The immune response in Coccidioidomycosis

Autoimmunity Reviews 10 (2010) 94–102

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Autoimmunity Reviews j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a u t r ev

Review

The immune response in Coccidioidomycosis Andrea T. Borchers, M. Eric Gershwin ⁎ Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis School of Medicine, Davis, CA 95616, United States

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Article history: Received 10 August 2010 Accepted 13 August 2010 Available online 20 August 2010

a b s t r a c t With the increasing use of biologics, clinical rheumatologists are becoming very well acquainted with opportunistic infections, including tuberculosis, histoplasmosis and Coccidiomycosis. In the great valleys of California as well as several other hot spots in the Southern areas of the United States and select pockets in South America, valley fever, also known as Coccidiomycosis, is an endemic infection. The vast majority of patients are asymptomatic following exposure, but are at risk for clinical disease in the case of immunosuppression. Additionally, although 60% of patients with infections are completely asymptomatic, nearly all patients have immunological evidence of exposure. Within some communities in the central valley of California, sero conversion approaches 100%, fortunately the vast majority remain asymptomatic. In this review we will place the context of the immune response to Coccidiomycosis in perspective and discuss not only the lymphoid response, but also recent data on antigenic analysis and bioinformatics of Coccidioides. This information is significant not only for a better understanding of Coccidiomycosis, but will also have utility in the management of patients within areas of the world who are treated with the biologics for autoimmune disease. © 2010 Elsevier B.V. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Innate immune responses . . . . . . . . . . . . . . . . . . . . 3. Dendritic cells . . . . . . . . . . . . . . . . . . . . . . . . . 4. Histopatholocigal studies of lymphocytes in Coccidioidomycosis . . 5. Cell-mediated immunity . . . . . . . . . . . . . . . . . . . . 6. T cell antigens . . . . . . . . . . . . . . . . . . . . . . . . . 7. Cellular immunity in mice. . . . . . . . . . . . . . . . . . . . 8. T cell antigens identified in immunization studies . . . . . . . . 9. B lymphocytes and the humoral immune responses to Coccidioides 10. B cell antigens . . . . . . . . . . . . . . . . . . . . . . . . . 11. Use of the biologics . . . . . . . . . . . . . . . . . . . . . . . Take-home messages . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Coccidioidomycosis is an infection caused by two members of the dimorphic soil-dwelling fungal genus Coccidioides, C. immitis and C. posadasii, which are genetically distinct, but virtually indistinguish-

⁎ Corresponding author. Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis School of Medicine, 451 Health Sciences Drive, Suite 6510, Davis, CA 95616, United States. Tel.: + 1 530 752 2884; fax: + 1 530 752 4669. E-mail address: [email protected] (M.E. Gershwin). 1568-9972/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.autrev.2010.08.010

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able at the phenotypic level. It is commonly called “Valley Fever” or “San Joaquin Valley Fever” because the major endemic area of C. immitis is the San Joaquin Valley of California. The endemic regions of C. posadasii are the arid and semi-arid zones of southern areas of Arizona, Utah, Nevada and New Mexico, western Texas and some pockets in South America. An estimated 150,000 new infections occur each year in the United States, and the incidence of reported cases in California and Arizona tripled between 1999/2000 and 2006 [1]. Although there are indications that the incidence decreased in the most recent years, it remains considerably higher than during the period 1995–2000.

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Although first described in 1892, the full spectrum of coccidioidomycosis was not appreciated until C.E. Smith and his colleagues investigated several thousand military personnel at various army air fields in the San Joaquin Valley in the 1940s [2]. They established that approximately 60% of Coccidioides infections remained asymptomatic, the only evidence of infection being provided by the conversion from a negative to a positive delayed-type hypersensitivity (DTH) reaction to skin testing with coccidioidal antigens [2]. The remaining 40% developed some form of pulmonary disease 7–28 days after exposure, the median incubation time being 2 weeks [3]. Note that the frequency of symptomatic disease can be considerably higher in outbreaks following dust storms, archeological digs or military training exercises, which is thought to relate to high levels of exposure [4,5]. Smith et al. [2] further found that the pulmonary disease resolved without complications in a majority of patients, but persisted in either stabilized or progressive form in approximately 5% of cases with symptomatic primary coccidioidomycosis. Approximately 1% of all clinically diagnosed cases developed extrathoracic dissemination. The incidence is again higher during outbreaks associated with high exposure levels (3–6%) [6]. Dissemination may be acute, chronic or progressive and may consist of a single lesion in virtually any tissue of the body or be multifocal. In cases of dissemination to a single extrathoracic site, the prognosis is generally good, except when the meninges are affected. Meningitis requires life-long therapy and is associated with considerable morbidity. Multifocal dissemination remains associated with N50% mortality. Risk factors for disseminated disease include Filipino, African American, and Mexican American ethnicity, gender, age, pregnancy and immunosuppression due to HIV infection, other immunosuppressive diseases, or drug therapy [6–8]. There are indications, however, that both the incidence and the severity of coccidioidomycosis in patients with HIV-1 infection have decreased markedly after the introduction of highly active antiretroviral therapy [9]. Typically, persons who have recovered from symptomatic coccidioidomycosis possess life-long immunity not only against the infecting strain, but also against geographically distinct strains [10,11]. However, the disease can be reactivated in the context of immunosuppression. Like several other pathogenic fungi, C. immitis and C. posadasii are dimorphic, but they are absolutely unique in that their parasitic stage consists of a complex morphogenetic cycle [12]. During the saprobic (soil) cycle, Coccidioides exists as a mycelial colony that forms arthroconidia (previously called arthrospores) in order to propagate. Airborne arthroconidia constitute the infectious form of the organisms. Once inhaled, they rapidly enter a second morphologic cycle that starts with their transformation into separated round cells, which rapidly enlarge in association with nuclear divisions and segmentation, ultimately resulting in the formation of spherules that contain between 100 and 300 endospores. Rupture of the spherule releases these endospores, each of which can in turn develop into a spherule. While the presence of spherules in tissue is diagnostic, it should be noted that mycelial forms, including septate hyphae and arthroconidia, have been detected in a substantial portion of patients with chronic coccidioidomycosis [13], in particular those with underlying type 2 diabetes [14]. 2. Innate immune responses The first cells to respond to the presence of arthroconidia are polymorphonuclear leukocytes (PMN) [15]. There are indications that this cellular influx occurs partly in response to soluble factors derived from the fungus that generate complement components with chemotactic activity [16]. Peripheral PMN from immune and nonimmune humans and animals are capable of phagocytosing arthroconidia [15,17,18]. Higher rates of phagocytosis have been observed in the presence of immune serum. As arthroconidia transform into round cells and progress towards the spherule stage, there is a progressive

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decline in phagocytosis by PMN [18]. Spherules are too large (60 to N100 μm in diameter) to be engulfed by phagocytes [18], and it is only the release of endospores that triggers another influx of PMN [15,19]. Like arthroconidia, endospores are quite readily phagocytosed, particularly in the presence of immune serum [18]. The interactions of PMN with fungal particles trigger an oxidative burst, the components of which are capable of inhibiting fungal growth [18,20,21]. The magnitude of the oxidative burst and the extent of cytostatic activity progressively decline during the development from arthroconidia to mature spherules [20]. However, even arthroconidia are not killed very efficiently, the rate being ≤5% in non-immune serum compared to 20%–30% in immune serum [15,17,18,22,23]. There are even indications that PMN induce the development of arthroconidia into spherules [24]. The extent of phagocytosis and killing of arthroconidia, round cells and endospores depends on the fungal strain [18], suggesting that fungal as well as host factors may influence the course of the disease. Monocytes/macrophages from humans and a variety of other species are capable of phagocytosing arthroconidia and endospores [25–28] and young spherules [22,29]. However, in the majority of these studies killing of the ingested fungal particles is minimal to non-detectable, and ingested endospores have been observed to develop into spherules that eventually lyse the host macrophage [27]. Studies in humans and animals indicate that Coccidioides arthroconidia and endospores inhibit phagosome–lysosome fusion in macrophages from non-immune hosts [27,30] and that this inhibition can be overcome by the addition of activated immune lymphocytes, but not immune serum or complement [31,32]. This effect was suggested to be mediated by the production of IFNγ and TNFα, since preincubation of monocytes with either of these cytokines enhanced the killing of engulfed endospores and arthroconidia by monocytes [28,30,31]. Note, however, that these treatments were only effective when applied before infection. Interestingly, the addition of IFNγ also inhibited the development of endospores into spherules [28]. In contrast, the results of another investigation suggest that human peripheral blood monocytes from both skin testpositive and skin test-negative subjects are capable of killing engulfed arthroconidia [25] and that the addition of TNFα or IFNγ does not significantly enhance this ability [22]. Of note, in this study, human peripheral monocytes were even capable of killing young spherules, but this ability decreased with increasing spherule maturation and was minimal when 96-h spherules were used [22]. These discrepant results may be largely attributable to methodological differences. The addition of immune serum did not significantly increase either the phagocytosis or the killing of arthroconidia and endospores in alveolar macrophages from rhesus macaques [27] or mouse peritoneal macrophages [32]. In contrast to these results obtained with the polyclonal antibody population present in immune serum, it was recently shown that a purified IgG fraction of the antiserum raised against a specific domain of the spherule outer wall glycoprotein (SOWgp) significantly augmented the ability of mouse alveolar macrophages to engulf and to kill spherules in the early stages of maturation [29]. Human peripheral monocytes/macrophages from both skin test positive and negative subjects were able to increase their production of TNFα, IL-1β and IL-6 upon incubation with formalin-killed and, to a lesser extent, viable spherules [33]. Similarly, others reported that killed arthroconidia or spherules stimulated TNFα production by monocytes from both immune and non-immune subjects [34]. However, skin test positive subjects produced significantly higher amounts biologically active TNFα, but similar levels of immunoreactive TNFα, in response to stimulation with killed spherules. To date there have been few investigations of the receptors involved in the recognition of Coccidioides. The mannose receptor was shown to mediate the binding of some fungal elements to human peripheral monocytes [35] and monocyte-derived dendritic cells (DCs) [36]. In

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mouse peritoneal macrophages, toll-like receptor (TLR)-2 and dectin, but not TLR-4, were shown to be necessary in order for spherules to trigger the production of cytokines and chemokines [37]. While others confirmed that TLR-4 does not contribute to primary host defense to Coccidioides in mice, they nonetheless found that this receptor is a crucial element in the prevention of fungal dissemination [38]. The role of natural killer (NK) cells in Coccidioidomycosis remains essentially undefined. There is a single study suggesting that NK cells make a significant contribution to killing C. immitis endospores and young spherules [39]. However, the Leu-11 (CD16) antibody that was used for the identification of the effector cell type is not specific to NK cells, making it difficult to clearly attribute the activity to NK cells. Although NK cells express intracellular IFNγ after stimulation with a coccidioidal antigen preparation, their contribution to the overall secretion of this cytokine is negligible [40]. Yet, the synthesis of both TNFα and IL-10 was markedly enhanced in the absence of NK cells, suggesting a role in regulating the production of these cytokines in response to coccidioidal antigens.

T27K, had a significantly higher number of polyfunctional CD4+ and CD8+ T cells compared to non-immune donors [43]. Incubation of PBMC from non-immune or immune subjects with T27K-pulsed DCs did not increase the frequency of polyfunctional CD4+ T cells or the frequency of CD4+ T lymphocytes positive for IFNγ, IL-2 or TNFα alone [43]. Nonetheless, it did augment the secretion of IFNγ and IL-2 when PBMC from immune and non-immune subjects were cultured in the presence of autologous T27K-pulsed DCs, similar to what had been reported from the previous study with T27K-pulsed DCs [42]. The ingestion of killed C. posadasii spherules by immature DCs derived from peripheral monocytes from non-immune donors induced the phenotypic and functional maturation of these DCs [36]. It also significantly stimulated the production of TNFα, IL-1β, IL8 and IL-10, but not IL-12p70. This would be expected to result in a Th2 polarization of the ensuing T cell response, which would be highly undesirable given the importance of Th1-dominated cell-mediated responses particularly in the early phases of the host defense against Coccidioidomycosis.

3. Dendritic cells

4. Histopatholocigal studies of lymphocytes in Coccidioidomycosis

Dendritic cells (DCs) are crucial in the activation of naïve T cells and the polarization of the ensuing T cell responses. The generation of a Th1 immune response is critically dependent on the ability of DCs to produce IL-12, a cytokine consisting of a p35 and a p40 subunit that form the biologically active dimer p70. The factors driving a Th2 polarization of the T cell response are not completely understood, but are thought to involve the relative lack of IL-12 production by DCs. It has been demonstrated that a toluene lysate of Coccidioides spherules (TSL) can induce the maturation of monocyte-derived DCs obtained from individuals without Coccidioides immunity, and the extent of maturation was similar to that seen with TNFα [41]. These TSL-pulsed DCs were capable of stimulating autologous lymphocyte proliferation and IFNγ production. In contrast, another antigen preparation from spherules called T27K did not induce DC maturation, but T27K-pulsed DCs elicited a proliferative response in autologous PBMC obtained from patients with disseminated Coccidioidomycosis who manifested anergy to T27K in the absence of such DCs [42]. As seen with TSL, T27K-pulsed DCs also expanded an antigen-specific population of Coccidioides-specific T cells from non-immune subjects, stimulated the production of significant amounts of IFNγ and, after a second round of stimulation, induced low but measurable amounts of IL-2 and TNFα, but also IL-4, IL-5 and IL-10. Note that, in healthy nonimmune donors, restimulation induced significantly higher levels of all of these Th2 cytokines compared to anergic subjects, although levels of IFNγ were still at least 10-fold higher. The reversal of Coccidioides-specific anergy with appropriate stimulation suggests a defect in antigen presentation. However, since some patients with Coccidioidomycosis are initially DTH positive and lose this reactivity during the course of the disease, the defect may not reside in DCs but rather in other antigen-presenting cells (APCs) capable of restimulating antigen-specific T cells, such as macrophages and B cells. It is generally accepted that a Th1 response is required for successful host defense against Coccidioides infection. Therefore, it remains to be established whether the induction of Th2 cytokine synthesis, as seen upon restimulation of PBMC from non-immune donors with T27Kpulsed DCs, represents a desirable outcome [42]. Another study examined the ability of T27K-pulsed DCs to increase the number of polyfunctional T lymphocytes, defined as simultaneously producing intracellular IFNγ, IL-2 and TNFα [43]. Polyfunctional T lymphocytes, more generally defined as exhibiting more than one function in response to stimulation with a specific antigen, have been associated with protection from a variety of diseases [44,45], although negative correlations have also been reported [46]. After stimulation with T27K, PBMC from subjects with coccidioidal immunity, as determined by in vitro cytokine release in response to

An investigation of lymphocyte subsets in organized necrotizing coccidioidal granulomas in 9 lung biopsy specimens revealed that T cells predominated in the mantle, although B cells were also observed [47]. The number of CD4+ and CD8+ T cells was approximately equal. In addition, clusters of small lymphocytes consisting of B cells surrounded by a somewhat lower number of CD4+ and CD8+ lymphocytes were seen at the margin of the granulomas. In these clusters, the ratio of CD4+ to CD8+ T cells was approximately 4:1. Of note, occasionally these clusters formed germinal centers, suggesting the occurrence of a local humoral immune response. There were about equal numbers of lymphocytes expressing IFNγ and IL-10 in the mantle and a substantial number of lymphocytes expressing IL-10 in the perigranulomatous clusters, suggesting a counter-regulation of the cellular immune responses. This pattern is quite different from that of a patient with cavity pulmonary Coccidioidomycosis, where CD4+ T cells were observed throughout the granuloma, while CD8+ T cells were found in the mantle, with CD4+ T cells constituting the major population overall [48]. In contrast, the skin of patients with disseminated disease contained granulomas where CD4+ and CD8+ T cells were localized predominantly in the mantle and CD8+ T cells outnumbered CD4+ lymphocytes. 5. Cell-mediated immunity Approximately 80% of patients with primary nondisseminating Coccidioidomycosis manifest skin test reactivity to Coccidioidal antigens, whereas only about 30% of patients with progressive or multifocal disseminated disease do [6,49]. When skin test reactivity is low or absent, the prognosis is frequently poor, whereas the acquisition of DTH responses is associated with recovery and possibly with remaining free of relapse after discontinuation of therapy [49]. There are examples of patients reverting from a negative to a positive skin test after recovery [50]. This suggests that, at least in some cases, the anergy is acquired during the course of the disease and is not pre-existing. Of note, the hallmark of “Valley Fever” as originally described is the development of erythema nodosum with or without erythema multiforme, which are thought to represent DTH responses, and skin reactivity to Coccidioidin usually develops within days of the appearance of erythema [3]. In keeping with the correlation between DTH reactivity and good prognosis, this manifestation of Coccidioidomycosis constitutes a benign form of the disease that results in spontaneous recovery in almost all cases and very rarely proceeds to progressive or disseminated disease. Since skin DTH reactions constitute a measure of cellular immunity, the observed correlation between the presence of strong skin reactivity to Coccidioidal antigens and a favorable prognosis suggests that cellmediated immunity plays a central role in the human host defense

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against Coccidioides. The importance of CD4+ T cells in providing protection from Coccidioidomycosis is further illustrated by the finding that the risk of developing symptomatic Coccidioidomycosis is associated with lower peripheral blood CD4+ T cell counts in patients with HIV-1 infection [8,9]. DTH responses to Coccidioidal antigens are long-lived and their measurement mainly has been used to distinguish immune from nonimmune subjects. The original antigen used for this purpose was Coccidioidin, a culture filtrate of the saprobic (mycelial) phase because appropriate methods for the in vitro culture of the parasitic phase had not been developed at the time. Once such culture methods became available, a spherule lysate called spherulin was found to be somewhat more sensitive in detecting skin reactivity than Coccidioidin [49]. Both of these preparations have also been used for in vitro tests of cellular immunity, such as lymphocyte transformation (LT), cytokine production and expression of activation markers. Such in vitro tests are at least equally sensitive as DTH tests in distinguishing immune from immune donors. Nonetheless, there generally is little correlation between the size of the induration and the magnitude of the in vitro LT or cytokine response [50–53]. Moreover, there are numerous instances where PBMC from some subjects who show DTH reactions to Coccidioidal antigens fail to respond to in vitro stimulation with the same or a related antigen and vice versa [53–57]. This suggests that skin reactivity and in vitro LT or cytokine production do not measure identical processes. In vitro assessments of cellular immunity generally reveal a similar pattern of responsiveness in the various clinical forms of Coccidioidomycosis as seen with DTH responses. Patients with active pulmonary or disseminated Coccidioidomycosis have significantly lower responses compared to healthy immune donors, while patients who have recovered from their infection have intermediate responses [50,51,54,58,59], although this is not an entirely consistent finding [60]. In response to various Coccidioidal antigen preparations, PBMC from healthy immune subjects produce significantly increased amounts of IFNγ, IL-2, IL-12 and TNFα, but the levels of IL-4 and IL10 are low and similar to those seen in non-immune controls [33,40,54,61]. Patients with active Coccidioidomycosis show the same pattern, but lower responses. Anergic patients do not secrete more IFNγ and other Th1 cytokines than non-immune controls, but synthesize similar amounts of IL-4 and IL-10 as all other groups [40,54,61]. It has also been shown that certain cytokines can modulate in vitro T cell responses to Coccidioidal antigens. For example, exogenous IL-10 significantly decreased the TSL-stimulated proliferation as well as IFNγ and IL-12 production of PBMC from healthy immune donors [61]. In contrast, exogenous IL-12 increased the synthesis of IFNγ in healthy immune donors and also in subjects with active Coccidioidomycosis and positive DTH responses. This effect was not seen in skin test-negative patients with active disease who showed little in vitro response to TSL. The neutralization of IL-10 did not enhance the TSL-induced IFNγ or IL-12 secretion in PBMC from DTH-positive or DTH-negative donors. In partial contrast, neutralization of IL-10 significantly increased the proportion of CD3 T lymphocytes producing intracellular IFNγ after incubation of whole blood with T27K [40]. It bears emphasizing again, however, that PBMC from anergic patients do not produce higher levels of IL-10 (or IL-4) in response to stimulation with Coccidioidal antigens compared to nonanergic subjects [54,61], although only a limited number of subjects has been tested to date. In vitro assessments of cellular immune processes have also provided some insights into the mechanisms potentially involved in the anergy seen mainly in patients with disseminated Coccidioidomycosis. This anergy is generally specific for Coccidioidal antigens, but can be more generalized in patients with multifocal dissemination [55,56]. Importantly, some of the patients who manifest anergy upon skin testing exhibit in vitro responses to Coccidioidal antigens [53–56]. In some cases, the use of heterologous rather than autologous

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serum restored the in vitro responsiveness [56,62]. In others, heterologous serum diminished the response to certain fungal antigens, while enhancing the response to others, with the overall pattern being different in each of the patients tested [60]. In addition, sera from some patients show antigen-specific or even non-specific suppression of LT responses of PBMC from healthy skin-reactive donors or other patients [56,63]. Together, these results suggest the involvement of multiple host and fungal factors. Since circulating immune complexes are frequently detected in patients with Coccidioidomycosis [64], with the highest levels seen in those with multifocal dissemination [65] it was examined whether immune complexes were responsible for the suppression of LT responses. The results implicated monomeric IgG, but not immune complexes, in the suppressor activity [63]. In addition, there are several reports that high concentrations of certain Coccidioidal antigens inhibit LT in vitro [52,66,67]. Other data suggest the induction of suppressor cells [68]. Indeed, two different Coccidioidal antigen preparations were found to activate a suppressor cell population that induces Coccidioidinspecific anergy in mice [69]. Importantly, it was demonstrated that these mice initially developed delayed footpad hypersensitivity to Coccidioidin after infection, but subsequently became anergic [70]. Of note, the antigen-induced suppressor activity could be transferred not only by viable spleen cells from antigen-injected donors, but also by filtered spleen cell lysates from infected donors. This transfer of suppression by non-viable spleen cells suggests that the induction of suppressor cells was not the only mechanism of suppression. 6. T cell antigens Some attempts to identify specific human T cell antigens started with the fractionation of Coccidioidal antigen preparations that had stimulated cellular immune functions in vitro, such as TSL [61,71], or an alkali-soluble water-soluble cell wall extract of C. immitis mycelia designated as C-ASWS [50,51]. A 33-kDa apoglycoprotein was isolated from TSL and subsequently identified as a proline-rich antigen (PRA) [72,73], while C-ASWS was shown to contain a protein designated as antigen 2 (Ag2). Both antigens were eventually cloned, shown to be identical, and are now referred to as Ag2/PRA [73,74]. The 33-kDa apoglycoprotein isolated from TSL after chemical deglycosylation of the crude extract stimulated LT in PBMC from skin test-positive donors, though to a lesser extent than the crude extract [72]. An extract of the membranous spherule outer wall (SOW) has also been shown to elicit a proliferative response from PBMC of skin testpositive, but not from skin test-negative, subjects [67]. In addition, it markedly enhanced the release of IFNγ in immune donor PBMC, while not significantly affecting IL-10 production. A glycoprotein (SOWgp) contained in this extract, which is expressed exclusively during the parasitic phase of Coccidioides, also stimulated the proliferation of PBMC from immune subjects. 7. Cellular immunity in mice Murine Coccidioidomycosis differs substantially from its human counterpart in that even low inocula lead to disseminated disease and death of all infected animals (if they belong to a susceptible strain), in contrast to the high rate of spontaneous resolution and very limited occurrence of dissemination in humans. In addition, the intraperitoneal (i.p.) route of infection has often been used, particularly in earlier studies. It has been documented that mouse strains not only differ in their disease susceptibility, but specific strains also manifest differential susceptibility to i.p. compared to intranasal (i.n.) infection. This suggests that the systemic and mucosal routes of administration activate different types of T cell responses, possibly due to the involvement of distinct types of DCs as APC. While there is mainly indirect or in vitro evidence for the importance of cell-mediated immunity in resistance to Coccidioides infection in

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humans, much more extensive and convincing evidence has come from studies of murine Coccidioidomycosis [6]. Thymectomized or T celldeficient animals are much more susceptible to infection, whereas B cell-deficient mice are not [75,76]. Increased susceptibility is also seen in mice lacking either CD4+ or CD8+ T cells, suggesting that both subsets play a role in primary immunity [6,76]. There have been intensive efforts to generate a vaccine for Coccidioidomycosis using mice as the experimental system. The protection afforded by immunization can be transferred via T cells, but not B cells or passive transfer of serum, and CD4+ T cells are sufficient for the transfer of protection at least after i.p. infection [6,76]. Note, however, that depletion of either CD4+ or CD8+ T cells from the adoptively transferred spleen cells partially abrogated the protective effect in i.n. challenged mice [75]. Genetically susceptible strains produce less IFNγ and more IL-4 and IL-10 in response to infection and IL-10 knockout mice are more resistant to infection than their wild-type animals [6]. Exogenous IFNγ or IL-12 is protective in susceptible strains, whereas neutralization of these cytokines exacerbates the disease in normally resistant strains. Note, however that some of these treatments are effective only after i.p. infection, but not after i.n. challenge, and the timing of the treatment also appears to be important in particular in the cases of IFNγ and IL-12. Importantly, the molecular and cellular mechanisms of vaccineinduced immunity differ from those occurring in primary infection. Neither CD4+ nor CD8+ T cells are essential for vaccine-induced protective immunity in i.p. challenged mice [76,77]. However, the results of one study indicate that MHC class II-restricted T cells are absolutely required [77]. The authors speculated that MHC class IIrestricted CD4-negative T cells might have been responsible for the protection. However, adoptive transfer experiments demonstrated that CD8+ T cells were able to partially compensate for CD4+ T cells in the induction of protective immunity in mice lacking this T cell population [76]. These results are consistent with reports of a certain plasticity in the immune system that makes it possible for CD8+ or other T cell subsets to replace some of the functions of CD4+ T cells in vaccine-induced immunity in other fungal diseases [78]. This has important implications for the development of vaccines with efficacy in immunocompromised hosts. It was recently demonstrated that differences in Coccidioidomycosis susceptibility between three mouse strains were associated with specific immunohistochemical patterns and that the pattern of vaccine-induced protection differed from that of innate resistance [79]. Most notably, vaccination of the most susceptible strain, C57BL/6 (B6), resulted in significantly improved clinical and histological control of the disease and a lower fungal burden than seen in naturally resistant Swiss Webster mice. This was associated with a marked and progressive increase in the number of CD3 T cells in the perivascular/peribronchiolar infiltrate in B6 mice, whereas the neutrophil count was sharply reduced. Similar, but not identical, responses allowed Swiss Webster to control their infection without vaccination. This further underscores the importance of T cells in the murine host defense against Coccidioides. 8. T cell antigens identified in immunization studies In recent years, there have been increased efforts to identify protein T cell antigens that are suitable candidates for Coccidioidomycosis vaccines. Given the importance of cell-mediated immunity in the human and murine host defense against Coccidioides infection, the focus has been on T cell antigens capable of inducing a Th1 response. The results of some recent immunization studies are summarized in Table 1. In the following, the emphasis will be on some insights provided by these studies with potential importance in human Coccidioidomycosis. Observations during the original isolation of Ag2/PRA suggested that this protein existed in a complex with one or more serologically related compounds. It has since been determined that C. posadasii contains at least seven additional proline-rich proteins with structural

homology to Ag2/PRA [80]. One of these, designated as Prp2, shares 70% identity and 86% similarity with Ag2/PRA, but is expressed only during the endosporulation phase, whereas Ag2/PRA is expressed throughout the parasitic cycle. Despite the extensive homology of Ag2/PRA and Prp2, it was demonstrated that all but one of the identified T cell epitopes were unique to each of the two proteins. Of note, the T cell epitopes of Ag2/PRA identified in this study of B6 mice were mostly located in the region spanning amino acids 89–140. It had previously been shown that the N-terminal subunit consisting of amino acids 1–106 (Ag2/PRA1–106) was essentially as protective as the full-length protein in BALB/c mice, and the most stimulatory the peptides for this mouse strain were reportedly located in this region [80]. However, it was subsequently found that the fungal burden remained very high and that some Ag2/PRA1–106-immunized animals became moribund after the initial 90-day observation period, similar to what was seen in B6 mice [80]. This suggests that the Nterminal region does not contain the major epitopes associated with protection in either of these mouse strains. Through the use of two-dimensional electrophoresis combined with immunoblot analysis and bioinformatics, another C. posadasii T cell antigen, aspartyl protease, was identified as a major protein component of a spherule cell wall extract capable of conferring protective immunity against Coccidioidomycosis in mice [81] (see Table 1 for results of the immunization study with the recombinant protein, rPep1). IFNγ ELISPOT assays indicated that two of the five peptides corresponding to predicted human T cell epitopes were recognized by immune T lymphocytes from B6 mice and transgenic mice expressing human HLADR4. Similar studies with predicted human T cell epitopes in αmannosidase (Amn1) and phospholipase B (Plb) revealed somewhat greater differences between B6 and transgenic mice, but showed that HLA-DR4 transgenic mice recognized 4 and 2 epitopes in Plb and Amn1, respectively [82] (see Table 1 for the vaccine efficiency of these proteins). This strongly suggests that Coccidioides Pep1, Amn1, and Plb represent human T cell antigens. A C. posadasii protein designated as Gel1p with high sequence homology to a b-1,3-glucanosyltransferase of Aspergillus fumigatus was also shown to protect mice from i.p. or i.n. Coccidioides infection [83] (see Table 1). When given at a vaccine dose of 1 μg, Gel1p significantly enhanced Th1 cytokine production as evidenced by significantly increased levels of IFNγ and IL-12, but markedly lower amounts of IL5 and IL-10, in bronchoalveolar lavage fluid compared to adjuvant alone [84]. At the higher dose (5 μg), however, the concentrations of IL-12 and IFNγ were substantially lower, while IL-5 and IL-10 were similar to the levels seen after the 1-μg dose. Note that this differs from the results obtained with splenic CD90+T cells restimulated in vitro, where the 1 μg dose induced significantly higher levels of IFNγ and lower amounts of IL-5 compared to the 5 μg dose. Spleen cells from infected unimmunized mice failed to produce IFNγ or IL-5 in response to stimulation with Gel1p, suggesting that this protein does not participate in the stimulation of murine immune responses to infection. T lymphocytes obtained from animals immunized with recombinant C. immitis urease (rURE) or heat shock protein (rHSP) 60 exhibited significant proliferative responses to stimulation with the respective antigen [85]. Immunization with rURE, but not with rHSP60 also promoted ex vivo proliferative responses to formalinkilled endosporulating spherules (FKES). Conversely, lymph node and spleen T cells isolated from mice that had been immunized with FKES proliferated in response to rURE, but not rHSP60, suggesting that urease constitutes a more relevant T cell antigen. This indicates that, after immunization with a single protein, ex vivo proliferative responses to a more complex Coccidioidal antigen preparation may provide first insights into the relevance of a T cell antigen. The cytokine mRNA patterns seen in T cells from immunized mice after restimulation with the respective antigens are summarized in Table 1. Interestingly, slightly different patterns of mRNA expression were observed in lung tissue of immunized mice. The rURE vaccine

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99

Table 1 Murine T cell antigens. Immunizing agent

IFNγ production

Th2 cytokine production

rAG2/PRA1-106 rCSA (Coccidioides-specific antigen) rAg2/PRA + rCSA

n.d. n.d.

Prp2 (proline-rich protein homolog of rAg2/PRA) rAg2/PRA + Prp2

↑ ↑ (N7-fold lower compared to rAg2/PRA) ↑ (4-fold higher compared to rAG2/PRA) ↑ (Number of IFNγ + CD4+ T cells) ↑ (Number of IFNγ + CD4+ T cells) n.d.

rPep1 (aspartyl protease)

↑

Multivalent (rPep1 + rPlb + rAmn1) rPep1 rPlb (Phospholipase B) rAmn1 (α-mannosidase) rGel1 1 μg (β-1,3-glucanosyltransferase) rGel1 5 μg

n.d.

↑ IL-5 ↑ IL-10 n.d.

n.d. n.d. n.d. ↑

n.d. n.d. n.d. ↑ IL-5

↑ (but significantly less compared to 1 μg dose)

↑ IL-5 (significantly higher than after 1 μg dose) ↓ IL-4, -5, 10 mRNA ±IL-4, -5, 10 mRNA

rAg2/PRA

rURE (urease) rHSP60

↑ IFNγ mRNA ↑ IL-2 mRNA ↑ IFNγ mRNA ↑ IL-2 mRNA (markedly less than with rURE)

Infective dose (approximate number of arthroconidia) 50

Route of infection

Survival after 90 daysa

Fungal burden (log10)

References

i.n.

83% ~ 30%

n.d. n.d.

[79]b

97%

n.d.

~ 65%

No statistical analysis provided n.d.

n.d. n.d.

70

i.n.

n.d.

~ 25%

n.d.

n.d.

↓ compared to rAg2/ PRA alone Down

90

i.n.

~ 70%

80

i.n.

~ 85%

i.n.

~ 55% ~ 60% ~ 65% 68-70%

Almost complete clearance 2.17 vs.7.07 2.62 vs. 7.07 1.3 vs. 7.07 2.0 vs. 4.5

35-50%

2.00 vs. 4.5

N 40%

1.3 vs.5.8

b 20%

5.5 vs 5.8

80

100

i.p.

[80]

[81] [82]

[83,84]

[85]

All experiments were conducted with CpG ODN as the adjuvant, except the first one, which used ISS-ODN. All experiments were conducted with B6 mice, except the last one, which used BALB/c mice. Survival of sham-immunized control mice was 0%. a Percentages preceded by “~” are estimates from graphic representations of the data. b In a later study, the authors reported that mice immunized with Ag2/PRA 1–106 before infection retained a high fungal burden and some animals succumbed after the initial 90day observation period [80].

enhanced IL-10 mRNA compared to unimmunized infected mice, while rHSP60 did not induce IFNγ transcripts, and decreased the levels of IL-4. Both the Gel1p study and this one underscore the importance of not relying exclusively on the ability of antigens to induce Th1 responses in vitro. 9. B lymphocytes and the humoral immune responses to Coccidioides Two types of antibody systems have played a significant diagnostic and prognostic role in Coccidioidomycosis–tube precipitins (TP) and complement fixing (CF) antibodies. While TP antibodies represent mainly IgM activity, CF activity resides in the IgG fraction [86,87]. Based on 39,500 serologic tests in more than 7000 patients, Smith and his colleagues [88,89] reported that TP antibodies were seen early during primary symptomatic infection and waned rapidly thereafter. CF antibodies appeared more slowly, but also persisted for much longer than TP antibodies, usually throughout the course of the infection. While TP antibodies could be demonstrated in almost every patient if tested early enough, at least half of all patients never developed CF antibodies, and their absence was associated with milder disease. On the other hand, there was a strong association between increasing CF titers and greater disease severity, i.e., more extensive dissemination, and poorer prognosis [88]. Conversely, waning CF titers were associated with recovery from disease. Although more sensitive methods for the detection of antibodies (e.g., immunodiffusion or latex agglutination) may detect antibodies earlier and in a greater percentage of patients, there are no studies on the temporal development of antibodies with the serological methods currently in use. And it seems unlikely that anybody will ever assemble a cohort of similar size.

This close association between CF titers and disease severity suggests that the humoral immune response in Coccidioidomycosis is not beneficial and may, indeed, be detrimental. It is increasingly recognized, however, that individual antibodies produced in response to a specific infectious agent can be protective, neutral, or even disadvantageous [78]. Beneficial effects could include opsonization, complement-mediated lysis, direct fungicidal or growth-inhibitory activity, augmentation of cell-mediated immunity, and modulation of the host cytokine response [78]. Many of these activities have been demonstrated for monoclonal antibodies directed against specific antigens of another pathogenic fungi, Histoplasma capsulatum [90–92]. Such activities were shown to crucially depend on the isotype, subclass and epitope of the individual antibodies. Similar studies have not been done in murine Coccidioidomycosis, but it has been shown that the coating of immature spherules with anti-SOWgp antibodies enhances their phagocytosis and killing by mouse alveolar macrophages [29]. Interestingly, the protein recognized by these antibodies is present on spherules, but is specifically cleaved from the surface of endosporulating spherules by a secreted metalloproteinase and consequently absent from endospores [29]. This is thought to represent a mechanism by which endospores, i.e., the developmental stage during which the parasite is most vulnerable to phagocytosis and killing, escape host detection. In support of this theory, mice immunized with the recombinant protein domain of SOWgp (which is recognized by B cells and was specifically used in order to optimize antibody production) were not protected when challenged with C. posadasii, but showed increased survival when infected with a mutant strain lacking the metalloproteinase activity. Of note, the results of a recent gene expression microarray study suggest that B cells may also have a role in vaccine-induced protection from Coccidioidomycosis [93].

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Table 2 Antigens recognized by human B cells, seropositivity rates and titers (or absorbance, Abs). Antigen

Primary Coccidioidomycosis frequency

33-kDa apoglycoprotein (Ag2/PRA)

10/11 (91%)

33-kDa apoglycoprotein (Ag2/PRA) rAG2/PRA rAG2/PRA19-96 rAG2/PRA (more purified than in Zhu et al) SOWgp

45% 4/11 (36%) 8/11 (73%) 0

Not reported (Abs 0.12) (Abs 0.16)

20/20 (disease form not specified) 10 of 10 (disease form not specified) 50%

Not reported

a

1:1600–1:12,800

0

Not reported

[29]

41%

1/160

[107]

SOWgp (repeat domain) Pmp1 (Peroxisomal matrix protein) a

Titers

Severe Coccidioidomycosis frequency

Titers

25/26 (96%)

1/640

13/17 (76%) 14/17 (82%) 37/42 (88%)

95%

(Abs 0.35) (Abs 0.69) 1:40–1:102,400

1:20,480

Healthy frequency

Titers

Reference

5/108

N1:160 IgM of N 1:80 IgG

[123]

a

2/77 (3%)

N1:40

[105] [106] [106] [104] [67]

Reactive was defined as mean ± 2 SD above controls.

10. B cell antigens Unfortunately, the identification of potentially protective antibodies in human Coccidioidomycosis is greatly hampered by the lack of information on the identity of B cells antigens, the development of the antibody profile over time, and the association between specific antibodies and various disease manifestations. There is only one small study suggesting that there may be an association between an antibody to a 70-kDa antigen and protection from dissemination [94]. Only a few human B cell antigens have been isolated, cloned and sequenced. These include the CF antigen, a chitinase with one or more B cell epitope(s) in the domain consisting of residues 20–310 [95–97], and the antigen recognized by IgM TP antibodies which has been identified as a βglucosidase [98–101]. In addition, there is SOWgp, which is expressed in greatest abundance during early spherule development and has been shown to function as an adhesin and to contribute to the virulence of C. immitis in experimental Coccidioidomycosis [102]. Note that the glycoprotein differs in size between C. immitis isolates, and this was found to stem from different copy numbers of a 41–47 residue tandem repeat rich in proline and aspartate. The repeat region contains an important human and murine B cell epitope [29]. Nonetheless, patient sera were able to recognize SOWgp from different fungal isolates with varying copy numbers [67]. As shown in Table 2, all patients with Coccidioidomycosis appear to elaborate anti-SOWgp antibodies, and high titers reportedly correlate with disease severity [103]. A 33-kDa apoglycoprotein isolated from TSL after chemical deglycosylation was eventually shown to be the same protein as Ag2/PRA. Both the apoglycoprotein and rAG2/PRA are recognized by sera from patients with Coccidioidomycosis, the titers being significantly higher in patients with severe disease compared to those with primary pulmonary Coccidioidomycosis (see also Table 2). However, conflicting results have been obtained concerning differences in the positivity rate between these groups of patients. Specifically, one study reported that none of the sera from patients with early self-limiting disease reacted with rAg2/PRA [104], even though antibodies to the apoglycoprotein previously had been detected in 13 of 18 of these sera [105]. There are data suggesting that at least two B cell epitopes localize to residues 19–96 and, interestingly, whereas only 4/11 patients with pulmonary disease reacted to the fulllength protein, 8/11 recognized Ag2/PRA19–96, as did 82% of the serum samples obtained from patients with disseminated disease [106]. Therefore, it seems possible that the extraction procedure used to obtain the apoglycoprotein unmasks the epitope that sera from patients with self-limiting primary Coccidioidomycosis preferentially recognize. Some B cells antigens have recently been identified as byproducts of searches for T cell antigens as vaccine candidates. These include a peroxisomal matrix protein that possibly functions as a yeast thiol peroxidase [107] (see also Table 2) and an α-mannosidase [108], both of which have been cloned and sequenced. The frequency of anti-αmannosidase antibodies in Coccidioidomycosis patients has not been

established yet. In addition, Tarcha et al. [82] found more than 40 seroreactive proteins in immunoblots of a parasitic cell wall fraction with pooled sera from patients with confirmed Coccidioidomycosis and provided putative protein identifications. 11. Use of the biologics It is not the purpose of this review to discuss the risk of Coccidiomycosis in patients treated with anti-TNF agents. There is already considerable literature on the role of TNF in response to infection and indeed the underlying immunosuppressive state of a number of autoimmune diseases [109–122]. Indeed, in people that live in endemic areas, evaluation for underlying infection is critical, much as it is for underlying mycobacterial infections. Although this review is primarily directed at rheumatologists, the data and discussion are equally true in the management of patients with any underlying immunological disease and especially those that are treated with immunosuppressive drugs. Take-home messages • Our understanding of the human immune response to Coccidioides infection is still rather limited. The focus so far has been on the intracellular phase of the fungus, i.e., the phagocytosis and killing of the pathogen. If this process were as ineffective in vivo as in vitro studies suggest, it would be unlikely to represent a major defense mechanism. • The majority of fungal cells in humans remains extracellular or returns to the extracellular space after having become so large as to lyse the host cell. This paradox leads to the question of why are cellmediated immune responses so important in the control of Coccidioides infection? • Antibodies appear to be detrimental rather than protective, although there have been few efforts to investigate the possible contribution of B cells to the human host defense. • The use of biologics and immunosuppressive agents as well as underlying autoimmune disease or secondary immunodeficiency, are all predisposing features for activation of underlying Cocciodioides infection.

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IVIg modulates BCR signaling through CD22 and promotes apoptosis in mature human B lymphocytes Among various mechanisms for interactions with B cells, intravenous immunoglobulin (IVIg) may operate through the insertion of its Fc part into the Fc-γ receptor, or the binding of its sialic acid (SA)- bearing glycans to the negatively regulating CD22 lectin. In this study, Seite J-F. et al (Blood 2010; 116: 1698-1704) show that IVIg reduces B lymphocyte viability in a dose-and time-dependent manner. Furthermore, they show by confocal microscopy that SA-positive IgG, but not SA-negative IgG bind to CD22. This interaction reduces the strength of B-cell receptormediated signaling through down-regulating tyrosine phosphorylation of Lyn and B-cell linker proteins, and up-regulating phospholipiase C γ2 activation. This cascade resulted in a sustained activation of Erk 1/2 and arrest of the cycle at G1 phase. These changes may be accounted for the efficacy of IVIg in autoimmune diseases.