Mucorales-specific T cells were investigated in 28 hematologic patients during the course of their treatment. Three developed proven invasive mucormycosis (IM), 17 had infections of known origin but other than IM, and 8 never had fever during the period of observation. Mucorales-specific T cells could be detected only in patients with IM, both at diagnosis and throughout the entire course of the IM, but neither before nor for long after resolution of the infection. Such T cells predominantly produced IL-4, IFN-γ, IL-10, and to a lesser extent IL-17 and belonged to either CD4+ or CD8+ subsets. The specific T cells that produced IFN-γ were able to directly induce damage to Mucorales hyphae. None of the 25 patients without IM had Mucorales-specific T cells. Specific T cells contribute to human immune responses against fungi of the order Mucorales and could be evaluated as a surrogate diagnostic marker of IM.

Invasive mucormycosis (IM), the second most common cause of invasive mold infections in hematologic patients, has mortality rates approaching 70% of affected individuals because of difficulties in obtaining an early definite diagnosis.1-5  In fact, a definitive diagnosis of IM relies exclusively on both histopathologic demonstration and cultural isolation of the pathogen from the involved organs.6  However, the obtainment of tissue specimens in hematologic patients is too often hampered by the presence of several comorbidities, and histologically proven IM may fail to grow in culture in at least one third of cases.7  Furthermore, neither serologic nor antigenic diagnostic methods exist, and the use of polymerase chain reaction has been limited almost exclusively to the identification and discrimination of fungal species.8,9 

Adaptive immunity has been reported to play a crucial role in the defense of the host against fungi, at least in the case of invasive aspergillosis and invasive candidiasis,10,11  and the recognition and enumeration of antigen-specific T cells has been demonstrated to be a useful tool for the diagnosis of definite infectious diseases, particularly of either active or latent tuberculosis.12  We therefore explored the possibility that Mucorales-specific T cells are elicited in patients with IM and that their detection may be of value in the diagnosis of active disease.

Twenty-eight hematologic patients were studied. Patients 1-3 had disseminated (pulmonary and splenic), tracheobronchial, and cerebral proven IM, respectively (supplemental Figures 1-2, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). The antifungal treatments of the 3 patients with proven IM are described in detail in supplemental Methods.

The remaining 25 cases included 17 patients who presented with infectious complications of proven origin on the basis of cultural or histologic examinations, but other than IM, and 8 who did not develop infectious complications during the course of their induction chemotherapy. Patients' clinical characteristics are given in supplemental Table 1. Informed consent was obtained in accordance with the Declaration of Helsinki, and the study was approved by the University of Modena and Reggio Emilia ethics committee.

An enzyme-linked immunospot (ELISpot) assay was performed to detect either Mucorales- or Aspergillus-specific T cells, as reported previously13  and described in detail in supplemental Methods, on 80 peripheral blood samples (range 2-6 per patient). Time points analyzed were as follows: in patient 1, at the beginning of induction chemotherapy, 20 days before the pulmonary biopsy, at the histologic and cultural identification of Rhizomucor pusillus infection, at the beginning of consolidation chemotherapy, and 16 weeks after resolution of the infection; in patient 2, on the day of cultural and histologic demonstration of Rhizopus oryzae infection and until death on 4 further occasions during the course of IM; in patient 3, on the day of histologic and molecular demonstration of Absydia corymbifera infection, on 3 occasions during the course of IM, and on complete resolution of the infection. All other patients were analyzed at least twice during the course of their infections or chemotherapeutic treatments (supplemental Table 1).

The phenotypic and functional characterization of Mucorales-specific T cells has been performed with a cytokine secretion assay, as reported previously14  and described in detail in supplemental Methods. Molecular studies, micromanipulation, and single-cell PCR to identify Mucorales species (supplemental Figure 2) were performed as reported previously15  and described in detail in supplemental Methods. Anti-Mucorales activity of specific T cells was assessed as reported in supplemental Methods.

Identification of Mucorales-specific T cells

In patient 1, the ELISpot was positive for the presence of Mucorales-specific T cells producing IL-10 in the second, third, fourth, and fifth samples and Mucorales-specific T cells producing IFN-γ in the second, third, and fourth samples. In contrast, no Mucorales-specific T cells could be detected before the occurrence of the infection (at day +1 of induction chemotherapy) or long after its resolution (day +238; Figure 1A).

Figure 1

Kinetics of Mucorales-specific T-cell responses by IFN-γ, IL-10, and IL-4 ELISpot assay in the 3 patients with invasive mucormycosis. (A) Patient 1. (B) Patient 2. (C) Patient 3. Yellow columns represent the number of Mucorales-specific T cells producing IL-10; blue columns, the number of Mucorales-specific T cells producing IFN-γ; red columns, the number of Mucorales-specific T cells producing IL-4; and dark gray background, T-cell responses in wells with phytohemagglutinin (PHA). Vertical axis shows the number of spot-forming cells (SFCs) per million peripheral blood mononuclear cells (PBMCs); horizontal axis indicates the time (in days) from the beginning of induction chemotherapy.

Figure 1

Kinetics of Mucorales-specific T-cell responses by IFN-γ, IL-10, and IL-4 ELISpot assay in the 3 patients with invasive mucormycosis. (A) Patient 1. (B) Patient 2. (C) Patient 3. Yellow columns represent the number of Mucorales-specific T cells producing IL-10; blue columns, the number of Mucorales-specific T cells producing IFN-γ; red columns, the number of Mucorales-specific T cells producing IL-4; and dark gray background, T-cell responses in wells with phytohemagglutinin (PHA). Vertical axis shows the number of spot-forming cells (SFCs) per million peripheral blood mononuclear cells (PBMCs); horizontal axis indicates the time (in days) from the beginning of induction chemotherapy.

In patients 2 and 3, the ELISpot showed the sole presence of Mucorales-specific T cells producing IL-10 in the first sample (on the day of cultural and histologic demonstration of IM) in both patients; increasing numbers of Mucorales-specific T cells producing IFN-γ in the second, third, and fourth samples in patient 2 and in the third and fourth samples in patient 3; and the occurrence of Mucorales-specific T cells producing IL-4 in the fourth sample in patient 2 and in the third and fourth samples in patient 3. The last examination demonstrated the sole presence of Mucorales-specific T cells producing IL-10 in patient 2, close to the time of the patient's death, and the absence of specific responses in patient 3 at the time of complete resolution of the infection (Figure 1B,C).

The differences in the median frequencies of Mucorales-specific T cells producing IL-10, IFN-γ, and IL-4 were not statistically significant in the 3 patients (P = .3), even when the results of the first 2 patients with more disseminated diseases were compared with those of the third patient, who had a more limited infection (P = .5). In the 25 control patients, the ELISpot never showed the presence of Mucorales-specific T cells. None of the analyzed patients demonstrated the occurrence of Aspergillus-specific T cells at any time point (supplemental Table 1).

Phenotypic and functional characterization of Mucorales-specific T cells

In patients 1-3, Mucorales-specific T cells were (1) predominantly CD8+ T cells (mean CD8+/CD4+ frequencies 3.62%/0.57%) of the CM (central memory) phenotype, producing IFN-γ; (2) predominantly CD8+ T cells (mean CD8+/CD4+ frequencies 4.35%/2.60%) of the EM (effector memory) phenotype, producing IL-4; or (3) either CD4+ or CD8+ T cells (mean CD4+/CD8+ frequencies 0.32%/0.26%), the former of either the CM or EM phenotype, and the latter mainly of the CM phenotype, producing IL-10. Mucorales-specific T cells producing IL-17 were also detectable, which were either CD4+ or CD8+ (mean frequency 0.44% and 0.56%, respectively), and exhibited predominantly the CM phenotype (Figure 2A-B).

Figure 2

Cytokine production profile and lytic activities of Mucorales-specific T cells. (A-B) The frequencies of Mucorales-specific T cells producing IFNγ, IL-10, IL-4, or IL-17, either as EM (light gray) or CM (dark gray), are shown as the mean percentage of positive cells, computed for the 3 patients with IM. Results are expressed as percentages of either CD4+ T cells (A) or CD8+ T cells (B). Mean frequencies of specific cytokine-producing T cells for individual patients are reported in each column, either as EM (■) or CM (○). (C-D) Hyphal damage at 2 (C) and 22 (D) hours to Rhizomucor pusillus and Rhizopus oryzae hyphae induced by anti-Mucorales T cells (T), polymorphonuclear leukocytes (PMNs), and antigen-presenting cells (APCs), alone or in combination, derived from patients 1 and 2 during the course of IM. E:T indicates effector/target cell ratio.

Figure 2

Cytokine production profile and lytic activities of Mucorales-specific T cells. (A-B) The frequencies of Mucorales-specific T cells producing IFNγ, IL-10, IL-4, or IL-17, either as EM (light gray) or CM (dark gray), are shown as the mean percentage of positive cells, computed for the 3 patients with IM. Results are expressed as percentages of either CD4+ T cells (A) or CD8+ T cells (B). Mean frequencies of specific cytokine-producing T cells for individual patients are reported in each column, either as EM (■) or CM (○). (C-D) Hyphal damage at 2 (C) and 22 (D) hours to Rhizomucor pusillus and Rhizopus oryzae hyphae induced by anti-Mucorales T cells (T), polymorphonuclear leukocytes (PMNs), and antigen-presenting cells (APCs), alone or in combination, derived from patients 1 and 2 during the course of IM. E:T indicates effector/target cell ratio.

Lytic activity of Mucorales-specific T cells

Mucorales-specific T cells from patients 1-3 were able to induce direct damage to the hyphae of the 2 clinical isolates, similar to that of either polymorphonuclear leukocytes or APCs. Only the combination of all 3 cell types resulted in significantly greater damage to the hyphae (P < .05; Figure 2C-D).

We have shown for the first time that Mucorales-specific T cells may occur during the course of infection in patients with IM and that they exhibit direct antifungal activity comparable, at least in vitro, to that of either polymorphonuclear leukocytes or APCs. The contribution of T cells to host defenses against these moulds could only be suspected on the basis of the enhanced fungicidal activity against Mucorales of polymorphonuclear leukocytes exposed to IFN-γ,16  but this has not yet been demonstrated formally.

The presence of Mucorales-specific T cells only during the course of IM and neither before infection nor after resolution of the infection in patients 1-3, as well as their absence in patients without infections or with infections other than IM, suggests that they are closely related to the occurrence of IM and may be a marker of overt disease. Of note, the presence of Mucorales-specific T cells was the only proof of IM in patient 1, before obtainment of the biopsy. The lower frequencies of specific T cells in patient 3 appear to suggest that a more confined IM could be associated with responses of an inferior magnitude; however, no statistically significant differences were observed in the median numbers of Mucorales-specific T cells in the 3 patients in the present study. Unfortunately, because all the samples were collected either when the patients were undergoing antifungal treatment or after withdrawal of the drug, no interaction between antifungal therapy and the occurrence of Mucorales-specific T cells could be determined in the present study.

The cytokine production profile of Mucorales-specific T cells in the present study was partially in line with what observed either in mice affected by invasive aspergillosis or in human T-cell clones stimulated with different Aspergillus antigens in vitro.17-19  The demonstration that CD8+Mucorales-specific T cells may produce either IL-4 or IL-10, predominantly in the late phase of the infection, is reminiscent of the type 2 cytokine shift of CD8+ lymphocytes, thus far reported only in patients with the cavitary phase of tuberculosis and in the late phase of HIV infection.20,21 

In conclusion, Mucorales-specific T cells emerge in the course of IM and contribute to human immune responses against Mucorales. The detection of Mucorales-specific T cells may be evaluated as a surrogate diagnostic marker of IM.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

This study was supported by the Associazione Italiana per la Ricerca sul Cancro, Milan, Italy (M.L.); the European Commission's FP6 Life Science Health Program (INCA project; LSHC-CT-2005-018704) (M.L.); the Associazione Italiana Lotta alle Leucemie, Linfoma e Mieloma-Sezione “Luciano Pavarotti”-Modena-ONLUS (L.P. and F.F.); the Programma di ricerca Regione-Università 2007-2009, Regione Emilia Romagna (M.L. and F.N.); and the Società Italiana di Ematologia Sperimentale (“Piero Martino” award, L.P.).

Contribution: L.P. and M.L. conceived and designed the study and wrote the manuscript; D.V., P.B., G. Riva, E.Z., C.Q., G. Rossi, F.R., and M.P. performed the ELISpot analysis, the cytokine secretion assay analysis, the XTT assays, the histologic examination, and the molecular characterization of fungi and interpreted the data; F.F., A.C., J.M., M. Morselli, M.C., A.P., M. Maccaferri, R.M., and F.N. provided well-characterized patient samples and critically revised the manuscript; C.D.G. and R.D. performed the statistical analysis and interpreted the data; and F.C. performed the radiologic studies and critically revised the manuscript.

Conflict-of-interest disclosure: M.L. received research funds from and serves on advisory boards for Merck Sharp & Dohme and Gilead Sciences and received honoraria from these 2 pharmaceutical companies and from Pfizer and Nanogen; L.P. serves on an advisory board for Merck Sharp & Dohme; A.C. serves on an advisory board for Merck Sharp & Dohme and received funds by Merck Sharp & Dohme, Gilead Sciences, and Pfizer; L.P., P.B., and M.L. have applied for a European patent regarding clinical applications of the ELISpot assay for the diagnosis of Aspergillus infection (PCT Nos. WO2008/075395A3, EP2094295, IT2007/000867); and L.P., D.V., P.B., F.F., and M.L. have applied for an Italian patent regarding clinical applications of the ELISpot assay for the diagnosis of Mucorales infection (No. MI2010A002224). The remaining authors declare no competing financial interests.

Correspondence: Mario Luppi, MD, PhD, Professor of Hematology, Chief, Division of Hematology, Department of Oncology, Hematology and Respiratory Diseases, University of Modena and Reggio Emilia, Azienda Ospedaliero-Universitaria, Policlinico, Modena, Italy; e-mail: mario.luppi@unimore.it.

1
Hibbett
 
DS
Binder
 
M
Bischoff
 
JF
, et al. 
A higher-level phylogenetic classification of the Fungi.
Mycol Res
2007
, vol. 
111
 
pt 5
(pg. 
509
-
547
)
2
Kontoyiannis
 
DP
Marr
 
KA
Park
 
BJ
, et al. 
Prospective surveillance for invasive fungal infections in hematopoietic stem cell transplant recipients, 2001-2006: overview of the Transplant-Associated Infection Surveillance Network (TRANSNET) Database.
Clin Infect Dis
2010
, vol. 
50
 
8
(pg. 
1091
-
1100
)
3
Pagano
 
L
Caira
 
M
Candoni
 
A
, et al. 
The epidemiology of fungal infections in patients with hematologic malignancies: the SEIFEM-2004 study.
Haematologica
2006
, vol. 
91
 
8
(pg. 
1068
-
1075
)
4
Chamilos
 
G
Luna
 
M
Lewis
 
RE
, et al. 
Invasive fungal infections in patients with hematologic malignancies in a tertiary care cancer center: an autopsy study over a 15-year period (1989-2003).
Haematologica
2006
, vol. 
91
 
7
(pg. 
986
-
989
)
5
Chamilos
 
G
Lewis
 
RE
Kontoyiannis
 
DP
Delaying amphotericin B-based frontline therapy significantly increases mortality among patients with hematologic malignancy who have zygomycosis.
Clin Infect Dis
2008
, vol. 
47
 (pg. 
503
-
509
)
6
De Pauw
 
B
Walsh
 
TJ
Donnelly
 
JP
, et al. 
Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group.
Clin Infect Dis
2008
, vol. 
46
 
12
(pg. 
1813
-
1821
)
7
Roden
 
MM
Zaoutis
 
TE
Buchanan
 
WL
, et al. 
Epidemiology and outcome of zygomycosis: a review of 929 reported cases.
Clin Infect Dis
2005
, vol. 
41
 
5
(pg. 
634
-
653
)
8
Bialek
 
R
Konrad
 
F
Kern
 
J
, et al. 
PCR based identification and discrimination of agents of mucormycosis and aspergillosis in paraffin wax embedded tissue.
J Clin Pathol
2005
, vol. 
58
 
11
(pg. 
1180
-
1184
)
9
Dannaoui
 
E
Schwarz
 
P
Slany
 
M
, et al. 
Molecular detection and identification of zygomycetes species from paraffin-embedded tissues in a murine model of disseminated zygomycosis: a collaborative European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Fungal Infection Study Group (EFISG) evaluation.
J Clin Microbiol
2010
, vol. 
48
 
6
(pg. 
2043
-
2046
)
10
Romani
 
L
Immunity to fungal infections.
Nat Rev Immunol
2004
, vol. 
4
 
1
(pg. 
1
-
23
)
11
Hebart
 
H
Bollinger
 
C
Fisch
 
P
, et al. 
Analysis of T-cell responses to Aspergillus fumigatus antigens in healthy individuals and patients with hematologic malignancies.
Blood
2002
, vol. 
100
 
13
(pg. 
4521
-
4528
)
12
Lalvani
 
A
Pathan
 
AA
Durkan
 
H
, et al. 
Enhanced contact tracing and spatial tracking of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells.
Lancet
2001
, vol. 
357
 
9273
(pg. 
2017
-
2021
)
13
Potenza
 
L
Barozzi
 
P
Vallerini
 
D
, et al. 
Diagnosis of invasive aspergillosis by tracking Aspergillus-specific T cells in hematologic patients with pulmonary infiltrates.
Leukemia
2007
, vol. 
21
 
10
(pg. 
578
-
581
)
14
Riva
 
G
Luppi
 
M
Barozzi
 
P
, et al. 
Emergence of BCR-ABL-specific cytotoxic T cells in the bone marrow of patients with Ph+ acute lymphoblastic leukemia during long-term imatinib mesylate treatment.
Blood
2010
, vol. 
115
 
8
(pg. 
1512
-
1518
)
15
Potenza
 
L
Luppi
 
M
Barozzi
 
P
, et al. 
HHV-6A in syncytial giant-cell hepatitis.
N Engl J Med
2008
, vol. 
359
 
6
(pg. 
593
-
602
)
16
Gil-Lamaignere
 
C
Simitsopoulou
 
M
Roilides
 
E
Maloukou
 
A
Winn
 
RM
Walsh
 
TJ
Interferon-gamma and granulocyte-macrophage colony-stimulating factor augment the activity of polymorphonuclear leukocytes against medically important zygomycetes.
J Infect Dis
2005
, vol. 
191
 
7
(pg. 
1180
-
1187
)
17
Bozza
 
S
Clavaud
 
C
Giovannini
 
G
, et al. 
Immune sensing of Aspergillus fumigatus proteins, glycolipids, and polysaccharides and the impact on Th immunity and vaccination.
J Immunol
2009
, vol. 
183
 
4
(pg. 
2407
-
2414
)
18
Ramadan
 
G
Davies
 
B
Kurup
 
VP
Keever-Taylor
 
CA
Generation of cytotoxic T cell responses directed to human leucocyte antigen class I restricted epitopes from the Aspergillus f16 allergen.
Clin Exp Immunol
2005
, vol. 
140
 
1
(pg. 
81
-
91
)
19
Chai
 
LY
van de Veerdonk
 
F
Marijnissen
 
RJ
, et al. 
Anti-Aspergillus human host defence relies on type 1 T helper (Th1), rather than type 17 T helper (Th17), cellular immunity.
Immunology
2010
, vol. 
130
 
1
(pg. 
46
-
54
)
20
van Crevel
 
R
Karyadi
 
E
Preyers
 
F
, et al. 
Increased production of interleukin 4 by CD4+ and CD8+ T cells from patients with tuberculosis is related to the presence of pulmonary cavities.
J Infect Dis
2000
, vol. 
181
 
3
(pg. 
1194
-
1197
)
21
Elrefaei
 
M
Burke
 
CM
Baker
 
CA
, et al. 
TGF-beta and IL-10 production by HIV-specific CD8+ T cells is regulated by CTLA-4 signaling on CD4+ T cells.
PLoS One
2009
, vol. 
4
 
12
pg. 
e8194
 

Author notes

*

L.P., D.V., P.B., G.R., and F.F. contributed equally to the study.