Expression of activation and cytotoxic molecules by peripheral blood lymphocytes of patients with paracoccidioidomycosis

Abstract

In a previous study, we reported an increased number of T CD8⁺ cells in the bronchoalveolar lavage (BAL) of patients with pulmonary paracoccidioidomycosis, suggesting a role for these cells in the local immune response. The aims of this study were to verify, by flow cytometry, the activation state, as well as the production of cytotoxic molecules by peripheral blood lymphocytes (CD8⁺ and CD4⁺). Specimens were obtained from patients with paracoccidioidomycosis (PCM), individuals with PCM-infection, i.e., healthy individuals with demonstrated strong cellular response against the fungus (PI) and controls, with studies conducted both ex-vivo and in vitro, after stimulation with Paracoccidioides brasiliensis yeast cells. The ex-vivo analysis demonstrated that PCM patients presented a lower frequency of granzyme A, B and perforin-positive cells, as compared to individuals with PCM infection (PI). P. brasiliensis stimulation led to a discrete increase in CD69⁺ cells and a reduction in cytotoxic granule expression in all groups. The addition of IL-15 induced an increase in the frequency of CD69⁺ cells only in PI individuals and controls. The effect of IL-15 on granzyme A and B expression was low, but a higher frequency of CD8⁺ perforin⁺ was detected in PI individuals than in patients with active PCM. IL-15Rα expression was lower in CD4⁺ T cells from patients, in relation to the PI group. Furthermore, low levels of granulysin were detected in sera from PCM patients, but a tendency for an increase in these levels was observed after antifungal therapy. Taken together, these results indicate that lymphocytes from PCM patients are poorly activated, express low levels of IL-15Rα and produce basal levels of cytotoxic granules. These findings may account for the defective cytotoxic activity in patients and, consequently, a low capacity to kill the fungus.

Keywords:

• Paracoccidioidomycosis
• CD8⁺ T cells
• perforin
• granulysin

Acknowledgements

We would like to thank Dr Carol Clayberger (Stanford University) and Dr Ana Paula Galvão for the anti-granulysin antibody and Dr Nicola Conran for English language editing. This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Pesquisas (CNPq).

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

This paper was first published online on Early Online on 17 February 2010.