Enhancement of human antigen-specific memory T-cell responses by interleukin-7 may improve accuracy in diagnosing tuberculosis

Abstract

Children and immunocompromised adults are at an increased risk of tuberculosis (TB), but diagnosis is more challenging. Recently developed gamma interferon (IFN-gamma) release assays provide increased sensitivity and specificity for diagnosis of latent TB, but their use is not FDA approved in immunocompromised or pediatric populations. Both populations have reduced numbers of T cells, which are major producers of IFN-gamma. Interleukin 7 (IL-7), a survival cytokine, stabilizes IFN-gamma message and increases protein production. IL-7 was added to antigen-stimulated lymphocytes to improve IFN-gamma responses as measured by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot (ELISPOT) assay. Antigens used were tetanus toxoid (n = 10), p24 (from human immunodeficiency virus [HIV], n = 9), and TB peptides (n = 15). Keyhole limpet hemocyanin was used as a negative control, and phytohemagglutinin was the positive control. IL-7 improved antigen-specific responses to all antigens tested including tetanus toxoid, HIV type 1 p24, and TB peptides (ESAT-6 and CFP-10) with up to a 14-fold increase (mean = 3.8), as measured by ELISA. Increased IFN-gamma responses from controls, HIV-positive patients, and TB patients were statistically significant, with P values of <0.05, 0.01, and 0.05, respectively. ELISPOT assay results confirmed ELISA findings (P values of <0.01, 0.02, and 0.03, respectively), with a strong correlation between the two tests (R(2) = 0.82 to 0.99). Based on average background levels, IL-7 increased detection of IFN-gamma by 39% compared to the level with antigen alone. Increased production of IFN-gamma induced by IL-7 improves sensitivity of ELISA and ELISPOT assays for all antigens tested. Further enhancement of IFN-gamma-based assays might improve TB diagnosis in those populations at highest risk for TB.

FIG. 1.

IL-7 enhances IFN-γ production from antigen-stimulated lymphocytes. Cells were incubated for 24 h with medium, 10 ng IL-7, KLH (negative-control antigen), KLH plus IL-7, antigen (tetanus toxoid, HIV p24, or TB peptides), or antigen plus IL-7 (n = 28). Supernatants were then assayed for IFN-γ using ELISA. IL-7 enhances antigen-specific responses 2- to 14-fold in all patients (P < 0.01), in 9 of 10 control patients (P < 0.05), in eight of the nine TB patients (P = 0.05), and in eight of the nine HIV⁺ patients (P = 0.01). Positive-control values (PHA, 1.0 μg/ml) are not shown, as all positive controls produced IFN-γ at levels above background with values ranging from 0.43 to 81.3 IU/ml. Statistically significant differences were also seen between the IFN-γ levels produced in response to antigen and the levels produced by unstimulated and negative controls (P < 0.05).

FIG. 2.

Addition of IL-7 increases ELISA sensitivity. The FDA-approved QFT-G defines a positive result as 0.35 IU/ml or greater. This graph displays the increased sensitivity in response to antigen stimulation after addition of IL-7 in all study subjects (10 controls, nine TB patients, and nine HIV⁺ patients). Increases with the addition of IL-7 were evenly distributed in each study group: TB response to TB peptides (range = 1.26 to 9.2, mean = 3.6, median = 2.1), HIV response to HIV-1 p24 (range = 1.0 to 7.3, mean = 3.5, median = 2.8), and control response to tetanus toxoid (range = 1.6 to 14.8, mean = 3.2, median = 3.1). When the QFT-G value of 0.35 IU/ml is used, IL-7 increased sensitivity, from 30% (7/23) to 70% (16/23).

FIG. 3.

Increase in effector cell number with IL-7 addition. Cells were incubated in ELISPOT plates for 20 h with medium alone, 10 ng IL-7, KLH (negative-control antigen), KLH plus IL-7, antigen (tetanus toxoid [TT], HIV p24, or TB peptides (ESAT-6 and CFP-10), or antigen plus IL-7 (n = 18). Plates were developed, and spots were counted using an automated reader. IL-7 enhances antigen-specific responses up to 16-fold (mean = 3) in all patients (P < 0.01), in all control patients (P < 0.01), in all TB patients (P = 0.03), and in all of the HIV⁺ patients (P = 0.02). Statistically significant differences were also seen between the IFN-γ levels produced in response to antigen and the levels produced by unstimulated and negative controls (P < 0.05).

FIG. 4.

Correlations between ELISA and ELISPOT results for two tetanus toxoid-stimulated control patients (A and B), two TB⁺ peptide-stimulated patients (C and D), and two HIV⁺ HIV-1 p24-stimulated patients (E and F). Correlations include ELISA and ELISPOT results for all conditions and are specific to the individual patients. Coefficient of determination values for graphs shown are two examples from each population sampled: controls (A [R² = 0.99] and B [R² = 0.99]), tetanus toxoid patients (C [R² = 0.99] and D [R² = 0.99]), and HIV⁺ patients (E [R² = 0.99] and F [R² = 0.99]).