Ontogeny of cytokine responses to PHA from birth to adulthood.

BACKGROUND: Altered production of cytokines is believed to contribute to early childhood susceptibility to infection. The aim of this study was to get further insight into the developmental patterns of cytokine responses from birth to adulthood. METHODS: The expression levels of 13 cytokines were compared in the supernatants of phytohemaggluttinin (PHA)-stimulated whole blood from healthy neonates (cord blood, n = 8), infants ( < 1-year-old, n = 20), and school-aged children (3-15 y; n = 20). Five adults were used as reference. RESULTS: While Th1, Th2, and Th17 cytokine levels increased progressively from birth to childhood (Mann-Whitney, p < 0.003), high IL-10 secretion at birth dropped to low adult levels in infants (p < 0.004) such that a negative correlation between IL-10 and Th1, Th2, and Th17 cytokine levels at birth (Spearman's correlation, r < -0.70, p < 0.01) converted to a positive correlation in infants (r > 0.60, p < 0.001). Finally, high IL-2, IL-7, and Granulocyte-Colony Stimulating factor (G-CSF) cytokine levels at birth decreased steadily over the first year of life (Mann-Whitney, p ≤ 0.001). CONCLUSION: The most noticeable result of the study is the rapid shift from enhanced IL-10 secretion capacity at birth toward balanced IL-10/Th1/Th2/Th17 cytokine levels early in life. This change appears an essential precondition to fight pathogens and at the same time to avoid overwhelming inflammatory reactions.

Altered vaccine-induced immunity in children with Dravet syndrome.

Dravet syndrome (DS) is a refractory epileptic syndrome. Vaccination is the trigger of the first seizure in about 50% of cases. Fever remains a trigger of seizures during the course of the disease. We compared ex vivo cytokine responses to a combined aluminium-adjuvanted vaccine of children with DS to sex- and age-matched heathy children. Using ex vivo cytokine responses of peripheral-blood mononuclear cells and monocytes, we found that vaccine responsiveness is biased toward a proinflammatory profile in DS with a M1 phenotype of monocytes. We provide new insight into immune mechanisms associated with DS that might guide research for the development of new immunotherapeutic agents in this epilepsy syndrome.

Performance of the QuantiFERON-TB Gold Assay Among HIV-infected Children With Active Tuberculosis in France.

BACKGROUND: Data regarding the use of QuantiFERON to assist the diagnosis of active tuberculosis (TB) in HIV-infected children are limited, especially in countries with low incidence of TB/HIV coinfection. METHODS: QuantiFERON results were analyzed in 63 HIV-infected children who presented to our hospital in Paris, France. Seventeen HIV-uninfected children with active TB (4 culture-confirmed) were included for comparison. RESULTS: The 63 HIV-infected children (median age: 11 yr) had 113 QuantiFERON tests. Thirty-four (54%) were born in sub-Saharan Africa. Vertical HIV transmission was documented for 50 of 52 (96%) and stage III HIV-infection for 30 of 50 children (60%). Over the study period, active TB was diagnosed in 7 of 63 HIV-infected children (3 culture-confirmed). Additional ongoing or previous opportunistic infections were present in 4 of 7. QuantiFERON results were positive in 2 of 7 HIV-infected children with active TB (sensitivity: 29%) and 16 of 17 HIV-uninfected children with active TB (sensitivity: 94%). At initial QuantiFERON testing of the 63 HIV-infected children, 8 (13%) had positive results (1, active TB; 5, latent TB; 2, previous TB) and 51 (81%) had negative results. Of 33 children with repeat testing after an initially positive or negative result, the only change was one conversion from a negative to a positive result at the onset of active TB. The 4 children (6%) with indeterminate quantiFERON results had a concomitant opportunistic infection. Results of repeat testing after clinical stabilization were negative in all 4. CONCLUSIONS: QuantiFERON testing performed poorly for active TB diagnosis in this series of children with advanced HIV infection.

Altered cytokine profiles in children with indeterminate quantiferon results and common infections.

OBJECTIVES: An increased rate of indeterminate quantiferon results (low IFN-γ release in the phytohemagglutinin-stimulated tube) has been reported in children with clinical signs compatible with tuberculosis but with the final diagnosis of infectious diseases different from tuberculosis. Here, we addressed the mechanisms involved and assessed potential alternative biomarkers to overcome indeterminate quantiferon results under these conditions. METHODS: Cytokine concentrations were measured in residual plasma from quantiferon assays performed in immunocompetent children (cases, median age: 3 years 9 months) with indeterminate results and community acquired pneumonia (n = 7) or meningoencephalitis (n = 1). Controls were age-matched immunocompetent children with determinate quantiferon results (infected with mycobacterium tuberculosis, n = 7 or not, n = 8). RESULTS: Lower IFN-γ expression in phytohemagglutinin-stimulated cultures from cases was accompanied by lower Th1 (IL-2, TNF-α, IP-10) and Th2 (IL-5, IL-13), but similar IL-10 secretion capacities as the controls. CONCLUSIONS: A state of hyporesponsiveness that resembles the concept of immunoparalysis in severe infection was observed in children with milder infections. Though IP-10, IL-2, IL-5 and IL-13 were confirmed as promising alternative biomarkers for discriminating controls with and without tuberculosis in this study, defective induction of these biomarkers by phytohemagglutinin in cases precluded their usefulness in overcoming quantiferon indeterminate results in the above-mentioned clinical conditions.

Relationship between cytomegalovirus (CMV) reactivation, CMV-driven immunity, overall immune recovery and graft-versus-leukaemia effect in children.

The interplay between immune recovery, cytomegalovirus (CMV)-reactivation, CMV-driven immunity and graft-versus-leukaemia effect (GVL) was analysed in 108 children (median age: 8 years) who underwent haematopoietic-stem cell transplantation (HSCT) for acute leukaemia. Follow-up was 2 years unless death or relapse occurred. CMV-polymerase chain reaction (PCR) was programmed weekly until month +3 post-HSCT. Immunomonitoring consisted of sequential lymphocyte subset enumerations and analyses of T-cell proliferative and γ-interferon responses to CMV and to adenovirus. In the 108 recipients, the 2-year relapse rate (RR) was 25% (median time to onset 4·5 months; range: 24 d-17 months). CMV reactivation occurrence was 31% (median time to onset 26 d). Donor/recipient CMV serostatus did not influence RR. Among the 89 recipients disease-free after day +120, i) early CMV-reactivation before day +30 was more frequent (P = 0·01) in the relapse recipient group opposed to the non-relapse group. ii) CD8(+) /CD28(-) and CD4(+) CD45RA(-) T-cell expansions induced by CMV did not influence RR, iii) Recovery of anti-CMV and also anti-adenovirus immunity and of naïve CD4(+) T-cells was faster in the non-relapse group (P = 0·008; 0·009 and 0·002 respectively). In contrast to adult acute myeloid leukaemia, CMV reactivation was associated with increased RR in this paediatric series. Accelerated overall immune recovery rather than CMV-driven immunity had a favourable impact on RR.

Cytokine responses to quantiferon peptides in pediatric tuberculosis: a pilot study.

OBJECTIVES: Detailed understanding of tuberculosis (TB) immunopathology and cytokine/chemokine responses can ultimately lead to the development of new diagnostic tools, especially useful in children where TB diagnosis remains challenging. METHODS: Nineteen cytokine/chemokine responses to Mycobacterium tuberculosis (M.tb) antigens were analyzed in 47 children distributed as follow: 28 with TB-disease (TD), 12 with latent TB and 7 uninfected controls. All the cytokines and chemokines were quantified in a multiplexed microsphere-based assay by using residual plasma from the quantiFERON kit (IFNγ release assay). RESULTS: IP-10, IL-2, IL-5 and IL-13 were among the best cytokines to diagnose infection as related by the area under ROC curve for IP-10 (0.96, 95%CI: 0.91-1.00), IL-2 (0.98, 95%CI: 0.93-1.02), IL-5 (0.91, 95%CI: 0.81-1.01) and IL-13 (0.97, 95%CI: 0.93-1.00). None of the 5 biomarkers, however, discriminated TB-disease from latent-TB. Finally, lower IL-5 (p = 0.02) and IL-13 (p = 0.02) levels were observed in severe opposed to non-severe TB. CONCLUSION: These results suggest that IP-10, IL-2, IL-5 and IL-13 may find a diagnostic application in pediatric tuberculosis and argue against the paradigm of a negative influence of Th2 responses in severe pediatric M.tb infection.

QuantiFERON to diagnose infection by Mycobacterium tuberculosis: performance in infants and older children.

OBJECTIVES: QuantiFERON value to diagnose tuberculosis (TB) in young children remains to be clarified. To this aim QF-TB-IT performance was evaluated in a large series of immunocompetent children that were stratified according to age and clinical conditions. METHODS: QF-TB-IT reactivity was analyzed in 226 immunocompetent children (0-15 years old): 31 were uninfected despite TB contact; 51 presented TB disease; 39 had Latent TB (LTBI) and 105 had TB disease suspected but an alternative diagnosis (TB excluded). RESULTS: QF-TB-IT specificity was 100% in TB excluded. In TB disease, low sensitivity of QF-TB-IT in infants (40%) increased with aging (77% in 1-<5 years and 82% in 5-<15 years old subgroups). In LTBI, agreement between TST and QF-TB-IT was 0% in infants, 40% in 1-<5 years and 57% in children >5 years old. Finally, the incidence of indeterminate results was high (24%) in children <5 years old with TB excluded, especially with non-TB pneumonitis (61%), but was low (0-6%) regardless of age group in TB disease, LTBI and uninfected contact cases. CONCLUSIONS: In our low burden country, i) QF-TB-IT specificity was 100%, ii) QF-TB-IT sensitivity was low in infants but commensurable to adult values in older children, and iii) indeterminate results mostly relied on ongoing infections unrelated to TB.

Cis-silencing of PIP5K1B evidenced in Friedreich's ataxia patient cells results in cytoskeleton anomalies.

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease characterized by ataxia, variously associating heart disease, diabetes mellitus and/or glucose intolerance. It results from intronic expansion of GAA triplet repeats at the FXN locus. Homozygous expansions cause silencing of the FXN gene and subsequent decreased expression of the encoded mitochondrial frataxin. Detailed analyses in fibroblasts and neuronal tissues from FRDA patients have revealed profound cytoskeleton anomalies. So far, however, the molecular mechanism underlying these cytoskeleton defects remains unknown. We show here that gene silencing spreads in cis over the PIP5K1B gene in cells from FRDA patients (circulating lymphocytes and primary fibroblasts), correlating with expanded GAA repeat size. PIP5K1B encodes phosphatidylinositol 4-phosphate 5-kinase ß type I (pip5k1ß), an enzyme functionally linked to actin cytoskeleton dynamics that phosphorylates phosphatidylinositol 4-phosphate [PI(4)P] to generate phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. Accordingly, loss of pip5k1ß function in FRDA cells was accompanied by decreased PI(4,5)P2 levels and was shown instrumental for destabilization of the actin network and delayed cell spreading. Knockdown of PIP5K1B in control fibroblasts using shRNA reproduced abnormal actin cytoskeleton remodeling, whereas over-expression of PIP5K1B, but not FXN, suppressed this phenotype in FRDA cells. In addition to provide new insights into the consequences of the FXN gene expansion, these findings raise the question whether PIP5K1B silencing may contribute to the variable manifestation of this complex disease.

Cellular and humoral immunity elicited by influenza vaccines in pediatric hematopoietic-stem cell transplantation.

Immunity induced by influenza vaccines following hematopoietic stem-cell transplantation (HSCT) is poorly understood. Here, 14 pediatric recipients (mean age: 6 years) received H1N1 (n=9) or H1N1/H3N2 (n=5) vaccines at a median of 5.7 months post-HSCT (HLA-identical related bone-marrow graft: 10/14). Fourteen clinically-matched non-vaccinated recipients were included as controls. Cellular response to vaccination was assessed by a T-cell proliferation assay. Humoral response was assessed by H1N1-specific antibody titration. IL2 and IFNγ responses to influenza were also evaluated by an intracellular cytokine accumulation method for some of the recipients. Higher proliferative responses to H1N1 (p=0.0001) and higher H1N1-specific antibody titers (p<0.02) were observed in vaccines opposed to non-vaccinated recipients. In some cases, proliferative responses to H1N1 developed while at the same time antibody titers did not reach protective (≥1:40) levels. Most recipients vaccinated with only the H1N1 strain had proliferative responses to both H1N1 and H3N2 (median stimulation index H1N1: 96, H3N2: 126 in responders). Finally, IL2 responses predominated over IFNγ responses (p<0.02) to influenza viruses in responders. In conclusion, H1N1 vaccination induced substantial cell-mediated immunity, and to a lesser extent, humoral immunity at early times post-HSCT. H1N1/H3N2 T-cell cross-reactivity and protective (IL2) rather than effector (IFNγ) cytokinic profiles were elicited.

TNF-α/IL-2 ratio discriminates latent from active tuberculosis in immunocompetent children: a pilot study.

BACKGROUND: Distinguishing latent tuberculosis (LTB) from tuberculosis (TB) disease may be challenging in children. Here, we analyzed cytokine profiles that can distinguish the two infection stages in a nonendemic country (France). METHODS: Immunocompetent children with LTB (n = 6) or TB disease (n = 8) (median age: 6.2 and 5.7 years, respectively) were analyzed. Four young uninfected children were included as controls. A Luminex assay evaluated cytokine responses to Mycobacterium tuberculosis antigens. RESULTS: Poor interleukin-4 (IL-4) and IL-10 responses precluded analysis of these cytokines. Interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), IL-2, and T-helper type 1 (Th1) cytokines and IL-5, IL-13, T-helper type 2 (Th2) cytokines were simultaneously induced by antigens in 14/14 infected but 0/4 uninfected children. Th1 cytokine levels were similar in LTB and TB disease: IFN-γ: 12,254 and 10,495 pg/ml; IL-2: 2,097 and 1,869 pg/ml; and TNF-α: 1,020 and 2,875 pg/ml, respectively. Th2 cytokine levels were similar and even higher in LTB than in TB disease: IL-5: 23 and 10 pg/ml; IL-13: 284 and 109 pg/ml, respectively. Positive correlation of cytokine levels, whether Th1 or Th2, was observed. Higher (P = 0.008) TNF-α/IL-2 ratios distinguished 6/8 active TB disease cases from 6/6 LTB cases. CONCLUSION: TNF-α/IL-2 ratio may discriminate TB disease from LTB in immunocompetent children. Larger studies in TB endemic settings must verify these results.

A long-term competent chimeric immune system in a dizygotic dichorionic twin.

We present here a rare case that involved the long-term coexistence of 2 mature, functional, and equilibrated immune systems in a single child after fetofetal transfusion between dizygotic twins. A dichorionic diamniotic pregnancy complicated by twin anemia-polycythemia sequence resulted in the demise of 1 twin. The detection of abnormal vessels on the dichorionic plate strongly suggested the existence of functional vascular anastomoses leading to blood chimerism in the survivor. Genetic, phenotypic, and immunologic analyses at 2 years revealed chimeric lymphoid and myeloid cells in the surviving twin, although no tissue mosaicism was detected, which indicates that early transfusion led to mutual immune tolerance.

Interferon-gamma release assay performance for diagnosing tuberculosis disease in 0- to 5-year-old children.

QuantiFERON-TB Gold In-Tube performance was evaluated in 19 French immunocompetent children (0.29-5.36 years; median: 1.52) with active tuberculosis. The rate of indeterminates results was 0/19 and the rates of positivity were 6/10 and 9/9 in <2 and 2- to 5-year-old children, respectively. QuantiFERON-TB Gold In-Tube in association with tuberculin skin test could improve diagnosis of tuberculosis even in young children.

Contribution of HLA-A/B/C/DRB1/DQB1 common haplotypes to donor search outcome in unrelated hematopoietic stem cell transplantation.

In unrelated hematopoietic stem cell transplantation (HSCT), the prediction of donor search outcome at the time of search initiation is of great value for the physicians to delineate the strategy of patient care. The probability of finding an unrelated donor is high for patients who carry at least 1 of the 10 most common HLA haplotypes in Caucasians. As only 10% to 20% patients respond to this criterion, here we aimed at finding additional common haplotypes to improve the prediction of a successful search. HLA broad HLA-A/B/DRB1 haplotypes that were observed with frequencies ≥0.19% in patient families of European origin and that split into ≤2 predominant 4-digit HLA-A/B/C/DRB1/DQB1 haplotypes were considered as common. Carriage of at least 1 of those in 168 patients of various geographic areas with no family donor was confronted to the chance of finding ≥9/10 HLA-matched unrelated donors. Fifty common 4-digit haplotypes were identified. A higher (P < 5 × 10(-6)) chance of finding a suitable donor was found for 55 of 170 (32%) recipients that carried at least 1 of these common haplotypes. Up to now, estimates classified patients into ≥3 groups of probability with ≥1 intermediate group of poor utility for the clinicians. Considering carriage of these common haplotypes together with the frequencies of alleles and of B/C and DRB1/DQB1 associations, which are carried by patient HLA haplotypes, we could classify the patients into 2 groups of probability with a 98% and 26% chance of finding a donor, respectively. Prediction of search outcome could be improved by including the 50 most common HLA haplotypes in the current approaches.

Outcome of children treated with haematopoietic-stem cell transplantations from donors expressing the rare C77G variant of the PTPRC (CD45) gene.

The uncommon C77G polymorphism of the Protein-Tyrosine Phosphatase (PTPRC) gene (PTPRC; previously termed CD45) could confer an increased risk of immunopathology. This study compared the outcome of children following human leucocyte antigen-matched unrelated haematopoïetic-stem cell transplantations (HSCT) from donors carrying (C77G cases: n = 8) or not (controls: n = 36) the PTPRC C77G polymorphism. Transmission of the PTPRC C77G polymorphism through the graft was suggested by unusual CD45RA phenotype in the donors and/or in the recipients after, but not before HSCT. Restriction-Fragment Length Polymorphism and sequencing confirmed the polymorphism. Overall survival rates were similar in C77G cases and controls (63% vs. 61%). Acute leukaemia relapse tended to be less frequent in C77G cases (0% vs. 32%; P = 0·09). Among recipients surviving ≥ 30 d, acute GVHD (aGVHD) ≥ grade 2 tended to be more frequent (100% vs. 58%; P = 0·07) and the rate of steroid-refractory or -dependant aGVHD higher (67% vs. 28%) in C77G cases. Finally, extensive chronic GVHD tended to occur more frequently (40% vs. 9%) in C77G cases. Recovery of lymphocyte subsets and virus-specific CD4 was similar in C77G cases and controls while interleukin 2 (IL2)-responses through CD3 stimulation were higher in C77G cases (P = 0·004). In conclusion, HSCT from PTPRC C77G donors could increase GVHD risk without compromising overall survival. Altered IL2-responses could be involved in this process.

Quantitative and qualitative CD4 T cell immune responses related to adenovirus DNAemia in hematopoietic stem cell transplantation.

The nature of adenovirus (AdV)-specific T cells that could best predict the capacity of immunocompromised host to fight AdV is unclear. To this aim, 47 pediatric patients were enrolled for at least 3 months either at allogeneic bone marrow transplantation (BMT) (23 genoidentical, 18 unrelated of which 9 were 10/10 and 9 were 9/10 HLA-matched) or at unrelated cord blood transplantation (n = 6). Enumeration of AdV-specific CD4 T cells secreting cytokines (flow cytometry) and proliferative responses to AdV ((3)HT-incorporation) were compared to AdV-DNAemia. A total of 44/47 patients did not evidence AdV-DNAemia. Thirty-two of 44 (73%) developed CD4-mediated interferon-gamma (IFN-γ) responses to AdV (median 0.36 CD4/µL of blood) since the first month post-HSCT (n = 11: 8 genoidentical and 3 unrelated) or the third month (n = 21 additional patients). At 3 months, both incidence and level intensities of AdV-specific CD4 appeared similar in genoidentical and unrelated BMT (70% and 80%; 0.36 and 0.21 CD4/µL, respectively) and not statistically different from age-matched controls (76%; 1.35 CD4/µL), whereas cord blood transplanted patients exhibited similar incidence but higher level intensities (67%; 1.49 CD4/µL). Polyfunctional (IL2 + IFN-γ) and proliferative responses appeared later, after the third month. Three of 4 9/10 HLA-matched unrelated HSCT that did not develop immunity to AdV presented chemotherapy-resistant AdV-DNAemia at 3 to 5 months post-hematopoietic stem cell transplantation (HSCT). Two were successfully treated with AdV-specific CTL infusion. Monitoring, since month 1 post-HSCT, of IFN-γ-secreting AdV-specific CD4 appears suitable for early detection of at-risk patients especially in 9/10 HLA-matched unrelated HSCT and preferable to monitoring of more delayed IL2- and proliferative responses.

Development of cytomegalovirus and adenovirus-specific memory CD4 T-cell functions from birth to adulthood.

Age-related changes in memory CD4 T cells (CD4) are poorly known. To address this issue, CD4 proliferative and cytokine responses to an anti-CD3 monoclonal (CD3), to cytomegalovirus (CMV), and to adenovirus (AdV) were assessed in 57 children (age, 0.07-17.16 y) and 17 adults. Results showed i) accumulation of memory CD4 with aging, with 2-3 times more central-memory T cell (TCM; CD45RA/CD62L) than effector-memory T cell (TEM; CD45RA/62L) CD4 at any age. ii) In children older than 2 y, CMV-specific CD4-secreting IFNγ alone predominated over CD4-secreting IL2 + IFNγ and a continuous increase, with aging, in IFNγ responses to the virus was observed. In contrast, in AdV infection, CD4-secreting IL2 + IFNγ predominated and IFNγ responses to the virus reached adult levels from 3 y of age. iii) In children aged 0-2 y, lower total IFNγ responses to CMV (p < 0.02), AdV (p = 0.05), and CD3 (p < 0.01) and lower IFNγ + IL2-responses (p = 0.1, p < 0.02, p < 0.05, respectively) contrasted with no decrease in CD4-secreting IFNγ alone. Defective proliferative responses to AdV (p = 0.03) were also observed. In conclusion, the development of memory CD4 differed in acute AdV and persistent CMV infections. Young age seemed to depress mostly polyfunctional (IL2 + IFNγ secreting) CD4 in both infections.

Nod2 regulates the host response towards microflora by modulating T cell function and epithelial permeability in mouse Peyer's patches.

Nucleotide oligomerisation domain 2 (NOD2) mutations are associated with susceptibility to Crohn's disease and graft-versus-host disease, two human disorders related with dysfunctions of Peyer's patches (PPs). In Nod2(-/-) mice transcellular permeability and bacterial translocation are increased in PPs. In this study, we show that both anti-CD4(+) and anti-interferon gamma (anti-IFNgamma) monoclonal antibodies abrogate this phenotype and reduce the expression of tumour necrosis factor (TNF) receptor 2 and the long isoform of myosin light chain kinase, thus demonstrating that immune T cells influence the epithelial functions. In turn, intraperitoneal injection of ML-7 (a myosin light chain kinase inhibitor) normalises the values of CD4(+) T cells, IFNgamma and TNFalpha. This reciprocal cross-talk is under the control of the gut microflora as shown by the normalisation of all parameters after antibiotic treatment. Toll-like receptor 2 (TLR2) and TLR4 expression were increased in Nod2(-/-) mice under basal conditions and TLR2 and TLR4 agonists induced an increased transcellular permeability in Nod2(+/+) mice. Muramyldipeptide (a Nod2 agonist) or ML-7 was able to reverse this phenomenon. It thus appears that Nod2 modulates the cross-talk between CD4(+) T cells and the epithelium recovering PP and that it downregulates the pro-inflammatory effect driven by the ileal microflora, likely by inhibiting the TLR pathways.