Jacqueline M. Cliff

Distinct Phases of Blood Gene Expression Pattern Through Tuberculosis Treatment Reflect Modulation of the Humoral Immune Response

BACKGROUND Accurate assessment of treatment efficacy would facilitate clinical trials of new antituberculosis drugs. We hypothesized that early alterations in peripheral immunity could be measured by gene expression profiling in tuberculosis patients undergoing successful conventional combination treatment. METHODS Ex vivo blood samples from 27 pulmonary tuberculosis patients were assayed at diagnosis and during treatment. RNA was processed and hybridized to Affymetrix GeneChips, to determine expression of over 47,000 transcripts. RESULTS There were significant ≥ 2-fold changes in expression of >4000 genes during treatment. Rapid, large-scale changes were detected, with down-regulated expression of 1261 genes within the first week, including inflammatory markers such as complement components C1q and C2. This was followed by slower changes in expression of different networks of genes, including a later increase in expression of B-cell markers, transcription factors, and signaling molecules. CONCLUSIONS The fast initial down-regulation of expression of inflammatory mediators coincided with rapid killing of actively dividing bacilli, whereas slower delayed changes occurred as drugs acted on dormant bacilli and coincided with lung pathology resolution. Measurement of biosignatures during clinical trials of new drugs could be useful predictors of rapid bactericidal or sterilizing drug activity, and would expedite the licensing of new treatment regimens.

Gene-expression patterns in whole blood identify subjects at risk for recurrent tuberculosis

BACKGROUND The majority of patients with tuberculosis who comply with appropriate treatment are cured. However, approximately 5% subsequently have a repeat disease episode, usually within 2 years of successful combination therapy. Presently, there is no way of predicting which patients will experience a relapse. METHODS We identified 10 subjects who had previously experienced recurrent tuberculosis and carefully matched them to cured subjects who had had only 1 episode of tuberculosis, to patients with active tuberculosis, and to latently infected healthy subjects. We compared their ex vivo whole-blood gene-expression profiles by use of DNA array technology and confirmed the results by use of quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). RESULTS The 4 clinical tuberculosis groups exhibited distinct patterns of gene expression. The gene-transcript profiles of the patients with recurrent tuberculosis were more similar to those of the patients with active tuberculosis than to those of the cured or latently infected subjects. Discriminant analysis of a training data set showed that 9 genes were sufficient to classify the subjects. We confirmed that measurement of the expression of these genes by qRT-PCR can accurately discriminate between subjects in a test set of samples. CONCLUSIONS A simple test based on gene-expression patterns may be used as a biomarker of cure while identifying patients who are at risk for relapse. This would facilitate the introduction of new tuberculosis drugs.

CCL2 responses to Mycobacterium tuberculosis are associated with disease severity in tuberculosis.

Background Leucocyte activating chemokines such as CCL2, CCL3, and CXCL8 together with proinflammatory IFNγ, TNFα and downmodulatory IL10 play a central role in the restriction of M. tuberculosis infections, but is unclear whether these markers are indicative of tuberculosis disease severity. Methodology We investigated live M. tuberculosis- and M. bovis BCG- induced peripheral blood mononuclear cell responses in patients with tuberculosis (TB) and healthy endemic controls (ECs, n = 36). TB patients comprised pulmonary (PTB, n = 34) and extrapulmonary groups, subdivided into those with less severe localized extrapulmonary TB (L-ETB, n = 16) or severe disseminated ETB (D-ETB, n = 16). Secretion of CCL2, IFNγ, IL10 and CCL3, and mRNA expression of CCL2, TNFα, CCL3 and CXCL8 were determined. Results M. tuberculosis- and BCG- induced CCL2 secretion was significantly increased in both PTB and D-ETB (p<0.05, p<0.01) as compared with L-ETB patients. CCL2 secretion in response to M. tuberculosis was significantly greater than to BCG in the PTB and D-ETB groups. M. tuberculosis-induced CCL2 mRNA transcription was greater in PTB than L-ETB (p = 0.023), while CCL2 was reduced in L-ETB as compared with D-ETB (p = 0.005) patients. M. tuberculosis –induced IFNγ was greater in L-ETB than PTB (p = 0.04), while BCG-induced IFNγ was greater in L-ETB as compared with D-ETB patients (p = 0.036). TNFα mRNA expression was raised in PTB as compared with L-ETB group in response to M. tuberculosis (p = 0.02) and BCG (p = 0.03). Mycobacterium-induced CCL3 and CXCL8 was comparable between TB groups. Conclusions The increased CCL2 and TNFα in PTB patients may support effective leucocyte recruitment and M. tuberculosis localization. CCL2 alone is associated with severity of TB, possibly due to increased systemic inflammation found in severe disseminated TB or due to increased monocyte infiltration to lung parenchyma in pulmonary disease.

The human immune response to tuberculosis and its treatment: a view from the blood

The immune response upon infection with the pathogen Mycobacterium tuberculosis is poorly understood, hampering the discovery of new treatments and the improvements in diagnosis. In the last years, a blood transcriptional signature in tuberculosis has provided knowledge on the immune response occurring during active tuberculosis disease. This signature was absent in the majority of asymptomatic individuals who are latently infected with M. tuberculosis (referred to as latent). Using modular and pathway analyses of the complex data has shown, now in multiple studies, that the signature of active tuberculosis is dominated by overexpression of interferon‐inducible genes (consisting of both type I and type II interferon signaling), myeloid genes, and inflammatory genes. There is also downregulation of genes encoding B and T‐cell function. The blood signature of tuberculosis correlates with the extent of radiographic disease and is diminished upon effective treatment suggesting the possibility of new improved strategies to support diagnostic assays and methods for drug treatment monitoring. The signature suggested a previously under‐appreciated role for type I interferons in development of active tuberculosis disease, and numerous mechanisms have now been uncovered to explain how type I interferon impedes the protective response to M. tuberculosis infection.

Extended Culture Enhances Sensitivity of a Gamma Interferon Assay for Latent Mycobacterium tuberculosis Infection

ABSTRACT To test the hypothesis that prolonged culture would enhance the sensitivity of latent tuberculosis detection by a gamma interferon release assay, blood samples from 33 household contacts of Gambian tuberculosis patients were stimulated with Mycobacterium tuberculosis-specific antigens. After 24 h of culture, 66% were positive, compared to 93% after 6 days of culture.

PD-1, PD-L1 and PD-L2 Gene Expression on T-Cells and Natural Killer Cells Declines in Conjunction with a Reduction in PD-1 Protein during the Intensive Phase of Tuberculosis Treatment.

Background The PD-1 axis is a cell intrinsic immunoregulatory pathway that mediates T cell exhaustion in chronic infection particularly in some viral infections. We hypothesized that PD-1, PD-L1 and PD-L2 would be highly expressed in untreated tuberculosis patients compared to controls due to their chronic infection and would decrease with successful TB treatment. Materials and Methods Untreated tuberculosis patients (n = 26) were recruited at diagnosis and followed up during treatment. Household contacts (n = 24) were recruited to establish baseline differences. Blood gene expression ex vivo was investigated using qRT-PCR. Flow cytometry was performed to establish protein expression patterns. Results PD-L1 gene expression was found to be elevated in active TB disease; however, this was not observed for PD-1 or PD-L2. The intensive phase of TB treatment was associated with a significant decline in PD-1, PD-L1 and PD-L2 gene expression. PD-1 protein expression on the surface of NK cells, CD8+ and CD4+ T cells was similar in patients with active TB disease compared to controls but declined with successful TB treatment, with the greatest decline occurring on the NK cells followed by CD8+ T cells and then CD4+ T cells. Granzyme B/PD-1 co-expression declined with successful intensive phase treatment. Conclusion Modulation of PD-1/PD-L1 pathway through TB treatment indicates changes in the peripheral T cell response caused by live Mycobacterium tuberculosis (Mtb) followed by the response to dead bacilli, antigen-release and immuno-pathology resolution. The PD-1 axis could be a host drug target for immunomodulatory treatments in the future.

Biogenesis of secretory organelles during B cell differentiation

The differentiation of B cells into Ig‐secreting plasma cells requires the expansion of secretory organelles to cope with the increased cargo load. To evaluate the timeline of this process, we have quantitated the kinetics of secretory organelle expansion relative to Ig secretion and examined regulatory components of secretory transport following in vitro activation of human B lymphocytes. Unstimulated B cells contain minimal endomembranes. After activation, ER membrane induction appears as tightly packed spherical structures of 0.5–1 μm diameter concentrated in a juxtanuclear position. When the cells differentiate into plasmablasts, there is dramatic cell‐size increase, but the ER remains concentrated close to the nucleus and only later fills the entire cell. In sharp contrast, previous studies in other cell types have found that the ER expands in synchrony with increasing cell size during interphase, by extension of ER tubules under the PM. In this study, the Golgi remains consistently as a single juxtanuclear structure but linearly expands sixfold in volume during B cell activation. Furthermore, following active cell proliferation, ER exit sites proliferate rapidly, increasing almost fourfold in number, in parallel with a sharp increase in Ig secretion. These findings demonstrate that the control of organelle biogenesis and expansion in primary human B cells are differentially regulated by cargo flux caused by Ig synthesis.

Differential Gene Expression Identifies Novel Markers of CD4+ and CD8+ T Cell Activation Following Stimulation by Mycobacterium tuberculosis

T cell activation in response to antigenic stimulation is a complex process, involving changes in the expression level of a large number of genes. We have used cDNA array technology to characterize the differences in gene expression between human CD4+ and CD8+ T cells. PBMC from six healthy donors were stimulated with live Mycobacterium tuberculosis, and the gene expression profiles of each donor’s CD4+ and CD8+ T cells were analyzed separately. ANOVA revealed 518 genes that were consistently differentially expressed between CD4+ and CD8+ T cells. These differentially expressed genes include a combination of well-known, previously characterized genes with a range of biological functions and unknown in silico predicted hypothetical genes. Where possible, the novel genes have been characterized using bioinformatics, and putative transcription factors, signaling molecules, transmembrane, and secreted factors have been identified. A subset of these differentially expressed genes could be exploited as markers of CD4+ and CD8+ T cell activation for use in vaccine trials. These observed differences in the gene expression profile of CD4+ and CD8+ T cells following activation by a human pathogen contribute to an increased understanding of T cell activation and differentiation and the roles these T cell subsets may play in immunity to infection.

Long-Lived Memory B-Cell Responses following BCG Vaccination

The role of T-cells in immunity against Mycobacterium tuberculosis (M. tuberculosis) infection has been extensively studied, however, that of B-cells still remains comparatively unexplored. In this study, we determined the presence and frequencies of mycobacteria-specific memory B-cells (MBCs) in peripheral blood from clinically healthy, Bacillus Calmette Guerin (BCG) vaccinated (n = 79) and unvaccinated (n = 14) donors. Purified protein derivative (PPD)-specific MBCs were present in most donors (both vaccinated and unvaccinated) but their frequencies were significantly higher in vaccinated than in unvaccinated donors. MBCs specific for other mycobacterial antigens [antigen-85A (Ag85A), antigen-85B (Ag85B), 6 kDalton early secretory antigenic target (ESAT-6) and the 10 kDalton-culture filtrate protein (CFP-10)] were less prevalent than those recognising PPD. Furthermore, PPD-specific MBCs were detected in BCG vaccinated donors without ESAT-6 and CFP-10 specific responses. Together, these results indicate that BCG vaccination induces long-lived MBC responses. Similar patterns of response were seen when we examined mycobacteria-specific antibody and T-cell responses in these donors. Our data show for the first time that BCG vaccination elicits long-lived mycobacteria-specific MBC responses in healthy individuals, suggesting a more substantial role of B-cells in the response to BCG and other mycobacterial infections than previously thought.

Recombinant ESAT-6-CFP10 Fusion Protein Induction of Th1/Th2 Cytokines and FoxP3 Expressing Treg Cells in Pulmonary TB

Background Early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) are Mycobacterium tuberculosis (Mtb)–specific antigens that are secreted by actively metabolising bacteria and contribute to the virulence of the bacteria. Their ability to induce Treg and Th2 responses, particularly during the first two weeks of treatment, has not been comprehensively examined to date. The purpose of this work was to characterise Th1, Th2 and Treg responses to rESAT-6-CFP10 fusion protein in TB patients before and during the intensive phase of treatment and in healthy M.bovis BCG vaccinated donors. Methods Forty-six newly diagnosed, HIV-negative, smear-positive pulmonary TB patients and 20 healthy donors were recruited in the UK and Ghana. Their peripheral blood mononuclear cells (PBMC) were used in ex vivo ELISPOT and in vitro cultures to identify immunological parameters of interest. Results The study confirmed that protective immune responses to rESAT-6-CFP10 are impaired in active TB but improved during treatment: circulating antigen-specific IL-4-producing T-cells were increased in untreated TB but declined by two weeks of treatment while the circulating antigen-specific IFN-γ producing T cells which showed a transient rise at one week of treatment, persisted at baseline levels at two months of treatment. In vitro T cell proliferation and IFN-γ production were reduced, while IL-4 and CD4+FoxP3+CD25hi cell expression were increased in response to rESAT-6-CFP10 fusion protein in untreated TB. These responses were reversed during early treatment of TB. Conclusions These observations support further investigations into the possible utility of these parameters as markers of active disease and favourable treatment outcomes.