Impaired Expression of Perforin and Granulysin in CD8 + T Cells at the Site of Infection in Human Chronic Pulmonary Tuberculosis

Abstract

ABSTRACT Protective immunity in tuberculosis is dependent on the coordinated release of cytolytic effector molecules from effector T cells and the subsequent granule-associated killing of infected target cells. In this study, we investigated the expression of cytolytic (perforin and granzyme A) and antimicrobial (granulysin) molecules at the single-cell level in cryopreserved lung tissue from patients with chronic, progressive tuberculosis disease. Quantification of protein-expressing cells was performed by in situ imaging, while mRNA levels in the infected tissue were analyzed by real-time PCR. Persistent inflammation, including excessive expression of inducible nitric oxide synthase in CD68 + macrophages and significant infiltration of CD3 + , CD8 + and CD4 + T cells, was evident in tuberculosis lesions in all patients. However, despite the accumulation of CD3 + T cells, perforin- and granulysin-expressing CD3 + T cells were detected at two- to threefold-lower ratios in the tuberculosis lesions than in distal lung parenchyma and uninfected control lungs, respectively. This was evident at both the protein and mRNA levels. Moreover, perforin- and granulysin-expressing CD8 + T cells were scarce in individual granulomas within the tuberculosis lesions. In contrast, significant up-regulation of granzyme A-expressing CD3 + T cells was evident in the lesions from all patients. Confocal microscopy revealed coexpression of perforin and granulysin, primarily in CD8 + T cells; however, this expression was lower in the tuberculosis lesions. These findings suggest that symptomatic, chronic tuberculosis disease is associated with insufficient up-regulation of perforin and granulysin coexpression in CD8 + T cells at the local site of infection.