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The Journal of Immunology, 2005, 174: 6282-6291.
Copyright © 2005 by The American Association of Immunologists

Dendritic Cells Cross-Present Exogenous Fungal Antigens to Stimulate a Protective CD8 T Cell Response in Infection by Histoplasma capsulatum1

Jr-Shiuan Lin, Chiao-Wen Yang, Dah-Wei Wang and Betty A. Wu-Hsieh2

Graduate Institute of Immunology, National Taiwan University College of Medicine, Taipei, Taiwan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The contribution of CD8 T cells in host defense against histoplasmosis is minor in the CD4 T cell-intact mouse, as it has been shown that depleting CD8 T cells only marginally affects fungal clearance. However, it remains to be determined whether the CD8 T cells are protective in a host lacking functional CD4 T cells. In this study, MHC class II-deficient mice infected with Histoplasma capsulatum (Histoplasma) kept the fungus in check for up to 16 wk, indicating that CD8 T cells are able to limit fungal replication. Ex vivo studies showed that CD8 T cells from Histoplasma-infected mice expressed both intracytoplasmic IFN-{gamma} and granzyme B. Furthermore, CD8 T cells exhibited cytotoxic activity against macrophage targets containing Histoplasma. We demonstrated that the macrophage, being the primary host cell as well as the effector cell, can also serve as Ag donor to dendritic cells. Histoplasma-specific CD8 T cells are stimulated by dendritic cells that present exogenous Histoplasma Ags, either through direct ingestion of yeasts or through uptake of apoptotic macrophage-associated fungal Ags, a process known as "cross-presentation." Based on these results, we present a model detailing the possible sequence of events leading to a cell-mediated immune response and fungal clearance in Histoplasma-infected hosts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Infection with HIV generates an immune response that usually contains but does not eliminate the virus. Both HIV-specific CD4 Th cells and CD8 CTL cells are activated. However, with progressive HIV infection, the CD4 T cell counts gradually decline and are finally lost, whereas total CD8 T cell counts increase and remain constant for a prolonged period of time (1, 2). Therefore, there is much to be learned about the function of CD8 T cells in the absence of CD4 T cells, especially in defense against opportunistic infections.

Histoplasma capsulatum (Histoplasma) 3 is an opportunistic fungal pathogen that kills AIDS patients (3). Depletion (4) and adoptive transfer (5) studies have established the vital role of CD4 T cells in clearing Histoplasma in the mouse model. The functional role of CD4 T cells is to produce IFN-{gamma} (6, 7, 8). Depleting CD4 T cells dramatically decreases IFN-{gamma} production and accelerates mortality in mice (7, 8). On the contrary, CD8 T cell depletion or {beta}2-microglobulin ({beta}2m) deficiency only marginally affects fungal clearance (8, 9). Because the role of CD8 T cells in a CD4 T cell-intact host against histoplasmosis is comparatively minor, not much is known about CD8 T cell function or how they are activated in Histoplasma infection. Considering the importance of CD8 T cells in defense against infections in patients with low CD4 T cell counts, a thorough study of CD8 T cells against this fungal pathogen is warranted.

The macrophages are the first cells to encounter Histoplasma in the host. Through phagocytosis, Histoplasma enters into the endocytic compartment of the macrophage (10). Control and clearance of this pathogen is dependent on an intact cell-mediated immune response. Ability of the host to produce IFN-{gamma}, which arms the macrophages, is critical to host defense against the infection (4, 11, 12). Without IFN-{gamma} activation, the intracellular parasitism causes macrophages to die (13). The macrophages are, by definition, professional APCs. It remains to be determined, besides being the host and the effector cell for Histoplasma yeasts, what role the macrophage plays in Ag presentation and priming of T cells (7, 10, 14). Recent studies demonstrate that dendritic cells (DCs) can process exogenous Ags into the MHC class I pathway, a function known as "cross-presentation" (15, 16, 17, 18). DCs can also acquire bacterial and viral Ags from their host cells that have become apoptotic and cross-prime sensitized CD8 T cells (15, 16, 17, 18). It remains a question whether cross-presentation occurs to intracellular fungal pathogens of the macrophage.

In this study, we first established the importance of CD8 T cells in protecting mice lacking functional CD4 T cells against histoplasmosis. Second, we showed that CD8 T cells function by both perforin-independent as well as perforin-dependent mechanisms, because CD8 T cells isolated from Histoplasma-infected mice produce IFN-{gamma} as well as kill Histoplasma-containing macrophage cell line IC-21 targets in vitro through a perforin-mediated mechanism. Third, we found that DCs can take up viable Histoplasma and stimulate sensitized CD8 T cells to express functions. Finally, we demonstrated that DCs can acquire Histoplasma antigenic materials from apoptotic infected macrophages, which induce MHC class I-restricted CD8 T cell response. Our data support the concept that apoptotic macrophages that are killed by the intracellular fungus can serve as Histoplasma-Ag donors for DCs, which in turn cross-present Histoplasma Ags to activate CD8 T cells. Based on our observations and previous studies, we propose a model to delineate cell-mediated immune response that involves CD4 and CD8 T cells in histoplasmosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Wild-type (WT), MHC class II-deficient (IIKO) (19), and C57BL/6-Prf1tm1Sdz perforin-deficient mice (20) and B6.129P2-B2mtm1Unc {beta}2m-knockout (21) mouse breeders on the C57BL/6 background were obtained from The Jackson Laboratory and were bred at the Laboratory Animal Center, National Taiwan University College of Medicine. Mice were housed in sterilized cages with sterilized bedding and filter cage tops and were fed with sterilized food and water. Mice at 8–10 wk of age were used in all of the experiments.

Infection and quantitation of fungal load

Histoplasma capsulatum strain 505 yeast cells were cultured at 37°C on brain-heart infusion agar supplemented with cysteine (1 mg/ml) and glucose (20 mg/ml). Mice were injected i.v. with 2.5 x 104 yeast cells, 1/100 of lethal dose (6). The spleen was homogenized in a tissue grinder with 1 ml of RPMI 1640 medium (Life Technologies). One to 10 serial dilutions were made and 0.1 ml was plated onto glucose-peptone agar. Histoplasma mycelial colonies were enumerated after incubation at 30°C for 10–14 days.

DC and macrophage cultures

Bone marrow cells were harvested from femurs of WT or {beta}2m–/– mice and cultured in RPMI 1640 complete medium (10% FCS) supplemented with 10% culture supernatants from transfected X-63 cell lines containing GM-CSF (100–200 ng/ml final concentration as determined by ELISA) (22, 23). At day 8, nonadherent cells were harvested, and 60–80% of these cells were CD11c+ as determined by FACS analysis. The resident peritoneal cells from normal intact mice without chemical treatment were used as a source for peritoneal macrophages (PMs). After incubation in 24-well plates at 37°C for 3–5 h, nonadherent cells were removed by gentle washes with warm HBSS. Approximately 70–80% of the adherent cells were F4/80+ macrophages. To isolate alveolar macrophages (AMs), mouse lungs were lavaged five to six times through a 24-gauge intratracheal catheter (TERUMO Corporation) with 37°C prewarmed PBS containing 5 mM EDTA (Sigma-Aldrich). The lavage fluids were pooled and cells were recovered by centrifugation (1000 x g for 10 min). Cells were seeded in plates and incubated at 37°C overnight. Nonadherent cells were removed by gentle washes with warm HBSS, and the adherent cells were used in the experiments.

Splenic T cell enrichment by nylon-wool passage and CD4 and CD8 T cell purification by MACS beads

Spleen cells were harvested from mice at day 14 of infection. Single-cell suspensions were applied to prerinsed nylon-wool columns. After incubation in an upright position at 37°C for 90 min, the nonadherent cells were eluted. To purify CD4 and CD8 T cells, nylon-wool-passaged cells were resuspended in MACS buffer (PBS with 0.5% FCS) containing anti-CD4 or anti-CD8 magnetic microbeads (Miltenyi Biotec) and incubated at 4°C for 30 min. The cells were positively selected by LS+/VS+ column. To release beads from cells, the cells were further incubated at 37°C for 90 min followed by centrifugation. In the resuspended cell pellet, ~95% of cells were either CD4+ or CD8+ T cells.

In vivo cell depletion

Mice were treated with concentrated anti-CD4 (clone GK1.5) or anti-CD8 (clone 2.43) hybridoma supernatant at the time of infection and twice weekly until concluding the experiment. The in vivo depletion efficiency was >95% (CD4) and >99% (CD8).

Preparation of Histoplasma for ingestion by macrophages and DCs

Fresh slants of Histoplasma yeast cells were prepared for each experiment. To kill the yeasts, an aliquot of the suspension was put into a 65°C water bath for 1.5–2 h. One hundred percent of yeast cells were confirmed dead by plating. To prepare yeast cell lysate, 0.5 ml packed volume of the yeast cells was suspended in 1 ml of Dulbecco’s PBS containing 10 µl of each protease inhibitor (2 µg/ml leupeptin, 5 µg/ml aprotinin, and 1 mM PMSF) and mixed with an equal volume of 0.5-mm glass beads (BioSpec Products). The mixture was vigorously vortexed for 10 s and cooled on ice immediately for 30 s, 60 cycles. After centrifugation (1000 x g for 10 min), the supernatant was collected and filtered through a 0.22-µm filter, and the protein concentration was quantified by Coomassie Brilliant Blue G-250 dye (Bio-Rad). The lysate was aliquoted and stored at –80°C.

Live or heat-killed yeast cells (2 x 104) or 25 µg of protein/ml yeast cell lysate were added to macrophage monolayers or DC cultures and incubated at 37°C for 2–3 h, followed by thorough washes to get rid of the extracellular free yeasts. In some experiments, lyticase (L-2524; Sigma-Aldrich) at 300 U/ml was added to the macrophage culture for 0.5 or 1 h, followed by thorough washes.

Intracytoplasmic cytokine, granzyme B staining and flow cytometry

One million nylon-wool-enriched T cells or MACS-purified CD8+ T cells were cultured in medium containing 2.5 x 104 live Histoplasma yeast cells for 24 h. At 6 h before harvest, monensin (2 µM; Sigma-Aldrich) was added. Cells were harvested and stained for intracellular IFN-{gamma} as previously described (24). To stain intracellular granzyme B, the cells were first treated with purified 2.4G2 Ab directed against mouse Fc{gamma}II/III receptors (Fc Block) and then stained with PE-labeled mouse anti-human granzyme B mAb (Caltag Laboratories), of which the specificity has been confirmed (25). PE-labeled mouse IgG1 was used as isotype control. Cells were acquired by FACSCalibur flow cytometer and analyzed by CellQuest (BD Biosciences). All Abs except granzyme B were purchased from eBioscience.

Cytotoxicity assay

Mouse macrophage cell line IC-21 at 1 x 104 per well was cultured in triplicate or quadruplicate in 96-well round-bottom plates. After 2-h adhesion, heat-killed (1 x 105) Histoplasma yeasts were added to the wells and incubated for another 2 h. Free extracellular yeasts were removed and the cultures were incubated for 16–18 h. Splenic CD8 T cells were purified from normal or infected mice pretreated with anti-CD4 mAb (clone GK1.5) 2 days before experiment. Different numbers of CD8 T cells were added to obtain different E:T ratios. After 6 h of incubation, 50 µl of culture supernatant was removed from the wells and transferred to an ELISA plate. An equal volume of substrate provided in CytoTox96 Non-Radioactive Cytotoxicity Assay kit (Promega) was added. The coupled-enzymatic assay detects lactate dehydrogenase in the culture supernatants. The plates were left at room temperature in the dark for 30 min. The reaction was stopped by Stop Solution and color absorbance was read at 492 nm. The amount of color formed is proportional to the number of lysed cells. Thus, percent cytotoxicity was calculated from the absorbance values as follows: % cytotoxicity = [(experimental – effector spontaneous – target spontaneous)/(target maximum – target spontaneous)] x 100%.

[3H]Thymidine incorporation assay and IFN-{gamma} ELISA

Nylon-wool-enriched T cells or MACS-purified CD4+ or CD8+ T cells (1 x 105/well) were added to wells containing macrophages or DCs (1 x 104/well). The irradiated macrophages or DCs (1000 rad) were pulsed with different forms of Histoplasma Ags before the experiment. The cultures were incubated at 37°C for 48 h (for MACS-purified CD4+ or CD8+ T cells) or 72 h (for nylon-wool-enriched T cells). At 18 h before harvest, 1 µCi of [3H]thymidine was added. Cells were harvested and [3H]thymidine incorportation was read by TopCount Microplate Scintillation and Luminescence Counter (Packard Instrument). {Delta}cpm was calculated as follows: {Delta}cpm = cpm in experimental sample – cpm in medium control.

The culture supernatants were collected at 24 h, aliquoted, and stored at –80°C. The level of IFN-{gamma} was determined by the OptEIA Set for mouse IFN-{gamma} (BD Biosciences) following the manufacturer’s instruction.

Detection and induction of macrophage and DC apoptosis

Live or heat-killed Histoplasma yeast cells (2 x 105) or 25 µg of protein/ml lysate were added to the PM and DC (1 x 105) cultures. For macrophage culture, free yeasts were washed away with warm HBSS after 2 h and then cultured for another 6 h. For DC culture, the yeast cells were left in the culture until harvest. DNA fragmentation was quantified by Cell Death Detection ELISAplus (Roche).

In samples where apoptosis was chemically induced, macrophages were treated with 1 µg/ml LPS for 4 h at 37°C, followed by incubation with 5 mM ATP for 45 min. The apoptotic macrophages were stained with propidium iodide and Annexin V to confirm cell death.

Phagocytosis assay

PMs (1 x 105) were labeled with 0.05 µM CFSE. Live or heat-killed yeast cells (2 x 105) or 25 µg of protein/ml yeast cell lysate were added, and the cells were cultured for 2–3 h at 37°C. Free yeasts were washed away, and the adherent cells were cultured for another 4–5 h. In those cultures where apoptosis was chemically induced, the macrophages were treated with LPS and ATP. In some experiments, DCs were treated with 10 µg/ml cytochalasin D or 4% paraformaldehyde in Dulbecco’s PBS at 4°C for 3 h. Macrophages were cocultured with DCs (1 x 106) for 2–3 h, then cells were harvested and DCs were labeled with PE-conjugated anti-CD11c mAb (BD Pharmingen).

TUNEL staining for detection of apoptosis

AMs and PMs were seeded on 12-mm circular coverslips (Assistent) in 24-well plates. After 3–5 h incubation, nonadherent cells were removed. Live or heat-killed Histoplasma yeast cells (at 1:2 cell:yeast ratio for PMs, 1:5 for AMs) or 25 µg of protein/ml lysate were added, and the cells were cultured for 2 h at 37°C. After free yeasts were removed, the adherent cells were cultured for another 24 h. Macrophage apoptosis was determined by TUNEL staining (In Situ Cell Death Detection Kit; Roche). The fluorescence signal was converted to brown color by use of converter-peroxidase and diaminobenzidine substrate (Roche).

Statistics

Student’s t test was used to compare the difference between groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD8 T cells are critical in host defense against histoplasmosis in the absence of CD4 T cells

To address whether CD8 T cells are protective against histoplasmosis, IIKO mice were infected with a sublethal dose of Histoplasma yeast cells. Results showed that the IIKO mice were able to contain the fungal burden at the same level as that at wk 1 through wk 16 (Fig. 1A). Furthermore, IIKO mice were depleted of CD8 T cells and infected with the same dose of Histoplasma yeast cells as was given to IIKO mice that were not depleted of CD8 T cells. Results show that depleting CD8 T cells not only significantly increased the fungal burden (p < 0.05; Fig. 1B) but also resulted in 100% mortality (Fig. 1C). Together, these results support the notion that CD8 T cells alone, although not able to clear the infection, are functional in limiting fungal replication and are protective in the absence of CD4 T cells.



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  		FIGURE 1. CD8 T cells are critical in controlling Histoplasma infection in the absence of CD4 T cells. A, Fungal counts in the spleen. B, Fungal counts in IIKO mice depleted of CD8 T cells. IIKO mice were treated with anti-CD8 mAb twice weekly from day 0 (*, p < 0.05, n = 3 in each group). C, Survival of IIKO mice with or without CD8 T cell depletion. IIKO mice were given the same dose of Histoplasma yeast cells as in A. n = 10 in each group.

 
CD8 T cell-mediated protection is perforin dependent as well as perforin independent

CD8 T cells could be contributing to holding Histoplasma in check by production of IFN-{gamma}, which would activate macrophages to limit its intracellular growth, and/or via a perforin/granzyme B-mediated cytotoxic effect on the infected macrophages. Comparing perforin-deficient (PKO) mice with those with MHC class I deficiency ({beta}2m–/–) in clearing the fungus, we found that both {beta}2m–/– and PKO mice had greater fungal burden than did WT controls (p < 0.05) (Fig. 2A). These results not only confirmed that CD8 T cells are involved in limiting fungal replication, but they also showed that perforin was important in controlling the fungus. However, fungal counts in {beta}2m–/– mice were higher than in PKO mice at day 21 (p < 0.05) of infection, suggesting that a CD8 T cell-mediated perforin-independent mechanism was also operative in controlling the fungus.



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  		FIGURE 2. CD8 T cells limit Histoplasma replication by perforin-dependent as well as -independent mechanisms. A, Perforin or {beta}2m deficiency impairs fungal clearance (*, p < 0.05, n = 3–5). B, Perforin-dependent mechanism contributes to fungal clearance in the absence of CD4 T cells. WT and PKO mice (three per group) were treated with anti-CD4 mAb twice weekly from day 0 (*, p < 0.05). Experiment was repeated two times.

 
The PKO mice were then treated with anti-CD4 Ab. The results show that CD4 T cell depletion increased fungal burden in both PKO mice and WT mice as it has been reported (p < 0.05; Fig. 2B) (4, 8). More importantly, PKO mice depleted of CD4 T cells had greater fungal counts than did WT mice with the same treatment, indicating that perforin-dependent cytotoxic activity was critical in restricting the growth of Histoplasma in mice without CD4 T cells (p < 0.05; Fig. 2B).

CD8 T cells from Histoplasma-infected mice have cytotoxic function and produce IFN-{gamma}

Furthermore, Fig. 3A shows that CD8 T cells from Histoplasma-infected mice expressed granzyme B, whereas CD8 T cells from normal uninfected mice did not. Importantly, CD8 T cells from infected WT mice at an E:T cell ratio of 160:1 killed 25% of macrophage IC-21 targets containing Histoplasma yeasts, whereas CD8 T cells from infected PKO mice did not exhibit cytotoxity (Fig. 3B). Additionally, CD8 T cells, similarly to CD4 T cells, produced IFN-{gamma} (Fig. 4), although the CD8 T cell response was less than that of CD4 T cells. Taken together, these data with those in Fig. 2A demonstrate that activated CD8 T cells have cytotoxic function and produce IFN-{gamma}.



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  		FIGURE 3. CD8 T cells in Histoplasma-infected mice are cytotoxic. A, Granzyme B expression. Spleen cells from Histoplasma-infected WT mice were stained with mouse anti-human granzyme B (grB) mAb or isotype control Ab. The numbers indicate the percentage of CD8 T cells expressing granzyme B (CD8+grB+/CD8+). The data shown are from one representative mouse. Three mice were used in each experiment and the experiment was repeated three times. B, CD8 T cells are cytotoxic against Histoplasma-containing IC-21 macrophages. CD8+ T cells were isolated from uninfected or infected WT (upper panel) or PKO (lower panel) mice. IC-21 macrophages with or without internalized heat-killed Histoplasma yeasts were used as targets. The data shown are representative of five separate experiments.

 


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  		FIGURE 4. Histoplasma infection induces CD8 T cell IFN-{gamma} production. Spleen cells were harvested from infected or uninfected WT mice and cultured in medium alone or medium containing live Histoplasma yeast cells for 24 h. The splenocytes were stained for intracytoplasmic IFN-{gamma}. The numbers at the upper right corner are the percentages of CD4 or CD8 T cells that are producing IFN-{gamma} (CD4+IFN-{gamma}+/CD4+ or CD8+IFN-{gamma}+/CD8+). The data shown are from one of eight independent experiments.

 
The next question was how the CD8 T cells are activated. Macrophages are the primary residence of Histoplasma yeasts and, like DCs, are by definition professional APCs. Therefore, it is important to evaluate how they are involved in presenting Histoplasma Ags to stimulate T cells.

Macrophages are not efficient in presenting Histoplasma Ags to stimulate sensitized T cells

Equal numbers (1 x 104/well) of macrophages and DCs were pulsed with the same dose of Histoplasma Ags in the form of lysate, heat-killed, or live yeasts, and their efficiency in stimulating T cell response was evaluated. Fig. 5A shows that at the same APC-to-T-cell ratio, T cell proliferative response was significantly less in cultures stimulated by macrophages than in those stimulated by DCs, showing that macrophages were poor APCs for endocytic Histoplasma Ags, whereas DCs were superior in presenting Histoplasma Ag to T cells.



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  		FIGURE 5. DCs are potent APCs in stimulating sensitized CD8 T cells to express functions. A, Bone marrow-derived DCs or PMs (1 x 104/well) pulsed with live yeasts (Live) or heat-killed yeasts (HK) or yeast cell lysate (Lysate) were cocultured with nylon-wool-enriched splenic T cells (1 x 105/well) for 72 h. Data presented are pooled from three to five experiments (*, p < 0.05, compared with macrophage cultured in the same condition). B–D, MACS-purified CD4 or CD8 T cells from infected mice were cocultured with DCs that were pulsed with different forms of Histoplasma in medium containing IL-2. B, CD4 and CD8 T cell proliferation. C, CD8 T cell IFN-{gamma} production. (**, p < 0.01, compared with those stimulated by live yeasts). D, Granzyme B expression. CD8 T cells were stained by anti-granzyme B mAb (grB) or PE-conjugated mouse IgG1 (isotype control). The numbers at the upper right corner indicate the percentage of CD8 T cells expressing granzyme B. The data shown are from one representative mouse. The experiment was repeated three times with three mice per group in each experiment.

 
DCs stimulate CD8 T cell proliferation, IFN-{gamma} production, and granzyme B expression

We further analyzed T cell subsets responding to stimulation by DCs ingesting different forms of Ags. T cell proliferative responses showed that CD8 as well as CD4 T cells responded to DC stimulation (Fig. 5B). Furthermore, Ag-pulsed DCs stimulated CD8 T cells to produce IFN-{gamma} (Fig. 5C) and to express granzyme B (Fig. 5D). It is worth noting that Fig. 5, A–D, also demonstrated a hierarchy of efficiency in CD8 T cell stimulation by DCs. DCs ingesting live Histoplasma yeasts were significantly more efficient than were DCs pulsed with heat-killed yeasts or DCs pulsed with lysate in stimulating CD8 T cell proliferation (Fig. 5B), IFN-{gamma} production (Fig. 5C), and granzyme B expression (Fig. 5D). However, there was no such difference between DCs pulsed with live yeasts and those pulsed with killed yeasts and lysate in stimulating proliferation of CD4 T cells (Fig. 5B). These data showed that DCs ingesting Histoplasma Ags, especially live yeasts, are capable of inducing sensitized CD8 T cell proliferative response as well as effector functions.

Engulfing live Histoplasma yeasts induces macrophage and DC apoptosis

We next determined whether the fungus causes primary macrophage apoptosis. PMs were allowed to ingest live, heat-killed yeasts or lysate, and DNA fragments were quantified. The results show that the level of DNA fragmentation in macrophages ingesting live yeasts was 7-fold higher than those in medium control (Fig. 6A). In contrast, the level of apoptosis induced by killed yeasts or lysate was comparable with medium control. These data demonstrated that live, but not killed, yeasts or lysate induced macrophage apoptosis. Similarly, DCs engulfing live yeasts also underwent apoptosis (Fig. 6B), although macrophages were more susceptible than DCs to live Histoplasma-induced apoptosis.



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  		FIGURE 6. Live Histoplasma yeasts induce macrophage and DC apoptosis. PMs (A) or DCs (B) were cultured in medium alone (Medium) or in medium containing heat-killed yeasts (HK) or live yeasts (Live) or yeast cell lysate (Lysate) for 6 h. Cell apoptosis was quantified by cell death detection ELISA. The enhancement factor is the absorbance at 405 nm of experimental groups divided by that of the medium control (**, p < 0.01, compared with Medium culture).

 
DCs take up apoptotic macrophages

We then determined whether DCs would take up Histoplasma-infected apoptotic macrophages. Dot plot analyses (Fig. 7A) show that ~20% of the DCs cocultured with macrophages that had taken up live yeasts internalized labeled macrophage materials. In contrast, <10% of DCs in the cultures with macrophages exposed to killed yeasts and <7% of DCs in cultures with macrophages pulsed with lysate had internalized CFSE-labeled material. These data suggest that DCs take up cellular materials from apoptotic macrophages whose death was induced by live yeasts.



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  		FIGURE 7. DCs internalize Ag-pulsed apoptotic macrophages. A, DCs were cultured with CFSE-labeled PMs that were incubated in medium alone (Medium) or medium containing heat-killed yeasts (HK) or live yeasts (Live) or yeast cell lysate (Lysate). B, DCs were cultured with LPS- and ATP-treated PMs (apoptotic PM). Cytochalasin D (CCD) or paraformaldehyde (PFA)-treated DCs (C and D) were cultured with untreated Ag-pulsed PMs. DCs were identified by PE-conjugated anti-CD11c mAb. The numbers at the upper right corner indicate the percentage of CD11c+CFSE+/CD11c+. Data shown are representative result of five independent experiments.

 
To prove that DCs more readily internalize cellular materials from apoptotic macrophages, we used LPS and ATP to induce macrophage apoptosis (18, 26, 27). Results show that the percentage of DCs that took up macrophage materials from the LPS- and ATP-treated macrophages cultured in medium and from those ingesting killed yeasts and lysate but not live yeasts increased compared with their untreated counterparts (Fig. 7, A and B).

Moreover, treatment with cytochalasin D or paraformaldehyde reduced DC uptake of macrophage materials, regardless of the form of Ag with which the macrophages were pulsed (Fig. 7, C and D). It demonstrates that through active phagocytosis DCs take up macrophage material.

DCs acquire Histoplasma antigenic materials from apoptotic macrophages to induce specific T cell proliferation

We further investigated whether DCs that take up cellular materials from apoptotic macrophages also acquire fungal Ags. The bar graphs in Fig. 8A show that DCs that were cocultured with LPS- and ATP-treated macrophages were much more efficient in stimulating sensitized T cell response than were those cocultured with untreated macrophages (p < 0.01), regardless of whether the macrophages were pulsed with killed yeasts or lysate. Furthermore, when DCs were treated with cytochalasin D or paraformaldehyde to inhibit phagocytosis, the DCs did not stimulate T cell proliferation (Fig. 8A), demonstrating that the fungal antigenic materials were phagocytosed and processed by DCs, but were not directly loaded onto preformed MHC molecules on the cell surface. Moreover, by use of an irrelevant Ag, we confirmed that DC-stimulated T cell response was Ag specific (Fig. 8B).



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  		FIGURE 8. Ingestion of Histoplasma antigenic materials from apoptotic macrophages is required for DCs to stimulate Histoplasma-specific T cell proliferation. PMs were allowed to uptake heat-killed yeasts (HK) or yeast cell lysate (Lysate) of Histoplasma (A and B) or heat-killed Candida albicans yeast cells (B) and were treated with LPS and ATP (apoptotic PM). DCs and splenic T cells from infected mice were then added to the macrophage cultures. The experiment was repeated six times. Data shown here are from one representative experiment (**, p < 0.01, compared with DC+(PM-Hc)).

 
DCs acquiring Histoplasma Ags from apoptotic macrophages efficiently activate CD8 T cells

Fig. 9A shows that DCs acquiring fungal Ags from apoptotic macrophages were far more efficient than were those from nonapoptotic macrophages or directly from ingested Ag in inducing CD8 T cell IFN-{gamma} production and proliferative response, regardless of whether the antigenic source was killed yeasts or lysate. However, DCs that acquired fungal Ags from apoptotic macrophages were not any more efficient than were those from nonapoptotic macrophage or directly from ingested Ag in stimulating CD4 T cell responses (Fig. 9B). Fig. 9C shows that {beta}2m–/– DCs cocultured with Ag-pulsed apoptotic WT macrophages stimulated CD4 T but not CD8 T cell response. The result confirmed that fungal Ags acquired along with apoptotic macrophages that stimulate CD8 T cell response were processed and presented through MHC class I pathway. Fig. 9D shows that lyticase treatment did not change the ability of DCs to stimulate CD8 T cell response, excluding the possibility that the DCs acquired fungal Ags from uningested, cell-attached yeasts (28).



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  		FIGURE 9. Engulfing apoptotoic macrophages increases the capacity of DCs to induce CD8 T cell response in an MHC class I-dependent manner. PMs pulsed with heat-killed (HK) or yeast cell lysate (Lysate) were treated with (apoptotic PM) or without (PMs) LPS and ATP. DCs from WT mice (A and B) as well as MACS-purified CD8 (A) or CD4 (B) T cells from infected mice were added to the macrophage cultures (*, p < 0.05; **, p < 0.01; compared with DC+(apoptotic PM-Hc)). C, DCs obtained from {beta}2m–/– mice were cocultured with yeast cell lysate-pulsed PMs and MACS-purified CD4 and CD8 T cells as described in A and B. Data shown are representative result of four independent experiments. D, PMs pulsed with heat-killed yeasts or yeast cell lysate were treated with lyticase (300 U/ml) for 0.5 or 1 h. After overnight incubation, PMs were treated with LPS and ATP to induce apoptosis. DCs from WT mice and MACS-purified sensitized CD8 T cells were added as described. Data shown are representative result of four independent experiments.

 
Taken together, these results suggest that ingesting Ag-pulsed apoptotic macrophages is an efficient pathway for DCs to acquire Histoplasma Ags for CD8 T cell cross-priming, a process that is MHC class I dependent.

AMs undergo apoptosis after ingestion of Histoplasma and serve as Ag donor for DCs to cross-present Ags to CD8 T cells

Because Histoplasma naturally enters the host through the pulmonary route, we examined whether ingestion of Histoplasma induces AM apoptosis. Results in Fig. 10, A–F, show that, like their peritoneal counterparts, the AMs were susceptible to live Histoplasma yeast-induced apoptosis, but not to heat-killed yeasts or yeast lysate (data not shown). Similar to what we observed in PMs, apoptotic AMs could also serve as Ag donor for DCs to cross-present Histoplasma Ags to stimulate sensitized CD8 T cells (Fig. 10G).



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  		FIGURE 10. AMs are susceptible to live Histoplasma yeast-induced apoptosis and serve as Ag donor cells for DCs to stimulate CD8 T cell response. AMs (A–C) and PMs (D–F) were seeded onto the coverslips and cultured with medium alone (A and D) or with medium containing live (B and E) or heat-killed (C and F) Histoplasma yeasts for 24 h. Apoptosis was detected by TUNEL. Magnification, x400. G, AMs pulsed with heat-killed (HK) or yeast cell lysate (Lysate) were treated with (apoptotic AM) or without (AM) LPS and ATP. DCs from WT mice and MACS-purified sensitized CD8 T cells were added to the macrophage cultures. The levels of IFN-{gamma} in the 24-h culture supernatants were quantified by ELISA. Data shown are representative of four independent experiments.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In CD4 T cell-intact mice, the contribution of CD8 T cells to defense against histoplasmosis is minor (8, 9). Thus, the functional role of CD8 T cells in Histoplasma infections has not been thoroughly studied. In the present study, we demonstrate that CD8 T cells alone in the absence of functional CD4 T cells, as in the situation of opportunistic infection in patients with low CD4 T cell counts, provide protection against Histoplasma infection.

It has been reported that perforin–/– mice exhibit increased fungal burden and accelerated mortality after infection (8). However, in vitro CD8 T cell cytotoxicity activity has never been demonstrated, nor has it been clarified whether perforin-dependent anti-Histoplasma activity was attributed to CD8 T cells or NK cells. In this study, we showed specifically that CD8 T cells from Histoplasma-infected mice express granzyme B and exhibit cytotoxic activity against Histoplasma-containing IC-21 macrophage targets. These results provide strong evidence that CD8 T cells in mice infected with Histoplasma have cytotoxic function. Histoplasma yeast cells are facultative intracellular organisms that reside within the macrophages (7, 10, 11). It has been reported that macrophages activated by IFN-{gamma} are armed to inhibit the intracellular growth of the fungus (29, 30). Thus, by producing IFN-{gamma} and exhibiting cytotoxic activity against macrophage targets, the CD8 T cells in Histoplasma-infected mice contribute to restricting intracellular growth and killing of the fungus within the macrophage.

Macrophages are the primary residence of Histoplasma yeasts and the effector cells that clear the fungus. Being the host cells and the effector cells of the fungus, whether macrophages also serve as APCs is an interesting question. Comparing DCs and macrophages in presenting Histoplasma Ags, we found that at the same APC-to-T-cell ratio, DCs are superior to macrophages in stimulating sensitized T cells to proliferate, a result similar to what was observed in an infection with Mycobacterium tuberculosis (31, 32). One possible explanation is that DCs are more efficient than macrophages are in supplying MHC class I and class II molecules and costimulatory signals to induce T cell responses (33). It is also possible that Histoplasma yeast cells enter through different receptors into DCs and macrophages, which results in different outcomes (14, 34). It may also be simply that a greater percentage of macrophages than DCs are killed by the intracellular parasitism (Fig. 6). Thus, higher percentage of DCs than macrophages is viable for Ag presentation to stimulate T cells.

The process of exogenous Ags being processed into the MHC class I pathway and then priming of CD8 T cells is known as "cross-presentation" (15). In the present study, we show that live Histoplasma yeast-derived Ags are presented by DCs to stimulate CD8 T cells. Histoplasma hemolysin has been reported (35). Therefore, it is possible that hemolysin in live Histoplasma yeast, like listeriolysin in Listeria, punctures phagolysosomal membranes, thereby allowing endocytic Histoplasma Ags to be delivered into the cytosol, where they find their way into MHC class I molecules in the DCs. In contrast, the DCs are known to be capable of exporting internalized Ags from the endocytic compartment to the cytosol (36, 37). On this account, Histoplasma Ags in live yeasts may be exported from endocytic compartment to cytosol for conventional MHC class I presentation.

Besides cross-presenting endocytic Histoplasma Ags, DCs can also acquire Histoplasma Ags from ingesting Ag-pulsed apoptotic macrophages to stimulate CD8 T cell responses. It has been noted that DCs but not macrophages are capable of cross-presenting cell-associated Ag derived from apoptotic cells, although both cell types are equally efficient in internalizing apoptotic material (17, 18). Our results showed that macrophages, being the host cells and the effector cells for Histoplasma yeasts in the host, do not present fungal Ags directly, but by dying through apoptosis-dependent pathway become Ag donors to DCs. DCs engulfing infected apoptotic macrophages and cross-presenting microbial Ags to stimulate CD8 T cells has been shown in vitro in infections with viruses and bacteria (16, 17, 18). Our results of this present study add fungus to the list of pathogens that induce macrophage apoptosis, which in turn are taken up by DCs for cross-presentation to CD8 T cells.

The mechanism of cross-presentation is a topic under intense investigation. It is reported that heat-shock proteins (HSPs) chaperoning antigenic peptides are involved not only in endogenous pathway of Ag presentation, but also in cross-presentation. In infection by Histoplasma, it is possible that the stress of ingesting the fungus induces HSPs in macrophages. The HSP-Histoplasma antigenic peptide complex released upon cell death is taken up by DCs. Interestingly, Histoplasma-dominant Ags His62 and His80 both belong to the HSP family (38, 39). Thus, like that in the bacteria, it is also possible that Histoplasma HSPs deliver HSP-bound fungal peptides during phagocytic processing of the fungus, thereby promoting MHC class I presentation of fungal Ags and inducing CD8 T cell response.

Schaible and colleagues (40, 41) have shown that infection-induced cell death is a crucial step in cross-priming of CD8 T cell response in tuberculosis. They showed that intracellular M. tuberculosis releases Ags to the extracellular vesicles of infected apoptotic cells. Apoptotic vesicles serve as a source of mycobacterial Ags for presentation by uninfected bystander APCs. These results, together with our findings, suggest that infection-induced apoptosis and Ag transfer from infected apoptotic cells to uninfected bystander APCs to cross-prime CD8 T cells are important host defense mechanisms against intracellular pathogens.

The importance of CD8 T cells was reported in vaccine-induced immunity against Blastomyces and Histoplasma (42). Mice are resistant to lethal challenge after vaccination by high inocula of live yeasts at s.c. sites, which induces efficient CD8 T cell-mediated immunity. Our study demonstrated that DCs cross-present exogenous Histoplasma through directly ingesting live yeasts and Ag-pulsed apoptotic macrophages to stimulate functional CD8 T cells. Therefore, it is conceivable that in hosts infected by Histoplasma, macrophages and DCs die after ingesting live Histoplasma yeasts (Fig. 6). Bystander DCs, while clearing apoptotic cells, acquire fungal antigenic materials, thereby activating CD8 and CD4 T cells, initiating cell-mediated immune response.

The conclusion of our study on DC cross-presentation of fungal Ag after ingesting Histoplasma Ag-pulsed apoptotic macrophages is mostly based on results from the study of PMs. Further studies showed that AMs, like the PMs, undergo apoptosis when exposed to live Histoplasma yeasts in vitro (Fig. 10). These results suggest that our observation reported herein is relevant in natural infection through the pulmonary route. Thus, we proposed a model to delineate the priming and activation of CD4 and CD8 T cells and their roles in clearing infection in experimental histoplasmosis (Fig. 11). After entering the host, Histoplasma is taken up by macrophages and some by DCs. Although DCs also phagocytose live yeasts, given the relative proportion and the distribution of DCs and macrophages, the chances of macrophages encountering Histoplasma in the first line of defense is far greater than for DCs (33). The yeast cells replicate uninhibitedly in unarmed phagocytes and induce apoptosis. Bystander DCs while clearing apoptotic cells acquire fungal antigenic materials. The fungal Ags that are associated with apoptotic macrophages are processed and presented through both the MHC class I and class II pathways and prime both CD4 and CD8 T cells. Releasing IFN-{gamma} by activated CD4 and CD8 T cells activates macrophages, which in turn inhibit and possibly kill the intracellular fungus. Activated CD8 T cell killing infected macrophage targets also contribute to the anti-fungal defense mechanism. Along the line of CD8 T cell priming, our study demonstrating cross-presentation by DCs of Histoplasma Ag(s) contributes to the understanding of the mechanism of CD8 T cell activation not only in infection by Histoplasma, but also by other nonviral intracellular pathogens.



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  		FIGURE 11. A model of the possible sequence of events leading to cell-mediated immune response and fungal clearance in Histoplasma-infected host. a, In an infected host, the macrophage is the first cell to encounter Histoplasma. b, The yeast cells cause macrophage cell death. c, DCs take up apoptotic macrophages and present fungal Ag (d) to activate CD4 T cells and cross-prime CD8 T cells. e, DCs can also encounter Histoplasma. f, Some DCs that take up live yeasts are killed, which are engulfed by other DCs (g). h, The DCs that take up live yeasts or apoptotic DCs present fungal Ag to activate CD4 T cells and cross-prime CD8 T cells. i, Activated CD4 T cells as well as CD8 T cells produce IFN-{gamma} to arm macrophages for fungicidal and fungistatic activities (j). k, Activated CD8 T cells are cytotoxic against Histoplasma-infected macrophage targets. The bold arrows indicate events that are demonstrated by experimental results presented in this study. Thin arrows indicate events that have been documented in the literature.

 

   Acknowledgments
 
We acknowledge Drs. D. H. Howard and J. W. Murphy for critical reading of the manuscript and Dr. R. Ahmed for helpful discussions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by funds from the National Science Council (Grants NSC 89-2320-B-002-195, NSC 90-2320-B-002-139, NSC 91-2320-B-002-096, and NSC 92-2320-B-002-178). Back

2 Address correspondence and reprint requests to Dr. Betty A. Wu-Hsieh, Graduate Institute of Immunology, National Taiwan University College of Medicine, No. 1 Jen-Ai Road, Section 1, Taipei 10051, Taiwan, Republic of China. E-mail address: wuhsiehb{at}ha.mc.ntu.edu.tw Back

3 Abbreviations used in this paper: Histoplasma, Histoplasma capsulatum; {beta}2m, {beta}2-microglobulin; DC, dendritic cell; WT, wild type; IIKO, MHC class II deficient; PM, peritoneal macrophage; AM, alveolar macrophage; PKO, perforin deficient; HSP, heat-shock protein. Back

Received for publication May 24, 2004. Accepted for publication February 28, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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