Immunity and Tolerance to Aspergillus Involve Functionally Distinct Regulatory T Cells and Tryptophan Catabolism 1 Claudia Montagnoli,* Francesca Fallarino,* Roberta Gaziano,* Silvia Bozza,* Silvia Bellocchio,* Teresa Zelante,* Wiswanath P. Kurup, † Lucia Pitzurra,* Paolo Puccetti,* and Luigina Romani 2 * The inherent resistance to diseases caused by Aspergillus fumigatus suggests the occurrence of regulatory mechanisms that provide the host with adequate defense without necessarily eliminating the fungus or causing unacceptable levels of host damage. In this study, we show that a division of labor occurs between functionally distinct regulatory T cells (Treg) that are coordinately activated by a CD28/B-7-dependent costimulatory pathway after exposure of mice to Aspergillus conidia. Early in infection, inflammation is controlled by the expansion, activation and local recruitment of CD4  CD25  Treg capable of suppressing neu- trophils through the combined actions of IL-10 and CTLA-4 on indoleamine 2,3-dioxygenase. The levels of IFN- produced in this early phase set the subsequent adaptive stage by conditioning the indoleamine 2,3-dioxygenase-dependent tolerogenic program of dendritic cells and the subsequent activation and expansion of tolerogenic Treg, which produce IL-10 and TGF-, inhibit Th2 cells, and prevent allergy to the fungus. The coordinate activation of Treg may, however, be subverted by the fungus, as germi- nating conidia are capable of interfering with anti-inflammatory and tolerogenic Treg programs. Thus, regulation is an essential component of the host response in infection and allergy to the fungus, and its manipulation may allow the pathogen to overcome host resistance and promote disease. The Journal of Immunology, 2006, 176: 1712–1723. A spergillus fumigatus, a termotolerant saprophyte, is asso- ciated with a wide spectrum of diseases in humans, rang- ing from severe infections to allergy (1, 2). The inherent resistance of immunocompetent and nonatopic subjects to As- pergillus diseases suggests the existence of regulatory mechanisms that efficiently oppose both inflammatory and allergic responses to the fungus. Most of the inhaled conidia are eliminated by exclusion mechanisms, through physical barriers and mediators with antimi- crobial and immunomodulatory properties (3). Polymorphonuclear neutrophils (PMN) 3 are the predominant immune cells in the acute stage of the infection and are essential in the initiation and exe- cution of the acute inflammatory response and subsequent resolu- tion of infection. However, pulmonary pathology may be reduced under conditions of PMN deficiency (1), which suggests that PMN may act as a double-edged sword, as the excessive release of ox- idants and proteases could be responsible for injury to organs and fungal sepsis. Th2 cell sensitization to fungal allergens is common in atopic subjects (2), yet respiratory exposure to inhaled conidia is a tolerogenic event in most individuals. It is known that respiratory tolerance is mediated by lung den- dritic cells (DCs) that produce IL-10 and thus induce the devel- opment of CD4 + T regulatory cells (Treg) (4, 5) expressing mem- brane-bound TGF- and the forkhead family transcription factor Foxp3 (6). Different types of CD4 + CD25 + Treg, including natu- rally occurring and inducible Treg, have been defined (7, 8) and so have the modes of their suppressive action, which range from the inhibitory cytokines IL-10 and TGF- to cell-cell contact via in- hibitory CTLA-4 (9). Both natural and inducible Treg have been described in infection (10, 11), their activation occurring through Ag-specific and nonspecific mechanisms. Treg with immunosup- pressive activity have also been described in fungal infections (12, 13). Consistent with the notion that signals emanating from CD28 and B7 are critical for thymic generation of Foxp3 + Treg (14) and for Treg self-renewal in the periphery (15, 16), the induction of CD4 + CD25 + Treg in candidiasis is strictly dependent on the lev- els of B7 costimulatory Ag expression by IL-10-producing DCs (13) and involves the IFN-/indoleamine 2,3-dioxygenase (IDO)- dependent pathway (17). In the present study, we explored the contribution of Treg to the balance between resistance and immunopathology associated with Aspergillus infection and allergy. We evaluated the occurrence of Treg in murine models of infection and allergy to the fungus, their relative dependency on costimulatory molecules, and the contri- bution of IDO and fungus to their induction and functional activity. Materials and Methods Animals Female BALB/c mice, 8 –10 wk old, were purchased from Charles River Breeding Laboratories. Homozygous, CD28 -/- , B7-1 -/- , B7-2 -/- , B7- 1 -/- /B7-2 -/- , and IFN- -/- mice on BALB/c background were bred un- der specific pathogen-free conditions in the animal facility of Perugia Uni- versity (Perugia, Italy). Procedures involving animals and their care were conducted in conformity with national and international laws and policies. *Department of Experimental Medicine and Biochemical Science, University of Pe- rugia, Italy; and † Medical College of Wisconsin, Veterans Affairs Medical Center, Milwaukee, WI 53285 Received for publication July 11, 2005. Accepted for publication November 9, 2005. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the National Research Project on AIDS, Contract 50F.30, Opportunistic Infections and Tuberculosis, Italy. 2 Address correspondence and reprint requests to Dr. Luigina Romani, Department of Experimental Medicine and Biochemical Sciences, Microbiology Section, University of Perugia, Via del Giochetto, 06126 Perugia, Italy. E-mail address: lromani@unipg.it 3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophil; DC, den- dritic cell; Treg, regulatory T cell; BAL, bronchoalveolar lavage; IDO, indoleamine 2,3-dioxygenase; pDC, plasmacytoid dendritic cell; RC, resting conidia; SC, swollen conidia; 1-MT, 1-methyl-dl-tryptophan; TLN, thoracic lymph node; WT, wild type; CCFA, cell culture filtrate Ag; PAS, periodic acid-Schiff. The Journal of Immunology Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00