Mucorales-specific T cells emerge in the course of invasive mucormycosis and may be used as a...

doi:10.1182/blood-2011-07-366526Prepublished online September 19, 2011;

Pecorari, Francesca Cavalleri, Roberto Marasca, Franco Narni and Mario LuppiAmbra Paolini, Monica Maccaferri, Cinzia Del Giovane, Roberto D'Amico, Fabio Rumpianesi, MonicaChiara Quadrelli, Anna Candoni, Johan Maertens, Giulio Rossi, Monica Morselli, Mauro Codeluppi, Leonardo Potenza, Daniela Vallerini, Patrizia Barozzi, Giovanni Riva, Fabio Forghieri, Eleonora Zanetti, and may be used as a surrogate diagnostic marker in high-risk patients

-specific T cells emerge in the course of invasive mucormycosisMucorales

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1

MUCORALES-SPECIFIC T CELLS EMERGE IN THE COURSE OF INVASIVE

MUCORMYCOSIS AND MAY BE USED AS A SURROGATE DIAGNOSTIC MARKER IN

HIGH RISK PATIENTS.

Leonardo Potenza1§

, Daniela Vallerini1§

, Patrizia Barozzi1§

, Giovanni Riva1§

, Fabio Forghieri1§

,

Eleonora Zanetti1

, Chiara Quadrelli1

, Anna Candoni2

, Johan Maertens3

, Giulio Rossi4

, Monica

Morselli1

, Mauro Codeluppi5

, Ambra Paolini1

, Monica Maccaferri1

, Cinzia Del Giovane1

, Roberto

D'Amico1

, Fabio Rumpianesi6

, Monica Pecorari6

, Francesca Cavalleri7

, Roberto Marasca1

, Franco

Narni1

, Mario Luppi1*

.

1

Section of Haematology, Department of Oncology, Haematology and Respiratory Diseases;

4

Section of Pathology, Department of Diagnostic and Laboratory Services and Legal Medicine;

5

Clinic of Infectious Diseases, Department of Internal Medicine and Medical Specialties;

6

Microbiology and Virology Unit, Department of Diagnostic and Laboratory Services and Legal

Medicine; 7

Section of Neuroradiology, Department of Neuroscience, all from the University of

Modena and Reggio Emilia, Modena; 2

Hematology and Bone Marrow Transplantation, Udine, all

from Italy.

3

Department of Hematology, Universitaire Ziekenhuizen Leuven, Campus Gasthuisberg, Leuven,

Belgium.

Short Title: Specific T cells to diagnose invasive mucormycosis

§

These authors equally contributed to the study

*Corresponding author:

Mario Luppi M.D., Ph.D.

Blood First Edition Paper, prepublished online September 19, 2011; DOI 10.1182/blood-2011-07-366526

Copyright © 2011 American Society of Hematology




2

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

Phone: ++39 059 422.4641 (office)- 5570 (mobile)

Fax: ++39 059 422.4429

e-mail: [email protected]




3

Summary

Mucorales-specific T cells have been investigated in 28 hematologic patients during the course of

their treatment. Three developed proven invasive mucormycosis (IM), 17 infections of known

etiologies but other than IM, and 8 never showed fever upon the period of observation. The

Mucorales-specific T cells may be detected only in patients with IM, at diagnosis and along the

entire course of the IM, but neither before nor long time after the resolution of the infection. Such T

cells produced predominantly interleukin-4, interferon-gamma (IFN-γ), interleukin-10, and to a

lesser extent also interleukin-17, and belonged to either CD4+ or CD8+ subsets. The specific T

cells producing IFN-γ were able to directly induce damage of Mucorales hyphae. None of the 25

patients without IM showed 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.

Word count = 150




4

Invasive Mucormycosis (IM), the second-most common cause of invasive mold infections in

hematologic patients, shows mortality rates approaching 70% of affected individuals, because of

difficulties in obtaining an early and undoubted diagnosis1-5. Actually, a definitive diagnosis of IM

relies exclusively on both histopathological demonstration and cultural isolation of the pathogen

from the involved organs6. However, obtaining 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 1/3 of cases7. Furthermore, neither serologic nor antigenic diagnostic methods

exist and the use of polymerase chain reaction (PCR) has almost exclusively been limited to the

identification and the discrimination of fungal species8,9.

Adaptive immunity has been reported to play a crucial role in the defence of the host against

fungi, at least in the case of invasive aspergillosis (IA) and invasive candidiasis10,11, and the

recognition and enumeration of antigen-specific T cells has been demonstrated a useful tool for the

diagnosis of definite infectious diseases, in particular of either active or latent tuberculosis12.

We have 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.




5

PATIENTS AND METHODS

Twenty-eight hematologic patients have been studied. Patients 1-3 had disseminated (pulmonary

and splenic), tracheo-bronchial and cerebral proven IM, respectively (Figures S1,S2).

The antifungal treatments of the three patients with proven IM have been described in details in

supplemental data.

The remaining 25 cases included 17 patients presenting infectious complications of proven etiology

on the basis of cultural and/or histologic examinations, but other than IM, and 8 who have not

developed infectious complications during the course of their induction chemotherapy. Patients'

clinical characteristics have been reported in Table S1. Informed consent was obtained in

accordance with the Declaration of Helsinki, and the study was approved by the University of

Modena and Reggio Emilia Ethical Committee.

The enzyme linked immunospot (ELISpot) assay has been performed to detect either Mucorales- or

Aspergillus-specific T cell, as reported13 and described in details in supplemental data, on 80

peripheral blood samples (range 2 to 6 per patients). Time points analysed were: in patient 1, the

beginning of induction chemotherapy, 20 days before the pulmonary biopsy, at the histological and

cultural identification of Rhizomucor pusillus infection, the beginning of consolidation

chemotherapy, and 16 weeks after the resolution of the infection; in patient 2, the day of cultural

and histologic demonstration of Rhizopus oryzae infection, and until death in four further occasions

during the course of IM; in patient 3, the day of histologic and molecular demonstration of Absydia

corymbifera infection, three occasions during the course of IM, and at the complete resolution of

the infection. All the other patients were analysed at least twice during the course of their either

infections or chemotherapeutic treatments (Table S1).

The phenotypic and functional characterization of Mucorales-specific T cells has been performed

with the cytokine secretion assay (CSA) as already reported14 and described in details in

supplemental data. Molecular studies, micromanipulation and single-cell PCR to identify Mucorales

species (Figure S2) were performed as previously reported15 and described in details in




6

supplemental data. Anti-Mucorales activity of specific T cells was performed as reported in

supplemental data.




7

RESULTS

Identification of Mucorales-specific T cells

In patient 1, the ELISpot resulted positive for the presence of Mucorales-specific T cells producing

interleukin (IL)-10 in the second, third, fourth and fifth samples, and Mucorales-specific T cells

producing interferon gamma (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) and long time after its resolution (day +238) (Figure 1A).

In patient 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; 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 death, and the absence of specific responses in

patient 3, at the time of complete resolution of the infection (Figure 1B,C).

The differences between the median frequencies of Mucorales-specific T cells producing IL-10,

IFN-γ and IL-4 did not result statistically significant in all the three patients (p = .3), not even when

the results of the first two patients with more disseminated diseases were compared with those of

the third patient, with 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 analysed patients demonstrated the occurrence of Aspergillus-specific T cell at any

time point (Table S1).

Phenotypic and Functional Characterization of Mucorales-specific T cells

In patients 1-3, Mucorales-specific T cells resulted: 1) predominantly CD8 + T cells (mean

CD8+/CD4+ frequencies 3.62%/0.57%) of CM phenotype, producing IFN-γ; 2) predominantly

CD8+ T cells (mean CD8+/CD4+ frequencies 4.35%/2.60%) of EM phenotype producing IL-4; 3)




8

either CD4+ or CD8+ T cells (mean CD4+/CD8+ frequencies 0.32%/0.26%), the former of either

CM or EM phenotype, the latter mainly of CM, producing IL-10. Mucorales-specific T cells

producing IL-17 were also detectable, being either CD4+ or CD8+ (mean frequency 0.44% and

0.56%, respectively), and exhibiting, predominantly, the CM phenotype (Figure 2A,B).

Lytic Activity of Mucorales-specific T cells

Mucorales-specific T cells from patients 1-3 were capable to induce direct damage to the hyphae of

the two clinical isolates, similar to that of either PMNs or APCs. Only the combination of all the

three cell types resulted in a significantly higher hyphal damage (p<0.05) (Figure 2C,D).




9

DISCUSSION

We have shown, for the first time, that Mucorales-specific T cells may occur during the infection

course in patients with IM and exhibit direct antifungal activity, comparable, at least in vitro, to that

of either PMNs or APCs. The contribution of T cells to host defences against these moulds could

only be suspected, based on the enhanced fungicidal activity against Mucorales of

polymorphonuclear leukocytes exposed to IFN-γ16, but it has not yet been formally demonstrated.

The presence of Mucorales-specific T cells only during the course of IM, but neither before nor

after the resolution of the infection in patients 1-3, and their absence in patients without infections

or with infections other than IM, suggest that they are closely related to the occurrence of IM and

may result a marker of overt disease. Of note, the presence of Mucorales-specific T cells has been

demonstrated the only proof of IM in patient 1, largely before the obtainment of the biopsy. The

lower frequencies of specific T cells in patient 3 seem to suggest that a more confined IM could be

associated with responses of inferior magnitude. However, no statistically significant differences

were observed between the median numbers of Mucorales-specific T cells in our three patients.

Unfortunately, as all the samples were collected either when the patients were undergoing

antifungal treatment or after the drug withdrawal, no interaction between antifungal therapy and the

occurrence of Mucorales-specific T cells could be determined in our study.

The cytokine production profile of Mucorales-specific T cells, in our study, is partially in line with

what observed either in mice affected by IA or in human T cell clones stimulated with different

Aspergillus antigens in vitro17-19. 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, so far reported only in patients with the cavitary phase

of tuberculosis and the late phase of human immunodeficiency virus infection20-21.

In conclusion, Mucorales-specific T cells emerge in the course of IM and contribute to the human

immune responses against Mucorales. The detection of Mucorales-specific T cells may be

evaluated as a surrogate diagnostic marker of IM.




1

ACKNOWLEDGEMENTS

This study was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan,

Italy (M.L.); the European Commission’s FP6 Life-Science-Health Programme (INCA project;

LSHC-CT-2005-018704) (M.L.); the Associazione Italiana Lotta alle Leucemie, Linfoma e

Mieloma (AIL)-Sezione “Luciano Pavarotti”-Modena-ONLUS (L.P.; F.F.), the Programma di

ricerca Regione-Universita` 2007–2009, Regione Emilia Romagna (M.L., F.N.), and the Societa`

Italiana di Ematologia Sperimentale (SIES; “Piero Martino” award to L.P.).




11

AUTHORSHIP CONTRIBUTIONS

LP and ML conceived, designed the study and wrote the manuscript; DV, PB, GiRi, GiRo, FR and

MP made the ELISpot analysis, the CSA analysis, the XTT assays, histological examination,

performed the molecular characterization of fungi and interpreted the data; AC, JM, FF, MoMo,

MC, AP, MoMa, RM, FN provided well-characterized patient samples and critically revised the

manuscript; CDG and RDA made the statistical analysis and interpreted the data; FC made the

radiological studies and critically revised the manuscript.




1

DISCLOSURE OF CONFLICTS OF INTEREST

ML received research funds by Merck Sharp & Dohme and Gilead Sciences. ML serves in Advisory

Boards for Merck Sharp & Dohme and Gilead Sciences, and received honoraria from these two

pharmaceutical industries and from Pfizer and Nanogen. LP serves in an Advisory Board for Merck

Sharp & Dohme. AC serves in Advisory Board for Merck Sharp & Dohme and received funds by

Merck Sharp & Dohme, Gilead Sciences and Pfizer.

ML, LP and PB have applied for a European patent regarding clinical applications of the ELISpot

assay for the diagnosis of Aspergillus infection [PCT: WO2008/075395A3, EP2094295,

IT2007/000867]. ML, LP, DV, PB and FF have applied for an Italian patent regarding clinical

applications of the ELISpot assay for the diagnosis of Mucorales infection (No. MI2010A002224).

All the other authors have no conflicts of interest to declare. In particular they have neither

employment, nor consultancy, including stock options in a start-up company, nor ownership interest

in a publicly traded company, nor research funding. They received neither honoraria nor paid expert

testimony. They have neither other potential financial relationship (e.g., holding a patent or

receiving royalties), nor membership on another entity’s Board of Directors or its advisory

committees.




1

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1

FIGURE LEGEND

Figure 1 A-C. Kinetics of Mucorales-specific T-cell responses, by IFN-γ, IL-10 and IL-4

ELISpot assay, in the three patients with invasive mucormycosis. A. Patient 1. B. Patient 2. C.

Patient 3. Yellow columns represent the number of Mucorales-specific T cells producing interleukin

10 (IL-10). Blue columns represent the number of Mucorales-specific T cells producing interferon

gamma (IFN-γ). Red columns represent the number of Mucorales-specific T cells producing

interleukin 4 (IL-4). The dark grey background represents the T-cell responses in wells with

phytohemagglutinin (PHA). Vertical axis shows the number of spot forming cells (SFCs) per

million of peripheral blood mononuclear cells (PBMCs). Horizontal axis indicates the time in days

from the beginning of induction chemotherapy.

Figure 2 A-D. 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 grey) or CM (dark grey), are shown as mean % positive cells, computed over the three

patients with IM. Results are expressed as percentages of either CD4+ T cells (A) or of CD8+ T cells

(B). Mean frequencies of specific cytokine-producing T cells for individual patients are reported on

each column, either as EM (�) or CM (O). EM = effector memory. CM = central memory.

C,D. Hyphal damage at 2 (C) and 22 (D) hours to Rhizomucor pusillus and Rhizopus oryzae

induced by anti-Mucorales T cells (T), polymorphonuclear leukocytes (PMNs), and antigen-

presenting cells (APCs), alone or in combination, deriving from patient 1 and patient 2 during the

course of IM. E:T = effector/target cells ratios.




1

Figure 1




1

Figure 2




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