Variability in Tuberculosis Granuloma T Cell Responses Exists, but a Balance of Pro- and Anti-inflammatory Cytokines Is Associated with Sterilization

Lung granulomas are the pathologic hallmark of tuberculosis (TB). T cells are a major cellular component of TB lung granulomas and are known to play an important role in containment of Mycobacterium tuberculosis (Mtb) infection. We used cynomolgus macaques, a non-human primate model that recapitulates human TB with clinically active disease, latent infection or early infection, to understand functional characteristics and dynamics of T cells in individual granulomas. We sought to correlate T cell cytokine response and bacterial burden of each granuloma, as well as granuloma and systemic responses in individual animals. Our results support that each granuloma within an individual host is independent with respect to total cell numbers, proportion of T cells, pattern of cytokine response, and bacterial burden. The spectrum of these components overlaps greatly amongst animals with different clinical status, indicating that a diversity of granulomas exists within an individual host. On average only about 8% of T cells from granulomas respond with cytokine production after stimulation with Mtb specific antigens, and few “multi-functional” T cells were observed. However, granulomas were found to be “multi-functional” with respect to the combinations of functional T cells that were identified among lesions from individual animals. Although the responses generally overlapped, sterile granulomas had modestly higher frequencies of T cells making IL-17, TNF and any of T-1 (IFN-γ, IL-2, or TNF) and/or T-17 (IL-17) cytokines than non-sterile granulomas. An inverse correlation was observed between bacterial burden with TNF and T-1/T-17 responses in individual granulomas, and a combinatorial analysis of pair-wise cytokine responses indicated that granulomas with T cells producing both pro- and anti-inflammatory cytokines (e.g. IL-10 and IL-17) were associated with clearance of Mtb. Preliminary evaluation suggests that systemic responses in the blood do not accurately reflect local T cell responses within granulomas

Impairment of IFN-Gamma Response to Synthetic Peptides of Mycobacterium tuberculosis in a 7-Day Whole Blood Assay

Studies on Mycobacterium tuberculosis (MTB) antigens are of interest in order to improve vaccine efficacy and to define biomarkers for diagnosis and treatment monitoring. The methodologies used for these investigations differ greatly between laboratories and discordant results are common. The IFN-gamma response to two well characterized MTB antigens ESAT-6 and CFP-10, in the form of recombinant proteins and synthetic peptides, was evaluated in HIV-1 uninfected persons in both long-term (7 day) and 24 hour, commercially available QuantiFERON TB Gold in Tube (QFT-GIT), whole blood assays. Our findings showed differences in the IFN-gamma response between 24 hour and 7 day cultures, with recombinant proteins inducing a significantly higher response than the peptide pools in 7 day whole blood assays. The activity of peptides and recombinant proteins did not differ in 24 hour whole blood or peripheral blood mononuclear cell (PBMC) based assays, nor in the ELISpot assay. Further analysis by SELDI-TOF mass spectrometry showed that the peptides are degraded over the course of 7 days of incubation in whole blood whilst the recombinant proteins remain intact. This study therefore demonstrates that screening antigenic candidates as synthetic peptides in long-term whole blood assays may underestimate immunogenicity

Studies on in vitro human T cell reactivity to antigens of mycobacterium tuberculosis

Studies on Mycobacterium tuberculosis (MTB) antigens are important to improve immunodiagnostics and vaccine efficacy. A novel genome based strategy for antigen discovery is to relate what is highly expressed by bacilli in vivo or in vitro, to what is recognized by human T cells as antigens. As hypoxia is a relevant stimulus that MTB encounters in vivo, whole genome based transcriptional profiles of M. tuberculosis subject to prolonged hypoxia (described as the Enduring hypoxic response (EHR) were analyzed, to guide the discovery of novel potential anitgens, by a combined bioinformatic and empirical approach and to determine evidence of infection stage specific recognition

SELDI-TOF mass spectrometry analysis of whole blood supernatant stimulated with ESAT-6 and CFP-10 peptide pools.

<p>(A) ESAT-6 peptide pool stimulated whole blood at Day 0 with expected masses from 1419 Da to 1738 Da. (B) ESAT-6 peptide pool stimulated whole blood harvested after 7 days of incubation. (C) CFP-10 peptide pool stimulated whole blood at Day 0 with expected masses from 1415 Da to 1804 Da. (D) CFP-10 peptide pool stimulated whole blood harvested after 7 days of incubation.</p

IFN-gamma response of PBMC to peptide pools and recombinant proteins in the overnight ELISpot assay (n = 29).

<p>“PP” indicates the peptide pools and “r” indicates recombinant proteins of ESAT-6 and CFP-10. No significant difference was found between the peptide pool and recombinant protein for either ESAT-6 or CFP-10 (Wilcoxon matched pairs test). Lines indicate median response.</p

QFT-IT results, representing the Antigen-Nil IU/ml INF-gamma values (n = 29, Panel A).

<p>The solid line represents the median. <b>IFN-gamma response to peptide pools (covering ESAT-6, CFP-10, and Rv2654) and recombinant ESAT-6 and CFP-10 in the 7 day WBA (n = 29, Panel B).</b> “PP” indicates peptide pool and “r” indicates the recombinant proteins. Recombinant proteins induced significantly higher responses than the peptide pools (p<0.0001 for both ESAT-6 and CFP-10). IFN-gamma concentrations are shown as pg/ml. Lines indicate median response.</p

Recombinant ESAT-6 protein profiles on SELDI-TOF mass spectrometry.

<p>(A) Recombinant rESAT-6 protein. (B) Recombinant ESAT-6 stimulated whole blood at Day 0 showing a peak at the predicted molecular weight of 11.7 kDa. (C) Recombinant ESAT-6 stimulated whole blood after 7 days of incubation showing that rESAT-6 cannot be detected, while a new peak appears at 10.8 kDa.</p