Host Immune Responses Against Pulmonary Fungal Pathogens

The lungs are a gateway for numerous airborne pathogens that are ubiquitous in our environment. Among these potential pathogens are fungi that can be found in the soil, bird excreta, air ducts, and many other places where their contact is unavoidable. Exposure to these fungal pathogens oftentimes goes unnoticed due to the activation of our robust immune systems which sequester and control these microbes before significant damage occurs. Still, there are many situations in which host immunity becomes compromised providing an opportunity for typically innocuous fungal organisms to become established and cause disease or for dormant infections to reawaken. Also, in certain cases disease may be exacerbated due to an over exuberant immune response. In this chapter, we will review the main aspects of innate and adaptive immune responses against pulmonary fungal pathogens. We will also discuss the potential for vaccines to prevent pulmonary fungal infections.


Introduction
The lungs are a gateway for numerous airborne pathogens that are ubiquitous in our environment. Among these potential pathogens are fungi that can be found in the soil, bird excreta, air ducts, and many other places where their contact is unavoidable. Exposure to these fungal pathogens oftentimes goes unnoticed due to the activation of our robust immune systems which sequester and control these microbes before significant damage occurs. Still, there are many situations in which host immunity becomes compromised providing an opportunity for typically innocuous fungal organisms to become established and cause disease or for dormant infections to reawaken. Also, in certain cases disease may be exacerbated due to an over exuberant immune response. In this chapter, we will review the main aspects of innate and adaptive immune responses against pulmonary fungal pathogens. We will also discuss the potential for vaccines to prevent pulmonary fungal infections.

Introduction to pulmonary fungal infections
Pulmonary fungal infections can be grouped into primary fungal pathogens and opportunistic fungal pathogens. Those organisms that can cause disease in immune competent hosts are considered primary pathogens including Histoplasma capsulatum, Coccidioides immitis, Paracoccidioides brasilensis, and Blastomyces dermatiditis. All of the primary pulmonary fungal pathogens are endemic to the United States and/or Central & South America. Histoplasma and Blastomyces are endemic to the Ohio River & Mississippi River Valleys of the United States and also to certain regions of Central and South America (Klein et al., 1986;Deepe, 2000). Coccidioides is prevalent in the desert southwest United States (Fisher et al., 2007), and Paracoccidioides is endemic in Central and South America, particularly in Brazil (Franco, 1987;Franco et al., 1989;Brummer et al., 1993). These 88 and IL-6 (Silva et al., 2011). P. brasilensis can proliferate within macrophages, but macrophage activation inhibits its growth (Brummer et al., 1988;Cano et al., 1994). Further, macrophages activated by interferon (IFN)- can kill P. brasiliensis (Cano et al., 1994;Gonzalez et al., 2000). In vitro stimulation of human monocytes and neutrophils with Paracoccidiodes yeast showed downregulation of toll-like receptor (TLR)2, TLR4, and dectin-1 on the surface of these cells. In addition, yeast cells induced the production of proinflammatory cytokines such as TNF- (Bonfim et al., 2009). Mice lacking TLR2 had a less severe pulmonary infection than wild-type (WT) mice and had decreased nitric oxide (NO) production. However, despite the differences in infection, both TLR2 -/-mice and WT mice had similar rates of survival and similar pulmonary inflammatory responses (Loures et al., 2009). Further, TLR2 deficiency skewed the adaptive response towards a T helper (Th)17 phenotype and caused a decrease in T regulatory cells. Increased neutrophils and eosinophils migrate to the lungs of mice susceptible to P. brasiliensis, (Cano et al., 1995), and this influx affects the disease outcome and the adaptive response induced to infection. In susceptible individuals recovered from Paracoccidiodes, neutrophils are able to phagocytose the organism, but this leads to degeneration of the neutrophils. These data suggest that susceptible individuals have an inherent neutrophil deficiency (Dias et al., 2008). Further, neutrophils from patients with P. brasiliensis have a digestive defect against the fungus (Goihman-Yahr et al., 1980), and also have a killing defect against the fungus (Goihman-Yahr et al., 1985;Goihman-Yahr et al., 1992).
B. dermatiditis interaction with DCs causes efficient upregulation of antigen presentation and costimulatory molecules and induces production of IL-12 and TNF- (Wuthrich et al., 2006). DCs can activate CD8 + T cells in the absence of CD4 + T cells, and the yeast alone is a sufficient inflammatory stimulus that can directly induce maturation of DCs and can induce production of TNF-, IL-1, and IL-12 (Wuthrich et al., 2006). Monocyte-derived dendritic cells can associate with yeast in the lung and transport them to the draining lymph nodes, but fail to present antigen to CD4 + T cells, however dermal DCs are capable of antigen presentation (Ersland et al., 2010). During B. dermatiditis infection, alveolar macrophages are only modestly able to ingest and kill the yeast form of the organism (Bradsher et al., 1987). Murine macrophages are only able to kill less than 5% of yeast (Brummer and Stevens, 1987;Brummer et al., 1988), and had a 25-30% reduction in respiratory burst compared to the respiratory burst induced by zymosan. The Blastomyces adhesion 1 (BAD1) molecule on the Blastomyces yeast surface is responsible for binding to CD11b/CD18 and CD14 on the macrophage surface and subsequent entry (Klein et al., 1993;Newman et al., 1995). Neutrophils rapidly infiltrate to pulmonary tissues following infection, and are responsible for the formation of pyogranulomatous lesions. Conidia are rapidly phagocytosed by neutrophils, but killing of conidia is inefficient. Similar to macrophages, the neutrophil respiratory burst induced by conidia is only 70% of that induced by zymosan (Drutz and Frey, 1985). In addition, yeasts are even more difficult for the neutrophils to phagocytose & kill than conidia (Drutz and Frey, 1985).
Pneumocystis interacts with DCs in vitro by MR, (Kobayashi et al., 2007) but the interaction does not lead to an increase in maturation markers such as MHC II, CD40, CD54, CD80, or CD86. Additionally, this interaction induces the production of IL-4 but not IL-12p40, IL-10, TNF-, or IL-6 (Kobayashi et al., 2007). However, Pneumocystis cell wall -glucans have the ability to induce costimulatory molecule upregulation on DCs, such as MHC II, CD80, CD86, and CD40. These DCs interacted with -glucans from Pneumocystis via dectin-1, and co-stimulatory molecule expression and Th1-type cytokine secretion by -glucan stimulated DCs was regulated by Fas-Fas ligand interaction (Carmona et al., 2006). In vivo administration of DCs pulsed with Pneumocystis induced specific T cell responses and release of IL-4 as well as specific IgG 1 , IgG 2a , and IgG 2b production (Kobayashi et al., 2007). Alveolar macrophages have been shown to directly kill both Pneumocystis trophozoites and cysts (Fleury et al., 1985;reviewed in Kelly and Shellito, 2010). Specifically, alternativelyactivated macrophages (aaMac) are important effector cells against Pneumocystis, and aaMac is enhanced by IL-33 (Nelson et al., 2011). In addition, Pneumocystis infection causes changes in gene expression of alveolar macrophages that included upregulation of genes involved in antigen presentation and antimicrobial peptides, but downregulation of genes involved in phagocytosis and uptake (Cheng et al., 2010). Pneumocystis major surface glycoprotein (MSG), which is a heavily glycosylated surface antigen, is recognized by MR on alveolar macrophages (Ezekowitz et al., 1991). Pneumocystis infection in HIV + patients induces shedding of the MR, which results in reduced alveolar macrophage phagocytosis of the microbe (Koziel et al., 1998;Fraser et al., 2000). Neutrophils can also interact with Pneumocystis, but the presence of neutrophils is correlated with inflammation and increased severity of disease (reviewed in Kelly and Shellito, 2010).
DCs bind and internalize A. fumigatus through DC-SIGN, and this binding triggers DC maturation (Serrano- Gomez et al., 2004). Mouse DCs can internalize conidia of A. fumigatus using MR and a C-type lectin receptor as well as FcR (Bozza et al., 2002). Upon exposure of DCs to A. fumigatus, DCs upregulate HLA-DR, CD80 and CD86 (Grazziutti et al., 2001;Bozza et al., 2002). Following A. fumigatus infection, DCs can release the chemokine CXCL8, which promotes migration of PMNs, can upregulate CCL19, which is important in migration of CCR7 + naïve T cells and mature DCs to lymph nodes, and can release soluble factors that increase CD11b and CD18 on PMNs (Gafa et al., 2007). DC phagocytosis of A. fumigatus conidia and hyphae occur by different means and through different receptors; conidia are phagocytosed by coiling phagocytosis and hyphae are phagocytosed by zippertype phagocytosis (Bozza et al., 2002). A. fumigatus killing by DCs is dependent on phagolysosomal fusion and a reduction in pH (Ibrahim-Granet et al., 2003). Plasmacytoid DCs (pDCs) have the ability to spread over A. fumigatus hyphae and inhibit their growth 90 (Ramirez-Ortiz et al., 2011), and antifungal activity does not require direct cell contact. Following interaction of pDCs with Aspergillus, pDCs release pro-inflammatory cytokines, such as IFN- and TNF-, and these are produced via a TLR9-independent mechanism (Ramirez-Ortiz et al., 2011). During the early stages of Aspergillus infection, alternatively activated macrophages are recruited to the lung and are important in host defense (Bhatia et al., 2011). Studies examining the interaction of Aspergillus conidia with alveolar macrophages showed that infectivity and inhibition of macrophage killing by the fungus were due to the presence of a siderophore system that allows the fungus to acquire iron (Schrettl et al., 2010). In neutropenic mice, inflammatory DCs are recruited to the lungs during Aspergillus infection, and this recruitment is dependent on the absence of neutrophils . This accumulation led to increased TNF-, CCL2, and CCL20, which resulted in further recruitment of inflammatory DCs. Neutrophils, when incubated with A. fumigatus hyphae, form neutrophil extracellular traps (NETs), which are antifungal, but mostly act in a fungistatic manner to limit spread of the hyphae (Bruns et al., 2010;Hasenberg et al., 2011).
In vitro studies of DCs with C. neoformans have shown that DCs are involved in detection, binding, phagocytosis, processing, antigen presentation, T cell activation, and killing of the organism (Bauman et al., 2000;Bauman et al., 2003;Wozniak et al., 2006;Wozniak and Levitz, 2008). DCs isolated from infected lungs presented cryptococcal mannoprotein (MP) to MP-specific T cells and induced T cell activation ex vivo (Wozniak et al., 2006). Depletion of DCs abrogated the T cell response (Mansour et al., 2006). Furthermore, DC phagocytosis of mannoprotein (MP) in the presence of the appropriate adjuvant induces production of Th1-type cytokines (Dan et al., 2008). Additional studies revealed that the interaction of C. neoformans with DCs, but not macrophages, induced the production of IL-12 and IL-23, two cytokines associated with protection against cryptococcosis (Kleinschek et al., 2010). Phagocytosis of encapsulated C. neoformans by DCs requires opsonization with either anticapsular antibody or complement, and the combination of these has an additive effect (Kelly et al., 2005). Also, both murine and human DCs are able to kill C. neoformans, by both oxidative and non-oxidative mechanisms (Kelly et al., 2005). Recognition and uptake of acapsular C. neoformans strains by DCs requires MR and FcR II (Syme et al., 2002). TLR2 and TLR4 are not important in uptake of C. neoformans or activation of DCs by the fungus (Nakamura et al., 2006). DCs stimulated with DNA from C. neoformans release IL-12p40 and express CD40, a costimulatory molecule associated with DC maturation, and thus was tied to recognition by TLR9 (Nakamura et al., 2008). Upon infection with C. neoformans, CCR2deficient mice, which are impaired in trafficking of monocyte-derived DCs, developed a non-protective Th2-type immune response and persistent infection, and had reduced DC recruitment, bronchovascular collagen deposition, and increased IL-4 production (Osterholzer et al., 2008). C. neoformans can also be phagocytosed by macrophages (Levitz et al., 1999;Del Poeta, 2004). Macrophage phenotypes are associated with differential immune responses against C. neoformans. Protection against infection is associated with the presence of classically-activated macrophages (caMac) Hardison et al., 2010a;Hardison et al., 2010b), while disease progression is associated with the presence of alternatively activated macrophages (aaMac) Muller et al., 2007;Arora et al., 2011;Chen et al., 2007;Guerrero et al., 2010). Also, macrophages can serve as a site of replication of C. neoformans (Tucker and Casadevall, 2002). Intracellular replication rates within macrophages correlated to virulence for C. neoformans strains (Voelz et al., 2009). In addition to replication, yeasts can be expulsed from macrophages by a non-lytic mechanism that leaves both C. neoformans and macrophages intact and capable of replication and growth (Alvarez and Casadevall, 2006;Ma et al., 2006;Alvarez and Casadevall, 2007;Johnston and May, 2010). C. neoformans can also be phagocytosed by activated neutrophils (Kozel et al., 1987). The capsule of C. neoformans induces neutrophils to release proinflammatory cytokines, such as IL-1, IL-6, IL-8 and TNF- (Retini et al., 1996). Neutrophils can kill C. neoformans by non-oxidative mechanisms, including neutrophil defensins and calprotectin (Mambula et al., 2000). Interestingly, induction of neutropenia in mouse models of infection reduces their susceptibility to infection (Mednick et al., 2003).
Although innate immune responses against Rhizopus, the main causative agent of mucormycosis, have not yet been fully characterized, recent work has shown that Rhizopus can trigger a common innate sensing pathway in DCs that leads to the production of IL-23 and drives Th17-type responses (Chamilos et al., 2010). This is due to interaction of dectin -1 with -glucans on the surface of Rhizopus hyphae.

NK cell activity
Another innate immune response to pulmonary fungal pathogens is due to recognition and action by natural killer (NK) cells. NK cells were thought to act primarily against viruses and tumors, but more recent studies have shown that NK cells have a wide variety of functions against bacteria, fungi, and parasites (Newman and Riley, 2007).
In H. capsulatum infection, there is little evidence of a protective role for NK cells. While beige mice (lacking functional NK cells) are more susceptible to H. capsulatum infection, T cells play a greater role in controlling infection (Patino et al., 1987). In studies evaluating both beige mice and mice depleted of NK cells, beige mice were still more susceptible to infection, while mice depleted of NK cells were no more susceptible to infection than WT mice, therefore indicating no major role for NK cells in protection (Suchyta et al., 1988). However, mice deficient in perforin, a major component of NK cell anti-microbial activity, had accelerated mortality and increased fungal burden (Zhou et al., 2001). Infection with Coccidioides during depletion of NK cells leads to increased susceptibility to infection (Petkus and Baum, 1987). Furthermore, NK cells have a direct cytotoxic effect on Coccidioides young spherule and endospore cells (Petkus and Baum, 1987). In Paracoccidioides, studies have shown increased NK cell activity in infected hamsters compared to uninfected controls. Impaired NK cell activity was associated with a decrease in cell-mediated immunity (CMI) and an increase in histopathologic lesions. However, after initial activation, NK cells alone were not able to control dissemination of Paracoccidiodes (Peracoli et al., 1995). In vitro NK cell activity correlated with growth inhibition of Paracoccidiodes yeast (Jimenez and Murphy, 1984).
In neutropenic mice with A. fumigatus infection, NK cells are the major cell type responsible for the production of IFN- early in the infection. Additionally, depletion of NK cells reduces IFN- levels and caused increased pulmonary fungal load (Park et al., 2009). NK cells have direct anti-fungal activity against hyphae but not against resting conidia (Schmidt et al., 2011). Killing is due to production of mediators by NK cells, including perforin. However, A. fumigatus can also down-regulate some cytokines induced by the NK cells, including IFN- and GM-CSF (Schmidt et al., 2011). In addition, recruitment of NK cells to the lung during A. fumigatus infection by the chemokine MCP-1 is required for optimal clearance of the organism from the lungs (Morrison et al., 2003). During Pneumocystis infection in SCID mice (lacking T and B cells), NK cells were responsible for production of cytokines such as IFN-, TNF-, TNF-, IL-10, and IL-12 (Warschkau et al., 1998). Recent studies have shown that NK cells are recruited to the lung during Pneumocystis infection and are important in fungal clearance of murine PCP (M. Kelly and J. Shellito, personal communication). Further, combined depletion of NK and CD4 + T cells resulted in increased pulmonary fungal burden compared to individual depletion of each subset. In vitro, NK cells have direct microbicidal activity against Pneumocystis, and this anti-fungal activity is significantly enhanced in the presence of CD4 + T cells, suggesting that both cell types are necessary for a protective response against Pneumocystis infection (M. Kelly and J. Shellito, personal communication). Early studies showed that NK cells can directly kill C. neoformans (Murphy and McDaniel, 1982). Further, IFN- production by NK cells enhances elimination of the fungus in murine models (Kawakami et al., 2001a;Kawakami et al., 2001b;Kawakami et al., 2001c). Depletion of NK cells using anti-asialo GM antibody resulted in increased fungal burden in mice (Hidore et al., 1991a;Hidore et al., 1991b). Increased fungal burden was seen in beige mice compared to wild-type mice, and in mice depleted of NK1.1 + NK cells, fungal burden was also increased compared to controls (Lipscomb et al., 1987;Salkowski and Balish, 1991). Human NK cells kill C. neoformans (Levitz and Dupont, 1993), and this killing is enhanced in the presence of anti-cryptococcal antibodies (Miller et al., 1990). Binding of NK cells is required for cryptococcal killing, and requires disulfide bonds and is dependent on magnesium (Nabavi and Murphy, 1985;Hidore and Murphy, 1989;Murphy et al., 1991). Killing of C. neoformans is due to perforin interaction with the organism (Ma et al., 2004;Marr et al., 2009). In summary, NK cells act as accessory cells in antifungal host defenses contributing to clearance of fungi by a variety of mechanisms.

Gamma/delta T cell activity
The role of gamma delta () T cells during the immune response to pulmonary fungal pathogens is diverse. During infection with Cryptococcus, mice genetically deficient or depleted in  T cells have reduced fungal burden compared to controls. Further, mice lacking T cells had increased levels of IFN- and decreased levels of TGF- compared to controls, therefore suggesting that T cells are detrimental to protective immunity during cryptococcal infection (Uezu et al., 2004). In Pneumocystis pneumonia, CD4 + T cells are necessary for protection against infection, but  cells are known to infiltrate into the lung during pneumonia (Kagi et al., 1993;Agostini et al., 1995;Steele et al., 2002). However, resolution of pulmonary Pneumocystis infection is augmented in  T cell-deficient mice, (Steele et al., 2002), suggesting that these cells are detrimental to clearance of the organism. Further, the absence of  T cells led to an increase in recruitment of CD8 + T cells and production of cytokines such as IFN-. Complete lack of all T cell subsets ( and ) during Pneumocystis infection led to lethal consequences (Hanano and Kaufmann, 1999). Thus, cells haveeither a limited role in antifungal protection or are detrimental to antifungal host defenses.

Innate anti-fungal defenses by non-immune cells
While innate immune cells and components of the innate immune system are the predominant innate immune defenses, it has also been shown that unconventional cells, such as epithelial cells can also play a role in anti-fungal innate host responses. Airway epithelial cells are capable of uptake and processing of antigens and initiation of Th-type immune responses (Gereke et al., 2009). P. brasiliensis interacts with and can be internalized by bronchial epithelial cells (Mendes-Giannini et al., 1994, and this internalization is due to activation of a tyrosine kinase pathway (Monteiro da Silva et al., 2007;Hanna et al., 2000). In addition, uptake of P. brasiliensis causes both cytoskeletal rearrangements as well as apoptosis of the epithelial cells (Mendes-Giannini et al., 2004). C. neoformans can bind to pulmonary epithelial cells by a mechanism believed to be due to carbohydrate moieties that can be a ligand for the yeast (Merkel and Scofield, 1997). C. neoformans interaction with bronchial epithelial cells causes the production of IL-8, but epithelial cells are also susceptible to damage by the organism (Guillot et al., 2008a). In A. fumigatus infection, conidia can be taken up by tracheal epithelial cells, alveolar type II cells, and endothelial cells (Paris et al., 1997). Further, cytokines such as IL-6 and IL-8 are released by epithelial cells in vitro following stimulation with A. fumigatus proteases (Borger et al., 1999) or by A. fumigatus hyphal fragments (Zhang et al., 2005) and nasal epithelium. And nasal epithelium can engulf A. fumigatus conidia (Botterel et al., 2008). Epithelial cells can also release antimicrobial peptides following stimulation with fungal organisms. Epithelial cells in vitro cultured with A. fumigatus conidia, swollen conidia, or hyphae produced large amounts of beta defensins (Alekseeva et al., 2009). Airway epithelial cells internalize A. fumigatus conidia, and a genome-wide analysis revealed differential gene expression in epithelial cells with conidia compared to cells without conidia. Genes that were upregulated with conidia included genes involved in repair and inflammation, such as matrix metalloproteinases and chemokines (Gomez et al., 2010). In Pneumocystis, several studies have shown that the organism interacts with pulmonary epithelial cells. The interaction of Pneumocystis with epithelial cells was shown to be one of the initial steps in infection (Lanken et al., 1980;Yoneda and Walzer, 1980;Long et al., 1986;Millard et al., 1990). The -glucan from the Pneumocystis cell wall can stimulate pulmonary epithelial cells to produce IL-8, and the organism can induce the production of MCP-1 and ICAM -1 (Yu and Limper, 1997;Evans et al., 2005;Wang et al., 2007;Carmona et al., 2010). Interaction of Pneumocystis and alveolar epithelial cells also leads to the production of the chemokine MIP-2 following NF-B signaling (Evans et al., 2005;Wang et al., 2005). These studies show that non-immune cells, such as epithelial cells, play a role in pulmonary anti-fungal immunity.

T cell and antibody mediated immune responses to fungal infections 4.1 Adaptive responses against pulmonary fungal pathogens
When challenged with pathogenic fungi, the adaptive immune system is capable of mounting an effective response against most fungal species to eliminate fungal infections and maintain immunological memory that prevents their reoccurrence. However, fungi are ubiquitous in the host's environment including the saprophytes and opportunists that survive on the host's body surfaces and thus, the adaptive immune system is constantly challenged by fungal antigens. Excessive response to these antigens could lead to allergic responses or other types of immunopathological reactions. The balance between day-to-day fungal antigen exposure and the immune system is thought to lead to a homeostatic state defined as protective tolerance. Protective tolerance allows the host to keep possible fungal pathogens "in check" while preserving integrity of the natural barriers, which are potential portals for fungal infections (Romani and Puccetti, 2008;de Luca et al., 2010a;Littman and Rudensky, 2010).
T cells are responsible for orchestration of adaptive immune responses and T cell derived signals produced in response to specific antigens lead to targeted expansion, recruitment, activation of leukocytes, and regulation of B cell antibody responses. T cells also serve as a pool of immunological memory. Additionally, T cells have been shown to directly act as the fungicidal effector cells and serve as regulators of inflammatory responses, contributing to the development and maintenance of the protective tolerance. These regulatory mechanisms are designed to limit the damage that the host immune system can inflict on host tissues during incorrect and/or excessive host responses. Thus, properly functioning T cells are responsible for building up protective immunity against fungal pathogens and play an essential role in maintaining normal homeostasis of the immune system in the context of normal presence of fungal antigens.
Both subsets of T lymphocytes, CD4 + and CD8 + cells, are involved in antifungal host defenses. CD4 + T cells classically represent the T helper cell population. The T helper function was defined by MHC II restricted antigen specific activation of B-cell clones needed for the generation of specific antibodies. The CD4 + cell function in cell-mediated immunity (CMI) likewise requires antigen presenting cells and MHC II restricted antigen presentation. Presentation of antigen to the reactive T cells by dendritic cells and/or macrophages results in cytokine production. Through generation of different cytokine spectra, CD4 + T cells orchestrate recruitment and activation of various leukocyte subsets. The cytokines produced by the effector T cells are essential for macrophage fungicidal function and granuloma formation, but also may support chronic inflammation and immunopathology Chen et al., 2008;Jain et al., 2009;Zhang et al., 2009). Thus, cytokine induction by differentially polarized T-cell lineages is the major determinant for fungicidal potential of distal effector cells. Although the effector CD4 + cell function relies predominantly on cytokine production, CD4 + T-cells are capable of fungal killing via direct cell contact. At least in some biological circumstances, the direct fungicidal effect of CD4 + T cells relies on granulysin as the fungicidal mediator (Zheng et al., 2007;Zheng et al., 2008).
In contrast with CD4 + T cells, CD8 + T cells are classically viewed as cytotoxic lymphocytes. These cells respond to antigen presentation in the context of MHC I, to enable their cytotoxic machinery. Such cytotoxic responses are particularly crucial in responses to viral infection and tumor cells, leading to elimination of the virally infected or tumor-transformed cells by cytotoxic lymphocytes. CD8 + cells also play an important role in host defenses to bacterial, parasitic and fungal infections (Oykhman and Mody, 2010). Numerous studies showed that CD8 + T cells significantly contribute to protection against Cryptococcus, Pneumocystis, Histoplasma and Blastomyces, even in the absence of CD4 + T cells. Depending on the type and virulence of the fungal pathogen, CD8 + cells could afford either partial or a complete protection against the major fungal pathogens in experimental models. In this context, CD8 + T cells could induce all the protective effector functions of CD4 + T cells including production of protective cytokines. Another important aspect of CD8 + T cell effector function is the direct fungicidal effect of CD8 + T cells. Such direct fungicidal activity of CD8 + T cells have been demonstrated for C. neoformans (Ma et al., 2002). The killing of C. neoformans requires direct cell contact; it is enhanced by IL-15 and is thought to be mediated by granulysin. The direct cytotoxic effects are most pronounced when lymphocytes from fungus-immunized mice are used, however, a relatively high rate of binding of T cells to the fungus suggests that these cytotoxic mechanisms are innate rather than adaptive.

Immune polarization in antifungal host defenses
T helper cell subsets characterized by differential cytokine production by differentially programmed T-cell lineages were initially defined as Th1 and Th2 (Mosmann et al., 1986;Cherwinski et al., 1987). The types of immune responses driven by each of these cell lineages are described as Th1 and Th2 immune responses, generate different types of immune effector responses, and show different spectra of effectiveness against different classes of pathogens. For effective control/clearance of the majority of fungal pathogens, Th1 is the required type of the immune response. The Th1 response is promoted by IL-12, IFN-, and TNF-. The two latter cytokines are also the major products of Th1 helper T cells (Cherwinski et al., 1987). Th1-type T-cells are responsible for the delayed-type hypersensitivity (DTH) reactions and CMI associated with vigorous proinflammatory responses and granuloma formation (Cher and Mosmann, 1987) and induction of IgG2a class antibodies in B cells . The Th2 immune response is characterized by T-cell production of IL-4, IL-5, IL-9, IL-10, and IL-13 (Cherwinski et al., 1987), IgG1 and IgE antibody production by B cells  and the presence of eosinophilic inflammation (Huffnagle et al., 1994;Cenci et al., 1999;Olszewski et al., 2001). The Th1 and Th2 responses counter-regulate each other predominantly via an IL-4/IFN-negative feedback loop Gajewski and Fitch, 1988); however other cytokines can be also involved in Th1/Th2 regulation. The oversimplified Th1/Th2 paradigm has further evolved as new T cell lineages were defined. Th17 and regulatory type T cells (Treg), are T-cell lineages that are distinct from Th1 and Th2 cells that possess distinct functions in host defenses. Th17 cells are generated following the priming with IL-6 and TGF-and sustained by the presence of IL-23. Th17 cells classically produce IL-17 and IL-22, however, a subset of Th17 cells can produce IFN-. Regulatory T-cells are thought to be responsible for tolerance that prevents autoimmune diseases and to contribute to resolution of inflammatory responses. These effects of Tregs are thought to be mediated by anti-inflammatory cytokines IL-10 and TGF-, which are signature cytokines for Treg cells. New Th-cell lineages continue to be described including Th22 (Eyerich et al., 2009;Fujita et al., 2009) and Th9 (Soroosh and Doherty, 2009). Just like CD4 + effector T-cells, CD8 + T cells can also display a polarization pattern and thus can be an important source of the polarizing cytokines. Thus, both CD4 + and CD8 + T cells contribute to the cytokine balance during the immune response (Huffnagle et al., 1994).
Unlike Th1-type responses, the Th2 response is non-protective and frequently results in pathological responses to fungal challenges. For most fungal species, Th2 responses and type 2 cytokines decrease clearance of fungus. This is attributed to: 1) a suppression of protective Th1 responses due to a mutual counterregulatory feedback loop (Cenci et al., 1999) and 2) a generation of alternatively activated macrophages that can harbor fungal organisms Jain et al., 2009;Osterholzer et al., 2009a;Zhang et al., 2009). Th2 cytokines such as IL-4, IL-13 are the major trigger of alternative activation of macrophages Jain et al., 2009;Zhang et al., 2009). These alternatively activated macrophages do not express nitric oxide synthase but induce arginase which metabolizes L-arginine without yielding fungicidal nitric oxide. In the Th2 biased experimental models of fungal infections, intracellular survival of fungus within macrophages parallels high induction of alternatively activated macrophage markers Zhang et al., 2009;.
Increased production of Th2-type cytokines has been associated with increased susceptibility to Cryptococcus Zhang et al., 2009; and Paracoccidioides (Ruas et al., 2009) infections and to invasive pulmonary aspergillosis (Cenci et al., 1997;Cenci et al., 1999). Fungus-triggered Th2-type responses in the respiratory system may also lead to allergic diseases such rhinitis/sinusitis, asthma, allergic bronchopulmonary mycosis. Th2 type responses are highly detrimental to the respiratory system by promoting mucus hypersecretion/goblet cell metaplasia, eosinophilic inflammation, and peribronchial fibrosis all of which contribute to impaired airway function. Cytokines IL-4, IL-5 and IL-13 are the major triggers of these pathologies as increased expression of these cytokines can be reproduced along with the allergic symptoms in the lungs challenged with fungi or their antigenic components (Blease et al., 2000;Arora et al., 2005;Jain et al., 2009;Zhang et al., 2009). Some of the fungal antigens can directly promote Th2 skewing. The secreted protein fraction from Aspergillus fumigatus promotes Th2 bias of the immune response . Th2 pathologies are also found in mouse models of C. neoformans infections (Abe et al., 2000;Jain et al., 2009;Osterholzer et al., 2009b) (Figure 1). Expression of enzymes phospholipase B and urease by C. neoformans promote Th2 bias in the infected mice (Noverr et al., 2003;Osterholzer et al., 2009b). While Th2-biased responses are clearly undesirable in most types of fungal infections the exception is Pneumocystis infection, in which the Th2 response can contribute to fungal clearance (Shellito et al., 2000;McKinley et al., 2006;Hu et al., 2009). The effects of Th17 responses and the IL-17 cytokine family in anti-fungal host responses may be protective or non-protective depending on fungal species and sites of infection ( Figure 2). Thus, Th17 responses may be beneficial for some types of fungal infections or response promotes control of most fungal infections; Th2 leads to severe pathology and fungal persistence; Th17 may support clearance or persistence of different fungal infections, but may promote chronic neutrophilic inflammation. Treg may limit pathology by promoting resolution of the inflammatory response, but may increase the risk of persistence. Correct balance between Th1 and Treg is thought to support protective tolerance. exhibit detrimental effects. In the H. capsulatum infection model, IL-17 neutralization increases pulmonary fungal burden in connection with increased Treg numbers, suggesting that Th17 is beneficial for clearance of Histoplasma (Kroetz and Deepe, 2010). Th17 responses also contribute to anticryptococcal protection and the development of the protective inflammatory response in C. neoformans infected lungs Hardison et al., 2010b;Wozniak et al., 2011a).
The IL-23/IL-17 axis contributes to clearance of Pneumocystis (Rudner et al., 2007). However, in IFN-deficient mice infected with Pneumocystis the development of strong Th17 response is detrimental, suggesting that a balance between IFN-and IL-17 is needed for optimal clearance of Pneumocystis (Hu et al., 2009). At other mucosal sites, the effects of Th17 are variable. Th17 cells and IL-17 receptor signaling are required for mucosal host defenses in oral candidiasis (Conti et al., 2009); whereas Th17 impairs antifungal resistance and promotes inflammation in gastric infection model (Zelante et al., 2007). Th17 responses impair antifungal resistance and promotes inflammatory damage in the lungs of mice infected with Aspergillus (Zelante et al., 2007;Bozza et al., 2009;D'Angelo et al., 2009).
Overall, the Th17 response may have beneficial effects for clearance of some fungal pathogens; however it also has high potential to produce undesirable effects, including inflammatory damage.
Excessive immune reaction and uncontrolled inflammation can result in serious damage of the inflicted organs and tissues. Anti-inflammatory or regulatory cytokines such as IL-10 and TGF-are an important part of the balance which prevents over exuberant inflammation during acute responses. These cytokines are also thought to be important components of resolution and tissue repair that occurs after elimination of the pathogen. Regulatory T cells are important sources of these cytokines and their role in inflammatory diseases and in the maintenance of healthy tissue homeostasis becomes increasingly appreciated (Romani and Puccetti, 2008;De Luca et al., 2010b;Littman and Rudensky, 2010). The importance of balance between pro-inflammatory processes and Treg cell regulation has r e c e n t l y b e e n d e m o n s t r a t e d i n m o d e l s o f Pneumocystis (McKinley et al., 2006) and Histoplasma infections (Kroetz and Deepe). The excessive/damaging inflammatory reaction can be exemplified by immune reconstitution inflammatory syndrome (IRIS). IRIS is characterized by uncontrolled inflammatory responses with high induction of IFN-, TNFand other pro-inflammatory cytokines (Mori and Levin, 2009). Overproduction of these cytokines, rather than having protective effects, contributes to tissue injury that leads to worsening of the patient condition and high mortality (Mori and Levin, 2009). Interestingly, occurrence of IRIS in HIV patients who undergo antiretroviral therapy is particularly high in patients with Cryptococcus and Pneumocystis infections (Singh et al., 2005;Singh and Perfect, 2007;Murdoch et al., 2008). The mechanism of inadequate inflammatory response in IRIS is not understood, however it has been proposed that the regulatory mechanisms that control the inflammation, including Tregs are not sufficiently mobilized to put a break on this inflammatory response (McKinley et al., 2006;Shankar et al., 2008). In fact, patients with IRIS showed reduced suppressor function and diminished secretion of anti-inflammatory IL-10 by Tregs in one of the studies (Seddiki et al., 2009). Tregs are critical for maintaining the proper homeostasis in the GI track, and such mechanisms of protective tolerance are likely to be critical in the respiratory tract which is constantly exposed to inhaled fungal antigens. Insufficiency of the regulatory mechanisms most likely contributes to the development of allergic diseases. Thus, Tregs cells are important for maintaining balance between appropriate clearance rate and the inflammatory tissue damage. Such balance can be disturbed and the excessive Treg function may promote fungal persistence. A detrimental role of IL-10 has been demonstrated in cryptococcal infection models (Blackstock et al., 1999;Arora et al., 2005). Future studies will be needed to evaluate the possible role of Tregs in fungal infections, especially in the patients who develop mycoses without apparent immunodeficiency.
The polarization of T cells to Th1, Th2, Th17 and Treg lineages is important for the development of protective immunity, protective tolerance, chronic/allergic syndromes, or overwhelming allergic reactions. The proper balance maintained by the mutual regulation between these arms of the immune system is necessary to optimize clearance and minimize inflammatory damage to the infected tissues in the context of fungal infection. Our present understanding of these responses evolved from an oversimplified polarized Th1/Th2 paradigm to a broader understanding of mutual regulation ongoing during the immune process. Recent studies show that the Th1, Th2, Th17 responses co-exist in a fungus infected lungs and the balance of cytokine production alters during different time points in a chronic fungal infection (Arora et al., 2011). Modulation of these responses can be achieved experimentally and therapeutically by use of cytokines and vaccination with different fractions of fungal antigens resulting in the induction of the proper and protective Th-cell polarization.

Vaccine-induced therapies targeting cellular mediated immunity
Currently, there are no standardized vaccines available for the prevention of fungal diseases in humans (as discussed earlier). A preponderance of evidence points to the development of cell-mediated immune responses, principally by Th1-type CD4 + T cells, as the predominant host defense mechanism against primary and opportunistic pulmonary fungal pathogens (Cutler et al., 2007). Further, ablation or neutralization of several Th1-type cytokines renders mice more susceptible to experimental infection with a number of fungal pathogens.
Consequently, there has been great interest in identifying antigens that elicit protective CMI against fungal infections; some of which will be discussed herein.
Vaccination with native or recombinant Hsp60 from H. capsulatum or a domain within Hsp60 conferred protection in mice given a sub-lethal challenge with yeast cells and prolonged survival in mice given a lethal challenge (Gomez et al., 1995;Deepe and Gibbons, 2002). Protection was CD4 + T cell dependent and associated with the induction of IFN-, IL-12 and, surprisingly, the Th2-type cytokine IL-10 ( Deepe and Gibbons, 2002;Scheckelhoff and Deepe, 2005). A similar vaccination strategy in mice using Hsp70 did not induce robust IL-12 or IFN-responses and protection against subsequent challenge with live yeast. Neutralization of IL-12 or IFN-abolished the protective efficacy of the Hsp60 vaccine in mice further highlighting the importance of these Th1-type cytokines in the induction of protection against H. capsulatum.
Similarly, vaccination of mice with recombinant Hsp60 derived from P. brasiliensis elicited protection against a lethal intranasal challenge with yeast (de Bastos Ascenco Soares et al., 2008). The protective effect of P. basiliensis Hsp60 was abrogated following the depletion of CD4 + T cells or neutralization of IFN-; similar to that observed for Hsp60 from H. capsulatum (Scheckelhoff and Deepe, 2005;de Bastos Ascenco Soares et al., 2008). However, IL-10 was not produced following antigen stimulation of splenocytes obtained from P. basiliensis Hsp60 immunized mice. While the efficacy of vaccination with forms of Hsp60 from H. capsulatum and P. brasiliensis are encouraging, immunization with recombinant Hsp60 derived from C. immitis resulted in predominantly Th2 cytokine responses and little protection against a subsequent intraperitoneal challenge (Li et al., 2001). Thus, the induction of Th1-type immune responses in the lungs appears critical for the development of protection following immunization with Hsp60.
Evaluation of live attenuated, recombinant, and DNA vaccines of C. immitis in murine models have also highlighted the importance of Th1-type cytokine production, particularly IFN-, in protection against this microbe (reviewed in (Cole et al., 2004;Cox and Magee, 2004;Xue et al., 2009)). Mice immunized with recombinant aspartyl protease (Pep1), alphamannosidase (Amn1), or phospholipase B (Plb) individually or together as amultivalent vaccine experienced a significant reduction in fungal burden and prolonged survival against a lethal pulmonary challenge with C. posadasii arthroconidia compared to controls (Tarcha et al., 2006). Approximately 85% of mice immunized with the multivalent recombinant vaccine survived to day 90 post-inoculation. Similarly, immunization of mice with two recombinant antigens, Coccidiodes-specific antigen (CSA) and the proline-rich cell wall protein Ag2/PRA, either as a mixture of two separately expressed proteins or as a single chimeric expression product was shown to protect mice from a lethal intranasal infection with C. posadasii (Shubitz et al., 2006). The protection observed with each vaccination strategy was associated with robust IFN-responses in protected mice, again showing the importance of Th1-type cytokines during protective host responses. Further, these studies highlighted the utility of a multivalent vaccination strategy that potentially evokes protective responses towards a broader set of T-cell epitopes.
The importance of CD4 + T cells and the generation of Th1-type responses towards eliciting protection against pulmonary fungal pathogens are also observed in vaccination models using a live C. neoformans strain engineered to express IFN- (Wormley et al., 2007) (Wozniak et al., 2009  ), a live attenuated strain of B. dermatitidis (Wuthrich et al., 2000), and recombinant A. fumigatus protein Asp f3 (Diaz-Arevalo et al., 2011). Immunization with recombinant Asp f3 of A. fumigatus protected cortisone acetate immune suppressed mice from an experimental pulmonary infection with A. fumigatus conidia (Diaz-Arevalo et al., 2011). The protection was dependent on CD4 + T cells as their depletion reduced the survival of vaccinated mice and adoptive transfer of Aspf3 primed CD4 + T cells into non-vaccinated mice enhanced their survival against experimentally induced pulmonary aspergillosis. Generation of sterilizing immunity in mice following pulmonary immunization with a C. neoformans strain engineered to express murine IFN-, designated H99 , was shown to require the induction of Th1-type cell-mediated immune responses (Wozniak et al., 2009). Interestingly, B-cell deficient mice immunized with H99 were protected from a subsequent lethal pulmonary challenge with WT C. neoformans (Wozniak et al., 2009;Young et al., 2009;Wozniak et al., 2011b). Also, vaccination of mice with an attenuated strain of B. dermatitidis containing a targeted deletion in the BAD1 locus resulted in prolonged survival that was chiefly mediated by IFN-and TNF-production by CD4 + T cells (Wuthrich et al., 2000;Wuthrich et al., 2002). Although these studies show that Th1-type CD4 + T cell responses are required for optimal host responses against these pulmonary pathogens, studies in H. capsulatum, B. dermititidis, and C. neoformans highlight the inherent plasticity of the host response against pulmonary fungal pathogens. That is that some elements of the immune response can compensate for the loss of other components.
Vaccine-induced immunity against B. dermatitidis was shown be mediated by CD4 + / T cell production of TNF-and IFN- (Wuthrich et al., 2002). Moreover, the initiation, but not maintenance, of protective memory responses to B dermatitidis required IL-12 production (Wuthrich et al., 2005). However, vaccine-induced immunity could be elicited and expressed in IFN-and TNF-deficient mice. The reciprocal cytokine or the presence of GM-CSF was shown to compensate for the loss of IFN-and TNF-showing some plasticity in the vaccine-induced host response to Blastomyces (Wuthrich et al., 2002). Furthermore, a role for Th17 cells in vaccine-induced protection against multiple pulmonary fungal pathogens has been shown (Wuthrich et al., 2011). Specifically, protection afforded by vaccination against C. posadassi, H. capsulatum, and B. dermatitidis was observed to be dependent on IL-17. In fact, IL-17 was shown to be indispensable since vaccinated IL-17A or IL-17RA deficient mice showed impaired anti-fungal resistance despite having normal Th1-type cytokine expression. In contrast, IL-17A was shown to contribute to but ultimately be dispensable for protection against experimental pulmonary cryptococcosis in C. neoformans strain H99 vaccinated mice (Hardison et al., 2010b;Wozniak et al., 2011a). Still, it appears imperative that vaccines strategies to prevent pulmonary mycoses be evaluated for their capacity to induce Th1 and Th17-type cytokine responses.
The induction of T cell-mediated immune responses is critical for optimal protection against pulmonary fungal pathogens (Cutler et al., 2007). Consequently, it may seem counterintuitive to suggest that vaccines designed to prevent fungal infections in patients with T cell deficiencies is possible. Nonetheless, vaccination studies using experimental models of H. capsulatum (Deepe and Gibbons, 2002), P. brasiliensis (de Bastos Ascenco Soares et al., 2008), H. capsulatum (Wuthrich et al., 2003), B. dermatitidis (Wuthrich et al., 2002;Wuthrich et al., 2003), C. immitis (Fierer et al., 2006), and C. neoformans (Wozniak et al., 2011b) have indicated that vaccine-induced protective immune responses can be elicited in immune deficient hosts. Cumulatively, the studies show that the presence of CD4 + or CD8 + T cells is essential for the induction (the period following vaccination) and expression (immune response following challenge) phases of the protective immune response. Protection is not induced in mice that are T cell deficient or depleted of both CD4 + and CD8 + T cell populations. Further, protection is lost in vaccinated mice following deletion of both T cell subsets. Interestingly, 80% of mice vaccinated with C. neoformans strain H99 and subsequently depleted of both CD4 + and CD8 + T cells were protected from a lethal pulmonary challenge with WT C. neoformans (Wozniak et al., 2011b). These studies highlight dynamic compensatory mechanisms that mediate vaccine-induced protection during both the induction and expression phases of the anti-fungal immune response. Altogether, the results demonstrating the plasticity of the vaccine-induced immune response to pulmonary fungal pathogens are particularly exciting as they highlight the potential for inducing protection in immune competent and immune compromised hosts.

Antibody-mediated immunity and therapeutics
The contribution of antibody mediated immunity (AMI) towards protecting individuals against pulmonary fungal infections remains uncertain. Individuals with humoral deficiencies such as autosomal hyper-IgM syndrome and IgA deficiency do not exhibit an increased susceptibility to fungal infections (reviewed in Antachopoulos, 2007Antachopoulos, , 2010. In contrast, patients with X-linked hyper IgM syndrome and common-variable immunodeficiency which are often accompanied by defects in T CMI have a higher risk of developing pulmonary and invasive fungal infections like cryptococcosis and histoplasmosis. The efficacious role for antibodies during the host immune responses against fungi is like that observed against bacterial and viral pathogens. Antibodies produced in response to fungal infection serve as opsonins to promote phagocytosis, participate in antibody-dependent cellular cytotoxicity, augment Th1-type polarization, help to eliminate immunosuppressive polysaccharide antigen from serum and tissues, inhibit biofilm formation, have direct antifungal activity, and modulate the immune response to prevent host damage (reviewed in Alvarez and Casadevall, 2007; reviewed in Zaragoza and Casadevall, 2004).
Most studies showing the efficacy of AMI against pulmonary fungal pathogens has involved experimental models of PcP and cryptocococcosis. The polysaccharide capsule of Cryptococcus, its main virulence determinant, is predominantly comprised of the polysaccharides glucuronoxylomannan (GXM) and galactoxylomannan (GalXM) and to a much lesser extent, <1%, mannoproteins (MP) (reviewed in Zaragoza et al., 2009). Conjugate vaccines consisting of GXM combined to either tetanus toxoid (TT) or Pseudomonas aeruginosa exoprotein A (rEPA) induce high antibody titers (Devi et al., 1991;Casadevall et al., 1992), enhanced antifungal activity of murine and human phagocytes (Mukherjee et al., 1995c;Pirofski, 1996, 1998) and conferred some protection against cryptococcosis in mice (Devi, 1996;Fleuridor et al., 1998;Nussbaum et al., 1999). Unfortunately, the profound suppressive effects on immune responses induced by cryptococcal polysaccharides and the highly variable immune responses observed in response to the intact GXM portion of the conjugate vaccine renders it an unlikely choice for future vaccine development (reviewed in Zaragoza et al., 2009;reviewed in Pirofski, 2001). A strategy using small peptide mimotopes (peptides which are able to induce antibodies that are capable of binding to the native antigen when administered as an immunogen) that mimic defined GXM epitopes was attempted to elicit protective antibody responses where using total GXM was unsuccessful. Zhang et al. described a peptide mimetic (P13) of GXM that was recognized by human anti-GXM antibodies  and showed that vaccination with P13-protein conjugates in mice resulted in prolonged survival after a lethal C. neoformans challenge compared to controls (Fleuridor et al., 2001) or following establishment of a chronic infection (Datta et al., 2008).
Casadevall et al. developed a murine monoclonal antibody (MAb), 18B7, to C. neoformans polysaccharide that underwent phase I clinical studies in HIV + patients with cryptococcal antigenemia (Casadevall et al., 1998). A modest reduction in serum cryptococcal antigen titers was observed in patients receiving singular doses of 1 and 2 mg/kg up to 10 weeks post-treatment before returning to baseline (Casadevall et al., 1998). To date, no follow-up clinical studies have been published. A new approach using MAb 18B7 currently being investigated in mice involves conjugation of the MAb to the therapeutic radioisotopes 188 Rhenium or 213 Bismuth (Dadachova et al., 2003;Bryan et al., 2010). Studies have shown that administration of radiolabeled MAb 18B7 to lethally infected mice results in prolonged survival, reduced organ fungal burden, and was a more effective therapy compared to mice treated with amphotericin B. Radioimmunotherapy can be applied using MAb derived against multiple pulmonary fungal pathogens and thus may evolve into an attractive option for the treatment of other pulmonary mycoses. Lastly, while most studies have examined passive administration with antibodies targeting C. neoformans polysaccharide, other cryptococcal targets for passive antibody therapy under experimental investigation include melanin (Rosas et al., 2001), -glucan (Rachini et al., 2007), heat shock protein (HSP) 90 (Nooney et al., 2005) and glucosylceramide (Rodrigues et al., 2007).
Mice deficient in B cells, either due to a targeted disruption of the IgM constant region (µMT mice) or using depletion antibodies, are more susceptible to PcP infection (Harmsen and Stankiewicz, 1991;Marcotte et al., 1996) (Lund et al., 2003;Lund et al., 2006). These studies showed that B cells were able to provide protection against PcP not only by producing Ab but also by amplifying the CD4 + T cell-mediated immune response. Passive administration of an IgM mAb shown to be directed against a surface antigen present on rat-, rabbit-, ferret-, and human-derived P. carinii induced partial protection against PcP in animal models (Gigliotti and Hughes, 1988). Subsequent studies showed that the passive administration of mAbs recognizing kexin-like molecule (KEX1) via the intranasal route prior to experimentally induced PcP resulted in a significant reduction in pulmonary fungal burden (~99%) (Gigliotti et al., 2002).
Contrasting studies have shown that B cell deficient µMT mice have lower pulmonary fungal burden following intranasal infection with A. fumigatus (Montagnoli et al., 2003) or B. dermatitidis (Wuthrich et al., 2000) and are not more susceptible to experimental pulmonary histoplasmosis infection (Allendorfer et al., 1999) compared to WT controls. Also, passive transfer of polyclonal serum or MAbs obtained from A. fumigatus, B. dermatitidis, or C. immttis vaccinated mice did not enhance protection against a subsequent intranasal challenge with these pathogens (Kong et al., 1965;Beaman et al., 1977;Frosco et al., 1994;Wuthrich et al., 2000;Beaman et al., 1979). The role of antibodies in the host defense against fungal infection remains controversial because of its complexity. The current consensus is that antifungal antibodies can mediate protective, nonprotective, or disease-enhancing effects on host defenses during infection (Mukherjee et al., 1995a;Mukherjee et al., 1995b;Yuan et al., 1998a). Thus, resistance to disease may depend upon the proportion of protective antifungal antibodies produced during infection. In support of this concept, nonprotective and protective MAbs to C. neoformans has been described (Mukherjee et al., 1995a;Maitta et al., 2004). Also, Nosanchuk et al. demonstrated that mice passively administered MAbs targeting a histone H2B-like protein on the surface of H. capsulatum before infection experienced a reduction in fungal burden and prolonged survival (Nosanchuk et al., 2003). These studies were somewhat surprising in light of previous studies showing no increased susceptibility to experimental histoplasmosis infection in B cell deficient mice (Allendorfer et al., 1999). Studies in C. neoformans has shown that the efficacy of MAbs appears to be dependent on several variables including host genetics (Rivera and Casadevall, 2005), Ab isotype (Yuan et al., 1995;Yuan et al., 1998b), T cell function (Yuan et al., 1997), and the presence of Th1-and Th2-related cytokines (Beenhouwer et al., 2001). AMI during the protective response to pulmonary fungal pathogens is broad and divergent, but it is clear that specific antibodies are efficacious for the host in the resolution of infection.
Studies also support the potential of using antibodies that target antigens common among multiple fungi to mediate cross-protection. Passive immunization using anti -glucan MAbs or vaccination with -glucan (laminarin) conjugated with the genetically-inactivated diphtheria toxin CRM197 (Lam-CRM vaccine) has been shown to confer protection against C. neoformans, C. albicans and A. fumigatus (Torosantucci et al., 2005;Rachini et al., 2007). Since -glucans are found in the cell wall of fungi, the efficacy of anti--glucan antibodies can be very broad. Cenci et al. used a killer anti-idiotype MAb reacting to a yeast killer toxin to protect mice from a lethal A. fumigatus challenge during experimental bone marrow transplantation (Cenci and Romani and 2375). Killer toxin is also expressed by multiple fungal species. Mycograb (NeuTec Pharman plc.), a recombinant antibody targeting an epitope within the HSP90 of Candida albicans that is conserved with the corresponding protein in C. neoformans, has been shown to act in adjunct with amphotericin B against multiple Candida species and C. neoformans. Altogether, these studies highlight the possibility that antibodies targeting "universal' antigens common to multiple fungal species such as -glucans, killer toxins, or Hsps may extend protection to multiple disparate fungal pathogens. Casadevall and Pirofski has published an elegant commentary on the emergence of cross-protective targets for fungi (Casadevall and Pirofski, 2007).

Conclusion
The principal route of entry for several of the primary and opportunistic fungal pathogens is via inhalation of infectious propagules into the lungs. Consequently, exposure to these fungi is unavoidable. Nevertheless, most encounters are asymptomatic due to the quick resolution of the fungi by resident effector cells within the lung. On those occasions that the fungal insult cannot be quickly eradicated, T cells, predominantly CD4 + T cells, preside over orchestrating the adaptive responses and provide help for antibody production. T cell responses are also influenced by cytokine production by innate effector cells. Nonetheless, T cells mediate various cellular responses at the sites of infection and are ultimately responsible for either resolution or pathological reactions associated with these infections. Furthermore, T cells are important players in homeostasis and protecting integrity of natural barriers. Recent advances in experimental animal models support the premise that antifungal vaccines may be effective in immune compromised hosts. The efficacy of anti-fungal vaccines in immune compromised populations is undoubtedly due to the inherent plasticity of host immunity. Altogether, it is clear that immune responses to pulmonary fungal infections are as diverse as the fungi themselves but that significant ground has been made towards its understanding.