The bone marrow represents an enrichment site of specific T lymphocytes against filamentous fungi

Bone marrow has already been described as an enrichment site for several antigen-specific T lymphocytes, but the presence of mould-specific T cells has never been investigated in the bone marrow. We have previously demonstrated that mould-specific T cells emerge in the peripheral blood of patients with invasive fungal infections (IFI) but tend to become undetectable after disease resolution. In seven patients with a history of IFI, we investigated the presence of mould-specific T cells secreting different cytokines in bone marrow and peripheral blood paired samples. The results showed that the frequencies of mould-specific T cells secreting the protective cytokine IFNI3 are significantly higher in bone marrow (BM) and are mainly represented by CD8+ T lymphocytes with effector phenotype. A putative disappearance of such protective BM responses after myeloablative therapy could contribute to the increased risk of IFI in hematologic patients

Mucorales-Specific T Cells in Patients with Hematologic Malignancies.

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

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

<p>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.</p

Clinical characteristics of the 21 patients positive for <i>Mucorales</i>-specific T cells.

<p>Clinical characteristics of the 21 patients positive for <i>Mucorales</i>-specific T cells.</p

Characterization of Specific Immune Responses to Different Aspergillus Antigens during the Course of Invasive Aspergillosis in Hematologic Patients

Several studies in mouse model of invasive aspergillosis (IA) and in healthy donors have shown that different Aspergillus antigens may stimulate different adaptive immune responses. However, the occurrence of Aspergillus-specific T cells have not yet been reported in patients with the disease. In patients with IA, we have investigated during the infection: a) whether and how specific T-cell responses to different Aspergillus antigens occur and develop; b) which antigens elicit the highest frequencies of protective immune responses and, c) whether such protective T cells could be expanded ex-vivo. Forty hematologic patients have been studied, including 22 patients with IA and 18 controls. Specific T cells producing IL-10, IFN-\u3b3, IL-4 and IL-17A have been characterized through enzyme linked immunospot and cytokine secretion assays on 88 peripheral blood (PB) samples, by using the following recombinant antigens: GEL1p, CRF1p, PEP1p, SOD1p, \u3b11-3glucan, \u3b21-3glucan, galactomannan. Specific T cells were expanded through short term culture. Aspergillus-specific T cells producing non-protective interleukin-10 (IL-10) and protective interferon-gamma (IFN-\u3b3) have been detected to all the antigens only in IA patients. Lower numbers of specific T cells producing IL-4 and IL-17A have also been shown. Protective T cells targeted predominantly Aspergillus cell wall antigens, tended to increase during the IA course and to be associated with a better clinical outcome. Aspergillus-specific T cells could be successfully generated from the PB of 8 out of 8 patients with IA and included cytotoxic subsets able to lyse Aspergillus hyphae. Aspergillus specific T-cell responses contribute to the clearance of the pathogen in immunosuppressed patients with IA and Aspergillus cell wall antigens are those mainly targeted by protective immune responses. Cytotoxic specific T cells can be expanded from immunosuppressed patients even during the infection by using the above mentioned antigens. These findings may be exploited for immunotherapeutic purposes in patients with I

Kinetics of specific T-cell responses to the seven recombinant antigens of <i>Aspergillus</i> by IFN-γ, IL-10, and IL-4 ELISpot assay in 22 patients with invasive aspergillosis (IA), patient 1 to patient 10.

<p>Kinetics of specific T-cell responses to the seven recombinant antigens of <i>Aspergillus</i> by IFN-γ, IL-10, and IL-4 ELISpot assay in 22 patients with invasive aspergillosis (IA), patient 1 to patient 10.</p

<i>Aspergillus</i>-specific T-cell responses to the 7 recombinant antigens during the course of Invasive Aspegillosis (IA).

<p><b>A,B,C,D. A.</b> Box plots showing specific immune responses producing IL-10 (yellow columns) to any single recombinant antigen. The horizontal axis represents the antigen, which the specific immune responses are directed to. <b>B.</b> Box plot showing specific immune responses producing IL-10 (yellow columns) against all the 7 recombinant antigens at the four phases of the IA. The horizontal axis represents the different phases of IA. <b>C.</b> Box plot showing specific immune responses producing IFN-γ (blue columns) to any single recombinant antigen. The horizontal axis represents the antigen, which the specific immune responses are directed to. <b>D.</b> Box plot showing specific immune responses producing IFN-γ (blue columns) against all the 7 recombinant antigens at the four phases of the IA. The horizontal axis represents the different phases of IA. The vertical axis shows the number of spot-forming cells (SFCs) per million peripheral blood mononuclear cells (PBMCs).The upper horizontal line represents the upper adjacent value. The upper hinge of the boxes represents the 75^th percentile. The middle horizontal line of the boxes represents the median value. The lower hinge of the boxes represents the 25^th percentile. The lower horizontal line represents the lower adjacent value. Yellow dots and blue dots are outrange values.</p

Cytokine production profile and lytic activities of <i>Aspergillus-</i>specific T cells.

<p><b>A, B.</b> The frequencies of <i>Aspergillus</i> specific T cells producing IFNγ, IL-10, IL-4 or IL-17A against the seven recombinant antigens, either as EM (dark gray portion of the columns) or CM (light gray portion of the columns), are shown as mean % positive cells ± standard deviation, computed over the 22 patients with IA. Results are expressed as percentages of either CD4⁺ T cells (A) or of CD8⁺ T cells (B). <b>C.</b> White columns represent rates of hyphal damage by <i>Aspergillus</i>-specific T cells expanded from PBMCs stimulated with heat killed <i>Aspergillus</i> conidia. Black columns represent rates of hyphal damage by <i>Aspergillus</i>-specific T cells expanded from PBMCs stimulated with <i>Aspergillus</i> recombinant antigens (GEL1, PEP1, α1–3 glucan, β1–3 glucan) at two and twenty two-hour cultures. E/T = effector/target cell ratio. <b>D.</b> Rates of hyphal damage by <i>Aspergillus</i>-specific T cells expanded with <i>Aspergillus</i> either heat killed conidia (white columns) or recombinant antigens (GEL1, PEP1, α1–3 glucan, β1–3 glucan) (black columns); by the supernatant of cultures of <i>Aspergillus</i>-specific T cell lines only; by antigen presenting cells (APCs); by polymophonuclears (PMNs) and by combinations of the different three cell fractions from three further patients with IA, at two-hour cultures. * = <i>P</i><.05. E/T = effector/target cell ratio. Results are expressed as mean+−Standard Deviation.</p