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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.

Active tuberculosis (aTB) remains a major killer from infectious disease, partially due to delayed diagnosis and hence treatment. Classical microbiological methods are slow and lack sensitivity, molecular techniques are costly and often unavailable. Moreover, available immuno-diagnostic tests lack sensitivity and do not differentiate between aTB and latent TB infection (LTBI). Here, we evaluated the performance of the combined measurement of different chemokines/cytokines induced by two different stage-specific mycobacterial antigens, Early-secreted-antigenic target-6 (ESAT-6) and Heparin-binding-haemagglutinin (HBHA), after a short in vitro incubation of either peripheral blood mononuclear cells (PBMC) or whole blood (WB). Blood samples were collected from a training cohort comprising 22 aTB patients, 22 LTBI subjects and 17 non-infected controls. The concentrations of 13 cytokines were measured in the supernatants. Random forest analysis identified the best markers to differentiate M. tuberculosis-infected from non-infected subjects, and the most appropriate markers to differentiate aTB from LTBI. Logistic regression defined predictive abilities of selected combinations of cytokines, first on the training and then on a validation cohort (17 aTB, 27 LTBI, 25 controls). Combining HBHA- and ESAT-6-induced IFN-γ concentrations produced by PBMC was optimal to differentiate infected from non-infected individuals in the training cohort (100% correct classification), but 2/16 (13%) patients with aTB were misclassified in the validation cohort. ESAT-6-induced-IP-10 combined with HBHA-induced-IFN-γ concentrations was selected to differentiate aTB from LTBI, and correctly classified 82%/77% of infected subjects as aTB or LTBI in the training/validation cohorts, respectively. Results obtained on WB also selected ESAT-6- and HBHA-induced IFN-γ concentrations to provided discrimination between infected and non-infected subjects (89%/90% correct classification in the training/validation cohorts). Further identification of aTB patients among infected subjects was best achieved by combining ESAT-6-induced IP-10 with HBHA-induced IL-2 and GM-CSF. Among infected subjects, 90%/93% of the aTB patients were correctly identified in the training/validation cohorts. We therefore propose a two steps strategy performed on 1 mL WB for a rapid identification of patients with aTB. After elimination of most non-infected subjects by combining ESAT-6 and HBHA-induced IFN-γ, the combination of IP-10, IL-2 and GM-CSF released by either ESAT-6 or HBHA correctly identifies most patients with aTB.

The new pandemic virus SARS-CoV-2 is characterized by uncontrolled hyper-inflammation in severe cases. As the IL-22/IL-22R1 axis was reported to be involved in inflammation during viral infections, we characterized the expression of IL-22 receptor1, IL-22 and IL-22 binding protein in COVID-19 patients. Blood samples were collected from 19 non-severe and 14 severe patients on the day they presented (D0), at D14, and six months later, and from 6 non-infected controls. The IL-22R1 expression was characterized by flow cytometry. Results were related to HLA-DR expression of myeloid cells, to plasma concentrations of different cytokines and chemokines and NK cells and T lymphocytes functions characterized by their IFN-γ, IL-22, IL-17A, granzyme B and perforin content. The numbers of IL-22R1+ classical, intermediate, and non-classical monocytes and the proportions of IL-22R1+ plasmacytoid DC (pDC), myeloid DC1 and DC2 (mDC1, mDC2) were higher in patients than controls at D0. The proportions of IL-22R1+ classical and intermediate monocytes, and pDC and mDC2 remained high for six months. High proportions of IL-22R1+ non-classical monocytes and mDC2 displayed HLA-DRhigh expression and were thus activated. Multivariate analysis for all IL-22R1+ myeloid cells discriminated the severity of the disease (AUC=0.9023). However, correlation analysis between IL-22R1+ cell subsets and plasma chemokine concentrations suggested pro-inflammatory effects of some subsets and protective effects of others. The numbers of IL-22R1+ classical monocytes and pDC were positively correlated with pro-inflammatory chemokines MCP-1 and IP-10 in severe infections, whereas IL-22R1+ intermediate monocytes were negatively correlated with IL-6, IFN-α and CRP in non-severe infections. Moreover, in the absence of in vitro stimulation, NK and CD4+ T cells produced IFN-γ and IL-22, and CD4+ and CD8+ T cells produced IL-17A. CD4+ T lymphocytes also expressed IL-22R1, the density of its expression defining two different functional subsets. In conclusion, we provide the first evidence that SARS-CoV-2 infection is characterized by an abnormal expression of IL22R1 on blood myeloid cells and CD4+ T lymphocytes. Our results suggest that the involvement of the IL-22R1/IL-22 axis could be protective at the beginning of SARS-CoV-2 infection but could shift to a detrimental response over time.

BackgroundSurfactant protein D (SP-D) and pulmonary club cell protein 16 (CC-16) are called “pneumoproteins” and are involved in host defense against oxidative stress, inflammation, and viral outbreak. This study aimed to determine the predictive value of these pneumoproteins on the incidence of acute respiratory distress syndrome (ARDS) or death in patients with coronavirus disease-2019 (COVID-19).MethodsThis retrospective study included 87 patients admitted to an emergency department. Blood samples were collected on three time points (days 1, 5, and 14 from hospital admission). SP-D and CC-16 serum levels were determined, and univariate and multivariate analyses considering confounding variables (age, body mass index, tobacco use, dyspnea, hypertension, diabetes mellitus, neutrophil-to-lymphocyte ratio) were performed.ResultsBased on the multivariate analysis, SP-D level on D1 was positively and slightly correlated with subsequent development of ARDS, independent of body mass index, dyspnea, and diabetes mellitus. CC-16 level on D1 was modestly and positively correlated with fatal outcome. A rise in SP-D between D1 and D5 and D1 and D14 had a strong negative association with incidence of ARDS. These associations were independent of tobacco use and neutrophil-to-lymphocyte ratio.ConclusionsOverall, our data reveal that increase in SP-D levels is a good prognostic factor for patients with COVID-19, and that initial CC-16 levels correlated with slightly higher risk of death. SP-D and CC-16 may prove useful to predict outcomes in patients with COVID-19.

Psoriasis is a skin inflammatory condition for which significant progress has been made in its management by the use of targeted biological drugs. Detection of latent M. tuberculosis infection (LTBI) is mandatory before starting biotherapy that is associated with reactivation risk. Together with evaluation of TB risk factors and chest radiographs, tuberculin skin tests (TST) and/or blood interferon-γ-release assays (IGRA), like the QuantiFERON (QFT), are usually performed to diagnose M. tuberculosis infection. Using this approach, 14/49 psoriatic patients prospectively included in this study were identified as LTBI (14 TST+, induration size ≥ 10mm, 8 QFT+), and 7/14 received prophylactic anti-TB treatment, the other 7 reporting past-treatment. As the specificity and sensitivity of these tests were challenged, we evaluated the added value of an IGRA in response to a mycobacterial antigen associated with latency, the heparin-binding haemagglutinin (HBHA). All but one TST+ patient had a positive HBHA-IGRA, indicating higher sensitivity than the QFT. The HBHA-IGRA was also positive for 12/35 TST-QFT- patients. Measurement for 15 psoriatic patients (12 with HBHA-IGRA+) of 8 chemokines in addition to IFN-γ revealed a broad array of HBHA-induced chemokines for TST+QFT- and TST-QFT- patients, compared to a more restricted pattern for TST+QFT+ patients. This allowed us to define subgroups within psoriatic patients characterized by different immune responses to M. tuberculosis antigens that may be associated to different risk levels of reactivation of the infection. This approach may help in prioritizing patients who should receive prophylactic anti-TB treatment before starting biotherapies in order to reduce their number.

Background: Improved diagnostic tests are needed for the early identification of Mycobacterium tuberculosis-infected young children exposed to an active TB (aTB) index case. We aimed to compare the diagnostic accuracy of new blood-based tests to that of the tuberculin skin test (TST) for the identification of all infected children and for a potential differentiation between aTB and latent TB infection (LTBI).Methods: 144 children exposed to a patient with aTB were included, and those who met all inclusion criteria (130/144) were classified in three groups based on results from classical investigations: non-infected (NI: n = 69, 53%, median age 10 months), LTBI (n = 28, 22%, median age 96 months), aTB disease (n = 33, 25%, median age 24 months). The first whole blood assay consisted of a 7-days in vitro stimulation of blood with four different mycobacterial antigens (40 μl/condition), followed by flow cytometric measurement of the proportions of blast cells appearing among lymphocytes as a result of their specific activation. Thresholds of positivity were determined by Receiver Operating Characteristic (ROC) curve analysis (results of NI children vs. children with LTBI/aTB) in order to identify infected children in a first stage. Other cut-offs were determined to discriminate subgroups of infected children in a second step (results from children with aTB/LTBI). Analysis of blood monocytes and dendritic cell subsets was performed on 100 μl of blood for 25 of these children as a second test in a pilot study.Results: Combining the results of the blast-induced CD3+ T lymphocytes by Heparin-Binding Haemagglutinin and by Culture Filtrate Protein-10 identified all but one infected children (sensitivity 98.2% and specificity 86.9%, compared to 93.4 and 100% for the TST). Further identification among infected children of those with aTB was best achieved by the results of blast-induced CD8+ T lymphocytes by purified protein derivative (sensitivity for localized aTB: 61.9%, specificity 96.3%), whereas high proportions of blood type 2 myeloid dendritic cells (mDC) were a hallmark of LTBI.Conclusions: New blood-based tests requiring a very small volume allow the accurate identification of M. tuberculosis-infected young children among exposed children and are promising to guide the clinical classification of children with aTB or LTBI.

BackgroundPeritoneal tuberculosis (TB) remains difficult to diagnose because of its non-specific clinical features and the lack of efficient microbiological tests. As delayed diagnosis is associated with high mortality rates, new diagnostic tools are needed.Methods and findingsWe investigated for 24 patients prospectively enrolled with a possible diagnosis of peritoneal TB, the diagnostic value of the analysis of IFN-γ production by peritoneal fluid lymphocytes in response to a short in vitro stimulation with mycobacterial antigens. The patients were classified in two groups: non-TB and confirmed or highly probable TB. Diagnosis of TB was based on microbiological and histopathological criteria and/or a favorable response to anti-TB treatment. The IFN-γ production by peritoneal CD4+ T lymphocytes was analyzed by flow cytometry after an overnight in vitro stimulation with three different mycobacterial antigens, purified protein derivative (PPD), heparin-binding haemagglutinin (HBHA) or early-secreted-antigen-target-6 (ESAT-6). The percentages of PPD-, HBHA- or ESAT-6-induced IFN-γ-producing peritoneal fluid CD4+ T lymphocytes were higher in the TB group than in the non-TB group (p = 0.0007, p = 0.0004, and p = 0.0002 respectively). Based on cut-off values determined by ROC curve analysis of the results from TB and highly probable TB compared to those of non-TB patients, the sensitivity of these three tests was 100% with a specificity of 92%.ConclusionsThe analysis of mycobacterial-induced IFN-γ production by peritoneal lymphocytes is a promising tool to reliably and rapidly diagnose peritoneal TB. Further studies should be performed on larger cohorts of patients in high-TB-incidence countries to confirm the clinical value of this new diagnostic approach for peritoneal TB.