Mycobacterium tuberculosis-specific T-cell responses in latent infection and active disease

Adaptive Immunantworten gegen Mycobacterium tuberculosis (M. tuberculosis) sind von entscheidender Bedeutung für die effektive Eindämmung des Erregers sowie den Schutz vor einer erneuten, sekundären Tuberkulose (TB). Obwohl Schlüsselfaktoren wie die Th1 Zytokine IFN-gamma und TNF-alpha bekannt sind, blieben Bemühungen zur Identifizierung eindeutiger immunologischer Parameter, welche ausschlaggebend für den Krankheitsverlauf sind, bislang erfolglos. Ein besseres Verständnis der zugrunde liegenden Immunprozesse sowie die Identifikation projektiver Biomarker für TB sind zentrale Ziele dieser Arbeit. Zur Bearbeitung dieser Fragestellungen wurden adaptive Immunantworten gegen M. tuberculosis in gesunden Probanden mit LTBI und Patienten mit aktiver TB analysiert. Hierfür wurde die Erkennung unterschiedlicher Proteine des Erregers durch die Messung IFN-gamma exprimierender CD4+ CD45RO+ Gedächtnis T Zellen untersucht. Eine Besonderheit war die Einbeziehung sogenannter Latenz-assoziierter Proteine, welche in Zusammenhang mit Dormanz und Reaktivierung des Bakteriums stehen. 7 Tage in vitro Inkubation in Verbindung mit einer zweimaligen Restimulation belegten eine spezifische Erkennung durch CD4+ CD45RO+ T Zellen für die Mehrheit der getesteten Proteine bei Spendern mit LTBI. Der darauf folgende Vergleich zwischen Patienten mit aktiver TB und Personen mit LTBI zeigte signifikant höhere T Zell Antworten für 7 der 35 M. tuberculosis Proteine während LTBI. Bemerkenswerterweise konnten spezifische T Zellen für eines der Protein, nämlich Rv3407, ausschließlich während LTBI gemessen werden und nicht bei Patienten mit aktiver TB. Diskriminanz Analysen zeigten, dass eine Unterscheidung zwischen LTBI und TB Patienten basierend auf T Zell Antwort gegen ausgewählte Latenz-assoziierte Antigene mit einer Genauigkeit von 82% möglich ist. Erneut erwies sich Rv3407 als der mit Abstand bedeutendste Faktor innerhalb der ausgewählten M. tuberculosis Proteine.Adaptive immune responses to Mycobacterium tuberculosis (M. tuberculosis) are crucial for an efficient containment of the pathogen and protection against secondary tuberculosis (TB). Although key mediators like the Th1 cytokines IFN-gamma and TNF-alpha released by M. tuberculosis-specific T cells are known, the immunological correlates determining the outcome of infection remain elusive. A better understanding of the underlying immune processes and the identification of protective biomarkers for TB are central aims of this thesis. To address these topics adaptive immune responses to M. tuberculosis were analyzed in healthy LTBI and patients with active pulmonary TB. The recognition of M. tuberculosis derived antigens was studied by measuring the expression of IFN-gamma in CD4+ CD45RO+ memory T cells. A special hallmark was the inclusion of latency proteins associated with dormancy, reactivation and resuscitation of the pathogen. Seven days in vitro incubation of PBMC and two rounds of restimulation followed by FACS analysis revealed T cell mediated recognition of the majority of tested latency-associated proteins in donors with LTBI. Comparison between active TB and LTBI documented significantly higher T-cell responses against 7 of 35 tested M. tuberculosis latency-associated antigens in LTBI. Notably, T cells specific for one M. tuberculosis antigen, namely Rv3407, were exclusively detected in the subgroup of LTBI. Discrimination analysis revealed that the T-cell response against selected antigens with our novel assay is capable of distinguishing TB patients and LTBI with 82% accuracy using cross-validation. Again Rv3407 was by far the most influential component present in this cluster. Peptide pool stimulation in a similar fashion identified single distinct candidate epitopes within Rv3407 in four LTBI

Efficient Construction of an Inverted Minimal H1 Promoter Driven siRNA Expression Cassette: Facilitation of Promoter and siRNA Sequence Exchange

RNA interference (RNAi), mediated by small interfering RNA (siRNA), is an effective method used to silence gene expression at the post-transcriptional level. Upon introduction into target cells, siRNAs incorporate into the RNA-induced silencing complex (RISC). The antisense strand of the siRNA duplex then "guides" the RISC to the homologous mRNA, leading to target degradation and gene silencing. In recent years, various vector-based siRNA expression systems have been developed which utilize opposing polymerase III promoters to independently drive expression of the sense and antisense strands of the siRNA duplex from the same template.We show here the use of a ligase chain reaction (LCR) to develop a new vector system called pInv-H1 in which a DNA sequence encoding a specific siRNA is placed between two inverted minimal human H1 promoters (approximately 100 bp each). Expression of functional siRNAs from this construct has led to efficient silencing of both reporter and endogenous genes. Furthermore, the inverted H1 promoter-siRNA expression cassette was used to generate a retrovirus vector capable of transducing and silencing expression of the targeted protein by>80% in target cells.The unique design of this construct allows for the efficient exchange of siRNA sequences by the directional cloning of short oligonucleotides via asymmetric restriction sites. This provides a convenient way to test the functionality of different siRNA sequences. Delivery of the siRNA cassette by retroviral transduction suggests that a single copy of the siRNA expression cassette efficiently knocks down gene expression at the protein level. We note that this vector system can potentially be used to generate a random siRNA library. The flexibility of the ligase chain reaction suggests that additional control elements can easily be introduced into this siRNA expression cassette

Identification of T-Cell Antigens Specific for Latent Mycobacterium Tuberculosis Infection

BACKGROUND: T-cell responses against dormancy-, resuscitation-, and reactivation-associated antigens of Mycobacterium tuberculosis are candidate biomarkers of latent infection in humans. METHODOLOGY/PRINCIPAL FINDINGS: We established an assay based on two rounds of in vitro restimulation and intracellular cytokine analysis that detects T-cell responses to antigens expressed during latent M. tuberculosis infection. Comparison between active pulmonary tuberculosis (TB) patients and healthy latently M. tuberculosis-infected donors (LTBI) revealed significantly higher T-cell responses against 7 of 35 tested M. tuberculosis latency-associated antigens in LTBI. Notably, T cells specific for Rv3407 were exclusively detected in LTBI but not in TB patients. The T-cell IFNgamma response against Rv3407 in individual donors was the most influential factor in discrimination analysis that classified TB patients and LTBI with 83% accuracy using cross-validation. Rv3407 peptide pool stimulations revealed distinct candidate epitopes in four LTBI. CONCLUSIONS: Our findings further support the hypothesis that the latency-associated antigens can be exploited as biomarkers for LTBI

Comparison of IFNγ-expressing CD4⁺ T cells specific for immunodominant and latency-associated M. tuberculosis antigens between patients with TB, LTBI, and TST-negative donors.

<p>(A). Percentages of IFNγ-expressing CD4⁺ CD45RO⁺ memory T cells are shown for stimulation with SEB, PPD from <i>M. tuberculosis</i>, and 11 latency-associated antigens after 7 days and two rounds of <i>in vitro</i> restimulation. T-cell responses from TST-negative donors are indicated as green circles, LTBI are indicated as blue squares, and TB patients are indicated as red triangles. Two-sided p-values for the Mann-Whitney U-test are indicated as follows: * <i>P</i><0.05, ** <i>P</i><0.01; and *** <i>P</i><0.001. (B) Classification of TB patients and LTBI based on random forest analysis using 11 latency-associated antigens as well as ESAT6_CFP-10, and PPD. Results from the cross validation are shown in a bar chart. Each bar represents an individual donor. TB patients are shown on the left (red bars), LTBI on the right side (blue bars). The y-axis indicates the prediction threshold calculated by random forest analysis. Negative bars predict a TB patient, positive bars an LTBI. The prediction probability is represented as the bar height. (C) Mean decrease of class impurity over all trees measured as Gini index (y-axis) indicates the relative importance of each factor (x-axis) for classification. PPD: purified protein derivative of <i>M. tuberculosis</i>; SEB Staphylococcus enterotoxin B.</p

IFNγ ELISA analyses after restimulation with immunodominant and latency-associated antigens of PBMC from LTBI and TST-negative donors.

<p>Analyses of IFNγ in the culture supernatant by ELISA after 7 days and two rounds of <i>in vitro</i> restimulation in PBMC from LTBI (<i>A</i>) and TST-negative donors (<i>B</i>) are shown. Scatter plots indicate mean and standard deviation. Background values of non-stimulated controls were subtracted. IFNγ concentrations in the supernatant are indicated on the y-axis for stimulation with SEB, PPD from <i>M. tuberculosis</i>, and tested antigens (x-axes). The most promising candidate Rv3407 is underlined.</p

List of proteins candidates.

*<p><i>M. tuberculosis</i> dormancy-related antigens.</p

Design of Rv3407 peptide pools.

1<p>Peptide number according to the position within the primary sequence from C- to N-terminus.</p

Overlapping peptide pools of latency-associated protein Rv3407 stimulate IFNγ-expressing CD4⁺ T cells after 7 days and two rounds of restimulation.

<p>PBMC from six LTBI (A–F) were restimulated with 15-mer synthetic peptide pools of Rv3407 for 7 days including two rounds of <i>in vitro</i> restimulation. IFNγ-expressing CD4⁺ CD45RO⁺ T cells are shown for stimulation with peptide pools 1 to 6 (grey bars) and pools 7 to 11 (black bars). Each peptide is constituent of one pool within pools 1 to 6 and of one pool within pools 7 to 11. Peptides inducing the most prominent responses are indicated for donors <i>A–D</i>. The horizontal line indicates the threshold for positive responses (0.2%). Background values of non-stimulated controls were subtracted.</p

Gating procedures of flow cytometry analyses to determine protein candidate specific T cell proportions.

<p>Representative analyses from a patient with Tb (A) and an LTBI (B) are shown. Open red circles and dot plot connected by red arrows indicate the sequence of analysis steps. First, lymphocytes were gated using size (forward scatter; FSC) and granularity (side scatter, SSC). These cells were then analyzed for CD4 expression. CD4⁺ T cells were analyzed for IFNγ CD45RO expression for each stimulation (without stimulus, w/o; proteine 3; protein 11; SEB). Proportions of CD45RO_high IFNγ expressing CD4⁺ T cells (upper right quadrants) were determined. The background of non-stimulated T cells (w/o) was subtracted for analyses.</p