Interferon (IFN)-gamma producing Natural Killer (NK) cells in differential diagnosis of tuberculous pleurisy. Virtues of a functional innate immune cell based assay.

SCHIERLOH P,; MUSELLA R,; CASTAGNINO J,; ABBATE E,; SASIAIN MC.

Virginia, USA

Simposio; Conference Immunodiagnosis of Tuberculosis:New Questions, New Tools; 2008

T and B cell based immunodiagnostics methods of tuberculosis (TB) are less efficient in populations with high coverage of BCG vaccination as well as high prevalence of healthy PPD+ and/or infected HIV+ individuals because of false positive and/or false negative test results (1). In this context, differential diagnosis between TB and not TB pleurisy remains as an unresolved clinical issue. In previous studies, employing TB patients pleural fluid mononuclear cells (PFMC), we found and characterised a “locally primed” NK cell population that, upon Mycobacterium tuberculosis (Mtb) stimulation, rapidly and consistently produce IFN-g (2,3). In the present preliminary retrospective work, we evaluated the potential diagnostic value of pleural NK and T cell mediated IFN-g response. Assays were carried out by Mtb-stimulation of PFMC for 24h and FACS analysis on CD3-CD56+ (NK) and CD3+ (T cell) gated cells. Samples from TB (N=26) and not TB (Malignant=6; Paraneumonic=3 and Helminthic=1) were employed. The specific response was calculated as:  D% IFN-g+ NK (or T cell) =% Mtb-stimulated IFN-g+ NK (or T cell) - % unstimulated IFN-g+ NK (or T cell) . The cut-off value determined by ROC analysis for D% IFN-g+ NK was 1.55%, sensitivity was 88.9% (51.8-99.7%, CI95%) and specificity was 95.2% (76.2-99.9%). The cut-off for D% IFN-g+ T cell was 0.50% with a sensitivity of 77.8% (39.9-97.2%) and specificity of 90.0% (68.3-98.8%). Besides, median D% IFN-g+ NK was 20.96% (7.22-42.18%; percentile25-75% range) and median D% IFN-g+ T cell was 2.26% (1.10-5.55%). These differences may reflect the polyclonal vs oligoclonal nature of IFN-g response in NK vs T cells within pleural space (4).   In spite of our limited casuistic, this niche specific NK cell based assay suggests better performance than a T cell assay. Hence, we conclude that pleurisy, the most common extrapulmonary clinical presentation of TB, constitutes a particular model where local innate immune response may have relevant properties with advantage in diagnosis, like short time requirements and high specificity. So, NK-IFN-g response may have a potential application field in high antigen exposed human populations.     1.        Perkins, M.D. 2000. New diagnostic tools for tuberculosis. Int. J. Tuberc. Lung Dis. 4(12):182-188. 2.        Schierloh, P. et al. 2005. Increased Susceptibility to Apoptosis of CD56dimCD16+ NK Cells Induces the Enrichment of IFN-g-Producing CD56bright Cells in Tuberculous Pleurisy. The Journal of Immunology. 175: 6852–6860. 3.        Schierloh, P. et al. 2007. Mycobacterium tuberculosis-Induced Gamma Interferon Production by Natural Killer Cells Requires Cross Talk with Antigen-Presenting Cells Involving Toll-Like Receptors 2 and 4 and the Mannose Receptor in Tuberculous Pleurisy. Infection and Immunity. 75 (11): 5325–5337. 4.        Medzhitov, R. & C. A. Janeway Jr. 1997. Innate immunity: the virtues of a nonclonal system of recognition. Cell. 91: 295-298.