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.

We and others have previously reported on the presence of specific T cells in patients with invasive infections caused by filamentous fungi [18][19][20][21][22]. These T cells contribute to human immune responses against these fungi, and seem to correlate with the outcome of the infection [19][20][21][22]. Specifically, we demonstrated that Mucorales-specific T cells could be detected in three patients with documented IM during the course of the infection. Such T cells could not be demonstrated before the infection or at complete resolution, and were completely absent in 25 controls without IM [20]. These findings suggest that the presence of Mucorales-specific T cells is strongly associated with the occurrence of IM.
In the present study, we have prospectively evaluated the feasibility of monitoring the presence of Mucorales-specific T cells in patients with hematologic malignancies undergoing treatment that puts them at high risk for invasive fungal diseases (IFD). Furthermore, we have investigated whether the presence of Mucorales-specific T cells correlates with specific clinical presentation of disease.
The median number [spot forming cells (SFCs) / 10 6 peripheral blood mononuclear cells (PBMCs) with their 25th and 75th percentiles] of Mucorales-specific T cells producing IFN-γ was: 65 (40-127.5); those producing IL-10: 20 (20-65) and those producing IL-4: 60 (20-114), respectively (Fig 2).  When we correlated the occurrence of Mucorales-specific T cells with the clinical outcome, patients with positive results could be grouped into two categories (Figs 1 and 2). The first group consisted of 15 patients (n = 1-15) with clinical, microbiological and radiological features unlikely to be related to IFD, because they met one of the following criteria: 1) they were diagnosed with proven non-fungal infections; 2) they never developed fever during the study period; 3) their infection resolved without the administration of antifungal agents ( Table 2).  The second group was composed of 6 patients with proven IM (n = 2) and possible IFD (n = 4), according to the EORTC/MSG criteria (Table 2 and Fig 3) [23].
In the first group, the median number (SFCs/10 6 PBMCs with their 25th and 75th percentiles) of Mucorales-specific T cells producing IFN-γ 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. In this group of patients the differences in the number of Mucorales-specific T cells producing IFN-γ and IL-10, and IFN-γ and IL-4 resulted significantly different (p = 0.001) (Fig 4A). These findings are indicative of Mucorales-specific immune responses polarized to the production of IFN-γ in this group.
The second group was additionally split in the two patients with proven IM and in the four patients with possible IFD. In the 2 patients with proven IM, the median number (SFCs/10 6 PBMCs with their 25th and 75th percentiles) of Mucorales-specific T cells producing IFN-γ was 132.5 (122.5-207.5); those T cells producing IL-10 was 43.5 (30-66); those T cells producing IL-4 was 232 (128-355). In these two patients only the difference between Mucorales-specific T cells producing IFN-γ-and IL-10 resulted statistically different (p = 0.005).
In the four patients with possible IFD the median number (SFCs/10 6 PBMCs with their 25th and 75th percentiles) of Mucorales-specific T cells producing IFN-γ was 40 (10-100); those T cells producing IL-10 was 80 (0-185); those T cells producing IL-4 was 80 (68-180) ( Fig 4B). When we compared the patients without IFD (group 1) with the two patients with proven IM, it resulted that the magnitude of specific immune responses, evaluated either as the number of samples with more than 100 SFCs or as median SFCs, was significantly greater in proven patients. In particular, the number of analysis with 100 SFCs/10 6 PBMCs was 10 (7.41%) out of 135 in the first group and 8 (66.6%) out of 12 in the proven patients, respectively (p < 0.001). Moreover, these latter patients showed also significantly higher number of Mucorales-specific T cells producing IL-4 and IL-10 than the 15 patients without IFD (232 vs 0, p = 0.01; 43.5 vs 0, p = 0.03), suggesting an immune response polarized in a non-protective fashion versus fungi of the order Mucorales (Fig 2).
To verify the hypothesis that Mucorales-specific T cells, and, in particular, those producing type-2 cytokines, were associated with IM, we calculated the proportion of patients correctly "diagnosed" by the ELISpot assay and performed the receiver-operating characteristic (ROC) analysis, between the cases with a defined diagnosis with or without Mucorales-specific T cells, namely 17 patients versus 167 patients (Fig 1). The sensitivity and specificity of the test resulted 100% and 92%, respectively, when all the three types of Mucorales-specific T cells were considered, and 100% and 94.5% when only Mucorales-specific T cells producing IL-10 and IL-4 were evaluated (S1 Table and S2 Table). These data suggested that Mucorales-specific T cells producing type 2 cytokine were more suitable to detect IM and were consistent with what observed in the three proven IM cases previously reported [20].
The following cut-offs were derived: 30 SFCs/10 6 PBMCs for Mucorales-specific T cells producing IL-10; 70 SFCs/10 6 PBMCs for Mucorales-specific T cells producing IL-4 and 30 SFCs/ 10 6 PBMCs when both Mucorales-specific T cells producing IL-10 and IL-4 were considered (S1 Fig). To evaluate whether the 4 positive possible IFD could be upgraded to a higher degree of suspicion for IM, we analysed the sample positivity rates in these patients and made the comparison with patients with a proven diagnosis, by applying the derived cut-offs. When Mucorales-specific T cells producing IL-10 or IL-4 or both in combination were considered, the rates of positive samples in the 4 patients with possible IFD resulted 45% (5 out of 11) for each analysis. Such rates were significantly higher than those obtained in the 15 patients without IFD, namely 14% (5 out of 36) (p = 0.04), 8% (3 out of 36) (p = 0.01), 5.5% (2 out of 36) (p = 0.004), respectively, while did not differ from those obtained in the 2 patients with proven IM, 100% (4 out of 4) (p = 0.10, 0.10 and 0.27, respectively) (S3 Table).
In the 4 possible IFD patients, the occurrence of Mucorales-specific T cells preceded the detection of the pulmonary lesions in 2 out of 4 patients, namely patients n°17 and 19; was contemporary in patient 18 and was demonstrated 4 days later in patient 16 (Table 2).
Of note, none of the patients with proven/probable IA, fusariosis and candidiasis showed the occurrence of Mucorales-specific T cells. Accordingly, neither the two patients with proven IM nor the 4 possible IFD patients, with Mucorales-specific T cells, demonstrated the occurrence of Aspergillus-specific T cells in all the tested samples.
4 (25%) out of the 16 possible IFD patients without Mucorales-specific T cells demonstrated the occurrence of Aspergillus-specific T cells in one or more samples, but only three showed such cells directed to more than one antigen or polarized in the production of type-2 cytokine, suggesting that such patients could be upgraded to a higher degree of suspicion for IA. The remaining subject demonstrated Aspergillus-specific immune responses similar to those reported in healthy donors [19,24] (S4 Table).

Discussion
We have previously demonstrated the presence of Mucorales-specific T cells in three patients with documented IM [20]. Herein, we describe for the first time-in a prospective study-the frequency of Mucorales-specific T cells producing IFN-γ, IL-10 and IL-4 in a large cohort of hematologic patients at high risk for IFD. Mucorales-specific T cells could be demonstrated in 10.3% of 204 patients. Based on their characteristics, patients with positive results could be divided in two groups: group 1 (n = 15), unlikely to have IFD, and group 2 (n = 6), having proven IM or possible IFD, according to revised EORTC/MSG criteria [23].
In group 1, Mucorales-specific immune responses were polarized in a protective manner versus fungi of the order Mucorales, with a median number of Mucorales-specific T cells producing IFN-γ that was significantly higher than the number of specific T cells producing either IL-10 or IL-4 (p = 0.001). Similarly, in the setting of IA, healthy subjects display higher frequencies of specific T cells producing IFN-γ to Aspergillus antigens when compared to IA patients [19,[24][25][26].
Patients unlikely to have IFD and the 2 patients with proven IM were significantly different in: 1) their magnitude of specific immune responses, with the strongest responses in group 2 (p < 0.001) and 2) their frequencies of Mucorales-specific T cells producing IL-10 and IL-4, which were significantly higher in group 2 (p = < 0.03 and < 0.01, respectively). These findings suggest 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. Both features are usually associated with high antigen loads [26,27].
These data are consistent 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 [19,20,22]. The increase of protective T cells occurs later and is associated with a favorable outcome [19].
Of note, the predominant type 2 cytokine secretion profile of Mucorales-specific T cells in the four patients with possible IFD in group 2, is similar to that of the two patients with proven IM in this study and to that of the three patients with proven IM previously reported [20], while significantly differed from the 15 positive patients without IFD.
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 [4,[11][12][13].
Of note, only three of the 16 remaining possible patients, without Mucorales-specific T cells, showed Aspergillus-specific T cells suggesting an increase likelihood of IA. This is not surprising as the whole cohort of patients have been extensively screened with the current cultural and non-cultural diagnostic methods (NCBDM) for the diagnosis of IA. Thus, it would have been very unlikely to upgrade a higher number of possible IFD patients to a higher degree of IA suspicion. Indeed, even if we hypothesize that the three possible IFD patients with Aspergillus-specific T cells are true IA cases, it should be acknowledged that such a common work-out correctly classified as IA 33 out of 36 (92%), with rates of infection matching those of the most recent epidemiological studies [8,28,29]. On the other hand, only 2 proven IM cases (0,9%) have been identified. Such rates are sensibly lower than the ones reported for IM in the same epidemiological studies [8,28,29]. These findings additionally emphasize the need for NCBDM for IM more than IA.
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.
Some limitations of this study should be discussed. First, the samples taken at T1, usually a period of low leukocyte/lymphocyte counts, demonstrated the absence of vital T cells in approximately 20% of cases, thus showing not informative results. This finding may diminish the usefulness of the assay at least in the first part of post-treatment aplastic phase. However, this drawback can be overcome by repeating the analysis or by analyzing fresh blood samples without thawing, which is a procedure that further reduces the number of viable cells (unpublished data). Secondly, the study was not designed to determine the diagnostic accuracy of detecting Mucorales-specific T cells for the diagnosis of IM. It should be recognized that designing such a rigorous study may represent a problem, considering the tissue-based diagnosis difficulties previously mentioned [11]. However, if we use the ELISpot assay to upgrade at a higher level of IM suspicion the 4 cases of possible IFD, the assay show positivity rates, which seem to match the incidence of IM reported in several cohort of hematologic patients [29][30][31]. On the other hand, given the high rates of negative patients, our assay could be of help at least to reduce the number of patients at high risk for IM undergoing high dose antifungal treatment based only on clinical clues.
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 NCBDM for the diagnosis of IM.

Patients
From October 2011 to January 2014, 4 tertiary care centers prospectively enrolled 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 allogeneic stem cell transplantation (alloSCT) into the study.
The study has been approved by the University of Modena and Reggio Emilia, Comitato Etico Provinciale di Modena (CE-protocol number 2414-63/11). Written informed consent was obtained from each patient, or from the next of kin, caretaker, or guardian on behalf of the minors enrolled in the study, according to the Declaration of Helsinki.
A sample of 30 mL of PB was drawn from patients at three pre-defined time-periods during their routine clinical examinations: T1 from day 15 to 30, T2 from day 30 to day 45, T3 from day 45 to 60, being T0 the start of chemotherapy or transplant procedure.
All the patients underwent a protocol-defined diagnostic work-up to define, when possible, the site and the etiology of the infectious/inflammatory process. The diagnostic work-up included cultures of blood, urine and feces specimens, molecular and serologic assays for diagnosing the most common viral pathogens, pulmonary and/or abdominal computed tomography (CT) scan, bronchoalveolar lavage (BAL) and, whenever feasible, biopsy of the affected tissue. BAL fluid was cultured for bacterial and fungal pathogens, mycobacteria, and Legionella, and was studied with immunofluorescent staining for Pneumocystis jiroveci, and with molecular and cultural analysis for viral pathogens. The galactomannan assay (Platelia) was performed twice a week on serum samples of acute leukemia patients and alloSCT recipients, and, at discretion of the attending physician, in all the other patients. When available, it was also tested in BAL. An index 0.5 and 1.0 were considered positive, for serum and BAL respectively. Patients were also surveyed for the development of sinonasal and cerebral infections. When they developed symptoms suggestive of such infections, brain CT scan and/or magnetic resonance imaging scans were obtained. Whenever possible autopsy was performed and specimens of organs involved by the infectious process were sent for histopathology and culture. Hematoxylin-eosine, periodic acid-Schiff, Grocott or Gomori methenamine silver stains were all used to detect hyphal invasion in tissue samples.
The EORTC-MSG criteria have been used to define IFD cases [23].

ELISpot Assay
Mucorales-specific T cells producing IFN-γ, IL-10 and IL-4 have been investigated through the Enzyme Linked ImmunoSpot (ELISpot) assay. The assay has been performed by three of the authors (DV, PB and GR), unaware of the clinical conditions of the patients, as previously described [19,20,22]. Briefly, PBMCs were co-cultured with specific antigens on 96-well flat bottom plates coated with anti-cytokine antibodies, namely anti-IFN-γ, IL-10 and IL-4, respectively (Mabtech AB, Nacka Strand, Sweden) [20]. The antigen preparation used was represented by heat-killed germinated conidia after four cycles of sonication at 280 W with a frequency of 24 KhZ, from fungi of the order Mucorales, at a concentration of 200,000 conidia/mL. All test conditions were carried out in duplicate or triplicate when possible. As previously reported, a sample was considered positive when all the following conditions were satisfied: 1) the presence of at least 5 SFCs; 2) the presence of more than 5 SFCs compared with the negative control; 3) the presence of a stimulation index of 2 (defined as ratio of SFCs in the sample versus the negative control). A sample was considered not informative when this last condition was not met [19,20,22]. Results have been reported as median number of SFCs/10 6 PBMCs with their 25th and 75th percentiles.
Aspergillus-specific T cells producing IFN-γ, IL-10 and IL-4 have been detected as previously reported [19]. The antigens used were the seven Aspergillus recombinant antigens previously reported plus the protein of the family of β-(1,3)-glucan-modifying enzymes, namely SUN1p [19].

Statistical Analysis
Median test was used to compare the median number of Mucorales-specific T cells producing IFN-γ-, IL-10 and IL-4, respectively, in positive patients, including those without IFD and those with proven IM.
Absolute numbers and percentages of the tests with 100 SFCs/10 6 PBMCs were determined and compared between the two groups of patients, by using the Fisher's exact test. In the same groups, the differences in the frequencies of Mucorales-specific T cells producing IFN-γ-, IL-10-and IL-4, respectively, have been evaluated.
To define the cut-off values allowing to discriminate between the "true positive" cases from the "false positive" cases, sensitivity, specificity, positive and negative predictive values were calculated and ROC analysis was performed between the cases with a defined diagnosis, with or without Mucorales-specific T cells. The first positive or negative sample has been considered for the analyses. Fisher's exact test has been used to compare the rate of positive samples, according to the identified cut-offs, between the groups. P values < 0.05 were considered statistically significant. Results were analyzed using STATA software (11.0, College Station, Texas, USA).  Table. Proportion of patients correctly "diagnosed" by the ELISpot assay when all the three types of Mucorales-specific T cells were considered. (DOCX) S2 Table. Proportion of patients correctly "diagnosed" by the ELISpot assay when Mucorales-specific T cells producing IL-10 and IL-4 were considered.