PLoS One. 2016 Feb 12;11(2):e0149108.
Mucorales-Specific T Cells in Patients with Hematologic Malignancies.
- 1Section of Hematology, Department of Surgical and Medical Sciences, University of Modena and Reggio Emilia, Azienda Ospedaliero-Universitaria Policlinico, Modena, Italy.
- 2Hematology and Bone Marrow Transplantation, Azienda Ospedaliero-Universitaria Santa Maria della Misericordia di Udine, Udine, Italy.
- 3Pediatric Hematology/Oncology, Policlinico GB Rossi, Verona, Italy.
- 4Department of Hematology, Universitaire Ziekenhuizen Leuven, Campus Gasthuisberg, Leuven, Belgium.
- 5Department of Pathology, Azienda Ospedaliero-Universitaria Policlinico, Modena, Italy.
- 6Clinic of Infectious Diseases, Integrated Department of Medicine, Emergency Medicine and Medical Specialties, Azienda Ospedaliero-Universitaria Policlinico, Modena, Italy.
- 7Section of Statistics, Department of Diagnostic, Clinical and Public Health Medicine, University of Modena and Reggio Emilia, Azienda Ospedaliero-Universitaria Policlinico, Modena, Italy.
- 8Division of Thoracic Surgery, Department of Surgical and Medical Sciences, University of Modena and Reggio Emilia, Azienda Ospedaliero-Universitaria Policlinico, Modena, Italy.
- 9Pediatric Hematology/Oncology and Transplantation, IRCCS Policlinico S. Matteo, Pavia, Italy.
- 10Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy.
- 11Unitè des Aspergillus, Pasteur Institut, Paris, France.
- 12Clinic of Infectious Diseases, Department of Internal Medicine, Geriatrics and Nephrologic Diseases, S. Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy.
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.
As clinicians working in the context of hematological malignancies, one of our major concerns consists in the insidious infections patients can develop during high-dose chemotherapy or allogeneic hematopoietic stemm cell transplant (alloSCT). Invasive Mucormycosis (IM) represents one of the most challenging fungal infection in this context, because of its difficult diagnosis and the crucial need to intiate a specific therapy as soon as possible to prevent fatal outcome. As a consequence, the lack of reliable and specific diagnostic tools for Invasive Mucormycosis is a major clinical problem. The analysis of the specific immune response against human pathogens represents nowadays a diagnostic tool for clinically relevant human infections, such as Tuberculosis . Searching for lymphocytes targeting specific infective agents allows to understand not only if there is an infection, but also how the immune system is fighting against it, more aggressively or more permissively.
Our group has already been demonstrated that Mucorales-specific T cells emerge in the course of documented Invasive Mucormycosis .
We have described in a prospective study the detection of T lymphocytes targeted against Mucorales in patients at high risk for developing such an infection. From October 2011 to January 2014, we have prospectively enrolled, into the study, 204 patients with acute myeloid or lymphoblastic leukemia undergoing induction chemotherapy, patients with other hematologic malignancies requiring either high dose chemotherapy or treatment with antithymocyte globulin, and patients undergoing alloSCT .
By using Elispot assay a total of 598 peripheral blood samples (average of 2.93 sample per patient) were analyzed in order to study Mucorales-specific T cells producing interferon-gamma (IFNγ), interleukin 10 (IL-10) and interleukin 4 (IL-4). Results of specific T cells were expressed as number of Spot Forming Cells (SPC) per million of peripheral blood mononuclear cells (PBMCs).
The ELISpot assay showed the presence of Mucorales-specific T cells in 21 of 204 patients (10.3%)
Figure 1: Mucorales specific T cells in patients without IFD are shown as absolute numbers of Spot Forming Cells per million PBMCs at different time points. Blue, yellow and red columns represent IFNγ-secreting, IL10 secreting and IL4 secreting T cells respectively.
We then correlated the finding of these specific cells to the clinical pictures of such patients, and analyzed the different “quality” of this immune defence. In facts, patients with positive results could be grouped into two categories according to the EORTC/MSG criteria . The first group consisted of 15 patients (n = 1-15) without clinical, microbiological and radiological features of Invasive Fungal Disease (IFD). In this group of patients the specific immune response against Mucorales are almost exclusively represented by IFNγ-secreting T cells as showed in Figure 1.
The second group was composed of 6 patients (n = 16-21) consisting of 4 possible IFD (patients n = 16-19) and 2 proven IM (n. 20 and 21). Of note, in this group of patients the Mucorales-specific immune repertoire are represented not only by IFNγ-secreting T cells but also by T cells secreting IL-10 and IL-4 as shown in Figure 2 with yellow and red columns respectively.
Figure 2: Mucorales-specific T cells in 2 patients with IM and 4 patients with possible IFD are shown as absolute numbers of Spot Forming Cells per million PBMCs at different time points. Blue, yellow and red columns represent IFNγ-secreting, IL10 secreting and IL4 secreting T cells respectively.
In the first group, the median number of Mucorales-specific T cells producing IFNγ was 62.5 (40-115); those T cells producing IL-10 was 0 (0-20) and those producing IL-4 was 0 (0-28) as well (Figure 3A). In this group of patients the differences between the number of IFNγ producing and IL-10 or IL-4 producing Mucorales-specific T cells resulted significantly different (p = 0.001).
These data resembling what observed in the setting of Invasive Aspergillosis (IA) where healthy subjects display higher frequencies of specific T cells producing IFNγ to Aspergillus antigens when compared to IA patients .
In the second group, the median number of Mucorales-specific T cells producing IFNγ was 100 (25-140); T cells producing IL-10 was 60 (30-140) and those producing IL-4 was 140 (76-180) (figure 3B). Of note, Mucorales-specific T cells producing IL-10 (p = 0.01) and IL-4 (p < 0.001) were significantly higher in the second group than in the first group, whereas no differences were shown for specific T cells producing IFNγ (p = 0.534), suggesting that group 2 patients had a stronger stimulation of the specific immune responses and such responses were shaped in a non-protective manner versus fungi of the order Mucorales.
These data are in line with previous reports: IM patients, as well as IA patients, have specific immune responses predominantly polarized to the production of IL-10 and/or IL-4 at the onset of the infection [2,6]. The increase of protective T cells occurs later and is associated with a favorable outcome .
These findings are important because most cases of IM are diagnosed as possible IFD, mainly due to the absence of sensitive and specific microbiological diagnostic tools and to the fact that tissue diagnosis, the gold standard, is only rarely performed in high risk patients, due to their several comorbidities. Our results further support the hypothesis that a type 2 cytokine profile is the immunological signature of IM. The concomitant absence of clinical signs of IFD in patients in the first group, characterized by a predominant type 1 cytokine profile, supports such a suggestion.
In conclusion, Mucorales-specific T cells may be routinely monitored in hematologic patients during chemotherapy/immunosuppressive regimens and alloSCT. The identification of high numbers of T cells producing IL-10 and/or IL-4 is associated with the diagnosis of proven IM. The detection of Mucorales-specific T cells should be explored as a Non-Culture-Based Diagnostic Method for the diagnosis of IM.
Figure 3: Comparison between frequencies of Mucorales-specific T cells in patients without IFD and with proven IM. (A). Box plots showing median value of specific immune responses in Group 1 patients (n=15) without IFD and (B) in the 2 patients with proven IM and 4 patients with possible IFD. The vertical axis shows the number of spot-forming cells (SFCs) per million PBMCs. The horizontal axis represents Mucorales-specific T cells producing IFNγ (blue), IL10 (yellow) and IL4 (red). The upper and the lower hinge of the boxes represents the 75th and 25th percentile respectively. The middle horizontal line of the boxes represents the median value. Blue, yellow and red dots are outrange values. * and ▲ = P< 0.05.
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