The Mycobacterium tuberculosis Phagosome Is a HLA-I Processing Competent Organelle

Mycobacterium tuberculosis (Mtb) resides in a long-lived phagosomal compartment that resists maturation. The manner by which Mtb antigens are processed and presented on MHC Class I molecules is poorly understood. Using human dendritic cells and IFN-γ release by CD8+ T cell clones, we examined the processing and presentation pathway for two Mtb–derived antigens, each presented by a distinct HLA-I allele (HLA-Ia versus HLA-Ib). Presentation of both antigens is blocked by the retrotranslocation inhibitor exotoxin A. Inhibitor studies demonstrate that, after reaching the cytosol, both antigens require proteasomal degradation and TAP transport, but differ in the requirement for ER–golgi egress and new protein synthesis. Specifically, presentation by HLA-B8 but not HLA-E requires newly synthesized HLA-I and transport through the ER–golgi. Phenotypic analysis of the Mtb phagosome by flow organellometry revealed the presence of Class I and loading accessory molecules, including TAP and PDI. Furthermore, loaded HLA-I:peptide complexes are present within the Mtb phagosome, with a pronounced bias towards HLA-E:peptide complexes. In addition, protein analysis also reveals that HLA-E is enriched within the Mtb phagosome compared to HLA-A2. Together, these data suggest that the phagosome, through acquisition of ER–localized machinery and as a site of HLA-I loading, plays a vital role in the presentation of Mtb–derived antigens, similar to that described for presentation of latex bead-associated antigens. This is, to our knowledge, the first description of this presentation pathway for an intracellular pathogen. Moreover, these data suggest that HLA-E may play a unique role in the presentation of phagosomal antigens

Human Neonatal Dendritic Cells Are Competent in MHC Class I Antigen Processing and Presentation

Neonates are clearly more susceptible to severe disease following infection with a variety of pathogens than are adults. However, the causes for this are unclear and are often attributed to immunological immaturity. While several aspects of immunity differ between adults and neonates, the capacity of dendritic cells in neonates to process and present antigen to CD8+ T cells remains to be addressed. We used human CD8+ T cell clones to compare the ability of neonatal and adult monocyte-derived dendritic cells to present or process and present antigen using the MHC class I pathway. Specifically, we assessed the ability of dendritic cells to present antigenic peptide, present an HLA-E–restricted antigen, process and present an MHC class I-restricted antigen through the classical MHC class I pathway, and cross present cell-associated antigen via MHC class I. We found no defect in neonatal dendritic cells to perform any of these processing and presentation functions and conclude that the MHC class I antigen processing and presentation pathway is functional in neonatal dendritic cells and hence may not account for the diminished control of pathogens

Negative Effect of Smoking on the Performance of the QuantiFERON TB Gold in Tube Test.

False negative and indeterminate Interferon Gamma Release Assay (IGRA) results are a well documented problem. Cigarette smoking is known to increase the risk of tuberculosis (TB) and to impair Interferon-gamma (IFN-γ) responses to antigenic challenge, but the impact of smoking on IGRA performance is not known. The aim of this study was to evaluate the effect of smoking on IGRA performance in TB patients in a low and high TB prevalence setting respectively. Patients with confirmed TB from Denmark (DK, n = 34; 20 smokers) and Tanzania (TZ, n = 172; 23 smokers) were tested with the QuantiFERON-TB Gold In tube (QFT). Median IFN-γ level in smokers and non smokers were compared and smoking was analysed as a risk factor for false negative and indeterminate QFT results. Smokers from both DK and TZ had lower IFN-γ antigen responses (median 0.9 vs. 4.2 IU/ml, p = 0.04 and 0.4 vs. 1.6, p < 0.01), less positive (50 vs. 86%, p = 0.03 and 48 vs. 75%, p < 0.01) and more false negative (45 vs. 0%, p < 0.01 and 26 vs. 11%, p = 0.04) QFT results. In Tanzanian patients, logistic regression analysis adjusted for sex, age, HIV and alcohol consumption showed an association of smoking with false negative (OR 17.1, CI: 3.0-99.1, p < 0.01) and indeterminate QFT results (OR 5.1, CI: 1.2-21.3, p = 0.02). Cigarette smoking was associated with false negative and indeterminate IGRA results in both a high and a low TB endemic setting independent of HIV status

QuantiFERON®-TB gold in-tube performance for diagnosing active tuberculosis in children and adults in a high burden setting.

To determine whether QuantiFERON®-TB Gold In-Tube (QFT) can contribute to the diagnosis of active tuberculosis (TB) in children in a high-burden setting and to assess the performance of QFT and tuberculin skin test (TST) in a prospective cohort of TB suspect children compared to adults with confirmed TB in Tanzania. Sensitivity and specificity of QFT and TST for diagnosing active TB as well as indeterminate QFT rates and IFN-γ levels were assessed in 211 TB suspect children in a Tanzanian district hospital and contrasted in 90 adults with confirmed pulmonary TB. Sensitivity of QFT and TST in children with confirmed TB was 19% (5/27) and 6% (2/31) respectively. In adults sensitivity of QFT and TST was 84% (73/87) and 85% (63/74). The QFT indeterminate rate in children and adults was 27% and 3%. Median levels of IFN-γ were lower in children than adults, particularly children <2 years and HIV infected. An indeterminate result was associated with age <2 years but not malnutrition or HIV status. Overall childhood mortality was 19% and associated with an indeterminate QFT result at baseline. QFT and TST showed poor performance and a surprisingly low sensitivity in children. In contrast the performance in Tanzanian adults was good and comparable to performance in high-income countries. Indeterminate results in children were associated with young age and increased mortality. Neither test can be recommended for diagnosing active TB in children with immature or impaired immunity in a high-burden setting

Whole Blood Interferon-Gamma Responses to Mycobacterium tuberculosis Antigens in Young Household Contacts of Persons with Tuberculosis in Uganda

Due to immunologic immaturity, IFN-gamma-producing T cell responses may be decreased in young children compared to adults, thus we hypothesized that IFN-gamma responses to mycobacterial antigens in household contacts exposed to Mycobacterium tuberculosis (Mtb) would be impaired in young children relative to adults. The objective of this study was to compare whole blood IFN-gamma production in response to mycobacterial antigens between children and adults in Uganda.We studied household contacts of persons with culture-positive pulmonary tuberculosis (TB) enrolled in a cohort study conducted in Kampala, Uganda. Whole blood IFN-gamma production in response to Mtb culture-filtrate antigens was measured by ELISA and compared between infants (<2 years old, n = 80), young children (2 <5 years old, n = 216), older children (5 <15 years old, n = 443) and adults (> or =15 years old, n = 528). We evaluated the relationship between IFN-gamma responses and the tuberculin skin test (TST), and between IFN-gamma responses and epidemiologic factors that reflect exposure to Mtb, and the effect of prior BCG vaccination on IFN-gamma responses. Young household contacts demonstrated robust IFN-gamma responses comparable to those of adults that were associated with TST and known risk factors for infection. There was no effect of prior BCG immunization on the IFN-gamma response.Young children in a TB endemic setting can mount robust IFN-gamma responses generally comparable to those of adults, and as in adults, these responses correlated with the TST and known epidemiologic risk factors for Mtb infection

Interpretation of ambiguous situations: evidence for a dissociation between social and physical threat in Williams syndrome

There is increasing evidence that Williams syndrome (WS) is associated with elevated anxiety that is non-social in nature, including generalised anxiety and fears. To date very little research has examined the cognitive processes associated with this anxiety. In the present research, attentional bias for non-social threatening images in WS was examined using a dot-probe paradigm. Participants were 16 individuals with WS aged between 13 and 34 years and two groups of typically developing controls matched to the WS group on chronological age and attentional control ability respectively. The WS group exhibited a significant attention bias towards threatening images. In contrast, no bias was found for group matched on attentional control and a slight bias away from threat was found in the chronological age matched group. The results are contrasted with recent findings suggesting that individuals with WS do not show an attention bias for threatening faces and discussed in relation to neuroimaging research showing elevated amygdala activation in response to threatening non-social scenes in WS

Human and murine clonal CD8+ T cell expansions arise during tuberculosis because of TCR selection

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRß bias. Using a retro genic model of TB10.44-11-specific CD8+ Tcells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-? production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity.This work was supported by the Portuguese Foundation for Science and Technology individual fellowship (CNA) www.fct.pt, a National Institutes of Health Grant R01 AI106725 (SMB) www.nih.gov, and a Center for AIDS Research Grant P30 AI 060354 (SMB) www.nih.gov. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript