Monitoring Anti-tuberculosis Treatment Response Using Analysis of Whole Blood Mycobacterium tuberculosis Specific T Cell Activation and Functional Markers.

Background: Blood-based biomarkers have been proposed as an alternative to current sputum-based treatment monitoring methods in active tuberculosis (ATB). The aim of this study was to validate previously described phenotypic, activation, and cytokine markers of treatment response in a West African cohort. Methods: Whole blood immune responses to Mycobacterium tuberculosis ESAT-6/CFP-10 (EC) and purified protein derivative (PPD) were measured in twenty adults at baseline and after 2 months of standard TB treatment. Patients were classified as fast or slow responders based on a negative or positive sputum culture result at 2 months, respectively. Cellular expression of activation markers (CD38, HLA-DR), memory markers (CD27), and functional intracellular cytokine and proliferation (IFN-γ, Ki-67, TNF-α) markers were measured using multi-color flow cytometry. Results: There was a significant increase in the proportion of CD4+CD27+ cells expressing CD38 and HLA-DR following EC stimulation at 2 months compared to baseline (p = 0.0328 and p = 0.0400, respectively). Following PPD stimulation, slow treatment responders had a significantly higher proportion of CD8+CD27-IFN-γ+ (p = 0.0105) and CD4+CD27+HLA-DR+CD38+ (p = 0.0077) T cells than fast responders at baseline. Receiver operating curve analysis of these subsets resulted in 80% sensitivity and 70 and 100% specificity, respectively (AUC of 0.82, p = 0.0156 and 0.84, p = 0.0102). Conclusion: Our pilot data show reductions in expression of T cell activation markers were seen with treatment, but this was not associated with fast or slow sputum conversion at 2 months. However, baseline proportions of activated T cell subsets are potentially predictive of the subsequent speed of response to treatment.

Analysis of cellular and soluble profiles in QuantiFERON nonconverters, converters, and reverters in the Gambia.

BACKGROUND: Tuberculosis (TB) is the leading cause of death from a single infectious agent worldwide. The immune system is capable of clearing the pathogen before establishment of latent infection but the mechanisms for this are not yet understood. METHODS: This study analysed highly exposed household contacts (HHC) of TB index cases who were categorised according to QuantiFERON (QFT) results at recruitment and 6 months. Seventeen (17) QFT nonconverters, 14 QFT converters, 18 QFT reverters and 18 latent TB infection (LTBI) were analysed. Supernatants generated following QFT stimulation at both time-points were analysed using a 64-plex cytokine array. Flow cytometry was performed on QFT converters and nonconverters at baseline only. RESULTS: Interleukin-2 (IL-2), IL-5, IL-13, APRIL, IL-17A, IP-10, MIP-1ß, sIL-6rb, OPN, and sTNFR2 were all significantly higher in the QFT converters compared with nonconverters at baseline. Levels of interferon-α2 (IFN-α2) and IL-2 were significantly lower in QFT reverters compared with nonconverters at baseline. Analysis of Ag-specific IL-2 levels resulted in an area under the curve (AUC) of 0.93 (95% confidence interval [CI], 0.84-1.00) for QFT converters compared to nonconverters and an AUC of 0.80 (0.65-0.95) for QFT reverters compared with LTBI. Purified protein derivative (PPD)-specific CD4 + CD26 + IFN-γ + cells were significantly increased (P = .0007) in QFT nonconverters compared with QFT converters at baseline. CONCLUSIONS: Our results provide insight into the underlying mechanisms of resistance to sustained Mycobacterium tuberculosis infection.

M. tuberculosis T Cell Epitope Analysis Reveals Paucity of Antigenic Variation and Identifies Rare Variable TB Antigens.

Pathogens that evade adaptive immunity typically exhibit antigenic variation. By contrast, it appears that although the chronic human tuberculosis (TB)-causing pathogen Mycobacterium tuberculosis needs to counter host T cell responses, its T cell epitopes are hyperconserved. Here we present an extensive analysis of the T cell epitopes of M. tuberculosis. We combined population genomics with experimental immunology to determine the number and identity of T cell epitope sequence variants in 216 phylogenetically diverse strains of M. tuberculosis. Antigen conservation is indeed a hallmark of M. tuberculosis. However, our analysis revealed a set of seven variable antigens that were immunogenic in subjects with active TB. These findings suggest that M. tuberculosis uses mechanisms other than antigenic variation to evade T cells. T cell epitopes that exhibit sequence variation may not be subject to the same evasion mechanisms, and hence vaccines that include such variable epitopes may be more efficacious.

Sequence diversity in the pe_pgrs genes of Mycobacterium tuberculosis is independent of human T cell recognition.

UNLABELLED: The Mycobacterium tuberculosis genome includes the large family of pe_pgrs genes, whose functions are unknown. Because of precedents in other pathogens in which gene families showing high sequence variation are involved in antigenic variation, a similar role has been proposed for the pe_pgrs genes. However, the impact of immune selection on pe_pgrs genes has not been examined. Here, we sequenced 27 pe_pgrs genes in 94 clinical strains from five phylogenetic lineages of the M. tuberculosis complex (MTBC). We found that pe_pgrs genes were overall more diverse than the remainder of the MTBC genome, but individual members of the family varied widely in their nucleotide diversity and insertion/deletion (indel) content: some were more, and others were much less, diverse than the genome average. Individual pe_pgrs genes also differed in the ratio of nonsynonymous to synonymous mutations, suggesting that different selection pressures act on individual pe_pgrs genes. Using bioinformatic methods, we tested whether sequence diversity in pe_pgrs genes might be selected by human T cell recognition, the major mechanism of adaptive immunity to MTBC. We found that the large majority of predicted human T cell epitopes were confined to the conserved PE domain and experimentally confirmed the antigenicity of this domain in tuberculosis patients. In contrast, despite being genetically diverse, the PGRS domains harbored few predicted T cell epitopes. These results indicate that human T cell recognition is not a significant force driving sequence diversity in pe_pgrs genes, which is consistent with the previously reported conservation of human T cell epitopes in the MTBC. IMPORTANCE: Recognition of Mycobacterium tuberculosis antigens by T lymphocytes is known to be important for immune protection against tuberculosis, but it is unclear whether human T cell recognition drives antigenic variation in M. tuberculosis. We previously discovered that the known human T cell epitopes in the M. tuberculosis complex are highly conserved, but we hypothesized that undiscovered epitopes with naturally occurring sequence variants might exist. To test this hypothesis, we examined the pe_pgrs genes, a large family of genes that has been proposed to function in immune evasion by M. tuberculosis. We found that the pe_pgrs genes exhibit considerable sequence variation, but the regions containing T cell epitopes and the regions of variation are distinct. These findings confirm that the majority of human T cell epitopes of M. tuberculosis are highly conserved and indicate that selection forces other than T cell recognition drive sequence variation in the pe_pgrs genes.