Mucorales-Specific T Cells in Patients with Hematologic Malignancies

Abstract

Background: Invasive mucormycosis (IM) is an emerging life-threatening fungal infection. It is difficult to obtain a definite diagnosis and to initiate timely intervention. Mucorales-specific T cells occur during the course of IM and are involved in the clearance of the infection. We have evaluated the feasibility of detecting Mucorales-specific T cells in hematological patients at risk for IM, and have correlated the detection of such cells with the clinical conditions of the patients.

Methods and findings: By using an enzyme linked immunospot assay, the presence of Mucorales-specific T cells in peripheral blood (PB) samples has been investigated at three time points during high-dose chemotherapy for hematologic malignancies. Mucorales-specific T cells producing interferon-γ, interleukin-10 and interleukin-4 were analysed in order to detect a correlation between the immune response and the clinical picture. Twenty-one (10.3%) of 204 patients, accounting for 32 (5.3%) of 598 PB samples, tested positive for Mucorales-specific T cells. Two groups could be identified. Group 1, including 15 patients without signs or symptoms of invasive fungal diseases (IFD), showed a predominance of Mucorales-specific T cells producing interferon-gamma. Group 2 included 6 patients with a clinical picture consistent with invasive fungal disease (IFD): 2 cases of proven IM and 4 cases of possible IFD. The proven patients had significantly higher number of Mucorales-specific T cells producing interleukin-10 and interleukin-4 and higher rates of positive samples by using derived diagnostic cut-offs when compared with the 15 patients without IFD.

Conclusions: Mucorales-specific T cells can be detected and monitored in patients with hematologic malignancies at risk for IM. Mucorales-specific T cells polarized to the production of T helper type 2 cytokines are associated with proven IM and may be evaluated as a surrogate diagnostic marker for IM.

Fig 1. Classification of the patients according with the results of the ELISpot assay.

IFD = invasive fungal disease. IM = invasive mucormycosis; IA = invasive aspergillosis. Grey boxes indicate the patients with a defined diagnosis considered for the computation of sensitivity, specificity and receiver-operating characteristic analysis.

Fig 2

(A-C). Kinetics of T-cell responses in the patients showing Mucorales-specific T cells. (A) Kinetics of Mucorales-specific T cells producing IFN-γ (blue); (B) IL-10 (yellow); (C) IL-4 (red) in the 15 patients without IFD (light colours) and in the 6 patients with IFD (dark colours), 2 proven IM and 4 possible IFD, at each time period of the study. The central box represents the values from the lower to upper quartile (25 to 75 percentile). The middle line represents the median. The horizontal line extends from the minimum to the maximum value. Triangles and dots show "outside" and "far out" values, respectively. The vertical axis shows the number of spot-forming cells (SFCs) per million PBMCs producing a specific cytokine. The horizontal axis represents the three time periods, when peripheral blood samples have been obtained.

Fig 3

(A-F). Radiologic findings of patients from group 2 with possible IFD (A–D) and proven IM (E and F). (A) Patient 16. Well shaped nodular lesion of 4.5 cm, with surrounding area of ground glass, and air crescent sign in the posterior part of the upper left pulmonary lobe at HRCT. (B) Patient 17. Pulmonary HRCT demonstrating a small well shaped nodular lesion of the left upper lobe. (C) Patient 18. Pulmonary HRCT showing multiple bilateral well shaped nodular lesions, sometimes surrounded by ground-glass attenuation. Bilateral pleural effusions. (D) Patient 19. HRCT showing a large (4.7 cm) nodular lesion surrounded by an area of ground glass attenuation in the posterior part of the upper left pulmonary lobe. (E) Patient 20. Sagittal sinus CT scan. Soft tissue obliteration of ethmoid and middle meatus, erosion of ethmoid bony lamellae and spheno-etmoidal floor. Obliteration of frontal and sphenoidal sinus. (F) Patient 21. CT of the chest showing a central consolidation surrounded by a rim of ground-glass opacity within a large left hilar pulmonary consolidation (reverse halo-sign appearance). HRCT = high resolution computed tomography.

Fig 4

(A,B) Comparison between frequencies of Mucorales-specific T cells in patients without IFD and with proven IM. (A). Box plots showing specific immune responses producing IFN-γ (blue column), IL-10 (yellow column) and IL-4 (red column) in Group 1 patients (n = 15) without IFD. (B). Box plots showing specific immune responses producing IFN-γ (blue column), IL-10 (yellow column) and IL-4 (red column) in the 2 patients with proven IM. The vertical axis shows the number of spot-forming cells (SFCs) per million PBMCs. The horizontal axis represents the cytokine produced by Mucorales-specific T cells. The upper horizontal line represents the upper adjacent value. The upper hinge of the boxes represents the 75th percentile. The middle horizontal line of the boxes represents the median value. The lower hinge of the boxes represents the 25th percentile. The lower horizontal line represents the lower adjacent value. Blue, yellow and red dots are outrange values. * and ^▲ = P< 0.05.