s2fieldsofstudy: Biology Medicine

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The Impact of T Cell Intrinsic Antigen Adaptation on Peripheral Immune Tolerance

Overlapping roles have been ascribed for T cell anergy, clonal deletion, and regulation in the maintenance of peripheral immunological tolerance. A measurement of the individual and additive impacts of each of these processes on systemic tolerance is often lacking. In this report we have used adoptive transfer strategies to tease out the unique contribution of T cell intrinsic receptor calibration (adaptation) in the maintenance of tolerance to a systemic self-antigen. Adoptively transferred naïve T cells stably calibrated their responsiveness to a persistent self-antigen in both lymphopenic and T cell–replete hosts. In the former, this state was not accompanied by deletion or suppression, allowing us to examine the unique contribution of adaptation to systemic tolerance. Surprisingly, adapting T cells could chronically help antigen-expressing B cells, leading to polyclonal hypergammaglobulinemia and pathology, in the form of mild arthritis. The helper activity mediated by CD40L and cytokines was evident even if the B cells were introduced after extended adaptation of the T cells. In contrast, in the T cell–replete host, neither arthritis nor autoantibodies were induced. The containment of systemic pathology required host T cell–mediated extrinsic regulatory mechanisms to synergize with the cell intrinsic adaptation process. These extrinsic mechanisms prevented the effector differentiation of the autoreactive T cells and reduced their precursor frequency, in vivo.

Introduction
The efficient clonal expansion of pathogen-specific T cells plays a crucial role in determining the success of an immune response against a rapidly replicating infectious challenge. The ability of this expanded lymphocyte pool to effectively combat the pathogen also relies on the extent of effector functions that it acquires and maintains. Differentiated helper T cells produce cytokines and cell surface ligands that regulate the subsequent generation of cytotoxic and humoral responses. This differentiation process is often correlated with proliferative expansion, but there is evidence to suggest that the two processes can be independently regulated [1][2][3]. Subsequent to clearance of the pathogen, most members of these expanded populations of antigen-specific lymphocytes are eliminated and the few that survive often typically demonstrate better responsiveness.
In cases where a T cell response is initiated against a chronic nonclearable pathogen or a persistent self-antigen, the immune system evokes many regulatory mechanisms aimed at containing the potentially damaging chronic T cell activity. One such mechanism has been called adaptive tolerance [4]. This process is a T cell-intrinsic downregulation of responsiveness, likely mediated by the recruitment of negative feedback in signaling pathways downstream of the T cell receptor (TCR). The consequent hyporesponsiveness of the T cell is proportional to the strength of the ambient antigen presentation and is reversible upon removal from the antigen-bearing host [5][6][7]. Such a dynamic state is broadly consistent with the tunable activation threshold model originally proposed by Grossmann and Paul [8] and may allow for the persistence of autoreactive T cells that are potentially useful against foreign antigens [9]. We have earlier shown that this antigen adaptation primarily aims to restrict the turnover of T cells in vivo to a minimal basal level, despite the persistence of antigen [6]. The T cells that enter the hyporesponsive state, however, have undergone significant differentiation and can produce effector cytokines at levels higher than naïve T cells (albeit lower than memory T cells) after an in vitro restimulation. This raises the possibility that antigen-adapted T cells may continue to chronically display effector functions against the persistent antigen despite the restriction of their proliferative ability.
The downregulation of the proliferative potential of helper T cells, while maintaining their ability to mediate effector functions, has been reported in the case of T cells surviving an acute antigen exposure in the absence of adjuvant [10]. In this model, the tolerizing antigen does not persist and therefore the effector potential of the T cells is unlikely to be stimulated to induce pathology. It is therefore not clear if continued persistence of antigen would result in the elimination of the T helper cell's effector function as well. Furthermore, CD8 þ T cells that undergo adaptation to chronic lymphocytic choriomeningitis virus (LCMV) infection or a self-antigen downregulate both their proliferative and effector functionalities [11,12]. In this case, the ability to produce interleukin (IL)-2 was often downregulated rapidly, while various effector functions required extended stimulation through chronic viral exposure [13]. CD8 þ T cells experiencing chronic antigen in a transgenic model, however, retained the ability to mediate cytolytic activity in vivo despite anergy induction [14]. In the early phases of a chronic LCMV infection, CD4 þ T cells specific for the virus were able to help antigen-expressing (infected) B cells polyclonally, leading to serum hypergammaglobulinemia [15]. This antibody production correlates with the acute viremia and suggests that after several weeks of chronic viral infection, CD4 þ T cells lose the ability to help B cells [16]. Nonetheless, the fluctuations in the antigen load due to viral replication, clearance and tissue redistribution also makes such models less suited to examination of the in vivo functionality of stably antigen-adapted T cells.
We have previously described a model system for adaptive tolerance that uses transgenic mice constitutively expressing the antigen pigeon cytochrome C (PCC), driven by the MHC class I promoter and an Ig enhancer, in a variety of cellular compartments, including B cells and dendritic cells [6,7,17]. Antigen-specific naïve CD4 þ T cells adoptively transferred into such mice crossed onto a CD3e À/À background, adapt to the persistent antigen over the period of a week. In the absence of other competing T cells, these CD4 þ cells persist for extended periods of time in the adaptively tolerant state and show no evidence of having acquired a T-reg phenotype [6,7]. This system, therefore, allows us to examine the consequence of antigen adaptation on the T cell's ability to mediate effector functions in response to the ambient stimulation, for example, by helping B cells that express and present the cytochrome C antigen.
In this report, we demonstrate that the CD4 þ T cell expansion to the persistent self-antigen results in a polyclonal antibody response by the resident B cells. The antibodies are directed against a variety of self-antigens classically implicated in autoimmune disorders. The mice eventually develop a mild form of chronic arthritis. Interestingly, CD4 þ T cells that were first stably adapted to the antigen in the absence of B cells still retained the ability to help B cells and to cause autoimmune disease. In contrast, in the presence of an endogenous repertoire of T cells, the ability of the autoreactive T cell to induce pathology was severely compromised. This tolerance was correlated with a failure of the transferred transgenic T cells to complete effector differentiation and survive in the T cell-replete host. Overall, these data demonstrate that at least two distinct mechanisms-one cell intrinsic and the other in trans-must cooperate to ensure complete peripheral tolerance.

Adaptation Controls Autoreactivity Independent of Suppression
The exposure of naïve helper T cells to chronic peripheral antigenic stimulation leads to a reversible state of anergy known as adaptive tolerance. We have previously modeled this process by adoptively transferring Rag2-deficient 5C.C7 TCR transgenic T cells into CD3e knockout mice that express membrane-targeted PCC under the control of an H-2K promoter and an Ig enhancer [17]. After a robust initial expansion, the transferred T cells downregulate their ability to make cytokines to an in vitro restimulation and fail to clonally expand well upon retransfer into a fresh mPCC,CD3e À/À host [6,7].
Proliferative impairment in peripheral T cells after autoantigen encounter has often been attributed to the induction of a regulatory phenotype in T cells [18,19]. Since the host mice in our experiments do not have any endogenous T cells, by design (CD3e À/À ), we examined if the adaptively tolerant 5C.C7 T cells acquired a regulatory function in the host. As shown in Figure 1A, we could not detect foxp3 expression in the transferred cells at any time that we tested (days 4, 11, 22, and 28, of which day 11 is shown in Figure 1A, bottom row). Regulatory activity may still act through a foxp3 independent fashion, but would be expected to either impair the proliferation or differentiation of a new T cell response [20]. We tested this by adoptively transferring fresh Ly5.1 þ naïve 5C.C7 T cells into mPCC,CD3e À/À mice that already harbored Ly5.2 þ 5C.C7 T cells in an adaptively tolerant state. The resident population was unable to impair the first 3 d of expansion of the fresh naïve cohort ( Figure 1B, days 1-3). After this time, the new cohort did not expand significantly and could be recovered at similar numbers to that in a lymphopenic host (solid squares in Figure 1B). Finally, in order to examine if the differentiation of the second cohort was impaired by the presence of adaptively tolerant 5C.C7 T cells, we purified the expanded second cohort (Ly5.1 þ , 5C.C7) by FACS sorting. The sorted cells were assayed for their ability to produce interferon (IFN)c after in vitro restimulation. Ly5.1 þ 5C.C7 T cells that expanded in the presence of adapted Ly5.2 þ 5C.C7 T cells (solid triangles in Figure 1C) or in an empty mPCC,CD3e À/À host (open squares in Figure 1C) had differentiated similarly as evidenced by effector cytokine production in vitro. These data suggest by multiple criteria, that adaptive tolerance in this model, is a cellular phenomenon distinct from suppression. It is also independent of clonal deletion, since the adaptively tolerant T cells are not eliminated in the mPCC,CD3e À/À host for the life of the animal-solid squares in Figure 1B. Therefore, our model system allows for a direct evaluation of the impact of antigen adaptation on systemic immunological tolerance, unlike many other models in which multiple paradigms cannot be resolved sufficiently.

Adaptation Alone Is Insufficient to Generate Systemic Tolerance
The mPCC,CD3e À/À mice that harbor the tolerant PCCreactive 5C.C7 T cells do not display any obvious signs of acute graft versus host disease such as weight loss, lethargy, paralysis, or lethality. Interestingly, however, we began to observe swelling of the hindlimbs and forelimbs of a few mice, almost 2 mo after the adoptive transfer. These phenotypes were observed only in the PCC transgenic recipients and not in control CD3e À/À mice, despite a weak ''homeostatic'' expansion of the 5C.C7 T cells, reported earlier [6]. To further characterize this phenotype, we examined the animals by x-ray imaging (Figure 2A and 2B) and Immunohistochemistry ( Figure 2C-2E). Radiographs of the forelimb of an adoptive transfer recipient at day 86 showed bone loss with significant distortion of the phalanges (Figure 2A, right) relative to a control mouse (Figure 2A, left). The hindlimb ( Figure 2B) also showed increased bone density especially around the carpal joints. Hindlimbs of mPCC,CD3e À/À mice that were infused with autoreactive T cells 14 d earlier ( Figure  2D) or 60 d earlier ( Figure 2E) were stained by HEMATOX-YLIN AND EOSIN after decalcification and sectioning. The sections show the increased swelling at 60 d ( Figure 2E) compared to both the control ( Figure 2C) and the 14-d time point ( Figure 2D). The legs were extended maximally before being processed for sections and the lack of extension in the day 60 leg is also evident in Figure 2E. Furthermore, mild mononuclear cell infiltrates in the tibiotarsal regions were evident in magnifications of the day 60 sections (Figure 2E, bottom). These findings suggested that an arthritic process was developing in the 5C.C7 transfer recipients, despite the induction of adaptive tolerance in these T cells. This was surprising since the B10.A strain is not related to genotypes that are conventionally associated with the ability to develop arthritis, even after collagen immunization [21]. Although the severity of the pathology did not approach that of some arthritic models [22,23] or significantly hamper feeding or movement of the mice, we developed a scoring system to quantitate this swelling (described in Materials and Methods). Subtle alterations in joints or digits were visible in a few mice as early as 24 d after transfer and progressively increased over time ( Figure 2F). Although not completely penetrant, up to 80% of the transfer recipients developed disease scores over 5 in less than 2 mo.

Polyclonal Activation of B Cells Drives an Autoreactive Response during Adaptive Tolerance Induction
T cell-initiated autoimmune syndromes in mouse models often require B-cell-mediated antibody production or antigen presentation [24]. We examined whether this was the case in our model system, by assaying serum antibody levels in PCC transgenic mice at various days after the transfer of 5C.C7 T cells ( Figure 3). The total amount of IgM in the serum had increased to approximately 5-fold by 21 d of 5C.C7 T cell transfer ( Figure 3A, p , 0.0001 comparing normal serum values at day 0 with day 21 after T cell transfer). This increased level was maintained stably up to 200 d after the initial transfer. A similar pattern was observed for total amounts of IgG1 ( Figure 3B), IgG2a ( Figure 3C), IgG2b ( Figure 3D), IgG3 ( Figure 3E), and IgE ( Figure 3F). All increases were statistically significant over the levels detected in control mice (on day 21, p ¼ 0.0024 for IgG1, p , 0.001 for IgG2a, p , 0.001 for IgG2b, and p ¼ 0.01 for IgG3). The increase in these isotypes, however, was more dramatic, (C) Congenically marked Ly5.1 þ/þ ,5C.C7 T cells that expanded in a naive B10.A, PCC þ/þ , CD3e À/À host (open squares) or a PCC þ/þ , CD3e À/À host that had been injected with Ly5.2 þ/þ ,5C.C7 T cells 28 d in advance (solid squares) were purified by FACS sorting (Ly5.1þ, CD4þ) and restimulated in vitro to measure IFNc secretion. DOI: 10.1371/journal.pbio.0040340.g001 because little or no production was detected prior to the T cell infusion [open circles in (A)-(E), * indicates samples with undetectable levels]. The increase was also antigen specific since we did not see a comparable increase in Ig levels after the transfer of 5C.C7 T cells to B10.A,CD3e À/À mice that did not express PCC (unpublished data). The global increase in the amounts of all of these isotypes shows that there is no bias toward any ''class'' of immune response. This is also consistent with our earlier report of a global suppression of cytokine production in the adaptively tolerant T cells, without skewing toward any particular subset or class of response. The levels of IgA, however, were distinct from those of all the other isotypes ( Figure 3G). There was no increase in the fecal levels of IgA (triangles) after T cell transfer, and there was a reproducible decrease in the basal IgA levels detected in the serum (solid circles). Consistent with increased antibody production in these mice, we could also detect an increased amount of circulating antigen-antibody complexes as measured by a complement (C1q) binding ELISA ( Figure 3H).
We also examined the reactivity of antibodies generated in this system against a panel of mouse antigens. Antibodies could be detected against double-strand DNA ( Figure 3I) as well as ribosomal antigen extracts (unpublished data). The levels of anti-DNA antibodies in the serum of mice 56 d after 5C.C7 transfer were significantly enhanced over control mice in all three isotypes tested: IgM (p ¼ 0.01), IgG2a (p ¼ 0.02), and IgG2b (p ¼ 0.02). Sera from transfer recipients were tested on Western blots for reactivity against purified antigens. Although the private reactivities varied between individual mice (Table 1), most animals produced detectable antibodies against multiple antigens, including histones, glucose-6phosphate isomerase (GPI), fibrinogen, and collagen. Occasionally, antibodies could also be detected against PCC, consistent with the idea that B cells in this transgenic are not completely tolerant of PCC, in the presence of T cell help [25].
This broad spectrum of anticellular antibodies was also evident in immunofluorescence assays on test sections of healthy murine kidneys ( Figure 3J-3N). Sera obtained 4 wk after transfer consistently (n ¼ 22 mice) showed reactivity against cellular antigens. The staining showed a speckled subnuclear reactivity (shown by higher magnification in Figure 3O) that is consistent with patterns reported from mice demonstrating autoimmune B-cell pathology, including SLE. It is also noteworthy that although the total Ig levels were almost maximally induced by day 10 ( Figure 3A-3F), there were no detectable anticellular antibodies until after 4 wk ( Figure 3M). Thus, there was a qualitative change in the antibodies being made at later time points, even after the amount of secreted antibody had peaked ( Figure 3K-3N shows progressive changes in reactivity).

The Help for B Cells Is Available Even after the Stable Induction of Adaptive Tolerance
The antibody responses observed in the 5C.C7 transfer recipients could have been triggered by the robust, early (days 1-4) expansion of naïve T cells in the mPCC,CD3e À/À host ( Figure 1B, open squares), because stable adaptation to antigen (as measured by cytokine downregulation) takes approximately 1 wk to establish in this system [26]. We therefore devised an experimental strategy to test if T cells  Optical density values obtained from isotype specific ELISAs for IgM (solid circles), IgG2b (solid squares), and IgG2a (solid triangles) are shown. Empty circles (IgM), squares (IgG2b), and triangles (IgG2a) on day 0 represent sera obtained from three normal mice, assayed in the two experiments. (J-O) Sera from mPCC,CD3e À/À mice were assayed for anticellular antibodies of the IgM (green) and IgG (red) isotypes, by hybridizing with frozen tester sections of B10.A,Rag2 À/À kidneys. Numbers in the inset denote days after T cell transfer. (O) shows an enlarged area from (N) to illustrate speckled nuclear staining. (J) was probed with serum from a normal mPCC,CD3e À/À mouse without T cell transfer. Similar sera samples from mice 4 d (n ¼ 3) or 7 d (n ¼ 2) after T cell transfer did not show measurable IgG or IgM anti cellular antibodies. However, all mice tested after 28 d (n ¼ 22) scored positive for both IgG and IgM antibodies to various extents. DOI: 10.1371/journal.pbio.0040340.g003 could in fact mediate B cell help even after the stable induction of adaptive tolerance. We took advantage of the fact that naïve 5C.C7 T cells can be induced into a stable adaptively tolerant state in the absence of B cells, by using an mPCC,Rag2 À/À host for the adoptive transfers. (N. J. Singh, L. Chiodetti, and R. H. Schwartz, unpublished results). We waited 48 d after transfer of T cells into such hosts and then transferred 25 million naïve B cells intravenously ( Figure 4). B cells were purified from the spleens of mPCC,CD3e À/À mice (that express the antigen) or control CD3e À/À mice (that do not express PCC). Transfer recipients were bled at various days after B-cell transfer and serum immunoglobulins measured in an isotype-specific manner ( Figure 4A-4E).
Serum IgM levels in the transfer recipients increased steadily after B-cell transfer, and there was only a subtle increase afforded by antigen expression in the transferred B cells ( Figure 4A, solid triangles). In contrast, there were significant antigen-specific increases in IgG1 (p , 0.001 on day 14) and IgG2a levels (p , 0.001 on day 14) only observed in the presence of adapted T cells ( Figure 4B and 4C). In the case of IgG2b, although there was a 5-to 10-fold increase in antibody production with PCC-expressing T cells ( Figure  4D, solid triangles), the transfer of B cells alone resulted in increased total IgG2b levels in the absence of any T cells ( Figure 4D, open inverted triangles). However the antigenspecific increase was still significantly greater (p ¼ 0.05 on day 14, Figure 4D, closed triangles). In the presence of adapted T cells and antigen-expressing B cells, there was a sharp increase in IgE levels as early as day 8 and a consistently increased but lower level thereafter ( Figure  4E, solid triangles). We could not detect IgE production in the recipients of non-antigen-expressing B cells or in the absence of adapted T cells ( Figure 4E, open circles and triangles). These data demonstrate that even 48 d after T cell transfer into a self-antigen-expressing environment, adaptively tolerant T cells retain the ability to robustly help B cells in an antigen-specific fashion.
The increase in serum immunoglobulin levels in the T cell reconstituted B10.A,mPCC,CD3e À/À mice precedes the development of both anticellular antibodies ( Figure 3) and visible arthritis ( Figure 2). Therefore, it was still possible that the adapted T cells would be unable to aid the complete transition from increased serum titers to developing joint disease. We examined this possibility by introducing PCC transgenic B cells into mPCC,Rag2 À/À mice, 28 d after they had received a 5C.C7 T cell transfer. We then assayed serum anticellular antibodies and arthritis in the recipients at various days. The serum anticellular antibody levels in these mice were similar to those of B10.A,mPCC,CD3e À/À mice (compare Figure 4G to Figure 4H). However, the arthritis ( Figure 4I, solid triangles) was clearly delayed, since no significant joint swelling or rigidity was observed up to 60 d after transferring B cells (88 d after the T cell input). These mice did start to develop measurable disease by 80þ d; however, the severity was less than that of B10.A,mPCC,CD3e À/À mice at 60 d after T cell reconstitution ( Figure 4I, solid squares). These observations demonstrate that the antigen-adapted T cell population can still facilitate the maturation of the B-cell response that leads to the production of anti-self-reactive antibodies.

Adaptively Tolerant T Cells Mediate Help through Cytokines and CD40-CD40L Interactions
T cell help to B cells is mechanistically delivered through the secretion of cytokines (including IL-4, IL-5, IL-21, IFNc, etc.) and the engagement of cell surface receptors (including CD40). Since adaptation dampens TCR signaling in these T cells [27], the expression of these molecules may also be expected to be impaired. However, T cells have undergone significant differentiation to make effector cytokines by the time they are rendered tolerant [6]. The help that we observe could therefore result from either the low levels of effector  molecules that are made despite adaptive tolerance induction or alternatively by the unaffected synthesis of any one component, which is resistant to the dampening effects of adaptive tolerance. We tested these two possibilities by knockout and blocking strategies designed to ask if any one component was the dominant inducer of B-cell differentiation in this model system. The absence of IL-4 in the transferred 5C.C7 T cells ( Figure  5A-5C, gray bars) resulted in less than 2-fold decrease in serum IgG1 (p ¼ 0.08, in Figure 5A) and IgG2b (p ¼ 0.05, Figure  5C) levels. Similarly, IFNc knockout 5C.C7 T cells elicited approximately 2-to 3-fold lower levels of IgG2a (p ¼ 0.006, Figure 5B) with a slight increase in IgG1 (p ¼ 0.02). The cytokine IL-21 has been reported to act as a Th2 enhancer or plasma cell factor in differentiating B cells [28,29]. We used mPCC,CD3e À/À mice that were also deficient in IL-21 receptor as transfer recipients to examine the contribution of this cytokine to the hypergammaglobulinemia. The absence of IL-21 responsiveness in B cells decreased serum levels of all isotypes by 2-to 3-fold ( Figure 5A-5C, last three bars in each graph). Interestingly combining IL21R deficiency in B cells together with IL-4 or IFNc deficiency in T cells did not synergistically reduce serum Ig levels any further.
We then examined the role of CD40-CD40L interactions using the mPCC,Rag2 À/À model described in the previous section. Mice with resident adaptively tolerant T cells were treated with blocking anti-CD40L antibody (MR1) or a control antibody intraperitoneally (100 lg/mouse) at the same time as introducing naïve PCC-expressing B cells ( Figure 5D-5F). The blocking antibody treatments were continued every other day and the serum antibody titers measured 14 d after B-cell transfer. In contrast to the cytokine deficiency experiments, blocking CD40-CD40L interactions resulted in a profound decrease in IgG2a and IgG2b induction ( Figure 5E and 5F). The decrease in IgG1 was less pronounced but did show a 3-to 5-fold decrease. In a separate experiment, CD40L blockade was carried out for only a short period, from the time of B-cell transfer to 6 d after. Twenty-two days following the last treatment (28 d after B-cell transfer), IgG2a ( Figure 5G) and IgG2b ( Figure 5H) levels were still reduced in the treated groups, even though B cells were still detected in the hosts. These experiments suggest that B-cell help in this model is delivered by a combination of soluble and surface molecules and is not a consequence of a single, tolerance resistant factor or skewed differentiation of the adapted T cells.

Despite Help, Autoimmune Pathology Does Not Develop in a T Cell-Replete Host
The experiments so far clearly demonstrate that in a carefully constructed model system where adaptive tolerance is the only potential mechanism of tolerance to a peripheral self-antigen, T cell effector functions sufficient to mediate autoimmune pathology are still evident. We then asked if this pathology would still be initiated, if other mechanisms of immunoregulation are restored to the model system. This was tested by transferring congenic 5C.C7,Ly5.1 þ T cells to a B10.A,mPCC (Ly5.2 þ ) transgenic mouse that retained an intact repertoire of endogenous T cells (CD3e þ/þ ). Serum from these transfer recipients also showed a consistent increase in IgG1 ( Figure 6A), IgG2a ( Figure 6B), and IgG2b ( Figure 6C) levels. The titers of these antibodies, however, were approximately 5-to 10-fold lower than that observed in a T cell-deficient host (compare to Figure 3). Furthermore, the T cell-replete transfer recipients did not develop detectable titers of anticellular antibodies ( Figure 6F) compared to the T cell-deficient host ( Figure 6E). Consistent with these lowered titers and absence of anticellular antibodies, the transfer recipients that had an intact cohort of endogenous T cells did not develop detectable arthritic pathology even 120 d after T cell transfer ( Figure 6G).

5C.C7 T Cells Are Anergic in a T Cell-Replete Host, but Do Not Undergo Differentiation toward Effector Function
Given the striking difference in pathology observed in the presence or absence of endogenous T cells, we further dissected the fate of 5C.C7 T cells in the T cell-replete host. The transferred cells engrafted similarly in the T cell-replete host and initiated clonal expansion ( Figure 7A, open squares). However, this expansion lasted only for 3 d and resulted in just a 5-fold increase in cell number. This was a consequence of a failure to sustain cell divisions, as evidenced by the carboxy fluorescein succinimidyl ester profiles ( Figure 7B). Furthermore, the expanded T cells underwent a slow deletion process in the T cell-replete host, starting at day 4 and resulting subsequently in a approximately 3.5% per day loss of the expanded cells. This resulted in the reduction of the T cell numbers to below detection levels by approximately 2 mo. This disappearance phase was in sharp contrast to the fate of the autoreactive CD4 þ T cells Figure 5. Cytokine and CD40L Dependence of Help Rendered by Adaptively Tolerant T Cells (A-C) Serum from mice that received T cells 8 d before were assayed for total immunoglobulins as described. T cells were from 5C.C7 TCR transgenic mice (white bars) or similar mice also deficient in IFN-c (black bars) or IL-4 (gray bars). Host mice were mPCC,CD3e À/À (left three bars in each graph) or IL-21 receptor-deficient mPCC,CD3e À/À mice (right three bars). (D-F) B cells were infused into mPCC,Rag2 À/À mice that harbored stably adapted 5C.C7 T cells. The blocking anti-CD40L antibody MR1 (black bars) or a control hamster antibody (white bars) was administered at the same time and on every other subsequent day for 13 d, before assaying serum for antibody titers. in the CD3e À/À (T cell-deficient) PCC transgenic host ( Figure  7A, solid squares), which persisted for several months. We then asked if an adaptively tolerant form of anergy was in fact induced in the 5C.C7 T cells in the T cell-replete host, by measuring IL-2 production and TCR proximal signal transduction. Sorted 5C.C7 T cells 15 d after adoptive transfer into a B10.A,mPCCþ host showed an 85% downregulation of IL-2 secretion ( Figure 7D, gray bar, compared to naïve T cells in white bar) similar to the 93% downregulation in a CD3e À/À host. Over five separate experiments, 5C.C7 T cells in a T cell-replete host showed an 85 6 3.5% decrease in IL-2 production relative to naïve 5C.C7 T cells, while in the lymphopenic host, this was 91 6 7%. The CD3 proximal signaling machinery of the cells in either host was also tested by monitoring calcium influx in response to cross-linking with anti-CD3 ex vivo. The fluxes elicited by T cells from a T cell-replete host ( Figure 7C, red trace) were equally dampened in comparison to the fluxes from those in a T cell-deficient host ( Figure 7C, blue trace). Both were impaired in comparison to naïve T cells ( Figure 7C, green line) as previously reported [27]. Therefore, the adaptive tolerance induced in the 5C.C7 cells is T cell-intrinsic and independent of the presence of an endogenous host repertoire of other T cells.
Finally, we found that the adaptively tolerant 5C.C7 T cells recovered from a T cell-replete host showed no significant differentiation towards effector cytokine production. Upon restimulation in vitro, adaptively tolerant T cells from the CD3e þ/þ host ( Figure 7E, gray bar) made the same small amount of IFNc as undifferentiated naïve T cell cultures ( Figure 7E, white bar). T cells from a CD3e À/À host showed considerable differentiation to make this cytokine ( Figure 7E, black bar), although this was, as reported earlier, still greatly impaired relative to a memory 5C.C7 T cell [7]. We further confirmed that autoreactive T cells fail to differentiate and display effector functions in a T cell-replete host by assaying for IFNc secretion ex vivo, using a cytokine secretion assay ( Figure 7F). About 11% of the adaptively tolerant 5C.C7 T cells were secreting this cytokine 4 d after transfer to a CD3e À/À host ( Figure 7F, left), whereas only 0.6% of the cells could be scored positive in a CD3e þ/þ host. Similar to the lymphopenic host, 5C.C7 T cells in the T cell-replete host did not express detectable IL-4 or foxp3 or upregulate CD25, suggesting that they had not differentiated into a Th2 or T-reg lineage (data not shown). Therefore, although adaptive tolerance was primarily induced in every context, a superimposing influence of the endogenous T cells is critical to reduce the expansion, effector differentiation and pathology stemming from autoreactive CD4 þ T cells.

Discussion
Anergy, deletion, and regulation have been proposed as three mechanisms that the adaptive immune system uses to tolerize CD4 þ T cells reactive to peripheral self-antigens [4,30,31]. The relative significance of each of these processes to systemic tolerance and their synergism in vivo has been difficult to dissect, despite the existence of many model systems. It is formally possible that all of these mechanisms are in fact inseparable elements of a unified tolerance process. For example, the facts that regulatory cells display evidence of anergy and that anergic cells have been shown to regulate could be interpreted as evidence of similar biochemical machinery operating in both conditions. The conditions of assay or the quantitative gradation of the biochemical process may reveal anergy, regulation, or both. Therefore, it is critical to begin to separately quiz the roles of individual processes in peripheral tolerance and to decipher the network of interactions between these processes in vivo. This report represents a comprehensive examination of the role of in vivo anergy (adaptive tolerance) in the maintenance of peripheral tolerance. The adoptive transfer strategy we have used mimics the stimulation of a naïve T cell with a peripheral self-antigen or a chronic pathogen, albeit a systemic one. In most model systems, these responses are influenced by various noncell autonomous factors such as regulatory T cells, Th2 or Th3 cells, NK T cells, cd T cells, etc. The absence of these other endogenous T cells in the recipient we have used (CD3e À/À host) limits the regulatory mechanisms available to those intrinsic to the responding T cell population. We also eliminated the induction of trans-regulatory phenotypes within the population we have introduced, as has been found in other models [19,32]. Thus, our data clearly demonstrate that while tuning effectively constrains clonal expansion and quantitatively buffers cytokine synthesis, it is by itself unable to prevent the differentiation and survival of autoreactive T cells, two parameters that could very well be linked [33]. These parameters must be constrained by other non-cell-intrinsic regulators, so as to completely eliminate pathology.
The conclusions of this study are supported by earlier arguments that anergic T cells can help B cells generate a Th1 (IgG2a) class of response [10]. Those data, however, were consistent with the ''overcoming'' of anergy by a stronger antigenic challenge. In contrast, the helper activity in our model is continual and does not require an antigenic challenge exceeding the level of ambient presentation. Furthermore, the differentiation in this system does not skew the responses toward any particular class of antibody production. Antibodies typically associated with Th1 (IgG2a) as well as Th2 (IgG1 and IgE) helper function were all upregulated. The increased Ig levels did not just reflect specific antibody production against the cognate antigen (PCC, which was absent in most mice) but was of diffuse polyclonal reactivity (Figure 3). This is not surprising, since the expression of PCC under the control of the MHC-I promoter and Ig enhancer should result in antigen expression in all of the B cells [7,17]. Endogenous membrane bound antigens have been shown to gain access to the class II pathway during membrane recycling or by autophagy [34][35][36]. This should allow all B cells in this model system to express PCC peptide-MHC complexes and thus interact with the 5C.C7 T cells independent of the B cell's own receptor specificity. This in turn would generate an efficient mechanism for providing T cell help to any B cell whose Ig receptors also engaged their cognate antigen, including abundant self-antigens that might normally rely on the absence of T cell help to prevent autoantibody generation. In fact, among the antibodies generated in our system, some were reactive against DNA, histones, and GPI molecules implicated in B-cell-mediated autoimmune disorders like arthritis or lupus in other mouse model systems [37]. The transfer recipients in our model progress toward a mild form of arthritis (Figure 3). This disease did not develop in the absence of B cells (Rag2 À/À PCC þ transfer recipients) at least up to 110 d after T cell transfer (data not shown). Although the simplest interpretation of these data would be that T cell-dependent antibody production is the etiologic agent in the disease process, we have not excluded the possibility that the presence of B cells in the system merely enhanced a pathologic T cell response in this model. In preliminary experiments transfer of serum from arthritic mPCC,CD3e À/À mice to mPCC,RAG2 À/À mice did not result in the induction of arthritis in the recipients (unpub- lished data). A further complication is that in the mPCC,RAG2 À/À recipients the plateau levels of 5C.C7 T cells are approximately 4-fold lower than that in the mPCC,CD3e À/À host. Nonetheless, the disease process is clearly T cell initiated, whether through helping B cells or triggering other inflammatory cascades in the innate immune system [37,38]. In either case, it reflects continued effector responses from the peripherally ''tuned'' T cells.
Importantly, the B-cell-deficient system was useful to show that continued help is available to B cells even after the T cells have adapted (Figure 4). Interestingly, the arthritic pathology in this case was delayed by over a month and even then significantly reduced compared to that in the CD3e À/À host (Figures 2F and 4I). Again, although it is arguable that the delay in pathology represents a consequence of anergy induction, we cannot rule out the possibility that this was due to the 4-fold lower number of anergic T cells or the absence of continued B-cell generation in the same host resulting in a reduced ''repertoire'' of B cells. The ability of the B cells to receive help is still very surprising, given our earlier report that dendritic cells isolated from this antigen transgenic mouse present higher levels of antigen than resting B cells in an in vitro T cell proliferation assay [7]. One might have predicted that a quantitative adaptation to the level of antigen presentation by dendritic cells should have abrogated the T cell's ability to productively sense any antigen on B cells. Or observations therefore argue against quantitative receptor calibration being sufficient as a tolerance mechanism, at least for a high-affinity receptor-ligand pair in the periphery.
The ability of a broad repertoire of B cells to present antigen makes our system comparable to models of persistent viral infection, like the LCMV model. Interestingly, such infections also induce a polyclonal hypergammaglobulinemia that is strictly dependent on viral antigen-specific T cell help [15]. Virus-specific helper T cells during chronic infections also enter an anergic state within 2 wk and, in seeming progression of the adaptation, subsequently lose the ability to prime de novo B-cell responses [15,39]. This difference from our results might stem from intrinsic differences in the model systems. These include the generation of antibody escape variants due to viral replication, the presence of virus derived TLR ligands, and the possibility that constant fluxes in antigen dose may recruit mechanisms other than tuning to regulate immunopathology [40][41][42]. Furthermore, the use of mice with an intact repertoire of T cells in the LCMV infections allows for non-cell-autonomous regulatory mechanisms to be superimposed on the adaptive tolerance process (as we discussed earlier). Nonetheless, it is interesting to note that even during persistent viral infections, CD4 þ T cells are required for effective control of the virus [43,44]. In cases where chronic viral infections have been shown to abrogate effector functions in CD8 þ T cells, the viremia is still constrained progressively and the virus distribution localized to restricted tissues, arguing that biological activity in the CD4 þ or CD8 þ cells is being maintained despite the persistent infection [13]. These experiments, however, need to be reexamined in the absence of thymic output and transregulatory cells, using adoptive transfer systems similar to the one we have used.
The restoration of an intact polyclonal T cell compartment in our experimental model (Figures 6 and 7) was sufficient to constrain the autoimmune pathology that we observed. This allowed us to investigate the cellular paradigm that needs to superimpose on adaptive tolerance, in order to achieve ''complete'' tolerance. Interestingly, although adaptive tolerance (decreased TCR signaling and IL-2 production) was induced to equal extents without regard to the presence of other T cells, the subsequent elimination of autoreactive T cells only happens in the presence of other endogenous T cells. These data demonstrate that while anergy is chosen as a primary, cell-intrinsic fate, following tolerogenic antigen encounter in the periphery, the subsequent ''deletion'' is unlikely to be cell autonomous. The nature of the T-T interaction that results in the slow decay of the anergic T cell population is under investigation. Preliminary experiments using anti-CD25 and anti-IL-2 treatment to deplete CD25 þ T cells in the T cell-replete host suggest that such cells are unlikely to be sufficient to mediate this process. Alternatively, we favor a trophic competition process similar to the one that has been described for B cells [45]. The process is likely to be more than a mere competition for ''space,'' since preliminary experiments suggest that simply filling up the animal with a second TCR transgenic CD4þ population is insufficient to eliminate the adapted 5C.C7 T cell. However, the recent demonstration that clonal competition between T cells can affect proliferation and survival in vivo may be relevant to the control of autoimmune pathology in this regard [46,47]. A similar paradigm may also operate in the case of CD8þ T cells that are rendered tolerant to self-antigen [48].
Unlike thymic negative selection or activation-induced cell death, the ''deletional'' component of peripheral T cell tolerance seems to be a relatively slow process. While the reduction of T cell numbers as a result of it is indeed quite striking, it still does not prevent the persistence of a large number of autoreactive T cells in the animal for up to 2 mo. Thus, even in the T cell-replete host, the adaptive tolerance component must play a critical role in constraining the renewed recruitment and expansion of the high affinity autoreactive T cells. In addition, the inability of these CD4 þ T cells to fully turn on autoreactive B cells likely stems from the profound blockade in the differentiation of these cells in the T cell-replete host. T cell differentiation to acquire effector functions have previously been correlated with the extent of cell division [49]. The limited initial expansion of the T cells could therefore lead to poor expression of effector molecules (including IL-4, IFNc, IL-21, and CD40L) and handicap the ability of adapted T cells to help B cells. Such an impairment of effector differentiation (and migration) of pathogenic T cells can also be achieved by the action of trans-regulatory T cells as observed in other model systems [50,51].
Multiple theories have debated the merit of singular mechanisms that maintain peripheral tolerance (reviewed in [52]). Models of T cell tuning have argued that a quantitative dampening of the T cell's sensitivity to ambient antigen should be sufficient to prevent pathology [53,54]. The maintenance of such cellular tuning requires that the cell be constantly aware of the persistence of antigen. The prevention of pathology, on the other hand, requires the cell to avoid effector responses despite the constant interaction of the receptor with a strong endogenous agonist. The success of tuning as a tolerance mechanism therefore depends on the ability of the T cell to walk this biochemical tightrope efficiently. Our data would argue that over time, in the absence of additional mechanisms of peripheral tolerance, enough helper activity is able to break through, so as to mediate visible systemic pathology. It is quite likely that this inadequacy is not unique to the mechanism of adaptive tolerance, because it has been argued that other mechanisms of immune regulation, e.g., T-regs, also cannot control autoimmunity in isolation [55]. Nonetheless, quantitative dampening of TCR responsiveness (and the kinetic delay in the onset of pathology that it might afford [ Figure 4F]) could in fact be quite critical in vivo, allowing additional regulatory processes to effectively collaborate and enforce complete peripheral tolerance [56].
For adoptive transfer experiments involving T cells from 5C.C7 TCR transgenic mice, lymph node suspensions containing greater than 90% CD4þ T cells (1 to 3 million per recipient) were injected without further purification into recipients by the suborbital route. B cells were purified from the spleens of either B10.A,mPCC,CD3e À/À or B10.A,CD3e À/À mice using the mouse B-cell purification kit (Miltenyi Biotech, Auburn, California, United States) or sorted to greater than 98% purity after labeling with anti-B220 PE (BD Biosciences, San Diego, California, United States). Twenty-five million purified B cells were transferred in the experiments shown, but injections of up to 50 million B cells gave similar results.
In vivo blockade of CD40L was achieved by injecting 100 lg/d mAb MR1 and compared with a control hamster IgG (anti-TNP), both obtained as no azide/low endotoxin grade from BD Biosciences.
In vitro T cell cultures and functional assays. Transferred T cells stained with anti-CD45.1 PE and anti-CD4 APC (BD Biosciences) were sorted to greater than 99% purity using a BD Vantage or BD Aria sorter at the NIAID flow cytometry facility. Ten thousand sorted T cells were cocultured with 2 to 5 3 10 5 irradiated B10.A,CD3e À/À splenocytes and titrations of Moth Cytochrome C (MCC) 88-103 peptide (American Peptide Co., Sunnyvale, California, United States). Culture supernatants taken 48 h later were assayed by ELISA for IL-2 and IFNc secretion as per manufacturer's instructions (R&D Systems, Minneapolis, Minnesota, United States). Ex vivo analysis of cytokine secretion was carried out using the IFNc secretion assay (Miltenyi Biotec) as previously described for IL-2 [57]. Analysis was restricted to viable cells based on the exclusion of the vital dye 7-AAD (BD Biosciences). Intracellular foxp3 expression was measured using the FKJ-16s staining kit (eBioscience, San Diego, California, United States) per the manufacturer's instructions. Calcium responses in T cells were measured by loading cells with 5 lg/ml Indo-1 (Molecular Probes, Eugene, Oregon, United States) at 37 8C before stimulation. Loaded cells were washed in cold HBSS containing 1 mM CaCl 2 and 1 mM MgCl 2 , before labeling with Ly5.1-PE and CD4-APC at 4 8C. The washed cells were briefly warmed to 37 8C before analyzing on a BD LSR II cytometer. After 30 s, prewarmed biotinylated anti-CD3 was added to a final concentration of 10 lg/ml, and 2 min later, a warm solution of streptavidin (5 lg/ml final) was added and the data were collected for a further 400 s. Collected data were analyzed using the FlowJo software (Treestar, Ashland, Oregon, United States).
Antibody measurements. Serum samples were assayed for various antibodies using sandwich ELISAs. For determination of total globulin levels, anti-mouse kappa or anti-mouse lambda (Caltag, Burlingame, California, United States) was coated on NUNC Maxisorp plates (Nalge Nunc, Rochester, New York, United States) at 1 lg/ml concentrations in carbonate buffer (pH 9.0). Binding was blocked with 0.25% BSA in Tris-buffered saline þ 0.1% Tween-20 (TBS-Tween). Serum dilutions made in TBS-Tween were incubated with the plates and the bound antibodies detected with isotypespecific antisera coupled to HRP (Caltag). The concentrations of each isotype were calculated from a standard curve using purified Ig isotypes (CaltagA). Anti-DNA antibodies were assayed similarly, after coating with calf thymus DNA (10 lg/ml) instead of anti-lambda/ kappa.
Western blots. Purified proteins were loaded at 1 lg/well and separated on a 5% to 12.5% PAGE gel (Bio-Rad, Hercules, California, United States). Separated proteins were transferred onto nitrocellulose and bound with serum samples diluted 1:1,000 in TBS-Tween. Bound antisera were detected with anti-mouse Ig labeled with HRP (Bio-Rad).
Immunohistochemistry. Formalin-fixed and decalcified limb samples were mounted on paraffin blocks and sectioned to 15-lm thickness before processing for hematoxylin and eosin staining. For ANA analysis, healthy B10.A,Rag2 À/À kidneys were cryosectioned onto silanized slides by Histoserv Inc. (Gaithersburg, Maryland, United States). Each slide had duplicate sections of such kidneys and were fixed in Histochoice solution (Amresco, Solon, Ohio, United States) for 2 to 3 min. Nonspecific binding was blocked with 1% BSA and donkey serum, followed by incubations with serum samples diluted 1:3,000. Bound antibody was detected with Fab fragments of donkey anti-mouse IgM and donkey anti-mouse IgG, labeled with Cy3 and FITC, respectively (Jackson ImmunoResearch, West Grove, Pennsylvania, United States). Stained sections were scored as positive on a Leica DMRX fluorescent microscope if both duplicate sections on the slide were stained positive. Images were collected using a CCD camera and Slidebook software (Intelligent Imaging, Petaluma, California, United States). The range of colors observed depended on the relative amounts of IgM (red) and IgG (green) antibodies in the tested serum that bind self-antigens in the kidney sections. Overlap of red and green signals at high intensity resulted in the yellow colors on the composite images.
Arthritic score. An arbitrary arthritic score was developed to quantitate the extent of disease progression in the mice. Swelling in individual digits was assigned a score of 1 (per digit) with an additional score of 1 added if the digits were inextensibly curled up. Thus each leg could get a maximum theoretical score of 10 based on the health of the digits (counting the polex as the fifth toe in the front legs). Swelling of the feet and lower legs were assigned scores of one each increasing the maximum score per leg to 12. In addition, the inability to extend the elbow or knee joints fully was given a score of one per leg, while the failure to extend this joint beyond a approximately 45-degree angle was scored as 2. As a result, the maximum score per animal in this system was 56 (14 3 4). The shoulders and thighs were not scored in these mice.
X-ray imaging. Mice were killed and taped to film before being exposed to 30-kV x-rays for 55 seconds at the NICHD faxitron facility (Bethesda, Maryland, United States).
Statistical analysis. All statistical analyses were done using the unpaired, two-tailed Student's t-test and Prism 4 software (Graph-PAD, San Diego, California, United States).


extfieldsofstudy: Biology Medicine

text:
Transcription Factor STE12α Has Distinct Roles in Morphogenesis, Virulence, and Ecological Fitness of the Primary Pathogenic Yeast Cryptococcus gattii

ABSTRACT Cryptococcus gattii is a primary pathogenic yeast, increasingly important in public health, but factors responsible for its host predilection and geographical distribution remain largely unknown. We have characterized C. gattii STE12α to probe its role in biology and pathogenesis because this transcription factor has been linked to virulence in many human and plant pathogenic fungi. A full-length STE12α gene was cloned by colony hybridization and sequenced using primer walk and 3′ rapid amplification of cDNA ends strategies, and a ste12αΔ gene knockout mutant was created by URA5 insertion at the homologous site. A semiquantitative analysis revealed delayed and poor mating in ste12αΔ mutant; this defect was not reversed by exogenous cyclic AMP. C. gattii parent and mutant strains showed robust haploid fruiting. Among putative virulence factors tested, the laccase transcript and enzymatic activity were down regulated in the ste12αΔ mutant, with diminished production of melanin. However, capsule, superoxide dismutase, phospholipase, and urease were unaffected. Similarly, Ste12 deficiency did not cause any auxotrophy, assimilation defects, or sensitivity to a large panel of chemicals and antifungals. The ste12αΔ mutant was markedly attenuated in virulence in both BALB/c and A/Jcr mice models of meningoencephalitis, and it also exhibited significant in vivo growth reduction and was highly susceptible to in vitro killing by human neutrophils (polymorphonuclear leukocytes). In tests designed to simulate the C. gattii natural habitat, the ste12αΔ mutant was poorly pigmented on wood agar prepared from two tree species and showed poor survival and multiplication in wood blocks. Thus, STE12α plays distinct roles in C. gattii morphogenesis, virulence, and ecological fitness.

immunodeficiency virus-AIDS patients from Thailand, parts of Africa, and South and Central America (4,37,42,71). Similarly, our analysis of a collection of Cryptococcus isolates from AIDS patients in Southern California revealed about 12% C. gattii strains (12). More recently, an ongoing C. gattii outbreak in healthy humans and animals on Vancouver Island (British Columbia, Canada) has caused multiple fatalities; this is the first documented outbreak in North America (43,83). Thus, C. gattii continues to pose serious public health problems for immunocompromised and healthy individuals worldwide. Concomitant reports of severe and often fatal disease in animals also raise important concerns for the health of pets and wildlife. Along similar lines, a number of recent reports described the natural isolations of C. gattii from multiple tree species other than the Eucalyptus in Canada, Brazil, and India, thereby expanding the known geographical range and ecological niche of this pathogen around the globe (50,51,73). Still, the mechanisms behind host predilection and geographical distribution of C. gattii remain largely unknown, in part due to a paucity of systematic investigations.
STE12 was first identified among a group of Saccharomyces cerevisiae sterile mutants defective in sexual conjugation and related processes (30). Subsequent studies have shown that STE12 is a transcription factor downstream of the mitogenactivated protein kinase (MAPK) cascade that controls mating, filamentation, and cell wall integrity (25,29). The STE12 is activated by two MAPKS, KSS1 and FUS1; has a transcription partner, TEC1; and has two negative regulators, DIG1 and DIG2 (59). More recently, STE12 was reported to directly regulate expression of over 29 yeast genes that mediate a range of cellular processes, cell cycle, mating projections, cell fusion, polarized growth and budding, stress and/or starvation, and signal transduction (31,75). The roles of STE12 homologues in various biological processes have thus so far been characterized in few fungi other than S. cerevisiae. In the filamentous model fungus Aspergillus nidulans, the steA mutant is sterile without ascogenous tissue and fruiting body, but there are no effects on either the sexual cycle-specific Hülle cells or a number of asexual developmental programs (87). Among the few pathogenic fungi studied, STE12 homologues have been implicated in the pathogenic process itself. In the rice blast pathogen Magnaporthe grisea, MST12 disruption caused a serious loss of virulence, as the mutant failed to infect rice leaves or onion epidermal cells, even through wound sites (68,69). The mutation of the STE12 homologue CST1 in Colletotrichum lagenarium, the causal agent of anthracnose disease of cucumber, led to failure of infectious hyphal production from the penetration structure appressoria. This caused a nonpathogenic phenotype on intact leaves but produced disease on wounded leaves (86). The STE12 homologue CPH1 from the common human yeast pathogen Candida albicans was found to be only partially involved in hyphal formation, when a mutant strain was tested on solid medium. However, a double mutation in CPH1 and another gene, PHD1, caused complete loss of filamentation as well as attenuation of virulence in mice (56,58). In contrast, STE12 from the haploid yeast Candida glabrata, which is increasingly implicated in drug-resistant candidiasis, was found necessary for nitrogen starvation-induced filamentation and for a wild-type level of virulence in mice (6). In the opportunistic human pathogen Penicillium marneffei, stlA gene mutation caused no defects in growth, asexual division, or dimorphic switching, while CLS12, the STE12 homologue in Candida (Clavispora) lusitaniae, was required for mating but dispensable for filamentation (5,94).
In attempts to understand hyphal formation, a C. neoformans var. neoformans STE12␣ homolog of the S. cerevisiae STE12 gene was identified. Its overexpression caused hyphal projections and induction of the MF␣ pheromone gene, important for mating reaction, and CnLAC1, the gene that encodes the important virulence factor laccase; the possibility is therefore raised that STE12␣ provides a bridge between C. neoformans mating type and virulence via melanin production (90). Subsequent study of C. neoformans var. grubii by gene knockout revealed that STE12␣ was essential for haploid fruiting but was not essential for mating and virulence (95). The results of an STE12␣ overexpression study by Chang and coworkers were subsequently verified by gene knockout in C. neoformans var. neoformans to show that STE12␣ was essential for haploid fruiting, melanin production, and virulence but was dispensable for mating (11). A second homolog of C. neoformans var. neoformans STE12 was subsequently identified as MATa specific; it was essential for mating and virulence (10). Interestingly, C. neoformans var. grubii STE12␣ was later shown to be involved in reversal of hypervirulence of a crg1 mutant, which encodes the regulator of G protein signaling (89). Taken together, these findings suggested that STE12 is co-opted to perform distinct regulatory control functions be-tween closely related C. neoformans varieties. Therefore, we surmised that C. gattii STE12␣ is potentially a valuable target in studies aimed at unraveling the genetic basis for functional divergence among pathogenic Cryptococcus species.
C. gattii STE12␣ gene characterization and mutation. Primers V564 and V565 were used to PCR amplify a 420-bp fragment of the STE12␣ gene from the genomic DNA of C. gattii NIH444 (see Table S1 in the supplemental material). The resulting 420-bp fragment was used as a probe for colony hybridization analysis of the C. gattii cosmid library (75). After selection of positive clones, a primer walk strategy was used to obtain the complete sequence of STE12␣. This sequence was deposited in GenBank (see "Nucleotide sequence accession number" below). To obtain the amino acid sequence of C. gattii Ste12␣p, we confirmed the C terminus of Ste12␣p by establishing the polyadenylation site of the STE12␣ transcript by 3Ј rapid amplification of cDNA ends. The resulting 1.8-kb product of PCR rapid amplification of cDNA ends was ligated into the TOPO-2.1 TA vector (Invitrogen) and was sequenced.
Phylogenetic analysis of deduced Ste12␣p amino acid sequences was done with the PAUP v4.0b4a program (84), using a bootstrap method with a neighborjoining or maximum parsimony search.
The ste12␣⌬ mutant was generated by homologous recombination of the ste12␣::URA5 disruption cassette (see Fig. S1A in the supplemental material). The transformants were selected on complete synthetic medium without uracil medium and confirmed for gene deletion at the homologous site by PCR and Southern blotting. The reconstituted strain was generated by homologous integration of the 5.3-kb fragment containing the full-length STE12␣ gene. Transformants were selected on 5-FOA medium to produce uracil auxotrophic strains resulting from replacement of URA5 at the ste12␣ locus with the wild-type copy of STE12␣. The homologous reconstitution of STE12␣ in the ste12␣⌬ mutant was again confirmed by PCR and Southern blotting (see Fig. S1B and C in the supplemental material). The original ura5 mutated by selection on 5-FOA of the reconstituted STE12␣ strain was restored as described previously (62). The reverse transcription (RT)-PCR was performed to confirm the absence and presence of the STE12␣ message in mutant and reconstituted strains (see Fig.  S1D in the supplemental material).
Phenotypic characterization. Capsule production was assayed by incubating cultures in YPD broth overnight at both 30°C and 37°C or in LIM medium for 7 days at 30°C with shaking (180 rpm). The size of the capsule was assessed qualitatively under the light microscope using India ink mounts. The urease, phospholipase B and Cu,Zn superoxide dismutase (SOD) enzyme activities of the test strains were determined as described previously (22,23,61).
In vitro growth assessment of all the strains were determined by growing yeast cells in the YPD broth at 30°and 37°C with shaking (180 rpm). An aliquot of cell suspension was withdrawn at 3-h intervals, and the A 600 was recorded.
A commercial yeast identification system was used to compare assimilation patterns (API 20C AUX; BioMerieux). Additionally, the YT MicroPlate (BiOLOG) was used for the comparison of 94 biochemical tests. The API ZYM test (BioMerieux) was performed per the manufacturer's instructions to compare various enzymatic activities of the test strains.
Melanin production and laccase expression and regulation. Melanin production by the WT, ste12␣⌬ mutant, and ste12␣⌬ plus STE12␣ strains was assayed on Niger seed agar (46). Five microliters of cell suspension (10 7 cells/ml) from each strain was spotted on the agar surface, and cultures were incubated at 30°C for 3 to 6 days. Copper or cyclic AMP (cAMP)-mediated reversal of melanin pigmentation was tested on Niger seed agar supplemented with either 10 M to 200 M CuSO 4 (97) or 2 mM to 50 mM cAMP (3). The laccase activity in the test strains was determined by a previously published method (95). In brief, equal numbers of glucose-starved cells of each strain were used to determine the oxidation of the diphenolic substrate 2,2Ј-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS) (IU of activity ϭ 0.01 A 420 absorbance unit in 30 min). The transcription of LAC1 was estimated with semiquantitative RT-PCR using the primer pair V1380 and V1381 (78).
Mating and haploid fruiting. The WT, ste12␣⌬, and ste12␣⌬ plus STE12␣ strains were mixed with the compatible mating strain C. gattii NIH198 (MATa, serotype B) on V8 juice agar (pH 7.0) and were incubated at room temperature in the dark for up to 14 days (45,48). Plates were periodically checked for the appearance of hyphae at the edge of the fungal growth. These edges were examined under the light microscope for the presence of characteristic basidia and basidiospores. Fifty nonoverlapping areas were selected to estimate the extent of mating. V8 juice agar plates supplemented with 2 mM to 50 mM cAMP (Sigma) were used to rescue any mating defect in test strains (1). To further characterize the mating reactions, we used a scanning electron microscope (SEM). The petri plates with mating cultures were flooded with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) and were allowed to stand for 3 to 4 h. The fixed cell cultures were flooded with 0.1 M sodium cacodylate buffer once, and blocks, about 4 mm 3 in size, of the agar showing hyphal growth on the surface were cut out. These blocks were dehydrated using a graded ethanol series, and they were then critical point dried using liquid carbon dioxide. The dried agar blocks were sputter coated with pure gold and viewed in the SEM.
For the haploid fruiting assay, test strains were patched individually on dry, nitrogen-limited filament agar and incubated for 3 weeks at room temperature in the dark. The edges of fungal growth were photographed under the light microscope (91).
Virulence assays. The pathogenic potentials of the WT, ste12␣⌬, and ste12␣⌬ plus STE12␣ strains were compared in mice models of meningoencephalitis (16,61). BALB/c and A/Jcr mice (male, 6 to 8 weeks) were procured from Charles River Laboratories, Inc. All procedures for safe and pain-free handling of animals were followed, per the recommendations of the Institutional Animal Care and Use Committee. The log-phase cultures of test strains were resuspended in sterile phosphate-buffered saline, pH 7.4, at a concentration of 1 ϫ 10 7 cells/ml. Groups of five or six mice were injected intravenously (i.v.) with 10 6 cells of each strain. The animals were given food and water ad libitum and were observed twice daily for any sign of distress. Mice that appeared moribund or in pain were sacrificed using CO 2 inhalation and cervical dislocation. Survival data from the mouse experiments were analyzed by Kaplan-Meyer survival curves using the SAS system (SAS Institute, Inc.).
Histological lesions were examined in groups of three mice infected with 1 ϫ 10 5 cells (16). The mice were sacrificed on day 7 postinfection, and the whole brains were removed and fixed in Bouin's fixative. After 24 h of fixation, tissues were sectioned, and brain slices were washed in distilled water for several hours. Processing was done in a vacuum infiltration processor, the Tissue-Tek VIP 5 (Sakura Finetech), starting with 70% alcohol and proceeding through a series of dehydrating alcohols and xylenes into paraffin, for 15 min per station. Tissues were then embedded in paraffin blocks and sectioned at a 7-m thickness. Sections were stained with hematoxylin and eosin and mucicarmine (Richard Allen Scientific).
To determine the in vivo capsule size of the test strains, 2 mice per strain were injected with 10 6 cells i.v. At 3 days postinfection, mice were sacrificed using CO 2 inhalation, and the brain tissues were smeared on a glass slide, digested with KOH, and examined under a light microscope using Nomarski optics (Olympus).
Neutrophil (PMN) fungicidal activity. Polymorphonuclear leukocytes (PMNs) were isolated from the peripheral blood of healthy human volunteers by Ficoll-Paque (Pharmacia LKB Biotechnology) centrifugation, as described previously (17). One-hundred-microliter aliquots of PMN, 100 l of viable yeasts (5 ϫ 10 3 ), and 20 1 of pooled human serum were added to the wells of a 96-well tissue culture plate and incubated at 37°C in 5% CO 2 -95% air. After incubation for 4 h, the plates were centrifuged at 2,000 rpm for 10 min, and the supernatants were carefully aspirated through 27-gauge needles. The PMN were lysed by the addition of 100 l of 0.05% Triton X-100, and the yeasts were serially diluted and plated on YPD agar. The YPD agar plates were incubated at 30°C for 2 to 4 days for quantitation of CFU. The results were expressed as the percentage of C. gattii killed [ϭ 1 Ϫ (CFU of experiment ϫ CFU of inoculum) ϫ 100]. Values of zero indicated no killing. The study involving human subjects was performed under the guidance of a protocol approved by the Institutional Review Board.
Assessment of pigmentation and growth on wood-based media. The pigmentation and growth of the test strains were assessed on both wood chip and wood extract agar media. Black cherry (Prunus serotina Ehrh) chips were received courtesy of Roger Dziengeleski, Finch, Pruyn & Co., Inc., Glens Falls, NY. Chips were also prepared from Juniperus virginiana (red cedar), Tsuga canadensis (eastern hemlock), Populus tremuloides (trembling aspen), and Acer saccharum (sugar maple) collected from a local forest. These materials were not pretreated with any physical or chemical processes.
The wood agar was prepared by autoclaving 2.0 g of chips at 110°C for 30 min, followed by mixing with 100 ml of autoclaved 2% agar solution. Twentyfive-milliliter aliquots were dispensed in sterile petri plates and were designated wood agar.
Wood extract agar was prepared by mixing of 5.0 g chips in 250 ml of water. This mixture was stirred with magnetic stirrer for 3 h in the cold room, followed by centrifugation at 13,000 rpm for 30 min at 4°C. It was filter sterilized through a 0.22-m membrane, mixed with 250 ml of autoclaved water containing 4% agar, and dispensed in 25-ml aliquots into sterile petri dishes. Both wood agar and wood chip agar plates were inoculated with a 5-l suspension of yeast cell suspensions (10 7 cells/ml), were incubated for 10 days at 30°C, and were observed periodically for growth and pigmentation.
Wood section microscopy. Black cherry wood blocks (ϳ1-cm cubes) were prepared from freshly pruned branches. The blocks were autoclaved at 110°C for 30 min. Sterile blocks were set on 2% sterile water agar in petri plates so that the vessel elements were vertically oriented. The blocks were inoculated with 5 l of cell suspension (optical density at 600 nm [OD 600 ] ϭ 1.0), incubated at 30°C, and observe for up to 8 weeks. Adequate moisture was maintained throughout the incubation period. Finally, the blocks with fungal growth were removed, fixed in formalin for 24 h, and embedded in paraffin, and tangential sections (5 to 7 m) were cut on a rotary microtome. Sections were deparaffinized, covered with glass coverslips, and examined without any staining under the light microscope.
Nucleotide sequence accession number. The complete sequence of STE12␣ was deposited in GenBank under accession number AY168185.

RESULTS
STE12␣ gene characterization. The cloned C. gattii STE12␣ gene was a single copy with approximately 85% nucleotide identity to that of STE12␣ from C. neoformans var. grubii and C. neoformans var. neoformans (GenBank accession no. AAD 44111 and AAN 75715, respectively). The phylogenetic analysis of protein alignment of the C. gattii STE12␣ sequence with that of STE12␣ from C. neoformans var. neoformans, C. neoformans var. grubii, and 11 other fungal species revealed that this protein is segregated into distinct clades of ascomycetous yeasts, molds, and basidiomycetous yeasts, with good bootstrap support. Among basidiomycetes, C. neoformans var. grubii and C. neoformans var. neoformans formed one sister clade distinct from C. gattii. Interestingly, this clustering pattern was not evident for STE12a homologs (Fig. 1). Detailed characterization of the C. gattii STE12␣ protein revealed STE (amino acids 91 to 203) and C 2 H 2 -Zn 2ϩ motifs (amino acids 543 to 565) (see Fig. S2 in the supplemental material). The STE motif is common in STE12 homologs reported from all fungi, while two C 2 H 2 -Zn 2ϩ motifs have been reported only from STE12 homologs of ascomycetes (6,30,53,66,86). In silico analyses with deduced amino acid sequences using the PROSITE program (79) found a number of putative phosphorylation sites, including protein kinase C phosphorylation (11 sites), casein kinase II (19 sites), and cAMP-and cGMP-dependent protein kinase (3 sites). Transcriptional activation of STE12 by phosphorylation has been extensively studied in S. cerevisiae (64,80,93). The presence of phosphorylation sites has also been reported by bioinformatic analyses of STE12 of Magnaporthe grisea and Colletotrichum lagenarium (68,69,86).
STE12␣ is required for efficient mating but not for haploid fruiting. Mating among compatible strains is an important attribute of C. neoformans strains, with potential relevance in epidemiology and virulence (32,33,45,47,48). Although a good mating reaction between NIH444 (MAT␣) and NIH198 (MATa) is generally seen in 4 to 5 days, we observed all mating tests for up to 14 days, to include any poor or delayed reaction on V8 juice agar. Both WT and ste12␣⌬ plus STE12␣ strains showed mycelial elements at the edge of the colonies as early as 5 days postincubation ( Fig. 2A, G). Under the SEM, these elements revealed well-formed basidia with chains of basidiospores (Fig. 2B, H). In contrast, mating reactions of ste12␣⌬ did not show any mycelial formation at the edges until 11 days postincubation under light microscopy (Fig. 2C). However, analysis under SEM revealed isolated formation of hyphae with basidia and basidiospores (Fig. 2D), which were clearly visible at 11 days postincubation (Fig. 2E and F). Semiquantitative analysis using random examination of edges revealed 2 of 50 edges with positive mating reactions in ste12␣⌬ strains compared to 42 to 45 of 50 edges positive for mating reactions in WT and ste12␣⌬ plus STE12␣ strains. C. neoformans var. grubii mating is regulated by both G-protein alpha subunit Gpa1-cAMP and Gpb1-MAPK (39). However, the addition of cAMP to V8 juice agar did not alter the mating reaction of the ste12␣⌬ mutant (Fig. 3). This indicated that either STE12␣ is required for efficient mating of C. gattii through the pathway downstream of cAMP and/or independent of cAMP.
C. gattii STE12 was not involved in the haploid fruiting, as all the strains were positive in the morphological assay (Fig. 4). The appearances of rough edges with aerial hyphae were visible in light microscopy of three strains on filament agar. Further examination of these structures with SEM showed hyphae, basidia, and basidiospores. This observation is in contrast to the reported essential role of STE12␣ in haploid fruiting of C. neoformans var. grubii and C. neoformans var. neoformans (10,83).
STE12␣ is not required for several cellular functions. Capsule formation is an important C. neoformans virulence attribute, and the ste12␣⌬ mutants of C. neoformans var. grubii and C. neoformans var. neoformans revealed smaller capsules than did the corresponding WT strains (9,11,95). Interestingly, the capsule size examined by microscopic mounts of India ink preparations of the C. gattii ste12␣⌬ mutant was identical to that of WT and ste12␣⌬ plus STE12␣ strains when grown in YPD broth at 30°C or at 37°C or in capsule induction medium (LIM) at 30°C, indicating that C. gattii STE12␣ is dispensable for capsule growth. Similarly, C. gattii ste12␣⌬ did not exhibit any defect in the expression of other virulence factors, including urease, phospholipase, and Cu,Zn SOD (data not shown). In contrast, the C. neoformans var. neoformans ste12␣⌬ showed impairment of phospholipase and Cu,Zn SOD enzyme activities (11,22,23). To unmask any unexpected phenotype, we used the API 20C AUX, API ZYM, and YT MicroPlate systems to compare assimilation and enzymatic activities of the C. gattii WT, ste12␣⌬, and ste12␣⌬ plus STE12␣ strains, but no differences were observed (data not shown). Similarly, the C. gattii ste12␣⌬ mutant did not show any defect in growth in the presence of chemicals used as C. neoformans cell wall or cell membrane inhibitors (44). Alto- gether, these results indicated that C. gattii STE12␣ did not regulate several of the putative virulence factors assayed in this fungal pathogen. STE12␣ is required for melanin pigmentation and laccase expression. Melanization via C. neoformans laccase is an important virulence attribute, and it is down regulated in C. neoformans var. neoformans ste12␣⌬ mutants (10,11,76,92). Interestingly, the C. gattii ste12␣⌬ mutant was also defective in melanization and showed weak yellowish pigmentation compared to the dark brown pigmentation formed by WT and ste12␣⌬ plus STE12␣ strains on Niger seed agar at 3 days postincubation (Fig. 5A). Supplementation of Niger seed agar with CuSO 4 , a metal important in laccase induction, did not restore melanin pigmentation in the mutant strain (data not shown). However, supplementation of the medium with cAMP, important in transcriptional regulation of laccase via FIG. 4. STE12␣ is not required for C. gattii haploid fruiting. The light microscopic (left panel; magnification, ϫ100) and SEM (right panel; magnification, ϫ6,000) analyses of haploid fruiting of the WT, ste12␣⌬, and ste12␣⌬ plus STE12␣ strains are shown. The C. gattii strains were patched on dry, nitrogen-limited filament agar medium. The plates were incubated for 3 weeks at room temperature in the dark. The morphological assay showed characteristic filaments, basidia, and basidiospores.

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CRYPTOCOCCUS GATTII STE12␣ 1071 parallel or multipath pathways (39), restored melanin pigmentation in the C. gattii ste12␣⌬ mutant (Fig. 5A). The reduced melanin pigmentation in the C. gattii ste12␣⌬ mutant was caused by defects in laccase enzyme activity and laccase transcript levels under glucose repression condition ( Fig. 5B and C). These results indicated that STE12␣ regulates the expression of laccase in C. gattii. STE12␣ is required for a wild-type level of virulence in mice. As C. gattii causes disease in both immunocompromised and healthy individuals, we utilized immunocompetent BALB/c and C5-deficient A/Jcr mice strains for comparison of pathogenicity. The Kaplan-Meyer survival curves for mice infected with three strains are shown in Fig. 6. A/Jcr mice infected with the wild-type strain survived for 4 days, while similarly infected BALB/c mice survived for 10 days. The survival pattern after infection by the reconstituted strain was 11 days for A/Jcr and 14 days for BALB/c mice. In contrast, mice of both strains infected with the C. gattii ste12␣ mutant strain survived as long as 29 and 30 days, respectively (P Ͻ 0.0005). Thus, a significant reduction in the virulence of the ste12␣ mutant strain was observed for both complement-deficient A/Jcr mice and immunocompetent BALB/c mice.
The ste12␣⌬ mutant produced much smaller cysts in the brains of both BALB/c and A/Jcr mice, and these cysts contained fewer yeast cells. On the contrary, WT and ste12␣⌬ plus STE12␣ strains produced large cysts containing several to many yeasts. It was interesting to note that all strains tested divided relatively rapidly and produced more lesions in the brains of infected A/Jcr mice than in those of the infected BALB/c mice (Table 1), indicating that A/Jcr mice are relatively more susceptible than BALB/c to C. gattii infection. Light microscopy of brain smears revealed that all test strains FIG. 5. STE12␣ is required for melanin production, laccase enzyme activity, and LAC1 transcript expression. (A) C. gattii strains grown in YPD broth overnight were washed with phosphate-buffered saline, and a 5-l suspension of 10 7 cells/ml was spotted on Niger seed agar with or without cAMP and incubated for 3 days at 30°C for melanin production. (B) Laccase enzyme activity from equal numbers of glucose-starved cells (5 h) was determined by measuring the oxidation of the diphenolic substrate ABTS (IU of activity ϭ 0.01 A 420 absorbance unit in 30 min). Results are the means Ϯ standard deviations from three individual experiments. The absence of laccase activity in the mutant is consistent with the initial loss of melanization in this strain. (C) Semiquantitative RT-PCR to examine the expression of the LAC1 gene in all of the test strains. Total RNA from each strain was isolated and reverse transcribed (cDNA) with 100-ng aliquots in 1:5 serial dilutions. Actin was used as a control.

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REN ET AL. EUKARYOT. CELL produced large capsules in the infected mice (Fig. 6B). This observation was consistent with the large capsule size observed in vitro. Interestingly, the ste12␣⌬ mutant was significantly more susceptible to in vitro PMN killing than the WT and ste12␣⌬ plus STE12␣ strains (Fig. 7). However, in vitro H 2 O 2 sensitivity was similar in all three strains, which raised the possibility that highly reactive oxygen intermediates may be responsible for enhanced killing of the ste12␣⌬ mutant strain by human PMN. Collectively, these results indicated that functional STE12␣ is critical for C. gattii survival and multiplication against mammalian host defense mechanisms.
STE12␣ is involved in wood utilization. Many reports have described natural isolations of C. gattii from wood hollow and detritus of trees from around the world (28, 36, 50, 51, 73, 88).
Additionally, experimental inoculations of almond tree seedlings with C. gattii showed that fungus remained viable and could be recovered from plant tissues 100 days postinfection, indicating its potential for survival in planta (38). It has been previously proposed that C. neoformans laccase plays an important role in lignin degradation in wood hollows (67,72,96). It was interesting that the ste12␣⌬ mutant was defective in pigmentation and appeared almost white, as opposed to the dark brown to red pigment produced by wild-type and reconstituted strains on water agar containing wood chips from black cherry and eastern hemlock trees (Fig. 8A). The loss of pigmentation in the mutant was not due to poor growth, as serial dilutions of all the strains on water agar containing either 2% wood chips or 2% wood chip extract exhibited similar growth patterns (Fig. 8B). It is important that the pigmentations ob-FIG. 6. STE12␣ is required for virulence. (A) C. gattii strains were grown overnight in YPD broth, washed with phosphate-buffered saline, and counted, and 100 l of suspension containing 10 6 cells was injected intravenously into 5 (each) BALB/c and A/Jcr mice. Mice were monitored twice daily until moribund. (B) Two mice infected i.v. with 10 6 cells were sacrificed at 3 days postinfection, and brain tissues were smeared on microscope slides and examined under a microscope using Nomarski optics (magnification, ϫ380).

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CRYPTOCOCCUS GATTII STE12␣ served on the wood chips from black cherry and eastern hemlock were not a universal phenomenon, as wood chips from red oak, balsam fir, sugar maple, or trembling aspen did not induce pigmentation in any of the strains tested. This indicated that these tree species either lacked specific substrate(s) required for pigmentation or the condition for pigmentation was not appropriate in the laboratory setting. Next, we examined the role of C. gattii STE12␣ in tree colonization by using simulated conditions. Sections of black cherry wood blocks surface inoculated with WT and ste12␣⌬ plus STE12␣ strains showed abundant yeast cells in the vessels with heavy pigmentation deep within the wood block. A few budding cells were also seen ( Fig. 9A and B). Similar wood block sections inoculated with the ste12␣⌬ mutant showed very few yeast cells, with negligible brown pigment (Fig. 9C). Furthermore, the total numbers of yeast cells, counted from 10 representative sections, were approximately 40 Ϯ 16 to 19 for WT and ste12␣⌬ plus STE12␣ cells and 13 Ϯ 10 for ste12␣⌬ mutant cells (P Ͻ 0.05). These results indicated that pigmentation might be crucial for C. gattii survival, multiplication, and possibly, invasion in the face of plant host defense mechanism(s). Further experimental studies are imperative to test this possibility.

DISCUSSION
We have carried out a detailed characterization of the transcription factor STE12␣ in C. gattii biology. Currently, this pathogen is the cause of serious public health concerns due to its infectivity for AIDS patients and healthy individuals, its fatal outbreaks involving humans and animals, and its expanding geographical range, especially in North and South America (8,12,43). Experimental studies to determine what makes C. gattii such a potent pathogen are sparse. STE12␣ is among the very few transcription factors studied by the use of knockout mutants in both saprobic and pathogenic fungi. Therefore, STE12 is a valuable target for molecular pathogenesis studies to define C. gattii properties that are either distinctive or shared with other pathogens.
Previously, it was reported that STE12␣ was located within the MAT␣ locus of the C. gattii genome, along with elements of the MAPK signaling cascade, pheromones, receptors involved in mating, housekeeping genes, and putative open reading frames of unknown function (31,75). The overall organization of the mating locus in C. gattii is quite similar to that in C. neoformans var. grubii and in C. neoformans var. neoformans (31,41,75). However, the order and orientation of STE12␣ vis-à-vis other genes in the locus are not conserved in these fungi, raising the strong possibility that changes in STE12␣ expression can have occurred due to the positional effect of neighboring genes, as has been reported for other eukaryotic genomes (2,20,35,85).
C. gattii STE12␣ was required for robust mating, as the ste12␣⌬ mutant showed poor mating compared to the wildtype and reconstituted strains. Thus, unlike S. cerevisiae and other ascomycetes, the C. gattii ste12␣⌬ mutant was not sterile but merely mating impaired; it shared this property with similar mutants from C. neoformans var. grubii and C. neoformans var. neoformans (11,95). Taken together, our study and the two studies just cited provide a unique demonstration of a gene function conserved across all three pathogenic Cryptococcus forms ( Table 2). Future studies with a congenic pair of C. gattii strains (MAT␣ and MATa) and genetic epistasis experiments are clearly warranted to define the extent of the conserved role of STE12␣ in C. neoformans and C. gattii. Conventionally, mating studies in C. neoformans are carried out over a week, a length of time which, in our experience, proved insufficient for the knockout strain. However, when the observation period was extended to 2 weeks and when SEM was used for closer scrutiny of cells at edges of cocultures, a few spots with sparse mating reactions were found in mutant strain. Almost all fungal STE12 knockout mutants to date are reported to have mating or morphogenetic defects, a fact which affirms the central role of this transcriptional regulator in developmental programs. Our current data, taken together with the published literature from C. neoformans var. grubii and C. neoformans var. neoformans, do not identify the mechanism behind the observed partial defect in mating in the mutant strains. Perhaps other transcription factors play an important role in this process, either in association with or independent of STE12.
Another Cryptococcus developmental program, termed haploid or monokaryotic fruiting, involves filamentation and sporulation by MAT␣ strains; it is reported to be defective in ste12␣⌬ mutants of C. neoformans var. grubii and C. neoformans var. neoformans (11,95). This observation is similar to FIG. 7. STE12␣ is required for fungicidal activity of human neutrophils (PMNs). Human PMN at the effector-to-target cell ratio of 10:1 were inoculated with opsonized C. gattii strains (5 ϫ 10 3 ) for 4 h at 37°C in 5% CO 2 -95% air. The percent C. gattii killed was determined by using the following equation: 1 Ϫ (CFU of experiment ϫ CFU of control) ϫ 100. Results are means Ϯ standard deviations from PMNs of three individual donors. The asterisk denotes a P value of Ͻ0.05 compared to wild-type and reconstituted strains. a Mean Ϯ standard deviation of foci counted in 9 brain sections of 3 mice in each group.
b Mean Ϯ standard deviation of yeast cells counted in all of the foci divided by the number of foci. the complete or partial inhibition of filamentation seen in S. cerevisiae and Candida albicans ste12⌬ mutants (55,56). Interestingly, the C. gattii ste12␣⌬ mutant showed normal haploid fruiting, suggesting either that STE12␣ is redundant or that there is some independent regulatory control of this pathway. Thus, unlike the role of STE12␣ in mating, the functional role of this gene in filamentation differs between C. gattii and C. neoformans and thus represents a gene function that has diverged in these closely related pathogens. The importance of this observation is difficult to assess at present, as we do not know how widespread and relevant the phenomenon of haploid fruiting is for C. gattii biology and virulence. Recently, it has been suggested that C. neoformans var. grubii haploid fruiting represents a self-fertilizing form of sexual reproduction in the absence of the out-crossing mode of reproduction due to the rarity of the occurrence of MAT␣ mating partners (54).
The profound defects in laccase expression, enzyme activity, and melanin production in the C. gattii ste12␣⌬ mutant strain are consistent with earlier reports from C. neoformans var. neoformans on down regulation or up regulation of laccase levels due to knockout or overexpression of C. neoformans var. neoformans STE12␣ (11,90). In contrast, melanin production was reported to be unchanged in the C. neoformans var. grubii ste12␣⌬ mutant (95). The latter observation highlights another instance in which gene regulation has been conserved between C. gattii and C. neoformans var. neoformans but not in C. neoformans var. grubii, even though C. neoformans var. neoformans and C. neoformans var. grubii are closely related phylogenetically. The results with ste12␣⌬ mutants in C. gattii and C. neoformans var. neoformans raise the strong possibility that as-yet-undefined activators and/or suppressors of melanin participate in direct or indirect interactions with STE12␣. Importantly, the addition of cAMP reversed the melanin defect in the C. gattii ste12␣⌬ mutant strain; a possible interpretation is that cAMP acts to rescue laccase repression by STE12␣ via parallel or multipath pathways (39). An important role for the cAMP signaling pathway in regulation of C. neoformans melanin production has already been established by means of a number of gene knockout mutants (3,72). Overall, there is a strong possibility of cross talk between elements of the MAPK and cAMP pathways, which are known to play important roles in mating. A recent discovery of C. neoformans DEAD box RNA helicase VAD1 as a regulator of multiple virulence-associated genes is also highly relevant in this context (67). The vad1⌬ mutant was melanin deficient due to up-regulation of NOT1 (CDC39); the latter acted as an intermediary transcriptional repressor of laccase. NOT1 is part of the Ccr4-Not complex involved in global control of gene expression (21). The involvement of NOT1 in the transcriptional regulation of laccase raised the possibility of direct or indirect interactions with STE12 because some earlier studies have described involvement of CDC36 and CDC39 as negative regulators of the pheromone response in yeast (24,63). Thus, multiple lines of evidence suggest that regulatory elements in the pheromone response pathway are involved in regulation of laccase expression. Further examinations will be imperative to define the regulatory network for C. gattii laccase. The evidence for other regulatory controls of melanization in fungi comes from the plant pathogens Colletotrichum lagenarium and Magnaporthe grisea. Their respective ste12⌬ mutants have no defects in melanin, which is uniquely produced via polyketide synthesis (34). Melanin biosynthesis is controlled in these two organisms via novel transcription factors containing a Zn(II) 2 Cys 6 binuclear cluster motif and a Cys 2 His 2 zinc finger similar to motifs present in STE12 (86). The C. gattii ste12␣⌬ mutant was significantly less virulent than the wild-type and reconstituted strains in both immunocompetent (BALB/c) and C5-deficient (A/Jcr) mice. This observation was intriguing because we did not observe a down regulation of SOD, phospholipase, and urease or a smaller capsule size, all prominent characteristics of the less-virulent C. neoformans var. neoformans ste12␣⌬ mutant, although the C. gattii ste12␣⌬ mutant had a pronounced melanin defect (10,11). Even though the virulence defect was not seen in the C. neoformans var. grubii ste12␣ mutant, this mutant also pro-duced a smaller capsule; capsule size is a very important virulence factor (95). The reduced virulence of C. gattii ste12␣⌬ was one more instance in our study when C. gattii and C. neoformans var. neoformans display an important role for STE12␣ in animal pathogenesis, functionally diverged from C. neoformans var. grubii. Additionally, we noticed that the C. gattii ste12␣⌬ strain was severely defective in replication and in cyst formation in the brains of infected mice. The mutant also showed heightened sensitivity to oxidative and nonoxidative killing by phagocytic cells, as evident from experiments with purified human PMN. A common theme in all of the virulencerelated observations was the reduced laccase and melanin levels in the C. gattii ste12␣⌬ mutant in vitro. Laccase in C. neoformans var. neoformans and C. neoformans var. grubii plays important roles in resistance to stress, phagocytic killing, and conversion of dopamine to immunomodulatory products in the host brain (57,65). By implication, laccase and melanin are likely to play similar roles in C. gattii. The fact that albino strains are not usually seen in clinical cases of cryptococcosis is further evidence for an essential role of melanin in vivo.
We strongly suspected that environmental/nutritional sensing by STE12␣ by itself is an important fitness attribute missing from ste12␣⌬ strains, perhaps sufficient to account for their poor pathogenic potential. Support for this assumption comes from mutants of the human pathogen Candida glabrata and the plant pathogens Colletotrichum lagenarium and Magnaporthe grisea, which are all less pathogenic than their wild-type counterparts, in a melanin-independent manner (6,68,69,86). We FIG. 9. STE12␣ is involved in C. gattii utilization of wood. The black cherry wood blocks (ϳ1-cm cubes) were surface inoculated with a 5-l suspension of 10 8 cells/ml of each C. gattii strain. These blocks were placed on water agar in petri plates and incubated for 8 weeks at 30°C. Adequate moisture was maintained throughout the incubation period. Blocks with fungal growth were removed and processed for sectioning as illustrated in the accompanying schematics. The diagonal sectioning and selection of wood sections away from the inoculated surfaces allowed an estimation of fungal invasion (schematics). (a, b) WT-inoculated wood sections showing several pigmented yeast cells; (c, d) ste12␣⌬ mutantinoculated wood section with few yeast cells with negligible brown pigment; (e, f) wood section inoculated with the ste12⌬␣ plus STE12␣ strain showing abundant brown-pigmented yeast cells. V, vessels; P, parenchyma; F, fiber; R, rays. Both rays and vessels showed fungal growth.

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CRYPTOCOCCUS GATTII STE12␣ carried out extensive phenotypic tests to find additional fitness defects in the mutant strain, but these efforts were not successful, possibly due to reliance on laboratory media that are unlikely to reveal subtle STE12␣ regulatory control over functional genes in vivo. More refined studies are needed to identify diverse gene functions regulated by C. gattii STE12␣, as was achieved for S. cerevisiae STE12 by means of an exquisite genome-wide location and expression profiling approach (74).
A remarkable observation in this study was the inability of the ste12␣⌬ mutant strain to produce robust pigmentation on wood chips simulating C. gattii's environmental niche. To our knowledge, this is the first instance in which a gene function has been directly related to the specialized environmental niche of C. gattii or C. neoformans. Our wood chip agar model for testing environmental fitness was validated by the fact that the wild-type and reconstituted strains exhibited robust survival and multiplication on these substrates and produced copious amounts of melanin-like pigment. The poor pigmentation of the ste12␣⌬ mutant strain could be due to its impaired ability to produce laccase. It may be relevant to recall that filamentous basidiomycetes are the major cause of wood damage in nature (white rot). This process involves breakdown of ligninocellulose by laccase working in concert with other fungal enzymes, mediators, and toxic radicals (52). The essential role of laccase in lignin degradation has also been confirmed by the use of laccase-deficient mutants of the white rot fungus Pycnoporus cinnabarinus (27). A corollary of these destructive fungal activities is represented by the industrial applications of purified laccase in bioremediation (40,60). The results of our experiments suggest a model in which C. gattii STE12␣ senses appropriate nutrient/environment cues on the wood surface and responds by up regulation of laccase and melanization for cooperative utilization of substrate. This response was suboptimal in the mutant, due to the absence of Ste12p. Additionally, ste12␣⌬ mutant cells appeared defective in penetration and proliferation inside wood blocks compared to the WT and ste12␣⌬ plus STE12␣⌬ strains. We interpreted this observation as another manifestation of the ste12⌬-associated fitness defect, paralleling the observed defect in colonization and proliferation within mouse tissues (Fig. 9). Thus, STE12␣ appears to be an important regulatory gene for control of both the saprobic and the pathogenic aspects of the life cycle of Cryptococcus gattii.