Immunodominant Tuberculosis CD8 Antigens Preferentially Restricted by HLA-B

CD8+ T cells are essential for host defense to intracellular bacterial pathogens such as Mycobacterium tuberculosis (Mtb), Salmonella species, and Listeria monocytogenes, yet the repertoire and dominance pattern of human CD8 antigens for these pathogens remains poorly characterized. Tuberculosis (TB), the disease caused by Mtb infection, remains one of the leading causes of infectious morbidity and mortality worldwide and is the most frequent opportunistic infection in individuals with HIV/AIDS. Therefore, we undertook this study to define immunodominant CD8 Mtb antigens. First, using IFN-γ ELISPOT and synthetic peptide arrays as a source of antigen, we measured ex vivo frequencies of CD8+ T cells recognizing known immunodominant CD4+ T cell antigens in persons with latent tuberculosis infection. In addition, limiting dilution was used to generate panels of Mtb-specific T cell clones. Using the peptide arrays, we identified the antigenic specificity of the majority of T cell clones, defining several new epitopes. In all cases, peptide representing the minimal epitope bound to the major histocompatibility complex (MHC)-restricting allele with high affinity, and in all but one case the restricting allele was an HLA-B allele. Furthermore, individuals from whom the T cell clone was isolated harbored high ex vivo frequency CD8+ T cell responses specific for the epitope, and in individuals tested, the epitope represented the single immunodominant response within the CD8 antigen. We conclude that Mtb-specific CD8+ T cells are found in high frequency in infected individuals and are restricted predominantly by HLA-B alleles, and that synthetic peptide arrays can be used to define epitope specificities without prior bias as to MHC binding affinity. These findings provide an improved understanding of immunodominance in humans and may contribute to a development of an effective TB vaccine and improved immunodiagnostics

Human mucosal associated invariant T cells detect bacterially infected cells

Control of infection with Mycobacterium tuberculosis (Mtb) requires Th1-type immunity, of which CD8+ T cells play a unique role. High frequency Mtb-reactive CD8+ T cells are present in both Mtb-infected and uninfected humans. We show by limiting dilution analysis that nonclassically restricted CD8+ T cells are universally present, but predominate in Mtbuninfected individuals. Interestingly, these Mtb-reactive cells expressed the Va7.2 T-cell receptor (TCR), were restricted by the nonclassical MHC (HLA-Ib) molecule MR1, and were activated in a transporter associated with antigen processing and presentation (TAP) independent manner. These properties are all characteristics of mucosal associated invariant T cells (MAIT), an "innate" T-cell population of previously unknown function. These MAIT cells also detect cells infected with other bacteria. Direct ex vivo analysis demonstrates that Mtb-reactive MAIT cells are decreased in peripheral blood mononuclear cells (PBMCs) from individuals with active tuberculosis, are enriched in human lung, and respond to Mtb-infected MR1-expressing lung epithelial cells. Overall, these findings suggest a generalized role for MAIT cells in the detection of bacterially infected cells, and potentially in the control of bacterial infection. © 2010 Gold et al

Upaya peningkatan/pencapaian sasaran mutu dengan metode six sigma dan perancangan quality plan di PT Mulcindo

PT Mulcindo Steel Industry merupakan perusahaan yang bergerak di bidang fabrikasi baja, jasa dan produk forming serta pemrosesan galvanis. Permasalahan yang terjadi di PT Mulcindo adalah adanya pengukuran sasaran mutu yang belum tercapai di departemen Hollow sehingga akan dilakukan peningkatan pencapaian sasaran mutu. Selain itu pada PT Mulcindo belum terdapat departemen quality control, sehingga akan dilakukan perancangan quality plan untuk meningkatkan kualitas produk yang dihasilkan. Perancangan quality plan ini mencakup penentuan karakteristik kualitas, jenis dan kriteria kecacatan, standar penerimaan, aktivitas kritis, dan check sheet. Pengukuran sasaran mutu di departemen Hollow menggunakan metode Six Sigma. Metode ini terdiri dari fase define, measure, analyze, improve,dan control. Setelah dilakukan pengukuran terhadap sasaran mutu dan implementasi di departemen Hollow didapatkan bahwa terjadi penurunan tingkat kecacatan dari 1.6837% menjadi 0.6024%

Spectroscopy of Li+·Rg and Li+–Rg transport coefficients (Rg = He–Rn)

High-quality [CCSD(T), large basis sets] ab initio potential energy curves are calculated for the series of Li+·Rg species. These curves are employed to calculate spectroscopic parameters for these species, and are used to calculate transport properties for Li+ moving through a bath of the relevant inert gas. The transport results obtained are statistically compared to previous ones. The present potentials appear to be the best available for Li+·Ar, Li+·Kr and Li+·Xe and they rival the best ones for Li+·He and Li+·Ne. In the case of the Li+·Rn system, these are the first reported results

Mtb-reactive MAIT cells in the human lung.

<p>Single cell suspensions were prepared from the lung and adjacent LNs with minor modifications <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000407#pbio.1000407-Lewinsohn5" target="_blank">[52]</a>. The intracellular cytokine staining assay was performed using magnetic-bead purified CD8⁺ T cells from the lung and LNs as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000407#pbio-1000407-g006" target="_blank">Figure 6</a> legend. Only in the case of donor B were anti-MR1 or IgG2a isotype control antibodies added (2.5 µg/ml).</p

Mtb-specific NC CD8⁺ T cells are restricted by MR1.

<p>(A–E) Results of ELISPOT assays shown as IFN-γ spot forming units (SFU)/10,000 T cells in response to DCs (25,000/well) treated as described. (A) TLR agonist stimulation of DCs does not stimulate Mtb-reactive NC-restricted clones. DCs were treated (24 h) with TLR agonists specific for TLR2 (lipoteichoic acid, 10 µg/ml) and TLR4 (LPS; 100 ng/ml) at concentrations known to induce activation and cytokine production by DCs <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000407#pbio.1000407-Gold1" target="_blank">[14]</a>. (B) TLR2 (5 µg/ml) or TLR4 (10 µg/ml) blocking antibodies were added to DCs that were uninfected or infected 1 h prior to the addition of Mtb-reactive NC T-cell clones. (C) Mtb-infected DCs were incubated with blocking antibodies (5 µg/ml) to NKG2D, ULBP1, MICA, CD94 for 1 h prior to the addition of the T-cell clones. (D) The pan HLA–I (W632) and CD1a, b, c, and d blocking antibodies were added to Mtb-infected DCs prior to the addition of T cells. (E) DCs infected with Mtb overnight were incubated with anti-MR1 blocking antibody (clone 26.5) or a mouse IgG2a isotype control (both at 5 µg/ml) for 1 h prior to the addition of T cells. (F–H) Cell surface phenotypic analyses of MR1-restricted clones and control clones. For cell surface detection, cells were incubated with antibodies specific for Vα7.2 (clone 3C10) (F), or CD8α, CD8β (G), or CD161 (H), and analyzed by flow cytometry. For (F) and (H), filled histograms represent the isotype control, bold lines represent antibody-specific staining. Columns 1, 2, and 3 represent MR1-restricted clones from different TB exposure groups: D470B1 (uninfected), D426B1 (latent), D466F5 (active), respectively. Column 4 represents HLA-E restricted clone D160 1–23 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000407#pbio.1000407-Heinzel1" target="_blank">[16]</a>. Column 5 represents HLA-B08-restricted clone D480C6 specific for the Mtb antigen CFP-10_3–11. Column 6 represents CD4⁺ HLA-II–restricted clone D454E12 specific for the Mtb antigen CFP-10. Error bars represent the mean and standard error from duplicate wells. N.D., not done.</p

Phenotypic characterization of MR1-restricted Mtb-reactive T-cell clones.

<p>U, untypable indicates the Vβ chain could not be determined using the flow cytometric assay.</p><p>Abbreviations: N.D., not done.</p

MR1 presents a protein-containing antigen from the mycobacterial cell wall.

<p>(A) CFP or CW from the Mtb strain H37Rv were added (5 µg/ml) to DCs (25,000/well) for 1 h prior to the addition of one of 21 NC clones (5,000/well) followed by IFN-γ ELISPOT assay. (B) DCs (25,000) loaded with CW overnight were incubated with anti-MR1 blocking antibody (clone 26.5) or a mouse IgG2a isotype control (both at 5 µg/ml) for 1 h prior to the addition of T-cell clones (10,000/well). (C) dCW from Mtb was treated with proteases (subtilisin, trypsin, chymotrypsin, pronase, Glu-C) and added (5 µg/ml) to DCs (25,000/well) for 1 h before the addition of 21 NC clones (5,000/well) that were tested for their ability to produce IFN-γ in an ELISPOT assay. Reversed phase- high performance liquid chromatography (RP-HPLC) chromatogram analyses were used to confirm the inactivation of proteases. No responses were detected in the absence of DCs. (D) DCs were infected with <i>S. typhimurium</i>, <i>L. monocytogenes</i>, <i>and S. aureus</i> for 1 h with a calculated moi of 145, 6, and 15, respectively. DCs were washed, antibiotics added, and DCs (25,000) were incubated with three different Mtb-reactive MAIT-cell clones (10,000/well) that were tested for their ability to produce IFN-γ in an ELISPOT assay. Results shown are similar to a minimum of three independent experiments where <i>S. typhimurium</i>, <i>L. monocytogenes</i>, <i>and S. aureus</i> were tested at a variety of moi ranging from 5 to 150.</p