Frequency of Circulating CD4+Ki67+HLA-DR− T Regulatory Cells Prior to Treatment for Multidrug Resistant Tuberculosis Can Differentiate the Severity of Disease and Predict Time to Culture Conversion

Abstract

Identifying a blood circulating cellular biomarker that can be used to assess severity of disease and predict the time to culture conversion (TCC) in patients with multidrug resistant tuberculosis (MDR-TB) would facilitate monitoring response to treatment and may be of value in the design of future drug trials. We report on the frequency of blood Ki67+HLA-DR− CD4+ T regulatory (Treg) cells in predicting microbiological outcome before initiating second-line treatment for MDR-TB. Fifty-one patients with MDR-TB were enrolled and followed over 18 months; a subset of patients was sputum culture (SC) negative at baseline (n = 9). SC positive patients were divided into two groups, based on median TCC: rapid responders (≤71 days TCC; n = 21) and slow responders (>71 days TCC; n = 21). Whole blood at baseline, months 2 and 6 was stimulated with M tuberculosis (Mtb) antigens and Treg cells were then identified as CD3+CD4+CD25hiFoxP3+CD127−CD69− and further delineated as Ki67+HLA-DR− Treg. The frequency of these cells was significantly enlarged at baseline in SC positive relative to SC negative and smear positive relative to smear negative patients and in those with lung cavitation. This difference was further supported by unsupervised hierarchical clustering showing a significant grouping at baseline of total and early differentiated memory Treg cells in slow responders. Conversely, there was a clustering of a lower proportion of Treg cells and activated IFNγ-expressing T cells at baseline in the rapid responders. Examining changes over time revealed a more gradual reduction of Treg cells in slow responders relative to rapid responders to treatment. Receiver operating curve analysis showed that baseline Mtb-stimulated Ki67+HLA-DR− Treg cells could predict the TCC of MDR-TB treatment response with 81.2% sensitivity and 85% specificity (AUC of 0.87, p < 0.0001), but this was not the case after 2 months of treatment. In conclusion, our data show that the frequency of a highly defined Mtb-stimulated blood Treg cell population at baseline can discriminate MDR-TB disease severity and predict time to culture clearance.

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

“…As we expected, the CD4 + CD27 − T-cell subset was the predominantly activated population for all stimulation conditions, as demonstrated through HLA-DR, CD38, IFN-γ, and TNF-α expression levels, which is consistent with previous studies (18,21). CD4 + T-cells are important in controlling Mtb infection and those that are CD27 − are mostly TEMRA, EM, and effector cells which exert the quickest and strongest activated effector response (18,26). However, this subset is also the most likely to undergo activation induced cell death and to be exhausted from persistent antigen stimulation in vivo which will reduce their frequency (27).…”

“…Consequently, CD27 is expressed on central memory (CM) T-cells, variably expressed on effector memory (EM) T-cells and is not expressed on terminal effector memory (TEMRA) T-cells ( 22 – 25 ). As we expected, the CD4 + CD27 – T-cell subset was the predominantly activated population for all stimulation conditions, as demonstrated through HLA-DR, CD38, IFN-γ, and TNF-α expression levels, which is consistent with previous studies ( 18 , 21 ). CD4 + T-cells are important in controlling Mtb infection and those that are CD27 – are mostly TEMRA, EM, and effector cells which exert the quickest and strongest activated effector response ( 18 , 26 ).…”

“…CD38 and HLA-DR expression on CD4 + cells declines rapidly within the first month of treatment below the cut-off for ATB, while CD27 and Ki67 expression declined more slowly and this is correlated with mycobacterial load ( 3 , 10 ). PPD-specific CD4 + CD27 – T-cell frequencies have also been shown to distinguish healthy BCG vaccinated individuals, LTBI and ATB patients, suggesting exposure associated differentiation ( 14 , 18 ).…”

Emerging patterns of regulatory T cell function in tuberculosis

“…In contrast to animal model studies, where changes in circulating T reg frequency can impact infection levels, reports of T reg frequencies in human TB are varied. Some studies show an increase in peripheral T reg frequencies in TB [75–78]. However, our study [64] and others [79,80] found no differences in peripheral T reg frequencies between pulmonary TB patients and healthy controls.…”

IL-4 subverts mycobacterial containment inMycobacterium tuberculosis-infected human macrophages

“…While IL-4 has been implicated in the development [52] and maintenance of Tregs [53], the data are conflicting and this relationship has never been demonstrated in the context of human TB. There is substantial evidence on the detrimental role of Tregs in drugsensitive TB [25,[54][55][56], drug-resistant TB [26,57], and TB/HIV co-infection [58], including attenuation of mycobacterial containment in vitro [25,26], and downregulating Th1 responses [25,56]. There is substantial evidence on the detrimental role of Tregs in TB [25,26,56] including attenuation of mycobacterial containment in vitro [25,26], and downregulating Th1 responses [25,56].…”