Immunization with Apoptotic Phagocytes Containing ... · Histoplasma-infected host is associated...

INFECTION AND IMMUNITY, Nov. 2011, p. 4493–4502 Vol. 79, No. 110019-9567/11/$12.00 doi:10.1128/IAI.05350-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Immunization with Apoptotic Phagocytes Containing Histoplasma capsulatumActivates Functional CD8� T Cells To Protect against Histoplasmosis�

Shih-Hung Hsieh,1 Jr-Shiuan Lin,1† Juin-Hua Huang,1 Shang-Yang Wu,1 Ching-Liang Chu,1,2,3

John T. Kung,4 and Betty A. Wu-Hsieh1*Graduate Institute of Immunology, National Taiwan University College of Medicine, Taipei, Taiwan1; Immunology Research Center,

National Health and Research Institutes, Miaoli, Taiwan2; Yeastern Biotech Co., Taipei County, Taiwan3; andInstitute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan4

Received 10 May 2011/Returned for modification 26 May 2011/Accepted 24 August 2011

We have previously revealed the protective role of CD8� T cells in host defense against Histoplasmacapsulatum in animals with CD4� T cell deficiency and demonstrated that sensitized CD8� T cells arerestimulated in vitro by dendritic cells that have ingested apoptotic macrophage-associated Histoplasma anti-gen. Here we show that immunization with apoptotic phagocytes containing heat-killed Histoplasma efficientlyactivated functional CD8� T cells whose contribution was equal to that of CD4� T cells in protection againstHistoplasma challenge. Inhibition of macrophage apoptosis due to inducible nitric oxide synthase (iNOS)deficiency or by caspase inhibitor treatment dampened the CD8� T cell but not the CD4� T cell response topulmonary Histoplasma infection. In mice subcutaneously immunized with viable Histoplasma yeasts whoseCD8� T cells are protective against Histoplasma challenge, there was heavy granulocyte and macrophageinfiltration and the infiltrating cells became apoptotic. In mice subcutaneously immunized with carboxyfluo-rescein diacetate succinimidyl ester (CFSE)-labeled apoptotic macrophages containing heat-killed Histo-plasma, the CFSE-labeled macrophage material was found to localize within dendritic cells in the draininglymph node. Moreover, depleting dendritic cells in immunized CD11c-DTR mice significantly reduced CD8�

T cell activation. Taken together, our results revealed that phagocyte apoptosis in the Histoplasma-infected hostis associated with CD8� T cell activation and that immunization with apoptotic phagocytes containingheat-killed Histoplasma efficiently evokes a protective CD8� T cell response. These results suggest thatemploying apoptotic phagocytes as antigen donor cells is a viable approach for the development of efficaciousvaccines to elicit strong CD8� T cell as well as CD4� T cell responses to Histoplasma infection.

Histoplasma capsulatum is an opportunistic fungal pathogenthat threatens the life of immune-compromised individuals,especially those infected with HIV (8). Upon entry into therespiratory system, the fungus transforms into yeast cells and isphagocytosed by the macrophage. The yeast cells of Histo-plasma reside and replicate primarily in the phagosome ofmacrophages (16). Gamma interferon (IFN-�) produced byCD4� and CD8� T cells can activate macrophages to producereactive oxygen species and nitric oxide for fungal clearance (3,14, 16, 31). A previous depletion study established the vital roleof CD4� T cells in clearing Histoplasma in intranasal infectionof wild-type mice (3). Depletion of CD8� T cells or a defi-ciency in major histocompatibility complex class I (MHC-I), onthe other hand, has little effect on fungal clearance (3, 5).However, the protective role of CD8� T cells is prominent ininfection of mice with MHC-II deficiency (14), demonstratingthat CD8� T cells can be protective against histoplasmosis inthe absence of functional CD4� T cells. In HIV-infected indi-viduals whose CD4� T cell responses gradually decline, theCD8� T cells become the major effectors defending against

opportunistic infections. Thus, developing vaccines that aim toinduce functional CD8� T cell immune responses would bevaluable.

Vaccines designed for nonviral pathogens often focus oneliciting CD4� T cell and B cell immune responses (15, 24).The CD8� T cell response receives relatively little attention.Recently, increasing evidence demonstrated that exogenous ornonviral antigens can be presented on MHC-I molecules toprime CD8� T cell responses, processes referred to as “cross-presentation” and “cross-priming” (12). Exogenous antigenscoupled with heat shock protein (25), exosomes (29), immunecomplexes (20), and latex beads (22) all can be cross-presentedto prime CD8� T cells. Immunizing mice with antigen-contain-ing dead cells or with dead cell-pulsed dendritic cells is awell-recognized strategy in the development of cancer vaccinesto elicit strong CD8� T cell responses (6, 9). In their studies ofinfectious diseases, Albert et al. were the first to report thatapoptotic monocytes deliver influenza antigens to dendriticcells and trigger CD8� T cell immune responses (1). Nonviralintracellular pathogens such as Salmonella and Mycobacteriuminduce macrophage apoptosis, and the apoptotic cell blebsshuttle the bacterial antigens to uninfected bystander antigen-presenting cells to cross-prime CD8� T cells (21, 33). Wepreviously showed that sensitized CD8� T cells were restimu-lated in vitro by dendritic cells that acquired Histoplasma an-tigens through phagocytosis of heat-killed Histoplasma-con-taining apoptotic macrophages (14). In addition, Winau et al.showed that subcutaneously immunizing mice with apoptotic

* Corresponding author. Mailing address: Graduate Institute of Im-munology, National Taiwan University College of Medicine, No. 1,Ren-Ai Road, Section 1, Taipei, Taiwan. Phone: 886-2-321-7510. Fax:886-2-2321-7921. E-mail: [email protected].

† Present address: Trudeau Institute, 154 Algonquin Avenue, Sara-nac Lake, NY 12983.

� Published ahead of print on 12 September 2011.

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vesicles prepared from Mycobacterium bovis BCG-ovalbumin-infected macrophages not only induces adoptively transferredOT-1 CD8� T cell division and IFN-� production in vivo butalso protects mice from tuberculosis (28). The results of thesestudies together raise the possibility that a similar strategyaiming to cross-prime strong CD8� T cell responses could beapplied to develop fungal vaccines.

Here we show that immunization with apoptotic phagocytes(macrophages or neutrophils) containing heat-killed Histo-plasma efficiently activated functional CD8� and CD4� T cells.Inhibiting apoptosis during the early phase of infection weak-ened the CD8� T cell but not the CD4� T cell response topulmonary Histoplasma infection. We also demonstrate that, inmice subcutaneously immunized with viable Histoplasma yeastsin which CD8� T cells are protective, there was heavy granu-locyte and macrophage infiltration and the infiltrating cellsbecame apoptotic. While the carboxyfluorescein diacetate suc-cinimidyl ester (CFSE)-labeled macrophage material localizedwithin dendritic cells in the draining lymph node after immu-nization with CFSE-labeled apoptotic peritoneal macrophages(pMac) containing heat-killed H. capsulatum [pMac (HK Hc)],depleting dendritic cells in immunized CD11c-DTR mice sig-nificantly reduced CD8� T cell activation. Our results revealedthat phagocyte apoptosis during the early growth phase in aHistoplasma-infected host is associated with CD8� T cell acti-vation and that immunization with apoptotic pMac (HK Hc) orwith apoptotic peritoneal neutrophils (pNeu) containing heat-killed H. capsulatum [pNeu (HK Hc)] efficiently evokes a pro-tective CD8� T cell response. Thus, such an approach is aviable option to exploit apoptotic phagocytes as antigen donorcells in the development of vaccines that elicit strong protec-tive CD8� T cell as well as CD4� T cell responses to Histo-plasma.

MATERIALS AND METHODS

Mice. Wild-type, inducible nitric oxide synthase-deficient (iNOS�/�), and �2

microglobulin-deficient (�2m�/�) mice were originally obtained from the Jack-son Laboratory (Bar Harbor, MA) and bred at the Laboratory Animal Center,National Taiwan University College of Medicine. CD11c-DTR transgenic mice(10) were provided by A. DeFranco (University of California at San Francisco,San Francisco, CA). All mice used were of the C57BL/6 background and werehoused in sterilized cages fitted with filter cage tops and fed with sterilized foodand water. Mice at 8 to 10 weeks of age were used in all the experiments in thisstudy. The present study was carried out in strict accordance with the recom-mendations in the Guidebook for the Care and Use of Laboratory Animals (4a).The protocol was approved by the Committee on the Ethics of Animal Experi-ments of the National Taiwan University College of Medicine (permit 20040318).

Abs and medium. Allophycocyanin (APC)-rat-anti-mouse CD8 (clone 53-6.7),fluorescein isothiocyanate (FITC)-rat-anti-mouse CD4 (clone RM4-5), phyco-erythrin (PE)-hamster-anti-mouse CD3 (clone 145-2C11), FITC-rat-anti-mouseB220 (clone RA3-6B2), PE-rat-anti-mouse F4/80 (clone BM8), PE-rat-anti-mouse Gr-1 (clone RB6-8C5), and PE-rat IgG1 isotype (for immunofluorescencestaining) antibodies (Abs) were purchased from eBioscience (San Diego, CA).FITC- and PE-hamster-anti-mouse CD11c (clone HL3) were from BD Pharmin-gen (San Diego, CA). Purified hamster-anti-CD3 (clone 145-2C11), purifiedhamster-anti-mouse CD28 (clone 37.51), and PE-rat-anti-mouse IFN-� (cloneXMG1.2) antibodies were from BioLegend (San Diego, CA). PE-mouse-anti-human granzyme B (clone MHGB04) antibody was from Caltag Laboratories(Burlingame, CA). RPMI 1640 medium (Gibco BRL, Gaithersburg, MD) wassupplemented with 10% heat-inactivated fetal calf serum (Biological Industries,Kibbutz Beit Haemek, Israel), 2 mM L-glutamine, 0.1 mM nonessential aminoacid, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 �g/ml streptomycin, 5 �10�5 M 2-mercaptoethanol, and 25 mM HEPES buffer. The supplementedmedium was used as RPMI 1640 complete medium. All of the supplementsexcept serum were obtained from Gibco-BRL.

Fungus and infection. H. capsulatum strain 505 yeast cells were cultured at37°C on brain heart infusion agar (Difco, Sparks, MD) supplemented with 1mg/ml cysteine and 20 mg/ml glucose. Fresh yeast cell suspensions were preparedin RPMI 1640 medium for infection. For intratracheal infection, a small incisionwas made on the skin of mice under anesthesia to expose the trachea. Histo-plasma yeasts (200,000 [2 � 105]) in 40 �l of RPMI 1640 medium were injectedinto the trachea through a 29-gauge insulin syringe. The skin was sutured, andthe mice were kept under a warming lamp until they reawakened. For subcuta-neous infection, 2,000,000 (2 � 106) Histoplasma yeasts were suspended in 100 �lof RPMI 1640 medium. Mice were anesthetized, and each side of the base of thetail was inoculated with 50 �l of Histoplasma suspension through a 29-gaugeinsulin syringe. Heat-killed Histoplasma yeast cells were prepared by heatingfresh prepared yeasts at 65°C for 120 min.

Intracellular IFN-� and granzyme B staining. Single-cell suspensions of theinguinal or mediastinal lymph nodes were prepared. One million lymph nodecells in RPMI 1640 complete medium were cultured with 1 � 105 heat-killedHistoplasma yeast cells and 1 � 106 irradiated (1,000 rads) spleen cells fromnaive mice for 24 h. At 6 h before harvest, monensin (Sigma-Aldrich, St. Louis,MO) (2 �M) was added to the culture to block intracellular protein transport.Cells were collected and stained to reveal surface CD4 and CD8 and intracellularIFN-� as previously described (13). For granzyme B staining, the cells were firstincubated with 2.4G2 Ab (BioLegend) directed against mouse Fc� II/III recep-tors before staining with PE-anti-granzyme B monoclonal antibodies (Mabs) wasperformed as previously described (14). PE-mouse IgG1 was used as an isotypecontrol.

Inguinal lymph node cells harvested from CD11c-DTR transgenic mice werestimulated with plate-bound anti-mouse CD3 (0.5 �g/ml) and anti-mouse CD28(0.5 �g/ml) MAbs for 5 h in the presence of monensin. Cells were collected andstained to reveal surface CD4 and CD8 and intracellular IFN-� as describedabove.

Single-cell preparations from lung and subcutaneous injection sites. Thesubcutaneous tissues were collected at day 5 after subcutaneous inoculation ofHistoplasma. A portion of the tissue was snap-frozen in liquid nitrogen. Anotherportion was cut into small pieces in 500 �l of Dulbecco’s phosphate-bufferedsaline (PBS) (Biological Industries, Kibbutz Beit Haemek, Israel) with scissorsand treated with 5 mg/ml of type I collagenase (Sigma-Aldrich) at 37°C for 1 to1.5 h. The debris was removed by filtration through nylon mesh. Lungs wereharvested at day 4 after intratracheal inoculation and ground between twofrosted glass slides. A single-cell suspension was obtained by passing the suspen-sion through cotton wadding packed in a Pasture pipette.

Immunofluorescence staining for confocal microscopy and flow cytometricanalysis. Subcutaneous tissues from the area at the base of the tail where micewere immunized were cryopreserved. The sections were fixed in acetone for10 min and dried in air. The slides were blocked for 20 min with Dulbecco’sPBS (Biological Industries) containing 5% inactivated fetal calf serum andstained with PE-anti-F4/80, PE-anti-Gr-1, PE-anti-CD3, FITC-anti-CD11c,or FITC-anti-B220 MAb at 4°C in a humidified chamber in the dark over-night. After washing was performed, Hoechst 33258 reagent was added andallowed to stain for 3 to 5 min. The sections were viewed under a confocalmicroscope (Leica, Germany). The single cells isolated from subcutaneoustissue prepared as described above were stained with FITC-anti-CD11c, PE-anti-F4/80, or PE-anti-Gr-1 at 4°C for 30 min. Cells were acquired with aFACSCanto system and analyzed with FACSDiva software (Becton Dickin-son, San Diego, CA).

TUNEL staining (in situ cell death detection). The cryosections were fixed with4% paraformaldehyde (Merck, Darmstadt, Germany) for 20 min. The sectionswere then treated with 0.1% Triton X-100 (Sigma-Aldrich) at 4°C for 5 min andincubated with a terminal deoxynucleotidyltransferase-mediated dUTP-biotinnick end labeling (TUNEL) reaction mixture containing FITC-dNTP (Roche,Basel, Switzerland) at 37°C in the dark for 1 h. After being washed with PBS, theslides were stained with PE-anti-F4/80 or PE-anti-Gr-1. The sections were ex-amined under a confocal microscope.

For flow cytometric analysis, cells isolated from subcutaneous tissues and lungswere first stained with PE-anti-F4/80 or PE-anti-Gr-1 MAbs at 4°C for 30 minfollowed by the addition of the TUNEL reaction mixture containing FITC-dNTPand incubated at 37°C in the dark for 1 h. The cells were acquired with aFACSCanto system and analyzed with FACSDiva software.

Preparation of and immunization with apoptotic phagocytes containing heat-killed Histoplasma. Wild-type or �2m�/� mice were injected intraperitoneallywith 4% thioglycolate (Difco). To obtain macrophages, peritoneal cells wereharvested 4 days later. The cells (2 � 106) were allowed to adhere in RPMI 1640medium overnight. Nonadherent cells were removed by washing with prewarmedHanks’ balanced salt solution (HBSS). Twenty million heat-killed Histoplasma

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yeast cells were added to the monolayer. After 2 h of incubation at 37°C, themonolayer was washed with prewarmed HBSS to remove free Histoplasma yeastcells. The culture was left at 37°C overnight. Apoptosis was induced by treatmentwith lipopolysaccharide (LPS) (Sigma-Aldrich) (1 �g/ml) at 37°C for 4 h, fol-lowed by addition of 5 mM ATP (Sigma-Aldrich) for another 45 min. The cellsbecame nonadherent. They were harvested by gentle pipetting and washed withRPMI 1640 medium two times. One hundred percent of the cells were apoptoticas confirmed by a TUNEL assay. Apoptotic macrophages were suspended in 50�l of RPMI 1640 medium and injected subcutaneously into mice on two sides ofthe base of the tail (1 � 106 cells/injection) by two injections administered 2weeks apart.

To obtain neutrophils, peritoneal cells were harvested 4 h after thioglycolateinjection. Seventy-five percent of the cells were Gr1�. Ten million of the heat-killed Histoplasma yeasts were added to culture containing 1 � 106 neutrophils(multiplicity of infection [MOI � 10]). After overnight incubation, the cells wereexposed to UV irradiation at 350 mJ/cm2 to induce apoptotic cell death (7).Ninety percent of the cells were apoptotic after the treatment as confirmed byannexin V (BD Pharmingen) staining and flow cytometry analysis. Apoptoticcells were suspended in 50 �l of RPMI 1640 medium and injected subcutane-ously into mice on two sides of the base of the tail (1 � 106 cells/injection) by twoinjections administered 2 weeks apart.

To prepare CFSE-labeled apoptotic macrophages, thioglycolate-elicited peri-toneal macrophages were allowed to take up heat-killed Histoplasma as de-scribed previously. Before induction of apoptosis, CFSE (Invitrogen, Carlsbad,CA) at a final concentration of 5 �M was added to the macrophage culture andlet stand for 5 min. The monolayers were washed free of CFSE and treated withLPS and ATP to induce apoptosis. The detached cells were collected, washed,and injected subcutaneously into mice.

Injection of apoptosis inhibitor. Boc-D-FMK (pan-caspase inhibitor; Sigma-Aldrich) (0.2 �mol) or Z-FA-FMK (control peptide; Sigma-Aldrich) (0.2 �mol)was dissolved in 10 �l of dimethyl sulfoxide (DMSO) for storage and diluted with190 �l of sterilized Dulbecco’s PBS immediately before injection. Mice wereinjected intraperitoneally with Boc-D-FMK or Z-FA-FMK at the time of intra-tracheal infection with Histoplasma and daily afterwards until the day of theexperiment (2).

In vivo cell depletion. To deplete CD11c� dendritic cells, CD11c-DTR trans-genic mice and wild-type control mice were injected with 0.1 �g of diphtheriatoxin–200 �l PBS (Sigma-Aldrich) intraperitoneally 1 day befor and 2 days afterimmunization with apoptotic pMac (HK Hc). Diphtheria toxin treatment de-pleted �86% of the CD11c� in the spleen as determined by flow cytometry at 3to 4 days after the last dose of diphtheria toxin. To deplete CD4� and/or CD8�

T cells, mice were injected intraperitoneally with concentrated supernatants fromhybridoma GK1.5 (anti-CD4) or 2.43 (anti-CD8) or both at 1 day before andtwice weekly after challenge with Histoplasma. The protein concentrations ofconcentrated hybridoma supernatants were 4.0 mg of GK1.5 and/or 2.5 mg of2.43 per injection. The treatment depleted �95% of CD4� and �95% of CD8�

T cells in the spleen as determined by flow cytometry at 3 to 4 days after the lastdose of the antibody treatment.

Determination of fungal burden. Mice immunized subcutaneously with liveHistoplasma, apoptotic pMac (HK Hc), or RPMI 1640 medium were challengedintravenously with 2.5 � 104 live Histoplasma yeast cells at day 5 after the secondimmunization. Spleens were harvested and homogenized at day 10 after chal-lenge; the homogenates were plated on cysteine-blood-glucose agar and incu-bated at 30°C. Mycelial colonies were counted at 10 to 14 days after incubation.Fungal burdens in the spleens were expressed as the log10 value of CFU perspleen as previously described (14).

Lungs from mice treated with caspase inhibitor or control peptide or leftuntreated were collected at day 10 after intratracheal inoculation of 2 � 105

Histoplasma yeast cells. The lungs were homogenized, and the homogenates wereplated on cysteine-blood-glucose agar and incubated for 10 to 14 days. The CFUlevel in the control mice was assigned a value of 100%, and the CFU levels in theexperimental groups were compared to the control level. The data are presentedas percent CFU change.

H and E staining. The cryosections were fixed with 4% paraformaldehyde andstained with 0.1% hematoxylin and 2% eosin for 5 min.

Statistics. The comparisons between multiple groups were analyzed by one-way analysis of variance (ANOVA) followed by a post hoc Tukey test, performedusing Prism 4.0 software (GraphPad Software). The comparisons between twogroups were analyzed by Student’s t test. The level of statistical significance wasdefined as P 0.05.

RESULTS

Apoptotic macrophages as antigen donors are efficient indelivering Histoplasma antigen to induce functional CD8� andCD4� T cell activation in vivo. In previous in vitro studies, weshowed that apoptotic pMac (HK Hc) can deliver fungal an-tigens contained within them to dendritic cells, which in turncross-present Histoplasma antigens to activate sensitized CD8�

T cells (14). Here we extended the study and immunized micewith apoptotic macrophages that had taken up Histoplasmayeasts. IFN-� production as determined by intracellular cyto-kine staining was used as an indicator of functional activationof both CD8� and CD4� T cells (13). The results presented inFig. 1A show that apoptotic pMac (HK Hc) at 2 � 106 and 4 �106 efficiently induced activation of functional CD8� andCD4� T cells. Although the numbers of IFN-�-producingCD8� and CD4� T cells did not reach the levels seen with themice receiving live Histoplasma, they were significantly higherthan those seen with mice receiving heat-killed Histoplasmaalone (Fig. 1A) (P 0.001 for CD8� and P 0.05 for CD4�).Moreover, CD8� T cells expressed not only IFN-� but alsogranzyme B (Fig. 1B). These results indicate that heat-killedHistoplasma antigens contained within apoptotic macrophagesare efficiently delivered to cross-prime CD8� T cells and acti-vate CD4� T cells in vivo.

The results presented in Fig. 2A further revealed that im-munization with apoptotic macrophages mixed with heat-killedHistoplasma apoptotic (pMac � HK Hc), viable macrophageswith ingested heat-killed Histoplasma pMac (HK Hc), apop-totic macrophages (apoptotic pMac), or viable macrophages(pMac) alone did not induce T cell responses at a level com-parable to that of immunization with apoptotic pMac (HK Hc).It is noteworthy that apoptotic pMac simply mixed with heat-killed Histoplasma before injection (apoptotic pMac � HKHc) did not induce a strong CD8� or CD4� T cell response(Fig. 2A), suggesting that Histoplasma antigens must be con-tained within apoptotic macrophages for antigen presentation.Moreover, immunization with apoptotic neutrophils contain-ing heat-killed Histoplasma [apoptotic pNeu (HK Hc)] was asefficient as apoptotic pMac (HK Hc) at inducing a strongCD8� or CD4� T cell response (Fig. 2B), which indicates that,after Histoplasma yeasts are taken up by the phagocytes, thefungal antigen contained within the phagocytes can be pre-sented to activate CD8� and CD4� T cells. Interestingly, im-munizing mice with apoptotic pMac (HK Hc) lacking MHC-Imolecules (�2m�/�) activated CD8� T cells as efficiently aswild-type macrophages did (Fig. 2C), indicating that apoptoticcells serve as antigen donor cells to deliver Histoplasma anti-gens rather than as antigen-presenting cells in the immunizedhost.

CD8� and CD4� T cells make equal contributions to hostdefense after immunization with apoptotic macrophages con-taining heat-killed Histoplasma. Next, mice immunized withapoptotic pMac (HK Hc) were challenged with Histoplasmaintravenously. Figure 3A shows that immunized mice had sig-nificantly lower fungal burdens in the spleen compared withunimmunized mice (CM; P 0.01) at day 10 after challenge.Depleting either the CD8� population T cell or the CD4� Tcell population in mice immunized with apoptotic pMac (HKHc) at the time of challenge significantly increased the fungal

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burden by 61-fold or 74-fold, respectively, and depleting bothpopulations increased T cell fungal burden by 490-fold (Fig.3B). These results, together with those presented in Fig. 1 and2, demonstrate that immunization with apoptotic pMac (HKHc) effectively induces activation of protective CD8� cells andthat the contribution of CD8� T cells to host defense againsthistoplasmosis is comparable to that of CD4� T cells.

Reduction of macrophage apoptosis is due to iNOS defi-ciency decreases CD8� T cell activation. To explore the rela-tionship between macrophage cell death and CD8� T cellactivation, iNOS�/� and wild-type mice were intratracheallyinfected with Histoplasma. Figure 4A shows that the number ofapoptotic F4/80� cells in the lungs of infected iNOS�/� micewas significantly lower than in the wild-type mice (P 0.05).Coinciding with lower numbers of apoptotic F4/80� cells in thelungs, the percentage of IFN-�-producing CD8� T cells wasalso significantly lower in iNOS�/� mice than in wild-type mice(Fig. 4B) (P 0.05). Interestingly, the CD4� T cell response iniNOS�/� mice was not affected by the reduction in numbers ofapoptotic F4/80� cells (Fig. 4B). The results of the macro-phage phagocytosis assay and stimulation of CD8� T cells withanti-CD3 and anti-CD28 antibodies showed that macrophagesand CD8� T cells from iNOS�/� mice were functionally com-parable to those of wild-type mice (data not shown). Addition-ally, bone marrow-derived dendritic cells from iNOS�/� miceexpressed more MHC-I and CD40 but fewer MHC-II mole-cules on the surface than cells from the wild-type mice afterinfection with Histoplasma yeasts (data not shown). These ob-servations excluded the possibility of an intrinsic defect ofmacrophages or of the antigen presentation functions of den-dritic cells in the iNOS�/� mice affecting CD8� T cell activa-tion. These results together indicated that iNOS, possiblythrough production of nitric oxide, is the cause of macrophageapoptotic cell death and that macrophage death is importantfor CD8� T cell activation in cases of pulmonary infection byHistoplasma.

Inhibition of apoptotic cell death by a caspase inhibitordecreases CD8� T cell responses and exacerbates pulmonaryhistoplasmosis. To further demonstrate the importance ofapoptosis of antigen donor cells in activation of CD8� T cellresponses after pulmonary infection, we treated infected micewith the caspase inhibitor Boc-D-FMK (26). The number ofapoptotic F4/80� cells seen at the early phase after infectionwas significantly (P 0.05) reduced in mice treated with Boc-D-FMK compared with the number seen after treatment with

FIG. 1. Immunization with apoptotic macrophages containing His-toplasma induces CD8� and CD4� T cell activation. Thioglycolate-elicited peritoneal macrophages were allowed to ingest heat-killedHistoplasma before treatment with LPS and ATP to induce apoptosis.Mice were inoculated subcutaneously with live Histoplasma (Live Hc)(2 � 106), heat-killed Histoplasma (HK Hc) (2 � 106), or apoptoticmacrophages containing heat-killed Histoplasma [Apoptotic pMac(HK Hc)] (4, 2, 1, or 0.2 � 106) on days �14 and 0. Control mice weregiven medium (CM) alone. At day 5, cells were isolated from inguinallymph nodes and stimulated with heat-killed Histoplasma for 24 h.(A) Lymph node cells were stained with FITC-anti-CD4, APC-anti-CD8, and PE-anti-IFN-� antibodies and analyzed by flow cytometry.Bar graphs indicate the percentages of IFN-�� CD8� or IFN-��

CD4� T cells in the total CD8� or CD4� T cell population. The datashown represent the means standard deviations (SD) of the resultsobtained with a total of 4 mice used in 3 separate experiments, and thedata from all 4 mice are presented. The P values were obtained bycomparing the percentages of IFN-�-producing cells determined forthe mice receiving HK Hc and 4 different doses of apoptotic pMac(HK Hc) by a post hoc Tukey test. *, P 0.05; **, P 0.01; ****, P 0.001; NS, not significant. (B) Lymph node cells were stained withAPC-anti-CD8 and PE-anti-granzyme B (grB) antibodies or PE-mouseIgG1 as an isotype control. The percentages of granzyme B-producingCD8� T cells in the total CD8� cell population were analyzed (*, P 0.05; NS, not significant). The data shown represent the means SDof the results obtained with 5 mice used in 3 independent experiments.



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FIG. 2. Apoptotic phagocytes containing Histoplasma induce T cell responses independent of donor cell MHC-I. (A) Thioglycolate-elicitedperitoneal macrophages were allowed to ingest heat-killed Histoplasma before treatment with LPS and ATP. Mice were subcutaneously inoculatedwith 2 � 106 of apoptotic pMac (HK Hc) [Apoptotic pMac (HK Hc)] or nonapoptotic pMac [pMac (HK Hc)] containing heat-killed Histoplasma,apoptotic pMac [Apoptotic pMac] or nonapoptotic pMac (pMac) without ingested heat-killed Histoplasma, or apoptotic pMac mixed withheat-killed Histoplasma [Apoptotic pMac � HK Hc] at days �14 and 0. Cells from inguinal lymph nodes were harvested at day 5 and stained withFITC-anti-CD4, APC-anti-CD8, and PE-anti-IFN-� antibodies. IFN-�-producing CD8� and CD4� T cells were analyzed by flow cytometry. Datashown represent the means SD of the results obtained with 4 mice used in 3 independent experiments. The P values were obtained by comparingthe percentages of IFN-�-producing cells in the five groups by a post hoc Tukey test. *, P 0.05; **, P 0.01; ****, P 0.001. (B) Peritonealmacrophages (pMac) or neutrophils (pNeu) were allowed to ingest heat-killed Histoplasma [pMac (HK Hc)] or [pNeu (HK Hc)] or mix withheat-killed Histoplasma ([pMac � HK Hc] or [pNeu � HK Hc]) before apoptosis was induced by LPS and ATP treatment or UV irradiation. Micewere subcutaneously inoculated with the apoptotic cells as described for panel A, and the CD8� and CD4� T cell responses elicited were analyzed.Mice inoculated with PBS or live Histoplasma served as experimental controls. Data shown represent the means SD of the results obtained with4 mice used in 2 independent experiments. The P values were obtained by comparing the percentages of IFN-�-producing cells in the four groupsof mice receiving apoptotic cells by a post hoc Tukey test. *, P 0.05; NS, not significant. (C) Peritoneal macrophages from wild-type (WT) or�2 microglobulin-deficient (�2m�/�) mice were allowed to ingest heat-killed Histoplasma before treatment with LPS and ATP. Wild-type mice wereimmunized with apoptotic pMac (HK Hc) that had been obtained from either wild-type or �2 microglobulin-deficient mice. The immunizationschedule, time of experiment, and staining were the same as described for panel A. Cells harvested from mice receiving wild-type macrophagescultured in medium only (CM) served as controls. Data shown represent the means SD of the results obtained with 4 mice used in 3 independentexperiments. ****, P 0.001; NS, not significant.

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Z-FA-FMK control peptide (23) (Fig. 5A). Interestingly, andconsistent with what we observed in iNOS�/� mice, the CD8�

T cell response but not the CD4� T cell response was reducedin mice whose macrophage apoptosis was inhibited (Fig. 5B).Notably, Boc-D-FMK treatment resulted in a higher fungalburden than Z-FA-FMK treatment (Fig. 5C). These resultstogether indicate that macrophage apoptosis is critical forCD8� T cell activation and subsequent fungal clearance inHistoplasma infection.

CD8� T cell responses in mice subcutaneously immunizedwith viable Histoplasma yeasts correlate with infiltrating celldeath. It was previously reported that subcutaneous immuni-zation with live Histoplasma yeast cells elicits protective CD8�

T cell responses even in the absence of CD4� T cells (32).Whether induction of a protective CD8� T cell response cor-relates with antigen donor cell death in this mouse model wasa question to be addressed. Immunofluorescence analysis re-vealed that the cellular infiltrates in the subcutaneous tissues

FIG. 3. Immunization with apoptotic macrophages containing His-toplasma confers CD8� and CD4� T cell-dependent protection againstchallenge. (A) Wild-type mice were inoculated subcutaneously withlive Histoplasma (Live Hc) (2 � 106 yeast cells/mouse), apoptoticmacrophages containing heat-killed Histoplasma [Apoptotic pMac(HK Hc)] (2 � 106 cells/mouse), or medium (CM) as described for Fig.2A. At day 5, mice were challenged intravenously with 2.5 � 104 liveHistoplasma yeasts. Fungal burden in the spleen was assessed 10 daysafter challenge. (B) Wild-type mice were inoculated subcutaneouslywith apoptotic pMac (HK Hc) as described for panel A. At the daybefore challenge and twice weekly thereafter, mice were treated withdepleting antibodies against either CD4 or CD8 or both. Fungal bur-den in the spleen was assessed 10 days after challenge. (A and B) Datarepresent the means SD of the results obtained with 5 or 6 mice pergroup. The P values were obtained by comparing the results by a posthoc Tukey test (**, P 0.01; ****, P 0.001; NS, not significant).

FIG. 4. iNOS deficiency reduces F4/80� cell apoptosis and weak-ens CD8� T cell response in pulmonary histoplasmosis. Wild-typeand iNOS�/� mice were infected intratracheally with 2 � 105 liveHistoplasma. (A) At day 5 after infection, lung cells were isolatedand stained with PE-anti-F4/80 antibody and TUNEL reagents con-taining FITC-dNTP. F4/80� TUNEL� apoptotic cells were ana-lyzed by flow cytometry. Data shown represent the means SD ofthe total numbers of apoptotic F4/80� cells determined for 3 miceused in 3 independent experiments. (B) At day 10 after infection,the mediastinal lymph nodes were harvested and cells were stainedwith PE-anti-IFN-� and FITC-anti-CD4 or APC-anti-CD8 antibod-ies. Data shown represent the means SD of the percentages ofIFN-�-producing CD8� or CD4� T cells in the total CD8� or CD4�

T cell population determined for 6 mice used in 3 independentexperiments. Cells harvested from uninfected wild-type mice servedas controls. The P values were obtained by comparing the resultsdetermined for pairs of groups (linked by a bracket) using Student’st test (*, P 0.05; NS, not significant).



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(Fig. 6A) were mostly Gr-1� and F4/80� cells, some CD11c�

cells, and very few B220� and CD3� cells (Fig. 6B and C). Asingle-cell suspension prepared from the digested subcutane-ous tissues contained mainly Gr-1� (68.9%) and F4/80�

(18.9%) cells (Fig. 6B), confirming that granulocytes and mac-rophages represent the major cell populations at the site ofsubcutaneous inoculation. It is noteworthy that both infiltrat-ing granulocytes and macrophages became apoptotic (Fig. 6D)and that granulocytes and macrophages constituted 77.6% and17.2%, respectively, of the total apoptotic cell populations atday 5 after inoculation (Fig. 6E). Moreover, subcutaneousinoculation of iNOS�/� mice with viable Histoplasma inducedCD4� T cell but not CD8� T cell activation (Fig. 6F). To-gether, these results point to the importance of apoptotic celldeath during the early phase of Histoplasma infection in in-ducing protective CD8� T cell responses and fungal clearance.

CD11c� cells are required for CD8� T cell cross-priming.We further investigated the role of host dendritic cells in pre-senting antigens contained within apoptotic macrophages toactivate CD8� T cells. Mice were subcutaneously immunizedwith CFSE-labeled apoptotic pMac (HK Hc). Five days afterimmunization, CFSE-labeled apoptotic macrophage cytoplas-mic materials were found inside CD11c� cells in the draininglymph nodes (Fig. 7A). Furthermore, depleting CD11c� cellsin CD11c-DTR transgenic mice by diphtheria toxin treatmentbefore inoculation of apoptotic pMac (HK Hc) significantlyreduced the percentage of IFN-�-producing CD8� by 6.5-foldcompared with the results seen with wild-type mice treatedwith diphtheria toxin (Fig. 7B). These results indicate that hostCD11c� dendritic cells at the inoculation site take up thefungal antigens contained within the apoptotic macrophagesafter immunization with apoptotic pMac (HK Hc) and migrateto the draining lymph nodes to cross-prime CD8 T cells.

DISCUSSION

Histoplasma capsulatum is a facultative intracellular fungalpathogen of the macrophage (16). It has long been recognizedthat the macrophage serves as the host cell as well as the

FIG. 5. Inhibition of F4/80� cell apoptosis reduces CD8� T cellresponse and increases fungal burden. Wild-type mice were treateddaily with Boc-D-FMK apoptosis inhibitor or Z-FA-FMK control pep-

tide or left untreated starting at the day of intratracheal inoculation of2 � 105 live Histoplasma. (A) Lung cells isolated from infected mice atday 4 after infection were stained with TUNEL reagents containingFITC-dNTP and PE-anti-F4/80 antibodies. Apoptotic F4/80� cells inthe lungs were analyzed by flow cytometry. Data shown represent themeans SD of the total numbers of F4/80� cells determined for 3mice used in 3 independent experiments (*, P 0.05; NS, not signif-icant by Student’s t test). (B) Mediastinal lymph node cells were har-vested at day 10 after infection. Cells were stained with PE-anti-IFN-�and APC-anti-CD8 or FITC-anti-CD4 antibodies and analyzed by flowcytometry. Data shown represent the means SD of the percentagesof IFN-�-producing CD8� and CD4� cells in the total CD8� or CD4�

T cell population determined for 5 mice used in 4 independent exper-iments (**, P 0.01; NS, not significant by Student’s t test). (C)Fungal burdens in the lungs of infected mice were assessed at day 10after intratracheal infection. The CFU levels determined for infectedmice treated with Z-FA-FMK and those treated with Boc-D-FMKwere compared to the CFU levels determined for infected mice with-out treatment. The data are presented as percent change of CFU andshown as the means SD of the results obtained with a total of 5 or6 mice per group (***, P 0.005 by Student’s t test).



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effector cell for Histoplasma yeasts (17, 30, 31). The results ofour in vitro studies showed that ingestion of Histoplasma yeaststriggers macrophage apoptotic cell death and that the apop-totic macrophage serves as a Histoplasma antigen donor tocross-prime CD8� T cells (14). In this study, we showed thatimmunization with apoptotic pMac (HK Hc) or apoptoticpNeu (HK Hc) activates both CD8� and CD4� T cells. Re-markably, immunization with apoptotic macrophages or neu-trophils mixed with killed yeasts (apoptotic pMac � HK Hc orapoptotic pNeu � HK Hc) or with viable macrophages con-

taining killed yeasts [pMac (HK Hc)] does not efficiently acti-vate CD8� and CD4� T cells (Fig. 2A and B). In infection byHistoplasma, iNOS deficiency and apoptosis inhibitor treat-ment decrease the number of apoptotic macrophages andweaken CD8� T cell but not CD4� T cell responses. Theresults of our study demonstrate that apoptosis of phagocytesis an immune mechanism that is of critical importance toCD8� T cell activation in histoplasmosis.

Infection-induced apoptosis is a newly recognized immunefunction important to antimicrobial immunity, especially in

FIG. 6. CD8� T cell responses in mice subcutaneously immunized with Histoplasma correlate with infiltrating cell death. (A to E) Wild-typemice were injected subcutaneously with live Histoplasma at day 0, and subcutaneous tissues were collected at day 5. (A) The subcutaneous tissuesat the inoculation site were harvested. The tissues were subjected to cryosectioning and stained with hematoxylin and eosin (H&E) (magnification,�40). The circled area shows cellular infiltration in the inoculation site. (B) Subcutaneous tissues of the inoculation sites were treated with typeI collagenase to obtain single cells. Isolated cells were stained with FITC-anti-CD11c and PE-anti-Gr-1 or PE-anti-F4/80 antibodies and analyzedby flow cytometry. The numbers indicate the percentages of CD11c�, Gr-1�, or F4/80� cells in the total cell population. The data shown are fromone mouse and are representative of the results of two independent experiments. (C) Cryosections of the subcutaneous tissues at the inoculationsite were prepared, and the cell populations were determined by staining with PE-anti-Gr-1, PE-anti-F4/80, or PE-anti-CD3 (red) and FITC-anti-CD11c or FITC-anti-B220 (green) antibodies and Hoechst stain 33258 (blue). Images were viewed under a confocal microscope. Bars, 40 �m(magnification, �630). (D) Cryosections of the subcutaneous tissues at the inoculation site were stained with TUNEL reagents containingFITC-dNTP and PE-anti-Gr-1 or PE-anti-F4/80 antibodies. Images were viewed under a confocal microscope. The arrowheads point to apoptoticgranulocytes or macrophages where TUNEL-reactive nuclei are adjacent to Gr-1� or F4/80� staining. Bars, 40 �m (magnification, �630). (E) Cellsisolated from subcutaneous tissues at days 3, 5, and 7 after inoculation of Histoplasma were stained with TUNEL reagents and PE-anti-F4/80 orPE-anti-Gr-1 antibodies. The percentages of apoptotic macrophages (TUNEL� F4/80�) and granulocytes (TUNEL� Gr-1�) were analyzed by flowcytometry. The bar graphs show the means SD of the percentages of apoptotic macrophages or granulocytes in the total TUNEL� apoptoticcell populations. (F) Wild-type and iNOS�/� mice were injected subcutaneously with live Histoplasma at days �14 and 0. Cells were isolated andstained as described for Fig. 1. Data shown represent the means SD of the results obtained with 6 mice used in 3 independent experiments (***,P 0.005; NS, not significant by Student’s t test).



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defense against phagosome-enclosed pathogens (21, 27, 33).Different host and fungal factors are known to be involved inHistoplasma-induced macrophage death. Our unpublisheddata showed that Histoplasma yeasts induced macrophageiNOS expression. Coinciding with those results, we have shownin the present report that Histoplasma infection-induced mac-rophage apoptosis is significantly reduced in iNOS�/� micecompared to wild-type mice (Fig. 4). These findings togetherdemonstrate that iNOS induction contributes to macrophagedeath. On the fungus side, both the yeast cell wall component�-(1,3)-glucan and the secreted calcium-binding protein (CBP)have been previously identified as Histoplasma virulence fac-tors (19). Silencing �-(1,3)-glucan biosynthesis reduces theability of Histoplasma yeast to cause macrophage-like P388D1cell death (18), and a cbp1-null mutant lost the ability to killP388D1 cells in culture in vitro (11). Although it is not clear

whether Histoplasma yeast cell �-(1,3)-glucan and secretedCBP cause macrophages to produce nitric oxide, it appearsthat a built-in system exists in the interaction between thefungus and macrophage to ensure macrophage death, whichwe showed is essential to CD8� T cell cross-priming.

We have showed in the present report that macrophages andgranulocytes in subcutaneous tissues as well as macrophages inthe lungs constitute the major cell populations during the earlyphase of subcutaneous and pulmonary infection, respectively,and undergo apoptosis (Fig. 6 and 4). The percentages ofinfiltrating CD3� T cells, in contrast, are low at early timepoints (data not shown). Allen and Deepe analyzed the apop-totic cell populations in the lungs of Histoplasma-infected miceat days 7, 14, and 21 after infection (2), which represent theactivation and contraction phases of T cell response (13), andfound that apoptotic CD3� T cells were the major apoptoticcells (2, 13). Their study showed that inhibition of apoptosis isassociated with elevation of interleukin-4 (IL-4) and IL-10levels and that the release of those cytokines exacerbates theseverity of infection. We found that administration of Boc-D-FMK at the beginning of infection reduced the number ofapoptotic macrophages by about 50% at day 4 and dampenedthe subsequent CD8� T cell but not the CD4� T cell response(Fig. 5A and B). This causal relationship demonstrates thatmacrophage apoptotic cell death at the early phase of infec-tion, the time when antigen processing, presentation, andcross-presentation take place, modulates the protective CD8�

T cell response. Our results and those of Allen and Deepetogether stress that apoptosis at the antigen processing andpresentation phases as well as the T cell contraction phase iscritical for protective immune responses in histoplasmosis.

It has been reported that immunization of mice with apop-totic vesicles prepared from BCG-OVA-infected macrophagesinduces adoptively transferred OT-1 CD8� T cell activationand that such mice are protected against challenge by Myco-bacterium tuberculosis (28). Injection of apoptotic antigen-pulsed dendritic cells induces both humoral and cellular im-mune responses and confers protection against Toxoplasmagondii presented as an oral challenge (4). In this study, weshowed that immunization with apoptotic pMac (HK Hc) in-duces activation of functional CD8� and CD4� T cells andthat CD8� and CD4� T cells contribute equally to efficientclearance of Histoplasma. These results together suggest thatapoptotic cells delivering antigens to elicit functional and pro-tective CD8� and CD4� T cell responses can be used toimmunize against intracellular bacterial and parasitic as well asfungal pathogens.

After subcutaneous injection of live Histoplasma, there ismassive granulocyte infiltration and 60 to 80% of the cells areapoptotic at days 3 to 7 after inoculation (Fig. 6A to E).Apoptotic neutrophils are as efficient as apoptotic macro-phages at delivering Histoplasma antigens to activate CD8�

and CD4� T cells (Fig. 2B). Granulocyte infiltration in thespleen after systemic infection or in the lungs after intratra-cheal infection is not as pronounced as it is in the subcutaneoustissues after local infection (S.-H.H. personal observation).The contribution of apoptotic neutrophils to cross-presenta-tion in infection through the natural routes may not be asimportant as that of the macrophages. However, utilizing

FIG. 7. CD11c� cells are critical for CD8� T cell activation afterimmunization with apoptotic macrophages containing Histoplasma.(A) CFSE-labeled peritoneal macrophages were allowed to ingestheat-killed Histoplasma before LPS and ATP treatment. Wild-typemice were inoculated subcutaneously with apoptotic pMac (HK Hc).Five days after inoculation, the inguinal lymph nodes were snap-frozenand subjected to cryosectioning. The sections were stained with PE-anti-CD11c MAb and viewed under a confocal microscope. The ar-rowheads point to CFSE-labeled macrophage cytoplasmic materialsthat were contained within CD11c� cells. (B) Wild-type mice andCD11c-DTR transgenic mice were intraperitoneally administered 100ng of diphtheria toxin at days �1 and 2 and inoculated subcutaneouslywith apoptotic pMac (HK Hc) at day 0. Draining lymph node cellswere harvested at day 5 and stimulated with suboptimal concentrationsof plate-bound anti-CD3 (0.5 �g/ml) and anti-CD28 (0.5 �g/ml) anti-bodies for 5 h. IFN-�-producing CD8� T cells were analyzed by flowcytometry. Data shown represent the means SD of the results ob-tained with 4 mice used in 3 independent experiments. Lymph nodecells from wild-type mice inoculated with medium only (CM) served ascontrols. ***, P 0.005 by Student’s t test.



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apoptotic granulocytes or neutrophils as antigen donors forimmunization is also a feasible option.

In summary, we showed that phagocyte apoptosis during theearly phase of Histoplasma infection is important for activationof functional CD8� T cells. Immunization with apoptoticpMac (HK Hc) or, alternatively, apoptotic pNeu (HK Hc)activates both CD8� and CD4� T cells and protects againstfungal challenge. Thus, exploiting the role of apoptotic phago-cytes as antigen donor cells is a viable strategy for the devel-opment of efficacious vaccines against histoplasmosis.

ACKNOWLEDGMENTS

The work was supported by National Science Council grants NSC94-2320-B-002-064, 95-2320-B-002-027, and 96-2320-B-002-011 toB.A.W.-H., National Science Council Fellowship grants NSC 94-2811-B-002-076, 95-2811-B-002-073, and 96-2811-B-002-069 to J.-S.L., andSouthern Taiwan Science Park grant EY13151099 to C.-L.C.

We acknowledge the support provided by the Seventh Core Labo-ratory of the Department of Medical Research of the National TaiwanUniversity Hospital.

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