Highly Multiplexed Proteomic Analysis of Quantiferon Supernatants To Identify Biomarkers of Latent Tuberculosis Infection

Abstract

The tests for diagnosing latent tuberculosis infection (LTBI) are limited by a poor predictive value for identifying people at the highest risk for progressing to active tuberculosis (TB) and have various sensitivities and specificities in different populations. Identifying a more robust signature for LTBI is important for TB prevention and elimination. A pilot study was conducted with samples from immigrants to the United States that were screened for LTBI by the three commercially approved tests, namely, the tuberculin skin test (TST), the Quantiferon-TB Gold in-tube (QFT-GIT), and the T-SPOT.TB (T-SPOT). QFT-GIT supernatants from 13 people with concordant positive results and 26 people with concordant negative results were analyzed via the highly multiplexed SOMAscan proteomic assay. The proteins in the stimulated supernatants that distinguished LTBI from controls included interleukin-2 (IL-2), monocyte chemotactic protein 2 (MCP-2), interferon gamma inducible protein-10 (IP-10), interferon gamma (IFN-γ), tumor necrosis factor superfamily member 14 (TNFSF14, also known as LIGHT), monokine induced by gamma interferon (MIG), and granzyme B (P <0.00001). In addition, antigen stimulation increased the expression of heparin-binding EGF-like growth factor (HB-EGF) and activin AB in LTBI samples. In nil tubes, LIGHT was the most significant marker (P <0.0001) and was elevated in LTBI subjects. Other prominent markers in nonstimulated QFT-GIT supernatants were the complement-3 components C3b, iC3b, and C3d, which were upregulated in LTBI and markedly decreased upon stimulation. We found known and novel proteins that warrant further studies for developing improved tests for LTBI, for predicting progression to active disease, and for discriminating LTBI from active TB.

Keywords: biomarkers; diagnosis; immunity; latent infection; proteomics; tuberculosis.

FIG 1

Correlation of Qiagen QFT-GIT ELISA with SOMAscan data for differential IFN-γ signals between antigen and nil tubes. QFT-GIT supernatants in LTBI are in red and supernatants from HC are in blue. All samples from HC had <0.35 IU/ml and <400 RFU of IFN-γ released upon stimulation. Measurements of IFN-γ release in LTBI samples correlated well between ELISA and SOMAscan data, though ELISA has a smaller dynamic range with an upper limit at 10 IU/ml.

FIG 2

Volcano plot of the differential expression of proteins measured in stimulated QFT-GIT supernatants in LTBI compared with those in HC. The most statistically significant markers are shown toward the top, and proteins with the largest median up- or downregulation are toward the right and left, respectively.

FIG 3

(A) Top markers distinguishing LTBI and HC in stimulated QFT-GIT supernatants. Box plots show IL-2, MCP-2, IP-10, and IFN-γ levels in the supernatants from nil and stimulated tubes in LTBI and HC, along with the median upregulation of these proteins in LTBI (arrows). The association of the paired nil and stimulated samples from the 26 HC and 13 LTBI subjects is indicated by the gray connectors. (B) Models calculating the probability of LTBI were fit using the log-transformed differences between the nil and stimulated tubes. Using only IFN-γ RFU data, there is no decision boundary that perfectly separates the LTBI from HC (left). However, simply adding IL-2 to the model creates perfect separation (right).

FIG 4

Top markers distinguishing LTBI and HC in nonstimulated (nil) QFT-GIT supernatants. Box plots of LIGHT, C3b, and Holo-TC I levels in QFT-GIT supernatants from nil and stimulated tubes in LTBI and HC, along with the median upregulation of these proteins in LTBI (arrows). The association of the paired nil and stimulated samples from the 26 HC and 13 LTBI subjects is indicated by the gray connectors.

FIG 5

(A) Volcano plot of markers that distinguish LTBI from HC based on the fold change (FC) of differential expression in stimulated versus unstimulated samples. The most statistically significant markers are shown toward the top, and proteins with the largest median ratios of up- or downregulation in LTBI compared with those in HC are toward the right and left, respectively. (B) Box plots of two additional markers, activin AB and HB-EGF, in QFT-GIT supernatants from nil and stimulated tubes in LTBI and HC (IL-2, MCP-2, IP-10, and IFN-γ box plots are shown in Fig. 3).