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

Interleukin-12 Therapy Reduces the Number of Immune Cells and Pathology in Lungs of Mice Infected with Mycobacterium tuberculosis

Alternate modalities for the treatment of Mycobacterium tuberculosis are needed due to the rise in numbers of immunosuppressed individuals at risk for serious disease and the increasing prevalence of multidrug-resistant isolates. Interleukin-12 (IL-12) has been shown to improve immune responses against M. tuberculosis infection in both humans and mice. Previous studies using high-dose IL-12 in various disease models reported a paradoxical immunosuppression. We demonstrate here that exogenous administration of IL-12 for 8 weeks after an aerosolized low dose of M. tuberculosis results in increased survival and decreased pulmonary bacterial loads for CD4-T-cell-deficient mice, most likely due to an early increase in gamma interferon. IL-12 treatment did not impair or enhance the ability of the wild-type mice to control infection, as measured by bacterial numbers. Two novel findings are reported here regarding exogenous IL-12 therapy for M. tuberculosis infections: (i) IL-12 treatment resulted in decreased numbers of immune cells and reduced frequencies of lymphocytes (CD8(+), CD4(+), and NK cells) in the lungs of infected mice and (ii) IL-12 therapy reduced the pathology of M. tuberculosis-infected lungs, as granulomas were smaller and less numerous. These studies support an immunoregulatory role for IL-12 in tuberculosis

One Center's Guide to Outpatient Management of Pediatric Cystic Fibrosis Acute Pulmonary Exacerbation

Cystic fibrosis (CF) is a chronic disorder characterized by acute pulmonary exacerbations that comprise increased cough, chest congestion, increased mucus production, shortness of breath, weight loss, and fatigue. Typically, severe episodes are treated in the inpatient setting and include intravenous antimicrobials, airway clearance therapy, and nutritional support. Children with less-severe findings can often be managed as outpatients with oral antimicrobials and increased airway clearance therapy at home without visiting the specialty CF center to begin treatment. Selection of specific antimicrobial agents is dependent on pathogens found in surveillance culture, activity of an agent in patients with CF, and the unique physiology of these patients. In this pediatric review, we present our practice for defining acute pulmonary exacerbation, deciding treatment location, initiating treatment either in-person or remotely, determining the frequency of airway clearance, selecting antimicrobial therapy, recommending timing for follow-up visit, and recognizing and managing treatment failures

Mtb proteins require retrotranslocation for presentation.

<p>(A,B) DC were treated with exoA or cycloheximide for one hour prior to infection with H37Rv-eGFP (A) or addition of CFP10 and CFP10_3–11 (B). DC were harvested, fixed, and assessed for their ability to stimulate T cell clones by IFN-γ ELISPOT as described. Each bar reflects the mean±SEM of at least three experiments. ND, not done. (C) DC were treated with exoA, exoA/PJ34, or BSA/PJ34 for one hour prior to infection with vaccinia virus expressing eGFP. After 16–18 hours, DC were harvested and GFP expression analyzed by flow cytometry. Data are representative of three experiments. (D,E) DC treated with exoA, BSA, exoA/PJ34, or BSA/PJ34 for one hour were subsequently infected with H37Rv-eGFP (D) or pulsed with antigen (E) overnight, harvested, fixed, and assessed for their ability to stimulate T cell clones by IFN-γ ELISPOT. Data are representative of two experiments. (F) DC were treated with exoA, exoA/PJ34, or BSA/PJ34 for one hour prior to infection with vaccinia virus expressing HIV p24. After 16–18 hours, DC were harvested, fixed and used to stimulate the HIV p24_306–316-specific CD8⁺ clone 16A7 in an IFN-y ELISPOT assay. Data are representative of two experiments.</p

HLA-I, loading machinery, and HLA-I:peptide complexes are present in highly pure Mtb phagosomes.

<p>(A) Phagosomes were isolated by percoll gradient or magnetic purification and prepared for electron microscopy as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000374#s4" target="_blank">Materials and Methods</a>. (B) Magnetic bead-isolated phagosomes were analyzed by flow cytometry to assess HLA-II-PE (plasma membrane) and H37Rv-eGFP fluorescence as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000374#s4" target="_blank">Materials and Methods</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000374#ppat-1000374-g006" target="_blank">Figure 6</a>. The events shown represent a small proportion of the population of phagosomes isolated and the experiment is representative of three experiments. (C) DC were pulsed with magnetically-labeled H37Rv-eGFP for 20 minutes, washed, and incubated for 18 hr. After magnetic separation of Mtb phagosomes, flow organellometry was performed as described previously. Data are representative of three experiments. (D) Magnetically-isolated Mtb phagosomes were freeze-thawed and tested for their ability to stimulate D160 1-23 CD8⁺ T cell clones in the absence of additional APC. IFN-γ production was measured using ELISPOT. Data are representative of two experiments.</p

Mtb antigens are processed by the cytosolic pathway.

<p>(A,B) Human monocyte-derived DC were treated with epoxomicin, BFA, or bafilomycin for one hour before infection with Mtb H37Rv-eGFP (A) or addition of CFP10 and CFP10_3–11 (B). After 15–16 hours in the presence of the inhibitor, DC were harvested, fixed, washed extensively, and used as APC in an IFN-γ ELISPOT assay where T cell clones are effectors. DC were added to an excess of T cells so that antigen was the limiting factor (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000374#s4" target="_blank">Materials and Methods</a>). The mean number of spots produced by each clone was: D160 1-23 (228.4±39.3 to Mtb–infected DC, 19.6±6 to uninfected DC), D480 F6 (623.5±73.7 to Mtb–infected DC, 6.7±2.5 to uninfected DC), D454 E12 (453±52.4 to Mtb–infected DC, 13±4.4 to uninfected DC). Data have been normalized to the untreated control, and each bar reflects the mean±SEM of at least three experiments per clone (*p<0.05, **p<0.01 using two-tailed Student's t test compared to untreated controls, except where indicated). (C,D) DC were transduced with either empty vector or adenoviral ICP47 using Lipofectamine 2000. After 6–26 hours, DC were washed and infected with H37Rv-eGFP (C) or pulsed with antigen (D). Following overnight incubation, T cell clones were added and IFN-γ production was assessed by intracellular cytokine staining. The mean percentage of IFN-γ⁺ clones was: D160 1-23 (8.2±1.1 to Mtb–infected DC, 1.1±0.2 to uninfected DC), D480 F6 (46.4±3.9 to Mtb–infected DC, 1.5±0.4 to uninfected DC), D454 E12 (64±6 to Mtb–infected DC, 0.7±0.3 to uninfected DC). Each bar represents the mean±SEM of seven independent experiments.</p

The Mtb phagosome contains HLA-I loading accessory molecules.

<p>(A) DC were pulsed with H37Rv-eGFP for 20 minutes, washed, and incubated for 40 minutes. Phagosomal fractions were prepared as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000374#ppat-1000374-g003" target="_blank">Figure 3</a> and stained with the indicated antibodies (top panel). Intact DC were fixed, permeabilized, and stained with the indicated antibodies (bottom panel). Data are representative of three experiments. (B) Quantitative analysis of Mtb phagosomes over time. The percent positive number represents Overton cumulative histogram subtraction of the isotype control from the indicated stain. Each bar represents the mean±SEM from three experiments per timepoint.</p