Diagnosis of Mycobacterium tuberculosis infection using ESAT-6 and intracellular cytokine cytometry

Abstract

Diagnosis of infection with Mycobacterium tuberculosis (MTB) using tuberculin skin testing (TST) is often hampered by prior Bacille Calmette-Guérin (BCG) vaccination. ESAT-6 is a protein that is expressed by MTB but absent in BCG. It has been postulated that it might be useful in distinguishing MTB-specific immune responses. This study measured CD4 T cell responder frequencies specific for ESAT-6 and the TST reagent purified protein derivative (PPD) in patients with tuberculosis (n = 16), controls with non-tuberculous pneumonia (n = 8) and normal subjects (n = 7). Responses were identified using the intracellular cytokine staining technique and flow cytometry on whole blood samples, and performed blinded to the patient condition. Antigen-specific CD4 cells were defined by CD69 positivity and one or more cytokine [interleukin (IL)-2, IL-4, IL-10, interferon (IFN)-gamma] and/or CD40L positivity. With ESAT-6 stimulation it was found that TB patients had significantly higher frequencies of IFN-gamma and CD40L-positive CD4 T cells compared to the normal group, while no significant differences were measured with PPD stimulation. A responder frequency of 0.01% or higher for at least one of the measured cytokines/CD40L was defined as a positive response. Using this criterion to compare the two patient groups, PPD had 100% sensitivity but 0% specificity while ESAT-6 had 100% sensitivity and 88% specificity. Use of MTB-specific proteins such as ESAT-6 in combination with intracellular cytokine staining and flow cytometry has the potential to identify individuals with MTB infection.

Fig. 1

Each of the cytokine/CD40L responses to purified protein derivative (PPD) (left column) and ESAT-6 (right column) for the three groups studied; normal subjects, controls and cases. Note that responses are seen to each cytokine/CD40L but none produces a response in all subjects.

Fig. 2

Representative histograms detailing purified protein derivative (PPD) and ESAT-6 induced interferon (IFN)-γ and CD40L staining in CD4 T cells from subjects in the tuberculosis patient group (a–c) and control patient group (d–f). Histograms (b, c, e and f) were gated on CD4⁺ CD69⁺ lymphocytes (R3) in histograms (a) and (b), respectively. The percentage in each histogram is the frequency of CD4 cells positive for CD69 and IFN-γ or CD40L after subtracting staining from the non-stimulated control cells.