 23F were measured in 94 KTR on Day 0 (T0) and 1 year (T12) after renal transplantation and in 49 healthy controls. RESULTS: 1) At T0, TAnAb were detected in only 71% of patients vs. 98% of controls (p < 0.0001) and the titers were significantly lower in KTR (1.46 UI/ml vs. 2.74 in controls, p = 0.01); they further decreased between T0 and T12 (1.46 UI/ml to 0.31, p < 0.0001). The calculated half-life (t1/2) of TAnAb was 7.7 months, as compared to more than 10 years in a normal population. 2) In KTR, PnPsAb titers decreased significantly between T0 and T12 (p < 0.005); the t1/2 of the different PnPsAb ranged from 9.2 to 11.9 months. CONCLUSIONS: In KTR treated by MMF and CNI, the TAnAbs and PnPsAbs titers decrease significantly and profoundly during the first year. Immunization pre-transplantation should be encouraged to maintain adequate post-transplant Abs levels.

A semi high-throughput whole blood-based flow cytometry assay to detect and monitor Bordetella pertussis-specific Th1, Th2 and Th17 responses.

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-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. 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 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. 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.

Analysis of a Combined HBHA and ESAT-6-Interferon-γ-Release Assay for the Diagnosis of Tuberculous Lymphadenopathies.

BACKGROUND AND OBJECTIVES: The incidence of tuberculosis lymphadenopathy (TBLA) is increasing, and diagnostic procedures lack sensitivity and are often highly invasive. TBLA may be asymptomatic, and differential diagnosis with other adenopathies (ADPs) is difficult. We evaluated a blood-cell interferon-γ release assay (IGRA) with two different stage-specific mycobacterial antigens for the differential diagnosis of ADP suspected of mycobacterial origin. METHODS: Twenty-one patients were included and divided into three groups: (1) cervical/axillar ADP (n = 8), (2) mediastinal ADP (n = 10), and (3) disseminated ADP (n = 3). The mycobacterial antigens used for the IGRA were the heparin-binding haemagglutinin (HBHA) and the early-secreted antigenic target-6 (ESAT-6), a latency-associated antigen and a bacterial replication-related antigen, respectively. Diagnosis of TBLA based on microbiological results and/or response to anti-TB treatment was obtained for 15 patients. RESULTS: An IGRA profile highly suggestive of active TB (higher IFN-γ response to ESAT-6 compared to HBHA) was found for 3/6 TBLA patients from group 1, and for all the TBLA patients from groups 2 and 3, whereas this profile was not noticed in patients with a final alternative diagnosis. CONCLUSION: These results highlight the potential value of this combined HBHA/ESAT-6 IGRA as a triage test for the differential diagnosis of ADP.

HBHA-IGRA and cytotoxic mediators release assays for the diagnosis of cervical tuberculous lymphadenitis.

IMPORTANCE: Cervical tuberculous lymphadenitis (CTL), the most frequent extrapulmonary form of tuberculosis, is currently a major health problem in Tunisia and in several regions around the world. CTL diagnosis is challenging mainly due to the paucibacillary nature of the disease and the potential misdiagnosis as cervical non-tuberculous lymphadenitis. This study demonstrates the added value of the heparin-binding hemagglutinin-interferon-gamma release assay as an immunoassay in the context of CTL.

Both CD4⁺ and CD8⁺ lymphocytes participate in the IFN-γ response to filamentous hemagglutinin from Bordetella pertussis in infants, children, and adults.

Infant CD4⁺ T-cell responses to bacterial infections or vaccines have been extensively studied, whereas studies on CD8⁺ T-cell responses focused mainly on viral and intracellular parasite infections. Here we investigated CD8⁺ T-cell responses upon Bordetella pertussis infection in infants, children, and adults and pertussis vaccination in infants. Filamentous hemagglutinin-specific IFN-γ secretion by circulating lymphocytes was blocked by anti-MHC-I or -MHC-II antibodies, suggesting that CD4⁺ and CD8⁺ T lymphocytes are involved in IFN-γ production. Flow cytometry analyses confirmed that both cell types synthesized antigen-specific IFN-γ, although CD4⁺ lymphocytes were the major source of this cytokine. IFN-γ synthesis by CD8⁺ cells was CD4⁺ T cell dependent, as evidenced by selective depletion experiments. Furthermore, IFN-γ synthesis by CD4⁺ cells was sometimes inhibited by CD8⁺ lymphocytes, suggesting the presence of CD8⁺ regulatory T cells. The role of this dual IFN-γ secretion by CD4⁺ and CD8⁺ T lymphocytes in pertussis remains to be investigated.